University of Southern California Dissertations and Theses

Trafficking of targeted elastin‐like polypeptide nanoparticles in the lacrimal gland

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Trafficking of targeted elastin‐like polypeptide nanoparticles in the lacrimal gland

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Content USC SCHOOL OF PHARMACY

TRAFFICKING OF TARGETED ELASTIN-LIKE
POLYPEPTIDE NANOPARTICLES IN THE
LACRIMAL GLAND

By
Aaron Pang-Yu Hsueh

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
August 2015

Copyright Aaron Pang-Yu Hsueh

i

Dedication
This dissertation is dedicated to my parents, family, mentors, and friends.

ii

Acknowledgements
I would like to offer my most sincere gratitude to my mentors, Dr. Sarah Hamm-Alvarez
and Dr. J. Andrew MacKay, for their advice and encouragement that carried me through
difficult times and assisted me to shape my research skills.
I also thank all of my committee members, Dr. Curtis T. Okamoto and Dr. Wei-Chiang
Shen, for their valuable feedback and time spent in my research design and reviewing my
dissertation. I would like to show my gratitude to Dr. Honggang Cui, Dr. Yi-An Lin, and
the USC/Norris Cell and Tissue Imaging Core Facility (E. Barron, D. Hauser and A.
Rodriguez) for their suggestions, assistance, and help in my research. I would like to
thank all my lab members for their support through the entire project. In particular, I wish
to thank Frances Yarber and Hua Pei for the preparation of rabbit LGACs and Dr.
Guoyong Sun and Dr. Maria Edman-Woolcott for their valuable advices. I also thank the
help from Dr. Ben Xu, Dr. Pu Shi, Dr. Wan Wang, Zhen Meng, Mihir Shah, Jugal
Dhandhukia, Zhe Li, Dab Brill, and Srikanth R. Janga. I appreciate all the funding
resources for my research: the USAMRMC/TATRC grant W81XWH1210538, NIH
EY011386 and EY017293-S1, NIH R21EB012281, and P30 CA014089.

iii

Table of Contents
Dedication i
Acknowledgements ii
Table of Contents iii
List of Tables vii
List of Figures viii
Abbreviations xi
Abstract xii
Chapter 1: Introduction 1
1.1 Physiology of the lacrimal gland and lacrimal functional unit 1
1.2 Inflammatory disorders of the lacrimal gland 3
1.3 The immunology and pathophysiology of lacrimal gland 4
1.4 Elastin-like polypeptide 6
1.5 Goals and experimental design 7
Chapter 2: Tear-mediated delivery of nanoparticles through transcytosis
of the lacrimal gland 9
2.1 Introduction 9
2.2 Material and Methods 12
2.2.1 Materials and reagents 12
2.2.2 Biosynthesis and characterization of ELPs 13

iv

2.2.3 Dynamic light scattering and zeta potential measurement of ELPs 14
2.2.4 Fluorescent labeling of ELPs 14
2.2.5 Animals and Animal Procedures 15
2.2.6 Acinar cell isolation and primary culture 15
2.2.7 Adenovirus and baculovirus transduction and real-time fluorescence
Imaging 16
2.2.8 Quantification of fluorescence signal 17
2.2.9 Transmission electron microscopy 18
2.2.10 Statistics 19
2.3 Results and Discussion 19
2.3.1 Characterization of ELP diblock copolymer nanoparticles 19
2.3.2 Dimensions and size homogeneity of ELP nanoparticles with and
without the knob domain 22
2.3.3 Internalization and transcytosis of ELP fusions in LG acini 24
2.3.4 Characterization of KSI intracellular trafficking in LGACs 27
2.3.5 Characterization of KSI intracellular trafficking in vivo 35
2.3.6 Hypothesized trafficking model of Knob-ELP 39
2.4 Conclusion 46
2.5 Acknowledgements 46
Chapter 3: Epithelial Targeting using Elastin-Based Protein Polymers 48
3.1 Introduction 48
3.2 Materials and Methods 51
3.2.1 Materials and Reagents 51
3.2.2 Animals 52
3.2.3 Construction of ICAM-1 SI ELP nanoparticles 52
3.2.4 Characterization of ICAM-1 SI phase transition behavior and
nanoparticle formation 54
3.2.5 Fluorescent labelling of ELP nanoparticles 54
3.2.6 Lipofectamine transfection for ICAM-1 expression 55
3.2.7 Baculovirus transduction 55
3.2.8 Primary mouse LGAC culture 55
3.2.9 In vitro cell uptake and intracellular trafficking study 56
3.2.10 Reverse transcription (RT) and real-time polymerase chain
reaction (PCR) 57
3.2.11 Western blot analysis with mouse LG lysate 58
3.2.12 Immunohistochemistry of mouse LG 58

v

3.2.13 In vivo ICAM-1 targeting of ELP nanoparticles 59
3.2.14 microPET imaging 59
3.2.15 Statistics 60
3.3 Results and Discussion 61
3.3.1 ELP fusions form nanoparticles at physiological temperature 61
3.3.2 ELP nanoparticles target epithelial ICAM-1 in vitro 67
3.3.3 Internalized ELP nanoparticles were trafficked via a lipid raft
independent pathway 72
3.3.4 ICAM-1 is upregulated in inflamed LGs 77
3.3.5 In vivo targeting and pharmacokinetic profile of ELP
Nanoparticles 81
3.4 Conclusion 86
3.5 Acknowledgment 87
Chapter 4: Development of multifunctional nanoparticles for
immune-mediated inflammation 88
4.1 Introduction 88
4.2 Materials and Methods 90
4.2.1 Co-assembly of ELP nanoparticles 90
4.2.2 Tear fluid collection 91
4.2.3 Enzyme activity assay 92
4.2.4 ICAM-1 expression 93

4.3 Results and Discussion 93
4.3.1 ELPs sharing similar parent structures co-assemble above the low
critical micelle temperatures 93
4.3.2 Peptide from the pro region of cathepsin S exhibit potent
inhibitory capacity against cathepsin S 96
4.3.3 ICAM-1 expressions in a Sjögren's syndrome-susceptible
mouse, NOD 98
4.4 Conclusion 100

vi

Chapter 5: Conclusions 102
5.1 Significance 102
5.2 Conclusions 106
References 109

vii

List of Tables
Table 1: Elastin-like polypeptides evaluated in Chapter 2 22
Table 2: Elastin-like polypeptides evaluated in Chapter 3 67

viii

List of Figures
Figure 1: Schematic diagram illustrating the tear drainage system and three
layers of the tear film. 2
Figure 2: Schematic representation of KSI nanoparticles and their phase
transition behavior 21
Figure 3: Genetic fusion of the Ad5 knob domain to SI has minimal
impact on the morphology of ELP nanoparticles. 23
Figure 4: KSI is internalized and accumulates in the cytosol and lumena of
rabbit LGAC expressing GFP-actin. 26
Figure 5: ELP nanoparticles traffic to low pH compartments in rabbit LGACs 29
Figure 6: Intracellular trafficking of KSI nanoparticles in LGACs 31
Figure 7: Overexpression of a dominant negative Myosin Vb tail impairs
basolateral-to-apical transcytosis of KSI in LGACs 33
Figure 8: Distribution of CAR in LG from BALB/c mice 35
Figure 9: Comparison of in vivo ELP nanoparticle accumulation in LG from
BALB/c mice 36
Figure 10: Transmission electron microscopy confirms KSI transcytosis in
LGs from BALB/c mice 38
Figure 11: Working model for intracellular trafficking of KSI ELPs in LGACs.
KSI is internalized via CAR-mediated endocytosis 41
Figure 12: Schematic diagram of human ICAM-1 showing domain structures and
binding partners 48

ix

Figure 13: Structural maps of expression plasmid pET-25b(+) containing mouse
ICAM-1 targeting peptide or ICAM-1 SI ELP genes 62
Figure 14: Amphipathic protein polymers designed to target the ICAM-1 receptor 63
Figure 15: MALDI-TOF mass spectra of SI and ICAM-1 SI 64
Figure 16: ELP protein polymer with or without a mouse ICAM-1 targeting
peptide forms nanoparticles at physiological temperature 65
Figure 17: ICAM-1 SI internalizes in mouse ICAM-1 expressing HeLa cells 69
Figure 18: ICAM-1 SI exhibits a cross-species targeting activity to human
ICAM-1 71
Figure 19: ICAM-1 SI displays a similar but not identical intracellular
trafficking to cholera toxin B subunit, a marker for
CLIC-mediated endocytotic pathway 73
Figure 20: Internalized ICAM-1 SI nanoparticles traffic to early
endosomes and lysosomes 75
Figure 21: Schematic diagram showing the intracellular itinerary of
ICAM-1 SI in mouse ICAM-1 expressing HeLa cells 76
Figure 22: ICAM-1 is upregulated in lacrimal glands (LG) from
male non-obese diabetic (NOD) mice 78
Figure 23: Distribution of ICAM-1 in the LG of BALB/c and NOD mice 80
Figure 24: ICAM-1 SI targets inflamed LGs in vivo 81
Figure 25: Addition of mouse ICAM-1 targeting peptide to SI enhances
the ICAM-1 targeting specificity and internalization of SI
in NOD LG acini 82

x

Figure 26: Preliminary data showing the pharmacokinetic profile of
ELP nanoparticles in NOD mice. 83
Figure 27: Elastin based biopolymers with distinct functional moieties
assemble above their phase transition temperatures 95
Figure 28: Cathepsin S (CTSS) inhibitor peptide and its activity against
mouse and human CTSS 98
Figure 29: Expression levels of ICAM-1 in organs from BALB/c and NOD mice 100

xi

Abbreviations
Ad5, adenovirus serotype 5; Ad, adenovirus; AM, apical membrane; BEE, basolateral
early endosomes; BLM, basolateral membrane; CME, clathrin-mediated endocytosis;
CTSS, cathepsin S; CTSSIP, cathepsin S inhibitor peptide; CAR, coxsackievirus and
adenovirus receptor; cryo-TEM, cryogenic transmission electron microscopy; DLS,
dynamic light scattering; DN, dominant negative; EE, early endosome; ELP, elastin-like
polypeptide; GFP, green fluorescent protein; Ig, immunoglobulin; ICAM-1, intercellular
adhesion molecule-1; KSI, knob fusion to an ELP diblock copolymer; LG, lacrimal gland;
LGAC, lacrimal gland acinar cell; PCM, Peter’s complete medium; RFP, red fluorescent
protein; Rh, rhodamine; SI, ELP diblock copolymer; SV, secretory vesicle; SS, Sjögrens’
syndrome; TEM, transmission electron microscopy; Tt, transition temperature; YFP,
yellow fluorescent protein.

xii

Abstract
Rapid clearance from the tears presents a formidable obstacle to the delivery of peptide
drugs to the eye surface. This impedes therapies for ocular infections, wound healing, and
dry-eye disease that affect the vision of over four million Americans. To overcome this
challenge, this thesis first explores a novel nanoparticle strategy to reach the ocular
surface via receptor-mediated transcytosis through the lacrimal gland (LG), responsible
for the bulk of human tears. The LG abundantly expresses the coxsackievirus and
adenovirus receptor (CAR); furthermore, we recently reported a peptide-based ELP
nanoparticle (KSI) that targets CAR on liver cells. This thesis reports the unexpected
finding that KSI both targets and transcytoses into the LG acinar lumen, which drains to
tear ducts. When followed using ex vivo live cell imaging KSI rapidly accumulates in the
lumen formed by LG acinar cells. LG transduction with a Myosin Vb tail, which is a
dominant negative inhibitor of transcytosis, inhibits lumenal accumulation. Transcytosis
of KSI was confirmed in vivo by confocal and TEM imaging of LG tissue following
administration of KSI nanoparticles. These findings suggest that it is possible to target
nanomaterials to the tears by engaging receptors prevalent on the LG. This design
strategy represents a new opportunity to overcome barriers to ocular delivery.
Autoimmune diseases cause considerable morbidity, mortality, and healthcare cost
each year in the United States. Although effective, current clinical treatment approaches
which blunt all immune responses can depress beneficial immunity, increase rates of
infections, cancer, and oxicity. Sjögren's syndrome is thought to be an autoimmune
disease accompanying with the moderate-to-severe destruction of exocrine glands such as
the lacrimal gland (LG) and salivary gland. Recent advances suggest that maintaining a

xiii

balance of regulatory immune functions and effectors is critical for avoiding
autoimmunity. ICAM-1 is considered a disease initiator for T-cell activation and
migration to inflamed tissues in autoimmunity. This thesis characterizes a bio-polymeric
nanoparticle assembled from a diblock copolymer ELP fused with a mouse intercellular
adhesion molecule (ICAM)-1 targeting peptide. This material may form the basis of a
new drug delivery platform for the targeting of therapeutic agents to intracellular
compartments of diseased LG acinar cells (LGACs). Interestingly, this nanoparticle
forms a multivalent and monodisperse nanoparticle with a radius of 21.9 nm, which was
characterized by dynamic light scattering (DLS). This nanoparticle exhibits cross-species
activity, targeting human and mouse ICAM-1, internalizing into HeLa cells and mouse
ICAM-1-expressing HeLa cells. It is first trafficked to early endosomes before it
accumulates in lysosomes. Similar targeting activity was observed in ex vivo and in vivo
LGACs from male NOD mice, an established Sjögren’s Syndrome mouse model. This
nanoparticle has a blood half-life of 3.8 h compared with 7.2 h for its plain counterpart.
Therefore, by targeting to ICAM-1, this biopolymer has potential applications to deliver
pharmaceuticals to intracellular compartments of cells in inflamed tissues.

1

Chapter 1: Introduction
1.1 Physiology of the lacrimal gland and lacrimal functional unit
Lacrimal glands (LGs), which in humans are composed of a main gland and small
accessory glands (Figure 1), function as tear protein and fluid producers and have a
critical role in maintaining the integrity of a healthy ocular surface (Mathers, 2000). LGs
consist of plentiful lobules separated by fibrovascular septa. Each lobule consists of
numerous tubuloacinar structures with secretory acini connecting to ducts. 80% of the
mass of the LG are composed of LG acinar cells (LGACs). LGACs are highly active
secretory cells with polarized cell membranes delineated by the organization of actin
filaments, which are especially dense at the apical membrane. LGAC apical surfaces
connect to form central lumen and their basal surfaces are surrounded by a discontinuous
layer of flattened myoepithelial cells. Nuclei of LGAC sit basally and are surrounded by
mitochondria, endoplasmic reticulum, and Golgi apparatus (Hsueh et al., 2014). Mature
secretory vesicles beneath the apical surface containing tear proteins are mobilized and
exocytosed in response to stimuli (Chiang et al., 2011) while other cargo moves from
basolateral to apical domains through transcytosis to be secreted into tears (Evans et al.,
2008; Xu et al., 2013). Tear fluid secreted into lobular ducts drain to the excretory ducts
that drain on to the ocular surface.

2

Figure 1. Schematic diagram illustrating the tear drainage system and three layers of the
tear film. Tears, produced by (main and accessory) lacrimal glands, are secreted to the
ocular surface via excretory ducts. With blinking, the tear fluid drains down the lacrimal
canals and enters the lacrimal sac, where it drains to the nasolacrimal duct and eventually
enters the nasal cavity. Alterations in tear-film homeostasis may cause ocular disorders.

The tear film is a tri-laminar fluid comprised of an outermost lipid layer, an
intermediate aqueous layer, and an innermost mucous layer and covers the entire ocular
surface, including the conjunctiva and cornea (Dartt and Willcox, 2013; Johnson and
Murphy, 2004) (Figure 1). The lipid layer, produced by the Meibomian gland of the
eyelid in humans, provides a smooth optical surface for the cornea and retards
evaporative tear loss. The mucous layer allows the tear film to adhere to the eye. The
aqueous layer, constituting approximately 98% of the tear film, is secreted by the LG and
contains water, electrolytes, nutrients, and proteins for several functions, including
antimicrobial (e.g. secretory IgA, lysozyme, and lactorferrin), moisturizing, and
biological (e.g. EGF, lacritin, cytokines, and growth factors) activities (Seal et al., 1986).

3

The tear secretion is well maintained by the lacrimal functional unit which is defined as
an integrated system consisting of (main and accessory) LGs, ocular surface (cornea,
conjunctiva, and meibomian glands), and the interconnecting innervation including
parasympathetic, sympathetic, and sensory nervous system (Botelho et al., 1966; Sibony
et al., 1988; Stern et al., 2004). In addition, the lacrimal gland secretion is also regulated
by hormones released from the hypothalamic-pituitary-gonadal axis. These hormones can
be adrenocorticotropic hormone (ACTH), androgens, estrogens, arginine vasopressin
(AVP), glucagon, glucocorticoids, insulin, α-melanocyte stimulating hormone ( α-MSH),
progestins, prolactin and retinoic acid (Leiba et al., 1990; Mircheff et al., 1992; Rocha et
al., 2000; Sullivan, 2004; Sullivan et al., 1996; Ubels et al., 1994).
1.2 Inflammatory disorders of the lacrimal gland
The LG has a complex vascular system which enables to receive sufficient blood
supply and at the same time causes high susceptibility to systemic infections from
bacteria and viruses and the deposition of leukemic or neoplastic cells (Zoukhri, 2006).
The inflammation of the LG causes a swelling of the outer and upper lid and occurs as a
result of an acute dacryoadenitis attributed to bacterial or viral infection, chronic
dacryoadenitis resulting from an autoimmune disease, for instance Sjögren's Syndrome
(SS), induced inflammation in tissues adjacent to LGs, for instance nonspecific orbital
inflammation, or other factors such as the graft versus host disease, aging, etc (Zoukhri,
2006).
As a common factor causing dry eye syndrome, SS is a systemic autoimmune disease
affecting primarily exocrine glands. It can come with the inflammation of exocrine

4

glands alone (primary SS) or be accompanied by other autoimmune diseases (secondary
SS) like rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or systemic
sclerosis (Fox and Kang, 1992). The LG inflammation decreases the quantity or quality
of tear proteins and fluid secreted into the aqueous layer of tear film leading to dry eye
syndrome, so called aqueous-deficient dry eye, and eventually can cause blindness in
advanced cases. Dry eye syndrome can also be a result of Meibomian gland diseases
(Zoukhri, 2006). Due to the shortage of a targeted therapy to the LG (Hsueh et al., 2014) ,
current treatments for the above diseases are symptomatic therapies and rely only on
surgical procedures and/or systemic therapeutics (e.g. antibiotics, analgesics,
corticosteroids, antimetabolites, alkylating agents, cytotoxic agents, and
immunosuppressive agents); often accompanied with unnecessary side effects.
1.3 The immunology and pathophysiology of lacrimal gland
The LG is an important immunological tissue for the secretion of soluble
immunoglobulin (Ig) antibodies like IgA to the ocular surface (Saitoh-Inagawa et al.,
2000). These secreted Ig proteins can inhibit epithelial colonization, invasion by
pathogens, and mucosal penetration of hazard soluble materials. This secretory immunity
is believed to be mediated by the polymeric immunoglobulin receptor (pIgR) (Brandtzaeg
et al., 1999; Johansen et al., 2001; Norderhaug et al., 1999), constitutively expressed or
stimulated by cytokines, in LGAC. One of the best characterized examples is the
secretion of secretory IgA. The plasma B cells located in small interstitial spaces of the
LG (Brandtzaeg et al., 1999) secrete dimeric IgA, two monomeric IgA coupled by a
joining chain, for local production. The pIgR in LGACs transports dimeric IgA by a

5

basal-to-apical transcytosis (Xu et al., 2013). The dimeric IgA is processed to soluble IgA
before being released into the tear fluid.
As described previously, the LG is known to play a crucial role in tear and tear film
stability. LG dysfunction leads to severe ocular disorders. One of the common disorders
associated with LG dysfunction is dry eye syndrome, also known as keratoconjunctivitis
sicca (KCS), a multifactorial disease of the ocular surface and tears due to tear deficiency
or excessive evaporation which results in tear film instability and visual disturbance
(2007). These risk factors can be aging, sex, hormone imbalance, autoimmune diseases,
etc. An intriguing factor that we investigate is autoimmune disease, which causes SS-
associated dry eye. SS-associated dry eye results from a chronic inflammation of the LG
in which body’s white blood cells attack and destroy the LG and is characterized by focal
lymphocytic infiltration and loss of the integrity of the LG. The pathogenesis remains
controversial; however, the most widely accepted hypothesis is that inflamed LG
epithelial acinar cells mis-sort proteins, which can enhance proteolytic activity in late
endosomes and lysosomes (Li et al., 2010; Wu et al., 2009). As one example of how this
mis-sorting influences autoimmune disease, the protease cathepsin S (CTSS) appears to
increase MHC II-mediated antigen presentation (Mircheff et al., 1991; Mircheff et al.,
1994; Wood et al., 1997). The inflamed LGACs increasingly secrete inflammatory
cytokines to the apical and basal sites of LGACs to activate T-cells. The majority of these
T-cells are autoreactive CD4
+
T-cells that are surrounded by B-cells (Bradley, 2008).
Activated T-lymphocytes then secrete more pro-inflammatory cytokines (e.g. IL-1, IL-6,
TNF- α, etc), which exacerbates inflammation in the LG (Gao et al., 1998; Meggs, 1993;
Mircheff et al., 1998). Tumor necrosis factor (TNF) was originally identified as a

6

circulating factor causing necrosis of tumors, but it recently has been identified as a key
regulator in the inflammatory development (Bradley, 2008). Moreover, the inflammatory
cytokines and proteases secreted from LGACs drain to the ocular surface, where they
promote the inflammation including T-cell infiltration, activation of conjunctival
epithelium to express adhesion molecules, and degradation of extracellular matrix
(Afonso et al., 1999; Baudouin et al., 1997; Schenke-Layland et al., 2010; Sobrin et al.,
2000; Tishler et al., 1998).
1.4 Elastin-like polypeptide
Elastin-like polypeptides (ELPs) are a class of thermally sensitive biopolymers
inspired by human tropoelastin, a major protein widespread in the skin and tissues that
require elasticity such as lungs, bladder, arteries, cartilage, and elastic ligaments (Urry et
al., 2002). ELPs are composed of many repeats of a short motif that recapitulates some
physicochemical properties of tropoelastin. ELP undergo a pseudo-first order phase
transition, to a secondary aqueous phase above a lower critical solution temperature
(LCST). Such temperature-dependent phase separation is accompanied by
conformational change from single chained β-sheets and random coils to insoluble type II
β-turn spirals (Yamaoka et al., 2003). ELPs are composed of a repeating pentapeptide
sequence Val-Pro-Gly-Xaa-Gly, where the identity of the guest residue, Xaa, and n
determines the peptide’s phase behavior. In natural tropoelastin, guest residues are
primarily alanine, isoleucine, phenylalanine, and valine. Changes in guest residues may
endow ELPs with distinct hydrophobicity, temperatures triggering phase separation in
aqueous solutions, etc.

7

Considering their physicochemical properties and potential biocompatibility, ELPs
may have extensive in vitro and in vivo applications as molecular switches, drug delivery
vehicles, wound healing materials (Anderson et al., 2014; Koria et al., 2011; Mastria et
al., 2015; Urry et al., 2010; Wang et al., 2014b), and tissue engineering scaffolds
(Amruthwar and Janorkar, 2013; Zhu and Marchant, 2011), with emerging impact in the
fields of life science and pharmaceutical science (Hassouneh et al., 2012). Recently, our
collaborators discovered that ELPs can be fused with functional proteins and expressed
within mammalian cells; furthermore, their phase separation can deactivate target
molecular pathways, such as clathrin-mediated endocytosis (Pastuszka et al., 2012;
Pastuszka et al., 2014). As a drug delivery vehicle, MacKay and Hamm-Alvarez et al.
were the first group to decorate an ELP diblock copolymer with a folded protein domain
(Sun et al., 2011). These ELP diblock copolymers form monodisperse nanoparticles with
the diameters 50-100 nm (Janib et al., 2013; Shah et al., 2013; Sun et al., 2011). By
fusion to functional protein domains, these biopolymers can serve as nanobiological
particles that target lymphoma, breast cancer (Aluri et al., 2014; Shi et al., 2013), SS-
associated dry eye (Shah et al., 2013), age-related macular degeneration (Wang et al.,
2014c), and autoimmune inflammation (Hsueh et al., in preparation).
1.5 Goals and experimental design
In this study, I focused on developing a LGAC targeted nano-drug delivery system
(DDS), which is able to accumulate in endosomal/lysosomal compartments of target cells
where the DDS is degraded and encapsulated therapeutic agents are released. This study
was aimed at developing a molecularly targeted therapy for SS-associated dry eye;
however, to our surprise, one design behaves other than expected, which may impact the

8

development of ocular drug delivery significantly in unexpected ways. Another
formulation not only achieved our initial goals but has potential applications for other
inflammation-mediated diseases. This thesis begins with the construction of two ELP
nanoparticles targeting coxsackievirus and adenovirus receptor (CAR) and intercellular
adhesion molecule-1 (ICAM-1) on the cell surface of LGACs, follows the
characterization of physicochemical properties and intracellular trafficking of these
nanoparticles, and finally explores strategies used to construct multifunctional
nanoparticles by co-assembling ELP with distinct functional moieties.

9

Chapter 2: Tear-mediated delivery of nanoparticles through transcytosis of the
lacrimal gland
2.1. Introduction
Controlled drug delivery to the ocular surface seems intuitively straightforward, yet
remains a major challenge. Many drugs are delivered as topically-added eye drops,
leading to dilution of the added drug due to lacrimation, low contact time caused by rapid
tear turnover, poor penetration through native barriers such as the ocular surface mucin
layer, and rapid drainage of added drug through the nasolacrimal ducts (Kompella et al.,
2010). Studies show that applied drug is washed away within 15-30 sec, while less than
5% of the drug administered through eye drops reaches the target tissue (Ahmed, 2003).
In addition to the natural clearance barriers limiting drug absorption, treatment of acute
ocular surface disorders such as keratitis

(Shukla et al., 2008) as well as chronic diseases
including glaucoma

(Quigley, 2011) may require addition of eye drops up to hourly,
challenging patient compliance (Olthoff et al., 2005). In many cases, ocular diseases such
as scleritis (Galor and Thorne, 2007) and fungal (Thomas and Kaliamurthy, 2013),
bacterial and viral keratitis (Wilhelmus, 2010) must be treated systemically through oral
or intravenous drug delivery to get enough drug to the target area, resulting in significant
drug exposure at non-target sites. To overcome these challenges, various strategies,
including invasive and noninvasive approaches (Cholkar et al., 2013; Weiner and Gilger,
2010), have been developed to increase ocular bioavailability, improve precorneal
residence time, and prolong therapeutic efficacy after topical application. The
noninvasive strategies frequently focus on in situ gelling systems and nanoparticle
technologies. Several mucoadhesive and viscocity enhancing polymers, such as
polyacrylic acid- (Carbopol
®
) and polysaccharide-, including gellan gum (Timoptic XE
®
)

10

and xanthan gum (Timolol Gel Forming Solution
®
), based polymers (Ludwig, 2005),
have been incorporated into ophthalmic formulations now approved by the United States
Food and Drug Administration. In addition, colloidal dosage forms have also been
developed to increase drug stability, overcome drug efflux in conjunctival cells, and
reduce dosing frequency(Cholkar et al., 2013). Invasive strategies include the
developments of eroding and noneroding implants, such as collagen shields (Sawusch et
al., 1988) and pumps, have been reported to continuously deliver to the ocular surface.
While promising, these approaches can compromise vision during treatment. Each
strategy has its own advantages and drawbacks; furthermore, the choice of strategy
depends on the envisaged therapeutic use. Thus, there is a need to explore alternative
ophthalmic drug delivery strategies.
The natural source of tear fluid and proteins is the lacrimal gland (LG), an exocrine
gland composed largely of acinar epithelial cells (LGAC), polarized epithelial cells that
produce and secrete many of the proteins present in tears (Edman et al., 2010). Tear
protein release largely occurs from mucous and serous secretory vesicles sequestered in
the acinar cells which are mobilized upon stimulation by neurotransmitters released by
parasympathetic and sympathetic innervating neurons (Hodges and Dartt, 2003).
Alternatively, some tear proteins are of serum or paracrine origin and are secreted into
tear fluid through a vesicular transport process called transcytosis, which involves
vesicular transport through the acinar cells. There are two major transcytosis pathways,
nonspecific and receptor-mediated. Nonspecific transcytosis mainly applies to abundant
macromolecules in plasma (Simionescu, 1978). Receptor-mediated transcytosis, on the
other hand, is responsible for the uptake and transport of specific protein moieties and

11

their peptide constituents across cellular barriers such as the endothelium or epithelium,
and can be utilized for delivery of receptor-targeted drug molecules (Zhang et al., 2000;
Zhu et al., 2004).
An alternative strategy for delivery to the ocular surface might harness the body’s
own mechanisms in the LG for capturing tear constituents from the blood and releasing
those constituents into tears via transcytosis. To explore this strategy, this thesis describes
genetically engineered elastin-like polypeptides (ELP) targeted to the LG via the
coxsackievirus and adenovirus receptor (CAR). ELPs are composed of the repeated
amino acid sequence (Val-Pro-Gly-Xaa-Gly)n. These biocompatible and biodegradable
(Shah et al., 2012) protein polymers assemble a secondary aqueous phase, known as a
coacervate, above a transition temperature (Tt). This Tt can be precisely tuned by
selection of the hydrophobicity of Xaa and the number of repeats, n, of the pentamer
sequence. When ELPs with different Tts are combined in the same polymer, they can
assemble stable protein nanoparticles at temperatures between the Tt of the two ELPs
(Sun et al., 2011). ELPs can also be fused to targeting proteins that retain their cell-
binding or drug-binding abilities (Chilkoti et al., 2002; Shah et al., 2013; Shi et al., 2013;
Sun et al., 2011). This manuscript explores a specific ELP nanoparticle comprised of the
diblock copolymer, SI, which has 48 serine (Xaa=Ser) pentamers at the amino terminus
and 48 isoleucine (Xaa=Ile) pentamers at the carboxy terminus.
We have previously shown that the LG expresses CAR at one of the highest levels in
the body (Xie et al., 2006). CAR is a cell adhesion protein (Walters et al., 2002) targeted
by the fiber capsid protein of adenovirus serotype 5 (Ad5). Tissues with high surface
expression of CAR, including the LG and the liver, are highly transducible with Ad5,

12

which suggests that under certain conditions CAR mediates internalization (Xie et al.,
2006). Although our group was the first to suggest this entry mechanism in the LG,
endocytosis of CAR is supported by another study (Chung et al., 2005). We and others
have subsequently shown that the affinity of fiber protein for CAR can be replicated by
truncations of its terminal domain, called knob. In a previous cell-culture study using
only liver-derived cells, fusion of the knob domain to SI nanoparticles (KSI) conferred
CAR-mediated internalization (Sun et al., 2011). In contrast, for the first time this study
demonstrates in vivo that KSI nanomaterials can be endocytosed into the LG and,
surprisingly, that a subpopulation of these nanoparticles are efficiently trancytosed into
the lumen.

2.2. Material and Methods
2.2.1. Materials and reagents
Terrific broth dry powder growth medium was from MO BIO Laboratories, Inc.
(Carlsbad, CA). NHS-Rhodamine was from Thermo Fisher Scientific (Rockford, IL).
Sulfo-Cy5 NHS ester was from Lumiprobe Corp. (Hallandale Beach, FL). Copper
Chloride, Isopropyl-beta-D-thiogalactopyranoside, and Polyethylenimine were from
Sigma-Aldrich (St. Louis, MO). The knob domain gene sequence cloned into vector
pUC57 was from Integrated DNA Technologies (Coralville, IA). The pET-25b(+) vector
was from Novagen (Madison, WI). LysoTracker® Red DND-99, fluorescein 10,000 MW
dextran (anionic), and CellLight® RFP-Rab5a BacMam2.0 reagent were from Life
Technologies (Grand Island, NY). The QIAprep Spin Miniprep Kit and QIAquick Gel

13

Extraction Kit were from Qiagen (Valencia, CA). Matrigel
TM
was from Collaborative
Biochemicals (Bedford, MA). Doxycycline was from Clontech (Mountain View, CA).
35 mm glass-bottomed culture dishes were from MatTek Corp. (Ashland, MA). 4-20%
PAGEr Precast Gels were from Lonza (Rockland, ME). Tissue-Tek
®
O.C.T™ Compound
was from Sakura Finetek USA (Torrance, CA).
2.2.2. Biosynthesis and characterization of ELPs
Recombinant plasmids encoding the ELP diblock copolymers, SI and KSI, were
synthesized using plasmid recursive directional ligation (McDaniel et al., 2010; Sun et al.,
2011). The KSI protein polymer consists of a N-terminal 22
nd
β-repeat of the Ad5 fiber
shaft (15 amino acids) which is necessary for structural folding (Green et al., 1983), the
full-length Ad5 knob domain (GenBank Number: AB361382.1), a thrombin cleavage site
(Gly-Leu-Val-Pro-Arg-Gly-Ser), and a C-terminal (Val-Pro-Gly-Ser-Gly)48(Val-Pro-
Gly-Ile-Gly)48Y (SI), in order. ELP gene construction was carried out in a pET25b(+)
vector in TOP 10 competent cells followed by protein expression in the BLR (DE3) E.
coli strain. E. coli encoding SI was amplified as reported previously (Sun et al., 2011). E.
coli expressing KSI was first grown in 5 ml terrific broth medium supplemented with
ampicillin (100 µg/ml) at 37 °C at 250 rpm overnight. 0.5 ml of overnight culture was
then incubated in 1 L of terrific broth containing ampicillin. Isopropyl-beta-D-
thiogalactopyranoside induction was initiated when the optical density (OD 600 nm)
reached 0.5. KSI expression was induced by isopropyl-beta-D-thiogalactopyranoside (0.5
- 1 mM) at 25
o
C for 6 hrs. ELPs were purified using inverse transition cycling
(Hassouneh et al., 2010). In general, at least five rounds of cycling were needed to obtain

14

pure ELP samples. The purity of ELP protein polymers was assessed by SDS-PAGE
using 4–20% gradient gels stained with a 10% (w/v) CuCl2 staining solution.
2.2.3. Dynamic light scattering and zeta potential measurement of ELPs
Hydrodynamic diameters and polydispersity for each construct were measured using
dynamic light scattering (DLS). Samples were prepared at 25 μM in PBS and filtered
through a Whatman Anotop filter with a 0.02 μm pore size at 4 °C. 90 μl of each sample
was transferred to a pre-chilled 384 well microplate, centrifuged at 4 °C to remove air
bubbles, and covered with 20 μl of mineral oil to prevent evaporation. Samples were then
measured by a Wyatt DynaPro plate reader (Santa Barbara, CA) over a range of
temperature from 10 °C to 37 °C in 1 °C increments. Surface charge (zeta potential) of
ELP protein polymers was determined on a Zetasizer (Malvern, Worcestershire, UK).
Similarly, samples were prepared at 25 μM in PBS and passed through a 0.02 μm filter at
4 °C. 1 ml of each sample was applied to the disposable measurement cell. Zeta potential
was estimated from the electrophoretic mobility across an applied electric current at
temperatures above and below the assembly temperature for the SI and KSI nanoparticles
(Table 1).
2.2.4. Fluorescent labeling of ELPs
ELP samples were labeled with rhodamine (Rh) or sulfo-Cy5 using N-
hydroxysuccinimide chemistry. For Rh and Cy5 conjugation of ELPs, reactions were
performed in 0.1 M sodium bicarbonate solution (pH 8.3-8.5) at 4 °C for 3 hrs for KSI or
overnight for SI, and the conjugated ELPs were separated by size exclusion
chromatography on a PD10 desalting column (GE Healthcare, Piscataway, NJ).

15

2.2.5. Animals and Animal Procedures
Female New Zealand White rabbits weighing between 1.8 and 2.2 kg were obtained
from Irish Farms (Norco, CA). Male BALB/c mice aged 12-14 weeks were purchased
from Charles River Laboratories (Hollister, CA). All animal procedures were approved
by the University of Southern California Institutional Animal Care and Use Committee
and followed the Guide for the Care and Use of Laboratory Animals (NIH publication No.
85-23, Revised 1996). For intra-lacrimal injection of SI and KSI and evaluation of
fluorescence distribution by confocal fluorescence microscopy, male BALB/c mice were
anesthetized with an i.p injection of xylazine 8 mg/kg and ketamine 60 mg/kg. The LG
was exposed by a small incision along the axis between the lateral canthus of the eye and
the ear. 5 µl of 50 µM Rh-labeled SI or KSI and 50 µg of fluorescein 10k dextran were
injected directly into the LG using a NanoFil syringe with a 33 gauge needle (World
Precision Instruments, (Sarasota, FL). For analysis of fluorescence distribution by
confocal fluorescence microscopy, the injected LGs were removed after 1 hr, placed in
Tissue-Tek
®
O.C.T™ compound , snap frozen in liquid nitrogen, cut into 10-µm-thick
sections, mounted to slides, and stored at -80
o
C. For intra-lacrimal injection of SI and
KSI and evaluation of nanoparticle distribution by transmission electron microscopy
(TEM), male BALB/c mice were similarly anesthetized and injected with 5 µl of 50 µM
Rh-KSI, 50 µM Rh-SI, or 5 µl of free rhodamine dye in PBS. The LG were removed and
processed for TEM as described below.

16

2.2.6. Acinar cell isolation and primary culture
Primary acinar cells were collected from LG from female New Zealand white rabbits
using previously established protocols (Gierow et al., 1996). The isolated LGACs were
plated on 35 mm glass-bottomed dishes, coated with Matrigel
TM
diluted 1:50 in
Dulbecco’s PBS, at a density of 6.0x10
7
cell/dish and cultured for 2-3 days in Peter’s
complete medium (PCM) before analysis. Rabbit LGACs prepared in this way
reconstitute to form acinus-like structures with distinct basal-lateral and apical domains, a
defined actin network enriched beneath the apical plasma membrane (AM) and produce
mature secretory vesicles located in the subapical region beneath the lumena (Hamm-
Alvarez et al., 1997).
2.2.7. Adenovirus and baculovirus transduction and real-time fluorescence imaging
Adenoviral (Ad) constructs used in this study includes genes that are constitutively
expressed (e.g. Ad mCherry-myosin Vb tail DN and Ad YFP-Rab27b) and others which
are inducible upon addition of doxycycline (e.g. Ad GFP-actin and Ad mCherry-Rab3D).
The inducible Ad constructs required co-transduction with the Tet-on Ad helper virus and
addition of doxycycline to express regulatory proteins recognizing the reverse Tet
repressor and allowing expression of the gene of interest. Transduction of LGACs with
Ad constructs was done on the second day of culture. All Ad constructs were used at a
multiplicity of infection of 5 and analyzed 16-18 hrs after transfection. To study the
internalization of KSI, reconstituted rabbit LGACs co-transduced with Ad GFP-actin and
Ad Tet-On at 37 °C were utilized to highlight the basolateral membrane (BLM) and
apical membrane (AM) regions, as described previously (Jerdeva et al., 2005). To inhibit

17

the transcytosis of KSI, Ad mCherry-myosin Vb tail DN was utilized, a truncated mutant
of myosin Vb N’-terminally fused with a mCherry fluorescent protein tag (Xu et al.,
2011). Other constructs were also utilized to label specific intracellular trafficking
pathways. To label early endosomes, LGACs were incubated with Cell Light RFP-Rab5a
BacMam 2.0, a modified baculovirus expressing a fusion construct of the early endosome
marker, Rab5a, and red fluorescent protein (RFP), at a final concentration of 30 particles
per cell on day 2 of culture followed by 16 to 18 hrs incubation at 37 °C. The transduced
cells could be identified visually by the expressed vesicular red fluorescence. For
intracellular trafficking studies, these transduced LGACs were incubated with 30 µM of
Cy5-KSI at 37 °C for 60 min before imaging or, alternatively, pulsed with 30 µM of Cy5-
KSI at 37 °C for 10 min. The Cy-KSI was then removed and LGACs were chased for 45
min with simultaneous imaging by confocal fluorescence microscopy utilizing a Zeiss
LSM 510 Meta NLO imaging system (Thornwood, NY) equipped with Argon, red HeNe,
and green HeNe laser, and a Coherent Chameleon Ti-Sapphire laser mounted on a
vibration-free table.
2.2.8. Quantification of fluorescence signal
For evaluation of the percentage of fluorescence recovered within the cellular area
versus the lumenal region, the lumenal and cytosolic areas within each LGAC cluster
were selected by defining the regions of interest. The fluorescence intensities within these
regions were analyzed using ImageJ v1.43u (US National Institutes of Health, Bethesda,
MD); fluorescence intensity within each region of interest was determined by calculating
the integrated fluorescence intensity corrected for background. The ratio of fluorescence
intensity in the lumen to that in the cytosol was calculated. For optimal resolution, each

18

fluorescent image was converted from an RGB to an 8-bit grayscale image before
analysis. The fluorescent intensities, presented in the X-axis of Figure 4, 7 and 9 are
expressed as pixels/area.
2.2.9. Transmission electron microscopy
The morphology of the SI and KSI nanoparticles was observed by cryogenic
transmission electron microscopy (cryo-TEM). ELP solutions were kept in an ice bath (4
°C) before processing and then raised to 37 °C immediately prior to cryo-TEM sample
preparation using an FEI Vitrobot. A typical procedure involves several steps as
described below. In brief, ~6 μL of the sample solution was first loaded on a TEM copper
grid coated with a lacey carbon film, and the grid was placed in the Vitrobot chamber
with controlled temperature and humidity. After blotting of the excess solution using
preset Vitrobot parameters, the grid containing a thin solution layer (~less than 300 nm)
was plunged into a liquid ethane reservoir that was cooled and surrounded by liquid
nitrogen. After approximately 30 s, the sample was carefully transferred to a liquid
nitrogen Dewar and stored in liquid nitrogen temperature before imaging. Throughout the
imaging process, the cryo-TEM samples were kept at a temperature below -170
o
C. For
analysis of ELPs in vivo by transmission electron microscopy (TEM) the LG in male
BALB/c mice was injected with Rh-KSI, Rh-SI, or free rhodamine dye as described
above. The LG were removed and fixed in half-strength Karnovsky’s fixative solution at
4 °C overnight. After fixation, the samples were carefully minced into 1-mm
3
pieces,
rinsed three times in 0.1M cacodylate buffer, postfixed in 2% osmium tetroxide on ice for
2 h, and stained en bloc with 1% uranyl acetate overnight. The samples were dehydrated
in serially graded ethanol and infiltrated in eponate resin prior to embedding. The

19

sections were cut at a thickness of 75 nm, placed on copper grids and examined at 100
kV. To compare the size of two types of nanoparticles obtained from cryo-TEM and
TEM, SI and KSI were randomly selected and quantified with ImageJ.
2.2.10. Statistics
Values are presented as mean ± SD. Data from different experiments with only two
groups were analyzed using an unpaired two-tailed student’s t-test (GraphPad Prism
5.0.1). Experiments with four groups were compared with a global ANOVA followed by
the Tukey post-hoc test. To satisfy the homogeneity of variance assumption, the raw
intensity values were transformed by the Log10 function prior to ANOVA. The criterion
for statistical significance was p ≤ 0.05.

2.3. Results and Discussion
2.3.1. Characterization of ELP diblock copolymer nanoparticles
The immediate goal of this study was to explore the internalization pathways of ELP
nanoparticles displaying the Ad5 fiber knob domain in LGACs. SI and KSI are diblock
copolymer ELPs, consisting of the N-terminal hydrophilic (Val-Pro-Gly-Ser-Gly)48
moieties and C-terminal hydrophobic (Val-Pro-Gly-Ile-Gly)48 moieties. Above a critical
micelle temperature, both constructs assemble monodisperse nanoparticles (PDI < 0.1)
with slightly negative zeta potentials (-5.9 to -7.9 mV) (Table 1, Figure 2A). At neutral
pH the amino and carboxy termini of SI carry positive and negative charges respectively;
however, nanoparticle assembly has no effect on zeta potential. As SI lacks charged

20

amino acids, this suggests that SI nanoparticles are stabilized by steric repulsion provided
by the hydrophilic ELP, and not by electrostatic repulsion. With the addition of the
adenoviral knob protein, KSI showed a slightly negative shift in zeta potential, which
suggests it may be stabilized by a combination of steric and electrostatic forces. Since
cell surfaces are negatively charged, the stabilization of KSI may help prevent
nonspecific electrostatic adsorption and promote CAR-mediated specificity. Then, SDS-
PAGE was used to characterize the molecular weights of ELP constructs (Figure 2B)

21

Figure 2. Schematic representation of KSI nanoparticles and their phase transition
behavior. (A) Schematic representations summarizing physical properties of ELPs used
in this study. SI and KSI exist as soluble monomeric peptides below the critical micelle
temperature, as protein nanoparticles above the first transition temperature but below the
second transition temperature, and as insoluble coacervates above the second transition
temperature. (B) Copper-stained SDS-PAGE gel of SI and KSI, showing MW of 39.6
and 61.4 kDa, respectively.

22

revealing a molecular mass of ~38 kDa for SI and ~60 kDa for KSI, consistent with the
theoretical molecular masses reported in Supplementary table 1. The image was analyzed
using ImageJ to reveal the purity as 98.5% for SI and 94.5% for KSI.
Table 1. Elastin-like polypeptides evaluated in Chapter 2.
Label Amino acid sequence
a
Tt1
b
(°C)
Tt2
c
(°C)
Molecular
Weight
d
(kDa)
Hydrodynamic
diameter
e
(nm)
SI G(VPGSG)48(VPGIG)48Y 26.8 76.0 39.6 47.1±1.8
KSI GAITVGNKNNDKLTLWTTPAPSPNCRLNAEKDAKLTLVLTKCG
SQILATVSVLAVKGSLAPISGTVQSAHLIIRFDENGVLLNNSFLDP
EYWNFRNGDLTEGTAYTNAVGFMPNLSAYPKSHGKTAKSNIVS
QVYLNGDKTKPVTLTITLNGTQETGDTTPSAYSMSFSWDWSGH
NYINEIFATSSYTFSYIAQEGLVPRGSG(VPGSG)48(VPGIG)48Y
19.9 64.8 61.4 43.2±0.6
a) Ad5 Knob amino acid sequence; Underlined: thrombin cleavage site
b) Critical micelle temperature determined using optical density (25 µM, pH 7.4).
c) Bulk phase temperature determined using optical density (25 µM, pH 7.4).
d) Expected molecular weight estimated from the open reading frame.
e) Particle sizes between Tt1 and Tt2 (25 µM, pH 7.4) were measured by DLS (Sun et al.,
2011),

presented as mean ± SD (N=10), and confirmed by cryo-TEM (Figure 2).

2.3.2. Dimensions and size homogeneity of ELP nanoparticles with and without the
knob domain
We next determined the size homogeneity and morphology of KSI compared to that
of its SI counterpart. The hydrodynamic diameters of nanoparticles assembled by SI and
KSI, determined by dynamic light scattering (DLS), were 47.1 ± 1.8 nm and 43.2 ± 0.6
nm, respectively (p < 0.0001) (Table 1). In prior reports, the addition of the Ad5 knob
domain reduced the critical micelle temperature slightly; however, the hydrodynamic
diameter and stability at physiological temperatures was nearly unaffected by the fusion
of Ad 5 knob domain (Sun et al., 2011). Despite the increased molecular weight for
monomers of KSI compared to SI, the resulting nanoparticles have nearly identical sizes.
This suggests that KSI nanoparticles stabilize at a lower number of polymers per particle

23

compared to SI. If so, this might result from a larger hydrophilic fraction for KSI (66% vs.
48% for SI), which could result in a larger radius of curvature per polymer (Alexandridis
and Hatton, 1995; Hurter et al., 1993).

Figure 3. Genetic fusion of the Ad5 knob domain to SI has minimal impact on the
morphology of ELP nanoparticles. (A) Cryo-TEM micrographs of ELP nanoparticles
with and without the Ad5 knob domain. (B) A comparison of diameters from ELP
nanoparticles imaged by cryo-TEM and DLS. For cryo-TEM, the average diameters of SI
and KSI, measured with ImageJ, were 32.4 ± 4.2 nm and 32.0 ± 3.5 nm, respectively.
Values are expressed as mean ± SD (n=15). For DLS, the average diameters of SI and
KSI were 47.1 ± 1.8 nm and 43.2 ± 0.6 nm, respectively. Values are expressed as mean ±
SD (n=10). p value < 0.0001 (Student t-test). Statistical comparison was not performed
between cryo-TEM and DLS techniques because they measure different aspects of
particle formation.

24

To explore any confounding difference between SI and KSI nanoparticles, cryo-
TEM was employed (Figure 3A). For this analysis, particles suspended in PBS were
shock-frozen in liquid ethane. The suspension was supercooled to form a vitrified thin
layer so that the particles could be directly studied in situ. Figure 3A shows that SI and
KSI form nearly spherical particles with similar size homogeneity. Particle sizes of SI
and KSI, measured by DLS and cryo-TEM, are presented in Figure 3B. The diameter of
the KSI nanoparticles (32.0 ± 3.5 nm) from cryo-TEM was similar to that of the SI
nanoparticles (32.4 ± 4.2 nm) and there is no statistically significant difference between
the two constructs (Figure 3B). The particle sizes determined by cryo-TEM are slightly
smaller than the sizes reported from DLS, a difference possibly resulting from the
presence of the hydration layer on the surface of core-shell ELP nanoparticles. This shell
can be detected by DLS but is not visible by cryo-TEM. A similar phenomenon is
observed for soft colloidal nanoparticles but not hard shell particles (Cui et al., 2007).
Moreover, the hydrodynamic diameter from DLS measures an average that is also
influenced by the slight irregularity in the shape of the particles. Despite the slight
differences in size, morphologies of both KSI and SI from cryo-TEM are generally
consistent with the results from DLS. These observations suggest that fusion of Ad5 knob
domain to the SI core nanoparticle minimally influences the nanoparticle diameter and
morphology and that differences in their cellular trafficking result from receptor-
mediated interactions.
2.3.3. Internalization and transcytosis of ELP fusions in LG acini
KSI exhibits a fiber knob-dependent and CAR-mediated endocytosis in transformed
mouse hepatocytes; however, prior to this report it was unknown if these particles would

25

interact with the LG. Similarly to hepatocytes, cells of the LG abundantly express CAR;
therefore, LG CAR could be an excellent target for the selective delivery of KSI. To
explore this hypothesis, three-dimensional cultures obtained from rabbit LGs were used
to investigate the targeting and internalization of KSI. As documented in previous studies,
isolated primary LGACs assemble into ovoid clusters after two days in culture,
mimicking the acinar-like structures present in the LG (Evans et al., 2008). A schematic
of a typical reconstituted acinus comprised of LGACs is shown in Figure 4A (right) with
several reference points, including a central lumen bounded by the apical membrane (AM)
of adjacent cells, mature secretory vesicles (SVs) in the sub-apical region, beneath the
basolateral membrane (BLM).

26

Figure 4. KSI is internalized and accumulates in the cytosol and lumena of rabbit LGAC
expressing GFP-actin. (A) Live cell imaging comparing the intracellular distribution of
ELPs with and without Ad5 knob after 1 hr incubation at 37 °C. Green, GFP-actin; Red,
Rh-conjugated SI or KSI; *, lumenal space; Scale bar indicates 10 µm. Schematic
diagram on the top right depicts reconstituted rabbit LGACs shown on the first row
(Control), and indicates the presence of apical plasma membrane (AM), secretory
vesicles (SV) and the basolateral membrane (BLM). *, acinar lumen. (B) Quantification
of fluorescent intensity in LGACs treated with SI and KSI. ***, p value<0.005 (ANOVA
followed by Tukey post-hoc test). (C) Quantification of the fluorescence intensity
expressed as a ratio of lumenal to cytosolic fluorescence. **, p value=0.009 (Student T-
test). For (B) (C), fluorescence intensity was analyzed using ImageJ. Data are presented
as mean ± SD (n=5).

As shown in Figure 4A, fluorescently labeled Rh-SI or Rh-KSI were incubated with
LGACs transduced with Ad GFP-actin. GFP-actin was used to delineate the apical lumen
of reconstituted LG clusters due to its incorporation into the dense sub-apical meshwork
of actin filaments proximal to the lumen. In addition to detection of intracellular puncta,

27

presumed to be membrane compartments, Rh-KSI was clearly observed within the
lumenal area of LG clusters, compared with control Rh-SI. This suggests an active
transport mechanism moves KSI from the basolateral to the apical membrane in these
polarized LGAC cultures. The confocal fluorescence images of LGAC clusters were
quantified by ImageJ to compare average fluorescent intensities in cytosol and lumen
(Figure 4B) and estimate the ratio of nanoparticles between the lumen to cytosol (Figure
4C). The fluorescence intensity in the lumen of acini exposed to KSI was much higher
than that in cytosol, while for acini exposed to SI, the fluorescence intensity in the lumen
was lower than that in the cytosol (p=0.001). When the lumen intensity was normalized
by the cytosolic intensity, the ratio for KSI was significantly (p = 0.009) higher than for
the SI control. This finding suggests that the addition of the knob domain to SI enhances
the basolateral- to-apical transcytosis of the ELP nanoparticles, which possibly paves a
way to utilize the KSI nanoparticle to selectively deliver therapeutic agents to lacrimal
epithelial cells, tear ducts, and the ocular surface.
2.3.4. Characterization of KSI intracellular trafficking in LGACs
Previous reports suggested that the Ad5 knob domain administered through
intravenous injection is internalized in the liver and sorted into acidic compartments of
hepatocytes for degradation (Awasthi et al., 2004). We hypothesized that the uptake of
KSI in rabbit LGACs should involve the classical endosomal pathway and further that
KSI remaining in acini that did not appear to pass to the lumen would be recovered in
acidic lysosomal compartments. To evaluate this, Rh-SI or Rh-KSI was incubated with
rabbit LGACs at 37 °C for 60 min before confocal fluorescence microscopy imaging
(Figure 5). LysoTracker green was used as an acidic compartment marker for late

28

endosomes and lysosomes in LGACs. As can be seen in Figure 5, LGACs without any
ELP treatment showed no fluorescence signal (absence of red label). LGACs incubated
with Rh-SI showed significant surface association as well as enhanced labeling of single
cells present in the preparation. Some internalization was seen in puncta that colocalized
with LysoTracker-labeled compartments (arrowheads). In contrast, almost no surface
labeling of acini was seen for Rh-KSI which instead mainly internalized to puncta
partially co-localized with LysoTracker green; furthermore, Rh-KSI again showed
significant lumenal accumulation. Based on these findings, we propose that the
internalized SI and KSI are partially transported to late endosomes and lysosomes, but
that KSI internalization occurs more efficiently and is further transported via transcytosis.

29

Figure 5. ELP nanoparticles traffic to low pH compartments in rabbit LGACs. 30 µM of
Rh-coupled SI or KSI were incubated with rabbit LGACs at 37 °C for 1 hr, and imaged
using confocal fluorescence microscopy. KSI (red) exhibited significant co-localization
with acidic compartments labelled by LysoTracker green (green) as well as lumenal (*)
accumulation. SI showed significant surface binding and also some internalization to
acidic compartments labelled by LysoTracker green. Arrowheads indicate the co-
localization of SI or KSI with acidic compartments. White lines delineate the BLM of LG
acinar clusters. *, lumena. Scale bar indicates 10 µm.

Early endosomes are well-established recipients of endocytosed material in many
cell types (Rizzoli et al., 2006). In epithelial cells internalized cargo is first trafficked into
basolateral early endosomes (BEE). BEE can either be transported to common recycling
endosomes and apical recycling endosomes consecutively before the release of cargo into
the apical lumen, or alternatively they may be delivered to late endosomes and then
lysosomes (Carvajal-Gonzalez et al., 2012). To investigate whether internalized KSI
traffics to BEE in the LG, reconstituted LGACs, transduced with the BEE marker RFP-

30

Rab5a were incubated with fluorescence labeled Cy5-KSI and analyzed by time-lapse
confocal fluorescence microscopy (Figure 6). Similarly to Rh-KSI, Cy5-KSI also
trafficked to the lumen of LGACs, indicating that this effect is dye independent. Figure
6A shows that the Cy5-KSI is enclosed by Rab5a-enriched vesicles by 60 min, indicative
of the delivery of internalized KSI into Rab5a-enriched BEEs. As seen in Figure 6B, a
pulse-chase experiment with labeled KSI over a time course of 45 min of incubation
showed the internalization of KSI to BEEs was observed by 10 min. In particular, the
internalized KSI underwent a dynamic continuous exchange and redistribution from
smaller BEEs to larger BEEs, possibly through homotypic fusion of early endosomes. A
loss of fluorescence signal, possibly due to photobleaching or transfer of some KSI from
early endosomes to proximal trafficking compartments was observed at later time points.
Combined with data in Figure 5, these results suggest that KSI internalized by receptor-
mediated endocytosis is first sorted into Rab5a-enriched BEEs, and then a fraction of this
endocytosed material is delivered to late endosomes and lysosomes.

31

Figure 6. Intracellular trafficking of KSI nanoparticles in LGACs. (A) Internalization of
KSI to early endosomes in live rabbit LGACs expressing RFP-Rab5a, a marker of early
endosomes. LGACs transduced with RFP-Rab5a (green) were treated with 30 µM of
Cy5-KSI (red) at 37 °C for 1 hr, washed with DPBS twice, and analyzed by confocal
fluorescence microscopy. Arrowheads indicate Cy5-KSI in early endosomes. (B) LGACs
transduced with RFP-Rab5a (green) were pulsed with 30 µM of Cy5-KSI (red) at 37 °C
for 10 min, rinsed and followed by a 45 min chase in fresh PCM at 37 °C. The yellow-
boxed region is expanded in time-lapse images shown successively to the right.
Arrowheads indicate early endosomes that include Cy5-KSI at earlier times but show
label dissipating at later times of incubation. White lines depict the periphery of the
reconstituted cluster of LGAC obtained by phase contrast imaging. (C) LGACs
expressing mCherry-Rab3D (red) or YFP-Rab27b (red) were pulsed with 30 µM of Cy5-
KSI (green) at 37 °C for 10 min and imaged for 45 min in fresh PCM at 37 °C.
Internalized KSI did not associate with either Rab3D or Rab27b in live rabbit LGACs.
Data were presented at the indicated time points. White lines depict the periphery of the
reconstituted cluster of LGACs. Arrowheads indicate internalized Cy5-KSI. Scale bars
indicate 5 µm.

32

Having identified the primary routing steps for endocytosed KSI in LGACs, we next
explored the subsequent trafficking steps involved in release of KSI into the apical lumen.
Previous studies from our laboratory have identified at least three pathways for apically
directed secretion of tear proteins in LGACs: the Rab11a/MyosinVb mediated
transcytotic pathway (Xu et al., 2011) and the Rab3D and/or Rab27b regulated secretory
pathways (Chiang et al., 2012; Chiang et al., 2011; Xu et al., 2013). Rab3D and Rab27b
are predominantly localized to distinct populations of secretory vesicles in the subapical
region of LGAC that originate from the trans-Golgi network, although a subpopulation
expressing both Rab proteins may exist. To investigate whether the Rab3D and Rab27b
regulated secretory pathways are involved in the release of KSI into the acinar lumen of
LG, LGACs were transduced with either YFP-Rab27b or mCherry-Rab3D followed by
imaging of cells subjected to pulse chase uptake experiments. Figure 6C shows that the
internalized Cy5-KSI (red) does not co-localize with YFP-Rab27b or mCherry-Rab3D
(green), indicating that the secretion of KSI is not mediated through secretory vesicles
enriched in Rab3D or 27b.

33

Figure 7. Overexpression of a dominant negative Myosin Vb tail impairs basolateral-to-
apical transcytosis of KSI in LGACs. (A) Reconstituted rabbit LGACs, co-transduced
with AdGFP-actin (green) to delineate morphology, Ad mCherry Myosin Vb tail DN
(red), and Ad helper virus, were incubated with 30 µM of Cy5-KSI nanoparticles (purple)
at 37 °C for 1 hr before analysis. *, lumenal region of LGACs. Scale bar indicates 10 µm.
(B) Quantification of fluorescence intensity in lumen and cytosol of LGACs with or
without the dominant negative myosin Vb tail, which inhibits transcytosis. ***, p
value<0.005 (ANOVA followed by Tukey post-hoc test). Data were expressed as Mean ±
SD (n=4). (C) Quantification of fluorescence intensity as a ratio of lumenal to cytosolic
fluorescence. Data were expressed as Mean ± SD (n=4). KSI, 0.83± 0.48; KSI with
inhibition, 0.13 ± 0.09; p value = 0.031 (Student T-test).

We next evaluated the role of the Rab11a/MyosinVb regulated transcytotic pathway.
We have shown that expression of dominant-negative (DN) myosin-Vb tail directly
impairs the movement of transcytotic vesicles and their cargo from Rab11a-enriched
apical endosomes to the apical membrane, thus inhibiting transcytosis in LGACs (Xu et
al., 2011). The Rab11a-enriched apical endosome is well established in the trafficking of
another transcytotic cargo receptor, the polymeric immunoglobulin A receptor,
responsible for transcytosis of dimeric IgA into tears (Xu et al., 2013). In Figure 7,

34

LGACs were transduced with recombinant adenovirus encoding GFP-actin (green) with
or without mCherry-myosin Vb tail DN (red), an N-terminal mCherry fluorescent protein
fused to a truncated mutant of myosin Vb motor, which associates with vesicle cargo but
does not have motor function. Transduced acini were then incubated with Cy5-KSI. As
can be seen in Figure 7A, the Cy5 fluorescent signal (purple) recovered within the
lumenal regions surrounded by apical GFP-actin was reduced in LGACs co-transduced
with Ad mCherry-myosin Vb tail DN compared to that with Ad GFP-actin alone. The
fluorescent KSI signal was quantified using ImageJ in the cytosol and lumena (Figure 7B)
and the ratio of lumenal to cytosolic fluorescence was calculated (Figure 7C). Although
the dual transduction of adenovirus slightly reduced the level of KSI internalization
compared to single transduction (Figure 7B), there is a statistically significant (p = 0.031)
reduction of the KSI signal calculated from the ratio of lumen to cytosol (Figure 7C). In
LGAC in which transcytotic trafficking has been selectively impaired, Cy5-KSI
accumulation in the lumenal region is inhibited, demonstrating that its transport to the
lumen is via the transcytotic pathway.

35

2.3.5. Characterization of KSI intracellular trafficking in vivo
To further evaluate the potential of KSI as a targeted vehicle in vivo, our next step
was to confirm the internalization and transcytotic behavior of KSI in a mouse model.
First, we confirmed the expression and biodistribution of CAR in the LG of BALB/c
mice by immunofluorescence, which revealed primarily basolateral enrichment with
traces at the apical membrane (Figure 8).

Figure 8. Distribution of CAR in LG from BALB/c mice. Cryosections of LG from 12
week BALB/c male mice were fixed, permeabilized, and labeled without (untreated) or
with goat anti-mouse CAR polyclonal primary antibody (R&D systems, Minneapolis,
MN), before addition of FITC-conjugated donkey anti-goat secondary antibody (green).
F-actin was labeled with rhodamine phalloidin (red) and nuclei were labeled with DAPI
(blue). Scale bar indicates 10 µm.

Rh-SI or Rh-KSI, mixed with fluorescein 10k dextran as a marker of fluid phase
uptake, was administered using intra-lacrimal injection. 1 hr after injection the LG was
retrieved and analyzed (Figure 9A). Although fluorescein 10k dextran is clearly present

36

in the LG around the acinar cluster, LGs injected with Rh-SI displayed weak to no
fluorescent signal in cytoplasm or lumena of LGAC, indicative of little uptake of SI in
vivo (Figure 9A left). However, in the mice injected with Rh-KSI, KSI puncta appear
inside the LGAC where fluorescein 10k dextran was recovered, suggesting that KSI was

Figure 9. Comparison of in vivo ELP nanoparticle accumulation in LG from BALB/c
mice. (A) Intra-lacrimal gland injection of Rh-coupled KSI (red) into LG of 12 week
BALB/c mice showed significant internalization, LG retention, and lumenal
accumulation compared to SI (red). Fluorescein 10k dextran (green) was used as a control
and fluid-phase marker. White arrowheads indicate internalized KSI close to or in the
apical/lumenal region of mouse LGACs. Scale bar indicates 10 µm. (B) Quantification of
fluorescence intensity for internalized SI and KSI. The fluorescent signals were analyzed
in LG acinar clusters and analyzed using ImageJ. ****, p value< 0.0001 (Student T-test).
Bar are expressed as mean ± SD (n=5). (C) Quantification of fluorescence intensity in the
subapical membrane and lumenal region of clusters of mouse LGAC. Data were
expressed as mean ± SD (n=5). *, p value= 0.01 (Student T-test).

37

internalized. Additionally, the KSI signal was also detected in the apical region of the
LGACs, suggesting that the basolateral to apical transcytosis found in vitro occurs in vivo
as well. The images in Figure 9A were quantified by ImageJ in terms of the fluorescent
signals in each LG whole acinus (Figure 9B) and lumenal region (Figure 9C). By
considering each LG cluster, KSI displayed a 4-fold increased internalization in acini
relative to SI (p < 0.0001) (Figure 9B). For those analyzed LG acini, the fluorescent
signal in the apical region of LGACs was >7-fold higher with KSI relative to SI (p = 0.01)
(Figure 9C). These results suggest that knob enhances the tissue affinity of SI in vivo, and
that internalized KSI can be transcytosed to the apical region of LGACs and released into
the lumen.

38

Figure 10. Transmission electron microscopy confirms KSI transcytosis in LGs from
BALB/c mice. Transmission electron microscopy images compared mouse LGs from 12
week BALB/c mice administered with free rhodamine dye, SI or KSI by intra-lacrimal
injection. Vesicle-enclosed black puncta close to the apical membrane (AM) of mouse
LG acini are KSI nanoparticles. L, lumen; SV, secretory vesicles; M, epithelial microvilli
projecting from the AM of LG acini. Scale bars indicate 1 µm. KSI diameters were
summarized in a box-and-whisker plot. Particle diameters are expressed as mean ± SD. n
= 178 and 205 for SI and KSI, respectively. For the box-and-whisker plot, the box
expresses mean ± SD and the whisker shows minimum and maximum values.

To further confirm the internalization and intracellular trafficking of SI and KSI, we
repeated the intra-lacrimal injections and analyzed the glands by TEM. The images in
Figure 10 show sections from both apical and basolateral regions of mouse LG after
injection with free rhodamine, Rh-SI, and Rh-KSI. The rhodamine label (not shown) was
utilized to identify the area around the needle track. LGs injected with free rhodamine

39

were used as a control group. Apical and basolateral membranes were identified based
upon the relative locations of mitochondria, nuclei, secretory vesicles, and epithelial
microvilli. As shown in Figure 10, no particles were observed in the control group
administered with free rhodamine dye (Figure 10, free dye panels). For LGs injected with
Rh-SI, SI nanoparticles could only be observed in extracellular spaces next to the
basolateral membrane of mouse LG; none of these particles were seen in the apical region
or lumen (Figure 10, SI panels), which is consistent with what we observed in vitro and
with fluorescent labeling in vivo. However, for LGs injected with Rh-KSI, we observed
internalized uniform nanoparticles enclosed in vesicular structures both at the basolateral
and apical membranes of mouse LGs as well as in the lumen (Figure 10, KSI panels),
again confirming the endocytosis and basolateral-to-apical transcytosis of KSI. Some KSI
nanoparticles were also observed in autophagosome-like structures close to the
basolateral membrane. The box-and-whisker plot shows that the diameter of SI
nanoparticles (21.1 ± 4.6 nm) is similar to KSI nanoparticles (20.2 ± 2.8 nm), consistent
with cryo-TEM imaging (Figure 3).
2.3.6. Hypothesized trafficking model of Knob-ELP
Based on the data collected previously (Sun et al., 2011) and in this manuscript, the
following model is proposed for trafficking KSI in LGAC (Figure 11). Endocytosis,
initiated by the binding of recombinant knob on the KSI nanoparticles to the CAR on the
basolateral membrane, is followed by transport to early endosomes enriched in Rab5.
Thereafter, KSI is sorted into vesicles utilizing Myosin-Vb motors for delivery to the
apical membrane and released into the lumena. Prior studies have shown that transcytosis

40

of Rab11a vesicles is Myosin-Vb dependent.(Xu et al., 2011) The remaining KSI is
sorted into late endosomes, autophagosomes and lysosomes.
In this study, we observed a distinct intracellular trafficking pathway of the KSI
nanoparticle in primary cells of the lacrimal gland. Prior studies of transformed
hepatocytes showed that KSI nanoparticles undergo CAR-dependent internalization and
are transported to lysosomes. In contrast, KSI in the LG is transported to the apical
membrane and lumen through an intact transcytotic pathway. The mechanisms behind the
distinct trafficking behavior in these two cell types remain to be investigated. Both
hepatocytes and LGACs are specialized epithelial cells, featuring apical-basal polarity
maintained by tight junctions via protein complexes, and are responsible for vectorial
transport of ions and solutes across the epithelium. CAR has been identified as a
regulator in tight junction permeability for many years, but its biological function and
trafficking mechanism in mammalian cells remain unknown. However it is possible that a

41

Figure 11. Working model for intracellular trafficking of KSI ELPs in LGACs. KSI is
internalized via CAR-mediated endocytosis. KSI is then delivered to Rab5a-early
endosomes where some KSI nanoparticles are transported to late endosomes and
lysosomes, whereas others are sorted to Rab11a-associated sub-apical intracellular
compartments for release to the apical lumen of LG acini. Some KSI remains in
intracellular acidic compartments without entering the transcytotic pathway.

disparity in CAR function in the hepatocytes and LGACs may play a role in the different
trafficking patterns of KSI-CAR complexes between hepatocytes and LGACs. CAR
splice-variants have been identified in many tissues (Chen et al., 2003; Huang et al.,
2007) and each of them may exhibit alternative functions. CAR is expressed as at least
two isoforms containing identical extracellular and transmembrane domains, which
predict identical serotype preference and adenovirus binding, and differ only in the last

42

26 (CAR
ex7
) or 13 (CAR
ex8
) amino acids of the cytoplasmic domain (Excoffon et al.,
2010). CAR
ex7
and CAR
ex8
share a similar class of PDZ-binding domain, but interact with
different PDZ-domain containing proteins, which may trigger distinct signaling and
sorting pathways (Kolawole et al., 2012). For example, CAR
ex8
interaction with PDZ-
domain containing protein MAGI-1b results in CAR
ex8
degradation, while this interaction
cannot be observed in CAR
ex7
.(Kolawole et al., 2012) In addition, the cytoplasmic tail of
CAR contains 9 lysines which may serve as ubiquitylation sites for endocytosis and
trafficking of CAR to lysosomes for degradation. Deletion of this protein sequence may
abolish ubiquitylation, causing resorting of CAR to the cell surface or other intracellular
compartments (Marvin and Wiethoff, 2012).
As model systems, we investigated the intracellular trafficking and transcytosis of
KSI nanoparticles in the rabbit LGAC ex vivo and evaluated the LG retention of KSI
nanoparticles in the mouse LG in vivo. Part of the rational for using two different species
was that the ex vivo system for LGAC production requires large sources of primary
tissue, which can be obtained from rabbits. In contrast, the mouse LG is easily accessible
for direct injection, more so than in a rabbit. While significant anatomical diversity exists
between species, many ocular elements are conserved, which suggest the translational
potential of these findings. For example, rabbits have a blink rate every six min (10 sec
-1
)
(Toshida et al., 2009). Mice and rats share a similar blink rate averaged every five min
each blink (12 sec
-1
), while human has an average blink rate in every five second (0.15-
0.2 sec
-1
) (Tutt et al., 2000). These differences partially result from different eyelid
configuration among species. A faster blink rate reduces the precorneal residence time on
the ocular surface, increases the tear drainage, and alters the drug absorption of a

43

topically applied therapeutics across the cornea in comparison with a slower blink rate
(Urtti and Salminen, 1993). Cornea is a transparent multilayered epithelium and serves as
the primary barrier to drug absorption, especially for hydrophilic drugs. The corneal
thickness varies with age, disease, external influences (e.g. contact lenses), damage, and
species. Recent research has reported that transporters expressed in the corneal
epithelium may involve the transport of some hydrophilic drugs (Mannermaa et al., 2006);
however, corneal transporter-mediated uptake and elimination can vary largely between
species. While parameters such as these clearly suggest major differences between
species in ocular pharmacokinetics, the KSI targets the CAR receptor, which is expressed
constitutively in the LG across rabbits, mice, and humans (Xie et al., 2006). Thus the fact
that KSI trancytoses through the LGAC in two different species, suggests the possibility
that it may also have this ability in the human LG.
Two different fluorescent dyes (rhodamine, Cy5) were evaluated as labels for KSI in
this study. The criteria for fluorescent dye selection were based on two factors: i) the
need to label KSI with either red or far red wavelength emissions compatible with other
double and triple label microscopy studies; and ii) to demonstrate that KSI transcytosis
was conserved across at least two distinct fluorescent probes. Both rhodamine (e.g.
TRAMA) and Cyanine dyes (e.g. Cy5) are small molecule derivatives that link to
primary amino acids. Rhodamine derivatives have high photostability and little
sensitivity in physiological pH range and are suitable for multicolor labeling experiments
(Wessendorf and Brelje, 1992). In comparison, sulfo-Cy5 shows comparable
photostability, more intense emission, and increased water solubility. Therefore, sulfo-
Cy5 is well suited for the labeling of proteins that easily denature in the presence of

44

organic co-solvents sometimes used for labeling reactions. Rhodamine is excited by
green light; however, Cy5 derivatives are excited by red light (650nm) and emit in the
far-red region (680nm). Cy5 is well suited for triple label live-cell microscopy
experiments alongside probes (RFP-Rab5a, mCherry Rab3D, YFP-Rab27b, mCherry
Myosin Vb tail) with emissions overlapping that of rhodamine. As KSI conjugated with
either rhodamine or sulfo-Cy5 displayed comparable internalization and lumenal
accumulation in rabbit LGACs, this further supports the contention that KSI mediates
transport across the polarized cells of the LG.
The fiber-knob protein used in this chapter is from the human adenovirus serotype 5
(Ad5). Human adenoviruses, second to lentiviruses, are widely used in gene based
therapies, with applications ranging from cancer treatments to vaccinations (Hendrickx et
al., 2014). Adenoviruses are medium-sized (90-100nm) and non-enveloped viruses with
an icosahedral nucleocapsid containing a single double stranded (ds) DNA genome ~36
to 38 kilobases in size (Chailertvanitkul and Pouton, 2010). Among 57 serotypes existing
in humans, Ad serotype 5 (Ad5) has attracted tremendous interest and been widely
exploited as gene, protein, and drug delivery platforms mainly due to its ability to
efficiently infect a variety of cells. The adenoviral capsid of Ad5 contains 252 proteins,
constituting 3 different types: hexon, penton, and fiber based proteins (San Martin, 2012).
The icosahedral shape is conferred by 240 hexon proteins forming the 20 triangular facets
and 12 penton base proteins constituting vertices connecting with fiber proteins. The
adenoviral fiber consists of a N-terminal tail region at the proximal end, an elongated
shaft containing repeating motifs of approximately 15 residues, and a C-terminal globular
knob located on the distal end (Rux and Burnett, 2004). Both adenoviral fiber and penton

45

base proteins are keys in receptor binding and internalization, whereas interactions
between Ads and host cells are complex and vary cell by cell (Beatty and Curiel, 2012;
Wolfrum and Greber, 2013). The commonly accepted mode of Ad5 internalization begins
with the host cell attachment mediated by binding of the capsid fiber knob to CAR on the
cell surface. The conserved Arg-Gly-Asp (RGD) sequence on the exterior of the penton
base interacts with cell-surface integrin, triggering virus internalization via clathrin-
mediated endocytosis (CME). Penton RGD motifs are also believed to play a role in the
endosome escape step of Ad infection, even though the detailed mechanisms are
controversial. Once in the cytosol, the virus travels along the microtubule network to the
nuclear membrane where the viral DNA is imported via the nuclear pore complex.
Macropinocytosis is another endocytic pathway employed by adenoviruses to gain access
to cells. Although studies in our lab indicated that Ad5 fiber-knob proteins could trigger
macropinocytosis as well, it remained to be determined whether this pathway played a
significant role in the uptake of Ad5 in LGACs (Xie et al., 2006). Ad5 may trigger
macropinocytosis in LGACs; however, it utilizes knob-mediated endocytic pathway for
its primary entry route in LGACs (Xie et al., 2006).
Herein, we report for the first time a protein polymer nanoparticle that selectively
enters LGAC in vitro and in vivo and undergoes a basolateral-to-apical transport for
secretion into the apical lumen of LGAC. LGAC lumena drain into lacrimal ducts that
delivery tear proteins and electrolytes to the surface of the eye. This unusual
transcytosing property of the nanoparticle provides a unique capability that may be
exploited for sustained delivery of drugs to the ocular surface for those diseases that are
currently difficult to treat and require continuous infusion of drug. Transcytosing

46

nanoparticles could be developed for delivery i.v. or s.c., or alternatively injected to form
a depot in case of acute infection or trauma.

2.4. Conclusion
As a model for targeted transcytosis to the tear ducts, this strategy has the potential to
generate new nanomaterials that act at the LG, the ducts, or the ocular surface. Based on
the ELP diblock copolymer template, KSI fusion proteins assemble into stable,
monodisperse, and biodegradable nanoparticles at physiological temperature. Genetic
fusion of the Ad5 knob domain to SI minimally affects the morphology of the particle but
significantly enhances its internalization efficiency. With efficient targeting to CAR on
cells of the LG, KSI nanoparticles are internalized and transported from basolateral to
apical membranes. KSI nanoparticles represent the first engineered delivery vehicle with
potential for selective delivery of therapeutic agents from serum, through the lacrimal
gland acinar cells, and to the tears bathing the ocular surface.

2.5. Acknowledgements
I sincerely appreciate the assistance of F. Yarber and H. Pei for rabbit LGAC preparation
and recombinant adenovirus purification. We thank Drs. S. Karvar (Medical University
of South Carolina) and L.X. Shu (SUNY-Buffalo) for kindly providing original
constructs for Ad-YFP-Rab27b and Ad-mCherry-MyosinVb tail DN, respectively. We
thank Dr. Arnold Sipos (Keck school of Medicine of University of Southern California)

47

for his assistance in Zeta potential measurement. We also gratefully acknowledge the
help of the USC/Norris Cell and Tissue Imaging Core Facility and their staff, E. Barron,
D. Hauser and A. Rodriguez for sample preparation and assistance in TEM imaging. This
study was supported by a Department of Defense grant (TATRC, DOD W81XWH-12-1-
0538) to J.A.M. The study was also supported by the following National Institutes of
Health grants: RO1EY017293 and RO1EY017293S1 to S.F.H.-A.; R21EB012281 to
J.A.M., and P30CA014089 to the USC Norris Cancer Center. The content is solely the
responsibility of the authors.

48

Chapter 3: Epithelial Targeting using Elastin-Based Protein Polymers
3.1 Introduction
Intercellular adhesion molecule-1 (ICAM-1, or CD54), a cell-surface glycoprotein
member of the immunoglobulin (Ig) superfamily, consists of five extracellular Ig-like
domains, a transmembrane domain, and a short cytoplasmic domain (Bella et al., 1998)
(Figure 12). There exist two forms of this receptor: membrane-bound and soluble ICAM-
1 (sICAM-1) (Giorelli et al., 2002). ICAM-1 is constitutively expressed at low levels on
the cellular surface of most cell types; however, its upregulation can be induced by
interleukin-1 (IL-1), interferon- γ, and tumor necrosis factor (TNF)- α (Min et al., 2005),
partly due to NF-

Figure 12. Schematic diagram of human ICAM-1 showing domain structures and
binding partners. Human ICAM-1 is a transmembrane protein consisting of five
extracellular immunoglobulin(Ig)-like domains (D1-D5), a transmembrane spanning
region, and a short carboxyl-terminal cytoplasmic domain. The ICAM-1 D1 domain
contains binding sites for Plasmodium falciparum-infected erythrocytes (PFIE), human
rhinoviruses (HRV), leukocyte integrin LFA-1 (lymphocyte function-associated antigen-
1; aL β2), and fibrinogen. The ICAM-1 D3 domain can interact with macrophage-1
antigen (Mac-1). Soluble ICAM-1 comprises only extracellular domains (~45 kDa). N-
Glycosylation motifs are heavily in D2-D4 domains. Numbers in this figure present the
encoded amino acid position of the ICAM-1 protein sequence (Bella et al., 1998).

49

κB and CREB activation (Hadad et al., 2011), in response to inflammatory stimuli. As the
counter receptor for leukocytes and macrophages, ICAM-1 plays a vital role in
inflammatory and immune responses and is involved in lymphocyte migration, co-
activation of T- and B - cells, and leukocyte extravasation into lymphoid and inflamed
non-lymphoid tissues through the interaction with β2 integrin lymphocyte function-
associated antigen-1 (LFA-1, αL β2, or CD11a/CD18) and macrophage 1 antigen (Mac-1)
(Long, 2011). Studies in ICAM-1/LFA-1 interaction as a strategy to develop novel anti-
inflammatory therapies have mainly focused on immunoregulatory diseases, such as graft
rejection, atopic dermatitis, psoriasis, and rheumatoid arthritis (Nicolls and Gill, 2006;
Yusuf-Makagiansar et al., 2002; Zhong et al., 2011). Sjögrens’ syndrome (SS) is an
autoimmune exocrinopathy resulting from the destruction of secretory tissues, including
salivary and lacrimal glands, by lymphocytic infiltrates and subsequently causing dry
mouth and dye eye. Although the etiology of this disease is complex and multifactorial,
emerging evidence supports the hypothesis that inflammation is an important factor in the
pathogenesis of SS (Nikolov and Illei, 2009; Stern et al., 2010). Currently, there is no
effective and specific therapy for SS. It is partly due to the poor understanding of the
pathological mechanisms of SS and also the lack of therapeutic targets. Patients with SS
are often treated with topical, oral, or intravenous immunosuppressants, including
steroids and cyclosporin A, to suppress T cell proliferation; however, those drugs cause
huge side effects after long-term treatment. Therefore, there is an urgent need to explore a
novel drug delivery strategy to address this issue.
ICAM-1 constitutes a promising target for delivery of drugs directly to inflamed
tissues in SS associated dry eye and other inflammatory disorders. ICAM-1 expression is

50

considered to be important in the early stages of inflammation and significantly
correlated to the progression of many inflammatory diseases. For example, monitoring
the concentration of circulating ICAM-1, sICAM-1, can be used to improve the
prediction of diseases, including atherosclerosis (Fotis et al., 2012; Gross et al., 2012),
diabetes (Jing et al., 2014; Mendivil et al., 2013), and cerebral malaria (Adukpo et al.,
2013). In most cases, sICAM-1 levels are thought to correlate with the expression levels
of membrane-bound forms. As for SS, biopsies from conjunctiva, LG, and SG of human
and SS-susceptible animal models (e.g. mouse, rat, and canine) exhibit lymphocytic
infiltration with increased expression of various inflammatory and immune activation
markers such as ICAM-1, LFA-1, and MHC II antigens (Gao et al., 2002; Stem et al.,
2002). Moreover, in endothelial cells, ICAM-1, bound with ICAM-1 targeted cargoes,
triggers nonclassical endocytosis and delivery to lysosomes (Muro et al., 2005).
For these reasons, I have explored an efficient strategy to bioengineer ICAM-1
targeting peptides (Belizaire et al., 2003) onto an elastin-like polypeptide (ELP)
biopolymer that assembles a nanoparticle. This was intended to facilitate binding and
internalization on inflamed epithelial cells, for instance LGACs. Mimicking the repetitive
hydrophobic domains of human tropoelastin, ELPs are composed of a repeating penta-
peptide motif (Val-Pro-Gly-Xaa-Gly)n, where Xaa can be substituted with amino acids
that possess different hydrophobicity scales (Urry, 1997). ELPs exhibit a reversibly
stimulus-responsive lower critical solution temperature (LCST) phase transition behavior
which can be tuned by selection of Xaa and n (Janib et al., 2014; Urry, 1997). ELP
nanoparticles have emerged as a popular drug delivery platform with potential
applications in therapeutics of cancer, type II diabetes, osteoarthritis, and

51

neuroinflammation (MacEwan and Chilkoti, 2014), due to their biodegradability,
biocompatibility, low immunogenicity, and accurate control of the protein sequence.
Here we generated a polymeric ELP nanoparticle carrying mouse ICAM-1 targeting
peptides that recognize the cross-species homologous ICAM-1 receptor expressed on
epithelial cells and found that this delivery platform is highly specific to certain epithelial
cells in vitro and in vivo.

3.2 Materials and Methods
3.2.1 Materials and Reagents
TB dry® powder growth media was from MO BIO Laboratories (Carlsbad, CA). NHS-
Rhodamine was from Thermo Fisher Scientific (Rockford, IL). Sulfo-Cy5 NHS ester was
from Lumiprobe Corp. (Hallandale Beach, FL). Polyethylenimine (PEI) and Copper
Chloride were from Sigma-Aldrich (St. Louis, MO). Alexa Fluor® 488 donkey anti-goat
IgG, DAPI (4’,6-Diamidino-2-Phenylindole, Dihydrochloride), Rhodamine Phalloidin,
CellLight® RFP-Rab5a, RFP-Lamp1, and RFP-Golgi BacMam2.0 reagents were from
Life Technologies (Grand Island, NY). QIAprep Spin Miniprep Kit and QIAquick Gel
Extraction Kit were from Qiagen (Valencia, CA). HeLa cells were from ATCC
(Manassas, VA). The plasmid expressing mouse ICAM-1 turboGFP was from Origene
(Rockville, MD). For RT and real-time PCR, the high capacity cDNA RT kit, TaqMan®
universal PCR master mix, MicroAmp® optical 384- well reaction plates, MicroAmp®
optical adhesive films, and TaqMan® gene expression assay for mouse ICAM-1
(Mm00516024_g1) and mouse Sdha (Mm01352366_m1) were from Applied Biosystems

52

(Foster City, CA). Mouse ICAM-1 polyclonal antibody was from R&D Systems
(Minneapolis, MN). IRDye700-conjugated donkey anti-goat and goat anti-mouse
antibody, as wells as IRDye800-conjugated donkey anti-goat antibody were from
Rockland (Gilbertsville, PA). Mouse anti-actin monoclonal antibody was from Millipore
(Temecula, CA). 35 mm Glass bottom dishes were from MatTek Corp. (Ashland, MA).
For cell culture, the Modified Eagle’s Medium (MEM) with Earle’s salts and L-
glutamine, Penicillin/Streptomycin, 0.05% Trypsin/ 0.53 mM EDTA were from Cellgro
(Manassas, VA). Nonessential amino acid (NEAA) was from Lonza (Gaithersburg, MD).
64
Cu
2+
, produced by the
64
Ni (p,n)
64
Cu nuclear reaction, was from the University of
Wisconsin (Madison, WI).
3.2.2 Animals
Male BALB/c and NOD mouse breeders were purchased from Charles River
Laboratories (Wilmington, MA) and Taconic Farms (Hudson, NY), respectively.
Animals were bred in the University of Southern California (USC) vivarium. All animal
procedures were approved by the USC Institutional Animal Care and Use Committee and
performed in accordance with the Guide for the Care and Use of Laboratory Animals 8
th

ed (2011). All mice used in this study were male mice with 12-14 weeks of age. The
mouse LGs from male BALB/c and NOD mice were removed after mice were
euthanatized via intraperitoneal injection with 55mg Ketamine and 14mg Xylazine per
kilogram of body weight and followed by cervical dislocation.

53

3.2.3 Construction of ICAM-1 SI ELP nanoparticles
The plasmid pET25b(+)encoding the ELP diblock copolymer, SI, was synthesized
using plasmid recursive directional ligation as described previously

(Sun et al., 2011).
Sense and antisense murine ICAM-1 specific peptide sequences, identified by the phage
display selection (Belizaire et al., 2003), were synthesized with NdeI (5’) and BamHI (3’)
overhangs (Integrated DNA Technologies Inc., Coralville, IA):
sense (5’-TATGGGTTTCGAAGGCTTCTCGTTCCTCGCATTCGAAGACTTCGTAT
CATCAATAGGTTACTGATCTCCTCGG-3’) and antisense (5’-GATCCCGAGGAGA
TCAGTAACCTATTGATGATACGAAGTCTTCGAATGCGAGGAACGAGAAGCCT
TCGAAACCCA-3’).
Complementary oligonucleotides were heated at 95 °C for 2 min and cooled to room
temperature for 3 h. The annealed oligonucleotides were ligated into the pET25b(+)
vector, previously digested with NdeI/ BamHI. The SI gene was then ligated to the
downstream of the murine ICAM-1 specific sequence using BseRI/ BssHII cutting sites
in both plasmids. The correct ligation clones were confirmed by DNA diagnostic
digestion and DNA sequencing. ELPs were expressed in the BLR (DE3) Escherichia coli
strain and purified using inverse transition cycling (Hassouneh et al., 2010). The purity of
ELP fusion proteins was determined by running 50 µg of samples on a 4-20% SDS-
PAGE gel stained with 10% (w/v) copper chloride. Protein concentrations were
determined by measuring the absorbance of protein polymers at 280nm and calculated
using the Beer-Lambert law ( εSI/mICAM-1 SI= 1285 M
-1
cm
-1
). Protein molecular mass was
further confirmed by MALDI-TOF mass spectrometry (AXIMA Assurance, Shimadzu).

54

3.2.4 Characterization of ICAM-1 SI phase transition behavior and nanoparticle
formation
The temperature-concentration phase diagram of ICAM-1 SI ELP was determined by
optical density measurement at 350 nm using a DU800 UV-Vis Spectrophotometer
(Beckman Coulter, Brea, CA). Tt was defined as the temperature point at 50% maximal
turbidity (Meyer and Chilkoti, 1999). Self-assembly and hydrodynamic radius (Rh) of
ELP nanoparticles was measured using dynamic light scattering (DLS) in a DynaPro-
LSR Plate Reader (Wyatt Technology, Santa Barbara, CA). For DLS measurement,
samples were prepared at 25 μM in PBS and filtered through a filter with a 0.02 μm pore
size at 4 °C. 90 μl of each sample was applied to a pre-chilled 384 well microplate,
centrifuged at 4 °C to remove air bubbles, and covered with 20 μl of mineral oil to
prevent evaporation. DLS data were recorded at regular temperature intervals (1 °C) as
solutions were heated from 10 to 37 °C. The results were fitted to a cumulant algorithm
based on the sum-of-squares value and analyzed with a Rayleigh sphere model. The Tt1,
critical micelle temperature (CMT), was defined as the lowest temperature at which the
Rh is significantly greater than the averaged monomer Rh.
3.2.5 Fluorescent labelling of ELP nanoparticles
For fluorescent visualization, SI and ICAM-1 SI ELPs were conjugated with NHS-
rhodamine ester and Sulfo-Cy5 NHS ester via a chemically covalent crosslinking of
fluorophore to the lysine at the N-terminus of ELP polypeptides. Conjugation reactions
were performed in 100mM borate buffer (pH 8.5) at 4 °C overnight. Excess fluorophores

55

were removed by size exclusion chromatography on a pre-packed G-25 desalting column
(GE Healthcare, Piscataway, NJ).
3.2.6 Lipofectamine transfection for ICAM-1 expression
HeLa cells were maintained in MEM medium supplemented with 10% (v/v) fetal
bovine serum (FBS), 100 U/ml Penicillin, 100 µg/ml Streptomycin, 1% L-glutamine, and
1% NEAA, and incubated in a humidified 5% CO2 atmosphere at 37 °C. Briefly, HeLa
cells were grown in a 35 mm glass-bottom plate with the cell density of 3 x 10
5
cells/dish
and cultured until 90% confluence or greater. On the following day, the transfection
complexes were prepared by mixing the recombinant plasmid encoding mouse ICAM-1
turboGFP ( μg) and Lipofectamine 2000® ( μl) with the ratio of 1:2 at room temperature
for 20 min. HeLa cells were then incubated with transfection complexes at 37 °C for 6 h,
and thereafter rinsed with warm phosphate-buffered saline (PBS) twice, and maintained
in the complete MEM medium at 37 °C for another 36-48 h prior to uptake and
intracellular trafficking experiments.
3.2.7 Baculovirus transduction
The fresh medium containing CellLight® BacMam 2.0 reagents transducing RFP-
Rab5a, RFP-Lamp1, or RFP-Golgi was added to ICAM-1 expressing HeLa cells with the
final concentration of 30 virus particles per cell. The transduced cells were gently shaken
at 60 rpm at 37 °C for 1 h to maximize transduction efficiency, and then cultured at 37 °C
for another 16 h before experiments were performed.

56

3.2.8 Primary mouse LGAC culture
Isolation and primary culture of mouse LGACs were developed on basis of our
established protocol for rabbit LGACs (Chiang et al., 2012). Briefly, LGs from male
NOD mice were removed and minced into 1 mm
3
pieces with scalpels and sequentially
grown in a serum-free culture media containing 5 µg/ml laminin, 0.1 µM carbachol, and
1nM thyroxine. Cells were seeded on a Matrigel
TM
coat for live cell imaging on 35 mm
glass bottom dishes at 6 × 10
6
cells per dish and incubated at 37 °C for 2-3 h prior to the
experiments. Light microscope and β-hexosaminidase secretion assay were used to
confirm that these cells functionally and structurally mimic LGACs in vivo.
3.2.9 In vitro cell uptake and intracellular trafficking study
HeLa cells transiently expressing mouse ICAM-1 turboGFP, called mouse ICAM-1
expressing HeLa cells, were used 36-48 h post-transfection. SI was used as a control
group to compare the internalization of ICAM-1 SI in HeLa cells, mouse ICAM-1
expressing HeLa cells, and NOD mouse LG acini. For the uptake study, mouse ICAM-1
expressing HeLa cells were incubated with 30 µM of Rh-SI or Rh-ICAM-1 SI at 37 °C
for 30 min and 120 min. Cells were then rinsed with warm PBS thrice, maintained in the
fresh culture medium, and imaged by confocal microscopy. To evaluate the targeting
effect of ICAM-1 SI against human ICAM-1, HeLa cells were incubated with 30 µM of
Rh- SI or Rh-ICAM-1 SI at 37 °C for 30 min and 120 min before they are imaged by
confocal microscopy. To evaluate the biological function of ICAM-1 SI, LG acini from
male NOD mice were incubated with 30 µM of Rh- SI or Rh-ICAM-1 SI at 37 °C for 1 h.
To study the ICAM-1 SI internalization through CLIC-mediated endocytosis, ICAM-1

57

expressing HeLa cells were incubated with either 30 µM of Rh-SI or Rh-ICAM-1 SI in
the presence of Alexa Flour 647 cholera toxin b subunit (CTx-B, 10 µg/ml) at 37 °C for
1h. To track the intracellular trafficking of internalized ICAM-1 SI, mouse ICAM-1
expressing HeLa cells were transduced with a recombinant baculovirus to express RFP-
Rab5a, RFP-Lamp-1, or RFP-Golgi. After 16-18 h of transduction, cells were incubated
with 30 µM of Cy5-ICAM-1 SI at 37 °C for 10 min, rinsed with warm PBS thrice, and
imaged for another 45 min. To investigate the accumulation of ICAM-1 in lysosomes,
mouse ICAM-1 expressing HeLa cells were transduced with a baculovirus to express
RFP-Lamp-1 for 16-18 h. These cells were first incubated with 30 µM of Cy5-ICAM-1
SI at 37 °C for 60 min, rinsed with warm PBS, maintained in culture medium, and then
imaged with confocal microscopy after 3 h of the incubation. Images were acquired using
a Zeiss laser scanning microscope 510 Meta NLO confocal imaging system equipped
with Argon, red HeNe, green HeNe laser, and a Coherent Chameleon Ti-Sapphire laser
(LSM) mounted on a vibration-free table (Carl Zeiss, Thornwood, NY). All images were
acquired using a Plan-Apochromat 63x Oil immersion lens with a working distance of
0.19 mm.
3.2.10 Reverse transcription (RT) and real-time polymerase chain reaction (PCR)
Total RNA from LGs of NOD mice (N=10) and BALB/c mice (N=10) was extracted
using 5 PRIME Perfect Pure RNA tissue kit (Gaithersburg, MD). The transcribed cDNA
was prepared from 1 µg of total RNA using TaqMan reverse transcription reagent, with
the PCR setting: 1 cycle at 94 °C

for 3 min, followed by 30 cycles at 94 °C for 45 s, 55
°C for 30 s, and 72 °C for 90 s. The real-time PCR reaction for mouse ICAM-1
expression was conducted using TaqMan gene expression assay (Mm00516024_g1),

58

TaqMan Master Mix, and ABI7900HT fast real-time PCR system. Succinate
dehydrogenase, subunit A (SDHA) was used as a control. The recorded data were
analyzed using ΔΔCt method. The fold change of mouse ICAM-1 expression was
calculated by ΔCt = Ct (ICAM-1) – Ct (SDHA), ΔΔCt = Ct (NOD) – Ct (BALB/c), and
the relative expression was calculated by 2
- ΔΔCt
.
3.2.11 Western blot analysis with mouse LG lysate
LGs from BALB/c and NOD mice were lysed in RIPA buffer (150 mM NaCl,
50 mM Tris/HCl, 0.5% sodium deoxycholate, 0.5 mM EDTA, 1% NP-40, 0.1% TX-100,
and protease inhibitor cocktail, pH 7.4) by PT2100 polytron homogenizer. Cellular
debris in the homogenate was removed by centrifugation at 10,000 rpm at 4 °C for 10
min and the concentration of resultant supernatant was determined by Pierce
TM
BCA
protein assay kit. 20 µg of each sample was loaded onto a 10% SDS-PAGE gel and
resolved by the electrophoresis. Protein samples in the gel were then transferred to a
polyvinylidene difluoride (PVDF) membrane, blotted with goat anti-mouse ICAM-1
primary antibody (0.2 µg/ml; R&D Systems), mouse anti-actin antibody (1 µg/ml;
Millipore), IRDye 700-labeled goat anti-mouse IgG (dilution, 1:10,000; Life
Technologies), and IRDye 800-labeled donkey anti-goat IgG (dilution, 1:10,000; Life
Technologies). The PVDF membrane was then imaged using a Li-Cor Odyssey Scanning
Infrared Fluorescence Imaging System (Lincoln, NE) and fluorescent signals in the
membrane were converted digitally to black-and-white images.

59

3.2.12 Immunohistochemistry of mouse LG
LGs, retrieved from NOD and BALB/c mice, were fixed with 4%
paraformaldehyde/PBS at 25 °C for 3 h before transferred to a 30% sucrose/PBS solution
at 4 °C overnight. The LGs were embedded in O.C.T. cryostat sectioning medium, snap
frozen using dry ice, and stored at -80 °C.

The LG was then cut into 5-µm-thick sections
mounted onto glass microscope slides (VWR, Radnor, PA). For immunostaining, the
slides with LG sections were quenched with 50 mM NH4Cl, permeabilized with 0.1% Tx-
100, permeabilized with 1% SDS, and blocked non-specific binding with 1% BSA. The
resulting slides were blotted with goat-anti mouse ICAM-1primary antibodies (20 µg/ml;
R&D Systems) at 4 °C overnight and then incubated with rhodamine phalloidin (dilution,
1:100; Life Technologies), DAPI (dilution, 1:1000; Life Technologies), and donkey-
derived Alexa Fluor 488-conjugated anti-goat IgG antibodies (dilution, 1:100; Life
Technologies) at 37 °C for 1 h. Samples were mounted with a ProLong® Gold antifade
reagent (Life Technologies) and analyzed with LSM
3.2.13 In vivo ICAM-1 targeting of ELP nanoparticles
Male NOD mice at 12-14 weeks of age were used to determine the in vivo targeting
specificity of ICAM-1 SI. LGs of male NOD mice fully develop lymphocytic infiltration
by approximately 12 weeks of age (Lee et al., 2009) and this pathological feature is
hypothetically correlated with the increasing ICAM-1 expression in NOD LGs. Briefly,
mice were administered intravenously 100 µl of 200 µM Rh- SI or Rh-ICAM-1 SI twice,
with 1 h time interval, and euthanized 1h after injection. Mouse LGs were removed,

60

imbedded in O.C.T. cryostat sectioning medium, and sectioned as described above.
Tissue sections were covered with coverslips and imaged by LSM.
3.2.14 microPET imaging
Details of the
64
Cu-labeling ELP procedure were reported previously.(Janib et al.,
2013) Briefly, the bifunctional chelator AmBaSar (Cai et al., 2009) was activated by
EDC and SNHS with a molar ratio of AmBaSar: EDC: SNH = 10:9:8 at pH 4.0 at 4 °C
for 30 min. The five-fold molar excess of activated AmBaSar was then mixed with SI or
ICAM-1 SI in 0.1 M borate buffer (pH 8.5) at 4 °C overnight. The AmBaSar-SI or
AmBaSar-ICAM-1 SI was purified using size exclusion chromatography on a PD-10
desalting column. Thereafter, the AmBaSar-ELP conjugates were incubated with 1 mCi
of
64
Cu
2+
in 0.1 N NH4OAc buffer (pH 5.5) at 40 °C for 60min before separated by PD-10
column. The radioactivity of
64
Cu-AmBaSar labeled SI or ICAM-1 SI was measured
using a gamma counter. For in vivo microPET imaging, 100-110 µCi of
64
Cu-AmBaSar-
SI or –ICAM-1 SI was systemically administered into age-matched male BALB/c or
male NOD mice. Mice were analyzed using a microPET Sophie Genisys 4 scanner
(Culver City, CA) at 5 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h of post-injection. The images
were reconstructed using a 2-dimensitional ordered-subsets expectation maximum (2D-
OSEM) algorithm on AMIDE 1.0.4 software. For the image-driven pharmacokinetics, the
regions of interest (ROI) in selected tissues were selected and each selection was
converted to a number relevant to the signal intensity of
64
Cu. To model the data, the data
were fitted to a well-developed 7-compatment model using SAAM II (University of
Washington, Seattle, WA) with the following compartments: blood, liver, kidney, spleen,
heart, muscle, and LG. The tissue flux parameters obtained from the model fit were

61

normalized by an estimation of vascular space to give the flux per hour per volume of
blood in each tissue.
3.2.15 Statistics
All experiments were replicated at least three times. Data were expressed as mean ±
SD. Means from each group were analysed by the unpaired two-tailed student’s t-test. A
p value of less than 0.05 was considered statistically significant.

3.3 Results and Discussion
3.3.1 ELP fusions form nanoparticles at physiological temperature
The genetically engineered protein polymer called ICAM-1 SI was produced by
recombinant DNA technique and purified from BLR E. coli lysate by inverse transition
cycling. The ICAM-1 SI consists of a mouse ICAM-1 targeting peptide coupled to the N-
terminus of SI ELP which is made of a hydrophilic peptide motif, (Val-Pro-Gly-Ser-
Gly)48, and a C-terminal hydrophobic peptide motif, (Val-Pro-Gly-Ile-Gly)48 (Figure 13;
Figure 14B).

62

Figure 13. Structural maps of expression plasmid pET-25b(+) containing mouse ICAM-
1 targeting peptide or ICAM-1 SI ELP genes. (A) The DNA cassette encoding murine
ICAM-1 targeting peptides, mICAM-1, was cloned into the NdeI-BamHI cloning region
of a modified pET-25b(+) vector. (B) The recombinant plasmid encoding ICAM-1 SI
was generated by ligation of the plasmid, previously digested with restriction enzyme
BseRI and BssHII, containing mICAM-1 or S48I48 DNA cassette.

63

Figure 14. Amphipathic protein polymers designed to target the ICAM-1 receptor. (A)
Purified ELP protein polymers were characterized on a 4-12% SDS-PAGE gel stained
with 10% (w/v) copper chloride solution, showing Mw of SI, 39.54 kDa, and ICAM-1 SI,
41.42 kDa. (B) A mouse ICAM-1specific peptide was appended at the amino terminus of
an amphipathic ELP containing equal amount of hydrophilic (Xaa= Ser) and hydrophobic
motifs (Xaa= Ile), called ICAM-1 SI. ICAM-1 SI undergoes temperature-mediated phase
transition from soluble monomers to insoluble nanoparticles or coacervates at
physiological condition (37 °C).

ICAM-1 SI was estimated to undergo a phase separation forming core-shell nanoparticles
or coacervates above its transition temperatures. The yield of the ICAM-1 SI construct
was typically > 200 mg/L of bacteria culture. The SDS-PAGE gel stained with 10% (w/v)
copper chloride was used to confirm the molecular mass and purity of ELPs employed in
this study and showed that the purity of ICAM-1 SI was 87.92% compared to 92.64% of
the SI counterpart (Figure 14A). Molecular weights of SI and ICAM-1 SI were confirmed
by Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass

64

Spectrometry and were 39.64kDa, and 41.54kDa, respectively which was only 1.5% less
than their expected weights (Table 2; Figure 15).

Figure 15. MALDI-TOF mass spectra of (A) SI and (B) ICAM-1 SI.

65

Figure 16. ELP protein polymer with or without mouse ICAM-1 targeting peptides forms
nanoparticles at physiological temperature. (A) Temperature-induced phase separation of
ELP fusion proteins was determined by optical density (OD350), as a function of
temperature and concentration. The SI and ICAM-1 SI (25 μM, PBS) have two obvious
phase transition points. Tt1 is attributed to the phase separation of hydrophobic ELP (Xaa=
Ile), whereas Tt2 is associated with bulk phase separation of hydrophilic ELP (Xaa= Ser).
ELP assembles nanoparticles between Tt1 and Tt2. For SI, Tt1 and Tt2 are 25.5°C and
73.7°C, respectively. For ICAM-1 SI, Tt1 and Tt2 are 25.7°C and 46.9°C, respectively. (B)
The concentration-temperature phase diagrams for SI and ICAM-1 SI follow a log-linear
relationship. Tts of each ELP was fitted to the equation Tt = m Log [CELP] + b, where m is
the slope, CELP is the concentration, and b is the transition temperature at 1 μM. (C) The
mouse ICAM-1 targeting peptide has minimal influences on the assembly and radius of
ELP nanoparticles. Dynamic light scattering was used to measure the self-assembly and
hydrodynamic radius of ELP nanoparticles (25 μM, PBS) starting with an increase from
10°C to 37°C. BSA was used as an internal control. SI and ICAM-1 SI form
nanoparticles with a hydrodynamic radius of 23.6±0.4 and 21.9±0.6, respectively, at
37°C. Data are presented as mean ± SD. (D) SI and ICAM-1 SI assemble to
monodisperse nanoparticles at 37°C, with polydispersity index 0.0006 and 0.1259,
respectively (Table 1).

66

Next, the thermal transition behavior (Tt1 and Tt2) of ELPs was determined using UV-
Vis spectrophotometry by monitoring the optical density at 350 nm, which was as a
function of temperature, in PBS. SI exhibited two steep thermal responses at 25.5 °C, Tt1,
and 73.8 °C, Tt2, which were conferred from the hydrophobic (Val-Pro-Gly-Ile-Gly)48 and
the hydrophilic (Val-Pro-Gly-Ser-Gly)48, respectively (Table 2; Figure 16A). The
transition temperatures of ELPs are highly associated with the hydrophobicity of guest
residue, Xaa, in the ELP sequence, Val-Pro-Gly-Xaa-Gly. In general, guest residues that
are more hydrophobic have low transition temperatures than those hydrophilic. SI
underwent the phase separation at Tt1 and turned from soluble monomers to insoluble
micelles. This change caused a sharp increase in solution turbidity, giving an increasing
optical density. SI stayed as nanoparticles between Tt1 and Tt2 and underwent bulk phase
transition to form coacervates when the temperature reached Tt2. Similar to SI, ICAM-1
SI showed two obvious phase transitions at 25.7 °C (Tt1) and 46.9 °C (Tt2). Both SI and
ICAM-1 SI have similar Tt1 but distinct Tt2. We then tested the concentration dependence
of Tts for used ELPs. The transition temperatures of SI and ICAM-1 SI are linearized
with their logarithm of concentrations (Figure 16B), which is in accord with other ELP
fusions reported previously (Hsueh et al., 2014; Janib et al., 2014; Wang et al., 2014a).
The hydrodynamic radii of ELPs were then determined using dynamic light scattering. SI
and ICAM-1 SI assembled to nanoparticles between their Tt1s and Tt2s, with a radius of
23.6±0.4 nm and 21.9±0.6 nm, respectively (Table 2; Figure 16C). SI and ICAM-1 SI
existed mostly as monodisperse nanoparticles (Table 2; Figure 16D). These results
suggest that the addition of mouse ICAM-1 targeting peptide to SI minimally influences
the thermal transition behavior and particle size of SI.

67

Table 2. Elastin-like polypeptides evaluated in Chapter 3.
Label Amino acid sequence
a
Tt1
b

(°C)
Tt2
c

(°C)
Expected
M. W.
d
(kDa)
Measured
M. W.
e
(kDa)
Hydrodynami
c radius
f
(nm)
Polydispersity
Index
g

SI G(VPGSG)48(VPGIG)48Y 25.5 73.8 39.54 39.64 23.6±0.4 0.0006
ICAM-1 SI GFEGFSFLAFEDFVSSIG(
VPGSG)48(VPGIG)48Y
25.7 46.9 41.42 41.54 21.9±0.6 0.1259
a) Underlined bold: mouse ICAM-1 targeting peptide identified by phage display
screening (Belizaire et al., 2003).
b) Critical micelle temperature (25 μM, pH 7.4) determined by optical density
measurements at 350nm.
c) Bulk phase temperature (25 μM, pH 7.4) determined by optical density
measurements at 350nm.
d) Expected M.W. was calculated using DNASTAR Lasergene Editseq (Madison,
WI).
e) Observed M.W. ([M+H]
+
) was determined by MALDI-TOF-MS.
f) Hydrodynamic radius between Tt1s and Tt2s in PBS (25 µM, pH 7.4) were
measured by DLS, expressed as mean ± SD (N=10).
g) The polydispersity index (25 µM, 37°C) was calculated from the second term of a
power series expansion for a Cumulants fit of the autocorrelation function, as
measured by DLS.

3.3.2 ELP nanoparticles target epithelial ICAM-1 in vitro
It has been shown that ICAM-1 can facilitate the transport of its ligands to lysosomes
via CAM-mediated endocytotic pathway in endothelial cells (Muro et al., 2003b).
Although we have previously demonstrated that ICAM-1 SI assembles to nanoparticles at
physiological temperature, it has not previously been shown if this biopolymer has the
biological function to selectively target ICAM-1 and internalize into the cells. Here, we
used HeLa cells transfected with and expressing fluorescently tagged mouse ICAM-1 as
an in vitro model for accessing ICAM-1 SI activity. The confocal microscopy was
utilized to track the internalization of Rh-materials. The cells were incubated with 30 µM
of Rh-ELPs at 37 °C for 30 min and 120 min before analysis. Cells incubated with Rh-SI
showed a lower extent to none fluorescent signals in the cytoplasm of cells, while cells
incubated with Rh-ICAM-1 SI exhibited much more punctate intracellular accumulation,

68

indicating that ICAM-1 SI has been internalized via ICAM-1 mediated endocytosis; to
our surprise, we have observed tubular and ring-like morphologies, a hallmark of CLIC-
mediated endocytosis (Howes et al., 2010; Khan et al., 2010), in those cells treated with
Rh-ICAM-1 SI (Figure 17). This result suggests that the internalization of ICAM-1 SI in
ICAM-1 expressing HeLa cells may be through the CLIC/GEEC endocytotic pathway.

69

Figure 17. ICAM-1 SI internalizes in mouse ICAM-1 expressing HeLa cells. HeLa cells
expressing mouse ICAM-1 turboGFP were incubated with rhodamine-labeled ELPs at 37

°C for 30 min or 120 min prior to imaging using confocal microscopy. At any
comparable time points, ICAM-1 SI treated cells displayed more accumulated red puncta
and tubular/ring- like morphologies than those with SI. Green, mouse ICAM-1
turboGFP; Red, ELP nanoparticles; White arrowheads, internalized ELP nanoparticles;
White arrows, tubular/ring- like structure in cells. Scale bar= 10 µm.

70

We next evaluated the cross-species activity of ICAM-1 SI against the human
ICAM-1. To test this, HeLa cells were used as an in vitro cell model because HeLa cells
constitutively express low levels of the human ICAM-1 (Grunert et al., 1997). Prior to
this experiment, immunostaning was also used to confirm the expression of human
ICAM-1 on the HeLa cell surface (Data not shown). As shown in Figure 18, HeLa cells
incubated with Rh-ICAM-1 SI exhibited much more red punctate internalization as the
incubation period increased compared to those with Rh-SI (Figure 18). Moreover,
internalized ICAM-1 SI showed partially co-localization with LysoTracker, a green
marker staining low pH intracellular compartments, suggesting that ICAM-1 SI was
initially trafficked to other intracellular compartments before reaching the late endosomes
and lysosomes. In addition, the endocytosis of ICAM-1 SI in HeLa cells was temperature
dependent and can be inhibited at 4°C (Data not shown). HeLa cells, pretreated with the
anti-human ICAM-1 antibody, showed low cell surface binding and uptake of ICAM-1 SI
than those without any treatment (Data not shown). Taken together, ICAM-1 SI is
recognized and internalized by both mouse and human ICAM-1(Figure 17 and Figure
18), which represents a key finding to further the utility of this targeting strategy in both
mouse and human models.

71

Figure 18. ICAM-1 SI exhibits a cross-species targeting activity to human ICAM-1.
HeLa cells were treated with rhodamine-labeled ELPs at 37

°C for 30 min or 120 min
prior to imaging using confocal microscopy. 70nM Lysotracker Green was used to
indicate the low pH compartments. At any comparable time points, ICAM-1 SI exhibited
significant internalization than SI. Green, low pH compartments (late endosomes and
lysosomes); Red, ELP nanoparticles; White arrowheads, internalized ELP nanoparticles;
White arrows, tubular/ring- like structure in cells. Scale bar= 10 µm.

72

3.3.3 Internalized ELP nanoparticles were trafficked via a lipid raft independent
pathway
Having demonstrated the cross-species activity of ICAM-1 SI against mouse and
human ICAM-1, we next explored the intracellular trafficking itinerary of ICAM-1 SI in
mouse ICAM-1 expressing HeLa cells. HeLa cells incubated with ICAM-1 SI exhibited
distinct tubular and ring morphologies at the leading edge of the cells (Figure 17), which
is a hallmark of CLIC-mediated endocytosis. Others also reported that human rhinovirus
14 infection, mediated by ICAM-1, may via a clathrin-, cavelin-, and dynamin-
independent pathway (Grunert et al., 1997; Khan et al., 2010)
.
However, up to date the
specific marker for identifying CLIC/GEEC pathway remains controversial. Here,
cholera toxin B subunit (CTxB), a widely accepted marker for CLIC/GEEC endocytosis,
was used to define CLICs although it also internalizes via caveolae-mediated and lipid
raft-dependent endocytosis in HeLa cells (Lundmark et al., 2008; Nonnenmacher and
Weber, 2011). We observed that ICAM-1 SI partially colocalized with CTxB (Figure 19).
Removal or sequestering of cholesterol profoundly inhibits entry of ligands internalized
via this route. Methyl- β-cyclodextrin (M βCD) sequesters cholesterol and lead to the
impairment of caveolin/lipid raft-dependent endocytosis. M βCD did not significantly
inhibit the entry of ICAM-1 SI (Data not shown). This confirms that caveolin/lipid rafts
have no major importance in the endocytosis of ICAM-1 SI.

73

Figure 19. ICAM-1 SI displays a similar but not identical intracellular trafficking to
cholera toxin B subunit, a marker for CLIC-mediated endocytotic pathway. HeLa cells
expressing mouse ICAM-1 turboGFP were coincubated with rhodamine-labeled ELPs
and Alexa Flour 647 conjugates of cholera toxin B subunit (CTx-B, 10µg/ml) at 37

°C for
1 h before imaged by confocal microscopy. ICAM-1 SI partially colocalizes with CTx-B.
White arrowheads indicate the colocalization of ICAM-1 SI with CTx-B. Scale bar = 10
µm.

To further investigate the intracellular itinerary of ICAM-1 SI, a pulse-chase study
was used to track the dynamic movement of ICAM-1 SI in early endosomes (EE),
lysosomes (Lys), and Golgi networks (Golgi). The HeLa cells were first transfected to
express the mouse ICAM-1 turboGFP (green), then transduced with baculoviruses
encoding Rab5a-, Lamp1-, or Golgi-red fluorescent protein (RFP) (red) to mark the EE,
Lys, and Golgi, respectively. ICAM-1 SI was labeled with the purple-emissive

74

fluorophore cyanine dye (Cy5) and incubated with cells for 15 min at a concentration of
30 µM/µL before recorded for another 45 min. The confocal imaging revealed that
ICAM-1 SI trafficked to EE after a 15 min incubation and the internalized ICAM-1 SI
was transported from smaller EE in the cell periphery to larger EE near nuclei of cells via
homotypic early endosomal fusion (Skjeldal et al., 2012) (Figure 20A). The fluorescent
signal of ICAM-1 SI was increasingly observed in Lys in the entire chase period (Figure
20B) but the Golgi (Figure 20C). ICAM-1 SI largely accumulated in the perinuclear
region by 1 h, in agreement with its known endo-lysosomal routing (Muro et al., 2003a).
In addition, the confocal imaging used to analyze ICAM-1 expressing HeLa cells 3 h
after 1 h incubation with Cy5-ICAM-1 SI showed that Cy5-ICAM-1 SI resided almost
exclusively in Lys (Figure 20D). These findings suggest that ICAM-1 SI internalized by
receptor-mediated endocytosis is first sorted into EE, and then a fraction of this
endocytosed material is delivered to and accumulates in Lys.

75

Figure 20. Internalized ICAM-1 SI nanoparticles traffic to early endosomes and
lysosomes. Mouse ICAM-1 expressing HeLa cells transduced with CellLight
®
(A) Rab5-
RFP, (B) Lamp1-RFP, or (C) Golgi-RFP Bacmam 2.0 reagent were pulsed with 30 µM
Cy5-labeled ICAM-1 SI for 10 min, with a 45-min chase period. ICAM-1 SI partially
colocalizes with Rab5-RFP and highly colocalizes with Lamp1-RFP but Golgi-RFP,
indicative of its delivery to early endosomes and lysosomes. D) ICAM-1 SI exhibits an
extremely high co-localization with Lamp1-RFP after 3h of the incubation. ICAM-1 SI
accumulates in lysosomes in a large degree. Scale bar= 10 µm.

76

Based on the data collected in this study, the following model is proposed for the
trafficking of ICAM-1 SI in HeLa cells (Figure 21). Endocytosis, initiated by the binding
of mouse ICAM-1 targeting peptides on the ICAM-1 SI nanoparticles to ICAM-1
expressed on the cell surface, is followed by CAM-mediated endocytosis. A ring/tubular-
like structure was observed in this step and ICAM-1 SI is transported to early endosomes.
Thereafter, ICAM-1 SI is sorted and resided in lysosomes.

Figure 21. Schematic diagram showing the intracellular itinerary of ICAM-1 SI in mouse
ICAM-1 expressing HeLa cells. Cholera toxin subunit b (CTx-B) functions as a marker
for clathrin-independent endocytosis, even though it also internalizes via caveolin-
mediated endocytosis, in HeLa cells. ICAM-1 SI first traffics to early endosomes where it
co-localizes with CTx-B and eventually accumulates in lysosomes. Internalized ICAM-1
SI is hypothesized to traffic to clathrin-independent carriers/GPI-enriched early
endosomal compartments (CLIC/GEEC) prior to early endosomes and lysosomes.

77

3.3.4 ICAM-1 is upregulated in inflamed LGs
The ICAM-1 SI nanoparticle was initially applied in male NOD mice, an
autoimmune disease mouse model for SS-associated dry eye. Male NOD mice have been
reported to fully develop dacryoadenitis with infiltrating lymphocytes in the LG (Lee et
al., 2009). In addition, emerging evidences support that ICAM-1 is overexpressed in the
LG of MRL-lpr/lpr mice, another SS-like autoimmune mouse model (Gao et al., 2002).
Therefore, the quantitative real-time PCR and western blot were used to determine the
ICAM-1 expression in NOD LG compared to that from BABL/c control prior to other in
vivo assays. Data from real-time PCR showed that ICAM-1 gene expressions in LGs of
BALB/c and NOD are 1.26±0.67 and 13.9±4.76, respectively (p < 0.0001) (Figure 22A).
The mRNA level of ICAM-1 in NOD LG is 11.03 fold higher than that in BALB/c LG.
Similarly, the protein expression of ICAM-1 was confirmed by western blot showing an
obvious protein band at 100 kDa in the denaturing SDS-PAGE gel in NOD LG lysate
compared to BALB/c LG lysate (Figure 22B).

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Figure 22. ICAM-1 is upregulated in lacrimal glands (LG) from male non-obese diabetic
(NOD) mice. The LG from 12-week aged male NOD mice and BALB/c control were
used in this experiment. (A) Quantitative real-time PCR and (B) Western blot were used
to determine the expression of ICAM-1 at mRNA and protein levels. ICAM-1 gene
expressions in LGs of BALB/c and NOD are 1.26±0.67 and 13.9±4.76, respectively.
Data are presented as mean±SD (N=10). Means were compared with Welch’s unpaired
two-tailed student t test. p-value < 0.0001; β–actin was used as a loading control in
western blot.

LG mainly consists of LG acinar cells, which are polarized epithelial cells with
basolateral membranes as well as apical membranes facing lumen. Membrane proteins
are sorted to polarized plasma membrane domains for different biological functions. The
immunofluorescence was further used to determine the distribution of ICAM-1 in
BALB/c and NOD LGs (Figure 23). The lymphoid foci have been observed in NOD LGs
instead of BALB/c LGs (Figure 23A). The ICAM-1 was observed in vascular
endothelium, infiltrating lymphocytes, and LG acini and its expression appeared to
depend on the severity of the disease. In particular, ICAM-1 was overexpressed in the
basolateral membranes of LGACs from NOD mice while ICAM-1 also expressed in

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leukocytes rarely scattered in BALB/c LGs (Figure 23B). Taken together, this
observation indicates that the ICAM-1 expression is closely associated with the
inflammation of the LG in male NOD mice.

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Figure 23. Distribution of ICAM-1 in the LG of BALB/c and NOD mice. Cryosections
of LGs from 12 week male BALB/c and NOD mice were processed and imaged at low
(A) and high (B) magnification using confocal microscopy. ICAM-1 was labeled with
goat-anti mouse ICAM-1 primary antibody and AF-488 conjugated donkey anti-goat
secondary antibody (green). F-actin was labeled with rhodamine phalloidin (red) and
nuclei were labeled with DAPI (blue). (A) The expression of ICAM-1 is in the
lymphocytic infiltration of the LG and correlated with the severity of the inflammation.
(B) The ICAM-1 expression was observed in vascular endothelium, lymphocytes, and
basolateral membranes of LG epithelial cells next to lymphocytes. White arrowheads: LG
acini; *: vascular lumen.

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3.3.5 In vivo targeting and pharmacokinetic profile of ELP nanoparticles
To evaluate the in vivo targeting effect of ICAM-1 SI, NOD mice were systemically
administered Rh-ELP nanoparticles twice with 1h time interval. LGs were removed and
processed for confocal imaging 1 h post-injection. The confocal images showed that Rh-
ICAM-1 SI mostly accumulated in LG acini whereas Rh-SI displayed weak to no
fluorescent signal in the LG (Figure 24A).

Figure 24. ICAM-1 SI targets inflamed LGs in vivo. 100 µl of 200 µM rhodamine-
labeled SI or ICAM-1 SI were administered intravenously twice, with 1 h time interval,
to 14-week-old male NOD mice. After 1 h of the second injection, LGs were removed,
fixed, cut to 10m sections mounted on glass slides, and imaged by confocal microscopy.
Quantification of internalized SI or ICAM-1 SI inside LG clusters was measured using
ImageJ. Data were analyzed by unpaired two-tailed student t test and presented as
mean±SD (SI: 0.03±0.04; ICAM-1 SI: 1.35±0.52). p= 0.00052. N=5. Green puncta
indicate the internalized rhodamine-labeled SI or ICAM-1 SI. Scale bar=10 µm.

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A similar result was also observed when ex vivo NOD LG acini were incubated with Rh-
ICAM-1 SI and Rh-SI (Figure 25).

Figure 25. Addition of mouse ICAM-1 targeting peptide to SI enhances the ICAM-1
targeting specificity and internalization of SI in NOD LG acini. ICAM-1 expression in
NOD LGs is hypothetically increased with the SS development. LG acini from 12-14
week male NOD mice were treated with 30 μM rhodamine-labeled (Rh)- SI or ICAM-1
SI at 37 °C for 1 h. Rh-ICAM-1 SI (red) exhibits significantly higher surface binding,
internalization, and co-localization with Lysotracker (green), a biomarker for low pH
compartments, in NOD LG acini than its SI counterpart. White arrowheads indicate the
co-localization of ICAM-1 SI and Lysotracker. Scale bar= 10 µm.

The confocal images were then quantified by ImageJ to compare the NOD LG acini
injected with Rh-ELPs. Rh-ICAM-1 SI exhibited a 41-fold increased internalization in
LG acini relative to Rh-SI (p=0.00052) (Figure 24B). Next, the image-driven
pharmacokinetic modeling was used to determine the clearance and blood half-life of
ELP nanoparticles in male NOD mice. The methodology has been described in 3.2.14
microPET imagin. SI nanoparticles had a similar hepatic (0.06 ml.h
-1
) and renal clearance
(0.04 ml.h
-1
) while ICAM-1 SI exhibited a dramatic difference in hepatic (0.08 ml.h
-1
)
and renal clearance (0.2 ml.h
-1
) (Figure 26A). This enhanced renal clearance drastically

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reduced the circulation time of ICAM-1 SI nanoparticles, with a blood half-life of 3.8 h
for ICAM-1 SI compared with 7.2 h for SI (Figure 26B).

Figure 26. Preliminary data showing the pharmacokinetic profile of ELP nanoparticles in
NOD mice. SI or ICAM-1 SI, labeled with radiotracer
64
Cu, was intravenously
administered. Mice were scanned at 5 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h post injection
by microPET scanner. microPET images were quantitatively analyzed and data were
fitted to a well-developed multi-compartment model for pharmacokinetic modeling. (A)
SI nanoparticles show a similar hepatic (0.06 ml.h
-1
) and renal clearance (0.04 ml.h
-1
)
while ICAM-1 SI exhibits a dramatic difference in hepatic (0.08 ml.h
-1
) and renal
clearance (0.2 ml.h
-1
). (B) Blood half-life of ELP nanoparticles was obtained from this
non-invasive estimation. The enhanced renal clearance drastically reduced the circulation
time of ICAM-1 SI nanoparticles, with a blood half-life of 3.8 h for ICAM-1 SI
compared with 7.2 h for SI. Data are presented as mean±SD with analysis of unpaired
tow-tailed student t test (N=3).

This is the first report of a biopolymer that targets the ICAM-1adhesion molecule in
vitro and in vivo. This protein polymer displays several unique features, compared to
other ICAM-1 specific drug delivery vehicles (Muro, 2014; Muro et al., 2006; Zhang et
al., 2008). First, elastin-like polypeptides (ELPs) are artificial peptides, inspired by
human tropoelastin, that reversibly coacervate above a critical temperature (Chilkoti et

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al., 2006). Biological polymers have been believed to behave much more structurally
rigid than synthetic polymers, such as polyethylene, nylon, and silicone (Gardel, 2013),
suggesting that this material maintain their shapes well when subjected to a wide range of
externally applied stress. The protein sequence and size of ELPs can be tuned precisely
through genetic engineering of recombinant plasmid that is incomparable by synthetic
polymers. ELPs with these biological compositions share high biocompatibility and
biodegradability and low immunogenicity, permitting their safe break down into peptides
and amino acids that can be easily cleared from the body (MacEwan and Chilkoti, 2014).
Moreover, the addition of an ICAM-1 specific peptide to SI ELP gives a minimal
opportunity to generate host immunity, especially after long-lasting treatment in vivo.
The major limitations of antibody-targeted therapeutic are immunogenicity induced from
the Fc regions of IgGs (Baiu et al., 1999) and nonspecific uptake of antibodies by
reticuloendothelial system, including liver and spleen (Aina et al., 2002). It is widely
believed that short peptides with sequence less than 20 amino acids in length should be
less immunogenic relative to native proteins (Camacho et al., 2008). Hence, conjugating
a 16-mer ICAM-1 targeted peptide to SI would be expected to arouse minimal host
immune response after a long-term in vivo treatment with this ELP nanoparticle.
The mouse ICAM-1 specific peptide used in this study was identified by screening a
phage displayed peptide library against the mouse ICAM-1 transfected COS-7 cells and
shown to retain highest avidity for domain-1 of mouse ICAM-1 and block the
lymphocyte function-associated antigen 1 (LFA-1)-associated interaction during the
antigen presentation process (Belizaire et al., 2003). Several ICAM-1affinity peptides,
mostly generated by phase display techniques, and ICAM-1 targeting nanocarriers have

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been developed in the past to block leukocyte-endothelial adhesion or intercellular
adhesion of immune cells during antigen presentation in vitro and in vivo (Garnacho et
al., 2012; Zhang et al., 2008). Some of them showed an interspecies targeting ability for
two or more species. In this study, we proved that this peptide sequence has a cross-
species activity in mouse and human ICAM-1. So far, multiple groups have developed
the targeting therapeutic particles using ICAM-1 as a molecular marker. For example,
Zhang et al. has developed a cLABL-conjugated PLGA nanoparticle with ~244nm in
diameter displaying an efficient binding to both human epithelial and endothelial ICAM-
1 (Chittasupho et al., 2011; Zhang et al., 2008). The cLABL is a
cyclo(1,12)PenITDGEATDSGC peptide derived from the αL-integrin I domain of LFA-1
and shown to block T cell adhesion and alleviate lymphocytic infiltration in islet
endothelium (Huang et al., 2005). Moreover, Muro and Muzykantov groups have also
developed targeting therapeutics binding to the endothelial ICAM-1 in vitro and in vivo
for treating lysosomal storage diseases, acute and chronic respiratory diseases, or other
vascular diseases (Muro et al., 2003a; Muro et al., 2005; Muro et al., 2006; Rossin et al.,
2008). By electrostatic adsorption of targeting antibodies onto the surface of this
polymeric nanosphere (Polysciences, Inc), these polystyrene latex microspheres with
particle size ranging from 100nm to 300nm in diameter were selectively delivered to
mouse or human ICAM-1 or other adhesion molecules. In particular, Garnacho in Muro
group described a 17-mer linear peptide derived from the ICAM-1-binding sequence of
fibrinogen (Garnacho et al., 2012). Since this fibrinogen-derived peptide share high-
degree DNA similarity in human, mouse, and chimpanzee, polystyrene nanoparticles

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adsorbing this peptide selectively target both human and mouse ICAM-1, which is
similar to ICAM-1 SI.

3.4 Conclusion
This manuscript describes a multivalent protein polymer that targets epithelial ICAM-1
as a mean to deliver candidate therapeutics to inflamed LGs. ICAM-1 SI was designed to
bind LGAC ICAM-1 that was increasingly over-expressed in the basolateral membrane
of inflamed LGACs. ICAM-1 SI displays a specific internalization in HeLa cells, mouse
ICAM-1 expressing HeLa cells, and mouse LG acini ex vivo and in vivo. Internalized
ICAM-1 SI traffics from early endosomes to lysosomes. MicroPET imaging showed that
ICAM-1 SI has a high renal clearance and a blood half-life of 3.8 h compared to 7.2 h for
its SI counterpart.
This delivery strategy was initially evaluated in inflamed LGACs for SS-associated
dry eye and will be extended to other autoimmune diseases, such as rheumatoid arthritis.
Compared to other ICAM-1 targeting carriers, ICAM-1 SI is the first biopolymer that (1)
targets epithelial ICAM-1 and internalizes in inflamed tissues in vitro and in vivo, (2)
self-assembles to multivalent and monodisperse nanoparticles, (3) displays cross-species
activity against mouse and human ICAM-1, (4) may exhibit low immunogenicity due to a
short targeting peptide and human friendly protein sequences, (5) is less toxic and
degradable due to its peptidic nature, (6) employs the recombinant DNA techniques to
accurately tune its protein sequence and easily scale-up, (7) represents a simple platform
easily assembling with other functional ELPs to form a multipurpose nanoparticles.

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3.5 Acknowledgment
This work was made possible by the University of Southern California, National
Institutes of Health grants RO1EY017293 and RO1EY011386 to S.F.H.-A.,
R21EB012281 to J.A.M., and P30 CA014089 to the USC Norris Cancer Center. Also, we
sincerely thank Zhen Meng for the in vitro mouse LGAC preparation and Srikanth Reddy
Janga for the assistance of real-time PCR.

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Chapter 4: Development of multifunctional nanoparticles for immune-mediated
inflammation
4.1 Introduction
With growing needs for accurate diagnosis and efficient treatment in uncontrolled
diseases, multifunctional nanoparticles are expected to aid greatly in reaching these goals.
Multifunctional nanoparticles have the potential to integrate various functionalities,
simultaneously offering (i) targeted delivery of therapeutic agents/gene, (ii) contrast for
in vivo multimodality imaging, and (iii) thermal therapies. Nanoparticles can show
similar sizes as antibodies, membrane receptors, nucleic acids and proteins. These
biomimetic features, together with a higher surface area to volume ratio and possibility of
surface engineering for therapeutic and diagnostic agents compared to similarly sized
liposomes, make nanoparticles attractive tools for imaging, diagnosis and therapy. It is
believed that these magic therapeutic vehicles can be used to improve solubility of
therapeutic agents under aqueous conditions, increase blood half-life of drugs to reduce
dosing frequency, and enhance targeting specificity to reduce nonspecific toxicity toward
normal cells and lower the overall administration dosage of the drug.
Synthesis of multifunctional nanostructures is never easy and requires various
organic reactions for modifying surface functionalities of these particles and their
composites (McCarthy et al., 2012). These reactions include but are not limited to amide
coupling, thiol and amine click chemistry, azide-alkyne click chemistry, amine-azide
conversion, and surface silanization (McCarthy et al., 2012). These complicated
processes for synthesis directly limit the scalability of manufacturing multifunctional

89

nanosystems. With unique phase-separation behavior and efficient synthesis, ELPs are
expected to improve the fabrication of multifunctional nanoparticulate systems by the
self-assembly strategy.
In this chapter, we evaluated our self-assembly strategy by co-assembling ICAM-1
SI with FSI, which is a diblock copolymer ELP consisting of 48 serine and isoleucine
pentamers, (VPGSG)48(VPGIG)48, genetically fused with a human FK-506 binding
protein 12 (FKBP12, hereafter called FKBP) on its N-terminus (Shi et al., 2013). FKBP
belongs to immunophilins that bind to the immunosuppressive drugs, such as FK-506
(Tacolimus), rapamycin (Sirolimus), and cyclosporine A (CsA), and show peptidylprolyl
cis/trans isomerase (PPIase) activity (Banaszynski et al., 2005). FKBP is a 12-kDa
protein and holds a hydrophobic ligand binding pocket (Yang et al., 2000). The
FKBP/rapamycin complex binds directly to the FKBP-rapamycin-binding domain of
mTOR, which inhibits the T cell lymphokine gene activation (Kang et al., 2008).
Previous studies demonstrated that rapamycin, binding to both shell and core of FSI
nanoparticles, exhibits low toxicity and potent efficacy against inflammation of LGs in
male NOD mice (Shah et al., 2013).
As a pilot study, we next investigated the inhibitory capacity of a peptide inhibitor,
CTSSIP, against mouse and human CTSS, as a prelude to appending this peptide to ELP
nanoparticles comprised of SI to LGACs. Cathepsin S (CTSS) is a cysteine endoprotease
that mediates the MHC class II invariant chain (Ii) processing and the degradation of
antigenic proteins to peptidyl fragments in specific antigen presenting cells (APCs). The
resultant antigenic peptides are loaded into the MHC class II. The MHC class II-antigenic
peptide complexes are transported to the cell surface of APCs for antigen presentation

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and subsequently triggers the activation of CD4
+
T cells (Bania et al., 2003; Hiltbold and
Roche, 2002; Hsieh et al., 2002). Continuous antigen presentation in both professional
and nonprofessional APCs has been thought to correlate with the maintenance of chronic
autoimmune diseases (Beers et al., 2005). Studies also report that CTSS activity can be
boosted with the stimulation of pro-inflammatory cytokines such as interferon- γ and is
upregulated in inflammatory conditions. Recently scientific evidence indicates that
inhibition of CTSS activity reduces the humoral immune processes without an impact on
innate immunity, thus, preventing autoantigen presentation and autoimmunity (Saegusa et
al., 2002). Since both ICAM-1 and CTSS are upregulated in pathophysiological
conditions, delivery of CTSS inhibitors to endosomal/lysosomal compartments of
nonprofessional APCs, such lacrimal gland acinar cells, via ICAM-1 would be an ideal
strategy to treat autoimmune diseases at the early stages of the diseases. We then
determined ICAM-1 expressions at gene and protein levels in organs from SS-susceptible
mice, NOD mice, compared to their BALB/c controls. This result furthers our
understandings about in vivo bio-distribution and targeting specificity of ICAM-1 SI in
NOD mice.

4.2 Materials and Methods
4.2.1 Co-assembly of ELP nanoparticles
Pure FSI and ICAM-1 SI ELPs were labeled with NHS-carboxyfluorescein (CF)
(Life technologies, Foster City, CA) and NHS-rhodamine (Rh), respectively, using the
conjugation chemistry. Both fluorescently labeled ELPs were mixed at same

91

concentrations, 100 µM, or at the concentration ratios of 1:3 (50 µM FSI: 150 µM ICAM-
1 SI) and 3:1 (150 µM FSI: 50 µM ICAM-1 SI) in 35 mm glass-bottomed dishes (MatTek
Corp., MA). The mixtures were imaged by a Zeiss LSM 510 Meta NLO confocal
microscope (Thornwood, NY) with an Instec’s HCS60 temperature control stage
(Denver, CO) attached to a thermal controller adjusting the temperature at 15 °C and 33
°C. All images were captured under a Plan-Apochromat 63x/1.40 oil immersion lens with
a working distance of 0.19 mm.
4.2.2 Tear fluid collection
For mouse tear collection, male NOD mice over the age of 12 weeks were
anesthetized using intraperitoneal ketamine (60 mg kg
-1
) and xylazine (8 mg kg
-1
). The
mouse eyes were washed with the Ocusoft® eye wash solution (Richmond, TX) prior to
tear collection. The LG was then exposed by cutting a small incision along the axis
between the lateral canthus of the eye and the ear. The tear fluid was stimulated by
adding the cholinergic agonist carbachol drops (50 μM, 3 μL) to the glands topically. The
tear fluid was collected with a 2 μL glass micro-capillary tube by gently placing the tip of
this tube at the inner canthus of the eye, moving along the palpebral conjunctiva and
holding just lateral to the inner canthus during the 1 min collection period. Collections
were performed in triplicate in both eyes with the frequency of 1 min interval every 5 min
stimulation. Six collections, three times per eye, were performed on each mouse. The
mouse tear samples were pooled and analyzed immediately for cathepsin S (CTSS)
activity. For the collection of CTSS in human tear fluid, tear samples from SS patients
were collected using Schirmer’s tear test strips in a standardized fashion (anesthetized
eyes with strips gently placed in the inferior fornix for 5 min) (Hamm-Alvarez et al.,

92

2014) from both eyes of each subject (N=4). All procedures adhered to the Declaration of
Helsinki. A topical anesthetic was utilized here to numb ocular irritations which cause the
reflex tear secretion. The residual fluid in the conjunctival cul-de-sac was removed prior
to tear collection. The moisture on the ocular surface was expressed as the length of
millimeter in Schirmer’s tear test strips. To extract CTSS from the tear fluid, the paper
strips were then placed into 5 ml Falcon tubes (BD labware, Franklin lakes, NJ)
containing CTSS reaction buffer (BioVision Inc., Milpitas, CA) with the volume ratio of
50 µl buffer for 1 mm of tear saturation. The tubes were subjected to centrifugation at
10,000 rpm for 10 s. Tear samples were placed on ice for immediate analysis.
4.2.3 Enzyme activity assay
Mouse and human CTSS activities were determined using a fluorescence-based
cathepsin S activity assay kit (BioVision Inc., Milpitas, CA). The rationale of this assay
kit utilizes a CTSS substrate peptide VVR labeled with a fluorescent marker, 7-amino-4-
trifluoromethyl-coumarin (AFC). CTSS in samples, such as tear and tissue lysates,
cleaves the synthetic substrate Z-VVR-AFC to release free AFC, which is quantified by a
fluorometer. For CTSS activity in the mouse tear, 2 μL of tear fluid per well was used for
the determination of CTSS activity. For CTSS activity in the mouse LG, mouse LGs were
homogenized with a Brinkman Polytron tissue homogenizer (Kinematica, Newark, NJ)
and followed by centrifugation to remove the cellular debris. Protein concentration of the
homogenate was determined using NanoDrop ND-1000 Spectrometer (Thermo
Scientific, Wilmington, DE). 25 µg of LG lysate proteins was loaded into each well of a
96-well plate for the determination of CTSS activity. For CTSS activity in the human
tear, CTSS was extracted from Schirmer’s test strips with the CTSS reaction buffer. Each

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well containing CTSS samples in a 96-well plate was loaded with an appropriate amount
of CTSS reaction buffer and 2 μL of Z-VVR-AFC (10 mM). The resultant wells in the
96-well plate were then added with or without CTSSIP or commercial CTSS inhibitor
(CI) and incubated at 37 °C for 1 h before analysis using a Gemini EM® microplate
spectrofluorometer (Molecular Devices, Sunnyvale, CA) with excitation of 400 nm and
emission of 505 nm.
4.2.4 ICAM-1 expression
Real-time qPCR and western blot were used to determine ICAM-1 expressions in
organs from male BALB/c and NOD mice at the age of 12 weeks of age. The detailed
procedures have been described previously (Wu et al., 2009) and ICAM-1 expressions
were compared in the LG, SG, liver, kidney, pancreas, and heart from both mouse strains.
For western blot, 20 μg of each sample was loaded into each well, and proteins were
separated out by a 4-12% SDS-PAGE gel at 4 °C. Proteins were electroblotted onto
PVDF membranes using the iBlot
®
dry blotting system (Life Technologies, Carlsbad,
CA). ICAM-1 and β-actin was identified by the goat anti-mouse ICAM-1 antibody (R&D
Systems, Minneapolis, MN) and mouse anti-actin antibody (EMD Millipore, Billerica,
MA), respectively.

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4.3 Results and Discussion
4.3.1 ELPs sharing similar parent structures co-assemble above the low critical
micelle temperatures
Our future goal is to develop multifunctional nanoparticles with targeting affinity for
diseased cells and therapeutic efficacy for disease-related biomarkers. We first tested this
ELP self-assembly strategy by co-assembling ICAM-1 SI with FSI, a SI ELP fused with
a N-terminal FKBP. Both ELP nanoparticles were tagged with fluorophores that have
different excitation/emission wavelengths.

95

Figure 27. Elastin based biopolymers with distinct functional moieties assemble above
their phase transition temperatures. (A) Same concentrations of two SI ELPs with
different functionalities, previously labeled with carboxyfluorescein (CF) or rhodamine
(Rh), were mixed at 1: 1 (v/v) ratio in a 35 mm glass bottomed dish and imaged by
confocal microscopy below (15 °C) and above (33 °C) their transition temperatures (Tts).
(B) SI ELPs with different functionalities can mix and assemble freely at different
concentration ratios above their Tts. Same volumes but different concentrations (1:3 or
3:1) of SI ELPs were mixed below their Tts and imaged by confocal microscopy at 33 °C.
Scale bar= 10 µm.

96

To make nanoparticles with targeting and therapeutic capacities, the co-assembly of
SI ELPs with different functional moieties were conducted and analyzed below (15°C)
and above (33°C) their transition temperatures using the fluorescent confocal microscopy.
Carboxyfluorescein (CF)-labeled FSI and rhodamine (Rh)-labeled ICAM-1 were mixed
at a concentration ratio of (1:1), (1:3), or (3:1). As shown in Figure 27A, CF-FSI self-
/co-assembled with Rh-ICAM-1 SI at 33°C, while solution containing these two SI ELP
nanoparticles remained clear at 15 °C. The fluorescently labeled ELPs were then mixed at
different concentration ratios. Interestingly, both CF-FSI and Rh-ICAM-1 SI exhibited a
high degree of colocalization at (1:3) and (3:1) ratios (Figure 27B). It is the first time to
show that ELP diblock copolymers with different functionalities co-assemble together,
although a similar result has been observed in mono-block ELPs such as V96 ELP, V192
ELP, and I24 ELP (Shi et al., 2014). This result suggests that the self-assembly of ELPs
with similar parent structures would be a feasible strategy to prepare the multifunctional
nanoparticles with ICAM-1 targeting specificity and rapamycin encapsulation capability.

4.3.2 Peptide from the pro region of cathepsin S exhibit potent inhibitory capacity
against cathepsin S
This strategy will be applied in SS-like NOD mice and other mouse models of
autoimmune diseases such as rheumatoid arthritis. Multifunctional nanoparticle with the
ICAM-1 targeting affinity and CTSS inhibitory capability will be built using the ELP co-
assembling strategy. As a pilot study, we first evaluated the inhibitory capacity of a CTSS
inhibitor peptide (CTSSIP), NHLGDMTSEEVMSLTSS, against mouse and human

97

CTSS. A commercial CTSS inhibitor was used as a positive control. CTSSIP is a 17
amino acid peptide from the pro region of nascent human CTSS (Figure 28A) and has an
inhibition constant (Ki) of 37.6 µM at pH 5.5. Inhibition studies conducted by Maubach
et al. have shown that CTSS propeptide has a Ki in the low nanomolar towards activated
CTSS and also has similar properties against both cathepsin L and cathepsin K at neutral
pH (Maubach et al., 1997). We then further evaluated the inhibitory capacity of CTSSIP
against mouse CTSS in both mouse tear (Figure 28B) and LGs (Figure 28C) and human
CTSS in human tear fluid (Figure 28D). Interestingly, compared to the commercial CTSS
inhibitor, CTSSIP exhibited a comparable inhibitory effect against both mouse and
human CTSS.

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Figure 28. Cathepsin S (CTSS) inhibitor peptide and its activity against mouse and
human CTSS. (A) CTSS inhibitor peptide (CTSSIP) is a 17 amino acid peptidic fragment
from the nascent human CTSS. (B) Inhibitory activity of CTSSIP against mouse CTSS in
tears from male NOD mice at 12 weeks of age. (w/o inhibitor: 211.8; 0.5 mM CASSIP:
89.3; CI: 64.0). (C) Inhibitory activity of CTSSIP against mouse CTSS in LGs of male
NOD mice at 12 weeks of age. N=10. (w/o inhibitor: 466.9±60.6; 0.5 mM CASSIP:
71.2±29.5; 1m M CASSIP: 24.6±14.5; CI: 29.0±6.7; ****<0.0001; ***=0.0003). (D)
Inhibitory activity of CTSSIP against human CTSS in tears from patients with SS-
associated dry eye. The lacrimation of the left and right eyes from each patient (N=4) was
determined by Schirmer’s tear test (STT) at the same time; thus, the number of samples
in each group is twice the number of patients recruited to each group. (w/o inhibitor:
652.5±505.3; 1 mM CASSIP: 178.3±110.3; CI: 50.8±27.5; *=0.03; **=0.01). Data were
presented as mean±SD. Means among groups were compared with one-way ANOVA
followed by Tukey’s post hoc test. RFU=relative fluorescence units; CI: commercial
inhibitor developed for cathepsins (BioVision Inc., Milpitas, CA).

4.3.3 ICAM-1 expressions in a Sjögren's syndrome-susceptible mouse, NOD
ICAM-1 plays an important role in the process of inflammation. ICAM-1-coupled
signal transduction cascades inside the cell trigger the secretion of TNF- α and lead to the
recruitment of inflammatory cells such as macrophages and granulocytes. It can be

99

induced by endotoxin and cytokines such as including IFN- γ, IL-1, and TNF- α both in
vitro and in vivo (Fotis et al., 2012; Li et al., 2012; Zhu et al., 2013). ICAM-1 is
upregulated in the early-onset inflammation and prevention of ICAM-1 expression has
been proved to reduce inflammation efficiently (Guan et al., 2013; Philpott and Miner,
2008). In this study, we evaluated the targeting specificity of ICAM-1 SI in a male NOD
mouse, which is a SS-susceptible mouse strain with fully developed infiltration of
lymphocytes in its LG at 12 weeks of age. Therefore, male NOD mice have been utilized
as a disease model for pathophysiological and pathogenic studies of SS-associated dry
eye. These mice may have inflammation accompanied by the overexpression of ICAM-1
in other organs. These upregulated ICAM-1 receptors may serve a targeted ligand for
ICAM-1 SI, compromising the LG targeting effect of ICAM-1 SI. Therefore, real-time
qPCR and western blot were used to determine ICAM-1 expressions in organs including
the LG, salivary gland (SG), spleen, liver, kidney, pancreas, and heart from 12-week-old
male NOD mice compared to their age/sex matched BALB/c controls. As shown in
Figure 29A, only NOD LG and pancreas are able to evidence statistically significant
differences of ICAM-1 (fold change: LG, 10.63; pancrease, 4.40; p value<0.0005; student
t test). ICAM-1 expressions in BALB/c mice are as follows: LG 1.1±0.6, SG 1.0±0.4,
spleen 1.0±0.1, liver 1.0±0.2, kidney 1.0±0.4, pancreas 1.2±0.8 (Mean±SD). ICAM-1
expressions in NOD mice are as follows: LG 11.9±3.9, SG 1.6±0.2, spleen 1.0±0.2, liver
2.6±0.4, kidney 1.2±0.2, pancreas 5.4±1.3, heart 1.2±0.4 (Mean±SD). Similar results of
ICMA-1 expressions were also concluded by western blot (Figure 29B). Taken together,
ICAM-1 is upregulated in the LG and pancreas from male NOD mice at 12 weeks of age.
The expressed ICAM-1 in pancreas may interfere the LG targeting of ICAM-1 SI.

100

Figure 29. Expression level of ICAM-1 in organs from BALB/c and NOD mice. Mouse
organs were taken from male NOD mice, a SS-like disease model, and BALB/c controls
at 12 weeks of age. (A) Quantitative real-time PCR was employed to determine relative
expressions of ICAM-1 at mRNA levels. ICAM-1 exhibits statistically significant
differential expression in NOD LG and pancreas. (LG, p value=0.0011; Pancreas, p
value=0.0002). Means were compared with Welch’s unpaired two-tailed student t test. R:
relative fold changes of ICAM-1 in NOD compared to BALB/c. N=6. (B) Western blot
was used to determine expressions of ICAM-1 at protein levels in different organs from
both mouse strains. β–actin was used as a loading control in western blot.

4.4 Conclusion
In summary, we have evaluated the ability of ELPs to self-assemble into multifunctional
nanoparticles with both tissue specificity and therapeutic capabilities. The targeted
ICAM-1 SI ELP is able to assemble with the therapeutic FSI which has been proved to
encapsulate rapamycin (Shah et al., 2013; Shi et al., 2013). It seems that ELP sharing
similar parent structures (e.g. SI diblock copolymer) are able to co-assemble into a
nanostructure. We also explored ICAM-1 expression at both gene and protein levels in
organs from NOD and BALB/c mice, which paves a way for future studies concerning in

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vivo targeting and bio-distribution of ICAM-1 SI ELP in combination with ELPs like FSI
for treatment of diverse inflammatory disorders. We also ran a pilot study to determine
the inhibitory capacity of CTSSIP. This 17-amino acid peptide showed a promising
ability against both mouse and human CTSS in comparison with the commercial CTSS
inhibitor. This peptide may be able to genetically link to the N-terminus of SI ELP,
developed as a therapeutic ELP used for ELP self-assembly. Taken together, the ELP
self-assembly strategy provides an efficient way for the non-covalent functionalization
with affinity moieties. With the utilization of phage display techniques to identify novel
peptide-targeting ligands, this strategy can be used to develop multifunctional
nanoparticles with targeting, therapeutic, and/or imaging capacities for specifically
targeting a wide range of disease cells.

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Chapter 5: Conclusions
5.1 Significance
Topical ophthalmic drug administration is the most favored route for treating disorders
associated with the ocular surface and anterior segment of the eye because of its local and
rapid drug action and patient compliance. However, conventional ophthalmic dosage
forms, such as eye drop solution, ophthalmic suspension, and ophthalmic ointment,
typically give a poor bioavailability (< 5%) (Urtti, 2006), causing short duration of
therapeutic response. This is mainly attributed to the unique physiological and anatomical
features of the eye. The normal eye has many protective mechanisms, including blinking
and tear drainage, to prevent it from being hurt. These mechanisms can be triggered
immediately once the drug has been applied. One of concerns causing such poor
bioavailability is the tear film which constitutes a muco-aqueous barrier incessantly
washing away the drugs on the ocular surface. The non-permeability of corneal
epithelium, stroma, and endothelium is also a major concern for poor bioavailability.
Corneal epithelium has been considered as a barrier for hydrophilic drug, although
stroma functions as a diffusion barrier for lipophilic drug and endothelium is lipophilic in
nature. Other concerns causing poor drug bioavailability could be the binding to tear
proteins, metabolism in corneal epithelium, and limited corneal surface. Additionally, the
systemic adverse effect is also an important issue for conventional ophthalmic dosage
forms (Bowman et al., 2004; Muller et al., 2006). Up to 80% of applied drugs
administered topically are absorbed systemically via the highly vascularized
nasopharyngeal mucosa.

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The major challenge for designing an optimal ophthalmic therapeutic agent is to
achieve an efficient concentration at target site for the duration to provide a therapeutic
efficacy (Raghava et al., 2004). An optimal therapeutic formulation to the eye should be
easy to use, well tolerated, less systemic absorption, and enhanced pre-corneal drug
retention time in the eye. Therefore, various ophthalmic delivery strategies have been
developed to improve ocular tolerability, improve ocular penetration, and reduce toxicity
(Perry et al., 2008; Yavuz et al., 2012). Among these developed delivery strategies, the
ophthalmic in situ gelling systems would be the most intriguing one for controlled and
sustained release of therapeutic agents to the ocular surface to compensate the drug loss
caused by nasolacrimal drainage and the systemic absorption caused by nasopharyngeal
mucosa. In situ gelling systems behave as liquid on instillation and then undergo a
solution-to-gel phase transition in the ocular cul-de-sac due to presences of the
environmental ionic strength, pH, or temperature. Unlike conventional eye drops (i.e.
solutions and suspensions) with short drug resident time and ophthalmic semisolid
formulations (e.g. gels and ointments) often causing blurred vision and sticky sensation
resulting eye irritation, in situ gel-forming systems can enhance contact time of drugs
with cornea efficiently, increase the bioavailability, maintain the accurate dose of drugs at
the target site, and get better patient acceptability even though they still slightly cause
blurred vision, irritating sensation, and variability in efficiency. Additionally, different
gel-forming materials have distinct intrinsic properties, such as pH, ionic strength, and
transition temperature, and envisaged therapeutic utility. Incorporation with therapeutic
agents and other excipients may change the rheological behavior of these materials in
some cases. The “tear-mediated drug delivery strategy” achieved by the KSI ELP

104

biopolymer not only can compete with the current in situ gel-forming systems but it also
is expected to have more pre-corneal drug residence time and fewer systemic effects by
delivery mimicking the naturally secretory mechanism, the later which would be the most
unique characteristic compared to other ocular delivery strategies. Due to this particular
delivery strategy, The KSI ELP biopolymer has a potential to be used to deliver
therapeutic agents to the tear duct.
Another innovation described in this study is the ICAM-1 SI ELP biopolymer, which
was designed to target inflamed LGs for the treatment of SS-associated dry eye. Although
its pathogenesis remains unclear, SS-associated dry eye has been widely considered as a
chronic LG inflammation resulting from the over-activated immune response. Patients
who are diagnosed with this disease show moderate-to-severe tear deficiency and tear
film instability. A hypothesis raised by Hsueh et al. (2010) suggested that the LG
inflammation may be triggered by over-activated LGACs at the early stages of this
disease and eventually lead to a vicious cycle of inflammation on the ocular surface in
patients with dry eye. Current treatments are symptomatic due to the lack of therapeutic
targets, particularly the one presented at the early-onset development of this disease.
Current therapies can be a topical artificial tear combined with the tetracycline,
corticosteroid, and/or cyclosporine A administered orally and topically. Among above
therapeutic agents, cyclosporine ophthalmic emulsion (Restasis® ) manufactured by
Allergan Inc. (Irvine, CA, U.S.A.) has been considered a “causative therapeutic approach”
in the treatment of dry eye because of its efficient interruption of inflammatory cascades
in both LG and conjunctival epithelium even though it has been reported to cause ocular
burning (17%) and conjunctival hyperemia, eye pain, epiphora, foreign body sensation,

105

scratchiness, and visual disturbance (1-5%) in the long-term regimen. Cyclosporine A is
promising for long-term use and it is the first agent targeting the pathogenesis of this
disease. Cyclosporine A inhibits T-cell activation, inflammatory cytokine production, and
apoptosis by blocking MPTP (Kunert et al., 2002). Orally administered cyclosporine has
potential side effects like high blood pressure and kidney problems in elderly people.
Tetracyclines such as doxycycline can be used to decrease ocular inflammation inhibiting
matrix metalloproteinases (MMPs) and interleukine-1 (IL-1) production for the treatment
of ocular rosacea and recurrent corneal epithelial erosions (Amin et al., 1996; Frucht-Pery
et al., 1993; Shlopov et al., 1999). Corticosteroids can be used to decrease ocular
inflammation inhibiting MMPs, adhesion molecule production, and inflammatory
cytokines. Corticosteroids should be applied for a short-term treatment because their
long-term use may provoke steroid response and raise IOP as well as cataractogenesis
(Pflugfelder et al., 2004). Adverse effects mentioned above are mainly due to the off-
target effects; therefore, there is a necessity to develop novel strategies for the accurate
drug delivery at an early-stage of this disease and for targeted therapies on basis of the
disease-specific markers.
Drug delivery vehicles that target ICAM-1 would be a feasible strategy to carry out
targeted therapy in the early phase of this disease. First of all, ICAM-1 is an important
mediator to recruit leukocytes to the site of inflammation. Even though the supportive
evidence in LGs and LGACs is still limited, current studies indicate that ICAM-1 can be
induced by pro-inflammatory factors (e.g. TNF- α) at an early stage of many diseases,
including Atherosclerosis, diabetes, and colonic ulcer (Ikeda et al., 2000; Javaid et al.,
2003). The upregulated ICAM-1 may serve as recruiters to attract immune cells and

106

amplify the inflammatory signals to exacerbate the inflammation. Additionally, this
strategy allows the targeted drug delivery system to reach its target site on basis of the
severe level of inflammation. Secondly, ICAM-1 delivers its ligands to lysosomes, where
its cargos meet overexpressed and mis-sorted proteases that are induced in the
inflammatory conditions. Although Muzykantov and Muro et al. identified the ICAM-1
mediated endocytotic pathway in endothelial cells (Muro et al., 2005), we are the first
group to develop the ICAM-1 targeting nanoparticles in epithelial cells and characterize
its intracellular trafficking pathway. Moreover, in the diseased condition inflamed cells
often exhibit enhanced protease activities in their intracellular compartments. These
proteases, such as CTSS, CTSL (cathepsin L), etc, promote the antigen presentation and
exacerbate the inflammation. Therefore, developing an ICAM-1 targeting nanoparticle
with degradability in lysosomes and drug released for specific disease markers in
subcellular compartments or cytoplasma would be an ideal therapy for this disease.
Thirdly, the pathogenesis of inflammation shares a similar mechanism; thus, by targeting
to ICAM-1, the ICAM-1 targeting carrier should have a potential to be applied in other
immune-mediated inflammations such as rheumatoid arthritis. Overall, ELP biopolymers
show compelling characteristics compared to other ICAM-1 targeting carriers.

5.2 Conclusions
This dissertation describes two innovative ELP biopolymers, KSI and ICAM-1 SI, which
target CAR and ICAM-1, respectively. Both ELP fusions retain original physicochemical
characteristics of plain ELPs, reversibly assembling into nanoparticles between their Tts.

107

By binding to LGAC CAR, KSI exhibits an unexpected basal-to-apical trafficking
pathway in LGACs compared to hepatocytes. Under the cryoTEM, KSI showed a similar
size and morphology as its SI counterpart, revealing the minimal influence from the
genetic linkage of the Ad 5 knob domain. The in vitro and in vivo KSI uptake assays
showed that part of Internalized KSI is trafficked via endosomal/lysosomal pathway;
another KSI is trafficked via Rab11a-regulated transcytotic pathway. To develop a
targeted therapy for SS-associated dry eye, ICAM-1 SI was designed to bind LGAC
ICAM-1 that is increasingly over-expressed in the basolateral membrane of inflamed
LGACs. ICAM-1 SI shows specific internalization in HeLa cells, mouse ICAM-1
expressing HeLa cells, and mouse LG acini ex vivo and in vivo. Internalized ICAM-1 SI
traffics from early endosomes to lysosomes. MicroPET imaging shows that ICAM-1 SI
has a high renal clearance and a blood half-life of 3.8 h compared to 7.2 h for its SI
counterpart. The ELP self-assembly strategy was evaluated and used to prepare bio-
functionalized targeted nanoparticles. We found that ELPs sharing similar parent
structures are able to co-assemble into multifunctional nanoparticles above the Tts.
To maximize the use of our developed ELP biopolymers, future studies are required
to optimize the in vivo LG targeting for SS-associated dry eye by (i) varying the targeting
moiety density, (ii) introducing non-biofouling/shielding moieties (e.g. PEG) onto
nanoparticle surfaces, and (iii) identifying novel peptide targeting ligands from the
binding regions of CAR and ICAM-1 using phage display techniques. The surface
coverage ratio and structural conformation of functionalized moieties are important
features for improving the targeting and shielding efficiencies. Although a well-believed
concept is that high surface ligand density and concentration of functionalized moieties

108

contribute to high targeting and shielding efficiencies and increase cellular uptake, a few
studies indicated that high ligand densities may limit the conformational flexibility of
these moieties, reducing its binding affinity, and can even increase nonspecific
interactions with other cells, triggering immunogenicity and opsonization mediated
clearance of the nanoparticles (Ferrari, 2008). Berkland et al. also reported that the
maximum uptake of his PLGA-based ICAM-1 targeting nanoparticles is occurred in the
50% surface coverage rate in A5489 cancer cells (Fakhari et al., 2011). It may be due to a
required ICAM-1 mobility on the cell surface for ligand binding and clustering and a high
density of the targeting ligands can disturb the clustering (Wulfing et al., 1998).
Moreover, scientific evidence supports that shielding materials can not only poise a steric
hindrance to avoid the nonspecific protein absorption, but they also prevent nanoparticles
from recognition by reticuloendothelial system (RES). Therefore, the surface coverage of
the nanoparticle must be optimized during the design of any nanodrug delivery system.
Furthermore, novel peptide-based targeting ligands for specific cells, tissues, and proteins
associated with specific signaling pathway should be explored. Since ICAM-1 SI was
designed to target ICAM-1 overexpressed in the inflammation, identification of novel
peptide-based targeting ligands using phage display techniques can extend the
applications of ICAM-1 SI to other inflammation-mediated autoimmune diseases.

109

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Abstract (if available)

Abstract Rapid clearance from the tears presents a formidable obstacle to the delivery of peptide drugs to the eye surface. This impedes therapies for ocular infections, wound healing, and dry‐eye disease that affect the vision of over four million Americans. To overcome this challenge, this thesis first explores a novel nanoparticle strategy to reach the ocular surface via receptor‐mediated transcytosis through the lacrimal gland (LG), responsible for the bulk of human tears. The LG abundantly expresses the coxsackievirus and adenovirus receptor (CAR)

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Asset Metadata

Creator Hsueh, Aaron Pang-Yu (author)

Core Title Trafficking of targeted elastin‐like polypeptide nanoparticles in the lacrimal gland

Contributor Electronically uploaded by the author (provenance)

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 07/22/2017

Defense Date 04/01/2015

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

Tag Coxsackievirus and adenovirus receptor,elastin‐like polypeptide,immune‐mediated inflammation,intercellular adhesion molecule-1,knob,lacrimal gland,multifunctional nanoparticle,OAI-PMH Harvest,transcytosis

Format application/pdf (imt)

Language English

Advisor Hamm-Alvarez, Sarah F. (committee chair), Mackay, John Andrew (committee member), Okamoto, Curtis Toshio (committee member), Shen, Wei-Chiang (committee member)

Creator Email pangyuhs@gmail.com,pangyuhs@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-602725

Unique identifier UC11301973

Identifier etd-HsuehAaron-3667.pdf (filename),usctheses-c3-602725 (legacy record id)

Legacy Identifier etd-HsuehAaron-3667.pdf

Dmrecord 602725

Document Type Dissertation

Format application/pdf (imt)

Rights Hsueh, Aaron Pang-Yu

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 a...

Repository Name University of Southern California Digital Library

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