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Conjunctival epithelial uptake of biodegradable nanoparticles: Mechanism, intracellular distribution, and absorption enhancement

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Conjunctival epithelial uptake of biodegradable nanoparticles: Mechanism, intracellular distribution, and absorption enhancement

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Content CONJUNCTIVAL EPITHELIAL UPTAKE OF BIODEGRADABLE NANOPARTICLES: MECHANISM, INTRACEULLAR DISTRIBUTION, AND ABSORPTION ENHANCEMENT by Mohamed Ghazi Qaddoumi A Dissertation Presented to the FACULTY OF GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHARMACEUTICAL SCIENCES) May 2004 Copyright 2004 Mohamed Ghazi Qaddoumi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3140540 Copyright 2004 by Qaddoumi, Mohamed Ghazi All rights reserved. INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 3140540 Copyright 2004 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProOuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION To my wife, parents, sisters, and daughter Layal for their uneonditional love and support 1 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I would like to express my gratitude and sincere appreciation to my mentor, Professor Vincent Lee for his guidance and assistance during the course of my graduate studies in his laboratory. Dr. Lee’s passion for work as well his leadership roles and excellence in pharmaceutical research contributed greatly to the scientific community in industry and academia. It was also the prime reason for my eagerness to join his lab. I would also like to thank my dissertation committee professors; Dr. Wei-chang Shen, Dr. Eric Lien, Dr. Chiang-peng Chang, and Dr. Robert Koda for their support and constructive criticism during the course of my research studies. I would also like to thank Dr. Vinod Labhasetwar for providing the nanoparticle formulation and for his useful guidance. I would also like to thank all my lab teammates for their assistance and useful discussions and for making my stay in the lab very enjoyable and worthwhile. I wish to acknowledge the financial support provided by the University of Kuwait that helped me get into the Ph.D. program; by the Graduate School of Pharmacy, University of Southern California, Los Angeles; by National Institute of Health; and by Association of Research in Vision and Ophthalmology/National Eye Institute. Finally, I wish to acknowledge the endless support, affection, and advice received from my wife, parents, and sisters to seek higher goals and values in this life and who made this possible. Ill Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS CONTENTS PAGE DEDICATION........................................................................................... ii ACKNOWLEDGEMENTS.................................................................... iii LIST OF TABLES.................................................................................... viii LIST OF FIGURES................................................................................... ix ABSTRACT............................................................................................... xiii I. INTRODUCTION.............................................................................. 1 1. FUNCTIONAL ANATOMY OF THE CONJUNCTIVA .... 2 2. IMPORTANCE OF THE CONJUNCTIVA.......................... 4 2.1. Electrolyte and fluid transport properties............................ 5 2.2. Transporters and drug transport properties......................... 8 3. OCULAR DRUG DELIVERY................................................. 10 3.1. Conventional ocular drug delivery constraints.................... 14 3.1.1. Pre-ocular retention............................................... 14 3.1.2. Comeal absorption................................................. 15 3.2. Formulation approaches to improve ocular dmg absorption 16 3.2.1. Bioadhesive hydrogels........................................... 16 3.2.2. Prodmgs.................................................................. 19 3.2.3. Collagen Shields or inserts.................................... 20 3.2.4. Penetration enhancers............................................. 20 3.2.5. Cyclodextrins......................................................... 21 3.2.6. Colloidal systems................................................... 22 A. Liposomes....................................................... 22 B. Nanoparticles.................................................. 23 4. POLYMERIC DRUG DELIVERY SYSTEMS.................... 24 4.1. Biodegradable polymers for dmg delivery.......................... 26 4.2. Poly (DL-lactide-co-glycolide) copolymers....................... 28 4.2.1. History................................................................... 28 4.2.2. Degradation profile.............................................. 29 4.2.3. Ophthalmic PLGA dmg delivery systems 30 4.3. General and ocular therapeutic applications of nanoparticles 32 IV Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3.1. Protection of unstable drugs.............................. 32 4.3.2. Absorption enhancement of poorly soluble drugs 34 4.3.3. Reduction of drug side effects............................ 34 4.3.4. Tumor and inflamed tissue targeting................. 36 4.3.5. Improvement of drug bioavailability in the body 37 A. Endocytic capability..................................... 38 B. Bioadhesive nature....................................... 38 C. Avoidance of efflux pum ps......................... 38 D. Overcome biological barriers...................... 39 4.3.6. Controlled release of drugs for chronic conditions 39 4.3.7. Stimulation of immune response for vaccine purposes............................................................... 40 4.4. Previous ocular studies with nanoparticles........................ 41 4.5. Nanoparticle uptake characteristics in other epithelia 43 4.5.1. Factors affecting uptake...................................... 43 4.5.2 Intestinal penetration pathway of nanoparticles .. 46 4.5.3 Systemic distribution............................................. 46 II. STATEMENT OF THE PROBLEM............................................ 48 1. GOALS AND OBJECTIVES................................................. 49 2. SPECIFIC A IM S..................................................................... 50 III. CHAPTER I. ELUCIDATION OF THE ENDOCYTIC PATHWAYS INVOLVED IN PLGA NANOPARTICLE UPTAKE 56 1. INTRODUCTION AND PURPOSE.............................................. 57 2. MATERIALS AND METHODS..................................................... 59 2.1. Materials................................................................................ 59 2.2. Animal M odel....................................................................... 61 2.3. Primary air-interfaced culture of rabbit conjunctival epithelial cells...................................................................... 61 2.4. Bioelectric measurements.................................................. 63 2.5. Nanoparticle uptake study and analjdical method 63 2.6. Evaluation of nanoparticle endocytosis............................ 65 2.6.1. Effect of energy depletion and endocytosis inhibitors........................................................... 65 2.6.2. Confocal microscopy......................................... 65 2.6.3 Stimulation of fluid-phase endocytosis............. 66 2.7. Involvement of clathrin and caveolae in nanoparticle endocytosis........................................................................ 66 2.7.1. Inhibition of clathrin- and caveolin-mediated endocytosis......................................................... 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.7.2. Immunofluorescence and confocal microscopy. 67 2.7.3. RNA isolation and reverse transcription- polymerase chain reaction................................. 68 2.7.4. Western analysis of clathrin HC and caveolin-1 69 2.7.5. Transfection with antisense oligonucleotide against clathrin H C ............................................. 70 2.8. Data analysis............................. ........................................ 70 3. RESULTS......................................................................................... 71 3.1 Evidence for nanoparticle endocytosis............................. 71 3.1.1. Effect of energy depletion and endocytosis inhibitors............................................................ 71 3.1.2. Confocal microscopy......................................... 72 3.1.3. Stimulation of fluid-phase endocytosis 73 3.2. Elucidation of the endocytic pathway........................... 75 3.2.1. Inhibition of clathrin- and caveolin-mediated endocytosis......................................................... 75 3.2.2. Immimofluorescence studies............................. 78 3.2.3. Molecular evaluation of clathrin and caveolin gene expression................................................... 78 3.2.4. Knockdown of clathrin HC by antisense oligonucleotides.................................................. 83 4. DISCUSSION.................................................................................... 87 4.1. Involvement of endocytosis............................................... 87 4.2. Elucidation of the endoc5dic mechanisms........................ 91 IV. CHAPTER II. INTRACEULLAR DISTRIBUTION AND TRAFFICKING OF PLGA NANOPARTICLES.............. 100 1. INTRODUCTION........................................................................... 101 2. MATERIALS AND METHODS................................................... 104 2.1. Materials............................................................................. 104 2.2. Immunofluorescence colocalization studies..................... 104 2.3. Effect of agents that disrupts the endosomes/lysosomes.. 105 2.4. Determination of nanoparticle residence within RCECs .. 106 2.5. Formulation of protein-loaded nanoparticles.................... 106 2.6 Characterization of HRP-loaded nanoparticles................. 108 2.7. In vitro release of HRP from nanoparticles...................... 108 2.8. Enhancement of HRP uptake............................................. 109 3. RESULTS........................................................................................ 110 VI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.1. Intracellular distribution and trafficking of nanoparticles 110 3.2. Sustained residence time of nanoparticles...................... 120 3.3. Enhancement of HRP uptake........................................... 121 3.3.1. Characterization of HRP-loaded nanoparticles 121 3.3.2. Nanoparticles as proteetive carriers of HRP ... 122 4. DISCUSSION................................................................................ 125 4.1. Intracellular distribution and trafficking.......................... 125 4.2. Nanoparticles as carriers of proteins............................... 132 V. CHAPTER III. ENHANCEMENT OF NANOPARTICLE ABSORPTION VIA LECTIN-MEDIATED RECEPTOR ENDOCYTOSIS.............................................................................. 135 1. INTRODUCTION...................................................................... 136 2. MATERIALS AND METHODS.............................................. 138 2.1. Materials........................................................................ 138 2.2. Characteristics of lectin binding and uptake................ 138 2.3. Confocal microscopy.................................................... 140 2.4. Effect of lectin on cytokine induction......................... 140 2.5. Conjugation of lectin to nanoparticles and uptake of conjugate....................................................................... 142 3. RESULTS.................................................................................... 144 3.1. Characteristics of lectin binding and uptake............. 144 3.2. Internalization of Solarium tuberosum lectin............. 147 3.3. Effect of lectin on cytokine induction........................ 150 3.4. Uptake of lectin-nanopartiele conjugate.................... 151 4. DISCUSSION............................................................................ 154 4.1. Lectins binding and uptake characteristics................ 154 4.2. Safety of leetin use in the e y e.................................... 158 4.3. Lectins as endocytic ligands for macromolecules .... 159 VI. OVERALL CONCLUSIONS.............................................. 163 1. SUMMARY OF FINDINGS....................................... 164 2. SIGNIFICANCE OF FINDINGS............................. 167 3. FUTURE DIRECTIONS............................................ 170 VII. REFERENCES...................................................................... 174 V ll Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES TABLE PAGE Table 1 Summary of transporters and their substrates in rabbit conjimctival epithelium......................................................... 9 Table 2 Classifications of current ocular drug delivery systems utilized.................................................................................. 17 Tables Biodegradation of lactide/glycolide polymers..................... 29 Table 4 Experimental use of PLGA for ocular drug delivery 33 Table 5 Examples of nanoparticles used in ocular drug delivery ... 44 Table 6 Physicochemical factors affecting epithelial nanoparticle uptake.................................................................................... 45 Table 7 Effect of nanoparticles on stimulation of fluid phase endocytosis of Lucifer yellow ............................................. 75 Table 8 Physiochemical characteristics of HRP-loaded nanoparticles....................................................................... 122 Table 9 Model plant lectins and their affinity parameters in RCECs.................................................................................. 144 Table 10 Summary of cytokine data in RCECs.................................. 152 V lll Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES FIGURE PAGE Figure 1 Sectional view of the anterior segment of human e y e 3 Figure 2 Summary of putative active ion and transport processes in pigmented rabbit conjunctival epithelial cells............... 6 Figure 3 Cross section of the human ey e........................................... 11 Figure 4 Ocular penetration routes for topically applied drugs 13 Figure 5 Schematic representation of different breakdown mechanisms of biodegradable polymers............................ 27 Figured Chemical structure of PLGA copolymer............................. 28 Figure 7 Cyclosporin A (CyA) concentration in the conjunctiva after topical administration in rabbits of CyA-loaded chitosan (CS) nanoparticles and control formulations consisting of a CyA suspension in a CS aqueous solution and a CyA suspension in w ater........................................... 35 Figure 8 Concentration of '"’C-poly (hexyl cyanoacrylate) nanoparticles in healthy (open symbols) or inflamed rabbit eyes (closed symbols)............................................... 37 Figure 9 Schematic diagram of the multiple endocytic pathways in mammalian cells............................................................. 51 Figure 10 Schematic representation of PLGA nanoparticles and composition........................................................................ 59 Figure 11 Effect of energy depletion and vesicle transport inhibitors on nanoparticle uptake in RCEC culture 72 Figure 12 Confocal microscopy of RCECs after nanoparticle uptake 73 Figure 13 Uptake of Lucifer yellow in rabbit conjunctival epithelial cells....................................................................................... 74 IX Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 14 Effect of pharmacological treatments on intemalization of PLGA nanoparticles in RCECs..................................... 76 Figure 15 Effect of pharmacological treatments on intemalization of transferrin in RCECs...................................................... 77 Figure 16 Effect of pharmacological treatments on intemalization of cholera toxin B subunit in RCECs................................ 77 Figure 17 Confocal microscopy of RCECs following uptake of PLGA nanoparticles............................................................ 79 Figure 18 RT-PCR analysis of clathrin heavy chain and caveolin-1 mRNA expression............................................ 80 Figure 19 Nucleotide sequence of amplified rabbit clathrin HC gene fragment...................................................................... 81 Figure 20 Westem blot analysis of clathrin HC and caveolin-1 expression........................................................................... 82 Figure 21 Knockdown of clathrin HC protein expression................. 84 Figure 22 Nanoparticle uptake in clathrin-knockout RCECs 85 Figure 23 Intemalization of transferrin in clathrin-knockout RCECs 86 Figure 24 Schematic representation of endocytic pathways in polarized cells................................................................... 102 Figure 25 Lack of colocalization of nanoparticles and early endosomes in RCECs...................................................... I l l Figure 26 Selective eolocalization of nanoparticles and early endosomes in ARPE-19 cells.......................................... 112 Figure 27 Lack of colocalization of nanoparticles and late endosomes in RCECs...................................................... 113 Figure 28 Selective colocalization of nanoparticles and lysosomes in RCECs.......................................................................... 114 Figure 29 Trafficking of nanoparticles to lysosomes is transient in RCECs.......................................................................... 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 30 Colocalization of nanoparticles and lysosomes in ARPE-19 cells............................................................... 116 Figure 31 Selective colocalization of nanoparticles and Beta-COP in RCECs...................................................... 117 Figure 32 Selective colocalization of nanoparticles and Golgi marker in RCECs............................................................ 118 Figure 33 Absence of colocalization of nanoparticles and endoplasmic reticulirai marker in RCECs..................... 119 Figure 34 Effect of disruption of endosomes and lysosomes on nanoparticle uptake......................................................... 120 Figure 35 Sustained release properties of PLGA nanoparticles .... 121 Figure 36 % Cumulative in vitro release of HRP from PLGA nanoparticles.................................................................... 123 Figure 37 PLGA nanoparticles as carriers of H R P......................... 124 Figure 38 A summary diagram of SearchLight''^'^ rat cytokine array technique................................................................ 141 Figure 39 Effect of incubation time on uptake/binding of FITC -lectins in RC EC ............................................................. 145 Figure 40 Effect of concentration on binding/uptake of FITC- lectins in MDCK cells.................................................... 146 Figure 41 Effect of concentration on specific binding/uptake of FITC-lectins in RCEC..................................................... 147 Figure 42 Amount ofFITC-lectin internalized in RCEC............... 148 Figure 43 Evidence for intemalization of Solanum tuberosum lectin in RCECs................................................................. 149 Figure 44 Evidence for intemalization of STL into intermediate cell layers....................................................................... 150 XI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 45 Effect of STL and histamine on cytokine levels in RCECs........................................................................... 151 Figure 46 ST lectin enhances apical uptake of HRP-nanoparticles 152 Figure 47 Effect of STL concentration on uptake of HRP- nnoparticles.................................................................. 153 XU Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT This investigation was prompted by the potential application of biodegradable PLGA nanoparticles as ocular carriers for protein and drug delivery. The overall goal is to improve the mechanistic understanding of nanoparticle transport and trafficking within the conjimctival epithelium to facilitate the design of successful nanoparticle systems capable of enhancing the delivery of proteins and dmgs to the conjunctiva and other intraocular tissues. Our data provided evidence for the endocytosis of PLGA nanoparticles in rabbit conjunctival epithelial cells (RCECs), as demonstrated by the uptake inhibition in the presence of microfilament inhibitors and metabolic poisons, the vesicular uptake pattern seen under confocal microscopy, and the stimulatory effect on endocytosis of the fluid phase marker, Lucifer yellow. Evidence is presented for the mechanism of PLGA nanoparticle uptake in RCECs. Based on clathrin knockout studies, we demonstrated lack of a direct involvement of clathrin heavy chain in endocytosis of PLGA nanoparticles. However, inhibition studies using pharmacological treatments (hypertonicity and intracellular depletion), the partial colocalization of clathrin staining seen under confocal microscopy, and the incomplete knockout of clathrin protein suggest a minor involvement of clathrin in nanoparticle endocytosis. We have provided the first evidence for the expression of clathrin heavy chain at the protein and gene levels in rabbit conjunctival epithelial cells, whereas caveolin-1 expression was not xiii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. detected. Since macropinosomes are involved in endocytosis of larger particles (> 500 nm), our findings implicate mainly non-coated vesicles and clathrin-coated vesicles partially in the intemalization of PLGA nanoparticles in RCECs. Immunofluorescence staining studies delineated the intracellular distribution and trafficking behavior of PLGA nanoparticles following endocytosis. Nanoparticles were shown to escape endosomal trafficking, reside for a short while in the lysosomes, and localize to the Golgi compartment during their trafficking in RCECs. We have also provided evidence for the utility of using PLGA nanoparticles for protein dmg delivery and controlled dmg delivery in the conjunctival epithelium, and probably to other intraocular tissues. Finally, the feasibility of using lectins fi'om Solanum tuberosum (potato) to augment nanoparticle absorption in RCECs has been highlighted and shown to be efficacious and safe of ocular use as dmg carriers. XIV Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I. INTRODUCTION Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. FUNCTIONAL ANATOMY OF THE CONJUNCTIVA The conjunctiva is a thin, transparent mucous membrane lining the inner surface of the eyelids that extends to the sclera (Figure 1). It functions as a protective barrier against the permeation of pathogens and exogenous drugs, in the exchange of nutrients and solutes with the cornea, and in the excretion of solutes and endogenous enzymes contributing to tear content (Dilly, 1985). The critical role of the conjunctiva (along with the cornea) in the defense of the oeular surface arises from its highly vascularized nature, its ability to initiate and contribute to inflammatory defense reactions, and the presence of immunocompetent cells. For instance, the conjunctiva-associated lymphoid tissue, which has unique struetural and functional characteristics, appears to be involved in generating rapid immune responses to ocular surface antigens (Chodosh et al., 1998). In addition, the conjxmctiva contains at least three phenotypically distinet populations of myeloid antigen-presenting eells that share the unusual characteristic of being both MHC class II- and B7-negative (Baudouin et al., 1997). The conjunctiva is made up of three different layers from outer to interior structure of the eyelid: (i) an outer epithelium that is many layers in thickness and is considered the major permeability harrier; (ii) the substantia propria containing stmctural and cellular elements, nerves, lymphatie systems, and blood vessels; and (iii) the submucosa, which provides loose attachment to the underlying sclera tissue. The epithelium layer is divided morphologically into three distinct epithelia: the bulbar, continuous with the eomea, the fornix, and the palpebral epithelium. The Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sections of the conjunctiva covering the posterior surface of the eyelids and anterior surface of sclera are termed the palpebral and bulbar conjunctiva, respectively (Figure 1). The fornix is the area where the palpebral and bulbar segments meet. The palpebral conjunctiva is firmly attached to the lids, while the bulbar portion is loosely attached to the underlying sclera and folded several times. Lacrimal gland Fornix _ conjunctiva Sclera Bulbar __ conjunctiva Cornea Palpebral__ conjunctiva Figure 1. Sectional view of the anterior segment of human eye. The conjunctival epithelium is adjacent to the comeal epithelium and is a stratified epithelium that is squamous in nature at the eyelids and columnar towards the cornea. The number of cell layers of the conjunctival epithelium varies from region to region, being 10-15 layers at the cornea, 5-6 layers on the lids, and about 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. layers in the bulbar and fomiceal areas (Latkovic and Nilsson, 1979b). Like the cornea, the conjunctiva has three main epithelial cell types: superficial, wing, and basal cells. Wedged within the superficial cells are goblet and Langerhans cells. Goblet cells fimction in secreting mucin and glycoproteins, which provide the protective layer for the conjunctiva and cornea. Whereas, Langerhans cells are involved in antigen recognition and processing and stimulation of T-cell lymphocytes. The superficial conjunctival epithelium has numerous microvilli covered by glycocalyx and mucin produced by goblet cells (Pfister, 1975). The conjunctival epithelium displays tight barrier properties to drugs attributed mainly to the presence of tight jimctions and desmosomes between adjacent epithelial cells and hemidesmosomes between epithelial cells and basal lamina layer. As a result of insult to the conjunctival epithelium, basal cells will move upwards to compensate for any cellular defects followed by mitosis (Geggel et al., 1984). 2. IMPORTANCE OF THE CONJUNCTIVA Tremendous research related to the physiology and biochemistry of the conjimctiva has been conducted in the last decade that led to a plethora of information. The fact that the conjunctival epithelium behaves as both secretory and absorptive tissue eneouraged studies aimed at identifying biological membrane carriers and drug transport properties and pathways involved. In addition, the conjunctiva fimctions as a conduit for topical and systemic drug delivery and may Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. have implication in some ocular diseases (Ahmed and Patton, 1985;Chang and Lee, 1987). The following section summarizes recent relevant findings in the field and their implication. 2.1. Electrolyte and fluid transport properties In 1973, Maurice was the first who used rabbit conjunctival sac in vivo to measure Na^ and CT transport (Maurice, 1973). He found that the conjunctiva was highly permeable to both these ions and that an electrical potential difference was developed across the conjunctiva that should cause these ions to be absorbed. Subsequent studies using voltage clamp technique, Kompella et al. (Kompella et al., 1993) demonstrated that the excised pigmented rabbit conjunctiva is a tight barrier epithelium capable of active chloride transport. The potential difference (PD) was 17.7 ± 0.8 mV (tear-side negative), the short eireuit current was 14.5 ± 0.7 pA/cm^, and the transepithelial electrieal resistance (TEER) was 1.3 ± 0.1 kQ.cm^. Utilizing similar technique and specific chaimel inhibitors, they indicated the presence of a Cl' conductive pathway on the mucosal side of the conjunctiva, whereas Na^/K^- ATPase, Na^/KV2Cl' eotransport process, and conductive pathways were present on its serosal side (Figure 2). In addition, their findings advocated that amiloride- sensitive Na^-conductive pathways do not appear to be present on either side of the pigmented rabbit conjunctiva. Furthermore utilizing similar teehniques, Kompella et al. (Kompella et al., 1995) demonstrated the possible existence of a Na^-amino acid co-transport system on the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. apical side of the pigmented rabbit conjunctiva. Moreover, the existence of a Na^- glucose co-transport system on the mucosal side of pigmented rabbit conjunctiva that contributes to sodium absorption and short circuit current was demonstrated (Hosoya et al., 1996;Horibe et al., 1997a). Sbiue et al. (Sbiue et al., 1998) indicated that Cl' enter the pigmented rabbit conjunctiva from the serosal fluid via Na^/KV2Cl' co transporter and is actively secreted via different channels from the mucosal side. This active chloride secretion was linearly correlated with short circuit current and subject to modulation by second messengers (such as cAMP, C a " * ^ ^ , and protein kinase C) and nucleotides (Sbiue et al., 1998;Hosoya et al., 1999). Apical P-gp substrates / Tight Junctions Basolateral Nuc eoside G ucose Monocarboxylate D ipephde Organic cation MRP substrates Figure 2. Summary of putative active ion and transport processes in pigmented rabbit conjimctival epithelial cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In addition, the pigmented rabbit eonjunctiva exhibited net fluid secretion properties with a baseline rate of 4.3 ± 0.2 pl/br/cm^ in the serosal to mucosal direction (Sbiue et al., 2000). This net fluid secretion was coupled to active Cl' secretion rate and affected by compounds that affect active chloride secretion. These findings may be of relevance in pathological conditions affecting conjunctiva fluid flow such as inflammation, infection, and dry eye disease where stimulation of transconjunctival fluid secretion may be of therapeutic value. Evidence for involvement of chloride and fluid secretion in ocular eye diseases came from the findings of Kulkami et al. (Kulkami et al., 2003). They demonstrated that in adenovirus-5 infected rabbit model, both net fluid and chloride secretions were markedly reduced as a consequence and that these levels were restored by mucosal application of pyrimidine nucleotides such as UTP. Contrary to its stimulatory effect on chloride transport in the rabbit comeal epithelium, 5- hydroxytryptamine (5-HT) elicited a prompt and sustained inhibition of transcellular chloride movement in the basolateral to apical direction due to independent downregulation of apical chloride and basolateral potassium conductances (Klyce et al., 1982;Alvarez et al., 2001). One membrane protein channel that is thought to contribute to active chloride secretion is that of cystic fibrosis transmembrane conductance regulator (CFTR). Recently, CFTR expression at the gene and protein levels in rabbit conjunctival epithelium and apical localization was demonstrated (Tumer et al., 2002;Shiue et al., 2002). It is believed to be regulated by cAMP and is thought to be implicated in dry 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. eye syndrome. Identification of acid base transporter protein (both Cl'/HCO^' and Na^/H^; AE-2 and NHE-1 exchangers) in both rabbit and porcine conjunctival epithelium was demonstrated using Westem blot analysis (Tumer et al., 2001). These proteins were localized to the basolateral side of plasma membrane using histochemical and immunostaining studies. These findings suggest that the conjimctiva is not involved actively in the regulation of tear pH, rather it contributes to intracellular pH regulation. 2.2. Transporters and drug transport properties The dynamic nature of the pigmented conjunctiva is represented by the existence of several receptors (Table 1) at the apical membrane for CFTR, epidermal growth factor (EOF), insulin-like growth factor I (IGF-I), and serotonin (5-HT-IB and ID) in the pigmented rabbit conjunctiva (Tumer et al., 2002;Shiue et al., 2002;Narawane and Lee, 1995;Tumer et al., 2003). IGF-1 receptor is also capable of binding to IGF-II and insulin, whereas EGF receptor is also capable of binding to TGF-a. The serotonergic receptor subtypes expressed in the rabbit conjunctiva were, however, fewer than that of the human conjunctiva, which expressed 5-HT(lF) and 5-HT(7) as well. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD ■ D O Q. C o CD Q. ■ D CD C/) W o' o o 5 CD O O ■ D c q ' o o o "n c o CD ■ D O Q. C a o o ■ O o CD Q. ■ D CD (/) (/) Table 1. Summary of transporters and their substrates in rabbit conjimctival epithelium Transporter type Localization Examples of substrates/drugs/ions Reference EGF Apical EGF and TGF-a (Narawane and Lee, 1995) IGF-1 Apical IGF-I, IFF-II, and insulin (Narawane and Lee, 1995) 5HT- IB /ID Unknown Serotonin (Tumer et al., 2003) CFTR Apical Cr and Na^ (Tumer et al., 2002) Monocarboxylate Apical NSAID, fluoroquinolones, and cromolyn (Horibe et al., 1998) Nucleoside Apical Uridine, antiviral drugs (Hosoya et al., 1998) Dipeptides Apical P-lactam antibiotics, bestatin, and ACE (Basu et al., 1998) Organic cations Apical Brominidine and carbacbol (Ueda et al., 2000) P-gP Apical Propranolol, progesterone, & cyclosporin A (Yang et al., 2000a) MRP 1 Basolateral Doxorubucin, vincristine, glutathione Yang et al. (unpublished) NaVsolute Apical Amino acids and glucose (Hosoya et al., 1996) >0 In addition, various active transport systems were reported on the apical surface of the rabbit pigmented conjunctiva (Table 1) for Na^-coupled solutes and substrates, nucleosides, monocarboxylates, dipeptides, glutathione, and organic transport cation drugs (Horibe et al., 1997a;Hosoya et al., 1997;Hosoya et al., 1998;Horibe et al., 1998;Basu et al., 1998;Gukasyan et al., 2002;Ueda et al., 2000). Examples of monocarboxylate drugs are non-steroidal anti-inflammatory compounds and fluoroquinolone antibacterial drugs, whereas the amines, anti-glaucoma drugs dipiverfme and brimonidine are examples of organic cation drugs. Moreover, multi-drug resistant (MDR) efflux pumps such as p-glycoprotein (P- gp) on the apical plasma membrane of the conjunctival epithelium was shown to exist in the pigmented conjunctiva, thus representing additional barriers to the transport of cyclosporin A and other lipophilic drugs such as propranolol, progesterone, and verapamil (Saha et al., 1998;Yang et al., 2000a). 3. OCULAR DRUG DELIVERY The human eye is a very special organ, containing vascularized areas such as the cillilary processes, conjunctiva, and choroid, and non-vascular areas such as the cornea and lens all of which have special functions. For treating anterior ocular disorders, topical administration into the conjimctival cul-de-sac is usually the preferred route of delivery (Figure 3). Topical ocular administration is usually aimed for two purposes: to treat superficial eye diseases, such as infection (i.e. conjunctivitis, belpharitis, keratitis sicca, dry eye), and to treat intra-ocular diseases 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. through comeal absorption such as glaucoma or uveitis. Oral or intravenous administration is often unsatisfactory because of the presence of the blood-aqueous barrier (BAB), which prevents dmgs from entering into the aqueous humor, and the blood-retinal barrier (BRB), which may limit the entry of dmg into the extravascular retinal space and the vitreous body of the eye from the blood (Cunha-Vaz, 1979). Furthermore, due to dilution effect of the blood, a high dose has to be administered in order to achieve an adequate concentration in the vitreous, and this may cause systemic toxicity. lit; p D M K liin Figure 3, Cross section of the human eye. Other routes of administration, such as intravitreal injection or intraocular delivery devices, are used for delivery of dmgs to posterior segment tissues such as the retina (Kurz and Ciulla, 2002). Although successful treatments of ocular diseases 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was achieved in many cases, however, such routes of administration require repeated injections or surgeries, provide discomfort and pain for patients, and do not encourage patient’s compliance. New research is indicating that trans-scleral drug delivery could be an effective new approach to get drugs into the posterior segment (Geroski and Edelhauser, 2001). Topically applied drugs can reach the intraocular tissues by either the comeal and/or the noncomeal (conjunctival-scleral) pathway (Figure 4). The major route for dmg absorption is traditionally believed to be the comea, despite the tight barrier properties of the epithelium. However, the conjunctiva does play an important role in both ocular (Ahmed and Patton, 1985) and systemic absorption (Chang and Lee, 1987;Lee et al., 1993) of topically applied ophthalmic dmgs. The bulbar conjunctiva is the first tissue across which a topically applied dmg must pass in order to reach the underlying tissues in the uveal tract via the non-comeal route (Ahmed and Patton, 1985). Nonetheless, the conjunctiva still shows higher permeability to dmgs, in general, than the comea for many reasons. First, the surface area of conjunctiva in both rabbit and human is 9 and 17 times greater than that of the comea, respectively (Watsky et al., 1988). Second, the paraceullar pore size in the conjimctiva is estimated to be 230 times larger than that of the comea, thus allowing higher molecular weight hydrophilic compounds such as inulin and others to pass through (Hamalainen et al., 1997). In addition, the metabolic activity of several enzymes in the conjunctival epithelium of rabbit is lower than that in the comeal epithelium, thus reducing the 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. barriers to drug transport in this tissue (Lee et al., 1988;Stratford and Lee, 1985;Lee, 1983). SCLERA CORNEA CONJUNCTIVA ANTERIOR CHAMBER PRECORNEAL AREA CONTRALATERAL EYE OCULAR CIRCULATION SYSTEMIC CIRCULATION EsFTRAOCULAR TISSUES Figure 4. Ocular penetration routes for topically applied drugs. Key: 1 = transcomeal pathway, 2 = noneomeal pathway, 3 = systemic retum pathway, 4 = lateral diffusion. From Ahmed and Patton (1985). 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.1. Conventional ocular drug delivery constraints Though topical administration offers many advantages to treat disorders of anterior structures of the eye, it suffers from a serious disadvantage of poor bioavailability due to several biological factors, which exist to protect the eye and consequently limit the entry of ocular drugs. This results in low ocular absorption (only 1-3% of drug absorbed) mainly due to low residence time in the eye (less than 5 min) and the low permeability to the comea (Lang, 1995). The constraints in topical delivery of the eye are discussed below in details. 3.1.1. Pre-ocular retention It has been estimated that the human eye can hold approximately 30 pi of an ophthalmic solution without overflow or spillage at the outer angle while the volume delivered by most commercial ophthalmic eye drop dispensers is approximately 50 pi (Mishima et al., 1966). Thus, a large proportion of the dmg is wasted due to administration of an excess volume. Following the removal of the excess solution from the front of the eye, a second mechanism of clearance prevails. The eye has an efficient system for tear tumover (~1 pi /min). The two mechanisms of clearance result in a biphasic profile for an instilled solution with a rapid initial clearance phase due to removal of excess fluid followed by a slower second phase due to tear tumover (Chrai et al., 1973;File and Patton, 1980). Another serious route for the elimination of topically applied dmgs from the precomeal area is the nasal cavity, with its larger surface area and a high 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. permeability of the nasal mucosal membrane as compared to that of the comea. Ocular drugs are prone to absorption into systemic circulation through the nasal mucosal lining, which is continuous with the conjunctival sac (Desai, 1994). The contribution of the nasal mucosa to systemic absorption of topically applied beta- adrenergic antagonists in pigmented rabbits was shown to exceed 50% and to diminish with increasing drug lipophilicity (Lee et al., 1993). Nonetheless, changes in the formulations of some ocular dmgs aimed at prolonging the retention time in the conjunctival cul-de-sac will probably be effective in reducing their systemic absorption. 3.1.2. Corneal absorption The main route for intraocular absorption is across the comea (Ahmed and Patton, 1985). Two features, which render the comea an effective barrier to dmg absorption, are its small surface area and its relative impermeability. For instance, the conjunctival and scleral tissues were 15 to 25 times more permeable to dmgs than the comea and the molecular size affected the conjunctival permeability less than that of the comea (Hamalainen et al., 1997). Thus, following topical administration to the pre-ocular area, conjunctival dmg absorption is an important loss factor that competes with comeal absorption (Lee and Robinson, 1979). Secondly, in terms of drag delivery, the comea can be considered to be comprised of three layers, which account for its poor permeability characteristics: (i) the outer epithelium, which is lipophilic in nature; (ii) the stroma, which constitutes 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. approximately 90% of the thickness of comea and is hydrophilic; and (iii) the inner endothelium consisting of a single layer of flattened epithelium-like cells. Since, the comea has both hydrophilic and lipophilic stractures, it presents an effective barrier to the absorption of both hydrophilic and lipophilic compounds. Age and disease can significantly alter drag penetration and ocular drag availability. For example, ageing of the comea alters topical drag delivery, and there is a breakdown in the blood- retinal barriers in several retinal diseases (Ke et al., 1999). The difference in permeability was more pronounced for large hydrophilic than small lipophilic compounds in the intact comeas. 3.2. Formulation approaches to improve ocular drug absorption Many non-invasive approaches have been proposed to increase ocular bioavailability of drags including the use of prodrags, penetration enhancers, bioadhesive hydrogels, cyclodextrins, collagen shield, and colloids such liposomes and nanoparticles. Examples of each system, their benefits, and disadvantages are shown in Table 2. 3.2.1. Bioadhesive hydrogels Bioadhesive polymers are usually macromolecular hydrocolloids with numerous hydrophilic functional groups (Robinson and and Mlynek, 1995). These include high molecular weight polymers such as the natural polysaccharide, hyaluronic acid (HA), and sjmthetic polymers such as sodium carboxymethylcellulose (CMC) and 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2. Classifications of current ocular drug delivery systems utilized. Ocular DDS Examples Advantages Disadvantages Reference Bioadhesive hydrogels HA* PAA* CMC PVA Chitosan Prolonged MRT, increased drug bioavail., non toxic*, anti oxidant, reduced toxic effects of preservatives*, commercially available, used as tear substitutes, lower systemic abs Discomfort & blurred vision, difficulty of accurate dosing, only modest dmg enhancement (Debbasch et al., 2002;Zignani et al., 1995;Snibson et al., 1990;Kyyronen and Urtti, 1990;Lehr et al., 1994;Felt et al., 1999) Prodrugs Nepafenac 0- palmitoyl tinsolol Enhanced comeal permeability, CNV treatment, prolonged MRT, protect against metabolism Tedious discovery, may not reduce systemic absorption (Takahashi et al., 2003;Kawakami et al., 2001;Chang et al., 1988) Collagen Shield or inserts Ocusert® Already on market, sustained drug release, therapeutically effective, improve bioavailability Not well tolerated, high cost, difficulty of inserting into eye (Robinson and Mlynek, 1995) Penetration enhancers EDTA, Brij®, bile salts, BAC Improve comeal & conjunctival absorption, reduce size of instilled drop Long ocular accumulation, irritant, damaging (Ashton et al., 1991;Saettone et al., 1996) CDs a and P CDs Beneficial for poorly soluble dmgs, eliminate local irritation, improve bioavailability Limited to lipophilic dmgs and by slow dissociation of CDs (Kanai et al., 1989;Reeretal., 1994) 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2. Classifications of current ocular drug delivery systems utilized (continued). Colloids Liposomes Biocompatible, already Liposomes (Pleyer et al.. Nanoparticles on market, ease of unstable in 1993;Losa et al.. formulation, incorporate vivo, only 1993;Calvo et al.. hydrophilic or stearylamine 1996a;Taniguchi et hydrophobic drugs. liposomes al., 1988;Zimmer et prolong MRT, enhance were toxic al., 1991 ;Liu et al.. drug absorption, reduce onlyPACA 1989) systemic side effects, no nanoparticles blurring, sustain drug were toxic to levels, biodegradable comeal cells Abbreviations: HA; hyaluronic acid; PAA; polyacrylic acid; CMC; carboxymethyl cellulose; PVA; poly vinyl alcohol; NPs; nanoparticles; CNV; choroidal neovascularization; BAC; benzalkonium chloride; Brij®; Polyoxyethylene fatty acid ether; DDS: drug delivery system; CDs: cyclodextrins; MRT: mean residence time (tear). polyacrylic acid (PAA). It is widely accepted that non-newtonian vehicles such as HA and PAA are more effective than viscous newtonian formulations containing polyvinyl alcohol or cellulose in a similar viscosity range (Greaves et al., 1992). These bioadhesive hydrogels (with the exception of polyvinyl alcohol, PVA) were shown to prolong residence time in the comea and led to higher drag bioavailability compared to commercial aqueous eye drop of the drug (Lehr et al., 1994;Snibson et al., 1990;Kyyronen and Urtti, 1990). Another bioadhesive polymer that has recently shown great potential is chitosan, which is a polycationic polymer. Chitosan is mucoadhesive, has penetration-enhancing properties, and favorable biological properties such as excellent biodegradability, ocular tolerance, and prolonged corneal residence (Lehr et al., 1992b;Dodane et al., 1999;Mi et al., 2002;Felt et al., 1999). 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Most ocular studies with chitosan involved either microspheres or nanoparticles, which will be discussed later. 3.2.2. Prodrugs An alternative to bioadhesive hydrogels is prodrugs. Prodrugs are bioreversible derivatives of drugs with the potential to alter absorption, decrease side effects, or prolong duration of action (Sinkula and Yalkowsky, 1975). Inactive as such, prodrugs rely on chemical or enzymatic conversion for expression of pharmacological activity. Wei and coworkers (Wei et al., 1978) were the first to utilize prodrugs in the eye when they formulated an analogue of epinephrine, dipivalyl epinephrine (DPE). This analogue was foimd to penetrate the comea 10- fold higher and reduced intraocular pressure (lOP) significantly at lower concentrations than epinephrine itself. Further studies with prodmgs by Chang and coworkers (Chang et al., 1988) demonstrated their superiority in enhancement of ocular timolol absorption compared to bioadhesive hydrogels, despite higher systemic absorption. Besides improvement of comeal penetration, prodmgs have the ability to protect dmgs fi*om enzymatic metabolism (Wang et al., 1991). The improved comeal penetration of prodmgs is attributed to either higher lipophilicity or higher affinity to membrane transporters such as dipeptide transporters (Wei et al., 1978;Dias et al., 2002). 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2.3. Collagen Shields or inserts Ocular inserts such as Ocusert® device (Alza Corp.) consists of a central reservoir of drug (e.g. pilocarpine) enclosed between two semipermeable membranes that allow drug diffusion for a week. This device has been successful in lowering intraocular pressure (lOP), but because of its insolubility it must be removed after use (Gurtler and and Gumy, 1995). This has led to the development of soluble inserts based on natural polymers such as collagen, made from porcine scleral tissue similar in composition to that of human comea. Collagen inserts were effective in improving the ocular absorption of several dmgs and providing adequate, sustained therapeutic levels as long as they are formulated with one dmg only (Lee et al., 1992;Mablberg et al., 1991;Unterman et al., 1988). However, due to difficulties related to the design and use of collagen shields, patient discomfort, and vision problems they are reserved to chronic ocular conditions requiring frequent therapy (Kaufman et al., 1994). 3.2.4. Penetration enhancers This approach consists of increasing transiently the permeability characteristics of the comea or conjunctiva with appropriate substances. There are two modes of action of penetration enhancers: Surface active agents are believed to increase permeability of the cell membranes, while calcium chelators act mainly on loosening tight junctions (although some enhancers may act on both). An example of calcium chelators is EDTA, which was shown initially by Grass and Robinson 20 Reproducecl with permission of the copyright owner. Further reproduction prohibited without permission. (Grass and Robinson, 1988) to double the ocular absorption of topically applied glycerol and cromolyn sodium. However, the effect of EDTA seems to be limited to hydrophilic drugs (Ashton et al., 1991). Examples of surfactants are non-ionic surfactants (Brij®35), bile salts (sodium deoxycholate), preservatives (benzalkonium chloride), glycosides (saponin), and fatty acids (capric acids). Most of these surfactants were shown to be effective in improving comeal and conjunctival permeability of several P-blockers, insulin, and peptides (Saettone, 1996;Hayakawa et al., 1992). However, these penetration enhancers can also penetrate the eye and may, therefore, lead to unknown toxicological complications, e.g. benzalkonium chloride (BAC) was found to accumulate in the comea for days, whereas bile salts and surfactants were found to cause irritation of the eye and nasal mucosa (Green, 1993;Merkus et al., 1993). 3.2.5. Cyclodextrins Cyclodextrins (CDs) are a group of homologous cyclic oligosaccharides with a hydrophilic outer surface consisting of several glucose units. Due to their solubility in water and lipophilic interior cavity, CDs is used to complex poorly soluble or imstable dmgs to improve their dissolution, distribution, and stability (Szejtli, 1994). They were introduced in ocular dmg formulations initially to increase the solubility of lipophilic dmgs in solution. Kanai et al. (Kanai et al., 1989) tested four different combinations of a-CDs with cyclosporin and found that 0.025% a-CDs-cyclosporin complex resulted in the least comeal toxicity and penetrated the comea 5-10 times 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. more than did the drug in a lipophilic vehicle. Other CDs, include hydroxypropyl-p- CD which was shown to increase the ocular permeability of a complex solution of diclofenac compared to commercial solution of Voltaren® (Reer et al., 1994). The mechanism of enhanced ocular permeability by CDs is unknown, but is attributed to increased viscosity of the solution and stabilization of drug. However, improvement of ocular absorption by CDs is limited to just lipophilic drugs and by the slow dissociation of the drug-CD complex in the lacrymal fluid causing release of the drug in the tear fluid (Usayapant et al., 1991). 3.2.6. Colloidal systems Colloidal carriers such as liposomes and nanoparticles have been widely studied with the objective of specific drug targeting, facilitate bioavailability of drugs, and to protect labile drugs against enzyme inactivation (Kaur and Smitha, 2002;Diepold et al., 1989a;Smolin et al., 1981). Colloidal carriers have demonstrated not only their ability to improve the ocular residence time of drugs but also increased permeability to either intra-ocular (aqueous and vitreous humor) or extra-ocular (conjimctiva and comea) tissues compared to other existing systems (Calvo et al., 1996a;De Campos et al., 2001;Pleyer et al., 1993). A. Liposomes Liposomes consist of one or more phospholipid bilayers enclosing an aqueous phase. They can be classified as large multilamellar liposomes (MLV), small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV), depending on 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. their size and the number of lipid bilayers. Water-soluble drugs can be included within the aqueous compartments, and lipophilic or amphiphilic compounds can be associated with the lipid bilayers (Gregoriadis and Ryman, 1971). It has been shown that the concerted action of duodenal enzymes and bile salts destroys the lipid bilayers of most types of liposomes, thus releasing the dmg (Woodley, 1985). Smolin and coworkers (Smolin et al., 1981) were the first to exploit the potential of liposomes in topical ocular dmg delivery and demonstrate the efficacy of doxuridine-liposome preparation over doxuridine solution in the treatment of acute and chronic herpetic keratitis. The benefits of liposome use over eonventional systems are their biodegradable and non-toxic nature, prolonged retetntion with the conjunctiva and comea surfaces thereby increasing dmg absorption, and their suitability for unstable, poorly absorbed dmgs (Schaeffer and Krohn, 1982;Liu et al., 1989). Positively charged liposomes seem to interact more favorably with comeal surface than neutral or negatively charged liposomes (Felt et al., 1999). Despite their potential for ocular dmg delivery, liposomes are not very popular due to their short half-life, limited dmg capacity, and problems in sterilization. B. Nanoparticles Nanoparticles are colloidal particles made from synthetic or natural polymers ranging in size from 10 nm to 1 pm. They consist of macromolecular materials in which the dmg is dissolved, entrapped, adsorbed, or dispersed (Kreuter, 1983). Nanoparticles is a colleetive name for nanospheres and nanocapsules. Nanospheres have a matrix type stmcture, where the active dmg can be adsorbed, entrapped, or 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dissolved in the matrix. Nanocapsules have a polymeric shell and an inner core and the active ingredient is usually dissolved in the core. Nanoparticles show some advantages over liposomes in terms of stability both during storage and in vivo. Other advantages of nanoparticles for ocular drug delivery include their prolonged retention in comeal and conjimctival surfaces, improvement of ocular dmg absorption, sustained dmg release, and reduced systemic toxicity (Wood et al., 1985;Calvo et al., 1996a;Losa et al., 1993;Giordano et al., 1993). The potential of nanoparticles for dmg delivery in the eye and other absorptive tissues will be explained in later sections. POLYMERIC DRUG DELIVERY SYSTEMS One of the primary objectives of dmg delivery systems is targeted controlled dmg delivery at an optimal therapeutic rate. This would result in improved efficacy of dmgs along with side effects reduction. Among the most promising systems to achieve this goal are colloidal dmg delivery systems. Colloidal dmg delivery systems include liposomes, niosomes, nanoparticles, micelles, and microemulsions. These systems are similar in size and mode of administration, but have different physicochemical properties. Liposomes, for instance, are made from natural body components such as lecithin and phospholipids making them biocompatible with the body. Nanoparticles are usually made of synthetic or natural polymer and are usually more stable. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Polymeric systems can be divided into two categories: (1) monolithic or homogenous systems in which the drug is dissolved or dispersed throughout the polymer matrix and release of the drug is controlled by surface erosion of polymer and (2) reservoir system in which the drug is entrapped hy a impermeable membrane layer, which governs the release of drug. Polymers may be classified into two types: non-degradable and biodegradable. Polymers based on poly (ethylene-co-vinyl acetate) are typical examples of the non-degradable polymeric drug delivery systems (Rawa et al., 1985) Examples of biodegradable systems for drug delivery (especially proteins) are nanoparticles or microspheres based on poly (lactic-co-glycolic acid) (PLGA). Use of biodegradable polymers relieves the patient from removing the drug delivery device system after the drug is depleted. Biodegradable polymers investigated for drug delivery purposes can be divided into synthetic or natural materials. The latter includes proteins (e.g. albumin and collagen), polysaccharides (e.g. chitosan and hyaluronic acid), and virus envelopes. Synthetic polymers include aliphatic polyesters of hydroxy acids [PLGA, poly (hydroxybutyric acid), poly (e-caprolactone)], poly (orthoesters), and poly (alkylcarbonates). Synthetic polymers are preferable since natural polymers vary in purity, require crosslinking in the microencapsulation method, and are hydrophilic causing rapid drug release. 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.1. Biodegradable polymers for drug delivery Biodegradable polymers are high molecular weight substances that can decompose into nontoxic monomers or low molecular weight fragments in the living organism. The term biodegradable is in some cases used interchangeably with hioerodihle and bioresorbable. However, the term bioerodible refers to polymers in which the degradation occurs at the surfaces of devices and the term bioresorbable is used in many cases where polymeric systems are removed via the normal metabolic pathway. Other terms such as bioabsorabable and bioassimilable are used to indicate polymers that degrade in biological environments. As shown in Figure 5, there are three types of degradation profiles for biodegradable polymers (Heller, 1984). Type I degradation refers to hydrolysis of water-insoluble hydrogels with unstable crosslinked bonds to yield water-soluble poljmiers. The cleavage of the linkages between the monomers result in disassembled polymer. Crosslinked poly(vinyl alcohol) can undergo type I erosion. In t)^e II degradation, water insoluble macromolecules are converted to water- soluble macromolecules by hydrolysis, ionization, and protonation of pendant groups. Because no backbone cleavage occurs, there materials are only useful for topical applications. Alkylmonoesters of poly(vinyl methyl ester/maleic anhydride) imdergo type II degradation. In type III degradation, high molecular weight, water- insoluble macromolecules are converted to small, water-soluble molecules by hydrolytic cleavage of the labile bonds in the polymer backbone. Since the degradation products of these polymers are completely non-toxic, they are used for 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. both systemic and topical administration. Polymers belonging to type III degradation category include aliphatic polyesters, polyamides, poly(cyanoacrylates), polyanhydrides, polyacetals, and poly(ortho esters). Among these, polylactic acid (PLA), polyglycolic acid (PGA), and PLGA are the most popular. Ifisalutelis aoltiblA r x T T T T Xamlialilo lE L S D X u J b l» s n a il sal-ubld im lcK SU liBkB ; Hydrolytically miBtable bands A i Hydrophobic subBtihuents Hydrolyala, ionization, ptrotonatlon Figure 5. Schematic representation of different breakdown mechanisms of biodegradable polymers. Adapted from Heller (1984). 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2. Poly (DL-lactide-co-glycoIide) copolymers 4.2.1. History Poly (DL-lactide-co-glycolide) copolymers (PLGA) were widely used as excipients for drug delivery as early as 1973. Their application as medical sutures in 1970’s demonstrated their beneficial features and potential in drug delivery. PLGA polymers are very lipophilic and dissolve in organic solvents. They can range in molecular weight from 10-150 kDa depending on the method of preparation. The chemical structure of such co-polymer is shown in Figure 6. O CH. O CH, C O CH C O n Figure 6. Chemical structure of PLGA copolymer. (n ~ 88-1306, however in our study n = 1263) Properties such as biodegradability, biocompatibility, ease of formulation, safety, and FDA approval of for commercial use attracted scientists to PLGA polymers (Gilding and Reed, 1979). In addition, the abundance of PLGA polymer materials has greatly expanded their scope as controlled drug delivery systems (Frazza and Schmitt, 1971). As a result, depot injectable microspheres of LH-RH agonists (leuprorelin acetate and tryptorelin) using biodegradable PLGA for the 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. treatment of advanced prostate cancer and endometriosis (Lurpron Depot and Decapeptil) are already on the market. 4.2.2. Degradation profile Since they fall under type III, PLGA polymers and others are highly desirable because they can be applied to the surface of the eye as well as given as implants. The biodegradation of PLGA polymers occur by hydrolysis of ester groups (could be enzymatic) producing monomeric acids, which are eliminated from the body through the Krebs cycle primarily as carbon dioxide and in urine. The 50:50 PLGA polymer mixture has the fastest degradation time, degrading in about 60 days, with higher lactide content in the polymer resulting in increased degradation time (Table 3) (Lewis, 1990). Table 3. Biodegradation of lactide/glycolide polymers Polymer Approximate time for biodegradation (months)^ Poly (L-lactide) 18-24 Poly (DL-lactide) 12-16 Poly (glycolide) 2-4 50:50 (DL-lactide-co-glycolide) 2 85:15 (DL-lactide-co-glycolide) 5 90:10 (DL-lactide-co-glycolide) 2 “ Biodegradation times vary depending on implant surface area, porosity, and molecular weight. (Adapted from Lewis, 1990) 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The degradation rate of PLGA matrices depends on several factors including the preparation method, the intrinsie properties of the polymer (moleeular weight, chemical structure, eopolymer composition, tacticity, hydrophobicity, and crystallinity), and extrinsic physicochemical properties such as pH, temperature, ionic strength of the environment, and the presence of other additives and stabilizers (Park, 1994;Park, 1995;0'Hagan et al., 1994;Tracy et al., 1999). For example, incorporation of lactic acid, which is a hydrophobic component, increases the duration of a polymer in the body by deereasing the polymer water uptake. Higher crystallinity results in lower water uptake, aeeordingly, resulting in slow degradation rate. PLGA degradation oeeurs through a process of autocatalytie hydrolysis of the ester bonds (Shive and Anderson, 1997). The acidic (lactic acid and glycolic acid) monomers and oligomers formed catalyze the further degradation of the parent polymer. Thus, any factor that influences the formation and/or retention of the acidic monomers in the particles could affect the polymer degradation rate and the in vitro release of the entrapped therapeutie agent. 4.2.3. Ophthalmic PLGA drug delivery systems PLGA-based systems were first investigated for potential use in ophthalmology in order to reduce the number of injeetions of drug into the vitreous body (Shell, 1978). Moritera and coworkers (Moritera et al., 1991) demonstrated the ocular safety of PLGA microspheres containing 5-fluorouracil injected into the vitreous eavity of rabbits. On average, the PLGA mierospheres were eompletely 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cleared by 48 days with no adverse effects. To evaluate the therapeutic potential of PLGA microspheres, Giordano and coworkers (Giordano et al., 1993) reported on the kinetics of retinoic acid release from PLGA microspheres and their antiproliferative effect after single injection in rabbit eyes. In vivo, the drug was released for more than 40 days and it significantly reduced the incidence of fractional retinal detachment in rabbits by 64% at the end of 8 weeks. Hashizoe and colleagues (Hashizoe et al., 1997) also demonstrated that intraviteal administration of a plug made of PLGA polymers and containing 25% ganciclovir to pigmented rabbits released ganciclovir up to a 10-week period. The vitreous therapeutic levels of the drug were shown to be adequate enough to treat cytomegalovirus retinitis for 12 weeks. In addition, no significant retinal toxicity was observed. These studies prove that it is feasible to deliver substances to the retina or the eye using biodegradable polymers made of PLGA and maintain adequate therapeutic levels. It also demonstrated that PLGA could be used to encapsulate very hydrophilic drugs, such as ganciclovir, into microspheres and remain stable. Another application of PLGA microspheres was to target drugs to retinal pigment epithelial (RPE) cells. Surface modification of PLGA with a fluorescent dye, rhodamine 6GX, resulted in phagocytosis by RPE cells and intracellular release (Moritera et al., 1994). Very few studies have utilized PLGA nanoparticles in ocular drug delivery with the exception of Marchal-Heussler and coworkers (Marchal-Heussler et al., 1992). Their findings showed that nanoparticles containing betaxolol based on 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. poly(epsilon-caprolactone) had the highest effect of lowering of intraocular pressure and the greatest potential for intraocular penetration compared with that of nanoparticles based on PLGA or polyisobutylcyanoacrylate (PBCA). Since that study, most literature studies have focused on other types of polymeric nanoparticles. However, the potential of PLGA nanoparticle for peptide, drug, and gene drug delivery has been reported in the intestine and other epithelial tissues (Kawashima et al., 2000;Panyam and Labhasetwar, 2003a;Lamprecht et al., 2001). Table 4 lists some examples of ophthalmic use of PLGA-based systems over the last 15 years. 4.3. General and ocular therapeutic applications of nanoparticles There are various potential applications of nanoparticles that depend on the route of administration of these colloidals systems and on the system studied. 4.3.1. Protection o f unstable drugs First for oral delivery of drugs, nanoparticles can be used to protect a labile drug from degradation in the gastrointestinal tract. Encapsulation of insulin in poly (alkylcyanoacrylate) nanocapsules was effective in reducing glycaemia in diabetic rats, which lasted up to 20 days (Damge et al., 1990). In vitro studies further confirmed these findings showing that nanocapsules protected insulin from degradation by digestive enzymes (Damge et al., 1997). 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4. Experimental use of PLGA for oeular drug delivery Dosage form Drug Results/response Reference Implant Fluorescein Na None (Olsen et al., 1989) Microspheres 5-fluorouracil Cleared in 48 days (Moritera et al., 1991) Microspheres Adriamycin Lower lOP (Kimura et al., 1992) Microspheres Cyclosporin A Therapeutic levels for 6 days in comea and AH (Harper et al., 1993) Matrix 5-fluorouracil Therapeutic levels for 21 days (Rubsamen et al., 1994) Scleral plug Gancyclovir Therapeutic levels for 12 weeks to treat CMV (Hashizoe et al., 1997) Nanoparticles; nanocapsules Betaxololo HCl Reduced lOP, but no prolonged residence (Marchal-Heussler et al., 1992) Microspheres Ara-C, 5-FU Detectable for 11 days (Peyman et al., 1992) Microspheres Retinoic acid Retained for 40 days and reduced TRD (Giordano et al., 1993) Implant Doxorubicin Remained in vitreous for 4 weeks, no toxicity (Hashizoe et al., 1994) Microparticles BSA Ocular immunization (Ridley Lathers et al., 1998) Microspheres implanted 5-FU Adequate levels for 1 week, low toxicity (Chiang et al., 2001) Microspheres PKC412 Remained for 20 days in retina and choroid (Saishin et al., 2003) Abbreviations: PKC412, a kinase inhibitor that blocks several isoforms of protein kinase C, Ara-C: cytosine arabinoside; CMV: cytomegalovirus retinitis; AH: aqueous humor; TRD: tractional retinal detachment; BSA: bovine serum albumin 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3.2. Absorption enhancement o f poorly soluble drugs A second application is the use of nanoparticles to improve the absorption of poorly water-soluble drugs. In absorptive epithelial tissues, the small submicron size of nanoparticles helps to increase the surface area and thereby facilitate drug dissolution. Topical instillation of 10 pi of cyclosporin A (CyA)-loaded chitosan (CS) nanoparticle formulation to rabbits in vivo achieved higher concentrations of cyclosporin A in external ocular tissues (comea and conjunctiva) compared to control formulation of either chitosan and CyA or CyA alone up to 48 hr after application (Figure 7) (De Campos et al., 2001). However, intraocular tissues (aqueous humor, iris ciliary body) had negligible amoxmts CyA levels indicating that the transport of chitosan nanoparticles across these two tissues may be limited. This is highly likely considering the impediment provided by the stromal layer and connective tissues to the diffusion of the hydrophilic chitosan nanoparticles. 4.3.3. Reduction o f drug side effects Thirdly, poly (D,L-Lactide) nanocapsules have been shown to be effective in protecting the GI mucosa from the ulcerating effects of non-steroidal anti inflammatory drags after oral administration (Guterres et al., 1995). In the eye, topical instillation of poly isobutylcyanoacrylate nanocapsules containing metipranolol base resulted in a drastic reduction of the drug's systemic side effects compared with the commercial eye drops (Losa et al., 1993). 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. C ^ A co««ralcw i m ti« oonjimctiva lug CyA/g cQipuKstiYal m m 4 c m m m m m fOOO '■ i^ te f td td ^ iw f io p a r td ^ CyA»ii}«»kmlniCS»liiti!iii t 4 TiDMsCW Figure 7. Cyclosporin A (CyA) concentration in the conjunctiva after topical administration in rabbits of CyA-loaded chitosan (CS) nanoparticles and control formulations consisting of a CyA suspension in a CS aqueous solution and a CyA suspension in water (* denotes statistically significant differences, P<0.05). (Adapted from De Campus et al., 2001). Similarly, the incorporation of carteolol into poly (epsilon-caprolactone) (PCL) nanocapsules produced a decline in the cardiovascular side effects in comparison with aqueous eye drops (Marchal-Heussler et al., 1993). This indicated that nanocapsules (or nanoparticles) were capable of reducing the undesired noncomeal absorption of carteolol. 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3.4. Tumor and inflamed tissue targeting Another application of nanoparticles is the passive targeting of anticancer drugs or antibiotics to solid tumors or inflamed tissues. This occurs due to the highly permeable nature of the tumor vasculature or the inflamed tissue that result in accumulation of the small sized nanoparticles into these sites. For example, intravenous administration of doxorubicin-loaded poly (isobutyl cyanoacrylate) nanoparticles to SI30 myeloma-bearing rats increased their survival rate significantly compared to free solution of doxorubicin (Couvreur et al., 1982). Similarly, entrapment of ampicillin in polyhexylcyanoacrylate nanoparticles was found to increase by 120-fold the efficacy of the antibiotic in experimental murine salmonellosis (Henry-Michelland et al., 1987;Fattal et al., 1989). In the eye, Diepold et al. (Diepold et al., 1989b) studied the association of poly (hexyl-2-cyanoacrylate) nanoparticles in normal and inflamed (using the betamethasone-induction model) conjunctiva and comeal tissues. The concentration of nanoparticles was four times higher in inflamed eyes and the ratio of nanoparticles in inflamed tissue to normal tissue was higher in conjunctiva than the comea between 60 and 240 min (Figure 8). Thus, dmg delivery through nanoparticles may be of therapeutic potential in the treatment of inflammation in extraocular tissues. 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N ic tita tin g M em brane 70 - Inf lamed eyes 5 0 - 30 - Normal o o 1 0 - 130 - Normal eyes C onjunctiva ^ C o rn e a o c = o 90 - 70 - ai o 50- Inf lamed eyes 30 10 A queous Humor Inflamed eyes Normal o 240 120 60 0 510 20 30 Time (min) Figure 8. Concentration of ^'*C-poly(hexyl cyanoacrylate) nanoparticles in healthy (open symbols) or inflamed rabbit eyes (closed symbols) (Diepold et al., 1989b) 4.3.5. Improvement of drug bioavailability in the body Moreover, nanoparticles are popular due to their ability to enhance the bioavailability of drugs in the body. This could be attributed to several properties, such as the their endocytic capability, their bioadbesive and retentive nature to epithelial surface, their proteolytic protection of unstable drugs, and their ability to overcome drug resistance and efflux phenomenon and barriers. 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A. Endocytic capability The endocytic capability of nanoparticles has been demonstrated by the increased indometbacin ocular bioavailability of 300% after instillation of nanoparticles made of poly-epsilon-caprolactone (PECL) (or emulsion) compared to microparticles of the same polymer system or commercial eye drops (Calvo et al., 1996a). The authors postulated that these nanoparticles penetrate the comeal epithelium by an endocytic mechanism as seen by confocal microscopy. In addition, a similar observation was shown for poly(isobutylcyanoacrylate) nanoparticles containing fluorescent rbodamine where they appeared in vesicles or granules suggesting endocjdosis by conjunctival epithelium (Zimmer et al., 1991). B. Bioadhesive nature The bioadbesive nature of nanoparticles was shown by the studies of Wood et al. (Wood et al., 1985) in which poly (bexyl-2-cyano-[3-C^'^] acrylate) nanoparticles bad an elimination half-life of 20 min in tear fluid following topical application to albino rabbit. It is this feature of nanoparticles along with their colloidal size that many believe contribute to drug delivery enhancement. C. Avoidance o f efflux pumps Interestingly, drug-loaded nanoparticle was shown to accumulate in cancer cell lines and appeared to avoid P-glycoprotein dependent efflux. For instance, the association of doxombicin with poly (alkylcyanoacrylate) nanoparticles reversed the 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. resistance to doxorubicin in a large number of multi-drug resistant cell lines (Soma et al., 1999;de Verdiere et al., 1997). This reversal was only observed in vitro, however, with poly (alkylcyanoacrylate) nanoparticles and was not the result of particle endocytosis. The authors concluded that multi drug resistance was overcome as a result of both the adsorption of nanoparticles to the cell surface and increased doxorubicin diffusion by the accumulation of an ion pair at the plasma membrane. D. Overcome biological barriers Another characteristic function of nanoparticles is their ability to delivery drugs across several biological barriers to the target site (Lockman et al., 2002;Fisher and Ho, 2002). The brain delivery of a wide variety of drugs, such as antineoplastics and anti-HIV drugs, is markedly hindered because they have great difficulty in crossing the blood brain barrier (BBB). Recently, it has been demonstrated that poly (butylcyanoacrylate) nanoparticles coated with polysorbate 80, are effective in transporting the hexapeptide delargin and other agents into the brain (Kreuter et al., 2003). The enhanced transport of delargin by poly butylcyanoacrylate nanoparticles was not due to their generalized toxic effects on brain endothelial cells as previously suggested (Olivier et al., 1999). 4.3.6. Controlled release o f drugs fo r chronic conditions Nanoparticles were also utilized to allow sustained drug release within the target site for days or even weeks. For instance, PLGA nanoparticles was first 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. investigated in ophthalmology in order to reduce the number of injections of drug (necessary to maintain therapeutic drug levels in diseases such as cytomegalovirus retinitis and endophthalmitis) into the vitreous body (Shell, 1978). Intravitreal administration of PLGA microspheres containing retinoic acid into rabbit eyes released therapeutic levels of retinoic acid for more than 40 days (Giordano et al., 1993). These microspheres significantly reduced the incidence of experimental tractional retinal detachment in rabbits by 64% at the end of 8 weeks. 4.3.7. Stimulation o f immune response fo r vaccine purposes Finally, several groups have reported the broader application of nanoparticles as a vaccine adjuvant to elicit mucosal immune response. This is because of their distinct uptake by M cells in the Peyer’s patches of the distal small intestine (Hussain et al., 2001). For example, Kim et al. demonstrated that multiple oral immunizations of mice with Helicobacter pylori-PLGA nanoparticles induced significantly H. pylori-specific mucosal IgA response as well as serum IgG response (Kim et al., 1999). This study confirmed that immrmological prevention of H. pylori infection using an oral vaccine is feasible. Similarly, the potential of PLGA nanoparticles for stimulation of anti-viral antibody responses following intranasal inoculation in mice was demonstrated. Mice immunized with PLGA nanoparticles containing proteins of bovine parainfluenza type 3 virus (BPI-3), developed higher levels of virus-specific antibody than mice immunized with the polymethylmethacrylate (PMMA) 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. nanoparticle vaccine eontaining BPI-3 or with soluble viral proteins alone (Shephard et al., 2003). 4.4. Previous ocular studies with nanoparticles Ticho et al. (Ticho et al., 1979) was the first to utilize pilocarpine-loaded nanospheres (Piloplex systems) using poly(methylmethacrylate-acrylic acid) copolymers. In clinical trials, Piloplex lowered intraocular pressure, but since these nanospheres were based on non-degradable polymers their further use was not investigated. Numerous ocular studies have investigated nanoparticles based on polycyanoacrylate polymer their derivatives with few studies focusing on nanoparticles made from chitosan and poly(lactic acid) polymers. Only one study was performed with PLGA nanoparticles to our knowledge (Marchal-Heussler et al., 1992). The potential of nanoparticles for ocular drug delivery developed as a result of their prolonged retention times in the comeal and conjunctival surfaces compared to commercial eye drops (Wood et al., 1985;Fitzgerald et al., 1987). However, only 0.1% of the initial amount of nanoparticles remained in the comea and conjunctiva after 6 hours. Interactions with the mucus layer coating the conjunctiva or comea was not likely to be the reason, as treatment of the eye with mucolytic agent (N- acetylcysteine) prior to nanoparticle application did not decrease nanoparticles concentration in either tissue (Wood et al., 1985). Further work by Harmia et al. (Harmia et al., 1986) on pilocarpine- loaded polybutylcyanoacrylate (PBCA) 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. nanoparticles demonstrated that the mitotie response was better when pilocarpine was adsorbed to the surface, rather than loaded into nanoparticles. This was attributed to slow degradation of PBCA nanoparticles in the tear. Li and coworkers (Li et al., 1986) confirmed this finding by showing that progesterone concentrations after administration of progesterone nanoparticles were lower than the control solution. Thus, it seems that a major problem with the use of alkylcyanoacrylate nanoparticles is the slow drug release. Marchal-Heussler and colleagues (Marchal-Heussler et al., 1990) studied the physicochemical properties affecting ocular administration of nanospheres and demonstrated that surface charge and binding type of the drug onto nanospheres were the most important factors in improving the therapeutic response to betaxolol. In another study, Marchal-Haussler and coworkers compared the efficiency of nanoparticles as a function of the type of polymer used (Marchal-Heussler et al., 1992). They tested three different nanoparticle formulations based on PLGA, poly(s- caprolactone), poly(isobutylcyanoacrylate) and showed that the decrease in intraocular pressure using the antiglaucoma loaded drug, betaxolol, was more pronounced with poly(s-caprolactone) nanoparticles. However, the mechanism seemed to be related to the agglomeration of poly(s-caprolactone) nanoparticles in the conjunctival sac. Furthermore, nanocapsules were shown to display better effect than nanospheres, probably because diffusion of the drug from the oily phase to the comea seemed to be more effective than diffusion from the internal matrix of the 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. nanospheres. Losa and coworkers (Losa et al., 1992;Losa et al., 1993) used poly(s- caprolactone) nanocapsules to encapsulate metipranolol and showed an enhanced therapeutic effect with lower drug concentration, thus minimizing systemic effects. Zimmer et al. (Zimmer et al., 1991) studied the transport pathway of poly (isobutylcyanoacrylate) nanospheres through the rabbit comea and conjunctiva using fluorescent dye. They observed that penetration of nanospheres was limited to just the first two layers in what appeared to be granules and vesicles and that a transcellular pathway, such as phagocytosis was likely to be involved. Similar findings was observed for the enhanced comeal absorption of cyclosporin-loaded poly(isobutylcyanoacrylate) nanocapsules, in that penetration was limited to superficial cells (Le Bourlais et al., 1997). Table 5 lists more ocular studies with nanoparticles and their findings. 4.5. Nanoparticle uptake characteristics in other epithelia 4.5.1 Factors affecting uptake The absorption of various nanoparticles prepared from different polymers was shown to depend on physicochemical characteristics and the non-specific interactions of the nanoparticles with the epithelia. Various factors have been shown to affect nanoparticle uptake including size, surface charge, Hydrophobicity, and stabilizer (surfactant) content (Table 6). 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD ■ D O Q. C o CD Q. ■ D CD C/) W o' o o Table 5. Examples of nanoparticles used in ocular drug delivery O O ■ D c q ' o o o " n c o CD ■ D O Q. C a o o ■ O o CD Q. ■ D CD ( / ) ( / ) Polymer Drug loaded Ocular results or findings Reference PBCA Amikacin Higher concentrations in comea and AH (Losa et al., 1991) PBCA Rhodamine-6G Particles seen in conjunctiva & comea top 2 layers (Zimmer et al., 1991) PBCA Pilocarpine Prolonged intraocular pressure reduction (Zimmer et al., 1994) Eudragit Flurbiprofen Inhibition of mitotic response, higher AH levels (Pignatello et al., 2002) Chitosan Cyclosporin A Therapeutic cone in comea & conjunctiva for 48 h (De Campos et al., 2001) PLA Rhodamine-6G Localized to RPE, NPs remained for 4 months (Bourges et al., 2003) PEG- Acyclovir 25-fold increase in AH dmg levels (Fresta et al., 2001) PBCA& Metipranolol Drastic reduction in systemic side effects (Losa et al., 1993) PLA& Acyclovir 7- & 12-folds higher therapeutic levels in AH (Giannavola et al., 2003) PHCA '"^C-tracer Residence time in inflamed tissue 4 times higher than healthy tissues of comea, conjunctiva, & AH (Diepold et al., 1989b) PBCA Pilocarpine Prolonged miosis response & reduction in lOP (Diepold et al., 1989a) PECL Indometbacin > 3-fold dmg levels in AH, comea, & iris-ciliary (Calvo et al., 1996b) 4^ Abbreviations: PBC A : polybutylcyano acrylate; lOP: intraocular pressure; A H : aqueous hum or; N P: nanoparticles; PLA : poly-d,l-lactic acid; RPE: retinal pigment epithelium ; PEG : poly(ethylene glycol); PECA : polyethyl-2-cyanoacrylate; PECL: poly-epsilon-caprolactone; PH CA : poly hexyl-2-cyanoacrylate. Table 6. Physicochemical factors affecting epithelial nanoparticle uptake Physical factors Model or Species Effect on nanoparticle uptake Reference Size Caco-2, COS-7 and HEK-293 cell lines, rat intestine Transfectivity and uptake increasing with decreasing particle diameter. Uptake by absorptive enterocytes is limited to < 500 nm particles. Smaller NPs adhere more highly to mucous than larger NPs (Desai et al., 1997; Prabha et al., 2002; Jani et al., 1989; Jani et al., 1990) Stabilizer content HASMC, IV injection in mice, PBCEC Higher amount of residual PVA in nanoparticles had relatively lower uptake. PEG modification of PLGA NP resulted in lower uptake by liver cells after systemic administration. Polysorbate-80 increased brain drug delivery of PBCA NPs (Kreuter et al., 2003; Gref et al., 1994; Sahoo et al., 2002) Hydrophobicity Rabbit intestine Uptake decreases with decreasing hydrophobicity (Jepson et al., 1993) Surface charge Rat intestine, intestinal bioadhesion assay Uptake higher for positively charged and neutral than negatively charged polystyrene NP. Negatively charged NPs made from poly (fumarie- co-sebaeic acid) copolymers demonstrated the highest adhesion. (Jani et al.,1989; Chickering and Mathiowitz, 1995) Abbreviations: HASMC: Human arterial smooth muscle cells; PVA: polyvinyl alcohol, NP: nanoparticles; PEG: polyethylene glycol; BPCEC: bovine primary cerebral endothelial cells; PBCA: polybutylcyanoacrylate 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.5.2 Intestinal penetration pathway o f nanoparticles A recent study carried using transmission electron microscopy after nanoparticle application to intestinal epithelium provided eogent evidence for the involvement of both paracellular and transcellular routes in the absorption of nanoparticles (Mathiowitz et al., 1997;Matsuno et al., 1983). However, a number of reports suggest uptake of particles (0.5-5 pm) in the intestinal epithelium is mostly via membranous epithelial eells (M cells, associated with antigen sampling) into the Peyer’s patches (Pappo and Ermak, 1989). The eut off point for intestinal absorption seems to be larger particles (> 5 pm), whieh lacked penetration into intestinal mucosa (mostly retained in ileal lumen in comparison with 1-5 pm particles) and were therefore devoid of any immune response (Damge et al., 1996). Other investigators have studied the uptake of nanoparticles in Caeo-2 cells and suggested that endocytosis as the main meehanism for internalization (Desai et al., 1997;Ma and Lim, 2003). 4.5.3 Systemic distribution It has been shown that intravenously injected nanoparticles are rapidly removed from the blood stream by the macrophages of the mononuelear phagoeyte system (MPS) of the liver (60-90% of injected dose) and spleen (2-10%) and the rest distribute to the lungs (3-20%) and the bone marrow (Kreuter, 1985). In addition, hydrophobie nanopartieles were shown to be taken up more by RES organs than hydrophilic nanoparticles. This prevents their application in controlled drug delivery 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and drug targeting other than MPS. Surface engineering, however, led the discovery of hydrophilic polymers such as polyethylene glycol (PEG) that conferred long circulation properties to poly(lactide), PLGA, and poly(caprolactone) nanoparticles (Gref et al., 1994;Stolnik et al., 1995;Bazile et al., 1995). The presence of the hydrophilic coating on nanoparticle surface is thought to sterically stabilize them against opsonization and phagocytosis. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II. STATEMENT OF THE PROBLEM 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. GOALS AND OBJECTIVES For treating anterior ocular diseases, topical administration of the drug into the conjimctival cul-de-sac using solutions, suspensions, gels, and ointments has been the preferred route. Topically applied drugs can reach the intraocular tissues by either the comeal and/or the noncomeal (conjunctival-scleral) pathway (Ahmed and Patton, 1985). However, ocular absorption of dmgs amounts to less than 5% of the dose, mainly due to low comeal permeability and short residence time (1-3 min) due to systemic absorption and tear tumover (Lee et al., 1993;Lang, 1995). One approach to improve ocular dmg absorption would be to use biodegradable polymers in the form of nanoparticles, as these have been shown to prolong dmg residence time and to enhance ocular absorption compared to a polymer-dmg solution or commercial eye drops (Wood et al., 1985;Calvo et al., 1996a). One attractive type of biodegradable polymer for ophthalmic use is poly(d,l- lactide-co-glycolide) copolymers (PLGA), which have been used for many years as surgical sutures due to their biocompatiblity, physical stability, and safety for medicinal use (Gilding and Reed, 1979). In addition, PLGA-based nanoparticles have been shown to promote dmg delivery in both the intestine and lung (Kawashima et al., 2000;Jiao et al., 2002). Since the conjunctiva has higher permeability to dmgs, in general, than the comea, we decided to evaluate the uptake characteristics of PLGA nanoparticles in primary cultured conjunctival epithelial cells (RCECs), which were shown to be useful as an in vitro model for evaluating dmg transport (Watsky et al., 1988;Hamalainen et al.,1997;Saha et al., 1996b). 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Our previous findings have indicated that uptake of PLGA nanopartieles in RCECs was size-, time-, concentration-, and temperature-dependent (Qaddoumi et al., 2000). It is known that many macromolecules are absorbed into epithelial cells by a process known as endocjdosis and then undergo degradation in the lysosomal compartment (Mellman, 1996). These observations led us to raise the following questions: 1) How does PLGA nanopartieles (or other types) bring about an enhancement in drug absorption in the eye? 2) What regulates this phenomenon? 3) Do these nanopartieles undergo intracellular delivery or degradation? 4) What strategies or physical characteristics can improve upon nanoparticle uptake? Therefore, we wanted to test the HYPOTHESIS that biodegradable PLGA nanopartieles enhance the conjunctival transport of drugs and proteins by stimulating vesicle formation (endocytosis) and avoiding the lysosomal degradation during its intracellular trafficking in rabbit conjunctival epithelial cells. In order to attain this goal, we have come up with three specific aims. 2. SPECIFIC AIMS Aim #1: To delineate the endocytic mechanisms involved in nanoparticle uptake in rabbit conjunctival epithelial cells (RCEC). In this aim, PLGA nanopartieles (100 nm in diameter) loaded with coumarin (as fluorescent marker) and bovine serum albumin (as a surfactant) was utilized. To confirm the involvement of endocytosis, we studied the effect of metabolic inhibitors and microfilament and microtubule inhibitors on PLGA nanoparticle uptake in 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RCECs. In addition, confocal laser scanning microscopy was used to confirm endocytic uptake of PLGA nanopartieles. Since nanopartieles were shown to enhance drug delivery in epithelial cells, we hypothesized that this may be due to stimulation of vesicle formations in cells. Therefore, we evaluated the effect of PLGA nanopartieles on endocytosis of fluid-phase marker to confirm this hypothesis. Based on morphological classifications (Figure 9), there are three distinct endocytic pathways that have been characterized in mammalian cells for the uptake of macromolecules (clathrin-mediated, caveolae-mediated, and non-coated vesicles). (a) C iathrm -coated vesicle (c) N o n -c o a te d v e sic le D ynam in C lathrin (d) C av eo tae 50- 80^ ( i n i I C aveofin V 9 9 V 9 9 Figure 9. Schematic diagram of the multiple endoeytie pathways in mammalian cells. In epithelial cells, clathrin- and caveolae-mediated endocytosis are the most common form of endocytosis in mammalian cells. Non-coated vesicles are also found, but are difficult to discern. Macropinosomes are not normally found in epithelial cells. Adapted fi*om Lamaze and Schmid (1995). 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. We dissected the involvement of each of these endocytic pathways on PLGA nanoparticle uptake in RCECs using pharmacological treatments aimed at inhibiting either clathrin- or caveolae-mediated endocytosis. We further validated the involvement of clathrin or caveolae in PLGA nanoparticle endocytosis by examining the staining pattern of coumarin-loaded PLGA nanoparticle with that of antibody markers of clathrin heavy chain or caveoin-1 proteins using confocal microscopy. The involvement of non-coated vesicles was determined by exclusion of the other two endocytic pathways, as no definitive methods are known to date. Finally, the molecular and protein expression of both clathrin heavy chain and caveolin-1 in RCEC was determined. This allowed us to conduct knockout experiments of either clathrin heavy chain or caveolin-1 proteins using antisense oligonucleotides against them. Aim # 2: Elucidate the intracellular distribution and trafficking of nanopartieles within conjunctival epithelial cells While the dynamics of endocytosis in vivo could be different from that observed in vitro, it is important to understand the intracellular trafficking and distribution of nanopartieles to further explore the drug delivery applications of nanopartieles. Several investigators have shown that microspheres based on PLGA copolymers were capable of releasing the active drug for a period ranging from 4-12 weeks either in vitreous fluid or the cytoplasm of retinal pigment epithelial cells following subretinal/intraviteal delivery (Hashizoe et al., 1997;Giordano et al.,1993). 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In general, maeromolecules and proteins (receptors or ligands) undergo degradation in the endosomal/lysosomal compartment or upon their endocjdosis resulting in reduced activity and half-life. We thought that this prolonged residence is due to the physiochemical properties of the poljmieric PLGA nanopartieles that allow it to disrupt (or escape) lysosomal degradation and release the drug in the cytoplasm in a controlled manner. Therefore, if nanopartieles are to be used for the delivery of drugs and proteins to the conjunctival epithelium, evidence for their controlled release properties and protective capability is needed. We evaluated the sustained release properties of nanopartieles by studying the release of 6-coumarin from nanopartieles in RCEC culture over several days. Immunofluorescence and colocalization studies with organelle markers were used to reveal the intracellular distribution and trafficking of nanopartieles in conjunctival epithelial cells. We assessed the contribution of lysosomes or endosomes to nanoparticle degradation by using agents that disrupt their pH gradients (ionophores). Finally, we demonstrated the protective ability of PLGA nanopartieles by comparing the apical uptake of HRP-loaded PLGA nanopartieles with that of free HRP in RCECs using the same concentration. Knowledge about the intracellular distribution and trafficking of nanopartieles will facilitate our understanding of the mechanism of their action, lead to the design of better targeted nanopartieles, and reveal the factors that regulate their trafficking and intracellular targeting. This understanding will eventually lead us to 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. better strategies to enhance the delivery of nanopartieles to the conjunctival epithelium and retina. Aim # 3: Enhance nanoparticle absorption by utilizing receptor-mediated endocytosis of lectins in conjunctival epithelial cells We sought to find a substrate with high affinity and rapid binding to RCECs, so we can utilize as a surface ligand to improve nanoparticle absorption efficiency. The extent of PLGA nanoparticle absorption in intestinal epithelium was variable and ranged from 1-39%. In our RCEC culture system, 6-10% of the total dose of PLGA nanoparticle was absorbed following 2 hr uptake studies. Based on the ease of chemical conjugation experiments and assay, the reported prolonged residence time in tear, and the demonstrated safety of lectins, we decided to focus on plant lectins (Nicholls et al., 1996;Smart et al., 1999b). We first analyzed three different plant lectins (Lycopersicon esculentum, TL; Solanum tuberosum, STL; and Ulex europeaus 1, UEA-1) that were reported to have excellent binding and endocytic capability in ocular tissues (Maeda et al., 1998;Nicholls et al., 1996;Rittig et al., 1990). Based on optimal binding affinity and endocytic capacity in RCEC, we selected Solanum tuberosum lectin (STL) as the best candidate to utilize for conjugation experiments with nanopartieles for enhanced absorption. Due to concerns regarding the immunogenicity and safety of lectin use in human, we have evaluated the effect of Solanum tuberosum lectin on cytokine induction in RCECs 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and on the bioelectric parameters of cells. Based on ocular tolerance, we conjugated Solanum tuberosum lectin to HRP-loaded PLGA nanopartieles using a covalent carbodiimide linkage and evaluated its absorption enhancement capability relative to control nanopartieles. 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. III. CHAPTER 1. ELUCIDATION OF THE ENDOCYTIC PATHWAYS INVOLVED IN PLGA NANOPARTICLE UPTAKE 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. INTRODUCTION AND PURPOSE We have previously shown that internalization of PLGA nanopartieles loaded with fluorescent dye (6-coumarin) in rabbit conjimctival epithelial cells was size-, time-, concentration-, and temperature-dependent (Qaddoumi et al., 2000). These earlier findings together with the reported equivalent pore radius of the conjunctival epithelium of 5.5 nm suggested that PLGA nanoparticle uptake occurs via a transcellular route, such as endocytosis (Horibe et al., 1997b). It is known that absorption of maeromolecules and nutrients into epithelial cells occurs by endocytosis, which are then engulfed in membrane invaginations and internalized as vesicles, with subsequent intracellular sorting (Mellman, 1996). It is also known that vesicle formation (endocytosis) at the apical membrane was dependent on the integrity of actin filament network and energy supply (Gottlieb et al., 1993;Smythe etal., 1992). Morphologically speaking, three distinct endocytic pathways have been characterized in mammalian cells and are illustrated in Figure 9 shown previously (Lamaze and Schmid, 1995). Coated pits around the epithelial cell membrane characterize clathrin-type vesicles, whereas caveolae are smooth invaginations of the plasma membrane that were first described in endothelial cells (Palade, 1953). Non- coated vesicles are hard to discern and their role is usually determined by exclusion of the other two pathways. Macropinosomes are not normally found in epithelial cells, therefore, their role on nanoparticle uptake will not be evaluated. Both clathrin and caveolin are thought to be ubiquitous proteins, although their existence and role 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in conjunctival epithelial cells have not been studied systematically. The localization of caveolin at the cell membrane of comeal epithelium was previously reported in the context of wound healing (Amino et al., 1997). In addition, the presence of both clathrin and caveolin in the retina and lens was described (Bloom and Puszkin, 1983;Brown et al., 1990;Bridges et al., 2001). Therefore the purpose of this aim was: a) to evaluate the effect of inhibitors of actin filament or energy metabolism on PLGA nanoparticle uptake. B) to examine the expression of clathrin heavy chain and caveolin-1 in rabbit conjunctival epithelial cells and investigate whether they have any role in PLGA nanoparticle endocytosis using pharmacological treatments aimed at dismpting the formation of clathrin- coated pits and caveolae. C) to evaluate the effect of transfection with specific antisense oligonucleotides designed against the rabbit clathrin isoform on nanoparticle uptake. Elucidation of the endocytic pathway (clathrin and/or caveolae-mediated or non-coated vesicles) involved in nanoparticle uptake is pivotal for further studies aimed at regulation of that pathway to enhance total nanoparticle uptake in these cells and to manipulate intracellular sorting and trafficking of these nanopartieles for specific targeting and avoidance of the degradative machinery. 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2. MATERIALS AND METHODS 2.1. Materials Nanopartieles of polylactic polyglycolic acid co-polymer (PLGA 50:50, inherent viscosity 1.31 measured in hexafluroisopropanol) of mean diameter 100 nm (actual diameters was 0.11 ± 0.05 pm) were obtained from Dr. Labhasetwar’s laboratory (Nebraska College of Pharmacy) and characterized with the methods reported by Davda and Labhasetwar (Davda and Labhasetwar, 2002). The nanopartieles contained bovine serum albumin (4% w/w) and polyvinyl alcohol (1%) as a surfactant and 6-coumarin (0.05% w/w) as a fluorescent marker (Figure 10). • • • • • • magnified Cluster of NPs 100 nm in mean diameter Poly (DL-lactide:glycolide) acid co-polymers (PLGA) 6-Coumarin (Fluorescent marker 0.05 % w/w) Bovine serum Albumin (BSA) 4% w/w (as a surfactant) Figure 10. Schematic representation of PLGA nanopartieles and composition. Nanopartieles (100 nm in diameter) were made from a 50:50 mixture of poly(dl- lactic.glycolic) acid co-polymers (PLGA) containing both bovine serum albiunin (4% w/w) and 6-coumarin as a fluorescent marker (0.05% w/w). Human epidermal growth factor, hydrocortisone (cell culture grade), protease (Type XIV from Streptomyces griseus), deoxyribonuclease I (Type IV from bovine pancreas), nystatin, cholera toxin P-subunit, filipin, nocodazole, cytochalasin D, and 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Triton-X 100, were obtained from Sigma Chemical Co. (St. Louis, MO). Ca^^-free Hanks’ balanced salt solution (HBSS), Ca^^-free minimum essential medium (S- MEM), gentamicin, penicillin-streptomycin 10,000 U, lOx trypsin-EDTA, TRJzol® reagent, Lipofectamine™ 2000 reagent, and sense and antisense oligonucleotide primers targeted against clathrin heavy chain and caveolin-1 genes were obtained from Invitrogen Corp. (Carlsbad, CA). Certified fetal bovine serum (FBS), cell strainer (40 pm), collagen (Type 1), amphotericin B (Streptomyces sp.), bovine pituitary extract (BPE), ITS™ (insulin, transferrin, and selenious acid), and Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) were obtained from VWR Intemational Co. (West Chester, PA). PC-1 serum-free, low protein, defined medium was purchased from Biowhittaker (Walkersville, MD). ClearwelF'^ filters (12 mm and 6.5 mm diameter, 0.4 pm pore size) were obtained from Costar (Cambridge, MA). HeLa (human adenocarcinoma) and A431 (human epidermoid carcinoma) cell lines were obtained from ATCC (Manassas, VA). Lucifer yellow, FITC-transferrin, and Prolong™ anti-fade mounting kit were purchased from Molecular Probes, Inc. (Eugene, OR). Mouse monoclonal antibodies against either clathrin heavy chain (HC) or caveolin-1 proteins were purchased from BD Biosciences (Lexington, KY). DC protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA). SuperSignal West Pico Chemiluminescent Substrate and ImmunoPure TMB Substrate Kit were obtained from Pierce Biotechnology, Inc. (Rockford, IL). QIAquick Gel Extraction kit was purchased 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. from Qiagen Inc. (Valencia, CA). pGem®-T Easy vector was obtained from Promega Corp. (Madison, WI). 2.2. Animal model Male Dutch-belted pigmented rabbits, weighing 2.0-2.5 kg, were obtained from Irish Farms (Norco, CA) and handled in accordance with Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80-23). 2.3. Primary air-interfaced culture of rabbit conjunctival epithelial cells Conjunctival tissue was isolated and prepared as described previously by Kompella and coworkers (Kompella et al., 1993). Rabbits were euthanized by an overdose injection of sodium pentobarbital solution (325 mg/kg) into the marginal ear vein. Conjunctival tissues from the excised eyeballs were carefully dissected and isolated for primary cell culture. Rabbit conjunctival epithelial cells (RCECs) were harvested using a protocol developed by Saha et al. (Saha et al., 1996a) and modified by Yang et al. (Yang et al., 2000b). Briefly, following excision, the conjunctiva was washed in ice-cold Ca^’ ^/Mg^^- free Hanks’ balanced salt solution. The conjunctiva was trimmed off to within approximately 4 mm from the limbus, freed of extraneous tissues, placed in a petri dish containing ice cold 0.2% protease type XIV in Minimum Essential Medium (S-MEM), and incubated for 60 min at 37 °C in 95% air/5% CO2, to dissociate the cells. Epithelial cells were scraped using sterile fine forceps and #10 scalpel blade. 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The isolated cells were immediately transferred to a pre-equilibrated S-MEM (at 37 °C) containing 10% fetal bovine serum (FBS), and 1 mg/ml deoxyribonuclease (DNAase I) to stop protease reaction. The isolated cells were then mixed and centrifuged at 700x g for 10 min at room temperature. The resulting cell pellet was suspended in S-MEM containing 10% FBS, filtered through a 40 pm cell strainer, and centrifuged again using the same settings. The final cell pellet was resuspended in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 100 U/ml penicillin-streptomycin, 0.5% gentamicin, 0.4% amphotericin B, 2 mM L-glutamine, 1% ITS^(6.5 pg/ml insulin, 6.5 pg/ml transferrin, 6.5 ng/ml selenious acid, 1.25 mg/ml BSA, and 5.35 mg/ml linoleic acid), 30 pg/ml bovine pituitary extract (BPE), 1 pM hydrocortisone, and 1 ng/ml epidermal growth factor (EGF). After cell viability testing based on exclusion of 0.1% trypan blue, cells were counted using a grid and light microscope and plated on Clearwells™ pre-coated with rat tail collagen type I at a density of ~ 1.2x 10^ cells/cm^ with the bottom-side of the insert bathed in DMEM/F12. Cells were cultured in a humidified incubator at 37 °C in 5% CO2 and 95% air. From day 2 onward, the growth medium was changed to PC-1 growth medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin- streptomycin, 0.5% gentamicin and 0.4% amphotericin B. Cells were switched to an air-interface (i.e., nominally fluid-free on the apical surface of the cell layers) on day 4 onward. On day 6 or 7 cells became confluent and were utilized for experiments. 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.4. Bioelectric measurements The transepithelial electrical resistance (TEER) and potential difference (PD) were monitored daily from day 4 of primary culture with a Voltohmmeter electrode (World Precision Instruments, Sarasota, FL). The values were corrected for background values contributed by the blank filter (coated with substratum) and culture medium and expressed as kQ.cm^ and mV, respectively. 2.5. Nanoparticle uptake study and analytical method The detailed uptake of nanoparticles and analysis method were described previously (Davda and Labhasetwar, 2002). Briefly, following confluency of cultured RCECs, the culture medium on both sides of the cells was replaced with a physiological bicarbonate Ringer’s solution (BRS) containing: 1.8 mM CaCli, 5.6 mM KCl, 0.8 mM MgS0 4 , 0.8 mM NaH2P04,116 mM NaCl, 25 mM NaHCOs, 15 mM HEPES, and 5.5 mM D-glucose and incubated for 30 min at 37 °C. The BRS was bubbled with air containing 5% CO2 and adjusted to pH 7.4 before usage. The osmolality of the solution was in the range of 290-310 mOsm. All nanoparticle suspensions were prepared in BRS solution. After 30 min incubation with BRS, the apical side of conjunctival epithelial cells was replaced with the nanoparticle suspension and incubated for 30 min at 37 °C. The apical solution was then aspirated and the cell filters were washed three times with ice-cold BRS buffer solution to remove nanoparticles not internalized. The cell filters were then cut off from the Transwell using a blade and transferred separately into covered disposable 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tubes filled with 1 ml BRS solution. For the determination of the amount of membrane-bound fraction of nanoparticles, washed cells were trypsinized with lOx trypsin-EDTA solution (Invitrogen Corporation, Carlsbad, CA) for 30 min to a total volume of 1 ml and centrifuged twice at lOOOx g for 10 min each. The resulting pellet was solubilized with 0.5 ml of 0.5% Triton-X 100 in BRS for 30 min at 37 °C and diluted to 1 ml with BRS. The filter samples and/or both the supernatant and dissolved pellet (1 ml each) of each sample were then frozen at -20° C and lyophilized overnight. Extraction of coumarin from nanoparticles was done by incubating the lyophilized samples with 1 ml methanol at 37 °C for 24 hr under gentle agitation. The samples were then centrifuged at lOOOx g for 10 min and the supernatant collected and analyzed using a spectrofluorometer F-2000 (Hitachi, Tokyo, Japan) set at an excitation wavelength of 450 nm and emission wavelength of 490 ran. Standard curve for each nanoparticle uptake experiment was obtained by spiking different concentrations of nanoparticles (12.5-200 pg/ml) in BRS and treated the same way as the nanoparticle samples from the experiments. The effect of the nanoparticles on the bioelectric parameters of the conjxmctival epithelial cell culture was examined by evaluating the change in both the transepithelial electrical resistance (TEER) and potential difference (PD) after two hours of incubation with nanoparticles 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.6. Evaluation of nanopartlcle endocytosis 2.6.1. Effect o f energy depletion and endocytosis inhibitors To evaluate if nanoparticle uptake occur through an active process, RCECs were pre-incubated with metabolic inhibitors (10 mM sodium azide and 0.2 mM 2,4- dinitrophenol) for 30 min, prior to nanoparticle uptake and throughout the 2-hr uptake period. The effect of microfilament and microtubule inhibitors (also known as vesicle formation or endocytosis inhibitors) on nanoparticle uptake in RCECs was evaluated by pre-incubating cells with 0.1 pg/ml cytochalasin D (microfilament inhibitor) and 1 pg/ml nocodazole (microtubule inhibitor) (both dissolved in DMSO), respectively, for 30 min prior to nanoparticle application and throughout the 2-hr uptake experiment. 2.6.2. Confocal microscopy Semi-confluent RCECs (70-80% confluent on day 5 or 6) were incubated from the apical side with 0.5 mg/ml suspension of 6-coumarin nanoparticles for two hours at 37 °C and then washed three times with ice-cold BRS buffer to remove excess nanoparticles. This concentration was chosen since it was optimal for distinguishing the fluorescent intensity of nanoparticles from that of the cell auto-fluorescence. Cells were then fixed with 4% paraformaldehyde in PBS solution for 30 min, permeabilized using 0.5% Triton-X 100 in water for 15 min, and incubated for 45 min with or without the nuclear marker, propidium iodide. Cells were then rinsed 3 times with PBS solution and the cell filter removed and mounted on a glass slide 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. using Prolong™ mounting anti-fade media (Molecular Probes, Oregon) and viewed under a confocal microscope (Ziess LSM 510, Germany) using FITC (wavelength 450-490 nm) and rhodamine filters (wavelength 550-570 nm). 2.6.3 Stimulation o f fluid-phase endocytosis The effect of nanoparticles on the uptake of Lucifer yellow (LY), a known fluid-phase marker in hepatocytes and Madin-Darhy canine kidney cells (MDCK), was carried out to evaluate if nanoparticles had any stimulatory effect on vesicle formation or endocytosis (Oka et al., 1989). First, the kinetics of internalization of 0.1 mM LY in RCEC culture was followed up to 45 min to ensure that it hehaves as a fluid phase tracer. Then, the uptake of LY at the 4-hr time period, in the presence and absence of 1 mg/ml nanoparticles was evaluated. 2.7. Involvement of clathrin and caveolae in nanoparticle endocytosis 2.7.1. Inhibition o f clathrin- and caveolin-mediated endocytosis Hypertonic challenge or intracellular depletion were performed according to Hansen et al. (Hansen et al., 1993) to disrupt clathrin-mediated endocytosis. Briefly, hypertonic challenge was carried out by incubating RCECs with BRS supplemented with 0.45 M sucrose for 15-20 min at 37 °C from both sides. Depletion of cytosolic was achieved by incubating primary cultured RCECs with hypotonic BRS medium (50%) containing ouabain (1 mM) for 5 min followed by ice-cold K^-ffee BRS medium for 20 min on both sides. Once treatment was 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. complete, RCECs were then incubated from the apical side with either PLGA nanoparticles (0.5 mg/ml) or FITC-transferrin (50 pg/ml, also used for basolateral uptake studies) for 30 min, washed three times with ice-cold BRS, and analyzed for fluorescence as described above. Treatments aimed at inhibiting caveolae-mediated endocytosis were evaluated by incubating RCECs from both sides with a sterol- binding agent, nystatin at 50 pg/ml or filipin at 5 pg/ml, for 30 min at 37 °C followed by apical uptake of nanoparticle (0.5 mg/ml) or FITC-cholera toxin P (5 pg/ml), a substrate known to be internalized by caveolae-mediated endocytosis in other cell systems. 2.7.2. Immunofluorescence and confocal microscopy Sub-confluent RCECs (80% confluence) were incubated from the apical side with 0.5 mg/ml suspension of PLGA nanoparticles loaded with 6-coumarin at 37 “C, and then washed three times with ice-cold BRS buffer. Cells were then fixed with 4% paraformaldehyde in PBS solution for 30 min, permeabilized using 0.5% Triton- X 100 in water for 15 min, blocked with 10% bovine serum albumin (BSA) in PBS solution for 30 min, and incubated with mouse monoclonal antibody against either clathrin HC or caveolin-1 for 2 hrs. Cells were then washed several times with PBS and incubated for 1 hr with rhodamine-labeled goat anti-mouse secondary antibody. Finally, cell filter was cut and mounted on a glass slide using Prolong™ anti-fade mounting kit and viewed under a confocal microscope (Ziess LSM 510, Germany) 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. using both FITC (wavelength 450-490 nm) and rhodamine filters (wavelength 550- 570 nm). 2.7.3. RNA isolation and reverse transcription-polymerase chain reaction Freshly isolated rabbit conjunctival epithelial cells were treated with 1 ml TRIzol® reagent and total RNA was obtained according to manufacturer’s direction. Isolated conjunctival RNA was reverse-transcribed to cDNA using oligo-dT primers (20 bp long) (Superscript™ II, Invitrogen Corp., Carlsbad, CA). PCR was performed using sense (5’-CGGTTGCTCTTGTTACGG-3’) and antisense (5’- AGAGCATTAAATTTCCGGGC-3’) primers based on conserved regions of clathrin HC genes cloned from both human (gi: 4758011) and rat (gi: 203301). The sense primers chosen correspond to 525-542 bp of human and 451-468 bp of rat clathrin HC gene, whereas the antisense primers correspond to 1265-1284 bp of human and 1191-1210 bp of rat clathrin HC gene. For caveolin-1, PCR was performed using sense (5’-CAACATCTACAAGCCCA-3’) and antisense (5’- AAACTTCTACACTAACG-3’) primers based on conserved regions of caveolin-1 gene cloned from human (gi: 38515), dog (gi: 943), rat (gi: 575379), mouse (gi: 603660), and chicken (gi: 211426). The sense primers correspond to 344-360 bp of human, 134-150 bp of dog, 94-110 bp of rat, 66-82 bp of mouse, and 135-151 bp of chicken. The antisense primers correspond to 480-496 bp of human, 270-186 bp of dog, 230-246 bp of rat, 202-218 bp of mouse, and 271-287 bp of chicken caveolin-1 gene. PCR conditions for clathrin were as follows: 94°C for 30 sec, 51°C for 30 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sec, and 70°C for 30 sec, all 30 cycles and PCR conditions for caveolin-1: 94°C for 30 sec, 42°C for 30 sec, and 70°C for 30 sec, all 30 cycles (even 50 cycles were performed to check for caveolin-1 gene fragment). The RT-PCR products were resolved under agarose gel electrophoresis and cDNA amplicons corresponding to predicted product sizes of clathrin HC purified using the QIAquick Gel Extraction kit. The resultant cDNA fragments were ligated into a pGem®-T Easy vector and the vectors were expanded by transformation of Escherichia coli DHSa competent cells. Positive clones were selected and analyzed by endonuclease (E'coRI) restriction assay. The resultant plasmid was then sequenced using infrared fluorescent dye-labeled M13 primers (GeneMed Synthesis Inc., San Francisco, CA). The homology of amplified jfragments was verified by comparison to known clathrin HC gene sequences using standard multialign program (Clustal W sequence alignment program). 2.7.4. Western blot analysis o f clathrin HC and caveolin-1 Western blot analysis was performed on cell lysates of confluent RCECs on day 7-9 using a mouse monoclonal antibody against either clathrin HC or caveolin-1. RCECs were incubated for 30 min at 37 °C with lysis buffer (1% SDS in PBS) containing 1% protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO) and 20 pi of cell lysate was used to determine the protein content using DC protein assay kit. Cell lysate of HeLa cells was used as positive control for clathrin HC expression, whereas A431 cells was used as a positive control for caveolin-1 expression. Rabbit 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cardiac muscle tissue was used as a control to demonstrate cross reactivity of mouse monoclonal caveolin-1 antibody for rabbit protein. About 50 pg of protein from each cell type (RCEC, cardiac muscle, HeLa, and A431) was then electrophoresed on 8% SDS-PAGE, followed by electrotransfer to a nitrocellulose membrane, frnmnoblot procedure utilizing the enhanced chemiluminiscence method (ECL) was performed according to the manufacturer’s protocol (Pierce, Rockford, IE). 2.7.5. Transfection with antisense oligonucleotide against clathrin HC RCECs grown on 12 mm coated Clearwells on day 7-8 of culture with TEER values of 1.1 ± 0.15 Q.cm^ were treated for 4-6 hrs with a 15-bp antisense oliognucleotide (5’-CTTCCGTCACCTACA-3’) directed against the rabbit clathrin HC mRNA (corresponding to fragment between 290-304 bp). The 15-mer antisense oligonucleotide was complexed with Lipofectamine 2000 reagent according to manufacturer’s direction (Invitrogen Corp., Carlsbad, CA) prior to use. HeLa cells were used as a positive control for transfection. Cell lysates were then processed after 24 hr transfection for Western blot analysis as described above to confirm the reduction of clathrin HC protein. 2.8. Data analysis Uptake data are presented as mean ± s.e.m. (n), where n is the number of observations. Both Student’s t-test and one-way and two-way ANOVA analysis (using Tukey-Kramer test) were utilized to evaluate significant differences (p < 0.05) in sample means, as appropriate. 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. RESULTS The average transepithelial electrical resistance (TEER) and potential difference (PD) values for RCEC culture (day 7) used in all uptake experiment were 1.20 ± 0.17 kOhm.cm^ and 3.7 ± 0.75 mV, respectively. Following 2-hr incubation with nanoparticles, the TEER and PD values were not significantly different from control (p > 0.05) with values of 1.02 ±0.1 kOhm.cm^ and 3.1 ± 0.32 mV (n = 12), respectively. 3.1. Evidence for nanopartlcle endocytosis 3.1.1. Effect o f energy depletion and endocytosis inhibitors Pre-incubating nanoparticles for 30 min with either sodium azide or 2,4- dinitrophenol reduced nanoparticle internalization into RCECs by 24 ±1.5% and 19 ±0.9%, respectively, compared to that of control (Figure 11). The internalized fi-action of nanoparticles represented 6% of the applied dose of nanoparticles (0.5 mg/ml), whereas the membrane-bound portion was only 1.5%. Fig. 11 also demonstrates that the 2-hr uptake of 0.5 mg/ml nanoparticles in RCECs decreased significantly when cells were pre-incubated with 0.1 pg/ml cytochalasin D, an actin filament inhibitor, but not in the presence of 1 pg/ml nocodazole, a microtubule inhibitor, despite a noticeable decrease in uptake pattern. 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cytochalasin D Nocodazole NaNj DNP Control Internalized Membrane-bound H * - 1 * H 0 10 20 Nanopartlcle amount In RCECs In pg/ml 30 Figure 11. Effect of energy depletion and vesicle transport inhibitors on nanoparticle uptake in RCEC culture. For energy depletion, RCECs were preincubated with either 10 mM sodium azide or 0.2 mM 2,4-dinitrophenol (metabolic inhibitors) in a glucose-free BRS at 37 °C for 30 min prior to 2-hr uptake experiment with 0.5 mg/ml nanoparticles. To determine the effects of vesicle transport inhibitors, cells were pre-incubated at 37 ° C for 30 min with either 0.1 pg/ml cytochalasin D (actin inhibitor) or 1 pg/ml nocodazole (microtubule inhibitor) before a 2-hr nanoparticle uptake study. The membrane- bound fraction was isolated by trypsinization of cells followed by centrifugation (see methods section). Bars represent mean ± s.e.m. (n = 4). Asterisk (*) denotes significant differences (P< 0.05). 3.1.2. Confocal microscopy Confocal microscopy revealed evidence of PLGA nanoparticle endocytosis. Fig 12a shows that nanoparticles were abundant in the intermediate layer of RCECs just below the apical compartment. Small amounts of nanoparticles were also seen in the deep layer of the cells (not shown). Nanoparticles were seen either distributed in 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a punctate manner (long arrows) around the cell membrane, in the perinuclear area, below the cell surface, and in distinct compartments (maybe an endosome), or seen in a diffuse pattern (arrowheads) all over the cytoplasm. Nanoparticles were not found in the nuclear region (stained red with propidium iodide). Control images, in the absence of nanoparticles, did not show fluorescence in the cells (Fig. 12b). Figure 12. Confocal microscopy of RCECs after nanoparticles uptake. A and B are X-Y images scanned below the apical plasma membrane (intermediate layer of conjunctival epithelial cells). (A) Represents uptake of nanoparticles by RCECs incubated for 2-hours. Nanoparticles are seen within the cytosol, undemeath the cell membrane, and in specific compartments (long arrows), but not within the nucleus (small arrows). (B) Control experiment without nanoparticle incubation. 3.1.3. Stimulation o f fluid-phase endocytosis Since PLGA nanoparticles have been shown to enhance drug absorption in other epithelia, we thought this could be mediated by stimulation of endocytosis (vesicle formation). We tested this by evaluating the effect of nanoparticles on the 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. uptake of Lucifer yellow (LY), a known fluid-phase marker in hepatocytes (Oka et al., 1989). Since LY has not been used previously with conjunctival epithelial cells, it was necessary to demonstrate that this molecule behaves as bona fide fluid phase molecule with these cells. Our observation of the linear uptake of LY (R^ = 0.85) at 37 °C over a 45 min period is consistent with it being a fluid-phase marker (Fig. 13). 30 n 20 - O ) I- S ® c 10- o .E 0 15 30 45 Time (min) Figure 13. Uptake of Lucifer yellow in rabbit conjunctival epithelial cells. RCECs were incubated at 37 °C with 0.5 mM Lucifer yellow (LY) in BRS for 45 min (R^ = 0.85). Cells were washed and LY content was calculated as described in the methods section. Values represent mean ± s.e.m. (n = 4). Table 7 shows that simultaneous co-administration of LY with nanoparticles enhanced the apical uptake of LY in RCECs at 37 °C at 4 hr period by 39% as compared to LY applied alone. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 7. Effect of nanoparticles on stimulation of fluid phase endocytosis of Lucifer yellow Chemical used Total uptake amount (ng/ml/mg of protein) % Relative difference Lucifer yellow (As control) 119.7 ±5.5 Control Lucifer yellow nanoparticles 166.4+12.9* 39% increase in Lucifer yellow uptake Values represent mean ± s.e.m. (n = 4). Asterisk (*) indicates statistically significant differences (P< 0.05). 3.2. Elucidation of the endocytic pathway 3.2.1. Inhibition o f clathrin- and caveolin-mediated endocytosis As shown in Fig. 14, apical uptake of PLGA nanoparticles in RCECs decreased by 45% and 35%, respectively, as a result of intracellular depletion or hypertonic treatment of cells for 30 min, respectively. However, 30 min pretreatment of RCECs with either nystatin or filipin, maneuvers known to interfere with caveolin-dependent endocytosis in other cells, did not affect apical uptake of nanoparticles. As a control for clathrin-mediated endocytosis, we also studied the apical and basolateral uptake of FITC-transferrin in RCECs. Fig. 15 shows that both apical and basolateral uptake of FITC-transferrin has decreased significantly by 22% and 70%, respectively, as a result of intracellular depletion or hypertonic media treatment of RCECs. Furthermore, both the apical and basolateral uptake of FITC- transferrin was not affected with pretreatment of cells using nystatin, suggesting 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. endocytosis of transferrin into RCECs does not occur via caveolae-dependent mechanisms. Filipin Nystatin Depletion Hypertonic media Control H I Internalized I Membrane-bound H - J — T " 10 —r~ 20 —I 30 Nanoparticle amount in pg/ml Figure 14. Effect of pharmacological treatments on internalization of PLGA nanoparticles in RCECs. Apical uptake of coumarin-loaded PLGA nanoparticles (0.5 mg/ml) in RCECs for 30 min at 37 °C under various treatments aimed at inhibiting endocytosis mediated by clathrin (K^ depletion and hypertonic challenge) or caveolae (nystatin and filipin). The asterisk denotes significant differences (p<0.05). Bars represent mean ± standard error of the mean (n=6). To further evaluate the role of caveolae in the endocytosis of molecules in RCECs we studied the apical uptake of cholera toxin P (CTB), a known substrate for caveolae-mediated endocytosis in other cell types (Montesano et al., 1982;Henley et al., 1998). Apical uptake of CTB in RCECs was not affected by pretreatment of cells with either filipin or nystatin (Fig. 16), indicating that caveolae may be absent in RCECs. 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. u u 9 A a u H I U H fl o > o u a D A B 'w o fl 1500 n 1000 - 500 - 0 Basolateral uptake Apical uptake X X u i Control Nystatin K depletion Hypertonic Figure 15. Effect of pharmacological treatments on internalization of transferrin in RCECs. Apical and basolateral uptake of FITC-transferrin (50 fig/ml) in RCECs for 30 min at 37 °C under treatments aimed at inhibiting clathrin- or caveolae-mediated endocytosis. The asterisk denotes significant differences from both basolateral and apical uptake (p<0.05). The plus sign denotes significant differences from apical uptake (p<0.05). Bars represent mean ± standard error of the mean (n=6). c 3 o E (0 o Control Filipin Nystatin Figure 16. Effect of pharmacological treatments on internalization of cholera toxin B subunit in RCECs. Apical uptake o f 5 jJLg/ml FITC-cholera toxin B subunit (CTB) in RCECs for 2 h at 37 °C under treatments aimed at inhibiting caveolae-m ediated endocytosis. Bars represent mean ± standard error o f the mean. (n=6). 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2.2. Immunofluorescence studies To further validate the possible involvement of clathrin-mediated processes in endocytosis of PLGA nanoparticles, we evaluated if the staining pattern of coumarin-loaded nanoparticle is associated with that of clathrin in RCECs. Fig. 17 shows confocal images of RCECs (40x magnification)) stained with clathrin E E C following 30 min apical uptake of PLGA nanoparticles. As seen in the double labeling studies (panel D), clathrin staining (red in panel A) is partially overlapped with that of 6-coumarin-loaded nanoparticles (green in panel B), indicating that nanoparticle uptake may partially associate with clathrin-mediated processes. The fluorescence colocalization pattern for both nanoparticles and clathrin was found both at the plasma membrane and intracellularly, probably due to internalized clathrin-coated vesicles, hnmunostaining for caveolae using mouse caveolin-1 monoclonal antibody did not reveal any staining pattern in RCECs (image not shown), fortifying the notion that caveolae-mediated processes are absent in RCECs. These findings were confirmed with magnification of up to lOOx, however, for best illustrative quality without compromising image resolution only the 40x magnified specimens are shown. 3.2.3. Molecular evaluation o f clathrin and caveolin gene expression The results in Fig. 18 illustrate RT-PCR products detected in both RCECs and HEK293 (as control) corresponding to an expected 744 bp in size. To verify that 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. this amplified product represented clathrin HC cDNA, the band for clathrin HC cDNA fragment was extracted, sequenced, and foimd to have 66% and 62 % identity Figure 17. Confocal microscopy of RCECs following uptake of PLGA nanoparticles. A and B represent x-y confocal images (40x magnification) scanned below the apical layers of sub-confluent RCECs (80% confluency). A) RCECs stained with clathrin HC antibody (red). B) Uptake of PLGA nanoparticles containing coumarin (green) after incubation for 30 min at 37 °C. C) Phase contrast image of RCECs. D) Merged image of A and B. Areas of overlap are denoted by arrowheads. These findings were confirmed with magnification of up to lOOx, however, for best illustrative quality without compromising image resolution only the 40x magnified specimens are shown. 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1000 bp B ) Clathrin HC gene fragment (744 bp) GAPDH (control housekeeping gene) Caveolin-1 gene fragment (152 bp) Figure 18. RT-PCR analysis of clathrin heavy chain and caveolin-1 mRNA expression. A) Primers designed from conserved sequences based on the clathrin HC gene cloned from rat and human. B) Primers designed from conserved sequences based on the known sequence of caveolin-1 genes cloned from rat, mouse, dog, ehicken, and human. The signal for GAPDH was also observed at 488 bp similarly for all the tissues tested in B for positive control since the RT-PCR samples in A and B were all analyzed in one reaction. to clathrin HC mRNA from human and rat, respectively, using Clustal W sequence alignment program (Fig. 19). To address whether caveolin cDNA was present in RCECs, RT-PCR amplification of mRNA transcript was performed using cDNA primer sequences designed from conserved caveolin-1 regions. Fig. 18b shows that the expected RT-PCR gene product for caveolin-1 (corresponding to 152 bp in size) was not detected in RCECs and rabbit brain tissue, but was detected in several others including the cornea, trachea, cardiac muscles, and HEK cells. The signal for 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. GAPDH was also observed at 488 bp similarly for all tissues tested in Fig. 18b for positive control. F A b b it TCGTWrCCAeCCTCQCMCOrOCCJKiATCAJCMTTACCGTKCMKrBCTAMBCAM W 7TrTTM A CTTC-CCAATA TCCCCCC*CCA AI8€AM ATK--<7rTSTSCeTGCOII 52 m w n a n P a t R a b b it QTGCTTA::TTCT9JkCT6&1ATATCTGCM::M;CAMATVSiX<n^eTG«GACCTXtCCMCT 720 CrrSSrr9CZCCT(*IUCTOGCJl7AfCT(iCACAlGCVkPPTC5CGTa6«TOCXCC7MOCAec:f 6 4 6 J t f M 8 C ? X i y < 1 « « 3 t i M g C g T C T 3 g T T O “ ~ ~ t» ^ a a iS l» ^ ^ 1 0 9 * * « » « ** * * < ^ i t * * ^ * « * • ♦»* ?so ttx m -----<aguieffl«c3 urra(yufcBci»— sciTtocJi-cc3 ^ ^ is? * * * * * * . * » * : » '> * * ' •» • « -* « Kuraan R a t Human mt R a b b it A T r r i u M y m M a A S G J U U t f C G O i K s a o i S i i A ^ ^ ? 6 « mwJv s a * J> A X T a c ia w fia w ^ ^ 2 1 7 * « < » * * * * m * * * * * * * * * * * * ■ » * * * * * * 895 CliGGCA£GG-G€K0Uli^fAC-ll2^Krc&7«r€liyU3 821 CiUUKrT6GM^i«;6MST19U:^CM7m:A77QAAS^2«»GCM:^^ 277 Human R4 t R a b b it mt b i t CCCTTTCCM A to c ? m s c c iu u k . ^ S i U U S O C A C ^ G G J O G I ^ ' G I U I G G C A C T G ^ I c x a m rrc c iu u M ^ c iM m y ^ iT e r rrrc T fi'c \TSmfTfCCTCOiG»MSCM:M^^ 994 CCI^C?lGJI»K:iyGKmT€l^^ 880 MSMGCGCJyUM^T^TmC 337 * * * * ■ * ■ * * * * * ’ * ■ * * * * * * Antisense sequence T 0 7 ? < K J A 7 ( K : 0 « ^ ’ r C A 6 T 0 M M G C P 7 t a K 7 S « 7 G & T S f f C 7 7 G A 7 i f M O A a 3 7 W ^ 1 0 1 4 T @ : P 7 9 C U A T & C P S ^ T C J U S T C 0 U U U U 5 C A ^ ^ a T & ^ ^ 9 4 9 T*m‘ OOW«^KyiSPSCJWTCOWWUMSCAT«3ATGTGGTJITTCTT^ 397 * * * * * * * * * * * * * * * * * » , * % «•**•»»•«• * * « ■ « * * * • •• ••****•* • * • * x u a m n Rat R a b b e t »•* ♦ ■ # « • « * # * * * • * * * * * * * * * * * * jyyCJIA lTlTt^A C1’ 0CA^C7CA7<Uy^H:CACR£C’ 2 O aP ^^ 1134 ju ^ c K T c iT T ® rr* C T !W ic c K M S iy T O a s c * B « re a » d M M U iT M S w a a « e » * ^ ^ i,o « o A iM m ic « W i0 5 « C A c c « :i« c a w x a w io ( r io a » * m M ! ^ ^ »17 M W ld t: * » t K a U b i t Gi?«iuauw;iTOBKEaaasTOTGTOS»j»im^M****eMA«rtccrr»ic^^ 1194 « (S * e M 8 T a im T a w T W O T T « )a g w i* M * s iiA a o * fc « w c T * )^ ^ iiso iass»ail»«*C16?«raST(»C T«aM G A M IU IA I«»*C*ITCCIT*T»IC»Ca»W l 977 **:«««»««* ** ***** *•*«-*«••••■ *«* ***** ******** *********** T C fJiC M kJkktC C Z Q hT rttiacm Q hlM T ^^X r^^ 1294 C^PCIUUU^TCCi;aATmK^TCTGJUilU^Te&Cl<iiT6CG€jyu:^ 1180 C^TJ«yOUUktCCtGAtmGCCTTOMMT«^CTGTiS:STAPaUWC13^^ 637 W m & n R a t ■ R a b b i t Rat R a b b it fiS 3 A C T cm cccc< :m 3 A T rn A 7 ^n x:fn i:^€ ^^^^ 1 3 3 4 — O f s c c e c 6 9 1 «»» *««»« ♦ )« * 4 t4 t( m < in n n ir * l« ” * •**•*• **•*•* *»♦ ♦ * * * ■ ■ * * ,ijg k O T s< jc T O » p is 3 c a a y g y y s s s A im x ftc © fiic ix ^ ^ 1374 . ,juusQ i^® scfJU iT 0ai^< ^kik6© 5jyiT ccm G ?r3w si^<a7% ccjffcc 1 3 0 0 --.-»Cf5CASe^GliaSA11kTNS5®l®R©CTCCCJUW33CCrTtSGPm3l2^ 744 Figure 19. Nucleotide sequence of amplified rabbit clathrin HC gene fragment. The nucleotide sequence of rabbit clathrin HC is compared with known sequences of human and rat. Residues identical to clathrin HC between species are labeled with an asterisk. The nucleotide sequence used for the design of the antisense utilized in clathrin HC knockdown is shown in a box (between 290-304 bp of rabbit clathrin 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To verify the existence of clathrin HC and caveolin-1 at the protein level, Western blot analysis of RCEC lysates was performed using mouse monoclonal antibodies. Fig. 20a shows that the clathrin HC antibody detected a 180 kDa-sized protein in RCEC and HeLa cells. On the other hand, caveolin-1 antibody could not detect any protein band in RCECs, but did detect a 21 kDa-sized protein in both A431 cells, known to express caveolin-1 abundantly, and rabbit cardiac muscle cells used to demonstrate cross reactivity of mouse caveolin-1 antibody against rabbit protein (Fig. 20b). A (kDa) HeLa RCEC 2 S 0 - ' ^ ^ ^ C la th rtn H C 1 5 0 - C180 icDa) B MW C kD a) Cardiac RCEC A431 Myscia 1 9 - (22 kDa) Figure 20. Western blot analysis of clathrin HC and caveolin-1 expression. A) Expression of clathrin HC in RCECs and HeLa cells (positive control) probed with clathrin HC monoclonal mouse antibody. B) Expression of caveolin-1 in A431 cells (positive control) and rabbit cardiac muscle cells, but not in RCECs, was found when probed with caveolin-1 monoclonal mouse antibody. All cell lysates were prepared from confluent cells grown on tissue culture-treated substratum. 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2.4. Knockdown o f clathrin H C by antisense oligonucleotides In order to ascertain the role of clathrin in nanoparticle endocytosis, we devised an antisense oligonucleotide-based strategy to knockdown clathrin HC transcript levels in RCECs and HeLa cells. Fig. 21a demonstrates that RCECs transfected with antisense oligonucleotides using Lipofectamine^'^ reagent inhibited clathrin protein expression in a dose dependent manner, with an apparent IC50 of 0.99 ± 0.01 pg/ml. Similar treatment with sense oligonucleotides did not cause any inhibition at the same concentrations tested (not shown). A similar pattern of clathrin protein inhibition occurred in HeLa cells as a result of transfection with antisense oligonucleotides (data not shown). To demonstrate that the antisense oligonucleotide knockdown of clathrin HC was specific, we evaluated the expression of P-actin protein in treated cells. Fig. 21a shows that P-actin protein expression is unaffected with clathrin HC antisense oligonucleotide treatment at all applied concentrations. Fig. 21b is a semi- quantitative analysis of clathrin protein expression normalized by P-actin expression in RCECs. It demonstrates that maximal inhibition of clathrin protein expression (ranging from 75-900%) occurred at 2 pg/ml of antisense oligonucleotides. Additionally, transfection with antisense oligonucleotides at 4 pg/ml did not cause significant changes in transepithelial resistance (0.9 ±0.12 kQ.cm^) and potential difference (10 ± 2 mV apical negative), assuring cell viability. 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) 250 - 150 - 50 - 37 " MW (kDa) 0 1 1.5 2 3 Amount of antisense oligonucleotide in |L ig - ^ n n p u p s i ^ p i l j ^ p Clathrin HC (180 kDa) Actin (40 kDa) B) £ < / > ■ 5 % 0.8 c « | . 2 0.6 ■ K “ > S 1 0 .Q ( ] ) .2 ^0 4 ■ 5 X 2 0 ) fi 0-2 I ? - ™ 0 0 1 1.5 2 3 Amount of antisense oligonucleotide In Figure 21. Knockdown of clathrin HC protein expression. A) Western blot analysis of RCEC lysate following 24 h transfection using Lipofectamine reagent (10 /tg/ml) with various concentrations of antisense oligonucleotides targeted against clathrin HC mRNA. Represents one typical western blot (n=l). B) Quantitative analysis of clathrin expression normalized against actin protein expression as a function of increasing doses of antisense oligonucleotides in fig. Images and bands were scanned and quantified using Scion Image software and indicated as percent inhibition with respect to clathrin HC levels in untreated samples (mock transfection). Bars represent mean±standard error of the mean (n=4). 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In contrast to earlier pharmacological treatments aimed at disrupting clathrin coated formation, apical uptake of PLGA nanoparticles in these transfected RCECs was unchanged at all antisense oligonucleotides concentrations tested (Fig. 22). Fig. 23b demonstrates that basolateral endoctyosis of transferrin was reduced significantly by 40% in these transfected RCEC compared to mock transfection (with sense oligonucleotide), which is commensurate with clathrin protein reduction in these cells (Fig. 23 a). O 3 ® U t re a o c re z 150 c ' r e o L _ 100 0.0 1.0 1.5 2.0 3.0 Amount of AS oligonucleotide in Figure 22. Nanoparticle uptake in clathrin-knockout RCECs. Apical uptake of PLGA nanoparticles (0.5 mg/ml) in RCECs at 37 °C for 30 min following 24 h transfection with antisense oligonucleotides against clathrin HC (see previous figure for clarifications). Bars represent mean ± standard error of the mean (n=4). 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) 250 150 0 1 2 3 Amount of antisense oligonncleotide in pg : ■ -r# ---- Clathrin HC (180 kDa) 50 - 37 “ M W (kDa) Actin (40 kDa) B) a 9 O B 9 9 a ’S t ® cS a 2 W > i E > 'e® H s 100C 750 500 250 0 * * * 0.0 1.0 2.0 3.0 Amount of AS oligonucleotide in (A g Figure 23. Internalization of transferrin in clathrin-knockout RCECs. A) Western blot analysis of RCEC lysate following 24 h transfection with various concentrations of antisense oligonucleotides targeted against clathrin HC mRNA. B) Basolateral uptake of FITC-transferrin (50 /rg/ml) in RCECs at 37 °C for 30 min following 24 h transfection with antisense oligonucleotides against clathrin HC. Bars represent mean ± standard error of the mean (n=4). The asterisk denotes significant differences from control (zero antisense oligonucleotide concentration, p<0.01). 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4. DISCUSSION 4,1. Involvement of endocytosis Confirmation of endocytosis involvement in PLGA nanoparticle uptake was obtained by the significant reduction of uptake as a result of pretreatment of RCECs with microfilament inhibitors and metabolic energy poisons (Fig. 11) as well as by the punctate distribution pattern seen imder confocal microscopy (Fig. 12). In addition, our findings for the stimulatory effect on fluid phase endocytosis in rabbit conjunctival epithelial cells corroborate the role of endocytosis in PLGA nanoparticle uptake. Our first evidence for endocytosis of PLGA nanoparticles came from uptake studies done in the presence of micro filament inhibitors. At the apical membrane of epithelial cells, vesicle formation for both membrane-bound and fluid-phase markers was shown to depend on the activity and polymerization of an actin-microfilament network (Gottlieb et al., 1993), which can be specifically disassembled by cytochalasin D. Treatment of RCECs with cytochalasin D reduced PLGA nanoparticle uptake significantly (Fig. 11). This supported the notion that nanoparticles induce vesicle formation (endocytosis) in RCECs. Our cytochalasin inhibition data are in agreement with a previous report for the uptake of isobutylcyanoacrylate nanoparticles loaded with actinomycin in rat glumerular kidney cells (Manil et al., 1994). We have only studied the effect of cytochalasin D on apical uptake of PLGA nanoparticles, and not basolateral uptake, since the 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reported inhibitory effect of cytochalasin D on endocytosis of macromolecules and nutrients was postulated to be only at the apical membrane (Jackman et al., 1994). In contrast, nocodazole, a microtubule inhibitor, did not cause any significant reduction in PLGA nanoparticle uptake (Fig. 11). This confirmed that microtubules are involved in vesicle transport and not endocytosis or receptor recycling at the plasma membrane, as suggested by Hunziker et al. (Hunziker et al., 1990) for the transport of dimeric immunoglobulin A across MDCK cells. This finding is in contrast to the previous report by Pratten and Lloyd (Pratten and Lloyd, 1997) for the uptake of '^^I-labelled Percoll nanoparticles (which comprises 30-nm silica particles coated with polyvinylpyrrolidone) in rat visceral yolk sac. However, these authors used colchicine, which is a different microtubule inhibitor. In addition, this discrepancy could be attributed to the higher endocytotic activity in visceral yolk sac epithelial cells, as part of their nutritional support function to the embryo (Jollie, 1986). The use of metabolic energy poisons resulted in reduced PLGA nanoparticle uptake in RCECs, which demonstrates that uptake is an active process and support the role of endocjdosis (Fig. 11). Since the metabolic inhibitors (sodium azide and 2,4-dinitrophenol) only inhibit endocytosis but not binding of the ligand (Douglas and King, 1985), the membrane-bound fraction is unchanged in the presence or absence o f these metabolic inhibitors. Therefore, we have separated the internalized portion of PLGA nanoparticles in RCECs from the membrane bound portion by trypsinization and centrifugation. Internalization of invaginated coated pits was 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. shown to be dependent on the energy level in the cell (Smythe et al., 1992). Our findings are in concert with the findings by Panyam and Labhasetwar (Panyam and Labhasetwar, 2003b) demonstrating that exocytosis of PLGA nanoparticles containing 6-coumarin was reduced after treatment of vascular smooth muscle cells with sodium azide and deoxyglucose. Confocal microscopy (Fig. 12) showed that PLGA nanoparticles were internalized in RCECs rather than adsorbed on the cell surface and were distributed in both a diffused manner in the cytoplasm and in a punctate manner in subcellular, possibly endosomal, compartments. This pimctate vesicular distribution supports endocytic uptake of these PLGA nanoparticles. The diffused distribution of PLGA nanoparticles may indicate that some of these nanoparticles do not reside in subcellular compartments long enough, thus implicating their potential for cytoplasmic or intracellular drug delivery. A similar observation for the vesicular uptake of polyisobutyl cyanoacrylate (PBC A) nanoparticles containing fluorescent marker in the conjunctival tissue was reported (Zimmer et al., 1991). However, these authors observed these nanoparticles in the first two layers only, and reported that no full tissue penetration occurred. In addition, these authors postulated that either endocytosis of nanoparticles occurred or lysis of conjunctival cell wall by nanoparticle metabolic degradation products are possible explanations of their data. Although the degradation products of poly (alkylcyanoacrylate) nanoparticles were ascribed to doxorubicin diffusion and accumulation in tumor cells due to formation of ion pair at the plasma membrane (de Verdiere et al., 1997), this scenario has not 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. been reported and is unlikely to occur with PLGA nanoparticles. Therefore, direct endocytosis of these lipophilic PLGA nanoparticles into RCECs is a logical explanation of the data. Since the uptake of Lucifer yellow has not been studied in the conjunctival epithelium, it was imperative to validate its behavior as a fluid phase molecule before making any conclusions. The linear uptake pattern of Lucifer yellow over time qualified it as a marker for fluid-phase endocytosis in the conjunctival epithelium (Fig. 13). Lucifer yellow (LY) has been used to measure fluid-phase endocytosis in macrophages (Swanson et al., 1985), proximal tubular cells (Goligorsky and Hruska, 1986), and hepatocytes (Oka et al., 1989). LY seems well suited for use since it is highly fluorescent, is impermeable to membranes, and does not bind to membranes (Mir et al., 1988). Our findings are consistent with previous observation in hepatocytes (Oka et al., 1989). The significant increase in Lucifer yellow uptake (39%) when co-administered with nanoparticles implicated nanoparticles in the induction of endocytosis (vesicle formation) in RCECs (Table 7). This may explain the mechanism of drug delivery enhancement by nanoparticles seen in ocular epithelial tissues and others. All the present findings coupled with our previous findings (Qaddoumi et al., 2000) for the saturable, temperature-dependent, and competitive inhibition of PLGA nanoparticle uptake in RCECs advocate endocj^osis as the main pathway for internalization. Ultrastructural evidence for endocytosis (or phagocytosis) of proteins and latex nanospheres (800 nm in diameter) in the conjunctival epithelium has been 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reported previously (Steuhl and Rohen, 1983;Latkovic and Nilsson, 1979a). Uptake of particulate systems could occur through various processes, such as by fluid-phase pinocytosis, adsorptive-mediated endocjdosis, phagocytosis, and receptor-mediated endocytosis (Foster et al., 2001 ;Suh et al., 1998). It has been previously suggested that a linear uptake process is indicative of fluid phase pinocytosis, whereas saturable uptake is an indication of receptor- or adsorptive-mediated endocytosis (Catizone et al., 1993). Because PLGA nanoparticle uptake in RCECs exhibited a saturable pattern and competitive inhibition, fluid-phase endocytosis is not likely to be involved (Qaddoumi et al., 2000). On the other hand, both adsorptive and receptor- mediated endocytosis involve active and saturable uptake processes, which depend on binding to specitic/non-specitic binding sites and receptors, respectively (Fillebeen et al., 1999). Since macromolecules transported by the adsorptive pathway have affinity constants in the micromolar range (our Km is 420 pg/ml, (Qaddoumi et al., 2000)) compared to nanomolar range in receptor-mediated endocytosis (Goldstein et al., 1985;Sai et al., 1998), we postulate that PLGA nanoparticle uptake in RCECs occur through adsorptive-mediated endocytosis. 4.2. Elucidation of the endocytic mechanisms Our most significant finding in this study is that rabbit conjunctival epithelial cells express clathrin heavy chain (HC) at the gene and protein level, but not caveolin-1. In addition, we have obtained evidence that endocytosis of PLGA nanoparticles in rabbit conjunctival epithelial cells occurs independently of both 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. clathrin HC and caveolin-1, although PLGA nanoparticle uptake may in part take place via clathrin-mediated processes. The existence of an endocjdic mechanism for the internalization of PLGA nanoparticles in rabbit conjunctival epithelial cells was previously suggested by the uptake inhibition by microfilament inhibitors and metabolic poisons as well as the punctate distribution pattern shown under confocal microscopy. Similar endocytic processes were also described for the uptake of PLGA nanoparticles in other cell types, including vascular smooth muscle cells (Panyam and Labhasetwar, 2003b). However, the mechanism of endocytosis of polymeric nanoparticles into epithelial- type cells specifically has not been elucidated to our knowledge. Panyam et al. (Panyam et al., 2002) reported that uptake of PLGA nanoparticles in vascular smooth muscle cells was significantly reduced after inhibition of clathrin-mediated pathways, but not for the caveolae-dependent ones. Similar findings were also reported by Huang et al. (Huang et al., 2002) for the uptake of FITC-chitosan nanoparticles by A549 cell line. However, these studies lacked control markers for either clathrin- or caveolae-mediated endoc3dosis, were not supported by another evidence, and were studied in non-epithelial type cells. As a result of intracellular depletion or treatment of RCECs with hypertonic solution, pharmacological treatments aimed at indirectly inhibiting clathrin-mediated endocytosis (Hansen et al., 1993), we initially confirmed the involvement of clathrin in nanoparticle uptake (Fig. 14). Disruption of clathrin- related endocytic mechanisms in RCEC resulted in 35-45% maximal inhibition of 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PLGA nanoparticle uptake. In contrast, disruption of caveolae-mediated processes with either nystatin or fillipin (Lobie et al., 1999), substrates affecting cholersterol function in cells and caveolae structure, did not affect PLGA nanoparticle uptake (Fig. 16). Besides, the partial colocalization of clathrin staining with coumarin- loaded nanoparticles suggested a similar conclusion. Our cytochalasin D inhibition data also substantiate the role of clathrin- mediated endocytosis in nanoparticle uptake, as visualization of actin filaments surrounding the fusion ring of coated pits was shown by irmmmogold electron microscopy (Snigirevskaya and Cottier, 1999). It is possible that PLGA nanoparticles enter epithelial cells through a process similar to adsorptive endocytosis, with nanoparticles being concentrated on membrane pits that are non coated rather than floating in cell fluid (such as fluid-phase markers). Such complex pathways are poorly understood, but have been attributed in the entry of influenza virus and internalization of interleukin-2 receptors in HeLa cells and lymphocytes, respectively (Sieczkarski and Whittaker, 2002;Lamaze et al., 2001). However, it may be pointed out that treatments such as hypertonic and intracellular depletion may not be specific toward interrupting formation of clathrin-coated vesicles and may affect other endocytic processes in general, such as adsorptive endoc3dosis and fluid phase endocytosis. That may be the case as intracellular depletion was shown to significantly diminish the luminal to basolateral transfer of the fluid phase marker, Lucifer yellow, in cultured canine proximal tubular cells (Goligorsky and Hruska, 1986). Similarly, hypertonic 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. treatment of rat hepatocytes significantly reduced the uptake of radiolabeled bovine serum albumin (BSA), a marker for fluid phase pinocytosis (Synnes et al., 1999). However, the extent of inhibition for BSA averaged 20-60% (depending on time point), whereas that of radiolabeled asialoorosomucoid, a marker for clathrin- mediated endocytosis, averaged 40-90%. These studies suggest that these pharmacological treatments (hypertonic and intracellular depletion) may affect other types of endocytosis, not just that mediated by clathrin. We have obtained uptake inhibition for PLGA nanoparticles as a result of RCEC treatment with hypertonic and intracellular depletion averaging 40-45% (Fig. 14). According to the findings by the previous investigators (Goligorsky and Hruska, 1986;Synnes et al., 1999), our endocytosis of PLGA nanoparticles may only be partially dependent on clathrin-coated vesicles. Our data for the marked inhibition of basolateral endocytosis of transferrin (70%), as a result of this similar treatment, may corroborate this notion (Fig. 15). In addition, our confocal microscopy data suggested only partial colocalization of PLGA nanoparticles staining with that of clathrin HC (Fig. 17). A more definitive conclusion can be drawn from our knockout studies of clathrin HC protein in RCECs (Fig. 21). These data showed almost complete inhibition of clathrin HC protein expression (ranging from 70-90%) and have allowed us to elucidate the role of clathrin HC in PLGA nanopartiele endocytosis. Our inhibition data for the basolateral endocytosis of transferrin (45%) in clathrin HC-knockout RCECs justified the validity of the antisense oligonucleotide 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transfection model (Fig. 23). Our results from endocytosis of PLGA nanoparticle in rabbit conjunctival epithelial cells suggested elathrin heavy chain-independent mechanisms (Fig. 22). These findings may indicate that endocytosis of PLGA nanoparticles in rabbit conjunctival epithelial cells occur through multiple mechanisms, out of which clathrin-coated pits may not eontribute significantly. However, the extent of inhibition of transferrin endocytosis was much lower than that reported by other investigators (80-90%) in similar knockout models of clathrin HC (Llorente et al., 2001;Motley et al., 2003). Although one might argue that inhibition of clathrin heavy chain may not entirely affeet clathrin-mediated endocytosis (due to the presence of clathrin light chain), several studies have pointed that knockout of clathrin heavy chain is sufficient for marked reduction of clathrin- mediated endocytosis (Iversen et al., 2001;Llorente et al., 2001). It is also possible that clathrin expression was not knocked out completely. However, one cannot rule out non-speeific, random assoeiation of nanopartieles in clathrin-coated vesicles. It is possible that nanopartieles are taken up non-specifically through clathrin-coated vesieles (mediated by van der Waals forces), rather than through a sorting sequence. Evidence for this comes from transmission electron microscopy images were osmium oxide-loaded PLGA nanopartieles were seen in the center of a newly formed endocytic vesicles of vascular smooth muscle cells instead of being attached to the membrane wall (Panyam et al., 2002). Evidence for clathrin involvement in internalization of fluorescent latex beads in non phagocytic B16 cells was demonstrated recently (Rejman et al., 2004). These investigators demonstrated that 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. there is a size dependent internalization of fluorescent latex beads in that particles < 2 0 0 nm was primarily taken up by clathrin-mediated processes, whereas a shift to internalization by caveolae was shown for particles in the size range of > 500 nm. Since our nanopartiele size is 100 nm, these studies support our conclusion of a partial involvement of clathrin in PLGA nanopartiele endocytosis in RCECs. Clathrin plays an important role in endoeytosis of macromoleeules and nutrients as well as in membrane trafficking steps, particularly in sorting events in the trans-Golgi network. Although clathrin is thought to be a ubiquitous protein, the existence of clathrin in ocular tissues has only been described in the rat retina and lens epithelial cells (Bloom and Puszkin, 1983;Brown et al., 1990). Our current study is the first report describing the presence of clathrin mRNA and protein in the rabbit conjimctival epithelium (Figures 18 and 20). Earlier studies have highlighted the important role played by clathrin heavy chain, as demonstrated by the decrease in pinocytosis and receptor-mediated endocytosis in clathrin HC deficient CV-1 cells (Doxsey et al., 1987). Previous attempts at disrupting clathrin HC involved targeted gene mutation studies in single cell level in eukaryotes or cell lines (Lemmon and Jones, 1987;Ruscetti et al., 1994;Wettey et al., 2002). In addition, the use of stably transfected cell lines with either mutant dynamin or antisense overexpression of clathrin HC has been entertained recently in estimating the role of clathrin (Iversen et ah, 2001;Llorente et al., 2001). A clathrin knockout model using an antisense approach in primary 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cultures of epithelial cells is a better approximation of physiological situations than the use of cell lines, as we have shown in this study. Caveolae are small flask-shaped invaginations of the plasma membrane that approximately 50-70 nm in diameter (Lamaze and Schmid, 1995). They are particularly abundant in endothelial cells, adipocytes, vascular smooth muscle, fibroblasts and lung alveolar type I epithelial cells (Smart et al., 1999a). The functions of caveolae are relatively poorly understood, but they have been implicated in the transcytosis of some molecules (e.g., albumin), cholesterol homeostasis, and signal transduction. For instance, Griffoni et al. (Griffoni et al., 2000) have shown that caveolin-1 knockdown impairs angiogenesis in vitro and in vivo. Since caveolin-1 is the most widely investigated of the caveolin proteins (3 isoforms in all) and because over 90% of caveolin-1 is found associated with caveolae, it can be reliably used as a marker for caveolae structures (Rothberg et al., 1992). Our data indicate that rabbit conjunctival epithelial cells do not express caveolin-1 mRNA or protein, as assessed by RT-PCR and Western analysis, respectively (Figures 18 and 20). Our finding for the absence of caveolin-1 in rabbit conjunctival epithelial cells is in contrast to the report by Boulton et al. (Boulton et al., 2001) of positive staining of caveolin-1 in human conjimctival tissue. Stmctural differences of conjunctival and comeal epithelial tissues between human and rabbit species may explain the apparent species differences (Watsky et al., 1988), and/or the use of whole conjunctival tissue by Boulton and coworkers, which may have resulted from caveolin-1 staining of non-epithelial origin. When we 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. performed Western blot analysis on whole conjunctival tissue obtained from pigmented rabbits, the expression of caveolin-1 could be visualized (data not shown). By contrast, carefully isolated conjunctival epithelial cells do not display the expression of caveolin-1 both at the gene and protein levels, unlike the intact tissue. It is worthwhile to point out that gene expression of caveolin-1 in rabbit comeal epithelial tissue (Fig. 18) is in agreement with previous report (Amino et al., 1997) and that the absence of caveolin-1 gene expression in rabbit brain tissue (Fig. 18) is consistent with reports of Tang et al. (Tang et al., 1996), who used rat brain tissue. The expression of caveolin-1 protein in rabbit cardiac muscle cells mles out the possible lack of cross reactivity of mouse caveolin-1 monoclonal antibody with rabbit protein. Our pharmacological data suggest that other caveolins (caveolin-2 or 3) are not likely to be involved in nanopartiele endocytosis in rabbit conjunctival epithelial cells. In sunamary, our findings indicate the absence of clear correlation between endocytosis of PLGA nanopartieles in rabbit conjimctival epithelial cells and the involvement of clathrin HC or caveolin-1 proteins. However, partial intemalization of PLGA nanopartieles via clathrin-mediated endocytosis is likely to be the case. The absence of a caveolin-1 specific signal in these cells coupled with the lack of changes in PLGA nanopartiele uptake under clathrin HC protein knockdown conditions of RCECs requires further investigation to offer some physiological relevance. It can be suggested that adsorptive endocytosis or macropinocytosis (less likely) may be involved in nanopartiele endocytosis into rabbit conjunctival 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. epithelial cells, which may resemble the pathway of lipid and particulate absorption reported in the intestinal epithelium (Sanders and Ashworth, 1961). As a corollary to our findings for the existence of clathrin in conjunctival epithelial cells, future studies aimed at dissecting the endocytic pathway used by different molecules and the role of clathrin-mediated endocytosis in conjunctival physiology may benefit from our data presented herein. 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV. CHAPTER II. INTRACELLULAR DISTRIBUTION AND TRAFFICKING OF PLGA NANOPARTICLES 1 0 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. INTRODUCTION The development of an efficient therapy based on macromolecular drugs such as genes and proteins depends on their safe and efficient intracellular delivery. The benefits of using nanopartieles as carriers for proteins and genes are their controlled release properties providing steady drug concentration, their protective nature of proteins from degradation, and their enhanced permeability in epithelial cells (Li et al., 1997;Johnson et al., 1997;Femandez-Urrusuno et al., 1999). The degradation time for PLGA nanopartieles was reported to be 60 days in solution, but this may vary in the body (Park, 1995). If nanopartieles are to be applied for the delivery of proteins to the conjunctival epithelium, evidence for their protective capability and controlled release properties is needed. Many macromolecules and proteins (receptors or ligands) undergo degradation in the lysosomal compartment upon their endocytosis resulting in reduced activity and half-life of the protein or macromolecule (Riezman et al., 1997;Wattiaux et al., 2000). Little is known about the trafficking pattern of molecules or macromoleeules in the conjunctival epithelium. Knowledge about the intracellular distribution and trafficking of nanopartieles will facilitate our understanding of the mechanism of their action, lead to the design of better targeted nanopartieles, and reveal the factors that regulate their endocytosis and trafficking. This understanding will eventually lead us to better strategies to enhance the delivery of nanopartieles to the conjunctival epithelium and other intraocular tissues. 1 0 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The various steps in the endocytic process are mediated by specialized organelles (Figure 24). For instance, in most epithelial cells, endocytosed vesicles form primary endosomes, which then carry their cargo to early endosomes. The early endosomes serve as a sorting station that can carry their cargo to multivesicular bodies, late endosomes, and lysosomes where ligand degradation occurs by acid hydrolases; to recycling endosomes where cargo return to plasma membrane; and to a central compartment where cargo may be targeted to trans-Golgi network and transcytosis into the contralateral plasma membrane (Swaan, 1998). APtCAL ft V ^ V. BAmATmu. Figure 24. Schematic representation of endocytic pathways in polarized cells. Abbreviations: coated pit (CP), coated vesicle (CV), apical recycling compartment (ARC), apical sorting endosome (ASE), central recycling compartment (CRC), late endosomes (LE), lysosomes (dark small circle), multivesicular bodies (MVB), Golgi (G), trans-Golgi network (TGN), basolateral recycling compartment (BLRC), basolateral sorting endosome (BLSE), nucleus (N), tight junctions (TJ). Adapted from (Swaan, 1998). 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Since drug-loaded PLGA nanopartieles and micro spheres have heen shown to accumulate in retinal epithelial cells ranging from 20-40 days (Giordano et al., 1993;Yasukawa et al., 2002), we believed that this may be attributed to their lysosomal or endosomal escape from degradation (or lack of complete degradation). Furthermore, flow cytometry studies on the uptake of fluorescent-labeled oligonucleotide-loaded poly(lactic acid) nanopartieles in prostate cancer cells showed no accumulation in acidic compartments, whereas uptake of the free oligonucleotide resulted in localization in acidic compartments (Berton et al., 1999). However, these studies were based on indirect observation after monensin addition (endosomal ionophore) rather than direct observation in acidic compartments. In addition, cancer cells may behave differently than epithelial cells in general. Therefore, we studied the intracellular distribution and trafficking of PLGA nanopartieles in RCECs using organelle antibody markers and confocal microscpy. In addition, the sustained delivery of molecules from PLGA nanopartieles in RCECs was also investigated. Finally, we demonstrate the protective capability of PLGA nanopartieles for proteins against intracellular degradation using encapsulated horseradish peroxidase as a protein marker. 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2. MATERIALS AND METHODS 2.1. Materials Horseradish peroxidase type VI-A, poly (dl-lactide-co-glycolide) 50:50 (PLGA, Molecular weight 75,000), monensin, TRITC-labeled phalloidin, and mouse monoclonal anti-Golgi 58K protein antibody was obtained form Sigma Chemical Co (St. Louis, MO). Coumarin 6 was obtained from Polysciences, Inc. (Warrington, PA). Rabbit polyclonal anti-Beta-COP peptide antibody and rabbit polyclonal anti- calreticulin protein antibody were obtained from Affinity BioReagents (Golden, CO). Mouse anti-lysosome-associated membrane protein 1 (LAMP 1) monoclonal antibody, rabbit anti-Rab4 polyclonal antibody, and rabbit anti-Rapl polyclonal antibody were all purchased from Stressgen Biotechnlogies (Victoria, BC, Canada). ImmunoPure TMB substrate kit was obtained from Pierce Biotechnology Inc. (Rockford, IL). Human ARPE-19 cell line (retinal pigmented epithelium, CRL-2302) was purchased from American Type Culture Collection (Manassas, VA). 2.2. Immunofluorescence colocalization studies Sub-confluent RCECs (80% confluence) or ARPE-19 cell lines (retinal pigmented epithelium) were incubated from the apical side with 0.5 mg/ml suspension of PLGA nanopartieles loaded with 6 -coumarin at 37 °C for various time periods (2, 5, 10, 15, and 30 min), and then washed three times with ice-cold BRS buffer. Cells were then fixed with 4% paraformaldehyde in PBS solution for 30 min, permeabilized using 0.5% Triton-X 100 in water for 15 min, blocked with 10% 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bovine serum albumin (BSA) in PBS solution for 30 min, and incubated for 2 hr with mouse monoclonal antibody against either the lysosome-associated membrane protein 1 (LAMP 1) or Golgi58 protein or with rabbit polyclonal antibody against any of the following proteins: Beta-COP, calreticulin, Rapl, and Rab4. Cells were then washed several times with PBS and incubated for 1 hr with either rhodamine- labeled or Cy5-labeled goat anti-mouse or donkey anti-rabbit secondary antibody (depending on the antibody used). Cells were then incubated with TRITC-phalloidin for 45 min at room temperature and washed thoroughly. Finally, cell filter was cut and mounted on a glass slide using Prolong^*^ anti-fade mounting kit and viewed under a confocal microscope (Ziess LSM 510, Germany) using FITC (wavelength 450-490 nm), rhodamine filters (wavelength 550-570 nm), and Cy5 filter (wavelength 650-670 nm). 2.3. Effect of agents that disrupts the endosomes/lysosomes To evaluate if coumarin-loaded PLGA nanopartiele traffick to either the endosomal or lysosomal compartment following their endocytosis, RCECs were pre incubated with either 0.1 mM chloroquine (CQ, disrupts lysosomal pH gradient) or 10 pM monensin (disrupts endosomal pH gradient) for 1-hr before nanopartiele uptake at 37 °C for either 1 or 4 hr. After the designated uptake period, RCECs were washed with ice-cold BRS and then analyzed for the content of 6 -coumarin from PLGA nanopartieles as described previously in the methods section of chapter III. 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.4. Determination of nanopartiele residence within RCECs To justify the controlled release property of comarin-loaded PLGA nanopartieles in cells (not just in vitro release), we incubated semi-confluent RCECs at 37 °C with 0.5 mg/ml coumarin-loaded PLGA nanopartieles on days 4, 5, 6 , and 7 of culture for 2 hr. During each day, RCECs were then washed thoroughly with BRS and then incubated at 37 °C until day 7 of culture. Next the content of coumarin in RCECs at day 7 was determined (after 1,2, 3, and 4 days of incubation) as described previously in the methods section of chapter III. 2.5. Formulation of protein-loaded nanopartieles Nanopartieles were formulated according to a protocol developed by Davda and Labhasetwar (Davda and Labhasetwar, 2002) with slight modifications. In detail, nanopartieles were prepared using a water-in-oil-in-water (w/o/w) emulsion solvent evaporation technique. In a typical procedure, 100 mg PLGA was dissolved in 10 ml of methylene chloride. A 1.5% solution of PVA was prepared in cold distilled water, saturated with 25 pi of chloroform, and centrifuged at 1000 rpm for 5 min and then filtered through a 0.22 pm hydrophilic polysulfonic membrane syringe filter (25 mm Nalgene® filter unit, Nalge Co, Rochester, NY) to remove any undissolved PVA. A 1-ml aqueous solution of horseradish peroxidase (HRP, 1% w/v) was added, in drop wise portions, to the 10 ml PLGA-methylene chloride solution with vortexing for 1 min after each addition. It was then placed on an ice bath for 5 min and emulsified using a microtip probe sonicator set at 55 W of energy output (Microprobe 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sonicator , Misonix, Inc, Farmingdalc, NY) for 30 s, to obtain a primary water-in-oil emulsion. The primary emulsion was then added in two portions to 100 ml of the PVA solution with intermittent vortexing to obtain the multiple w/o/w emulsion. The emulsion was placed on an ice bath for 5 min and then sonicated for 2 min. The w/o/w emulsion was stirred for 4 hr on a magnetic stir plate to allow for the evaporation of chloroform and formation of the nanopartieles. The suspension of nanopartieles was stirred in a warmed water bath connected to a rotavap for an additional 2 hrs to ensure complete removal of the organic solvent. The suspension was transferred into Ultra-Clear™ centrifuge tubes and centrifuged at 21000 rpm (using SW28 rotor) for 20 min at 4 °C in an ultracentrifuge (Beckman Optima™ LE-80K, Beckman Instruments, Inc, Palo Alto, CA). The pellet was resuspended in distilled water and sonicated using microprobe sonicator for 30 s on an ice bath to disperse any aggregates. Ultracentrifugation was repeated two more times at 21000 rpm for 20 min each. This washing step was meant to remove PVA and unencapsulated HRP from the formulation. The supematant and the washings were stored to determine the amount of HRP that was not entrapped in the nanopartieles. From the total amount added and the amount not entrapped into nanopartieles, the protein loading in the nanopartieles was determined. After the last centrifugation, the nanopartieles were resuspended in 10 ml of distilled water and sonicated as above for 30 s on an ice bath. The nanopartieles were then centrifuged at 1000 rpm for 10 min at 4 °C to remove any large aggregates. The supematant was collected, frozen using ice-cold acetone 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. solution, and subsequently lyophilized for 2 days (VirTis Co, Inc, Freeze Dryer, Gardiner, NY). The lyophilized nanopartieles were stored desiccated at 4 °C afterwards. 2.6. Characterization of HRP-loaded nanopartieles The diameter of the nanopartieles, their size distribution, and their zeta potential were determined by sending a sample of HRP-loaded nanopartieles to Malvern Instruments, Inc. (Southborough, MA). 2.7. I n vitro release of HRP from nanopartieles 15 ml nanopartiele suspension (1 mg/ml) prepared in bicarbonate Ringers’ solution (BRS) was placed on a shaker for 14 days at 37 °C. Periodic samples (1 ml) were then taken and replaced with fresh BRS buffer of equivalent volume. Each sample was then subjected to centrifugation at 16,000x g for 2 min. The supematant was collected and analyzed for the released horseradish peroxidase (HRP) content using ImmunoPure® TMB substrate kit according to manufacturer’s direction. Briefly, equal volume of solution 1 (TMB solution) is mixed with peroxide solution 2 (H2O2, 0.02% in citric acid buffer). Of this solution mixture, 100 pi is placed into each ELISA 98-well plate. Add 20 pi of HRP samples to each well and incubate at room temperature for 5 min. Then stop the color reaction with 100 pi 2 M H2SO4 and read absorbance at 490 nm using a microplate reader. To determine the amoimt of HRP released from PLGA nanopartieles, we constracted a standard curve of HRP- 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. loaded nanopartieles (concentrations rcinging from 0.1-500 (xg/ml) in BRS with to which 0.1 M NaOH was added to break the nanopartieles apart and then analyzed as done for the in vitro released samples. 2.8. Enhancement of HRP uptake RCECs were incubated at 37 °C from the apical side for various time periods (1,2, and 4 hr) with either a suspension 1 mg/ml of HRP-loaded PLGA nanopartieles in BRS or a solution of 50 pg/ml of free HRP. Following uptake, RCECs were washed 3 times with ice-cold BRS solution. The transwell filter was then cut with a blade, placed in glass tube containing 1ml BRS and lyophilized overnight. Lyophilized nanopartiele samples containing HRP were extracted with 0.5 ml water following shaking in an oven set at 37 °C for 6 hrs. 20 pi of the extracted cell solution was then used for protein assay measurement using DC protein kit. In addition, 20 pi was used for HRP assay measurement as described above. A standard curve of HRP solution (0.5-1000 ng/ml) dissolved in BRS was constructed to determine the concentration of HRP samples from the uptake study. 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. RESULTS 3.1. Intracellular distribution and trafficking of nanopartieles As shown earlier under aim # 1, coumarin-loaded PLGA nanopartieles do not accumulate in the nucleus as demonstrated by the absence of colocalization with the nuclear marker, propidium iodide (Fig. 12). Using the early endosomal marker anti- Rah-4 rabbit polyclonal antibody, the results in Figure 25 demonstrates the absence of any colocalization of staining pattern of coumarin-loaded nanopartieles with that of early endosomes in RCECs at 15 min. We also looked at different time points (2, 5, 10, and 30 min), but could not find any colocalization of staining (Figures not shown to avoid redundancy). However, partial colocalization of nanopartiele staining with the early endosomal marker (Rab4) was found in retinal-pigmented epithelial cell line (ARPE-19), used as a control, at 5 min (Fig. 26). Similarly, Figure 27 shows that nanopartiele staining did not overlap with that of the late endosomal marker, anti-Rapl rabbit polyclonal antibody, in RCECs at all time points (only 15 min time point shown). To assess if coumarin-loaded PLGA nanopartieles are associated with lysosomal accumulation, we looked at nanopartiele staining in the presence of mouse anti-lysosome-associated membrane marker (LAMP-1) at different time points. The results in Figure 28 shows that nanopartiele staining pattern partially overlapped with that of LAMP-1 lysosomal marker only at 5 min time point or less, but not at longer time periods (10, 15, and 30 min) (Figure 29). Figure 30 demonstrates that a similar behavior was displayed by ARPE-19 cells. 1 1 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. where nanoparticles staining overlapped with that of LAMP-1 only at time points 5 min and less. Figure 25. Lack of eoloealization of nanoparticles and early endosomes in RCECs. Panel A, B, and C represent x-y confoeal images (40x magnification) scanned below the apical layers of sub-confluent RCECs (80% confluency). A) RCECs stained with rabbit anti-Rab4 polyclonal antibody (blue). B) RCECs following 15 min uptake of coumarin-nanoparticles (green) at 37 °C. C) RCECs stained with TRITC-pballoidin (Red). D) Merged image of A, B, and C. These findings were confirmed at other time points (2, 5,10, and 30 min). Ill Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 26. Selective colocalization of nanoparticles and early endosomes in ARPE-19 cells. Panel A, B, and C represent x-y confoeal images (40x magnification) scanned below the apical layers of sub-confluent RCECs (80% confluency). A) ARPE-19 stained with rahhit anti-Rab4 polyclonal antibody (blue). B) ARPE-19 following 15 min uptake of coumarin- nanoparticles (green) at 37 °C. C) RPE-19 stained with TRITC- phalloidin (Red). D) Merged image of A, B, and C. These findings were confirmed at other time points (2, 5, and 10 min). Arrows indicate areas of overlap in staining. 1 1 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 27. Lack of colocalization of nanoparticles and late endosomes in RCECs. Panel A, B, and C represent x-y confoeal images (40x magnification) scanned below the apical layers of sub-confluent RCECs (80% confluency). A) RCECs stained with rabbit anti-Rapl polyclonal antibody (blue). B) RCECs following 15 min uptake of coumarin-nanoparticles (green) at 37 °C. C) RCECs stained with TRITC-phalloidin (Red). D) Merged image of A, B, and C. These findings were confirmed at other time points (2, 5,10, and 30 min). 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 28. Selective colocalization of nanoparticles and lysosomes in RCECs. Panel A and B represent x-y confoeal images (40x magnification) scanned below the apical layers of sub-confluent RCECs (80% confluency). A) RCECs stained with mouse anti-lysosome-associated membrane protein 1 (LAMP-1) monoclonal antibody (blue). B) RCECs following 2 min uptake of coumarin-nanoparticles (green) at 37 °C. C) Phase contrast image of RCECs. D) Merged image of A and B. Areas of overlap in staining are indicated by arrows. These findings were only confirmed at 5 min time points and not at later time points. 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 29. Trafficking of nanoparticles to lysosomes is transient in RCECs. Panel A, B, and C represent x-y confoeal images (40x magnification) scanned below the apical layers of sub-confluent RCECs (80% confluency). A) RCECs stained with mouse anti-lysosome-associated membrane protein 1 (LAMP-1) monoclonal antibody (blue). B) RCECs following 15 min uptake of coumarin- nanoparticles (green) at 37 °C. C) RCECs stained with TRITC-phalloidin (red). D) Merged image of A, B, and C. These findings were confirmed at 10 and 30 min. 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 30. Colocalization of nanoparticles and lysosomes in ARPE-19 cells. Panel A, B, and C represent x-y confoeal images (40x magnification) scanned below the apical layers of sub-confluent RCECs (80% confluency). A) RPE-19 cells stained with mouse anti-lysosome-associated membrane protein 1 (LAMP-1) monoclonal antibody (blue). B) RPE-19 cells following 5 min uptake of coumarin-nanoparticles (green) at 37 °C. C) ARPE-19 cells stained with TRITC- phalloidin (red). D) Merged image of A, B, and C. Areas of overlap in staining are indicated by arrows. These findings were only confirmed at 2 min time point. 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. To evaluate if nanoparticles traffick from endoplasmic reticulum (ER) to the Golgi compartment, we used a Beta-COP rabbit polyclonal antibody, which is a coatomer protein involved regulating transport between the ER and the Golgi complex and intra-Golgi transport. Fig. 31 shows partial overlap of nanoparticle staining (green) with Beta-COP antibody staining (dark blue) after 15 min incubation of RCECs with nanoparticles. Figure 31. Selective colocalization of nanoparticles and Beta-COP in RCECs. Panel A and B represent x-y confoeal images (40x magnification) scanned below the apical layers of sub-confluent RCECs (80% confluency). A) RCECs stained with rabbit polyclonal Beta-COP peptide antibody (blue). B) RCECs following 15 min uptake of coumarin-nanoparticles (green) at 37 °C. C) Phase contrast image of RCECs. D) Merged image of A and B. Areas of overlap in staining are indicated by arrows. These findings were only confirmed at 30 min time point. 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Using both ER and Golgi monoclonal antibodies (calreticulin and Golgi58, respectively) as specific markers and confoeal microscopy, we show that nanoparticles staining pattern partially overlapped with that of the Golgi marker (Golgi58) at 15 min time point (Fig. 32), but not with the ER marker calreticulin (Fig. 33). Figure 32. Selective colocalization of nanoparticles and Golgi marker in RCECs. Panel A and B represent x-y confoeal images (40x magnification) scanned below the apical layers of sub confluent RCECs (80% confluency). A) RCECs stained with mouse monoclonal anti-GolgiS8K protein antibody (blue). B) RCECs following 15 min uptake of coumarin-nanoparticles (green) at 37 °C. C) Phase contrast image of RCECs. D) Merged image of A and B. Areas of overlap in staining are indicated by arrows. These findings were only confirmed at 30 min. 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 33. Absence of colocalization of nanoparticles and endoplasmic reticulum marker in RCECs. Panel A, B, and C represent x-y confoeal images (40x magnification) scanned below the apical layers of sub confluent RCECs (80% confluency). A) RCECs stained with rabbit polyclonal anti-caltreticulin protein antibody (blue). B) RCECs following 15 min uptake of coumarin-nanoparticles (green) at 37 °C. C) RCECs stained with TRITC-phalloidin (red). D) Merged image of A, B, and C. These findings were only confirmed at 10 and 30 min. To further confirm the localization of nanoparticles within the lysosomal compartment, we pretreated RCECs with 0.1 mM chloroquine (known to elevate the intralysosomal pH, thus inhibiting several lysosomal degradative processes). Figure 34 shows that choloroquine pretreatment of RCECs resulted significantly in a 37% increase in the apical uptake of coumarin-loaded nanoparticles following 1-hr. However, longer uptake of nanoparticles in RCECs (4 hr) following choloroquine pretreatment resulted in a modest 9% increase in the apical uptake of nanoparticles, 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. which was insignificant (Fig. 34). Figure 34 also shows that pretreatment of RCECs with 10 pM monensin (disrupts endosomal pH gradient) did not affect 1 hr apical uptake of nanoparticles. Further incubation of RCECs with nanoparticles (4 hr) following monensin pretreatment, however, did result in a 50% decrease in apical uptake of nanoparticles uptake (Fig. 34). 200 c o ^ 2 ” 100 H o O ) E O ) o E (0 ® u ■ ■ E a a o c n > T X I :: ::::::: ::: ill ::: ::::: iiii ■rnLrn 1-hr 4-hr Control Monensin CQ Figure 34. Effect of disruption of endosomes and lysosomes on nanoparticle uptake. RCECs were pre-incubated with either 10 mM monensin (disrupt endosomal pH gradient) or Chloroquine (lysosomotropic agent) for 1 hr prior to apical uptake of coumarin-loaded PLGA nanoparticle in RCECs for either 1 hr or 4 hr. The asterisk indicates significant difference from control (p<0.05). Bars represent mean ± standard error of the mean (n=6). 3.2. Sustained residence time of nanoparticles To evaluate if PLGA nanoparticles can be used for the controlled release of molecules, we evaluated the residence time of coumarin-loaded PLGA nanoparticles after different incubation period in RCECs. Figure 35 demonstrates that coumarin- 1 2 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. loaded nanoparticles were still remaining within RCECs for a period of 3 days following apical uptake, with 6% fraction of the dose applied remaining on day 3 (half of that on day 0). w ® o ■ ■ E (0 r- Q. .E 0 ® g s C a. 2 I 1 ? I 14% 13% 400 300 8.5% 6% 200 100 0 1 2 3 0 incubation period in days Figure 35. Sustained release properties of PLGA nanoparticles. RCECs were incubated at 37 °C at various culture days (4, 5, 6, and 7) with 0.5 mg/ml of coumarin-loaded PLGA nanoparticles for 2 hr. RCECs were then rinsed with culture medium and analyzed for content of coumarin- nanoparticles after 1, 2, 3, and 4 days as described previously. Bars represent mean ± s.e.m. (n = 6). Numbers above each bar represent the percent of dose absorbed or remaining. 3.3. Enhancement of HRP uptake 3.3.1. C haracterization o f H R P -lo a d ed n anoparticles PLGA nanoparticles containing 10% w/v horseradish peroxidase (HRP) were formulated according to an established protocol (Davda and Labhasetwar, 2002). 121 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 8 shows that the average diameter size of HRP-loaded nanoparticles was 296.5 nm with a polydispersity of 0.132. The zeta potential was -12.48 mV. These physiochemical characteristics of HRP-nanoparticles were measured using a Zetasizer® (analyzed by Malvern Instruments Inc., MA). Since the original loading of HRP in nanoparticles during formulation was 100 pg per mg of PLGA nanoparticles. The concentration of HRP remaining adsorbed or loaded in PLGA nanoparticles following formulation was 48.7 pg. Therefore, the encapsulated amount of HRP that retained activity in PLGA nanoparticles was 48.7% (Table 8). Table 8. Physiochemical characteristics of HRP-loaded nanoparticles Mean diameter Zeta potential Polydispersity HRP HRP encapsulation (nm) (mV) amount efficiency (%) (mg) 296.5 + 23.4 -12.48 + 2.73 0.132 + 0.02 100 48 The release profde of HRP-loaded nanoparticles was studied and shown to continually release HRP for a period of at least 2 weeks (32% released) using in vitro dissolution tests (Fig. 36). 3.3.2. Nanoparticles as protective carriers o f HRP To demonstrate the intracellular stability of nanoparticles (protective ability), we then studied the apical uptake of HRP-loaded nanoparticles in RCECs compared to 1 2 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a. a: o o > M (0 re 3 o w 40 “ 35 I 30 c 25 re C on < O _l Q . E o k. 10 5 15 Time (days) Figure 36. % Cumulative in vitro release of HRP from PLGA nanoparticles. 15 ml PLGA nanoparticle suspension in BRS (1 mg/ml) containing HRP (10% w/v) was placed in a vial on a shaker for 14 days at 37° C. Periodic samples (1 ml) were then taken and analyzed For the content of HRP as described in the methods section. Points represent mean ± s.e.m. (n = 4). that of free HRP using the same concentration for both (48.7 pg/ml). Our results show that nanoparticles enhanced apical uptake of loaded HRP in RCECs at all time points (1,2, and 4-hr) by at least 2-3 folds compared to that of free HRP application (Fig. 37). Apical uptake of HRP at 4-hr time point was much smaller than that at 1 or 2 hr (Fig. 37). 123 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 a a 0 Ph W 30 n 1 = 1 HRP ^ HRP-NP a 'S 20 - 10 - 1-hr 2-hr 4-hr Figure 37. PLGA nanoparticles as carriers of HRP. RCECs were incubated at 37 °C with either 1 mg/ml of HRP-loaded PLGA nanoparticles or free HRP both dissolved in BRS (using the same concentration of HRP of 48 mg/ml) for different time periods (1, 2, and 4 hr). The asterisk indicates significant difference from control (p<0.05). Bars represent mean ± standard 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4. DISCUSSION 4.1. Intracellular distribution and trafficking In this study, evidence was obtained to indicate that the trafficking of PLGA nanoparticles maybe entirely different than that of proteins and other macromolecules in their ability to escape endosomal trafficking, their short residence time in the lysosomal compartment, and their avoidance of sorting to endoplasmic reticulum (ER). These findings may explain the prolonged residence time of nanoparticles in epithelial tissues and other tissues following absorption and their protective ability of unstable drugs form intracellular degradation. In addition, these findings may corroborate the efficiency of nanoparticles in intracellular delivery of encapsulated drugs. Initially, we have shown that endocytosed vesicles containing coumarin- loaded nanoparticles may not be sorted to early endosomes (using rabbit anti-Rab4 antibody) during their trafficking pathway in RCECs (Fig. 25). Rab4 is a 25 kDa GTP-binding protein that regulates recycling of proteins from the early endosomes to the cell surface (Ayad et al., 1997). The accumulation of coumarin-loaded nanoparticles in the endosomal compartment of ARPE-19 cells and not RCECs suggested that the sorting and trafficking of nanoparticles might depend on the cell type studied (Fig. 26). This cell-type difference cannot be attributed to differences in the polarized nature of the cells, as both cell types (RCECs and ARPE-19) have been reported to exhibit polarized distributions of transporters or cell surface markers and are well differentiated (Turner et al., 2002;Dunn et al., 1998). We also showed that 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PLGA nanoparticles, once internalized, avoid accumulations in late endosomes as well using rabbit anti-Rapl polyclonal antibody as a marker (Fig. 27). Rapl is a member of the ras family of low molecular weight GTP-binding proteins that mediate recycling from late endosomes to multivesicular bodies or plasma membrane (Bos et al., 2001). Our findings for endosomal escape of nanoparticles are in agreement with earlier report by Berton et al. (Berton et al., 1999) for the uptake of fluorescently-labeled oligonucleotide poly(lactic acid) nanoparticles in DU 145 prostate cancer cells. They reported that upon addition of monensin, an increase in fluorescent signal intensity was observed by flow cytometry, indicating that free oligonucleotides were resident in an acidic intracellular environment, whereas oligonucleotides-loaded nanoparticles did not reside in an acidic compartment. However, these findings for avoidance of endosomal compartment by PLGA nanoparticles are in contrast to that by Panyam et al. (Panyam et al., 2002). They reported that in human arterial smooth muscle cells (HASMC), a partial co localization of staining between fluorescent PLGA nanoparticles and transferrin, a marker for early and recycling endosomes, was obtained (Peters et al., 2001). However, these authors later suggested that PLGA nanoparticles appeared to accumulate in compartments separate form the transferrin-labeled compartments, thus nanoparticles may be present in either later endosomes/lysosomes or the cytoplasm. Since these authors (Panyam et al., 2002) found a clear co-localization of nanoparticle staining with that of LysoTracker red (lysosomal marker) at 2 min time point, these findings point to lysosomal accumulation only (rather than endosomal 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. accumulation) which is consistent with our data. In addition, the failure of monensin, which is known to disrupt endosomal pH gradient, to increase apical uptake of PLGA nanoparticles in RCECs indicated lack of nanoparticle accumulation in endosomal compartment (Fig. 34). Prolonged treatment of RCECs with monensin (4 hr) was shown to decrease apical uptake of PLGA nanoparticles by 50% (Fig. 34). This could be attributed to the toxic effects of monensin on RCECs in a way to prevent accumulation of nanoparticles within the cell, as suggested by Sehested et al. (Sehested et al., 1988). Although most endocytosed cargo protein vesicles are destined to endosomal compartment following endocytosis, there is evidence to suggest that this trafficking behavior may not be universal to all endocytosed cargo. Endosomal escape has been reported for viral and non-viral vectors used in gene therapy (Wattiaux et al., 2000;Liu and Huang, 2002). For instance, viruses and peptide toxins use a fusogen peptide to cross endosomal membrane and reach the cytosol (Cotten et al., 1992;Wagner et al., 1992). In addition, polymers such as polyethyleneimine were thought to cause swelling and rupture of the organelles by sequestering protons and their counter-ions and creating an osmotic imbalance, making them function like lysosomotropic compounds or agents that disrupt the endosomes (Boussif et al, 1995). It is possible that PLGA nanoparticles may escape the endosomal compartment all together during their intracellular trafficking in RCECs. In such a scenario, nanoparticles trafficking through the cell may follow that of lipid and 127 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. particulate translocation through the intestinal epithelium, which accumulate in the Golgi apparatus following endocytosis prior to sorting to the lateral membrane for exocytosis (Sanders and Ashworth, 1961). However, one eannot exclude the possibility that PLGA nanoparticles may have trafficked to the endosomal compartment at later time points (>30 min), as we have only followed nanoparticle distribution up to 30 min. This is plausible since low rate of cellular processing have been documented for clathrin-mediated uptake of lipoplexes with a mean diameter of 200 nm (Zuhom et al., 2002). Interestingly, the shorter accumulation of PLGA nanoparticles in the lysosomal compartment (using LAMP-1 as marker) of RCECs or ARPE-19 cells (up to 5 min), may suggest that nanoparticles eventually escape this compartment to either the cytoplasm or another sorting compartment such as the Golgi (Figures 28- 30). LAMP-1, also known as Igpl20, is a type 1 integral membrane protein that is transported from trans-Golgi network to lysosomes (Chen et al., 1985). The observation for the short residence of nanopartieles in lysosomes is in agreement with our hypothesis for the ability of PLGA nanopartieles to avoid or escape the lysosomal compartment during their intracellular trafficking. Panyam et al. (Panyam et al., 2002) reported a similar observation, where PLGA nanopartieles were localized in the lysosomes as early as 2 min and them accumulated at 10 min in the cytoplasm. These authors suggested that nanopartieles escaped rapidly from lysosomal compartments into their cytoplasm following their uptake. 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The short residence of nanopartieles in the lysosomal compartment could be attributed to destabilization of lysosomal membrane by the hydrophobic PLGA nanopartieles leading to their cytoplasmic accumulation. It was reported that surface cationization of PLGA nanopartieles in the endosomal or lysosomal compartment is responsible for their escape, in a maimer similar to cationic lipids (Panyam et al., 2002). Cationization of PLGA microparticles or poly (lactide) with the change of pH was previously reported and was attributed to the transfer of excess protons form the bulk liquid to the nanoparticle surface or attributed to hydrogen bonding between carboxyl groups of PLGA and hydronium molecules in acidic pH (Stolnik et al., 1995;Makino et al., 1986). For instance, since early endosomes have a physiological pH (Mukheijee et al., 1997), PLGA nanopartieles would be expected to have a net negative charge, which would probably be repelled by negatively charged endosomal membrane. However, other physicochemical properties of PLGA nanopartieles (such as size and tacticity) may also be responsible for endosomaFlysosomal escape following their uptake in RCECs and trafficking. For instance, Laurent et al. (Laurent et al., 1999) found that intracellular hydrolysis of DNA was more rapid when poly-L-lysine was used compared to poly-D-lysine. These observations are explained by the fact that the translocation of DNA to lysosomes, where the degradation of the nucleic molecules takes place, is markedly slower when poly-D- lysine is used as carrier. In addition, it was reported that the size of the DNA- cationic vector complex is a determinant for its translocation to lysosomes (Wattiaux 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. et al., 2000). Rejman et al. (Rejman et al., 2004) evaluated the effect of particle size on the fate of particles to lysosomes or late endosomes in non-phagocytic mouse melanoma B16 cells. They noticed that only latex fluorescent particles < 200 nm colocalized with either FITC-dextran or LysoTracker Red (late endosomal/lysosomal markers), whereas larger particles > 500 nm did not. It would be interesting to evaluate the effect of different sizes of PLGA nanopartieles on lysosomal residence time and accumulation. The transient accumulation of nanopartieles in the lysosomal compartment is further confirmed by our inhibition study with chloroquine (Fig. 34). Besides disrupting lysosomal pH gradient, chloroquine is known to inhibit the transfer of an endocytosed molecule from endosomes to lysosomes (Mellman et al., 1986). Our data (Fig. 34) showed that short incubation of RCECs with chloroquine increased apical uptake of PLGA nanopartieles by 37%, while longer incubation (4 hr) resulted in a modest increase (9%). These findings emphasize that PLGA nanopartieles do not reside in the lysosomes for longer periods. Our study demonstrated that PLGA nanopartieles colocalized in RCECs with Beta-COP (Fig. 31), which is a coatomer protein involved in regulating transport between the endoplasmic reticulum (ER) and the Golgi complex and in intra-Golgi transport (Orci et al., 1997). Although our initial observation favored trafficking of nanopartieles to the ER, subsequent Immunofluorescence colocalization studies using calreticulin showed otherwise (Fig. 33). These findings cannot be argued against specificity of the antibody marker used, since calreticulin is a major calcium 130 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. binding protein found in ER membranes (Deiming et al., 1997). The ER is a network of tubules, vesicles and sacs that are interconnected. The major function of the ER is in synthesis and export of proteins and glycoproteins. This indicates that sorting of endocytosed vesicles containing PLGA nanoparticle to ER is not likely to be important for the cell. There are no literature reports, to our knowledge, that have looked at the intracellular distribution and trafficking of any type of polymeric nanopartieles or liposomes in the ER compartment. Out data pointed to the accumulation of PLGA nanopartieles within the Golgi network in RCECs, as indicated by the colocalization with the Golgi marker Golgi58 (Fig. 32). This marker, Golgi58, is found in the cytoplasmic face of the Golgi apparatus and binds to microtubules in vitro (Bloom and Brashear, 1989). Since the Golgi apparatus is the central compartment through which vesicle sorting to final destination and further processing of cargo occur (Donaldson et al., 1990), it is conceivable that PLGA nanopartieles might migrate to that compartment before arriving to its final destination or export to the basolateral membrane of RCECs. Such an intracellular fate would resemble that of lipid and particulate absorption in the intestine, which translocate to the Golgi apparatus before sorting to the lateral membrane for exocytosis (Sanders and Ashworth, 1961). This is the first report of an intracellular Golgi localization of PLGA nanopartieles or other types of nanopartieles. One might wonder whether the Golgi vesicular cargo of nanopartieles is released to the cytoplasm or is targeted to the basolateral membrane for exocytosis. 131 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2. Nanopartieles as carriers of proteins If nanopartieles are to be used for the delivery of proteins and drugs to the conjunctival epithelium and anterior segment of the eye, evidence for their controlled release properties and protective capability is warranted. Our study has demonstrated that coumarin-loaded PLGA nanopartieles were retained within RCECs for 3 days following apical uptake, with 6% fraction of the dose remaining on day 3 (Fig. 35). De Campos and coworkers (De Campos et al., 2001) have shown a similar sustained release profile for cyclosporin A in both comeal and conjunctival tissues for 48 hr following topical instillation of chitosan nanopartieles loaded with cyclosporin A to rabbits. Our data for the sustained accumulation of coumarin-loaded nanopartieles in RCECs over time point to the intracellular stability of nanopartieles in the cytoplasm and to lysosomal degradation during their short transit, as suggested earlier by immunofluorescence colocalization studies. We have also managed to fabricate a stable formulation of HRP-loaded PLGA nanopartieles with adequate size, loading, and release properties. The physiochemical characteristics of these nanopartieles were evaluated and shown to have favorable particle size (~ 300 nm) and in vitro release profile up to 14 days (Table 8). The cumulative amount of HRP released in vitro from PLGA nanopartieles after 14 days was 32% of the total amount (Fig. 36). These data indicate that our formulation had a small burst effect and was chemically stable. The burst effect has always been observed with nanoparticle systems formulated by double emulsion technique (w/o/w) and was prevented by improving the dmg 132 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. distribution throughout the polymer matrix (Fu et al., 2003). Another common problem with protein formulations into PLGA nanopartieles is that degradation of the polymer generates acidic monomers. Fu et al. (Fu et al., 2000) have shown the formation of a very acidic environment within the particles with the minimum pH as low as 1.5 in the center of the PLGA nanopartieles. This raised questions about protein or drug stability in PLGA nanopartieles upon storage. We have observed a 40% reduction in HRP activity in three months after formulation into PLGA nanopartieles (data not shown). One way to increase stability of proteins into PLGA nanopartieles during release is the incorporation of poorly water soluble basic inorganic salts, such as zinc carbonate (Johnson et al., 1997). Cleland et al. (Cleland et al., 1997) have reported on the successful encapsulation and release of recombinant human growth hormone in its fully bioactive from PLGA microspheres over 30 days. They pointed out that the chemical degradation rates of human growth hormone were not affected by the PLGA microspheres indicating the internal environment confers stability to proteins. In addition, Kim et al. (Kim et al., 2002) showed that single oral administration of PLGA nanopartieles containing collagen (300 nm in size) to mice had significantly suppressed the development of arthritis. This suggests that PLGA nanopartieles may serve as a powerful vehicle to promote protein drug delivery. Another problem encountered is that the release of proteins from the matrices of hydrophobic polyesters (like PLGA) occurs primarily through pore diffusion and 133 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the release rate is usually very slow for some proteins (Huatan et al., 1995). Owing to this reason, PLGA polymers have been blended with several hydrophilic polymers biocompatible to modify the hydration of the polymeric matrix of nanopartieles (Ibim et al., 1997;Wang et al., 1999). We have encountered no such problem since our PLGA nanoparticle formulation of HRP incorporates a hydrophilic polymer, polyvinyl alcohol, which tends to stabilize the formulation and aid in release of encapsulated drugs. We have shown that apical uptake of HRP was enhanced as a result of encapsulation in PLGA nanopartieles compared to HRP application (Fig. 37). The smaller uptake of HRP at 4-hr in comparison with 2-hr uptake (for both the free HRP and encapsulated HRP) could be attributed to intracellular degradation of free HRP in the cytoplasm or other cellular compartment. The enhancement of protein absorption was reported earlier for chitosan and poly (alkyl cyanoacrylate) nanopartieles in both the nasal and intestinal epithelium, respectively (Femandez- Urrusuno et al., 1999;Damge et al., 1997). Recently, PLGA nanopartieles were successfully utilized for the nasal and oral delivery of proteins and polypeptides (Vila et al., 2002;Sang and Gwan, 2004). Thus, our data and literature reports suggest the utility of PLGA nanopartieles for ocular delivery of proteins and peptides such as epidermal growth factor and interferon that may be of therapeutic relevance for certain ocular diseases or conditions. 134 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. V. CHAPTER III. ENHANCEMENT OF NANOPARTICLE ABSORPTION VIA LECTIN-MEDIATED RECEPTOR ENDOCYTOSIS 135 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. INTRODUCTION We sought to identify a substrate, which utilizes receptor-mediated endocytosis, i.e. as an endocytic ligand, for the purpose of promoting the ocular absorption of biodegradable PLGA nanoparticles loaded with a model protein. Based on their high binding affinity, rapid ocular binding to the conjunctival and comeal epithelium, and the demonstrated safety of lectins, we decided to evaluate the uptake characteristics of several plant lectins (Nicholls et al., 1996;Rittig et al., 1990;Smart et al., 1999b). Lectins are non-immunoglobulin glycoproteins that recognize and bind to specific sugar moieties on the cell membrane. They are involved in a variety of biological processes, such as cell-cell and host pathogen interactions and innate immune responses (Goldstein et al., 1977). Lectins are usually classified based on their saccharide specificity, although lectins in the same classification category, such as galactose-binding lectins, may have sugar binding pattems that are considerably different from one another (Virtanen et al., 1986). Since lectins demonstrated prolonged ocular retention and enhanced dmg delivery across epithelial tissues (Nicholls et al., 1996;Lehr, 2000), it is worthwhile to investigate their binding and intemalization kinetics as a means of using them as endocytic cell ligands to promote the ocular absorption of drug-loaded PLGA nanoparticles. The purpose of this aim was to investigate the binding and uptake characteristics of three model plant lectins (Solanum tuberosum, STL; Lycopersicon esculentum, TL; and Ulex europaeus, UEA-1) in primary cultured rabbit 136 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. conjunctival epithelial cells (RCECs). The conjugation of plant lectin to biodegradable PLGA nanoparticles and their uptake relative to unconjugated PLGA nanoparticles will be evaluated as a means of enhancing the conjunctival absorption of horseradish peroxidase (HRP). 137 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2. MATERIALS AND METHODS 2.1. Materials Fluorescently-labeled Solanum tuberosum (potato) lectin (FITC-STL, molar ratio fluorescein/protein 3.2), VYIC-Lycopersicon esculentum (tomato) lectin, FITC-Wex europaeus 1 (UEA-1, Gorse) lectin, and chitin hydrolysate (an inhibitor of potato and tomato lectins) were all obtained from Vector Laboratories (Burlingame, CA). L-a-fucose (an inhibitor of UEA-1 lectin) and l-ethyl-e-(3-dimethylaminopropyl) carbodiimide (EDAC) were obtained from Sigma Chemical Co. (St Louis, MO). Madin-Darby canine kidney cells (MDCK) type II cells (NBL-2) were obtained from American Type Culture Collection (Manassas, VA). 2.2. Characteristics of lectin binding and uptake Model lectins from three species were used: Solanum tuberosum (Potato lectin, STL), Lycopersicon esculentum (Tomato lectin, TL), and Ulex europaeus (gorse lectin, UEA-1). The sugar specificity and molecular weight of each are described in the results section. These lectins were examined with respect to their time-, concentration-, and temperature- dependent uptake and binding in primary cultured RCEC layers. For time-dependent uptake experiments, RCECs were incubated at 37 °C with 1 pM of FITC-STL, 1 pM FITC-TL, or 3.17 pM of FITC- UEA-1 lectin for various time intervals up to 2 hr. The concentration-dependency experiment was then carried out. To obtain the total binding and uptake in RCECs for each lectin, RCECs were incubated at 1 hr with various lectin concentrations, 138 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ranging from 0.1-6.34 p.M, in the absence or presence of an inhibitory sugar. Chitin (30 mg/ml) was used as an inhibitory sugar to correct for non-specific binding of FITC-TL or FITC-STL. L-a-fucose (10 mM) was used as inhibitory sugar for FITC- UEA-1 lectin. The specific binding/uptake was obtained by subtracting the total binding/uptake, in the absence of inhibitory sugar, from non-specific binding/uptake in the presence of excess amounts of the inhibitory sugar. The binding affinity parameters for each lectin (V m ax and Km values) were calculated from the specific binding/uptake curve fitted to Michaelis-Menten equation, and analyzed using GraphPad Prism software (GraphPad Software Inc., CA). The uptake/binding of various concentrations of FITC-STL (in the range of 0.25-2 pM) in MDCK cells was determined to provide a comparison between the binding affinity of STL in MDCK cells and the affinity in RCECs. To evaluate the amount of lectin internalized (endocytosed) into RCECs, the uptake of various concentrations of fluorescently labeled lectins by RCECs at 4 °C for 1 hr, at a range of concentrations similar to that used for uptake experiments at 37 °C, was evaluated. The uptake/binding curve obtained at 37 °C was then subtracted from the uptake/binding curve at 4 °C. This difference represented the active component of uptake (the amount internalized) and was expressed as nM lectin/mg total protein. The effect of lectins on the bioelectric parameters of RCECs was examined by incubating RCECs with lectins for 2 hr then evaluating the change in 139 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. both the transepithelial electrical resistance (TEER) and the potential difference (PD). 2.3. Confocal microscopy We used confocal laser scanning fluorescent microscopy to confirm the binding and intemalization of lectins in RCECs. Briefly, RCECs were incubated for 30-60 min at 37 °C with 1 pM FITC-STL, fixed with 3.7% formaldehyde for 20 min, permeabilized with 0.5% Triton-X 100 for 15 min, treated with 0.1 mM TRITC- phalloidin for 45 min, mounted on a cover slide using Prolong™ anti-fade kit (Molecular Probes Inc., OR), and viewed under the confocal microscope. In another set of experiments to confirm the specificity of lectin intemalization/binding in RCEC rather than the fluorophore FITC, RCECs were incubated at 37 °C for 1 hr with FITC-STL in the presence of excess amount of the inhibitory sugar, chitin, at 30 mg/ml. After RCECs were permeabilized, they were stained with fluorescent TRITC-phalloidin at 1 pM for 40 min to label actin filaments around the cell periphery. Confocal images (X-Y and X-Y gallery) of stained RCECs were viewed using FITC and TRITC filters on a Zeiss 510 LSM microscope (Carl Zeiss, Thomwood, NY). 2.4. Effect of lectin on cytokine induction The effect of Solanum tuberosum lectin on cytokine release in RCECs was evaluated using SearchLight rat cytokine array (Pierce Biotechnology Inc., Rockford, IL). This 140 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. array is a multiplexed sandwich ELISA for the quantitative measurement of 9 different cytokines per well: IL-la, IL-ip, IL-2, IL-4, IL-6, IL-10, GMCSF, IFNy and TNFa (Figure 38). Pre-spotted plate with 9 different cytokines/well i H S O ll w to a p i!* .' A<KSOi^SA-HRR WiSflDllt* fnemao til n iH iifiis usM artj^[» iw rs£ ieM l« « CCS ciTMra Figure 38. A summary diagram of SearchLight™ rat cytokine array technique. This proteome array is a sandwish ELISA for the quantitative measurements of nine different cytokines: IL-la, IL-lp, IL-2, IL-4, IL-6, IL-10, GMCSF, IFN-y, and TNF-a. The protocol was performed according to manufacturer’s direction. Histamine at 30 pM was utilized as a positive control for cytokine induction, as previously shown for cultured human conjunctival epithelial cells (Weimer et al., 1998). Briefly, RCECs 141 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. on day 6 of culture (1 day before confluency) were incubated at 37 °C with either 5 (ag/ml of Solanum tuberosum lectin (STL) or 30 |aM histamine in DMEM/F12 medium for different time periods (1, 2, 4, 8, and 24 hr). Following a simple ELISA procedure, the array generates a chemiluminescent signal that is imaged using a commercially available 12-bit cooled CCD camera. Using array software, the intensity of the spots for each unknown sample are compared with standard curves and exact values of each cytokine (pg/ml) are calculated. The optical density was calculated using an Array Vision™ software (Pierce Biotechnology Inc., Rockford, IL). 2.5. Conjugation of lectin to nanoparticles and nptake of conjugate Solanum tuberosum lectin was conjugated to PLGA nanoparticles using a protocol developed by Ertl et al. (Ertl et al., 2000) involving carbodiimide method. Briefly, 10 mg of HRP-loaded nanoparticles are washed three times in 1 ml 20 mM HEPES solution with subsequent centrifugation at 16,000 x g for 2 min. Next, nanoparticles are reconstituted in 0.5 ml HEPES solution to form a suspension and added to 1 ml mixture of 0.1 M l-ethyl-e-(3-dimethylaminopropyl) carbodiimide (EDAC) and 0.11 M N-hydroxysuccinimide in HEPES. The mixture is then rotated for three hrs at room temperature protected from light and centrifuged at 16,000 x g for 2 min to get rid of unreacted EDAC and N- hydroxysuccinimide. The pellet was reconstituted in 5 mg/ml HEPES solution of either Solanum tuberosum lectin (STL) or bovine serum albumin (BSA) and left to react at 4 °C for 1 hr to allow for 142 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chemical conjugation to occur. The unreacted STL and BSA were removed using overnight dialysis at 4 °C in BRS buffer (membrane cutoff of 70 and 80 kDa). The nanoparticle conjugate mixture was then centrifuged and washed five times before final reconstitution in 1 ml BRS buffer for uptake experiment. To confirm the amount of BSA and STL bound to nanoparticles was the same, FITC-BSA and FITC-STL were used in a similar conjugation experiments. RCECs were incubated at 37 °C for 1 hr with 1 mg/ml suspension of STL- nanoparticles conjugate, BSA-nanoparticle conjugate, or control nanoparticles (unconjugated). RCECs were then washed with ice-cold BRS buffer three times, cell filter cut with a blade, and incubated with 1 ml BRS before overnight lyophilization. Lyophilized nanoparticle samples containing FIRP were extracted with 1 ml water after 6 hr shaking in oven set at 37 °C. HRP activity was quantified using ImmunoPure^*^ TMB substrate kit as described previously under in vitro release method for HRP-loaded nanoparticles (Chapter 2). To evaluate if STL enhancing effect on apical uptake of HRP-loaded nanoparticles was dose dependent, we coated 1 mg nanoparticles with 1 ml different concentrations of STL (0, 1, 2, 4, and 6 pM) after rotating at 4 °C for 1 hr. The STL- coated nanoparticles were then centrifuged once and reconstituted with 1 ml BRS. RCECs were then incubated at 37 °C for 1 hr with various concentrations of ST- nanoparticle products in the presence or absence of 30 mg/ml chitin (inhibitory sugar for STL) and then cell washed and analyzed for HRP content as described above. 143 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. RESULTS The average transepithelial electrical (TEER) and potential difference (PD) values for RCEC culture (day 6 and 7) obtained in all uptake experiments were 0.90 ±0.18 kf2.cm^ and 2.9 ± 0.85 mV, respectively. Treatment of RCECs with lectin during uptake/binding did not significantly alter the TEER and PD values. 3.1. Characteristics of lectin binding and uptake As shown in Table 9, three model plant lectins with variable molecular weight and substrate specificity were chosen for this study. Table 9. Model plant lectins and their affinity parameters in RCECs. Lectin Name Source & Abbr. Mwt (kDa) Sugar specificity V m a x (nM/mg protein) K . (PM) Solanum tuberosum Potato (STL) 100 N-acetyl glucosamine oligomers 52.3 + 2.33 0.39 ± 0.06 Lycopersicon esculentum Tomato (TL) 100 N-acetyl glucosamine oligomers 15.0 + 0.76 0.48 ± 0.07 Ulex europeaus 1 Gorse (UEA-1) 63 a-L-fucose 53.7 ±2.20 4.81 ± 0.53 Note: Values represent mean + s.e.m. (n = 6). V m a x and K m values for STL in MDCK cells (chosen as control) were 37.1 ± 5.9 nM/mg protein and 0.38 + 0.14 pM, respectively. Fig. 39 shows that the binding and uptake of all three lectins in RCECs was time- dependent (reaching a plateau at 1-2 hr period). Since most reported studies used 1- hr period to evaluate lectin binding, we have chosen an incubation period of 1 hr for 144 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. all further binding/uptake studies. The binding/uptake of STL was first studied in MDCK cells (as a control) at 37 °C to evaluate its binding parameters (V m ax and Km) and the suitability of chitin as a lectin binding/uptake inhibitor at the concentration used. C O) c .E o I s S a I I I I 50 STL 40 UEA-1 30 TL 20 10 0 0 30 60 90 1 2 0 Time (min) Figure 39. Effect of incubation time on uptake/binding of FITC-lectins in RCEC. The concentrations of FITC-STL and FITC-TL used were 0.5 and 0.71 mM respectively, whereas the concentration of FITC-UEA-I was 3.17 mM. Uptake/binding of lectins was carried out at 37 °C up to 2 hr period. Symbols represent mean ± s.e.m. (n = 6). As shown in Fig. 40, the specific binding/uptake of STL to MDCK cells was concentration dependent with V m ax and Km values of 37.1 ± 5.9 nM/mg protein and 0.38 + 0.14 pM, respectively. The specific STL binding/uptake curve was deduced by subtracting the non-specific binding/uptake obtained in the presence of excess chitin from the total binding/uptake obtained in the absence of chitin. 145 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. O) c .E o 1 1 5 a II ! 1 60 Total Specific 40 20 Non-specific 0 0.5 1.0 1.5 2.0 2.5 [Solanum tuberosum] in pM Figure 40. Effect of concentration on binding/uptake of FITC- Icctins in MDCK cells. MDCK were incubated at 37°C for 1 hr using various concentrations for STL ranging from 0.1-2 mM, in the presence or absence of the inhibitory sugar chitin (30 mg/ml), to obtain the total and non-specific binding/uptake curves as described in the Methods section. Symbols represent mean ± s.e.m. (n = 6). As shown in Fig. 41, the specific binding/uptake of the three plant lectins to RCECs increased in a concentration-dependent manner when incubated at 37 °C, and followed a Michaelis-Menten equation fit. The specific binding/uptake curves were similarly deduced as in Fig. 40 (Total and non-specific curves not shown). As shown in Table 9 previously, STL and TL had higher affinity constants (Km values corresponding to 0.39 and 0.48 pM, respectively) than UEA-1 lectin, while STL and UEA-1 had higher maximal uptake/binding values (V m ax corresponding to 52.3 and 53.7 nM/mg protein, respectively) than TL. 146 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7 5 -1 c STL o (0 50- "S. 3 "O ) ® c E I s 25- .E c C Q EA-1 TL 0 5 10 15 20 25 [Lectin] in fxM Figure 41. Effect of concentration on specifie binding/uptake of FITC- lectins in RCEC. RCECs were incubated for 1 hr with various concentrations of FITC-lectins (STL, TL, and UEA-1) ranging from 0.1-31.6 mM at 37 °C in the presence or absence of an inhibitory sugar to obtain specific binding/uptake for each lectin as described in the Methods section (non-specific and total binding/uptake curve not shown). Symbols represent mean ± s.e.m. (n = 6). All three lectins display temperature dependent uptake, with higher cell association at 37 °C than at 4 °C (not shown). The uptake difference between the two temperatures represents the internalized (endocytosed) amount of lectins in RCECs (Fig. 42). STL had the highest amount of intemalization and the optimal affinity parameters of the three lectins in 1 hr uptake/binding study. 3.2. Internalization of S o la n u m tuberosum lectin Confocal microscopy demonstrates lectin binding and intemalization into RCECs. Fig. 43 shows that STL staining (green) was abundant in the sub-apical and 147 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30-1 c 'a> STL UEA-1 TL 0 1 2 3 4 5 6 7 [Lectin] in |iM Figure 42. Amount of FITC-lectin internalized in RCEC. Uptake of various concentrations of FITC-labeled lectins (with a similar range as that done at 37° C in RCECs) was carried at 4° C for 1 hr. The uptake/binding curve obtained at 37° C was then subtracted from that at 4° C to obtain the active component of uptake (amount of lectin intemalized), which is expressed as nM lectin/mg protein. Symbols represent mean ± s.e.m. (n = 6). intermediate layer of RCECs (see also Fig. 44). STL was distributed on the cell periphery, below the cell membrane, and in the cytoplasm (either in a diffused or punctate manner). STL staining was absent when RCECs were simultaneously treated with chitin (at 30 mg/ml) for 1 hr, indicating that it is due to lectin uptake/binding rather than FITC staining (Fig. 43 panel B). STL stain co-localized with phalloidin (red stain) indicating that it was staining the cell membrane (Fig. 43 panel D). Fig. 43C is a confocal image of RCECs treated with phalloidin only (red stain, as control). 148 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 43. Evidence for intemalization of Solanum tuberosum lectin in RCECs. Confocal images of RCECs following 30 min incubation with ImM of FITC-STL. A) Control. B) Presence of 30 mg/ml chitin (inhibitory sugar). C) Labeled with TRITC-phalloidin in the absence of FITC-STL applied. D) Merged image of A and C. Images represent x-y confocal images (40x magnification) scanned below the apical layers of sub-eonfluent RCECs (80% confluency). Fig. 44 is an x-y confocal image gallery of RCEC layers sectioned at 0.5 pm increments. It shows the distribution and staining of both STL (green) and phalloidin (red) stain in both the sub-apical and intermediate layers of RCECs. 149 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 44. Evidence for intemalization of STL into intermediate cell layers. Confocal image is an x-y gallery image of RCECs taken by 5 mm sections of cells following a 1-hr incubation with FITC- STL (potato lectin) at 37° C and staining with TRITC-phalloidin Abbreviations: AP: apical layer, SAP: Subapical layer, INT: Intermediate layer, BL: Basolateral layer. 3.3. Effect of lectin on cytokine induction As shown in Fig. 45, Solanum tuberosum lectin (STL) did not elicit any cytokine intracellular release for up to 24 hr after treatment compared to control (only 4 hr period shown). In contrast, histamine treatment significantly caused intracellular release of several cytokines including IL-la, IL-ip, IL-2, IL-6, and IL- 10 that occurred mostly at 4-hr treatment, although increases in other periods were also noticed such as 2 and 8 hrs. ISO Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3500 n 3000 ■ 5 2500 U ) a £ 2000 o > ® 1500 0 1 1000 o X , O 500 ■ control ■ STL □ Histamine A Figure 45. Effect of STL and histamine on cytokine levels in RCECs. Intracellular levels of several cytokines (9 in total) were measured after 4 hr exposure of RCECs to either 5 mM STL (potato lectin) or 30 mM histamine, cytokine concentrations were analyzed using SearchLight rat cytokine array technique (see methods for details). Cytokine levels were also measured up to 24 hr exposure to either agent (only 4 hr shown). Asterisk (*) indicates significant difference (P < 0.05), n = 4. Table 10 provides a summary of the basal levels of the main cytokines that got affected and their levels upon stimulation with histamine. STL levels were not shown, as they were not significantly different than control basal values. 3.4. Uptake of lectin-nanoparticle conjugate Figure 46 shows that conjugating nanoparticles with 5 mg/ml of Solanum tuberosum lectin (STL) significantly increased apical uptake of the loaded protein marker HRP by 3-fold compared to unconjugated nanoparticles (control) and 151 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 10. Summary of cytokine data in RCECs. Cytokine Basal (pg/ml) Histamine stimulated (pg/ml) Fold increase* I L -la 290 + 38.4 428 ± 27.3 1.5 IL -ip 1406 ± 2 3 3 .7 2522 ± 307.0 1.8 IL-2 1647 ± 119.4 2298 ± 177.1 1.4 IL-6 2400 ± 139.2 2942 ± 171.3 1.25 11-10 142 ± 9.6 178 + 11.3 1.24 Note: all of the histamine-stimulated cytokine increased levels were observed to occur at 4 hr time interval. Cytokine levels of Solanum tuberosum lectin (STL) were similar or less than control (untreated cells) at all time intervals. Asterisk (*) indicates that increases of > 15% was chosen, n = 4 for all samples. a fl a ’5 3 o B o a ( m O M ) u a u o fl a. A d 1 'o i) a 40 30 - 20 - 10 - 0 Control STL-NP BSA-NP Figure 46. ST leetin enhances apical uptake of HRP-nanoparticles. RCECs were incubated at 37 °C with uncoated nanoparticles (control), STL- modified nanoparticles, or BSA-modified nanoparticles for 1-hr. Nanoparticle concentration was determined by measuring the activity of the loaded HRP protein marker (methods section). Amount of STL and BSA used (acting as ligands) in conjugation was 5 mg/ml. Asterisk (*) indicates significant difference (P < 0.05). Bars represent mean ± s.e.m. n = 4 152 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. nanoparticles conjugated with bovine serium albumin of similar concentration to that of STL. To evaluate if lectin enhancement of apical uptake of HRP-loaded nanoparticles could be improved, we coated nanoparticles with different concentrations of STL ranging from 0-6 pM. Figure 47 shows that apical uptake of HRP-nanoparticle in RCECs at 1-hr (chosen due to maximal uptake time of STL) increased significantly in a dose-dependent manner when coated with STL ranging from l-6pM, although it did plateau at 2 pM concentration. Figure 47 demonstrates the specificity of STL enhancement of HRP-nanoparticles by the significant reduction in apical uptake of 6 pM STL-nanoparticles in the presence of 30 mg/ml chitin (inhibitor sugar for STL). 1501 50- 0 Chitin w .£ 0 o 1 |ioo- i Q. O) 'illL 0 1 2 4 6 6 [Solanum tuberosum lectin] In Figure 47. Effect of STL concentration on uptake of HRP-nanoparticles. RCECs were incubated at 37 °C with increasing concentrations of STL- HRP nanoparticle conjugate for 1 hr in the presence or absence of chitin (grey bar). HRP activity was analyzed as described in the methods section. Bars represent Mean ± s.e.m. (n = 6). 153 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4. DISCUSSION 4.1. Lectin binding and uptake characteristics Our studies determined the binding/uptake characteristics of three model plant lectins in a rabbit conjunctival epithelial cell culture model and showed that the lectin from Solanum tuberosum displayed the most optimal properties and was considered for further testing. These three lectins differ in their molecular weight and in their sugar specificity, with UEA-1 binding to L-flicose residues, and TL and STL binding to N-acetylglucosamine oligomer residues. Cytochemical and immunofluorescence studies have shown that both UEA-1 and STL display intense and rapid binding on the conjunctival epithelium surface (Nicholls et al., 1996;Rittig et al., 1990). Tomato lectin (TL) was chosen in this study to evaluate whether lectins with similar sugar specificity and size (with regards to STL) have similar uptake/binding pattern. In addition, TL is a bioadhesive glycoprotein that has been shown to bind selectively to the small intestinal epithelium and augment the oral intestinal absorption of macromolecules and nanoparticles (Carreno-Gomez et al., 1999;Hussain et al., 1997;Naisbett and and Woodley, 1994). Our study have shown that all three lectins (STL, TL, and UEA-1) display a time-dependent binding/uptake pattern at 37 °C in RCECs, with a maximal binding/uptake occurring at around 1-2 hr period, depending on the type of lectin. This is consistent with the findings of Lehr and coworkers (Lehr et al., 1992a) who studied tomato lectin binding to both isolated pig enterocytes and Caco-2 cells. In contrast, Wirth and coworkers (Wirth et al., 1998b) reported that binding of STL to 154 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the carcinoma cell lines HT-29 and Caco-2 cells was independent of incubation time up to four hrs. This difference could be attributed to slow binding kinetics of STL to these cell lines or to a negligible number of binding sites in these cell types. This is a plausible explanation, as differences in the binding capacity of the same lectin to various epithelial cell types have already been reported. For instance, lectins such as Griffonia simplicifolia agglutinin-I-B4 and Ricinus communis agglutinin had marked rapid reactivity towards the conjunctival epithelium compared with the comeal epithelium (Tuori et al., 1994). We have also shown that these three lectins exhibit specific, saturable binding/uptake pattem in RCECs, with affinity constants ranging from 0.39-4.81 pM. This binding/uptake was specific, as supported by the marked inhibition in the presence of competing sugars such as N-acetylglucosamine and L-a-fucose. . The non-linear binding/uptake pattem for lectins is consistent with that reported for Caco-2 cells and human alveolar epithelial cells (Ahu-Dahab et al., 2001;Haltner et al., 1997). The affinity constant values for lectins obtained in these cell systems are comparable to those obtained in RCECs. For instance, Haltner and coworkers (Haltner et al., 1997) reported Km value for TL in the range of 1-2 pM in Caco-2 cells Of the three lectins, STL displayed optimal binding/uptake potential to RCECs, with favorable affinity parameters (Km of 0.39 ± 0.06 pM and V m ax of 52.3 ± 2.3 nM/mg protein). We ranked the lectins' binding affinity to RCECs as STL > TL > UEA-1. The higher V m ax uptake/binding value for potato lectin compared to 155 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tomato lectin could be attributed to differences in the molecular structure and conformation of the two lectins that favor binding to RCECs. Lectin intemalization could be facilitated by additional protein-protein interaction between the lectin and membrane proteins. This is very likely, as chitin (the competing oligosaccharide) inhibited lectin uptake up to 60-85%, indicating that a fraction of the lectin binding to the cell surface is mediated by non-glycosidic interactions. It is interesting to note that UEA-1 lectin, the smallest of all lectins used which binds to L-fucose residues, has a similar V m ax value to STL but a lower Km value than either STL or TL. This supports the notion that other factors are involved in the cellular association of these lectins. One factor may be cell recognition of the lectin as a ligand for endocytosis or intracellular stability from lysosomal degradation following endocytosis. In our study, all three lectins demonstrated temperature dependence in their uptake/binding pattem (although that of tomato lectin was not significant), however, STL and UEA-1 were endocytosed to a greater degree than TL. The ranking of their capacity for intemalization was STL > UEA-1 > TL. In another study, lectins displayed both temperature dependence and temperature independence (Haltner et al., 1997). The absence of temperature dependence in some lectins was explained either by lack of endocytosis (only binding) or by transport of the intemalized lectins into acidic intracellular compartments, where they were degraded. Indeed, accumulation of wheat-germ agglutinin-BSA conjugate within lysosomal compartments followed by subsequent proteolytic degradation was demonstrated in Caco-2 cells (Gabor et al., 2002). 156 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In our findings, there was no significant difference in TL cell association at both 37 °C and 4 °C, indicating that the lectin may have been degraded within acidic compartments in RCECs. In fact, Lehr and coworkers (Lehr et al., 1992a) showed that tomato lectin binding to isolated, fixed pig enterocytes was reduced in an acidic environment. Thus, the greater amount of STL that was accumulated in RCEC compared to other lectins points to its higher endocytic rate and intracellular stability. We have measured the uptake/binding of STL at lower pH (pH range 4-5) and did not notiee any significant difference in binding/uptake in RCECs supporting our conelusion (data not shown). Several investigators have demonstrated the binding and intemalization of lectins in various epithelial cells using confocal microscopy (Lehr, 2000;Wirth et al., 1998b). These lectins were shown to stain the plasma membrane and cytoplasm, with some intensely stained granular regions indieative of vesicular endocytosis. Our confocal microscopy findings with STL staining of RCECs are in agreement with these reports. Lectin endocytosis may be occurring by adsorptive endocytosis as suggested previously for the transport of tomato lectin and others in Caco-2 cells (Lehr and Lee, 1993). However, the small affinity constant for STL (Km = 390 nM) in the nanomolar range indicates that endocytosis of STL in RCECs occurs through receptor-mediated endocytosis. 157 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2. Safety of lectin use in the eye Earlier studies with tomato lectin showed that it stimulated immune response in mice following oral absorption and raised fears about the safety of lectin use in human (Naisbett and Woodley, 1990). Similarly, a number of plant lectins such as TL and UEA-1 were reported to stimulate the production of specific serum IgG and IgA antibody after three intranasal or oral doses administered to mice (Lavelle et al., 2000). Therefore, we wanted to evaluate if STL would behave similarly to other lectins such as TL or UEA-1. We have observed no toxic effects of STL on RCECs, as evidenced by the lack of deleterious effect on tight jimctions (TEER) and ion transport properties (potential difference) by STL during a 4 hr uptake study. This is in agreement with previous findings evaluating the tolerance of STL in epithelial tissues (Nicholls et al., 1996;Smart et al., 1999b). In these in-vivo studies, no evidence of irritancy was observed when STL was instilled into the eye of rats or injected intradermally into rabbits. Since cytokine levels are known to increase after injury to the ocular surface and during would healing, we decided to monitor the release of several cytokines following incubation of rabbit conjunctival epithelial cells with STL for 24 hr (Sotozono et al., 1997). The ability of conjimctival epithelial cells to produce pro- inflammatory cytokines in response to ocular diseases is well-documented (Gamache et al., 1997). Our study (Fig. 45) demonstrated lack of cj^okine induction by STL in comparison with histamine as a positive control up to 24 hr treatment of RCECs. Only histamine was shown to stimulate the intracellular release of several cytokines 158 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (such as IL-la, IL-lp, IL-2, IL-6, and IL-10) at several time periods (only 4 hr was shown in Figure 45). The cytokine inducing effect of histamine on conjunctival epithelial cells is in concert with previous findings of Weimer and coworkers (Weimer et al., 1998). In addition, the upregulated levels of IL-6 in these studies were comparable to our study (1.67 fold compared to 1.4-1.6 fold increase). Similarly, the basal level of TNF-a in tear fluid of patients fotmd to be 967 pg/ml is compared to our data (Table 10) for 950 pg/ml in RCEC culture (Vesaluoma et al., 1999). However, reported values for released cytokines are usually for tear fluid or pathological tissues and have variable levels depending on technique used, so it is not easy to make comparisons. Based on these findings, our data suggest the safety and well tolerance of Solanum tuberosum lectin in the eye, although long-term in vivo studies are warranted to justify this. 4.3. Lectins as endocytic ligands for macromolecules Our study confirmed the utility of Solanum tuberosum lectin as an appropriate ligand to promote the absorption of biodegradable PLGA nanoparticles containing proteins in the conjunctival epithelium. By covalent immobilization of STL to PLGA nanoparticles loaded with horseradish peroxidase (HRP), we have managed to increase the apical absorption of HRP by 3 folds (Fig. 46). This absorption enhancement effect for nanoparticles (and HRP) was probably due to utilization of STL of receptor mediated endocytosis pathway, since conjugating 159 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. nanoparticles with bovine serum albumin was not different than control. This finding confirms the greater endocytic capacity of STL in conjunctival epithelial cells determined earlier (Fig. 42). In addition, we have shown that apical uptake of HRP-nanoparticles was dose dependent of STL concentrations, reaching a plateau at 2 pM. At this concentration, the apical uptake of HRP-nanoparticles increased by 8 folds compared to control non-coated nanoparticles. This is likely to be the case considering the affinity constant for STL is ~ 0.4 pM and higher concentrations would tend to display saturability in uptake, thus impeding further binding and internalizations of STL. If we express uptake as the endocjdic index, defined as the volume (in pi or pg of substrate) captured per mg cell protein/hr, the rate of uptake of STL coupled HRP-loaded nanoparticles was 30 ng/mg per h (Fig. 46), which was 3-fold higher than nanoparticles coupled to BSA or control (~ 12 ng/mg per hr). Our findings for the endocytic index of lectin-coupled HRP nanoparticles are similar to that reported by Carreno-Gomez and coworkers (Carreno-Gomez et al., 1999). These authors reported that rate of uptake of TL coupled polystyrene microspheres in everted gut sac model of the intestine was 41.5 ng/mg per hr, which was 4-fold higher than microspheres coupled to BSA (11.8 ng/mg per hr). The absence of any enhancement effect for nanoparticle uptake by BSA suggest that this protein enters rabbit conjunctival epithelial cells by either fluid-phase or adsorptive endocytosis, since its rate was similar to unconjugated nanoparticles. However, further studies are required to validate the use of BSA as a fluid-phase marker in these cells. 160 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Our findings for the uptake enhancement of HRP-nanoparticles by STL are in concert with that of Hussain and coworkers, who reported a 50-fold increase in the intestinal uptake of 500 nm fluorescent polystyrene nanoparticles when conjugated to tomato lectin (Hussain et al., 1997). However, the higher enhancement capacity reported is attributed to the frequent oral administration (over 5 days) of tomato lectin-nanoparticles to female Wistar rats. The specificity of STL enhancing effect was confirmed by the significant reduction in apical HRP uptake in the presence of an inhibitory sugar, chitin. Besides nanoparticles, several investigators suecessfully used lectins to target histochemical markers (HRP and biotin) to mouse intestinal M cells and enhance their subsequent absorption across intestinal epithelial barrier (Clark et al., 1995;Giaimasca et al., 1994). The authors postulated that lectins could serve as "receptors" for targeting of lectin-antigen conjugates to the mucosal immune system. In addition, Wirth and coworkers (Wirth et al., 1998a) demonstrated increased adhesion/transport of doxorubicin in Caco-2 cells following conjugation to wheatgerm agglutinin lectin. Furthermore, lectin conjugates were demonstrated to deliver and express a reported gene in human airway epithelial cells (Yin and Cheng, 1994). In the eye, the only report to our knowledge is by Schaeffer et al. (Schaeffer et al., 1982) in which ganglioside-containing liposomes with entrapped carbachol enhanced carbachol flux across isolated rabbit corneas when pre-treated with wheat germ agglutinin. Our data for the enhancement of protein (HRP) absorption by 161 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Solanum tuberosum lectin points to its effective role as an ocular carrier for the delivery of drugs to the conjunctiva and the anterior segment of the eye. 162 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VI. OVERALL CONCLUSIONS 163 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1. SUMMARY OF FINDINGS The potential application of biodegradable PLGA nanoparticles as ocular carriers for proteins and drug delivery has prompted the studies described in this dissertation. The overall goal is to improve the mechanistic understanding of nanoparticle transport and trafficking within the conjunctival epithelium to facilitate the design of successful nanoparticle systems capable of enhancing the delivery of proteins and drugs to the conjunctiva and other intraocular tissues. Our data provided evidence for the endocytosis of PLGA nanoparticles in rabbit conjunctival epithelial cells (RCECs). This was demonstrated by the uptake inhibition in the presence of microfilament inhibitors and metabolic poisons, the vesicular uptake pattern seen imder confocal microscopy, and the stimulatory effect on endocytosis of the fluid phase marker, Lucifer yellow. We further provided evidence for the mechanism of PLGA nanoparticle uptake in RCECs. Based on clathrin knockout studies, our data demonstrated lack of a direct major involvement of clathrin heavy chain in endocytosis of PLGA nanoparticles. However, since complete knockout of clathrin was not attainable (only 70-90%) and because the basolateral uptake of transferrin, a marker for clathrin- mediated endocytosis, was reduced only by 40%, we speculate that there may be enough clathrin expression remaining in the cell to mediate or regulate PLGA nanoparticle endocj^osis. These findings are not just based on speculations, as our pharmacological treatments (hypertonicity and intracellular depletion) revealed the dependence of nanoparticle uptake on clathrin-mediated endocytosis. These 164 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pharmacological treatments (hypertonicity and intracellular depletion) have been known to interfere specifically with clathrin distribution, assembly, and recycling at the cell periphery and inhibit clathrin-coated vesicle endocytosis (Larkin et al., 1986;Heuser and Anderson, 1989;Wang et al., 1993). However, several studies have also implicated these treatments in the inhibition of fluid-phase endocytosis in epithelial cells of the kidney and liver (Goligorsky and Hruska, 1986;Synnes et al., 1999). The partial colocalization of clathrin staining with that of coumarin-loaded PLGA nanoparticles seen imder confocal microscopy further implicated the minor involvement of clathrin in nanoparticle endocytosis. We have provided the first evidence for the expression of clathrin heavy chain at the protein and gene levels in rabbit conjunctival epithelial cells and showed that protein knockout of clathrin HC in primary cultured cells is attainable. Since the presence of a recognition motif for internalization is unlikely for nanoparticles, random non-specific association of clathrin-coated vesicles with nanoparticles is still plausible. These non-specific interactions could be mediated by Van der Waals forces. This is likely to be the case as evidence for accumulation of osmium-loaded PLGA nanoparticles in the center of a newly formed endocytic vesicles, instead of being attached to membrane wall, of vascular smooth muscle cells was demonstrated using transmission electron microscopy (Panyam et al., 2002). The work by Rejman and coworkers (Rejman et al., 2004) further highlights the role of clathrin in our studies. They showed that internalization of fluorescent latex beads with diameter < 200 nm in non-phagocytic 165 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B16 cells involved clathrin-coated pits, whereas with increasing size a shift to a mechanism that relied on caveolae-mediated internalization became apparent and predominant at particle size of 500 nm and higher. The absence of caveolin-1 at the gene and protein levels together with our pharmacological data with nystatin and filipin suggest that caveolae (even other isoformes of caveolin) are not likely to be involved in the endocytosis of PLGA nanoparticles. Caveolin-1 was chosen in this study as a marker for caveoalae since over 90% of caveolin-1 is found associated with caveolae and is the only isoform found in epithelial cells (Rothberg et al., 1992). Since macropinosomes are involved in endocytosis of larger particles (> 500 nm), our findings implicate mainly non coated vesicles and clathrin-coated vesicles partially in the internalization of PLGA nanoparticles in RCECs. Our work delineated the intracellular distribution and trafficking behavior of PLGA nanoparticles following endocytosis. Nanoparticles were shown to escape endosomal trafficking and reside for a short while in the lysosomes, thus aiding in avoiding intracellular degradation machinery of the cell and in accumulating nanoparticles in the cytoplasm for drug delivery. The localization of nanoparticles to the Golgi compartment suggests the possible subsequent sorting and movement of Golgi-derived vesicles containing nanoparticle to either the basolateral or lateral membrane for exocytosis. We have also provided evidence for the utility of using PLGA nanoparticles for protein drug delivery and controlled drug delivery in the conjunctival epithelium, 166 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and probably to other intraocular tissues. Finally, the feasihility of using plant lectins such as Solanum tuberosum to augment nanoparticle absorption in RCECs has been highlighted and shown to be efficacious and safe of ocular use as drug carriers. 2. SIGNIFICANCE OF FINDINGS The significance of this work is that it will (a) facilitate the discovery of ocular formulations aimed at improving ocular drug absorption and (b) will set the stage for future in vivo studies aimed at studying the efficacy of PLGA nanoparticles as carriers of drugs and proteins to the anterior segments of the eye. The present findings on the existence of clathrin heavy chain in conjunctival epithelial cells might be useful to dissect, at the cellular levels, the endocytic pathways utilized by different substrates in the conjunctiva and also help to elaborate the role of clathrin-mediated endocytosis in conjunctival physiology and pathological conditions such as dry eye syndrome. This will also facilitate in the selection and screening of ligands that are substrates for clathrin-mediated endocytosis. Our findings for the absence of caveolin-1 and caveolae in conjunctival epithelial cells might be useful in stirring efforts to find altemative isoforms of caveolins that may be implicated in endocytosis of macromolecules or signal transduction within the cells. A variety of hormones, nutrients, and macromolecules are taken in the cell by either clathrin- or cavoelae-mediated endocytosis (Bemier-Valentin et al., 1990;Ellington et al., 1999;Hanover et al., 1984;Anderson et al., 1977;Fan et al., 167 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1982;Lobie et al., 1999). The existence of EGF and IGF-I receptors on the apical membrane of pigmented rabbit conjunctiva has been demonstrated, but their role or mechanism in internalization of EGF has not been examined (Narawane and Lee, 1995). The internalization of EGF is a complex process in which caveolae and clathrin-coated pits are involved (Mineo et al., 1999). Since caveolae were shown to be absent in our RCEC system, it would be reasonable to conclude that internalization of EGF occurs via clathrin-mediated endocytosis or study its internalization focusing on clathrin-coated pits. Our present study might signify that clathrin-coated pit/vesicle system may not be the best-known entrance into RCECs and may be considered one of many endocytic mechanisms responsible for degradation, recycling, and to some extent, receptor-mediated transcytosis. These findings are also relevant for understanding the etiology and mechanism of ocular infections in the conjunctiva, as many viruses and bacteria were shown to utilize clathrin or caveolae as endocytic machineries to gain access to cells and begin their infections (Ellington et al., 1999;Pho et al., 2000;Norkin et al., 2002). It would be interesting to evaluate if viruses, such as simian virus 40, can still be endocytosed and infect conjunctival epithelial cells without the help of caveolae. In addition, our antisense oligonucleotide technique for knocking out clathrin protein is unique, as it has not been demonstrated in a primary culture model. The use of such technology may be extended to the potential use of therapeutic antisense oligonucleotides in ocular diseases to prevent viral replications and recurrent infections. One product already on the market is Vitravene®, which is based on a 168 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phosphorothioate antisense oligonucleotides that inhibit viral replication in cytomegalovirus retinitis by intravitreal injection. However, the use of frequent injections into the vitreous is complicated and discomforting for the patient, thus would highlight the importance of formulating a topical formulation of antisense oligonucleotides capable of reaching intra-ocular tissues. Our present study on the intracellular distribution of nanoparticles and avoidance of endosomal pathway and short residence in lysosomal compartment is interesting and might trigger further studies aimed at evaluating the properties of PLGA nanoparticles that determine such fate. For many proteins and nutrients, avoidance of degradative pathway is key to their delivery to target site. These findings will encourage efforts at elucidating the reasons for endosomal/lysosomal escape and in designing agents that mimic such nanoparticle systems. It may also signify the existence of an unknown endocytic pathway, whereby internalized molecules can avoid the degradative machineries of both the endosomes and lysosomes. Due to their efficient intracellular delivery, the development of an efficient therapy based on PLGA nanoparticles containing drugs such as genes and proteins will be ongoing and will likely supersede existing methods. Finally, our findings for the suitability and feasibility of plant lectins such as Solanum tuberosum to augment ocular drug absorption might provoke future attempts at designing drug-lectin conjugates and evaluating their in vivo efficacy and safety. In addition, such findings will likely stir efforts towards the identification of other endocytic ligands capable of promoting ocular drug absorption. 169 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. FUTURE DIRECTIONS The current project has laid the foundation for further studies to address some interesting, unanswered questions in the future: 1. What is the residence time of PLGA nanoparticles following topical instillation of drug-nanoparticle suspension into the conjunctival cul-de-sac area and what is their endocytic rate compared to other nanoparticles? Wood and coworkers (Wood et al., 1985) have evaluated the tear residence time of nanoparticles made from poly(alkycyanoacrylate) and indicated prolonged residence time up to 20 min. However, the residence time of PLGA nanoparticles has not been studied in the eye.. It would be vital to demonstrate the prolonged residence time of PLGA nanoparticles to justify their enhancing effect before any in vivo drug absorption studies are pursued. In addition, it is mandatory to compare the drug delivery efficacy properties of PLGA nanoparticles compared to other polymers such as chitosan and Polycyanoacrylate. Only one study evaluated the effect of different polymer-based nanoparticles on ocular absorption of betaxolol (Marchal-Heussler et al., 1992). However, that study looked at only a lipophilic drug such as betaxolol and utilized a different PLGA nanoparticle formulation (85:15), which has very slow drug releasing properties and is not expected to release the drug in the tear within 24 hr. 2. What is the effect of mucus on nanoparticle absorption in the conjunctiva? Although our primary culture model of conjunctival epithelial cells has been shown to be adequate for drug transport studies (Saha et al., 1996b), it is deficient of mucus. 170 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Since most polymers are known to be mucoadhesive, it would be wise to evaluate the effect of mucus on PLGA nanoparticle absorption. 3. Can nanoparticles be utilized for the delivery of therapeutic drugs to the conjunctiva and the anterior segments of the eye in vivo! Although PLGA nanoparticles were shown to promote drug absorption in primary cell culture of conjunctival epithelium, these applications need to be extended to in vivo situation to demonstrate the stability of the drug within nanoparticles, their absorption efficacy relative to drug formulation alone, and their ability to sustain therapeutic levels of the drug to treat an ocular disease model. We feel that acyclovir is a good model drug for encapsulation into PLGA nanoparticles, due to its low water solubility, susceptibility to enzymatic degradation by esterases, and its therapeutic potential of treating ocular herpes simplex viral diseases. In addition, the ability of acyclovir-loaded nanoparticles to reach intraocular tissues such as aqueous humor and iris-ciliary is essential so that they can be utilized for other drugs to treat anterior ocular diseases such as glaucoma and dry eye disease. 4. Can topical administration of nanoparticles be used for systemic administrations of peptides and proteins? Since systemic absorption of topically applied drugs was shown to exceed 50% and since nanoparticles were shown to protect unstable peptides from degradations, it would be of interest to evaluate the systemic absorption of topically applied 171 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. nanoparticles containing a peptide or protein (Lee et al., 1993;Damge et al., 1997). One candidate peptide would be elcatonin used to treat hypercalcaemia in blood. The oral delivery of elcatonin (administered intragastrically) to fasted Wistar rats using chitosan coated PLGA nanoparticles was shown to reduce blood calcium levels up to 48 hr (Kawashima et al., 2000). We believe that since the protease activity of the intestine is much higher than that of the conjunctiva, that topical delivery of peptides or proteins for systemic absorption is likely to show promise and is worthwhile. 4. Can PLGA nanoparticles interfere with efflux pump activity in conjunctival epithelium and/or enhance the absorption of efflux pump substrates? The existence of efflux pumps (MRPl and Pgp) in the conjunctival epithelium and their effects on limiting drug absorption have been demonstrated previously (Saha et al.,1998;Yang et al., 2000a). Poly (alkylcyanoacrylate) nanoparticles were shown to accumulate doxorubicin in several multi-drug resistant cell lines in comparison with doxorubicin alone and this effect was attributed to reversal of resistance (Soma et al., 1999;de Verdiere et al., 1997). The effect of PLGA nanoparticles on the apical- basolateral transport of propranolol (a Pgp substrate) or the basolateral-apical transport of vincristine (a MRPl substrate) compared to control solution of either drug would be investigated. 5. Would a topical formulation of antisense oligonucleotide targeted agains herpes simplex virus gene be stable and effective to prevent viral replication or slow infection? 172 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Our knockout studies with clathrin using antisense oligonucleotide demonstrate the utility and ease of this technology for use. A better therapeutic target would be to inhibit the transcription of viral genes and prevent viral replications in a number of ocular infections. One of these is herpes simplex virus, which can be a recurrent infection in some AIDS patients or those with compromised immune system. It would be interesting to explore the potential of designing a therapeutic antisense oligonucleotides and formulating it into nanoparticles or liposomes to evaluate its ocular permeation and efficacy and stability. 173 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VII. REFERENCES 174 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abu-Dahab, R. Schaefer U. F. and Lehr C. M. Interaction of lectins with epithelial cells of deep lung: human alveolar cells in primary culture versus A549 cell line. CRS meeting. 2001. Ahmed I and Patton TF (1985) Importance of the noncomeal absorption route in topical ophthalmic drag delivery. Invest Ophthalmol. Vis.Sci 26:584-587. Alvarez LJ, Turner HC, Zamudio AC, and Candia OA (2001) Serotonin-elicited inhibition of Cl(-) secretion in the rabbit conjimctival epithelium. Am. JPhysiol Cell Physiol 280:C581-C592. Amino K, Honda Y, Ide C, and Fujimoto T (1997) Distribution of plasmalemmal Ca(2+)-pump and caveolin in the corneal epithelium during the wotmd healing process. Curr.Eye Res. 16:1088-1095. Anderson RG, Brown MS, and Goldstein JL (1977) Role of the coated endocytic vesicle in the uptake of receptor-boimd low density lipoprotein in human fibroblasts. Ce//10:351-364. Ashton P, Podder SK, and Lee VH (1991) Formulation influence on conjunctival penetration of four beta blockers in the pigmented rabbit: a comparison with corneal penetration. Pharm.Res. 8:1166-1174. Ayad N, Hull M, and Mellman I (1997) Mitotic phosphorylation of rab4 prevents binding to a specific receptor on endosome membranes. EMBO 716:4497-4507. Basu SK, Haworth IS, Bolger MB, and Lee VH (1998) Proton-driven dipeptide uptake in primary cultured rabbit conjunctival epithelial cells. Invest Ophthalmol. Vis.Sci 39:2365-2373. Baudouin C, Brignole F, Pisella PJ, Becquet F, and Philip PJ (1997) Immunophenotyping of human dendriform cells from the conjunctival epithelium. Curr.Eye Res. 16:475-481. Bazile D, Prud'homme C, Bassoullet MT, Marlard M, Spenlehauer G, and Veillard M (1995) Stealth Me.PEG-PLA nanoparticles avoid uptake by the mononuclear phagocytes system. JPharm Sci 84:493-498. Bemier-Valentin F, Kostrouch Z, Rabilloud R, Munari-Silem Y, and Rousset B (1990) Coated vesicles from thyroid cells carry iodinated thyroglobulin molecules. First indication for an internalization of the thyroid prohormone via a mechanism of receptor-mediated endocjdosis. JB iol Chem. 265:17373-17380. Berton M, Benimetskaya L, Allemann E, Stein CA, and Guray R (1999) Uptake of oligonucleotide-loaded nanoparticles in prostatic cancer cells and their intracellular localization. Eur.JPharm Biopharm. 47:119-123. 175 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bloom GS and Brashear TA (1989) A novel 58-kDa protein assoeiates with the Golgi apparatus and microtubules. JBiol.Chem. 264:16083-16092. Bloom WS and Puszkin S (1983) Brain clathrin: immunofluorescent localization in rat retina. JHistochem.Cytochem. 31:46-52. Bos JL, de Rooij J, and Reedquist KA (2001) Rapl signalling: adhering to new models. Nat.Rev.Mol.CellBiol. 2:369-377. Boulton, M. E. Campbell L. Smith G. M. and Gumbleton M. Immunolocalisation of caveolin-1 expression in the human eye. Invest Ophthalmol.Vis.Sci. 2001. Ref Type: Abstract Bourges JL, Gautier SE, Delie F, Bejjani RA, Jeanny JC, Gumy R, BenEzra D, and Behar-Cohen FF (2003) Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles. Invest Ophthalmol. Vis.Sci. 44:3562- 3569. Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, and Behr JP (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc.Natl.Acad.Sci. U.S.A 92:7297-7301. Bridges CC, El Sherbeny A, Roon P, Ola MS, Kekuda R, Ganapathy V, Camero RS, Cameron PL, and Smith SB (2001) A comparison of caveolae and caveolin-1 to folate receptor alpha in retina and retinal pigment epithelium. Histochcm.J33:149- 158. Brown HG, Pappas GD, Ireland ME, and Kuszak JR (1990) Ultrastructural, biochemical, and immunologic evidence of receptor-mediated endocytosis in the crystalline lens. Invest Ophthalmol. Vis.Sci. 31:2579-2592. Calvo P, Alonso MJ, Vila-Jato JL, and Robinson JR (1996a) Improved ocular bioavailability of indomethacin by novel ocular drug carriers. JPharm Pharmacol. 48:1147-1152. Calvo P, Vila-Jato JL, and Alonso MJ (1996b) Comparative in vitro evaluation of several colloidal systems, nanoparticles, nanocapsules, and nanoemulsions, as ocular drug carriers. J Pharm Sci 85:530-536. Carreno-Gomez B, Woodley JF, and Florence AT (1999) Studies on the uptake of tomato lectin nanoparticles in everted gut sacs. Int JPharm 183:7-11. Catizone A, Medolago AL, Reola F, and Alescio T (1993) A quantitative assessment of non specific pinocytosis by human endothelial cells surviving in vitro. Cell Mol.Biol. 39:155-169. 176 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chang SC, Chien DS, Bundgaard H, and Lee VH (1988) Relative effectiveness of prodmg and viscous solution approaches in maximizing the ratio of ocular to systemic absorption of topically applied timolol. Exp.Eye Res. 46:59-69. Chang SC and Lee VH (1987) Nasal and conjunctival contributions to the systemic absorption of topical timolol in the pigmented rabbit: implications in the design of strategies to maximize the ratio of ocular to systemic absorption. J Ocul.Pharmacol. 3:159-169. Chen JW, Murphy XL, Willingham MC, Pastan I, and August JT (1985) Identification of two lysosomal membrane glycoproteins. J Ce// Biol. 101:85-95. Chiang CH, Tung SM, Lu DW, and Yeh MK (2001) In vitro and in vivo evaluation of an ocular delivery system of 5-fluorouracil microspheres. J Ocul.Pharmacol.Ther. 17:545-553. Chickering DE and Mathiowitz EJ (1995) Bioadhesive microspheres: I. A novel electrobalance-based method to study adhesive interactions between individual microspheres and intestinal mucosa. J.Control.Rel. 34:251-261. Chodosh J, Nordquist RE, and Kennedy RC (1998) Comparative anatomy of mammalian conjunctival lymphoid tissue: a putative mucosal immune site. Dev.Comp Immunol. 22:621-630. Chrai SS, Patton TF, Mehta A, and Robinson JR (1973) Lacrimal and instilled fluid dynamics in rabbit eyes. JPharm Sci 62:1112-1121. Clark MA, Jepson MA, Simmons NL, and Hirst BH (1995) Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo. Cell Tissue Res. 282:455-461. Cleland JL, Mac A, Boyd B, Yang J, Duenas ET, Yeung D, Brooks D, Hsu C, Chu H, Mukku V, and Jones AJ (1997) The stability of recombinant human growth hormone in poly(lactic-co-glycolic acid) (PLGA) microspheres. Pharm Res. 14:420- 425. Cotten M, Wagner E, Zatloukal K, Phillips S, Curiel DT, and Bimstiel ML (1992) High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles. Proc.Natl.Acad.Sci.U.S.A 89:6094-6098. Couvreur P, Kante B, Grislain L, Roland M, and Speiser P (1982) Toxicity of polyalkylcyanoacrylate nanoparticles II: Doxorubicin-loaded nanoparticles. J Pharm Sci 11:190-192. I l l Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cunha-Vaz J (1979) The blood-ocular barriers. Surv.Ophthalmol. 23:279-296. Damge C, Aprahamian M, Marchais H, Benoit JP, and Pinget M (1996) Intestinal absorption of PLAGA microspheres in the rat. JAnat. 189 ( Ft 3):491-501. Damge, C., Michel, C., Aprahamian, M., Couvreur, P., and Devissaguet, J. P. Nanocapsules as carriers for oral peptide delivery. J.Control.Rel. 13(2-3), 233-239. 1990. Damge C, Vranckx H, Balschmidt P, and Couvreur P (1997) Poly(alkyl cyanoacrylate) nanospheres for oral administration of insulin. JPharm Sci 86:1403- 1409. Davda J and Labhasetwar V (2002) Characterization of nanoparticle uptake by endothelial cells. Int JPharm 233:51-59. De Campos AM, Sanchez A, and Alonso MJ (2001) Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A. Int JPharm 224:159-168. de Verdiere AC, Dubemet C, Nemati F, Soma E, Appel M, Ferte J, Bernard S, Puisieux F, and Couvreur P (1997) Reversion of multidrug resistance with polyalkylcyanoacrylate nanoparticles: towards a mechanism of action. Br.J Cancer 76:198-205. Debbasch C, De La Salle SB, Brignole F, Rat P, Wamet JM, and Baudouin C (2002) Cytoprotective effects of hyaluronic acid and Carbomer 934P in ocular surface epithelial cells. Invest Ophthalmol. Vis.Sci 43:3409-3415. Denning GM, Leidal KG, Holst VA, Iyer SS, Pearson DW, Clark JR, Nauseef WM, and Clark RA (1997) Calreticulin biosynthesis and processing in human myeloid cells: demonstration of signal peptide cleavage and N-glycosylation. Blood 90:372- 381. Desai MP, Labhasetwar V, Walter E, Levy RJ, and Amidon GL (1997) The mechanism of uptake of biodegradable microparticles in Caco-2 cells is size dependent. Pharm Res 14:1568-1573. Desai SD and Blanchard J (1994) Ocular drug formulation and delivery, in Encyclopedia o f Pharmaceutical Technology (Swarbick J and Boylan JC ed) pp 43- 75, Marcel Dekker, New York. 178 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dias C, Nashed Y, Atluri H, and Mitra A (2002) Ocular penetration of acyclovir and its peptide prodrugs valacyclovir and val-valacyclovir following systemic administration in rabbits: An evaluation using ocular microdialysis and LC-MS. Curr.Eye Res. 25:243-252. Diepold R, Kreuter J, Himber J, Gumy R, Lee VH, Robinson JR, Saettone MF, and Schnaudigel OE (1989a) Comparison of different models for the testing of pilocarpine eyedrops using conventional eyedrops and a novel depot formulation (nanoparticles). Graefes Arch.Clin.Exp.Ophthalmol. 227:188-193. Diepold R, Kreuter J, Guggenbuhl P, and Robinson J (1989b) Distribution of poly hexyl-2-cyano-[3^'^C] acrylate na rabbit eyes. Int JPharm 54:149. hexyl-2-cyano-[3^'^C] acrylate nanoparticles in healthy and chronically inflammed Dilly PN (1985) Contribution of the epithelium to the stability of the tear film. Trans.Ophthalmol.Soc.U.K. 104 ( Ft 4):381-389. Dodane V, Amin KM, and Merwin JR (1999) Effect of chitosan on epithelial permeahility and stmcture. Int JPharm 182:21-32. Donaldson JG, Lippincott-Schwartz J, Bloom GS, Kreis IE , and Klausner RD (1990) Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action. J Cell Biol. I l l :2295-2306. Douglas GC and King BE (1985) Endocytosis and subsequent processing of 1251- labelled immunoglobulin G by guinea pig yolk sac in vitro. Biochem 7227:639-650. Doxsey SJ, Brodsky FM, Blank GS, and Helenius A (1987) Inhibition of endocytosis by anti-clathrin antibodies. Cell 50:453-463. Dunn KC, Marmorstein AD, Bonilha VL, Rodriguez-Boulan E, Giordano F, and Hjelmeland EM (1998) Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5. Invest Ophthalmol. Vis.Sci. 39:2744-2749. Ellington JK, Reilly SS, Ramp WK, Smeltzer MS, Kellam JF, and Hudson MC (1999) Mechanisms of Staphylococcus aureus invasion of cultured osteoblasts. Microb.Pathog. 26:317-323. Ertl B, Heigl F, Wirth M, and Gabor F (2000) Lectin-mediated bioadhesion: preparation, stability and caco-2 binding of wheat germ agglutinin-flmctionalized Poly(D,L-lactic-co-glycolic acid)-microspheres. JD rug Target 8:173-184. Fan JY, Carpentier JL, Gorden P, Van Ohberghen E, Blackett NM, Gmnfeld C, and Orel L (1982) Receptor-mediated endocytosis of insulin: role of microvilli, coated pits, and coated vesicles. Proc.Natl.Acad.Sci.U.S.A 79:7788-7791. 179 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fattal E, Youssef M, Couvreur P, and Andremont A (1989) Treatment of experimental salmonellosis in mice with ampicillin-bound nanoparticles. Antimicrob.Agents Chemother. 33:1540-1543. Felt O, Furrer P, Mayer JM, Plazonnet B, Buri P, and Gumy R (1999) Topical use of chitosan in ophthalmology: tolerance assessment and evaluation of precomeal XQi& aiion. Int J Pharm 180:185-193. Femandez-Urmsuno R, Calvo P, Remunan-Lopez C, Vila-Jato JL, and Alonso MJ (1999) Enhancement of nasal absorption of insulin using chitosan nanoparticles. Pharm Res \6A516-\5?,\. File RR and Patton TF (1980) Topically applied pilocarpine. Human pupillary response as a function of drop size. Arch. Ophthalmol. 98:112-115. Fillebeen C, Descamps L, Dehouck MP, Fenart L, Benaissa M, Spik G, Cecchelli R, and Pierce A (1999) Receptor-mediated transcytosis of lactoferrin through the blood- brainbarrier. J.Biol.Chem. 274:7011-7017. Fisher RS and Ho J (2002) Potential new methods for antiepileptic dmg delivery. CNS.Drugs 16:579-593. Fitzgerald P, Hadgraft J, and Wilson CG (1987) A gamma scintigraphic evaluation of the precomeal residence of liposomal formulations in the rabbit. JPharm Pharmacol. 39:487-490. Foster KA, Yazdanian M, and Audus KL (2001) Microparticulate uptake mechanisms of in-vitro cell culture models of the respiratory epithelixmi. JPharm Pharmacol. 53:57-66. Frazza EJ and Schmitt EE (1971) A new absorbable suture. J.Biomed.Mater.Res. 5:43-58. Fresta M, Fontana G, Bucolo C, Cavallaro G, Giammona G, and Puglisi G (2001) Ocular tolerability and in vivo bioavailability of poly(ethylene glycol) (PEG)-coated polyethyl-2-cyanoacrylate nanosphere-encapsulated acyclovir. JPharm Sci. 90:288- 297. Fu K, Harrell R, Zinski K, Um C, Jaklenec A, Frazier J, Lotan N, Burke P, Klibanov AM, and Langer R (2003) A potential approach for decreasing the burst effect of protein from PLGA microspheres. JPharm Sci. 92:1582-1591. Fu K, Pack DW, Klibanov AM, and Langer R (2000) Visual evidence of acidic environment within degrading poly(lactic-co- glycolic acid) (PLGA) microspheres. Pharm Res. 17:100-106. 180 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Gabor F, Schwarzbauer A, and Wirth M (2002) Lectin-mediated drug delivery; binding and uptake of BSA-WGA conjugates using the Caco-2 model. Int JPharm 237:227-239. Gamache DA, Dimitrijevich SD, Weimer LK, Lang LS, Spellman JM, Graff G, and Yanni JM (1997) Secretion of proinflammatory cytokines by human conjunctival epithelial cells. OculJmmunolJnflamm. 5:117-128. Geggel HS, Friend J, and Thoft RA (1984) Conjunctival epithelial wound healing. Invest Ophthalmol. Vis.Sci 25:860-863. Geroski DH and Edelhauser HF (2001) Transscleral drug delivery for posterior segment disease. Adv.DrugDeliv.Rev. 52:37-48. Giannasca PJ, Giannasca KT, Falk P, Gordon JI, and Neutra MR (1994) Regional differences in glycoconjugates of intestinal M cells in mice: potential targets for mucosal vaccines. Am. J Physiol 267:G1108-Gl 121. Giannavola C, Bucolo C, Maltese A, Paolino D, Vandelli MA, Puglisi G, Lee VH, and Fresta M (2003) Influence of preparation conditions on acyclovir-loaded poly- d,l-lactic acid nanospheres and effect of PEG coating on ocular drug bioavailability. Pharm Res. 20:584-590. Gilding DK and Reed AM (1979) Biodegradable polymers for use in surgery: poly(glycolic) / poly(lactic acid) homo- and copolymers. Polymer 20:1459. Giordano GG, Refojo MF, and Arroyo MH (1993) Sustained delivery of retinoic acid from microspheres of biodegradable polymer in PVR. Invest Ophthalmol. Vis.Sci. 34:2743-2751. Goldstein U and Hayes CE (1977) The lectins: carbohydrate-binding proteins of plants and animals. Adv.Carbohydr.Chem.Biochem. 35:150-189. Goldstein JL, Brown MS, Anderson RG, Russell DW, and Schneider WJ (1985) Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu.Rev. Cell Biol. 1:1-39. Goligorsky MS and Hruska KA (1986) Transcytosis in cultured proximal tubular cells. JMembr.Biol. 93:237-247. Gottlieb TA, Ivanov IE, Adesnik M, and Sabatini DD (1993) Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cqW s. J Cell Biol 120:695-710. 181 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Grass GM and Robinson JR (1988) Mechanisms of comeal dmg penetration. I: In vivo and in vitro kinetics. JPharm.Sci. 77:3-14. Greaves JL, Olejnik O, and and Wilson CG (1992) Polymers and the precomeal tear film. S.T.P.Pharm.Sci. 2:13-33. Green K (1993) The effects of preservatives on comeal permeability of dmgs., in Biopharmaceutics o f Ocular Drug Delivery. (Edman P ed) pp 43-49, CRC Press, Boca Raton. Gref R, Minamitake Y, Peracchia MT, Trabetskoy V, Torchilin V, and Langer R (1994) Biodegradable long-circulating polymeric nanospheres. Science 263:1600- 1603. Gregoriadis G and Ryman BE (1971) Liposomes as carriers of enzymes or dmgs: a new approach to the treatment of storage diseases. Biochem.J\1A:S'SP. Griffoni C, Spisni E, Santi S, Riccio M, Guamieri T, and Tomasi V (2000) Knockdown of caveolin-1 by antisense oligonucleotides impairs angiogenesis in vitro and in vivo. Biochem.Biophys.Res.Commun. 276:756-761. Gukasyan HJ, Lee VH, Kim KJ, and Kannan R (2002) Net glutathione secretion across primary cultured rabbit conjimctival epithelial cell layers. Invest Ophthalmol. Vis.Sci 43:1154-1161. Gurtler F and and Gumy R (1995) Patent literature review of ophthalmic inserts. Drug Dev.Indust.Pharmacy 21:1-18. Guterres SS, Fessi H, Barratt G, Puisieux F, and Devissaguet JP (1995) Poly(D,L- lactide) nanocapsules containing non-steroidal anti-inflammatory dmgs: gastrointestinal tolerance following intravenous and oral administration. Pharm Res. 12:1545-1547. Haltner EJ, Easson JH, and Lehr C-M (1997) Lectins and bacterial invasion factors for controlling endo- and transcytosis of bioadhesive dmg carrier systems. Eur.J.Pharm.Biopharm. 44:3-13. Hamalainen ICM, Kananen K, Auriola S, Kontturi K, and Urtti A (1997) Characterization of paracellular and aqueous penetration routes in comea, conjimctiva, and sclera. Invest Ophthalmol. Vis.Sci 38:627-634. Hanover JA, Willingham MC, and Pastan I (1984) Kinetics of transit of transferrin and epidermal growth factor through clathrin-coated membranes. Cell 39:283-293. 182 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hansen SH, Sandvig K, and van Deurs B (1993) Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification. J Cell Biol. 121:61-72. Harmia T, Kreuter J, Spieser P, Boye T, Gumy R, and Kubis A (1986) Enhancement of the myotic response of rabbits with pilocarpine-loaded polybutylcyanoacrylate nanoparticles. Int JPharm 33:187. Harper CA, III, Khoobehi B, Peyman GA, Gebhardt BM, and Dunlap WA (1993) Bioavailability of microsphere-entrapped cyclosporine A in the comea and aqueous of rabbits. Int Ophthalmol. 17:337-340. Hashizoe M, Ogura Y, Kimura H, Moritera T, Honda Y, Kyo M, Hyon SH, and Ikada Y (1994) Scleral plug of biodegradable polymers for controlled dmg release in the vitreous. Arch.Ophthalmol. 112:1380-1384. Hashizoe M, Ogura Y, Takanashi T, Kunou N, Honda Y, and Ikada Y (1997) Biodegradable polymeric device for sustained intravitreal release of ganciclovir in rabbits. Curr.Eye Res 16:633-639. Hayakawa E, Chien DS, Inagaki K, Yamamoto A, Wang W, and Lee VH (1992) Conjunctival penetration of insulin and peptide dmgs in the albino rabbit. Pharm.Res. 9:769-775. Heller J (1984) Biodegradable polymers in controlled dmg delivery. Crit Rev. Ther.Drug Carrier Syst. 1:39-90. Henley JR, Kmeger EW, Oswald BJ, and McNiven MA (1998) Djmamin-mediated intemalization of caveolae. J Ce// Biol. 141:85-99. Henry-Michelland, S., Alonso, M. J., Andremont, A., Maincent, P., Sauzieres, J., and and Couvreur, P. Attachment of antibiotics to nanoparticles: preparation, dmg release, and antimicrobial activity in vitro. Int J Pharm 35,121-127. 1987. Heuser JE and Anderson RG (1989) Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol 108:389-400. Horibe Y, Hosoya K, Kim KJ, and Lee VH (1997a) Kinetic evidence for Na(+)- glucose co-transport in the pigmented rabbit conjunctiva. Curr.Eye Res 16:1050- 1055. Horibe Y, Hosoya K, Kim KJ, and Lee VH (1998) Carrier-mediated transport of monocarboxylate dmgs in the pigmented rabbit conjimctiva. Invest Ophthalmol. Vis.Sci. 39:1436-1443. 183 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Horibe Y, Hosoya K, Kim KJ, Ogiso T, and Lee VH (1997b) Polar solute transport across the pigmented rabbit conjunctiva: size dependence and the influence of 8- bromo cyclic adenosine monophosphate. Pharm.Res. 14:1246-1251. Hosoya K, Horibe Y, Kim KJ, and Lee VH (1997) Na(+)-dependent L-arginine transport in the pigmented rabbit conjunctiva. Exp.Eye Res. 65:547-553. Hosoya K, Horibe Y, Kim KJ, and Lee VH (1998) Nucleoside transport mechanisms in the pigmented rabbit conjunctiva. Invest Ophthalmol. Vis.Sci. 39:372-371. Hosoya K, Kompella LFB , Kim KJ, and Lee VH (1996) Contribution of Na(+)- glucose cotransport to the short-circuit current in the pigmented rabbit conjunctiva. Curr.Eye Res. 15:447-451. Hosoya K, Ueda H, Kim KJ, and Lee VH (1999) Nucleotide stimulation of Cl(-) secretion in the pigmented rabbit conjunctiva. JFharmacol.Exp.Ther. 291:53-59. Huang M, Ma Z, Khor E, and Lim LY (2002) Uptake of FITC-chitosan nanoparticles by A549 cells. Pharm Res. 19:1488-1494. Huatan H, Collett JH, Attwood D, and Booth C (1995) Preparation and characterization of poly(epsilon-caprolactone) polymer blends for the delivery of proteins. Biomaterials 16:1297-1303. Hunziker W, Male P, and Mellman I (1990) Differential microtubule requirements for transcytosis inMDCK cells. EMBO J 9:3515-3525. Hussain N, Jaitley V, and Florence AT (2001) Recent advances in the understanding of uptake of microparticulates across the gastrointestinal lymphatics. Adv.Drug Deliv.Rev. 50:107-142. Hussain N, Jani PU, and Florence AT (1997) Enhanced oral uptake of tomato lectin- conjugated nanoparticles in the rat. Pharm Res 14:613-618. Ibim SE, Ambrosio AM, Kwon MS, El Amin SF, Allcock HR, and Laurencin CT (1997) Novel polyphosphazene/poly(lactide-co-glycolide) blends: miscibility and degradation studies. Biomaterials 18:1565-1569. Iversen TG, Skretting G, Llorente A, Nicoziani P, van Deurs B, and Sandvig K (2001) Endosome to Golgi transport of ricin is independent of clathrin and of the Rab9- and Rabl 1-GTPases. Mol.Biol.Cell 12:2099-2107. Jackman MR, Shurety W, Ellis JA, and Luzio JP (1994) Inhibition of apical but not basolateral endocytosis of ricin and folate in Caco-2 cells by cytochalasin D. J Cell Sci. 107 ( Ft 9):2547-2556. 184 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Jani P, Halbert GW, Langridge J, and Florence AT (1989) The uptake and translocation of latex nanospheres and microspheres after oral administration to rats. J Pharm Pharmacol. 41:809-812. Jani P, Halbert GW, Langridge J, and Florence AT (1990) Nanoparticle uptake by the rat gastrointestinal mucosa: quantitation and particle size dependency. JPharm Pharmacol. 42:821-826. Jepson MA, Simmons NL, O'Hagan DT, and Hirst BH (1993) Comparison of poly(DL-lactide-co-glycolide) and polystyrene microsphere targeting to intestinal M cells. JD rug Target V.2A5-2A9. Jiao Y, Ubrich N, Marchand-Arvier M, Vigneron C, Hoffman M, Lecompte T, and Maincent P (2002) In vitro and in vivo evaluation of oral heparin-loaded polymeric nanoparticles in rabbits. Circulation 105:230-235. Johnson OL, Jaworowicz W, Cleland JL, Bailey L, Chamis M, Duenas E, Wu C, Shepard D, Magil S, Last T, Jones AJ, and Putney SD (1997) The stabilization and encapsulation of human growth hormone into biodegradable microspheres. Pharm Res 14:730-735. Jollie WP (1986) Ultrastructural studies of protein transfer across rodent yolk sac. Placenta 7:263-281. Kanai A, Alba RM, Takano T, Kobayashi C, Nakajima A, Kurihara K, Yokoyama T, and Fukami M (1989) The effect on the comea of alpha cyclodextrin vehicle for cyclosporin eye drops. Transplant.Proc. 21:3150-3152. Kaufman HE, Steinemann TL, Lehman E, Thompson HW, Vamell ED, Jacob- LaBarre JT, and Gebhardt BM (1994) Collagen-based drag delivery and artificial iQ d n c s. J.Ocul.Pharmacol. 10:17-27. Kaur IP and Smitha R (2002) Penetration enhancers and ocular bioadhesives: two new avenues for ophthalmic drug delivery. Drug Dev.Ind.Pharm 28:353-369. Kawakami S, Nishida K, Mukai T, Yamamura K, Kobayashi K, Sakaeda T, Nakamura J, Nakashima M, and Sasaki H (2001) Ocular absorption behavior of palmitoyl tilisolol, an amphiphilic prodrug of tilisolol, for ocular drug delivery. J Pharm Sci 90:2113-2120. Kawashima Y, Yamamoto H, Takeuchi H, and Kuno Y (2000) Mucoadhesive DL- lactide/glycolide copolymer nanospheres coated with chitosan to improve oral delivery of elcatonin. Pharm Dev.Technol. 5:77-85. 185 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ke TL, Clark AF, and Gracy RW (1999) Age-related permeability ehanges in rabbit corneas. JOcul.Pharmacol.Ther. 15:513-523. Kim SY, Doh HJ, Jang MH, Ha YJ, Chung SI, and Park HJ (1999) Oral immunization with Helicobacter pylori-loaded poly(D, L-lactide-co- glycolide) nanoparticles. Helicobacter. 4:33-39. Kim WU, Lee WK, Ryoo JW, Kim SH, Kim J, Youn J, Min SY, Bae EY, Hwang SY, Park SH, Cho CS, Park JS, and Kim HY (2002) Suppression of collagen- induced arthritis by single administration of poly(lactic-co-glycolic acid) nanoparticles entrapping type II collagen: a novel treatment strategy for induction of oral tolerance. Arthritis Rheum. 46:1109-1120. Kimura H, Ogura Y, Moritera T, Honda Y, Wada R, Hyon SH, and Ikada Y (1992) Injectable microspheres with controlled drug release for glaucoma filtering surgery. Invest Ophthalmol.Vis.Sci. 33:3436-3441. Klyce SD, Palkama KA, Harkonen M, Marshall WS, Huhtaniitty S, Mann KP, and Neufeld AH (1982) Neural serotonin stimulates chloride transport in the rabbit comeal epithelium. Invest Ophthalmol. Vis.Sci. 23:181-192. Kompella UB, Kim KJ, and Lee VH (1993) Active chloride transport in the pigmented rabbit conjunctiva. Curr.Eye Res. 12:1041-1048. Kompella UB, Kim KJ, Shiue MH, and Lee VH (1995) Possible existence of Na(+)- coupled amino acid transport in the pigmented rabbit conjunctiva. Life Sci 57:1427- 1431. Kreuter J (1983) Evaluation of nanoparticles as dmg-delivery systems. Ill: materials, stability, toxicity, possibilities of targeting, and use. Pharm Acta Helv. 58:242-250. Kreuter J (1985) Evaluation of nanoparticlesl as dmg delivery systems II: Comparison of the body distribution of nanoparticles with the body distribution of microspheres (diameter >1 um), liposomes, and emulsions. Pharm Acta Helv 58:217. Kreuter J, Ramge P, Petrov V, Hamm S, Gelperina SE, Engelhardt B, Alyautdin R, von Briesen H, and Begley DJ (2003) Direct evidence that polysorbate-80-coated poly(butylcyanoacrylate) nanoparticles deliver dmgs to the CNS via specific mechanisms requiring prior binding of drag to the nanoparticles. Pharm Res. 20:409- 416. Kulkami AA, Trousdale MD, Stevenson D, Gukasyan HJ, Shiue MH, Kim KJ, Read RW, and Lee VH (2003) Nucleotide-induced restoration of conjunctival chloride and fluid secretion in adenovirus type 5-infected pigmented rabbit eyes. J Pharmacol.Exp. Ther. 305:1206-1211. 186 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Kvirz D and Ciulla TA (2002) Novel approaches for retinal drug delivery. Ophthalmol. Clin.North Am. 15:405-410. Kyyronen K and Urtti A (1990) Improved ocular: systemic absorption ratio of timolol by viscous vehicle and phenylephrine. Invest Ophthalmol. Vis.Sci 31:1827- 1833. Lamaze C, Dujeancourt A, Baba T, Lo CG, Benmerah A, and Dautry-Varsat A (2001) Interleukin 2 receptors and detergent-resistant membrane domains define a clathrin-independent endocytic pathway. Mol.Cell 7:661-671. Lamaze C and Schmid SL (1995) The emergence of clathrin-independent pinocytic pathways. Curr.Opin.CellBiol 7:573-580. Lamprecht A, Ubrich N, Yamamoto H, Schafer U, Takeuchi H, Maincent P, Kawashima Y, and Lehr CM (2001) Biodegradable nanoparticles for targeted drug delivery in treatment of inflammatory bowel disease. J Pharmacol.Exp. Thcr. 299:775-781. Lang JC (1995) Ocular drug delivery conventional ocular formulations. Adv.Drug Dcliv.Rcv. 16:39-43. Larkin JM, Donzell WC, and Anderson RG (1986) Potassium-dependent assembly of coated pits: new coated pits form as planar clathrin lattices. J Cell Biol 103:2619- 2627. Latkovic S and Nilsson SE (1979a) Phagocytosis of latex microspheres by the epithelial cells of the guinea pig conjimctiva. Acta Ophthalmol. (Copenh) 57:582- 590. Latkovic S and Nilsson SE (1979b) The ultrastructure of the normal conjunctival epithelium of the guinea pig. I. The basal and intermediate layers of the perilimbal zone. Acta Ophthalmol. (Copenh) 57:106-122. Laurent N, Wattiaux-De Coninck S, Mihaylova E, Leontieva E, Wamier-Pirotte MT, Wattiaux R, and Jadot M (1999) Uptake by rat liver and intracellular fate of plasmid DNA complexed with poly-L-lysine or poly-D-lysine. FEBSLett. 443:61-65. Lavelle EC, Grant G, Pusztai A, Pfuller U, and O'Hagan DT (2000) Mucosal immunogenicity of plant lectins in mice. Immunology 99:30-37. Le Bourlais CA, Chevanne F, Turlin B, Acar L, Zia H, Sado PA, Needham TE, and Leverge R (1997) Effect of cyclosporine A formulations on bovine comeal absorption: ex-vivo study. JMicroencapsul. 14:457-467. 187 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lee BL, Matoba AY, Osato MS, and Robinson NM (1992) The solubility of antibiotic and corticosteroid combinations. Am. J.Ophthalmol. 114:212-215. Lee VH (1983) Esterase activities in adult rabbit eyes. J Pharm Sci 72:239-244. Lee VH, Chien DS, and Sasaki H (1988) Ocular ketone reductase distribution and its role in the metabolism of ocularly applied levobunolol in the pigmented rabbit. J Pharmacol.Exp. Ther. 246:871-878. Lee VH and Robinson JR (1979) Mechanistic and quantitative evaluation of precorneal pilocarpine disposition in albino rabbits. J Pharm Sci 68:673-684. Lee YH, Kompella UB, and Lee VH (1993) Systemic absorption pathways of topically applied beta adrenergic antagonists in the pigmented rabbit. Exp.Eye Res. 57:341-349. Lehr CM (2000) Lectin-mediated drug delivery: the second generation of hioadhesvves. J Control Release 65:19-29. Lehr CM, Bouwstra JA, Kok W, Noach AB, dc Boer AG, and Junginger HE (1992a) Bioadhesion by means of specific binding of tomato lectin. Pharm Res. 9:547-553. Lehr CM, Bowstra JA, Schacht EH, and and Junginger HE (1992b) In vitro evaluation of mucoadhcsive properties of chitosan and some other natural polymers. IntJPharm 78:43-48. Lehr CM and Lee VH (1993) Binding and transport of some bioadhesive plant lectins across Caco-2 cell monolayers. Pharm Res. 10:1796-1799. Lehr CM, Lee YH, and Lee VH (1994) Improved ocular penetration of gentamicin by mucoadhesive polymer polycarbophil in the pigmented rabbit. Invest Ophthalmol. Vis.Sci 35:2809-2814. Lemmon SK and Jones EW (1987) Clathrin requirement for normal growth of yeast. Science 238:504-509. Lewis DH (1990) Controlled release of bioactive agents from lactide/glycolide polymers, in Biodegradable Polymers as Drug Delivery Systems (Chasin M and Langer R ed) pp 1 -42, Marcel Dekker, New York. Li JK, Wang N, and Wu XS (1997) A novel biodegradable system based on gelatin nanoparticles and poly(lactic-co-glycolic acid) microspheres for protein and peptide drug delivery. J Pharm Sci 86:891-895. 188 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Li VH, Wood RW, Kreuter J, Harmia T, and Robinson JR (1986) Ocular drug delivery of progesterone using nanoparticles. J Microencapsul. 3:213-218. Liu F and Huang L (2002) Development of non-viral vectors for systemic gene delivery. J Control Release 78:259-266. Liu KR, Peyman GA, and Khoobehi B (1989) Efficacy of liposome-bound amphotericin B for the treatment of experimental fimgal endophthalmitis in rabbits. Invest Ophthalmol. Vis.Sci 30:1527-1534. Llorente A, Prydz K, Sprangers M, Skretting G, Kolset SO, and Sandvig K (2001) Proteoglycan synthesis is increased in cells with impaired clathrin-dependent endocytosis. J Ce// Sci 114:335-343. Lobie PE, Sadir R, Graichen R, Mertani HC, and Morel G (1999) Caveolar internalization of growth hormone. Exp. Cell Res 246:47-55. Lockman PR, Mumper RJ, Khan MA, and Allen DD (2002) Nanoparticle technology for drug delivery across the blood-brain barrier. Drug Dev.Ind.Pharm 28:1-13. Losa C, Alonso MJ, Vila JL, Orallo F, Martinez J, Saavedra JA, and Pastor JC (1992) Reduction of cardiovascular side effects associated with ocular administration of metipranolol by inclusion in polymeric nanocapsules. J Ocul.Pharmacol. 8:191- 198. Losa C, Calvo P, Castro E, Vila-Jato JL, and Alonso MJ (1991) Improvement of ocular penetration of amikacin sulphate by association to poly(butylcyanoacrylate) nanoparticles. J Pharm Pharmacol. 43:548-552. Losa C, Marchal-Heussler L, Orallo F, Vila Jato JL, and Alonso MJ (1993) Design of new formulations for topical ocular administration: polymeric nanocapsules containing metipranolol. Pharm Res. 10:80-87. Ma Z and Lim LY (2003) Uptake of chitosan and associated insulin in Caco-2 cell monolayers: a comparison between chitosan molecules and chitosan nanoparticles. Pharm Res. 20:1812-1819. Maeda S, Ishikawa M, Abe T, and Sakuragi S (1998) Lectin cytochemistry of the rabbit conjunctiva and lacrimal sac. Jpn.J Ophthalmol. 42:443-449. Mahlberg K, Uusitalo RJ, Gebhardt B, and Kaufinan HE (1991) Prevention of experimental comeal allograft rejection in rabbits using cyclosporin-collagen shields. Graefes Arch. Clin.Exp.Ophthalmol. 229:69-74. 189 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Makino K, Ohshima H, and Kondo T (1986) Transfer of protons from bulk solution to the surface of poly(L-lactide) microcapsules. J Microencapsul. 3:195-202. Manil L, Davin JC, Duchenne C, Kubiak C, Foidart J, Couvreur P, and Mahieu P (1994) Uptake of nanoparticles by rat glomerular mesangial cells in vivo and in vitro. Pharm Res. 11:1160-1165. Marchal-Heussler L, Fessi H, Devissaguet J, Hoffman M, and and Maincent P (1992) Colloidal drug delivery systems for the eye: A comparison of the efficacy of three different polymers: Polyisobutylcyanoacrylate, poly(lactic-co-glycolic acid), poly (epsilon-caprolactone). STP Pharm.Sci. 2:98-107. Marchal-Heussler L, Maincent P, Hoffrnan M, Spittler J, and and Couvreur P (1990) Antiglaucomatous activity of betaxolol chlorhydrate sorbed onto different isobutylcyanoacrylate nanoparticle preparations. Int.JPharm 58:115-122. Marchal-Heussler L, Sirbat D, Hoffman M, and Maincent P (1993) Poly(epsilon- caprolactone) nanocapsules in carteolol ophthalmic delivery. Pharm Res 10:386-390. Mathiowitz E, Jacob JS, Jong YS, Carino GP, Chickering DE, Chaturvedi P, Santos CA, Vijayaraghavan K, Montgomery S, Bassett M, and Morrell C (1997) Biologically erodable microspheres as potential oral drug delivery systems. Nature 386:410-414. Matsuno K, Schaffher T, Gerber HA, Ruchti C, Hess MW, and Cottier H (1983) Uptake by enterocytes and subsequent translocation to internal organs, eg, the thymus, of Percoll microspheres administered per os to suckling mice. J Reticuloendothel.Sac. 33:263-273. Maurice DM (1973) Electrical potential and ion transport across the conjunctiva. Exp.Eye Res. 15:527-532. Mellman I (1996) Endocytosis and molecular sorting. Annu Rev Cell Dev.Biol 12:575-625. Mellman I, Fuchs R, and Helenius A (1986) Acidification of the endocytic and exocytic pathways. Annu.Rev.Biochem. 55:663-700. Merkus, F., Schipper, N., and and Hermeus, W. Absorption enhancers in nasal drug delivery: efficacy and safety. J.Control.Release 24, 201-208. 1993. Mi FL, Tan YC, Liang HF, and Sung HW (2002) In vivo hiocompatibility and degradability of a novel injectable-chitosan-based implant. Biomaterials 23:181-191. Mineo C, Gill GN, and Anderson RG (1999) Regulated migration of epidermal growth factor receptor from caveolae. JBiol.Chem. 274:30636-30643. 190 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mir LM, Banonn H, and Paoletti C (1988) Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol. Exp. Cell Res. 175:15-25. Mishima S, Gasset A, Klyce SD, Jr., and Baum JL (1966) Determination of tear volume and tear flow. Invest Ophthalmol. 5:264-276. Montesano R, Roth J, Robert A, and Orci L (1982) Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins. Nature 296:651-653. Moritera T, Ogura Y, Honda Y, Wada R, Hyon SH, and Ikada Y (1991) Microspheres of biodegradable polymers as a drug-delivery system in the vitreous. Invest Ophthalmol.Vis.Sci. 32:1785-1790. Moritera T, Ogura Y, Yoshimura N, Kuriyama S, Honda Y, Tabata Y, and Ikada Y (1994) Feasibility of drug targeting to the retinal pigment epithelium with biodegradable microspheres. Curr.Eye Res. 13:171-176. Motley A, Bright NA, Seaman MN, and Robinson MS (2003) Clathrin-mediated endocytosis in AP-2-depleted cells. /C e //Biol. 162:909-918. Mukheijee S, Ghosh RN, and Maxfield FR (1997) Endocytosis. Physiol Rev. 77:759- 803. Naisbett B and and Woodley F (1994) The potential use of tomato lectin for oral drag delivery: 1. Lectin binding to rat small intestine in vitro. Int.J.Pharm. 107:223- 230. Naisbett B and Woodley J (1990) Binding of tomato lectin to the intestinal mucosa and its potential for oral drag delivery. Biochem.Soc. Trans. 18:879-880. Narawane MA and Lee VH (1995) IGF-I and EGF receptors in the pigmented rabbit bulbar conjunctiva. Curr.Eye Res. 14:905-910. Nicholls TJ, Green KL, Rogers DJ, Cook JD, Wolowacz S, and Smart JD (1996) Lectins in ocular drag delivery; an investigation of lectin binding sites on the comeal and conjunctival surfaces. In tJ Pharm 138:175-183. Norkin LC, Anderson HA, Wolffom SA, and Oppenheim A (2002) Caveolar endocytosis of simian virus 40 is followed by brefeldin A-sensitive transport to the endoplasmic reticulum, where the virus disassembles. J Virol. 76:5156-5166. 191 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. O'Hagan D, Jeffery H, and Davis S (1994) The preparation and characterization of poly (lactide-co-glycolide) microparticles. III. Microparticles/polymer degradation rates and the in vitro release of a model protein. Int J Pharm 103:37-45. Oka JA, Christensen MD, and Weigel PH (1989) Hyperosmolarity inhibits galactosyl receptor-mediated but not fluid phase endocytosis in isolated rat hepatocytes. Chem. 264:12016-12024. Olivier JC, Fenart L, Chauvet R, Pariat C, Cecchelli R, and Couet W (1999) Indirect evidence that drug brain targeting using polysorbate 80-coated polybutylcyanoacrylate nanoparticles is related to toxicity. Pharm Res. 16:1836- 1842. Olsen KR, Parel JM, Lee W, Hernandez E, and Fliesler SJ (1989) Biodegradable mechanical retinal fixation. A pilot study. Arch. Ophthalmol. 107:735-741. Orci L, Stamnes M, Ravazzola M, Amherdt M, Perrelet A, Sollner TH, and Rothman JE (1997) Bidirectional transport by distinct populations of COPI-coated vesicles. Cell 90:335-349. Palade GE (1953) Fine structures of blood capillaries. J.Appl.Phys. 24:1424. Panyam J and Labhasetwar V (2003a) Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv.Drug Deliv.Rev. 55:329-347. Panyam J and Labhasetwar V (2003b) Dynamics of endocytosis and exocytosis of poly(D,L-lactide-co-glycolide) nanoparticles in vascular smooth muscle cells. Pharm Res. 20:212-220. Panyam J, Zhou WZ, Prabha S, Sahoo SK, and Labhasetwar V (2002) Rapid endo- lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. FASEB -1226. Pappo J and Ermak TH (1989) Uptake and translocation of fluorescent latex particles by rabbit Peyer's patch follicle epithelium: a quantitative model for M cell uptake. Clin.Exp Immunol. 76:144-148. Park TO (1994) Degradation of poly(D,L-lactic glycolic acid) microspheres: effect of molecular weight. J Rel 30:161-173. Park TG (1995) Degradation of poly(lactic-co-glycolic acid) microspheres: effect of copolymer composition. Biomaterials 16:1123-1130. Peters PJ, Gao M, Gaschet J, Ambach A, van Donselaar E, Traverse JF, Bos E, Wolffe EJ, and Hsu VW (2001) Characterization of coated vesicles that participate in endocytic recycling. Traffic. 2:885-895. 192 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Peyman GA, Conway M, Khoobehi B, and Soike K (1992) Clearance of microsphere-entrapped 5-fluorouracil and cytosine arabinoside from the vitreous of primates. Int Ophthalmol. 16:109-113. Pfister RR (1975) The normal surface of conjunctiva epithelium. A scanning electron microscopic study. Invest Ophthalmol 14:267-279. Pho MT, Ashok A, and Atwood WJ (2000) JC virus enters human glial cells by clathrin-dependent receptor- mediated endocytosis. J Virol. 74:2288-2292. Pignatello R, Bucolo C, Spedalieri G, Maltese A, and Puglisi G (2002) Flurbiprofen- loaded acrylate polymer nanosuspensions for ophthalmic application. Biomaterials 23:3247-3255. Pleyer U, Lutz S, Jusko WJ, Nguyen KD, Narawane M, Ruckert D, Mondino BJ, Lee VH, and Nguyen K (1993) Ocular absorption of topically applied FK506 from liposomal and oil formulations in the rabbit eye. Invest Ophthalmol. Vis.Sci 34:2737- 2742. Prabha S, Zhou WZ, Panyam J, and Labhasetwar V (2002) Size-dependency of nanoparticle-mediated gene transfection: studies with fractionated nanoparticles. Int JP/iarm 244:105-115. Pratten MK and Lloyd JB (1997) Uptake of microparticles by rat visceral yolk sac. Placenta 18:547-552. Qaddoumi MQ, Ueda H, Yang J, Davda J, Labhasetwar V, and Lee VHL (2000) Uptake characteristics of PLGA nanoparticles in rabbit conjunctival epithelial cells. Masters thesis. Rawa R, Siegel R, Marasca B, Karel M, and and Langer R (1985) An explanation for the controlled release of macromolecules from polymers. / Contro/Pe/1:259-267. Reer O, Bock TK, and Muller BW (1994) In vitro comeal permeability of diclofenac sodium in formulations containing cyclodextrins compared to the commercial product voltaren ophtha. J Pharm Sci. 83:1345-1349. Rejman J, Oberle V, Zuhom IS, and Hoekstra D (2004) Size-dependent intemalization of particles via the pathways of clathrin- and caveolae-mediated endocjdosis. Biochem.J 311:159-169. Ridley Lathers DM, Gill RF, and Montgomery PC (1998) Inductive pathways leading to rat tear IgA antibody responses. Invest Ophthalmol. Vis.Sci. 39:1005-1011. 193 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Riezman H, Woodman PG, van Meer G, and Marsh M (1997) Molecular mechanisms of endocytosis. Cell 91:731-738. Rittig M, Brigel C, and Lutjen-Drecoll E (1990) Lectin-binding sites in the anterior segment of the human eye. Graefes Arch.Clin.Exp.Ophthalmol. 228:528-532. Robinson JR and Mlynek GM (1995) Bioadhesive and phase-change polymers for ocular drug delivery. Adv.Drug Delivery Rev. 16:45-50. Rothberg KG, Heuser JE, Donzell WC, Ying YS, Glenney JR, and Anderson RG (1992) Caveolin, a protein component of caveolae membrane coats. Cell 68:673-682. Rubsamen PE, Davis PA, Hernandez E, O'Grady GE, and Cousins SW (1994) Prevention of experimental proliferative vitreoretinopathy with a biodegradable intravitreal implant for the sustained release of fluorouracil. Arch.Ophthalmol. 112:407-413. Ruscetti T, Cardelli JA, Niswonger ML, and O'Halloran TJ (1994) Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum. J Cell Biol. 126:343-352. Saettone MF, Chetoni P, Cerbai R, Mazzanti G, and Braghiroli L (1996) Evaluation of ocular permeation enhancers: in vitro effects on comeal transport of four beta- blockers, and in vitro/in vivo toxic activity. Int.J.Pharm. 142:103-113. Saha P, Kim KJ, and Lee VH (1996a) A primary culture model of rabbit conjunctival epithelial cells exhibiting tight barrier properties. Curr.Eye Res 15:1163-1169. Saha P, Uchiyama T, Kim KJ, and Lee VH (1996b) Permeability characteristics of primary cultured rabbit conjunctival epithelial cells to low molecular weight dmgs. Curr.Eye Res. 15:1170-1174. Saha P, Yang JJ, and Lee VH (1998) Existence of a p-glycoprotein dmg efflux pump in cultured rabbit conjunctival epithelial cells. Invest Ophthalmol. Vis.Sci. 39:1221- 1226. Sahoo SK, Panyam J, Prabha S, and Labhasetwar V (2002) Residual polyvinyl alcohol associated with poly (D,L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake. J Control Release 82:105-114. Sai Y, Kajita M, Tamai I, Wakama J, Wakamiya T, and Tsuji A (1998) Adsorptive- mediated endocytosis of a basic peptide in enterocyte-like Caco-2 cells. Am.JPhysiol 275:G514-G520. 194 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Saishin Y, Silva RL, Saishin Y, Callahan K, Schoch C, Ahlheim M, Lai H, Kane F, Brazzell RK, Bodmer D, and Campochiaro PA (2003) Periocular injection of microspheres containing PKC412 inhibits choroidal neovascularization in a porcine model. Invest Ophthalmol. Vis.Sci. 44:4989-4993. Sanders E and Ashworth C (1961) A study of particulate intestinal absorption and hepatocellular uptake. Exp Cell Res 22:137-145. Sang YH and Gwan PT (2004) Biodegradable nanoparticles containing protein-fatty acid complexes for oral delivery of salmon calcitonin. J Pharm Sci. 93:488-495. Schaeffer HE, Breitfeller JM, and Rrohn DL (1982) Lectin-mediated attachment of liposomes to cornea: influence on transcomeal drag flux. Invest Ophthalmol. Vis.Sci. 23:530-533. Schaeffer HE and Krohn DL (1982) Liposomes in topical drag delivery. Invest Ophthalmol. Vis.Sci. 22:220-227. Sehested M, Skovsgaard T, and Roed H (1988) The carboxylic ionophore monensin inhibits active drag efflux and modulates in vitro resistance in daunorabicin resistant Ehrlich ascites tumor cells. Biochem.Pharmacol. 37:3305-3310. Shell, J (1978) Ocular system made of bioerodible esters having linear ether. (Patent #4115544). Shephard MJ, Todd D, Adair BM, Po AL, Mackie DP, and Scott EM (2003) Immunogenicity of bovine parainfluenza type 3 virus proteins encapsulated in nanoparticle vaccines, following intranasal administration to mice. Res. Vet. Sci 74:187-190. Shiue MH, Gukasyan HJ, Kim KJ, Loo DD, and Lee VH (2002) Characterization of cyclic AMP-regulated chloride conductance in the pigmented rabbit conjunctival epithelial cells. Can.J Physiol Pharmacol. 80:533-540. Shiue MH, Kim KJ, and Lee VH (1998) Modulation of chloride secretion across the pigmented rabbit conjunctiva. Exp.Eye Res. 66:275-282. Shiue MH, Kulkami AA, Gukasyan HJ, Swisher JB, Kim KJ, and Lee VH (2000) Pharmacological modulation of fluid secretion in the pigmented rabbit conjimctiva. Life Set 66:C\05-C\n. Shive MS and Anderson JM (1997) Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv.Drug Deliv.Rev. 28:5-24. 195 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sieczkarski SB and Whittaker GR (2002) Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J Virol. 76:10455-10464. Sinkula AA and Yalkowsky SH (1975) Rationale for design of biologically reversible drug derivatives: prodrugs. JPharm.Sci. 64:181-210. Smart EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T, and Lisanti MP (1999a) Caveolins, liquid-ordered domains, and signal transduction. Mol.Cell Biol. 19:7289-7304. Smart JD, Nicholls TJ, Green KL, Rogers DJ, and Cook JD (1999b) Lectins in drug delivery: a study of the acute local irritancy of the lectins from Solanum tuberosum and Helix pomatia. Eur.JPharm Sci. 9:93-98. Smolin G, Okumoto M, Feiler S, and Condon D (1981) Idoxuridine-liposome therapy for herpes simplex keratitis. Am. J Ophthalmol. 91:220-225. Smythe E, Redelmeier TE, and Schmid SL (1992) Receptor-mediated endocytosis in semiintact cells. Methods Enzymol. 219:223-234. Snibson GR, Greaves JL, Soper ND, Prydal JI, Wilson CG, and Bron AJ (1990) Precorneal residence times of sodium hyaluronate solutions studied by quantitative gamma scintigraphy. Eye 4 ( Ft 4):594-602. Snigirevskaya ES and Cottier H (1999) Immunogold visualization of actin near the coated pits at the apical surface of human mammary epithelial cells. Tsitologiia 41:586-589. Soma CE, Dubemet C, Barratt G, Nemati F, Appel M, Benita S, and Couvreur P (1999) Ability of doxorubicin-loaded nanoparticles to overcome multidrug resistance of tumor cells after their capture by macrophages. Pharm Res. 16:1710-1716. Sotozono C, He J, Matsumoto Y, Kita M, hnanishi J, and Kinoshita S (1997) Cytokine expression in the alkali-burned cornea. Curr.Eye Res. 16:670-676. Steuhl P and Rohen JW (1983) Absorption of horse-radish peroxidase by the conjunctival epithelium of monkeys and rabbits 1. Graefes rch.Clin.Exp.Ophthalmol. 220:13-18. Stolnik S, Garnett M, Davies M, Ilium L, Bousta M, Vert M, and and Davis SS (1995) The colloidal properties of surfactant-free biodegradable nanospheres from poly (B-malic acid-co-benzyl malate) and poly (lactic acid-co-glycolide). Colloids Surfaces A Physicochem.Eng.Aspects 97:235-245. 196 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Stratford RE, Jr. and Lee VH (1985) Ocular aminopeptidase activity and distribution in the albino rabbit. Curr.Eye Res. 4:995-999. Suh H, Jeong B, Liu F, and Kim SW (1998) Cellular uptake study of biodegradable nanoparticles in vascular smooth muscle cells. Pharm Res. 15:1495-1498. Swaan PW (1998) Recent advances in intestinal macromolecular drug delivery via receptor- mediated transport pathways. Pharm Res 15:826-834. Swanson JA, Yirinec BD, and Silverstein SC (1985) Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J Cell Biol. 100:851-859. Synnes M, Prydz K, Lovdal T, Brech A, and Berg T (1999) Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes. Biochim.Biophys.Acta 1421:317-328. Szejtli J (1994) Medicinal applications of cyclodextrins. Med.Res.Rev. 14:353-386. Takahashi K, Saishin Y, Saishin Y, Mori K, Ando A, Yamamoto S, Oshima Y, Nambu H, Melia MB, Bingaman DP, and Campochiaro PA (2003) Topical nepafenac inhibits ocular neovascularization. Invest Ophthalmol. Vis.Sci 44:409-415. Tang Z, Scherer PE, Okamoto T, Song K, Chu C, Kohtz DS, Nishimoto I, Lodish HF, and Lisanti MP (1996) Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle. J Biol.Chem. 271:2255- 2261. Taniguchi K, Yamamoto Y, Itakura K, Miichi H, and Hayashi S (1988) Assessment of ocular irritability of liposome preparations. J Pharmacobiodyn. 11:607-611. Ticho U, Blumenthal M, Zonis S, Gal A, Blank I, and Mazor ZW (1979) A clinical trial with Piloplex— a new long-acting pilocarpine compound: preliminary report. Ann. Ophthalmol. 11:555-561. Tracy MA, Ward KL, Firouzabadian L, Wang Y, Dong N, Qian R, and Zhang Y (1999) Factors affecting the degradation rate of poly(lactide-co-glycolide) microspheres in vivo and in vitro. Biomaterials 20:1057-1062. Tuori A, Virtanen I, and Uusitalo H (1994) Lectin binding in the anterior segment of the bovine eye. Histochem.J. 26:787-798. Turner HC, Alvarez LJ, and Candia OA (2001) Identification and localization of acid-base transporters in the conjunctival epithelium. Exp.Eye Res. 72:519-531. 197 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Turner HC, Alvarez LJ, Candia OA, and Bernstein AM (2003) Characterization of serotonergic receptors in rabbit, porcine and human conjunctivae. Curr.Eye Res. 27:205-215. Tumer HC, Bernstein A, and Candia OA (2002) Presence of CFTR in the conjunctival epithelium. Curr.Eye Res. 24:182-187. Ueda H, Horibe Y, Kim KJ, and Lee VH (2000) Functional characterization of organic cation drug transport in the pigmented rabbit conjunctiva. Invest Ophthalmol. Vis.Sci 41:870-876. Unterman SR, Rootman DS, Hill JM, Parelman JJ, Thompson HW, and Kaufinan HE (1988) Collagen shield drug delivery : therapeutic concentrations of tobramycin in the rabbit cornea and aqueous humor. J. Cataract Refract.Surg. 14:500-504. Usayapant A, Karara AH, and Narurkar MM (1991) Effect of 2-hydroxypropyl-beta- cyclodextrin on the oeular absorption of dexamethasone and dexamethasone acetate. Pharm.Res. 8:1495-1499. Vesaluoma M, Rosenberg ME, Teppo A, Gronhagen-Riska C, Haahtela T, and Tervo T (1999) Tumour necrosis factor alpha (TNFalpha) in tears of atopic patients after conjunctival allergen challenge. Clin.Exp.Allergy 29:537-542. Vila A, Sanchez A, Tobio M, Calvo P, and Alonso MJ (2002) Design of biodegradable particles for protein delivery. J Control Release 78:15-24. Virtanen I, Kariniemi AL, Holthofer H, and Lehto VP (1986) Fluorochrome-coupled lectins reveal distinct cellular domains in human epidermis. J.Histochem.Cytochem. 34:307-315. Wagner E, Plank C, Zatloukal K, Cotten M, and Bimstiel ML (1992) Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proc.Natl.Acad.Sci.U.S.A 89:7934-7938. Wang LH, Rothberg KG, and Anderson RG (1993) Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J Cell Biol 123:1107-1117. Wang N, Wu XS, and Li JK (1999) A heterogeneously structured composite based on poly(lactie-co-glycolic acid) microspheres and poly(vinyl alcohol) hydrogel nanoparticles for long-term protein drug delivery. Pharm Res. 16:1430-1435. 198 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Wang W, Bundgaard H, Buur A, and Lee VH (1991) Comeal penetration of 5- fluorouracil and its improvement by prodmg derivatization in the albino rabbit: implication in glaucoma filtration surgery. Curr.Eye Res. 10:87-97. Watsky MA, Jablonski MM, and Edelhauser HF (1988) Comparison of conjunctival and comeal surface areas in rabbit and hiunan. Curr.Eye Res. 7:483-486. Wattiaux R, Laurent N, Wattiaux-De Coninck S, and Jadot M (2000) Endosomes, lysosomes: their implication in gene transfer. Adv.Drug Deliv.Rev. 41:201-208. Wei CP, Anderson JA, and Leopold I (1978) Ocular absorption and metabolism of topically applied epinephrine and a dipivalyl ester of epinephrine. Invest Ophthalmol. Vis.Sci. 17:315-321. Weimer LK, Gamache DA, and Yanni JM (1998) Histamine-stimulated cytokine secretion from human conjunctival epithelial cells: inhibition by the histamine HI antagonist emedastine. Int Arch.Allergy Immunol. 115:288-293. Wettey FR, Hawkins SF, Stewart A, Luzio JP, Howard JC, and Jackson AP (2002) Controlled elimination of clathrin heavy-chain expression in DT40 lymphocytes. Science 297:1521-1525. Wirth M, Fuchs A, Wolf M, Ertl B, and Gabor F (1998a) Lectin-mediated drag targeting: preparation, binding characteristics, and antiproliferative activity of wheat germ agglutinin conjugated doxorubicin on Caco-2 cells. Pharm Res. 15:1031-1037. Wirth M, Hamilton G, and Gabor F (1998b) Lectin-mediated drag targeting: quantification of binding and intemalization of Wheat germ agglutinin and Solanum tuberosum lectin using Caco-2 and HT-29 cells. J Drug Target 6:95-104. Wood R, Li V, Kreuter J, and Robinson J (1985) Ocular disposition of poly-hexyl-2- cyano [3-'‘ ^C] acrylate nanoparticles in the albino rabbit. Int J Pharm 23:175. Woodley JF (1985) Liposomes for oral administration of drags. Crit Rev. Ther.Drug Carrier Syst. 2:1-18. Yang JJ, Kim KJ, and Lee VH (2000a) Role of P-glycoprotein in restricting propranolol transport in cultured rabbit conjunctival epithelial cell layers. Pharm Res. 17:533-538. Yang JJ, Ueda H, Kim K, and Lee VH (2000b) Meeting future challenges in topical ocular drag delivery: development of an air-interfaced primary culture of rabbit conjunctival epithelial cells on a permeable support for drag transport studies. J Controlled Release 65:1 -11. 199 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Yasukawa T, Kimura H, Tabata Y, Miyamoto H, Honda Y, and Ogura Y (2002) Sustained release of cis-hydroxyproline in the treatment of experimental proliferative vitreoretinopathy in rabbits. Graefes Arch. Clin.Exp.Ophthalmol. 240:672-678. Yin W and Cheng PW (1994) Lectin conjugate-directed gene transfer to airway epithelial cells. Biochem.Biophys.Res.Commun. 205:826-833. Zignani M, Tabatabay C, and and Gumy R (1995) Topical semi-solid dmg delivery: kinetics and tolerance of ophthalmic hydrogels. Adv.Drug Deliv.Rev. 16:51-60. Zimmer A, Kreuter J, and Robinson JR (1991) Studies on the transport pathway of PBCA nanoparticles in ocular tissues. J Microencapsul. 8:497-504. Zimmer A, Mutschler E, Lambrecht G, Mayer D, and Kreuter J (1994) Pharmacokinetic and pharmacodynamic aspects of an ophthalmic pilocarpine nanoparticle-delivery-system. Pharm Res. 11:1435-1442. Zuhom IS, Kalicharan R, and Hoekstra D (2002) Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis. J Biol.Chem. 277:18021-18028. 200 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Creator Qaddoumi, Mohamed Ghazi (author)

Core Title Conjunctival epithelial uptake of biodegradable nanoparticles: Mechanism, intracellular distribution, and absorption enhancement

School Graduate School

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, pharmaceutical,Health Sciences, Pharmacy,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Lee, Vincent H.L. (committee chair), Chang, Chiang-peng (committee member), Koda, Robert T. (committee member), Lien, Eric J. (committee member), Shen, Wei-Chiang (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-524550

Unique identifier UC11335764

Identifier 3140540.pdf (filename),usctheses-c16-524550 (legacy record id)

Legacy Identifier 3140540.pdf

Dmrecord 524550

Document Type Dissertation

Rights Qaddoumi, Mohamed Ghazi

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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