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Chitosan nanoparticle mediated oral drug delivery of kidney targeting micelles for polycystic kidney disease
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Chitosan nanoparticle mediated oral drug delivery of kidney targeting micelles for polycystic kidney disease
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Content
CHITOSAN NANOPARTICLE MEDIATED ORAL DRUG DELIVERY
OF KIDNEY TARGETING MICELLES
FOR POLYCYSTIC KIDNEY DISEASE
by
Jonathan Wang
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
(Biomedical Engineering )
December 2021
Copyright 2021 Jonathan Wang
ii
Dedication
This dissertation is dedicated to firstly my PhD advisor, Dr. Eun Ji Chung. Thank you for
taking me on when starting the lab, and trusting that I could build a project from the ground up.
You have always been the model of professionalism that I strive to aim for, and will be a lifelong
role-model. The process of obtaining a PhD has been fruitful in more ways than I could have
imagined. I would also like to thank the members of the lab, especially Deborah Chin, Chris Poon,
and Noah Trac, who I have spent countless days working with, and alongside.
I would also like to acknowledge my parents, who have given me full support at every
stage of my life and have never let me down when I needed them. I would like to finally thank my
girlfriend, Fiona, who has given me even more motivation to strive for success. I could not have
accomplished this goal without all your love and support.
iii
Table of Contents
Dedication ................................................................................................................................... ii
List of Tables ............................................................................................................................ vii
List of Figures .......................................................................................................................... viii
Abbreviations ............................................................................................................................. ix
Abstract...................................................................................................................................... xi
Chapter 1: Introduction ............................................................................................................... 1
1.1 References .................................................................................................................. 3
Chapter 2: Hypothesis and Specific Aims ................................................................................... 4
2.1 Aim 1: ............................................................................................................................... 5
2.2 Aim 2: ............................................................................................................................... 5
2.3 Aim 3: ............................................................................................................................... 5
2.4 References ....................................................................................................................... 6
Chapter 3: Background .............................................................................................................. 7
3.1 History and Prevalence of Chronic Kidney Disease .......................................................... 7
3.2 Renal Physiology and Function ........................................................................................ 9
3.3 Autosomal Dominant Polycystic Kidney Disease (ADPKD) ..............................................10
3.3.1 ADPKD in the Context of the CKD Epidemic .............................................................10
3.3.2 Diagnosis and Detection of ADPKD ..........................................................................12
3.3.3 Genetics and Intracellular Signaling of ADPKD .........................................................13
3.4 Current Limitations in ADPKD Treatments .......................................................................16
3.4.1 Symptomatic Management ........................................................................................16
3.4.2 Disease Progression Modifying Treatments ..............................................................17
3.4.3 Tolvaptan Drawbacks ................................................................................................18
3.5 Drug Delivery Using Nanoparticles ..................................................................................19
3.5.1 Peptide Amphiphile Micelles .....................................................................................20
3.5.2 Nanoparticle Physiochemical Properties and Renal Clearances ...............................22
3.5.3 Targeting Ligands .....................................................................................................25
3.6 Oral Delivery ....................................................................................................................26
3.6.1 Barriers to Oral Delivery ............................................................................................27
3.6.2 Chitosan Nanoparticles .............................................................................................29
3.7 References ......................................................................................................................30
Chapter 4: Kidney Targeting Multimodal Micelles ......................................................................36
iv
4.1 Introduction, Objective, and Rationale .............................................................................36
4.2 Methods and Materials ....................................................................................................38
4.2.1 Micelle Synthesis ......................................................................................................38
4.2.2 Micelle Characterization ............................................................................................39
4.2.3 Cell Culture ...............................................................................................................40
4.2.4 In Vitro Binding ..........................................................................................................40
4.2.5 In Vitro Biocompatibility .............................................................................................41
4.2.6 In Vivo Renal Targeting .............................................................................................41
4.2.7 Histology and Immunohistochemistry ........................................................................42
4.2.8 Kidney Health Markers ..............................................................................................43
4.2.9 Statistical Analysis ....................................................................................................43
4.3 Results and Discussion ...................................................................................................43
4.3.1 Fabrication and Characterization of Micelles .............................................................43
4.3.2 KM Cell Binding to Renal Proximal Tubule Cells and Biocompatibility In Vitro ...........46
4.3.3 In Vivo Targeting and Biodistribution of KMs .............................................................47
4.3.4 Kidney Targeting to Megalin and Renal Tubule Epithelial Cells .................................50
4.3.5 Organ Morphology and Kidney Health .......................................................................52
4.4 Conclusion .......................................................................................................................53
4.5 References ......................................................................................................................54
Chapter 5: Chitosan Nanoparticles for Oral Delivery .................................................................57
5.1 Introduction, Objective, and Rationale .............................................................................57
5.2 Methods and Materials ....................................................................................................61
5.2.1 Synthesis of CS-NP ..................................................................................................61
5.2.2 Nanoparticle Characterization ...................................................................................62
5.2.3 Re-acetylation of Chitosan ........................................................................................63
5.2.4 Mucin-binding Assay .................................................................................................63
5.2.5 Drug Release and Morphological Response to pH ....................................................63
5.2.6 Cell Culture ...............................................................................................................64
5.2.7 In vitro Cell Compatibility ...........................................................................................64
5.2.8 Transepithelial Resistance (TER) Surveillance ..........................................................65
5.2.9 Cellular Uptake and Transport of CS-NP ...................................................................65
5.2.10 In Vitro Therapeutic Efficacy of CS-NP met Through ELISA and Epithelial Sodium
Channel (ENaC) Measurements ........................................................................................65
5.2.11 Ex vivo Imaging of Orally Administered CS-NP .......................................................66
5.2.12 In vivo Half-life of CS-NP .........................................................................................66
v
5.2.13 Histology and Immunohistochemistry ......................................................................67
5.2.14 Therapeutic Efficacy in ADPKD Mice ......................................................................67
5.2.15 Kidney Health in PKD Mice .....................................................................................68
5.2.16 Statistical Analysis ..................................................................................................68
5.3 Results and Discussion ...................................................................................................68
5.3.1. Fabrication and Characterization of Chitosan Nanoparticles ....................................68
5.3.2. Drug Release and Degradation Properties of CS-NP ...............................................71
5.3.3 In vitro Penetration Across Intestinal Epithelium ........................................................72
5.3.4 In vitro Therapeutic Efficacy of Metformin-loaded CS-NP ..........................................73
5.3.5 Ex vivo imaging of CS-NP in vivo and Intestinal Localization ....................................76
5.3.6 Therapeutic Efficacy of CS-NP met in PKD Mice.......................................................78
5.4 Conclusion .......................................................................................................................81
5.5 References ......................................................................................................................81
Chapter 6: Oral Delivery of Therapeutic Kidney Targeting Micelles for ADPKD .........................88
6.1 Introduction, Objective, and Rationale .............................................................................88
6.2 Methods and Materials ....................................................................................................90
6.2.1 Synthesis of Therapeutic KMs and Loaded Chitosan Nanoparticles ..........................90
6.2.2 Characterization of Micelle Loading into Chitosan Nanoparticles ..............................92
6.2.3 Drug Release of Metformin from Metformin-KMs .......................................................93
6.2.4 Cell Culture ...............................................................................................................93
6.2.5 3-D Matrigel Cell Culture of PKD1 Null and PKD1 Het Cells .....................................95
6.2.6 Transepithelial Resistance (TER) Surveillance ..........................................................95
6.2.7 Drug Release and Morphological Response to pH ....................................................95
6.2.8 In vitro Therapeutic Efficacy of CS-NP met Through ELISA and Epithelial Sodium
Channel (ENaC) Measurements ........................................................................................96
6.2.9 In vitro Therapeutic Effect of Metformin-KM and Western Blot ..................................96
6.2.10 Ex vivo Imaging of Orally Administered CS-NP .......................................................97
6.2.11 Therapeutic Efficacy in ADPKD Mice ......................................................................97
6.2.12 Histology and Immunohistochemistry ......................................................................98
6.3 Results and Discussion ...................................................................................................98
6.3.1 Loading of Micelles within Chitosan Nanoparticles ....................................................98
6.3.2 In Vitro Release of Metformin from KM met and Therapeutic Effect of KM met ....... 100
6.3.3 In Vitro Release and Therapeutic Efficacy of CS-NP KM ......................................... 102
6.3.4 Caco2 Permeation of CS-NP KM met ..................................................................... 103
6.3.5 Ex vivo Imaging of CS-NP for Biodistribution ........................................................... 104
vi
6.3.6 Therapeutic Efficacy of CS-NP KM met in Slowly Progressing ADPKD Mice .......... 105
6.4 Conclusion and Future Directions .................................................................................. 106
6.5 References .................................................................................................................... 108
Chapter 7: Epigenetic Therapeutics for Treatment of PKD: Preliminary Findings .................... 110
7.1 Introduction, Objective, and Rationale ........................................................................... 110
7.2 Methods and Materials .................................................................................................. 111
7.2.1 Synthesis of Therapeutic Micelles ........................................................................... 111
7.2.2 Cell Culture of PKD1 Null and Het Renal Cells for Cyst Studies .............................. 111
7.2.3 Biocompatibility for PKD1 Null and PKD1 Het cells ................................................. 111
7.3 Results and Discussion ................................................................................................. 112
7.3.1 Proliferation Dose Response of 5-Aza ..................................................................... 112
7.3.2 Proliferation Response of 5-Aza, Metformin, and Tolvaptan Combination Therapies
........................................................................................................................................ 113
7.4 Conclusions and Future Directions ................................................................................ 114
7.5 References .................................................................................................................... 115
Chapter 8: Conclusion ............................................................................................................. 116
8.1 Summary and Significance ............................................................................................ 116
8.2 References .................................................................................................................... 117
References ............................................................................................................................. 119
vii
List of Tables
Table 3-1 ADPKD mutation class, listed from most severe to least severe, and
expected age at ESRD
Table 4-1 Size and zeta potential of micelles
Table 4-2 Diseases with symptoms manifesting in both the kidney and liver
Table 4-3 BUN and urine creatinine levels in mice 24 h post-injection of KM, NT
micelle, or PBS
Table 5-1 Size and charge of unloaded CS-NP compared to CS-NP met
Table 5-2 Serum components, electrolytes, and kidney health markers for CS-NP met,
free met, and CS-NP orally gavaged mice
viii
List of Figures
Figure 3-1
Cross section of a human kidney, closeup of nephron structure, and cellular
composition of the renal corpuscle
Figure 3-2 Cyst formation at the level of the cell, nephron, and kidney
Figure 3-3 Schematic of primary cilium and the polycystin complex
Figure 3-4
Cellular pathways involved in ADPKD pathogenesis within a renal epithelial
cell
Figure 3-5 Tolvaptan biodistribution
Figure 3-6 Characteristic curve of the surface tension for aqueous surfactant solutions
Figure 3-7 Amphiphile structure and nanostructures based critical packing parameter
Figure 3-8 Typical size relation of nanoparticle biodistribution
Figure 3-9 In vivo biodistribution of mesoscale nanoparticles
Figure 3-10 Organ distribution study of labeled (KKEEE) 3K in mice
Figure 3-11 Anatomy of the stomach and small and large intestine barriers to oral delivery
Figure 3-12 Deacetylation of chitin to chitosan
Figure 4-1 Schematic of micelle self-assembly and the glomerular filtration barrier
Figure 4-2 Characterization of KM and NT micelles via DLS and TEM
Figure 4-3 In vitro binding of KMs vs. NT micelles
Figure 4-4 In vivo biodistribution of KM, NT, and PBS
Figure 4-5 Immunohistochemistry staining of KM and NT micelle-treated kidneys
Figure 4-6 Tissue morphology post-injection of KMs, NT micelles, or PBS
Figure 5-1 Schematic of the physiological barriers for orally delivered nanoparticles
Figure 5-2 Optimization of CS-NP synthesis parameters and mucin binding efficiency
Figure 5-3 In vitro pH response of CS-NP
Figure 5-4 In vitro transport mechanisms and therapeutic efficacy of CS-NP
Figure 5-5 Biodistribution of mice orally gavaged with CS-NP R and free R
Figure 5-6 Intestinal localization of CS-NP R and free R after oral gavage
Figure 5-7 In vivo therapeutic efficacy of CS-NP met
Figure 6-1 Schematic of an orally delivered therapeutic kidney targeting micelle system
Figure 6-2 Characterization of KM loading into CS-NP
Figure 6-3 Drug release and in vitro therapeutic effect of KM met
Figure 6-4 In vitro release of KM from CS-NP and Caco2 permeation
Figure 6-5 Biodistribution of CS-NP KM Cy7 met
Figure 6-6 In vivo therapeutic efficacy of CS-NP met
Figure 7-1 Proliferation dose response of PKD1 Het cells to 5 Aza
Figure 7-2
Proliferation of PKD1 Het cells treated with 5 Aza, RG 108, TSA combined
with either metformin or Tolvaptan
ix
Abbreviations
AC6 Calcium-inhibitable Adenylyl Cyclase
ACR Albumin to Creatine Ratio
ADPKD Autosomal Dominant Polycystic Kidney
Disease
AKI Acute Kidney Injury
AMPK AMP-Activated Protein Kinase
ANOVA Analysis of Variance
BSA Bovine Serum Albumin
BUN Blood Urea Nitrogen
cAMP Cyclic AMP
CFTR Cystic Fibrosis Transmembrane
Conductance Regulator
CKD Chronic Kidney Diseases
CMC Critical Micelle Concentration
CRISP Consortium for Radiologic Imaging Studies of
Polycystic Kidney Disease
CT Computed Tomography
DAPI 4’,6-diamidino-2-phenylindole
DLS Dynamic Light Scattering
DOX Doxorubicin Hydrochloride
DSPE PEG
2000
1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-methoxy-
poly(ethylene glycol 2000)
ECM Extracellular Matrix
eGFR Estimated Glomerular Filtration Rate
ESRD End Stage Renal Disease
FDA Food and Drug Administration
GFB Glomerular Filtration Barrier
GI Gastrointestinal
H&E Hematoxylin & Eosin
HALT-PKD Halt Progression of Polycystic Kidney
Disease
IV Intravenous
KM Kidney Targeting Peptide Amphiphile Micelle
MALDI-
TOF/TOF
Matrix-Assisted Laser Desorption Ionization
Time-of-Flight
MAPK Mitogen Activated Protein Kinase
MPS Mononuclear Phagocytic System
MRI Magnetic Resonance Imaging
NIH National Institutes of Health
NMD No Mutation Detected
OCT Optimum Cutting Temperature
x
PAM Peptide Amphiphile Micelle
PBS Phosphate Buffered Saline
PC1 Polycystin-1
PC2 Polycystin-2
PCR Urine Protein to Creatinine Ratio
PDE1 Calcium/Calmodulin-Dependent
Phosphodiesterase 1
PEEK Polyether Ether Ketone
PEG Polyethylene Glycol
PET/CT Positron Emission Tomography/Computed
Tomography
PI3K/AKT/mTOR Phosphoinositide 3-kinase, Protein Kinase B,
Mammalian Target of Rapamycin
PKA Protein Kinase A
PKD Polycystic Kidney Disease
PLGA Poly(lactic-co-glycolic acid)
PVDF Polyvinylidene Fluoride
QDs Quantum Dots
RAS Renin Angiotensin Aldosterone System
REPRISE Replicating Evidence of Preserved Renal
Function: An Investigation of Tolvaptan
Safety and Efficacy in ADPKD
RPTEC Renal Proximal Tubule Epithelial Cells
TBS Tris Buffered Saline
TEER Trans Epithelial Electric Resistance
TEM Transmission Electron Microscopy
TEMPO Tolvaptan Efficacy and Safety Study in
Autosomal Dominant Polycystic Kidney
Disease
TKV Total Kidney Volume
TPP Tripolyphosphate
WGA Wheat Germ Agglutinin
xi
Abstract
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited
disorder of the kidneys and affects 12 million people worldwide. It is characterized by uncontrolled
cyst growth in renal tissue, which destroys kidney filtration function and leads to kidney failure.
Traditional clinical solutions have focused primarily on management of the symptoms, while cysts
continued to grow. Only recently in 2018, Tolvaptan was approved by the Food and Drug
Administration (FDA) as the first drug to slow cyst growth. However, Tolvaptan is difficult to
tolerate, expensive, and may cause liver injury in patients, which has led to discontinuation of
treatment by many patients. We hypothesize that the utilization of nanomedicine strategies,
specifically a kidney targeting peptide amphiphile micelle (KM), can enhance accumulation of
drugs like Tolvaptan to the kidneys, increasing therapeutic efficacy while reducing off-target side
effects. Unfortunately, almost all FDA approved nanoparticles have relied on intravenous (IV)
injection. However, this method of administration is not suitable for chronic diseases that require
lifelong therapy like ADPKD. Oral delivery is the route associated with the highest patient
compliance, but it also presents physiological barriers that may degrade or limit drugs and other
payloads. Thus, we develop a biomaterials strategy using chitosan to deliver drugs and KMs into
systemic circulation via oral delivery. Together, we encapsulate drug-loaded KMs into chitosan
nanoparticles, which has promise in the treatment of ADPKD.
1
Chapter 1: Introduction
Chronic kidney disease (CKD) rates are rising in the United States and abroad. It is
predicted that CKD affects about 10% of the world’s adult population [1], and may increase to 16%
by 2030 [2]. If left unchecked, CKD patients will eventually reach kidney failure and need one of
two last resort treatments: Renal transplantation or dialysis. Less than half of the people in need
of renal replacement are receiving donor kidneys worldwide [3], but the cost of dialysis is $89,000
per patient annually in the United States. This means these solutions are simply inaccessible or
unaffordable in many parts of the world [4]. Therefore, massive healthcare burdens, both in terms
of economic load and patient quality of life, can be improved with new treatments to avoid kidney
failure.
One such disease under the category of CKD is autosomal dominant polycystic kidney
disease (ADPKD). ADPKD is the most common inherited genetic disorder of the kidneys, and
affects 12 million individuals worldwide [5]. It is characterized by uncontrolled cyst growth in the
kidneys which destroys kidney filtration function. Only one marketed drug, Tolvaptan, approved
by the Food and Drug Administration (FDA) in 2018, is available to slow the progression of this
disease. However, Tolvaptan is costly, difficult to tolerate, and can cause severe liver damage in
certain patients [6]. Thus, improving the pharmacokinetic properties of drugs like Tolvaptan to
increase its therapeutic efficacy and reduce off-target side effects has the potential to improve
patient outcomes in ADPKD.
Towards these goals, in Aim 1, we begin this project by describing the utilization of peptide
amphiphile micelles as a nanoparticle drug delivery system to target the kidneys. We evaluate
the biocompatibility and targeting specificity of kidney targeting peptide amphiphile micelles (KM)
in vitro. Furthermore, we show enhanced accumulation of this targeted nanoparticle in an in vivo
2
biodistribution study [7]. Nephron localization is probed, while biocompatibility and kidney health
markers are assessed.
While peptide amphiphile micelles have been primarily administered intravenously (IV),
such a delivery route is not ideal for translation to the clinic for chronic diseases such as ADPKD
[8]. ADPKD will require repeated dosing over the course of a lifetime and patient compliance has
been reported to decrease for difficult or painful administration routes [9, 10]. Oral delivery is the
most desired route with respect to patient compliance, convenience, and ease of clinical execution.
Towards oral delivery of KM, in Aim 2, we develop chitosan-based nanoparticles as an oral
delivery platform for ADPKD. Chitosan is an FDA approved polysaccharide for drug delivery and
tissue engineering applications, and has shown promising results as a gastrointestinal (GI) tract
permeation enhancer [11]. We investigate the ability of chitosan nanoparticles to penetrate an in
vitro intestinal model and probe the release profile of candidate payload drugs in pH conditions
relevant to the GI tract. We then present in vivo results on the delivery of a model drug payload
to mice via oral gavage. To conclude, a candidate PKD drug, metformin, is loaded into chitosan
nanoparticles to be orally delivered into a rapidly progressing model of PKD and is shown to be
more efficacious than orally delivered free metformin.
Finally, we combine the insights from our first and second aims, and encapsulate drug-
loaded, therapeutic kidney targeting micelles (KM met) within chitosan nanoparticles as an oral
delivery system for the treatment of ADPKD in Aim 3. We present the therapeutic efficacy of drug-
loaded micelles, and the characterization process of micelles loaded within chitosan nanoparticles.
Our studies will culminate with the treatment of PKD mouse models with orally delivered KM met
within chitosan nanoparticles, and findings regarding an improvement in cyst size reduction and
preservation of kidney function. Such results show promise for the translation of orally delivered
nanomedicine for the treatment of ADPKD in the clinic. Looking ahead, a variety of investigative
drugs can be incorporated into our orally delivered, kidney targeting nanomedicine strategies for
3
chronic kidney conditions beyond PKD. We set a framework towards a nanomedicine strategy
with a goal of reducing the global burden of chronic kidney disease.
1.1 References
1. Haileamlak, A., Chronic Kidney Disease is on the Rise. Ethiopian journal of health
sciences, 2018. 28(6): p. 681-682.
2. Ladan Golestaneh, M., MS; Paula J. Alvarez, RPh, , All-Cause Costs Increase
Exponentially with Increased Chronic Kidney Disease Stage. Am J Manag Care, 2017.
23(S0).
3. Jha, V., et al., Chronic kidney disease: global dimension and perspectives. The Lancet,
2013. 382(9888): p. 260-272.
4. 2015 USRDS annual data report: Epidemiology of Kidney Disease in the United States,
N.I.o.D.a.D.a.K.D. National Institutes of Health, Editor. 2015: Bethesda, MD.
5. Chapman, A.B., et al., Autosomal-dominant polycystic kidney disease (ADPKD):
executive summary from a Kidney Disease: Improving Global Outcomes (KDIGO)
Controversies Conference. Kidney international, 2015. 88(1): p. 17-27.
6. Torres, V.E., et al., Tolvaptan in Patients with Autosomal Dominant Polycystic Kidney
Disease. New England Journal of Medicine, 2012. 367(25): p. 2407-2418.
7. Wang, J., et al., Design and in vivo characterization of kidney-targeting multimodal
micelles for renal drug delivery. Nano Research, 2018. 11(10): p. 5584-5595.
8. Chung, E.J., et al., In vivo biodistribution and clearance of peptide amphiphile micelles.
Nanomedicine: Nanotechnology, Biology and Medicine, 2015. 11(2): p. 479-487.
9. Pridgen, E.M., F. Alexis, and O.C. Farokhzad, Polymeric nanoparticle technologies for oral
drug delivery. Clinical gastroenterology and hepatology : the official clinical practice journal
of the American Gastroenterological Association, 2014. 12(10): p. 1605-1610.
10. Bernkop-Schnürch, A., Reprint of: Nanocarrier systems for oral drug delivery: Do we really
need them? European Journal of Pharmaceutical Sciences, 2013. 50(1): p. 2-7.
11. Mohammed, M.A., et al., An Overview of Chitosan Nanoparticles and Its Application in
Non-Parenteral Drug Delivery. Pharmaceutics, 2017. 9(4): p. 53.
4
Chapter 2: Hypothesis and Specific Aims
The goal of emerging therapies for ADPKD is to develop treatments that reduce or reverse
the progression of cyst expansion and preserve function in the kidneys [12]. This will be a
paradigm shift from current clinical management which comprises mainly of symptomatic
management. There is only one FDA approved drug, Tolvaptan, which can be prescribed to
reduce the rate of cyst growth and protect kidney function [13]. However, this drug is not only
expensive but is prone to side effects such as liver toxicity, nausea, constant thirst and polyuria.
To develop alternative strategies for ADPKD treatment, peptide amphiphile micelles as a drug
delivery platform can be utilized as a solution to these shortcomings [14, 15]. We synthesize
micelles with physiochemical properties such as diameter and charge that favor kidney
accumulation and uptake into renal tubule cells [16]. We hypothesize that the targeting ability of
micelles can be improved by incorporating kidney targeting peptide ligands conjugated to the
surface of the particle, which can be used to enhance drug delivery to diseased kidneys.
However, like nearly all nanoparticles used clinically, micelles have been primarily
administered intravenously, which is impractical for chronic diseases like ADPKD, which often
take decades to progress and will need lifelong repeated dosing [17]. To meet this practical
demand for clinical translation, we aim to develop a chitosan nanoparticle suitable for delivering
payloads across the gastrointestinal (GI) tract. Chitosan is a widely available, biocompatible
polysaccharide that has shown promise in enhancing absorption by increasing para- and
transcellular GI tract permeability. We hypothesize our nanocarrier will successfully function as
an oral delivery platform by protecting payload drugs from enzymatic degradation and allow for
higher permeation of the intestinal epithelium compared to free drug.
Finally, we hypothesize by encapsulating micelles into larger chitosan nanoparticles, we
will develop a suitable oral delivery strategy to reduce cyst growth in PKD mice models.
5
To address these hypotheses, we present the following aims:
2.1 Aim 1:
We aim to synthesize kidney targeting peptide amphiphile micelles (KM) and evaluate
their binding and kidney accumulation properties in vitro and in vivo. Specifically, we 1) fabricate
KMs with a targeting peptide sequence, (KKEEE)3K, and characterize particle diameter and
charge. We assess 2) in vitro biocompatibility and targeting peptide specificity with renal proximal
tubule cells. 3) We then evaluate localization of our nanoparticle in vivo through intravenous (IV)
injection in wild type C57BL/6 mice, and nephron localization of our nanoparticles using
immunohistochemistry. The results from this aim are provided in Chapter 4.
2.2 Aim 2:
We aim to develop chitosan nanoparticles towards oral delivery of small molecule drugs
for autosomal dominant polycystic kidney disease (ADPKD). Specifically, we 1) synthesize and
characterize chitosan nanoparticle formulations and assess the loading efficiency, degradation,
and release profiles of a candidate ADPKD drug payload. We will 2) validate the feasibility for
successful oral delivery of our nanoconstructs through an in vitro Caco 2 Transwell model. 3) The
therapeutic potency of drug loaded chitosan nanoparticles administered via oral gavage to PKD
murine models is evaluated by cyst burden reduction. The results from this aim are provided in
Chapter 5.
2.3 Aim 3:
Combining the developments from Aim 1 and Aim 2, we provide a strategy for oral delivery
of kidney targeting micelles. Specifically, we 1) investigate the morphology and loading of micelles
in chitosan nanoparticles and probe the degradation and release profiles. We 2) evaluate the
effectiveness of drug-loaded micelles using an in vitro Caco 2 cell GI tract model as a barrier over
cortical collecting duct cells. 3) Moreover, we validate that oral gavage administration of CS-NP
6
KMs to PKD murine models reduces cyst burden to a greater extent than orally delivered free
drug. The results from this aim are provided in Chapter 6.
2.4 References
12. Chebib, F.T. and V.E. Torres, Recent Advances in the Management of Autosomal
Dominant Polycystic Kidney Disease. Clinical Journal of the American Society of
Nephrology, 2018. 13(11): p. 1765.
13. Bennett, H., et al., Modelling the long-term benefits of tolvaptan therapy on renal function
decline in autosomal dominant polycystic kidney disease: an exploratory analysis using
the ADPKD outcomes model. BMC nephrology, 2019. 20(1): p. 136-136.
14. Chung E, P.F., Nord K, Karczmar, Lee SK, et al., Fibrin- Targeting, Peptide Amphiphile
Micelles as Contrast Agents for Molecular MRI. J Cell Sci Ther, 2014. 5(181).
15. Poon, C., et al., Protein Mimetic and Anticancer Properties of Monocyte-Targeting Peptide
Amphiphile Micelles. ACS biomaterials science & engineering, 2017. 3(12): p. 3273-3282.
16. Zhou, P., X. Sun, and Z. Zhang, Kidney-targeted drug delivery systems. Acta
pharmaceutica Sinica. B, 2014. 4(1): p. 37-42.
17. Hamman, J.H., G.M. Enslin, and A.F. Kotzé, Oral Delivery of Peptide Drugs. BioDrugs,
2005. 19(3): p. 165-177.
7
Chapter 3: Background
3.1 History and Prevalence of Chronic Kidney Disease
The kidneys are crucial organs that maintain homeostasis in the body. By filtering the
blood, they remove wastes and maintain a balance of water and electrolytes. Additional hormonal
functions allow the kidneys to influence blood pressure, calcium metabolism, and red blood cell
production [18]. Thus, healthcare issues associated with such integral organs are of importance
in the United States and abroad.
Since the 1980’s, there has been a steady increase in the number of patients that need
renal replacement treatments, such as dialysis and kidney transplantation, due to renal failure
[19]. As hemodialysis treatment costs an average of $89,000 per patient annually in the United
States, any interventions that might lead to a decrease in end stage renal disease (ESRD), is
worthwhile economically [20]. Hemodialysis involves pumping a patient’s blood through an
external artificial filtration unit before blood is pumped back into the body. A typical hemodialysis
appointment is time consuming, averaging about three sessions per week, for 3-5 hours per
session at a medical facility. Dialysis is also physically exhausting for patients and is haunted by
eventual mortality, as the annual mortality rate of patients on hemodialysis in the United States is
24.3% [21].
While kidney transplantation offers freedom from dialysis, there is an intense shortage of
donor organs for ESRD patients. Well over 100,000 ESRD patients are on the U.S. transplant
wait-list, but only 20,161 kidney transplants were performed in the United States in 2016 [22]. The
need for donor kidneys will rise at an expected 8% per year, while donor availability will not match
the increase [22]. Additionally, transplant recipients must take immunosuppressants for the
remainder of their lives to avoid rejection of the donor organ, which increases risk of infections
and cancer.
8
Surprisingly, the growth of the chronic kidney disease (CKD) epidemic was unchecked
until the year 2005, when the Department of Health in the United Kingdom published The National
Service Framework for Renal Services [23]. This document mandated general practitioners
become involved in the management of kidney health. Prior to this policy, patients with blood work
showing kidney impairment were immediately referred to nephrologists, which administered care
without interaction with the general practitioner [19]. Since then, other organizations have
stressed the validity of early monitoring of kidney health in standard care to stem the growing
epidemic of ESRD. The Renal Association in 2010, and the international Kidney Disease
Improving Global Outcomes in 2012 have both published their own guidelines, demonstrating
global realization of this need [24].
Despite the improvement of early diagnosis and kidney health monitoring, CKD is still a
common disorder and has become a major public health concern leading to ESRD in the United
States, affecting an estimated 13.6% of the adult population. It is predicted that CKD prevalence
in adults aged ≥30 years will increase 16.7% by 2030 [2]. CKD patients, even in early disease
stages, carry a disproportionate burden of cardiovascular morbidity, mortality, healthcare
utilization and costs. If we are to slow or reverse CKD’s burden of disease over the next 25 years,
it is imperative that we develop new strategies towards kidney disease treatment. Unfortunately,
the National Institutes of Health (NIH) funding for kidney disease research is inadequate. NIH
spends far less on kidney research per patient (approximately $29/patient/year) than on cancer
($568/patient/year), and HIV/AIDS ($3064/patient/year) [25]. However, it is these novel treatment
breakthroughs, such as drug delivery platforms that halt or slow the progression of CKD, that can
provide tangible patient outcomes in quality of life and reduce rates of CKD [26]. Investigation of
such drug delivery solutions to reduce CKD burden is needed and are explored in this proposal.
9
3.2 Renal Physiology and Function
The ability of the kidneys to effectively filter blood ties closely with the structure of the
kidney, specifically with the repeating filtration unit of the kidney, the nephron. (Figure 3-1) [27].
Figure 3-1 Cross section of a human kidney (left), closeup of nephron structure (middle), and
cellular composition of the renal corpuscle (right). Major physiological structures are labeled.
Adapted and reprinted from and [27] and [28] with permission from Elsevier.
Nephrons are organized radially outwards from the center of each kidney. The first renal
structure that unfiltered blood from the renal artery encounters is the glomerulus. The glomerulus
is comprised of a cluster of capillaries surrounded by a structure composed of renal tubular cells
known as the Bowman’s capsule, which together, form the renal corpuscle. The renal corpuscle
is lined with parietal epithelial cells and serves as a reservoir for renal progenitor cells [29]. The
interstitial spaces within the glomerular capillaries are occupied with mesangial cells, which
provide the maintenance of capillary organization and support filtration processes for glomerular
endothelial cells [30].
The glomerulus, proximal, and distal tubules reside mostly in the outer layers, or cortex,
of the kidney. Nephrons are further subdivided based on which portion of the cortex the associated
10
renal corpuscle is located: The corpuscle of a superficial nephron is located in the outer region of
the cortex, while juxtamedullary types may exist near the outer portions of the medulla. (Figure
3-1). The Loop of Henle and collecting ducts of both types of nephrons extend into deeper layers
of the medulla.
The glomerular filtration barrier (GFB), which is responsible for the selective permeation
of components from the blood into the Bowman’s capsule, surrounds these endothelial and
mesangial cells. Podocytes form interdigitating foot processes along the glomerular basement
membrane, creating 4-11 nm slits that prevent the passage of large macromolecules. It is the
presence of this key physiochemical filtration at the GFB, paired with reabsorption of the filtrate
in the proximal and distal tubules, which allows the kidneys to constantly eliminate waste from the
blood [31].
3.3 Autosomal Dominant Polycystic Kidney Disease (ADPKD)
3.3.1 ADPKD in the Context of the CKD Epidemic
As previously mentioned, CKD is a major public health issue in the United States. The
epidemic of CKD has risen into the top ten leading causes of death in the United States over the
past 30 years. In 2017, one in seven US adults, or roughly 30 million individuals, were estimated
to have CKD [32]. Under the class of CKD, autosomal dominant polycystic kidney disease
(ADPKD) is the most common inherited cause of ESRD in adults and accounts for 10% of all
ESRD [33]. This condition manifests fluid fill sacs, or cysts, originating from diseased tubular cells
that enlarge and destroy healthy nephron architecture (Figure 3-2).
Currently, late stage CKD is diagnosed as either:
1. The estimated glomerular filtration rate (eGFR) is less than 60 ml/min on at least two
occasions over a period of no less than 90 days [34].
11
2. The urine albumin to creatine ratio (ACR) is greater than 30mg/mmol or the urine protein
to creatinine ratio (PCR) is greater than 50 mg/mmol [35].
This contrasts with acute kidney injury (AKI), which is usually caused by an event that
leads to kidney malfunction, such as dehydration, renal ischemia from major surgery or injury, or
nephrotoxic drugs causing drastic eGFR changes within the timescale of weeks. CKD, on the
other hand, is usually caused by a long-term disease, such as high blood pressure or diabetes,
tubulointerstitial diseases, glomerulonephritis, and cystic kidney diseases that slowly damage the
kidneys over time [36, 37].
With inadequate kidney function, excess wastes and fluid accumulate in the bloodstream
and tissues, which increases the comorbidity of heart disease, stroke, hypertension, and
respiratory issues [38]. To mitigate the symptoms of CKD, high doses of small molecule drugs
are often prescribed to overcome fast elimination times, which can also increase the risks of
adverse side effects. For instance, angiotensin-converting enzyme inhibitors used to treat
hypertension caused by CKD may induce hyperkalemia, or high blood potassium levels [39]. The
non-steroidal anti-inflammatory drugs used to manage pain can cause tubulointerstitial nephritis,
or inflammation to tubular cells [40]. Such drawbacks can be improved upon with drug delivery
systems that change the pharmacokinetic and pharmacodynamic properties of a drug, which will
be a focus of this work.
12
3.3.2 Diagnosis and Detection of ADPKD
Figure 3-2 Cyst formation at the level of the cell, nephron, and kidney, from right to left. Genetic
aberrations in the genes coding for PC1 or PC2 lead to increased cell proliferation, and fluid
secretion, which manifests fluid-filled cysts. As cysts enlarge, normal renal parenchyma is
compressed and reduced functional capacity. Adapted and reprinted from [41] with permission
from Rockefeller University Press.
Without prior knowledge of family history, ADPKD is often indicated upon the discovery of
either hypertension, the sudden onset of renal pain/hematuria, or the inadvertent discovery of
through physical or radiological examinations [42]. The decrease in kidney function usually only
occurs during late stages of cyst formation, when the patient is middle aged. Even though cyst
progression begins at birth, results from magnetic resonance imaging (MRI) and computed
tomography (CT) scans indicate that functioning renal tissue is lost many years before the actual
decline in GFR can be verified. Highly efficient compensatory hyperfiltration by surviving noncystic
nephrons maintain the GFR within a normal range, despite the massive buildup of cysts and the
loss of functioning parenchyma [43].
In the clinic, the presence of ADPKD when there is a known positive family history is
usually confirmed by renal ultrasound imaging. In the seminal Consortium for Radiologic Imaging
Studies of Polycystic Kidney Disease (CRISP) study, a strong relationship between kidney volume
at the beginning of the study and the subsequent change in the GFR was seen. Therefore, patient
height adjusted total kidney volume is now used for prognostication [44]. Furthermore, severity of
disease progression is classified in the Mayo Clinic Imaging Classification (MCIC) in 2014 [45],
13
which validated its classifier using the CRISP imaging data. This was the first of many clinical
studies to effectively give a predictor for the severity of ADPKD through imaging, which armed
clinicians with a tool to assess PKD progression in an affordable manner. Genetic testing,
although useful as an additional tool, is very costly in comparison, and may fail in identifying
mutations in about 10% of patients [12, 46]. Even though genetics can predict some phenotypic
aspects of the specific mutation, the eventual disease severity in individuals can vary quite
drastically from the average genetic trend. Even within families possessing the same type of
mutation, the age of ESRD can vary quite greatly [47].
3.3.3 Genetics and Intracellular Signaling of ADPKD
ADPKD has two main genetic loci identified, PKD1, which encodes for the polycystin-1
(PC1) protein, and PKD2 which encodes polycystin-2 (PC2) [48]. PKD1 mutations account for
approximately 78% of patients, and PKD2 for approximately 13%, with no mutation detected
(NMD) in approximately 9% of cases [49]. PKD2 mutation patients have a much milder phenotype
of kidney cysts compared to PKD1, and has a delayed onset of ESRD of ~20 years [50]. It is
unclear whether the NMD cases are caused by currently undetectable changes in PKD1 and
PKD2, or a third genetic loci exists [51, 52]. To date, there are more than 1,272 PKD1 and 202
PKD2 aberrant mutations reported [46], but there are five main categories that can give a
prediction of disease severity and time to onset of ESRD, listed in Table 3-1, ordered from most
severe to least severe cystic phenotype.
Mean Age at ESRD Ref
Protein truncating PKD1
mutation
53.4 [46]
In frame insertion deletion
mutation
58.6 [47, 51]
Non protein truncating PKD1
mutation (some PKD1
expression)
65.8 [46, 53]
PKD2 mutation 72.7 [46, 50, 53]
No detectable mutation 77.5 [47]
14
Table 3-1 ADPKD mutation class, listed from most severe to least severe, and expected age at
ESRD.
These polycystin proteins are essential to maintain the differentiated phenotype of renal
tubular epithelial cells in contact with filtrate flow. When either of these proteins are expressed at
too low a level, the renal cell undergoes a phenotypic switch resulting in the inability to maintain
planar polarity and increases rates of proliferation, apoptosis, fluid secretion, and extracellular
matrix (ECM) remodeling [54]. PC2 is a transmembrane, calcium-responsive cation channel [55].
PC1 also has a transmembrane portion, but additionally has domains extending into the
cytoplasm [48]. PC1 and PC2 are thought to interact via their C-terminal tails in a 1:3 (PC1:PC2)
ratio, forming the polycystin complex (PC) [56]. The PC complex structure was recently solved
using cryogenic electron microscopy (2018) (Figure 3-3) [57]. Several different subcellular
localizations of the PC complex have been studied, but it is currently hypothesized that the
pathways responsible for PKD pathogenesis stem from PC complexes residing on the primary
cilia of cells [58, 59]. Primary cilia are thin extensions of the plasma membrane that play a role in
sensing mechanical and chemical extracellular signals and drive intracellular calcium signaling.
Figure 3-3 A) Schematic of the primary cilium located on the outer membrane of renal tubule
cells. B) Topological illustration of PKD1 and PKD2 associating in the PC complex in a 1:3 ratio,
solved structure using cryo-electron microscopy. Adapted and reprinted from [60] and [57] with
permission from the American Association for the Advancement of Science.
15
Many downstream cellular signaling pathways are aberrant in ADPKD (Figure 3-4). Briefly,
low levels of the PC on the primary cilia and endoplasmic reticulum (ER) reduce the amount of
intracellular calcium available through the PC2 calcium channel. This triggers g-protein coupled
activation of calcium-inhibitable adenylyl cyclase 6 (AC6) and inhibition of calcium/calmodulin-
dependent phosphodiesterase (PDE), causing enhanced accumulation of cyclic AMP (cAMP) and
activation of protein kinase A (PKA) [61]. cAMP/PKA signaling regulates a network of downstream
pathways that affect cell cycle progression, cell proliferation, energy metabolism, and cell death
[62]. It is postulated that the upregulation of the mitogen-activated protein kinases (MAPK)
pathway, and phosphoinositide 3-kinases, protein kinase B, mammalian target of rapamycin
(PI3K/AKT/mTOR) pathways through the inhibition of tuberous sclerosis complex proteins (TSC1
and TSC2) drive aerobic glycolysis and cell cycle progression. The MAPK pathway also drives
caspase-dependent cell death [63], even though abnormally increased proliferation in renal tubule
cells is the hallmark of PKD [64]. The increased levels of apoptosis can destroy healthy nephron
filtration function, while heightened proliferation of cyst lining cells drives cyst progression and
expansion [65]. Downregulation of the AMP-activated protein kinase (AMPK) pathway stimulates
ion transport through the cystic fibrosis transmembrane conductance regulator (CFTR) and fluid
secretion, driving cyst formation [66]. The precise function of the PC complex on the cilium is an
ongoing debate and remains an unresolved issue; for a comprehensive review of molecular
mechanisms, refer to the textbook by Li et al. [66]. Additionally, for a current discussion of peptide
based treatment strategies to address misregulated cellular signaling and metabolic pathways in
PKD, refer to our review by Wang et al. [67].
16
Figure 3-4 A) Schematic of the primary cilium located on the outer membrane of renal tubule
cells. Cellular pathways involved in ADPKD pathogenesis within a renal epithelial cell. A) PC1 or
PC2 loss of function on the primary cilia and ER impairs the calcium transient response, resulting
in intracellular cAMP accumulation by preventing PDE degradation of cAMP. B) V2R activation is
a major source of cAMP production in the cell through g-protein coupled activation of AC-VI. C)
Ras dependent EGFR activation also lowers intercellular calcium. D) The response to abnormally
increased cAMP levels leads to an activation of B-Raf/MAP2K1/ERK and inhibition of TSC1 and
TSC2, driving the cell towards aerobic glycolysis and cell cycle progression. E) Other alterations
include loss of planar cell polarity through Wnt dysregulation, and downstream β-catenin-
dependent nuclear changes can drive cell de-differentiation into a proliferative phenotype. F)
Master regulators of cellular energy metabolism, including the AMPK and mTOR pathways are
mis-regulated, further enhancing proliferation. G) Altered CFTR behavior generates a secretory
cell phenotype, in contrast to the normal absorptive phenotype, while TNF-α is an upregulated
inflammatory mediator of the PKD phenotype. Proteins and receptors upregulated in PKD are
highlighted in blue, and those downregulated are highlighted in yellow. Potential therapeutic
targets have been listed in red next to the relevant receptor or misregulated protein. Adapted and
reprinted from [67].
3.4 Current Limitations in ADPKD Treatments
3.4.1 Symptomatic Management
Until very recently, measures to treat ADPKD followed generic chronic kidney disease
management strategies. These include a protein restricted diet, blood pressure control, and
inhibiting the renin angiotensin aldosterone system (RAS) [68]. However, clinical trials studying
17
these effects in ADPKD have shown minimal delays in the age of ESRD onset due to cyst
progression. In fact, a protein restricted diet was demonstrated by Klahr et al. [69] to have no
effect to postpone ESRD in ADPKD patients. Hypertension is associated with ADPKD as well, as
expanding cysts compress healthy nephrons which activates the RAS response to increase blood
pressure. First-line management of hypertension should include a drug which is a blocker of RAS
[70]. In patients with cyst infection, a positron emission tomography/computed tomography
(PET/CT) scan is recommended to enable determination of the location of cysts for the purpose
of diagnosis or drainage of cyst fluid through surgical aspiration [71]. Common antibiotics such as
ciprofloxacin are prescribed, but face the common drawbacks of widespread antibiotic use,
including hepatotoxicity as well as increasing the risk of bacterial resistance to antibiotics [72].
3.4.2 Disease Progression Modifying Treatments
While symptomatic management for ADPKD patients is necessary to improve quality of
life, it does not affect the progression of the disease and cannot prevent the eventual onset of
ESRD. Therefore, following the CRISP clinical trial in 2001, much effort has been given to finding
treatments that slow the growth of cystic kidneys and the deterioration of renal function. mTOR
pathway inhibitors have been prime candidates in clinical trials to slow the proliferation of aberrant
renal tubule cell proliferation. However, trials with Everolimus [73] and Sirolimus [74] appear to
show the slowing of increased total kidney volume, but did not correlate with any improvement in
GFR [75].
Vasopressin antagonist drugs such as Tolvaptan and Somatostatin have shown some
promise in slowing down cyst progression. The Tolvaptan Efficacy and Safety Study in Autosomal
Dominant Polycystic Kidney Disease (TEMPO) trials showed in a 3-year period, the increase in
total kidney volume in the tolvaptan treatment group was 2.8% per year, versus 5.5% per year in
placebo. Tolvaptan was also associated with a slower decline in kidney function, estimated by the
18
reciprocal of the serum creatinine concentration, which was −2.61 mg/mL per year in treated vs.
−3.81 mg/mL per year in the control group [6].
3.4.3 Tolvaptan Drawbacks
Tolvaptan is the only FDA approved drug currently available for ADPKD patients that has
shown to slow the decline of total kidney volume and kidney function. However, Tolvaptan is
restricted for patients that have a high risk for ESRD. The previously discussed MCIC imaging
classification gives some guidelines on the rate of total kidney volume (TKV) progression and
classification as high risk [45]. About 7% of the patient population will be excluded however as
the imaging classification is only reliable for patients with diffuse and uniformly distributed, or Type
1 cysts. Patients with a Type 2 presentation, or when cysts occur only in one kidney, or are
concentrated in one region of the kidney, are excluded from receiving Tolvaptan treatment. This
leaves the use of Tolvaptan as fairly subjective based on clinician discretion.
More importantly, the side effects of tolvaptan will be a major problem for some patients.
The Replicating Evidence of Preserved Renal Function: An Investigation of Tolvaptan Safety and
Efficacy in ADPKD (REPRISE) study, which investigated long term safety of the drug, pre-
selected their participants based upon ability to tolerate the drug from previous shorter length
trials, but still had a 9.5% dropout rate (versus 2.2% in the placebo group) [76]. The main reported
side effects included dry mouth, polyuria, hypernatremia, nausea/vomiting, and abdominal
pain/cramps [77]. In fact, the polyuria effects are so prominent that many clinicians do not
recommend Tolvaptan to patients living lifestyles without unrestricted access to the restroom,
such as truck drivers and nurses. In unselected ADPKD patients, this side effect and dropout rate
would presumably be substantially higher, as it was in the prior TEMPO study, which carried a
23% dropout [6]. Deranged liver function tests have also been detected in both the TEMPO and
REPRISE studies, which are potentially life-threatening side effects. Finally, there is only a
19
modest prediction of benefit: adherence to Tolvaptan treatment for 18 years is predicted to delay
the time to ESRD by 4.9 years [13].
Indeed, when one observes the 24 hour biodistribution of orally administered C14
radiolabeled Tolvaptan, accumulation in the liver is 23.8%, vs. a much lower 5.71% in the kidneys
(Figure 3-5) [78]. A large proportion of the drug also still resides in the intestines and partitioned
into the fat. Thus, a significant amount of free drug is not inducing a therapeutic effect on the
kidneys.
Figure 3-5 Oral biodistribution of Tolvaptan after 24 hours [78].
3.5 Drug Delivery Using Nanoparticles
Nanotechnology involves the engineering of materials at the submicron scale, and in the
context of medicine, the unique physiochemical properties of matter at this scale can be
harnessed to improve treatment outcomes [28]. Broadly, nanostructures can have tuned
physiochemical properties, such as size and surface charge, which allow for organ specific uptake
[7]. Nanocarriers can be synthesized from biodegradable substances and therefore can be
degraded or exit the body after performing its function [79]. Biodistribution of therapeutics can be
changed as the outer surface of nanoparticles are decorated with targeting moieties to obtain the
20
highest therapeutic potency with diminished off-target effects. One class of nanoparticle we will
focus on are peptide amphiphile micelles.
3.5.1 Peptide Amphiphile Micelles
Peptide amphiphiles are a class of amphiphilic molecule defined by the presence of a
hydrophilic peptide in the headgroup. The hydrophobic tail is typically an oily chain (i.e. alkyl or
fatty acid) or a hydrophobic peptide. The head and tail may be linked directly or linked by a spacer
moiety. The tuneability of the head vs. tail group of peptide amphiphile micelles provides many
potential applications.
The thermodynamics of peptide amphiphile micelle assembly are driven primarily by the
hydrophobic effect [80]. In general, a hydrophobic solute in aqueous solution will be surrounded
by a layer of water molecules with high surface tension, which increases the free energy of
solution, Gsoln due to the reduced entropy of the surrounding water layer. G soln, namely, is the free
energy of the assembled nanoparticle, relative to that of the dispersed monomers. Since the
enthalpy of the solution depends primarily on the volume of solute, which remains constant
regardless of solute-solute interaction, but the entropy depends on surface area of the solute-
water interface, G soln decreases as more solute molecules associate together into a separate
phase to minimize the surface area exposed to aqueous solution per solute molecule.
This decrease in entropy is the key to hydrophobic self-assembly of amphiphiles. The
presence of the hydrophilic moiety in the molecules provides a force favoring solution with the
water. The result is a micelle wherein the hydrophobic moiety acts as an oil phase while the
hydrophilic moiety maximizes contact with water and simultaneously minimizes the oil-water
contact, providing a cap on micelle size. The presence of the hydrophilic moiety also reduces Gsoln
for individual amphiphile molecules, creating a minimum concentration of amphiphiles below
which micelle formation is disfavored, known as the critical micelle concentration (CMC) (Figure
21
3-6). For derivation of CMC from the law of mass action, refer to work by Maibaum et. al. [80].
Generally, forces that strengthen the hydrophobic effect also lower the CMC. Because of this
correlation, the CMC is often taken as a proxy for the stability of the micelles in solution [81].
.
Figure 3-6 Characteristic curve of the surface tension for aqueous surfactants solution. A)
Schematic presentation of the peptide amphiphile with the hydrophobic C16 alkyl chain and the
hydrophilic peptide headgroup forming micelles by self-assembly above the CMC. B) Idealized
curve of surface tension as a function of surfactant concentration with the CMC representing the
point, where surfactant molecules start to form aggregates known as spherical micelles, with
further increasing concentration, the micelles may change the shape to cylindric forms. Adapted
and reprinted from Ref. [82] with permission from BioMed Central Ltd.
Micelle shape is determined by the packing parameter [83] of the amphiphile molecule.
The packing parameter P is given by the equation P = v/al, where v is the volume the molecule
occupies in solution, a the area of the hydrophilic headgroup when solvated, and l the length of
the hydrophobic tail in the oil phase. The critical packing parameter (Cpp) can be thought of as a
measurement of the shape an individual amphiphile preferentially takes in solution: For Cpp < 1/3,
the monomer shape is conical, and the resulting micelles are generally spheroids, while for Cpp >
1/2, the monomer shape is more cylindrical, driving formation of long wormlike micelles (Figure
3-7). Kinetic observations by Tirrell et al. suggest that most micelle formation begins with sphere
formation, even for higher packing parameters, but that the spheres then subsequently merge to
form the wormlike structures observed in the case of high Cpp [84]. According to these
22
observations, spherical micelle formation is relatively fast, and the packing parameter correlates
with the thermodynamic favorability of merging these micelles to form worms.
Figure 3-7 Amphiphile morphology and summary of the aggregate structures that can be
predicted from the critical packing parameter. Adapted and reprinted from Ref. [85] with
permission from Hindawi.
3.5.2 Nanoparticle Physiochemical Properties and Renal Clearances
Nanoparticle physiochemical properties, such as diameter and charge, can cause vastly
different biodistribution profiles after administration. In healthy kidneys, nanoparticles with
hydrodynamic diameters from 8-15 nm are able to pass through the glomerular membrane
barriers and are cleared via renal excretion [86, 87]. The vast majority of studies indicate both
23
rigid metal [88] or flexible polymer [89] nanoparticles in this size range are cleared by the kidney
(Figure 3-8) [90, 91]. In diseased states, breakdown of the glomerular endothelial cell lining and
podocyte architecture can lead to passage of much larger substances, leading to proteinuria [92].
Figure 3-8 Typical size relation to the distribution of nanoparticle accumulation in the lungs, liver,
spleen, and kidneys. Adapted and reprinted from [91] with permission from Nature Publishing
Group.
Deviations to this general size trend, however, have been reported. Choi et al. utilized Au-
PEG nanoparticles with varying core sizes between 5 and 98 nm and a polyethylene glycol (PEG)
“brush border” with molecular weight between 5000 and 20,000 g/mol to investigate the size
dependence of nanoparticles on kidney accumulation [93]. Optimal targeting to the mesangium,
or a thin membrane of cells that supports glomerular capillaries, of healthy BALB/c mice kidneys
was seen for nanoparticles approximately 75 nm in diameter. Particle accumulation inside renal
corpuscles revealed a strong function of size: particles on the order of 10 nm are suggested to
enter the mesangium only briefly, where they are not retained due to an absence of phagocytosis.
Particles larger than 75 nm are too large to pass through the glomerular endothelium pores.
Interestingly, accumulation in peritubular capillaries did not seem to correlate with size,
demonstrating distinct uptake characteristics in different portions of the nephron.
Traditionally, since the glomerular capillary wall is negatively charged, the surface charges
of nanoparticles have been hypothesized to play an important role in filtration ability. A positively
charged nanoparticle with a diameter of 6–8 nm can pass the kidney filtration barrier due to the
24
favorable charge interactions, whereas filtration through the kidney is difficult for the negatively
charged or neutral nanoparticles [94]. However, we again can find deviations to this trend:
Williams et al. constructed “mesoscale” nanoparticles, approximately 400 nm in diameter, which
accumulated seven times more efficiently in the kidney compared to the heart, lung, spleen, and
liver (Figure 3-9) [95]. These poly(lactic-co-glycolic acid) conjugated to polyethylene glycol
(PLGA−PEG) nanoparticles were synthesized in several forms, including anionic (ζ-potential = -
19.5± 0.6 mV), cationic (ζ-potential = -19.5 ± 0.6 mV), and neutral (ζ-potential = 0.38 mV) forms.
No significant difference was observed regarding organ distribution among all surface charge
variations. They were observed to localize in the basolateral region of proximal tubule epithelial
cells under histological analysis.
Figure 3-9 In vivo biodistribution of mesoscale nanoparticles, approximately 400 nm in diameter,
showing heightened accumulation in the kidneys compared to other organs. A) Dorsal image of
mice treated with PBS, 50 mg/kg Anionic-MNP (A-MNP), 50 mg/kg Cationic-MNPs (C-MNP), and
an equal molar weight of free dye. B) Ex vivo organ fluorescence from mice injected with MNPs,
dye, or PBS (mean ± SD). Adapted and reprinted from Ref. 23 with permission from Nature
American Chemical Society.
Negatively charged quantum dots (QDs) (∼3.7 nm) as a model system were used by Liang
et al. and found to accumulate in mesangial cells, with little found in urine [96]. In contrast, cationic
QDs of similar size (∼5.67 nm) were found to be readily excreted into urine shortly after injection.
25
We can see that it is crucial to fully understand the physiochemical properties of any nanoparticle
system and the relationship to renal clearance to achieve desired behaviors in vivo.
3.5.3 Targeting Ligands
Active targeting of nanoparticles typically involve ligands bound to the surface which
enables a higher degree of specificity compared to untargeted particles of the same physical
characteristics such as size and charge [97]. Peptide ligands take advantage of highly specific
interactions between the ligand and the target site to promote accumulation of nanoparticles.
Peptides are naturally degradable, easily synthesized, and custom tunable with a variety of linker
chemistries. By employing targeting peptides to nanoparticles, drugs have the potential to reduce
side effects and toxicity associated with current therapies for kidney disease, and can generate
a higher intrarenal drug concentration compared to that of free drug [98]. Notably, many potential
candidates for kidney targeting peptides from phage display technologies have been identified
[99], but relatively few nanoparticle studies for targeted drug delivery applications have been
attempted in recent years. For a review of potential peptide and antibody ligands used for renal
targeting, organized by cell target, refer to Wang et al. [100]
In the context of ADPKD, tubular epithelial cells are the cell type of interest that produces
cystic dysfunction. Building on previous work regarding lysine interactions with receptors on the
apical side of proximal renal tubule cells, Wischnjow et al. developed the peptide sequence
(KKEEE)3K [101]. They showed that the targeting peptide had high renal specificity and
accumulation in proximal tubule cells, making it an ideal kidney-specific carrier for drug delivery
and the treatment of kidney diseases (Figure 3-10). Immunohistochemistry showed that
(KKEEE)3K specifically accumulated in the renal cortex. (KKEEE) 3K was absorbed via megalin-
mediated endocytosis as demonstrated by the lack of (KKEEE) 3K accumulation in mice with
megalin-deficient kidneys. The peptide remained stable in serum after 24 hours with no signs of
degradation. In addition to having exceptional kidney accumulation and stability, (KKEEE) 3K
26
demonstrated renal clearance within a few hours to prevent toxicity due to long kidney retention.
It is this targeting peptide that we utilize in our nanomedicine strategy.
Figure 3-10 A) Organ distribution study of labeled (KKEEE)3K in mice at 1 hour post-injection. B)
γ-scintigraphy of (KKEEE)3K time-course. Adapted and reprinted from Ref. [101] with permission
from American Chemical Society.
3.6 Oral Delivery
Previous nanoparticle studies for kidney applications have heavily relied on intravenous
injection, providing 100% bioavailability of the compounds of interest. However, injection-based
therapies can suffer from poor patient compliance and reduced clinical performance due to the
pain and inconvenience associated with treatment regimens [9]. Instead, oral administration has
many advantages including increased patient convenience and compliance, especially in cases
of chronic disease treatment, which requires repeated dosing over long periods of time [10]. Oral
formulations are also cheaper to produce because they do not need to be manufactured under
27
sterile conditions [102]. However, development of successful drug delivery systems to negotiate
the GI tract is difficult, as there are substantial physiological barriers that need to be overcome.
3.6.1 Barriers to Oral Delivery
If nanoparticle-based systems are consumed, it must pass through multiple physiological
barriers to entry (Figure 3-11) [103]. The first barrier is the low pH environment of the stomach
and small intestine. The pH environments in the GI tract can range as low as 1 in the stomach to
as high as 8 in parts of the intestine, which can cause pH-induced oxidation, deamidation or
hydrolysis of protein therapeutics, leading to loss of activity [104]. Enzymatic degradation is due
to proteases, nucleases, and lipases secreted to digest biological molecules prior to absorption
begins in the stomach and continues through the small intestines. GI proteases generally digest
94–98% of orally administered proteins [105]. From the lumen of the small intestine, a therapeutic
must cross the mucosal layer coating microvilli to reach absorptive cells. Penetration of this mucus
barrier is necessary to reach the absorptive epithelial cells of the intestine. These epithelial cells
maintain tight junctions between each other and regulate the permeability of the monolayer.
28
Figure 3-11 Anatomy of the stomach, small and large intestine and magnification of the path
orally administered substances must overcome to reach systemic circulation. Adapted and
reprinted from [103] with permission from Informa UK Limited.
Once at the epithelial cell layer, substances can cross the barrier in two via paracellular
or transcellular mechanisms [102]. Absorption through each pathway is again dependent on
physiochemical properties, such as molecular weight, hydrophobicity, ionization constants, and
29
pH stability. Thus, an understanding of these mechanisms is important in designing delivery
systems for nanomedicines. Paracellular transport relies on substance passing through spaces
in between epithelial cells, and not contacting any cell cytoplasm. Claudins are well studied
transmembrane proteins that either tighten the paracellular pathway or function as paracellular
channels [106]. The dimension of the tight junction gap in which this occurs is on the order of 1
nm, and may be limited to macromolecules and ions [107]. Transcellular transport occurs through
specialized intestinal epithelial cells known as enterocytes and M cells. M cells take up
macromolecules, particles and microorganisms by adsorptive endocytosis, which can be via
clathrin-coated pits and vesicles, fluid phase endocytosis and phagocytosis [108]. Absorption of
the substance can be triggered by carrier-mediated or receptor-mediated transport, and
successful delivery to blood vessels depends on the exocytosis of materials to the basolateral
side of the epithelial layer [109].
3.6.2 Chitosan Nanoparticles
To address the challenges of oral delivery, nano-biomaterial approaches to navigate the
physiological barriers have emerged. One promising candidate is the usage of chitosan
nanoparticles, with their low toxicity, mucoadhesion, and tunable physical properties [11].
Chitosan is a naturally occurring polysaccharide derived from the chitin in crustacean shells such
as those from prawns and crabs. Chitosan is synthesized via the deacetylation of chitin under
basic conditions, which is depicted in Figure 3-12. It is cationic, basic, antimicrobial, and
biocompatible, and has been approved by the FDA for tissue engineering and drug delivery
applications [110]. In the context of oral delivery, chitosan acts as a GI tract permeation enhancer
by reversibly opening tight junctions in the intestinal epithelium, which has been shown to increase
both paracellular and transcellular transport [111]. The mucoadhesive property of chitosan can
be attributed to the positively charged chitosan complexing with negatively charged residues
(sialic acid) in the mucus [110].
30
Figure 3-12. Deacetylation of chitin to chitosan. Adapted and reprinted from Ref. [11] with
permission from MDPI.
Feng et al. have also recently reported a potential oral delivery strategy for anti-cancer
drugs. They have prepared nanoparticles of doxorubicin hydrochloride (DOX), a common
anticancer therapy, with chitosan and carboxymethyl chitosan. These nanostructures were found
to enhance the intestinal absorption of DOX throughout the small intestine [112]. Similarly, Pan
et al. have utilized chitosan nanoparticles roughly 300 nm in diameter to deliver insulin in diabetic
rat models. These rats exhibited a greater drop in glucose than was achieved using a control
insulin–chitosan solution, confirming the beneficial effect of nanoscale geometries. Orally
administered insulin would normally be degraded in the GI tract, but these chitosan nanoparticles
have been cited to protect their payloads from degradation [113, 114]. From these initial promising
results, we aim to develop an orally deliverable nanomedicine strategy for the treatment of
ADPKD.
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36
Chapter 4: Kidney Targeting Multimodal Micelles
The work in the following chapter was published as an article in the journal of Nano
Research, titled Design and in vivo characterization of kidney-targeting multimodal micelles for
renal drug delivery [7].
4.1 Introduction, Objective, and Rationale
Chronic kidney disease (CKD) results from any persistent condition that reduces kidney
function, which includes diabetic kidney disease, tubulointerstitial diseases, glomerulonephritides,
and cystic kidney diseases [20]. CKD has risen into the top ten leading causes of death in the
United States over the past 30 years. In 2017, 15% of US adults, or roughly 30 million individuals,
were estimated to have CKD [101]. Once a CKD patient is middle aged, they are often at risk for
end-stage renal disease (ESRD), currently affecting more than 660,000 Americans. In addition to
the eventual need for dialysis or kidney transplant, a host of other health complications may occur
with CKD. With inadequate kidney function, excess wastes and fluid accumulate in the
bloodstream and tissues, which increases the risks of heart disease, stroke, hypertension, and
respiratory issues [3]. In addition, as liver function is closely tied to kidney function, renal and liver
dysfunction are often present together in up to 85% of CKD patients [115]. For example, in
patients with autosomal dominant polycystic kidney disease (ADPKD), the most prevalent genetic
condition worldwide that is characterized by cyst formation mainly in the kidneys, more than 80%
of patients also form cysts in the liver impairing its function [5, 33, 116].
To mitigate CKD, high doses of small molecule drugs are often prescribed to overcome
fast elimination times, which can also increase the risks of adverse side effects. For instance,
angiotensin-converting enzyme inhibitors used to treat hypertension caused by CKD may induce
hyperkalemia, or high blood potassium levels [39], while the non-steroidal anti-inflammatory drugs
(NSAID) used to manage pain can cause tubulointerstitial nephritis, or inflammation to tubular
37
cells [40]. In ADPKD, several potential therapies have demonstrated cyst size reduction in mice
or in clinical trials, including metformin, a Food and Drug Administration (FDA) approved drug for
the treatment of type 2 diabetes [117]. However, mice studies of ADPKD utilize metformin at a
higher mg/kg dose than currently prescribed for patients with diabetes, in which 25% already
suffer from gastrointestinal (GI) discomfort and approximately 5% of the patients are unable to
tolerate the drug at all [118, 119]. In addition, metformin can cause more fatal side effects such
as lactic acidosis [120]. Therefore, to mitigate these off-target side effects, a targeted drug delivery
system that can decrease systemic toxicity without compromising therapeutic efficacy is needed.
Recent advances in nanomedicine and molecular engineering have allowed the design of
modular drug carrier systems such as peptide amphiphile micelles (PAMs) to address these
drawbacks by enhancing the blood circulation half-life and minimizing off-target organ
accumulation [121]. As mentioned in Chapter 3, PAMs are constructed from aggregations of
monomers that consist of a hydrophilic peptide “headgroup,” and a hydrophobic lipid “tail”. When
above the critical micelle concentration (CMC), these monomers self-assemble into micelles and
can be used as carriers for drug delivery [122, 123]. For kidney targeting, micelles are particularly
useful because they can be assembled to be less than 15 nm in diameter, which is optimal for
passage through the glomerular filtration barrier (Figure 4-1b) and thus accumulation in kidneys
[8, 93, 124]. In addition to tuning physiochemical properties such as size, targeting ligands can
be incorporated onto the surface of micelles to interact with cell surface receptors as well as
tubular epithelial cells responsible for many CKD conditions [100].
Towards renal drug delivery, we have selected the kidney-targeting peptide [(Lys-Lys-Glu-
Glu-Glu)3-Lys] ((KKEEE)3K) to incorporate into micelles. In previous work by Wischnjow et al.,
(KKEEE)3K was conjugated onto ciprofloxacin, an antibiotic normally excreted in the liver [101,
125] that was redirected to the kidneys upon peptide conjugation. Unlike this prodrug formulation,
our kidney-targeting multimodal micelles (KMs) consist of multiple monomers in a single
38
nanoparticle and can be decorated with numerous targeting ligands for multivalent and more
efficient cell targeting. This multivalent display can activate receptor-mediated endocytosis
pathways, potentially allowing for enhanced cell internalization [126, 127]. In addition, within a
micelle, targeted therapeutics can be combined with imaging agents to develop multifunctional
nanoparticles that include both diagnostic [128, 129] and therapeutic agents for tracking drug
efficacy and disease regression [15, 16, 130-132].
Due to the benefits micelles can provide, in this study we synthesized fluorescently-
labeled KMs with or without the (KKEEE)3K targeting peptide to test the hypothesis that KMs
would provide additional accumulation to the kidneys for future drug delivery applications. We
tested viability of human proximal tubule cells upon KM binding in vitro, and evaluated their ability
to target the kidney 24 hours post-injection into mice. Furthermore, we confirmed biocompatibility
in vivo through imaging and analysis of kidney function. These initial results lay the groundwork
for renal drug delivery applications using KMs in CKD.
4.2 Methods and Materials
4.2.1 Micelle Synthesis
The (KKEEE)3K targeting peptide was synthesized using standard Fmoc-mediated solid
phase peptide synthesis methods on rink Amide resin (Anaspec, Fremont, CA, USA) using an
automated benchtop peptide synthesizer (PS3, Protein Technologies, Tucson, AZ, USA). A
cysteine was added to the peptide sequence at the N-terminus to allow for a thioester linkage.
Peptides were cleaved from the resin and deprotected with 94:2.5:2.5:1 by volume trifluoroacetic
acid:1,2-ethanedithiol:H2O:triisopropylsilane and were precipitated and washed several times
with cold diethyl ether, dissolved in water, lyophilized, and stored as powders at 20°C. Crude,
peptide mixtures were purified by reverse-phase high performance liquid chromatography (HPLC)
(Prominence, Shimadzu, Columbia, MD, USA) on a C8 column (Phenomenex, Torrance, CA,
39
USA) at 50°C using 0.1% formic acid in acetonitrile/water mixtures and characterized by matrix-
assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectral analysis (Autoflex
Speed, Bruker, Billerica, MA, USA). Cysteine-containing peptides were conjugated to 1,2-
distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol 2000) (DSPE-
PEG(2000)-maleimide, Avanti Polar Lipids, Alabaster, AL, USA) by adding an equimolar amount
of the lipid to peptide in MilliQ water (pH 7.2). After gentle mixing for one week, the resulting
product was purified by HPLC on a C4 column as described above. The fluorophore-conjugated
monomer was synthesized by conjugating Cy7 via a peptide bond to DSPE-PEG(2000)-amine
(Avanti Polar Lipids, Alabaster, AL, USA) by adding an equimolar amount of Cy7 mono-N-
hydroxysuccinimide ester (GE Healthcare Life Sciences, Pittsburgh, PA, USA) to the lipid
dissolved in 10 mM aqueous sodium carbonate buffer (pH 8.5). After reaction at room temperature
for 24 hours, protected from ambient light, the mixture was purified on a C4 column and
characterized as described above.
Monomers were self-assembled into micelles via thin film evaporation. (Fig. 4-1a) The
appropriate DSPE-PEG(2000) amphiphiles were dissolved in methanol or chloroform, and
evaporated under a steady stream of air. The resulting film was dried under vacuum overnight,
and hydrated at 80˚C with either MilliQ water or PBS, vortexed and sonicated as needed to obtain
a clear solution, and allowed to cool to room temperature. There were two types of micelles
synthesized: (1) KMs with a monomer molar ratio of 10:45:45 consisting of DSPE-PEG(2000)-
Cy7:DSPE-PEG(2000)-(KKEEE)3K:DSPE-PEG(2000)-Methoxy; and (2) NT micelles with
monomer molar ratio of 10:90 consisting of DSPE-PEG(2000)-Cy7:DSPE-PEG(2000)-Methoxy.
4.2.2 Micelle Characterization
Dynamic Light Scattering (DLS): Stock solutions of PBS were titrated to pH 4.5, 5.5, 6.5,
or 7.4 with the addition of hydrochloric acid or sodium hydroxide. 1000 μM KM or NT micelle
solutions were filtered through Puradisc 0.2-μm Polyvinylidene fluoride (PVDF) membrane filters
40
(GE Healthcare Life Sciences, Pittsburgh, PA, USA) and measured immediately to confirm the
presence of monodisperse, spheroidal micelles. DLS measurements were determined at 163.5°
and 532 nm using a Wyatt Technology Möbiuζ system (Santa Barbara, CA, USA, N ≥ 3). All
measurements were carried out at 25°C after equilibrating for 5 minutes.
Zeta Potential: Zeta potential of 100 μM KMs and NT micelles dissolved in water and
filtered through Puradisc 0.2-μm PVDF membrane filters (GE Healthcare Life Sciences,
Pittsburgh, PA, USA) were measured using the same Möbiuζ system described above. Samples
were placed in a Quartz cuvette with a polyether ether ketone (PEEK) and platinum dip probe (N
≥ 3). All measurements were carried out at 25°C after equilibrating for 5 minutes.
Transmission Electron Microscopy (TEM): Negative stained samples for TEM were
prepared by placing 100 μM solution of KM or NT micelle in MilliQ water on 400 mesh lacey
carbon grids (Ted Pella, Redding, CA, USA) for 5 minutes. Excess liquid was wicked away with
filter paper and the grid was washed with MilliQ water before placing 2 wt.% uranyl acetate
solution for 2 minutes, then washing with MilliQ water. Dried samples were immediately imaged
on a JEOL JEM-2100F TEM (JEOL, Ltd., Tokyo, Japan).
4.2.3 Cell Culture
Human primary renal proximal tubule epithelial cells (RPTEC, PCS-400-010™, ATCC,
Manassas, VA, USA) were cultured following the manufacturer’s recommendations. Cells were
expanded in renal epithelial cell basal medium supplemented with renal epithelial cell growth kit
(ATCC, Manassas, VA, USA) at 37°C in a humidified incubator under 5% CO2. Cells at passage
3 were used for studies, and media was changed every 1-2 days.
4.2.4 In Vitro Binding
41
To assess cell binding of either KMs or NT micelles, RPTEC were cultured at a density of
5,000 cells/cm2. KM, NT micelle, or PBS treatment was administered at a final concentration of
100 μM for 15 minutes or 1 hour. To qualitatively assess binding and association, cells were
seeded on glass coverslips placed in 6-well plates for imaging. Cells were washed twice with pre-
warmed PBS and then fixed in 4% paraformaldehyde at various time points after micelle
incubation. Fixed cells were imaged in a LSM 700 confocal microscope (Zeiss, Oberkochen,
Germany, N ≥ 3). To quantitatively measure KM binding and association, cells were cultured on
96-well plates, and fluorescence was measured via a Varioskan LUX plate reader (Thermo Fisher
Scientific, Waltham, MA, USA), using an excitation wavelength of 730 nm and an emission filter
of 783 nm.
4.2.5 In Vitro Biocompatibility
Biocompatibility was assessed with an MTS cell proliferation colorimetric assay following
the manufacturer’s instructions (BioVision Incorporated, San Francisco, CA, USA). RPTEC (5,000
cells/well) were incubated with either 1, 10, or 100 μM of KMs or NT micelles, or PBS for 24 hours
on a 96-well plate before the addition of MTS reagent.
4.2.6 In Vivo Renal Targeting
To assess the targeting abilities of KMs, 6-7 week old male and female C57BL/6 mice
(Jackson Laboratories, Bar Harbor, ME, USA) were injected via tail vein with 100 μl of 1000 μM
KM, NT micelle, or PBS control. Mice were euthanized after 24 hours post-injection and organs
(e.g. brain, heart, lungs, liver, kidneys, spleen, intestines, and bladder) were excised and imaged
ex vivo on a Xenogen IVIS 200 imaging system (Caliper Life Sciences, Hopkinton, MA, USA).
Quantification of the fluorescence signal was achieved via Living Image software (PerkinElmer,
Downers Grove, IL, USA, N≥3), where PBS background was subtracted from both KM and NT
groups. Urine and blood samples were collected following organ excision and stored at -20°C
42
until further analysis. All animal procedures followed NIH guidelines for the care and use of
laboratory animals and were approved by the University of Southern California’s Institutional
Animal Care and Use Committee (Los Angeles, CA, USA).
4.2.7 Histology and Immunohistochemistry
Following optical imaging, the brain, heart, lungs, liver, kidneys, spleen, intestines, and
bladder were immediately frozen and embedded in optimum cutting temperature (OCT)
compound (Tissue Tek, Sakura Finetek, Torrance, CA, USA) before 6-μm samples were
sectioned in a CM3050 S Cryostat (Leica, Nussloch, Germany) and placed on positively-charged
Superfrost Plus slides (Fisherbrand, Waltham, MA, USA). Tissue sections were stained with
hematoxylin & eosin (H&E) and imaged with a Nikon Eclipse TS100 light microscope (Nikon,
Minato, Tokyo, Japan).
For immunohistochemistry and staining of megalin, tissue sections on slides were washed
with Tris Buffered Saline (TBS) with 0.025% Triton X-100. Samples were blocked with 1% bovine
serum albumin (BSA), 10% normal goat serum, 0.3 M glycine in 0.1% PBS Tween for 1 hour at
room temperature. Sections were then incubated overnight at 4°C in a humidified chamber with
an antibody against Lrp2/megalin (Abcam, Cambridge, United Kingdom, 1:100). The next day,
sections were incubated for 1 hour at room temperature using goat, anti-mouse IgG H&L Alexa
Fluor® 488 (Abcam, Cambridge, United Kingdom, 1:1000). Nuclei were then counterstained with
4’,6-Diamidino-2-phenylindole (DAPI) and samples were mounted using VectaMount™ aqueous
mounting medium (Vector Laboratories, Burlingame, CA, USA). Representative images are
presented, and colocalization analysis between Cy7 and Alexa Flour® 488 channels were
performed with the coloc2 feature in ImageJ.
To assess KM specificity to the glomerulus and tubule cells, tissue sections were washed
TBS with 0.025% Triton X-100. Samples were blocked with Carbo-Free Blocking Solution (Vector
43
Laboratories, Burlingame, CA, USA) for 30 minutes at room temperature. Samples were
incubated with Wheat Germ Agglutinin (WGA) Alexa Fluor® 488 Conjugate, Lectin PNA Alexa
Fluor® 488 Conjugate, or Lectin PHA-L Alexa Fluor® 488 Conjugate for 30 minutes at room
temperature (Vector Laboratories, Burlingame, CA, USA). Nuclei were then counterstained with
DAPI and samples were mounted using VectaMount™ aqueous mounting medium (Vector
Laboratories, Burlingame, CA, USA). Representative images are presented, and colocalization
analysis between Cy7 and Alexa Flour® 488 channels was performed with the coloc2 feature in
ImageJ.
4.2.8 Kidney Health Markers
To assess overall kidney functional health, blood urea nitrogen (BUN) in mouse serum
was analyzed using a commercial blood urea nitrogen enzymatic kit (Bioo Scientific, Austin, TX,
USA) immediately after in vivo imaging. The same was done for urine creatinine, using a
commercial mouse creatinine enzymatic kit (Crystal Chem, Elk Grove Village, IL, USA).
4.2.9 Statistical Analysis:
A Student’s t-test was used to compare means of pairs. Analysis of variance (ANOVA)
with Tukey’s multiple comparison test post-hoc analysis was used to determine significant
differences among three or more means. A corrected p-value of ≤ 0.05 was considered to be
significant.
4.3 Results and Discussion
4.3.1 Fabrication and Characterization of Micelles
To construct micelles for renal applications, the kidney-targeting peptide (KKEEE)3K was
used to decorate the nanoparticle surface as the peptide has been shown to have the ability to
redirect drugs to greater than 85% to the kidneys [101, 125]. We modified this sequence with a
44
terminal cysteine and conjugated it to DSPE-PEG(2000)-maleimide via a thioester linkage to
construct peptide amphiphile monomers. Micelles were prepared using thin film hydration and
self-assembled in water or PBS (Figure 4-1). KMs were composed of a monomer ratio of 10:45:45
consisting of DSPE-PEG(2000)-Cy7:DSPE-PEG(2000)-(KKEEE)3K:DSPE-PEG(2000)-Methoxy,
while NT micelles consisted of 10:90 of DSPE-PEG(2000)-Cy7:DSPE-PEG(2000)-Methoxy. Cy7
was chosen as the fluorophore as its emission wavelengths minimizes autofluorescence and a
molar ratio of 10:90 was used to maximize fluorescence intensity without quenching [133]. The
45 molar percentage of targeting DSPE-PEG(2000)-(KKEEE)3K was chosen to not fully saturate
the micelle surface and allow for the addition of therapeutic or other functional monomers in future
studies.
Figure 4-1 Schematic of (a) micelle self-assembly and the (b) the glomerular filtration
barrier.
KMs and NT micelles were shown to have an average diameter of 15.0 ± 0.0 and 12.0 ±
2.3 nm, respectively, via TEM and DLS (Figure 4-2a-c, Table 4-1) in physiological pH of 7.4. To
ensure our micelles can withstand the variation in pH found in the nephron, KM and NT micelles
were incubated in a range of pH values to test stability. The pH changes are mainly dictated by
the reabsorption of bicarbonate from the filtrate, and the largest amount of bicarbonate is
reabsorbed along the proximal tubule of the nephron, where the pH is approximately 6.8, but can
drop to approximately 4.4 during reabsorption in the distal tubule and collecting ducts [134]. As
45
the pH was varied between 4.5-7.4 pH, the hydrodynamic diameter remained relatively stable,
and KMs had a diameter between 13.9-14.9 ± 1.5 nm and NT remained between 11.0-12.8 ± 2.3
nm (Figure 4-2c). While our particles were slightly larger than the 10-nm cut-off of nanoparticles
that have been found to pass through the glomerular filtration barrier and enter the kidneys, there
are many examples in which “soft” organic, cationic, or larger mesoscale particles have
accumulated in the kidneys beyond this size [90, 100]. Nonetheless, planned future studies will
further tune the KM size by shortening the peptide repeats to (KKEEE) 2K or KKEEE.
Zeta potential of KMs and NT micelles were -7.8 ± 0.5 and -1.4 ± 0.6 mV, respectively
(Table 4-1). This is expected as the (KKEEE)3K peptide bears an overall charge of -2, due to the
repeating glutamic acids. The slight negative charge of the NT micelles can be traced to the
phosphate group of the DSPE tail. However, in the context of circulating nanoparticles, both KMs
and NT micelles were within the neutral range of ± 10 mV, which has been reported to have
minimal interactions with the mononuclear phagocytic system (MPS) as well as enhanced
circulation times [135]. The CMC was found to be approximately 2.0 x 10-6 M which is similar to
that reported of micellar systems composed of DSPE-PEG(2000) used in other studies [8, 133].
In mouse studies herein, 100 μL of 1000 μM micelle solution administered intravenously will
remain above the CMC when diluted in approximately 2 mL of circulating blood [136].
46
Figure 4-2 Characterization of KMs and NT micelles. TEM images of (a) KMs and (b) NT micelles
show spherical micelles of approximately 15.0 and 12.0 nm, respectively. (c) DLS measurements
show that both micelles are stable across the range of pH present in the kidney. (d)–(g)
Representative % number DLS curves for each pH condition are shown. Scale bar: 100 nm.
Table 4-1 Size and zeta potential of micelles
4.3.2 KM Cell Binding to Renal Proximal Tubule Cells and Biocompatibility In Vitro
To verify targeting and binding of KMs in kidney cells, RPTCs were treated with micelles
in vitro. Confocal fluorescence microscopy showed a 30% higher binding of KMs compared to NT
after 15 minutes (100 μM, Fig. 4-3a, 3b) of incubation at 37°C. To assess biocompatibility, KMs
and NT micelles were incubated with RPTCs over the course of 24 hours at 37°C, and viability
was assessed using an MTS assay at a micelle concentration of 1, 10, or 100 μM (Figure 4-3c).
47
Similar to results obtained from other PAM studies [14, 15], over 90% of cells were viable upon
treatment with either micelle type and were comparable to the PBS-treated control group.
Figure 4-3 In vitro binding of KMs vs. NT micelles in human RPTCs. (a) Representative confocal
images of Cy7 and DAPI. (b) Fluorescence quantification shows that KMs have 30% higher
binding 15 min after micelle administration as compared to NT micelles. (c) MTS assays confirm
both micelles are biocompatible, with ≥ 90% cell viability after 24-h incubation, normalized to
PBS treatment. Scale bar: 100 μm. * indicates p < 0.05.
4.3.3 In Vivo Targeting and Biodistribution of KMs
The targeting effectiveness of KMs as compared to NT micelles was assessed in healthy
C57BL/6J mice. Ex vivo optical imaging demonstrated accumulation of KMs mostly in the kidney
as well as the liver. Quantitative comparison between fluorescence levels in kidneys showed
3.3×109 ± 4.7×108 [p/s/cm²/sr]/ [µW/cm²] for KMs vs. 2.3×109 ± 4.8×108 [p/s/cm²/sr]/ [µW/cm²]
in NT micelles, which equates to 34.6 ± 5% of the total fluorescence measured from all organs
for KMs vs. 25.8 ± 6% in NT micelles 24 hours post tail-vein injection (p<0.01, Figure 4-4a). This
is in contrast to previous studies evaluating PAMs designed to target fibrin, which only found
approximately 10% accumulation in the kidneys 90 minutes post-injection [128, 129], confirming
48
the effectiveness of the kidney-targeting peptide, (KKEEE)3K. KM accumulation in the remaining
organs was below 10% and by 7 days, approximately 95% of both KMs and NT micelles were
cleared (not shown), which is in agreement with previous PAM studies [129]. Notably, inorganic
nanoparticles of the same size class (≤ 20 nm) composed of gold or silver accumulate mostly in
the liver and spleen in rodents, with no more than 15% accumulation in the kidneys [137] as
summarized by Yang et al. [138]. Hence, KMs demonstrate enhanced kidney-targeting beyond
size effects.
Interestingly, KMs were found to have a similar level of accumulation in the kidneys and
liver. We believe that the liver targeting is largely due to clearance via MPS. While the kidney-
targeting ability of KMs is a dramatic improvement compared to other nanoparticles that localize
mostly to the liver, many kidney diseases also have concomitant kidney and liver damage. Such
diseases may thus benefit from dual targeting to these two organs, as opposed to the over 85%
renal accumulation in the original (KKEEE)3K prodrug formulation [101]. Table 4-2 provides a list
of conditions with both renal and hepatic manifestations in which the biodistribution profile of our
KMs would be favorable [40-46]. For example, one treatment currently available for hepatorenal
syndrome, where liver cirrhosis reduces kidney perfusion, is misoprostol, a synthetic
prostaglandin. This drug provides minimal benefits to restore kidney blood flow and has common
side effects such as nausea and diarrhea [47]. Such GI tract issues have the potential to be
minimized if the therapy can be localized to the kidney and liver via KMs. However, it is important
to note that the underlying pathological mechanisms in the liver vs. kidney may be different in
these listed conditions, and careful consideration of drug(s) to be incorporated into the micelle will
be needed.
49
Figure 4-4 Biodistribution of KM, NT micelle, and PBS 24 h post tail-vein injection of 1,000 μM
micelle solution in C57BL/6 mice. (a) KMs accumulate in the kidneys to a greater extent as
compared to NT micelles. Quantitative comparison between fluorescence levels in kidneys
showed 3.3 × 109 ± 4.7 × 108 (p/s/cm²/sr)/(μW/cm²) for KMs vs. 2.3 × 109 ± 4.8 × 108
(p/s/cm²/sr)/(μW/cm²) in NT micelles, which equates to 34.6% ± 5% of the total fluorescence
measured from all organs for KMs vs. 25.8% ± 6% in NT micelles 24 h post tail-vein injection. (b)
Ex vivo organ fluorescence shows accumulation mainly in the kidney and liver. ** indicates p <
0.01.
50
Condition/Disease Liver Effects Kidney Effects Prevalence of
association
Reference
ADPKD Cyst formation Cyst formation ~75-90% [139]
Familial amyloidosis
polyneuropathy
Production of
mutated
transthyretin
protein
amyloid deposition
resulting in proteinuria
and declining GFR
~50% [139, 140]
Hepatitis C Liver fibrosis Glomerulonephritis ~40-60% [141]
sarcoidosis Hepatomegaly nephrocalcinosis ~30-70% [142]
Hepatorenal syndrome Liver cirrhosis reduced renal
perfusion and GFR
~39% [143]
Diabetes Steatohepatitis Diabetic nephropathy ~40% [144]
Table 4-2 Diseases with symptoms manifesting in both the kidney and liver
4.3.4 Kidney Targeting to Megalin and Renal Tubule Epithelial Cells
To understand the mechanisms of peptide targeting and micelle uptake, kidneys treated
with KMs and NT micelles were sectioned and stained for the multiligand receptor megalin.
Megalin, also known as LRP2, is located on many absorptive epithelial cells to facilitate
endocytosis and participates in classical internalization and endosome trafficking [127, 145]. One
known molecule associated with megalin uptake is albumin, which has a largest dimension of 15
nm [146], comparable to our KM nanoparticle diameter. Upon colocalization analysis, we found a
higher Pearson’s R value, corresponding to colocalization, of KMs to megalin (0.50 ± 0.07)
compared to NT micelles to megalin (0.35 ± 0.05) (Fig. 5a,b). This result is in agreement with the
initial study performed with the (KKEEE)3K peptide in prodrug form, where a megalin-deficient
mouse showed lower renal retention compared to wild type mice [101]. These findings suggest
(KKEEE)3K retains its interactions with megalin when conjugated into a micelle nanoparticle.
Furthermore, to differentiate which type of cells within the nephron our particle associates
with, we stained kidney sections with wheat germ agglutinin (WGA), a lectin that associates with
d-glucosamine and sialic acid [147] expressed in kidney glomeruli [148, 149]. A low colocalization
51
value was observed for both KMs and NT micelles to glomeruli (0.16 ± 0.05 and 0.15 ± 0.06,
respectively, Figure 4-5c,d), which is also in agreement with the free peptide in which little
retention was found in the glomerulus [2]. Hence, our particles are not binding to the glomerular
basement membrane, podocytes, mesangial cells, or fenestrated epithelium which comprise the
glomerular filtration barrier (Figure 4-1b). In contrast, larger nanoparticles on the order of 75 nm
have been reported to be retained by the mesangial cells within the glomeruli [18] and lack access
to tubule cells past the glomerular filtration barrier.
Figure 4-5 Immunohistochemistry staining of KM and NT micelle-treated kidneys 24 hours post-
injection. (a,b) Megalin staining shows a higher colocalization with KMs compared to NT micelles
(Pearson’s R values 0.50 ± 0.07 and 0.35 ± 0.05, respectively). (c,d) WGA lectin staining show
minimal colocalization between both KMs and NT micelles to glomeruli, but KM presence in
tubules cells (Pearson’s R values 0.16 ± 0.05 and 0.15 ± 0.06, respectively). Scale bar: 50 μm. *
indicates p < 0.05.
52
4.3.5 Organ Morphology and Kidney Health
To assess the safety of our nanoparticles, histopathological evaluations of the brain, heart,
lung, liver, spleen, intestine, kidney, and bladder were conducted and showed no signs of cellular
or tissue damage 24 hours post-injection, and there were no differences between the KM, NT
micelle, and PBS treatment groups (Figure 4-6). This is similar to other PAM systems and can
be attributed to the nontoxic nature of the components of the monomers [8, 93, 129, 133].
Figure 4-6 Tissue morphology 24 h post-injection of KMs, NT micelles, or PBS. Representative
sections stained with H&E show no morphological changes or lesions. Scale bar: 100 μm.
To assess kidney health, BUN levels were measured. Urea nitrogen is a normal metabolic
product of protein catabolism, and compromised kidney function results in a higher than normal
value in the blood. In all groups, BUN was found to be within the reported values in literature for
healthy C57BL/6 mice (25.0-75.0 mg/dL, Table 4-3) [150]. In addition, urine creatinine was
measured and also found to be within range of average healthy C57BL/6 mice (4.7 ± 3.1 mg/dL,
Table 4-3) [151]. In the acute period of a disease, creatinine levels in urine would be abnormally
low, as it is not being cleared from the blood at a sufficient rate. Since KMs do not alter normal
53
BUN and creatinine levels, this suggests that it can be used as a safe carrier for future therapeutic
studies. We acknowledge the urine volume and concentration may affect the relation of urine
creatinine to kidney injury, and will utilize a more relevant marker in future studies such as serum
creatinine.
*Healthy mice BUN reported to be approx. 25.0-75.0 mg/dL [53]
**Healthy mice urine creatinine reported to be approx. 4.7±3.1 mg/dL [54]
Table 4-3 BUN and urine creatinine levels in mice 24 h post-injection of KM, NT micelle, or PBS.
In many kidney diseases, the glomerular filtration barrier is disrupted and can become
more permeable. Hence, we expect KMs can further accumulate in the kidneys and renal tubule
cells in CKD patients [152]. This is analogous to many cancer-targeting nanoparticle studies that
use passive targeting to the “leaky” vasculature within tumors via the enhanced permeability and
retention effect (EPR) [153, 154]. Future studies will investigate KM accumulation in diseased
kidneys as well as explore a wider range of physiochemical properties such as charge, ligand
density, peptide length, sequence, and secondary structure [155]. Overall, our novel nanoparticle
platform has potential as a targeted drug delivery carrier for renal applications [7].
4.4 Conclusion
The kidney-targeting peptide (KKEEE)3K was incorporated into Cy7-labeled micelles as a
promising drug delivery carrier for kidney disease. These KMs have an average diameter of 15.0
± 0.0 and were slightly negatively charged with a zeta potential of -7.8 ± 0.5 derived from the
glutamic acid residues. Upon injection into mice, KMs showed higher accumulation in kidneys
compared to NT micelles after 24 hours. In vitro binding of KMs to RPTCs demonstrated targeting
effectiveness and upon intravenous injection in vivo, KMs were found to accumulate to a higher
54
degree in the kidneys compared to NT micelles. Specifically, KMs associated with megalin were
found to be present on tubular cells in the nephron. Future studies will test KMs in diseased CKD
mouse models and verify its potential to enhance therapeutic efficacy compared to free drugs as
well as limit off-target side effects.
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57
Chapter 5: Chitosan Nanoparticles for Oral Delivery
The work in the following chapter was published as an article in the Journal of Controlled
Release, titled Oral delivery of metformin by chitosan nanoparticles for polycystic kidney disease
[156].
5.1 Introduction, Objective, and Rationale
Since the Food and Drug Administration (FDA) approved liposomal doxorubicin (Doxil) for
cancer treatment in 1995, now more than 50 nano-pharmaceuticals are available for the clinic
[157, 158]. Nanoparticles have been shown to improve drug therapeutic efficacy, reduce toxicity,
and increase tissue selectivity compared to small molecule drugs [159]. Moreover, nanoparticles
can combine multiple functionalities, including therapeutic and diagnostic capabilities, onto a
single nanoparticle platform, [160, 161] and have the potential to provide feedback on treatment
effectiveness in real-time [162]. As such, nanomedicine has proven beneficial in the treatment of
cancer [163, 164], multiple sclerosis [165], and human immunodeficiency virus (HIV) [166].
Despite these advances, similar developments in nanotechnology to improve the standard
of care for chronic diseases is limited. For instance, autosomal dominant polycystic kidney
disease (ADPKD) affects up to 12.5 million individuals worldwide [167], but no nanomedicine
efforts have been developed [7, 100]. ADPKD is a slowly progressing, irreversible genetic
condition characterized by cyst formation and enlargement arising from aberrant tubular epithelial
growth and fluid secretion occurring throughout the kidney nephron, ultimately destroying kidney
function [116, 168]. Several potential therapies have demonstrated benefits in slowing cyst growth
in mice or clinical trials [169, 170], such as metformin (met), an FDA-approved drug for type 2
diabetes [117, 171], and tolvaptan, the only drug that was approved by the FDA in 2018. However,
ADPKD preclinical studies administered drugs such as met at a higher dose (300 mg/kg/day) than
currently prescribed (maximum 37.5 mg/kg/day for diabetic patients). When corrected for volume
58
of distribution and surface area-to-volume ratio differences between mice and humans, a ~38%
higher than typical maximum daily dose given to adult diabetic patients is derived. Subsequently,
adverse side effects such as gastrointestinal (GI) discomfort manifest in 25% of patients, and 5%
of patients develop complete intolerance for the drug [172-174]. In addition, more serious side
effects of met include hypoglycemia and lactic acidosis, that latter of which has been found to be
fatal in certain patients [120]. Thankfully, primary results from a clinical trial utilizing metformin for
PKD (TAME PKD), have shown metformin at therapeutic doses can be safely administered to
PKD patients [175]. Tolvaptan, which is specifically prescribed to ADPKD patients with a rapidly
progressing cyst phenotype [176], can also be a difficult drug to tolerate due to its many off-target
side effects such as nausea, polyuria, muscle cramps, and idiosyncratic liver toxicity [76, 77]. The
tolvaptan clinical trial dropout rate was significant at 23%, and projections show that after 18 years
of continuous tolvaptan treatment, only a modest benefit of 4.9 year delay is achieved until kidney
failure [177]. Therefore, new drug delivery approaches that can decrease systemic toxicity without
compromising therapeutic efficacy is imperative for chronic diseases such as ADPKD.
Previously, kidney-targeted nanoparticle drug carriers using intravenous (IV)
administration have been developed to enhance drug accumulation in the kidney and reduce such
systemic side effects [7, 8, 100]. However, IV administration is not practical nor feasible in many
cases for chronic diseases that progress over a lifetime, such as ADPKD [178]. Instead, oral drug
delivery is the most convenient route of self-administration, and results in the greatest treatment
adherence [179, 180], preferred by 70% of patients [181]. Particularly for chronic conditions, oral
delivery is attractive as it avoids needle complications such as infection, phlebitis, and pain [182,
183]. Additionally, patients with high needle fear and chronic life-threatening health conditions
(e.g., diabetes and multiple sclerosis) have been found to make important treatment decisions
based on their aversity to needles over medical expertise [184-186], and hence, achieving a
tolerable, self-administrable route is an important aspect to clinical success of future targeted
therapies [187, 188].
59
Although favorable for patient compliance, orally-delivered drugs or nanoparticles must
overcome unique physiological barriers that have historically limited their therapeutic efficacy
(Figure 5-1) [17]. These challenges include the acidic pH and enzymes present in the stomach
that can degrade pharmaceutically active drugs [103], as well as the intestinal epithelial barrier
that acts as a selectively permeable barrier to drugs for systemic circulation [189]. Furthermore,
enterocytes within this epithelium secrete a mucosal layer, presenting a continuously recycled
barrier hundreds of micrometers thick, that have been found to trap and remove nanoparticles,
substantially limiting drug bioavailability [190]. Moreover, even upon reaching the blood after
absorption in the intestines, the first pass effect can metabolize up to 70-90% of orally
administered drugs, rendering it therapeutically inactive through biotransformation [189, 191, 192].
Chitosan-based materials have been proposed for oral delivery, as they offer many
favorable properties such as biocompatibility, mucoadhesion, and tunability for controlled drug
delivery [11, 193]. Chitosan is derived from naturally occurring chitin found in the shells and
exoskeletons of many crustaceans and is the second most abundant polysaccharide [194-196].
The purification process of chitin also allows tuning of the resultant chitosan, such as molecular
weight, pKa (6–7.5) and degree of deacetylation properties, which provides a biomaterial that can
be tailored for a wide range of biomedical applications [197, 198].
Currently, chitosan is used in commercial biomedical products like the AQUANOVA
Super-Absorbent Dressing and is currently under clinical investigation for use as dental fillers
(NCT03237624) and wound dressings (NCT03719261). Chitosan is considered Generally
Recognized As Safe (GRAS) and edible by the FDA, but has not been directly approved for any
nanoparticle drug delivery usage. The bottleneck may lie in the poor correlation between specific
formulations or modifications of chitosan and the predicted in vivo response [199]. Hence,
systematic studies assessing chitosan properties such as nanoparticle size and degree of
acetylation are still needed to exploit the beneficial properties of chitosan for drug delivery
applications in the clinic [183, 200, 201].
60
Herein, we take advantage of these properties to develop chitosan nanomaterials that
have optimal size, stability, and mucoadhesion to navigate through the GI tract and achieve
efficient systemic delivery compared to free drugs [202]. Specifically, to form chitosan
nanoparticles, several methods have been studied including polyelectrolyte complexation [203],
covalent cross-linking [204], complex coacervation [205], and ionotropic gelation [206-208]. We
selected ionotropic gelation for our studies as the mild and aqueous processing conditions, non-
toxic reagents, and ease of production is suitable for eventual clinical scale-up [209-212].
Moreover, chitosan nanoparticles have been previously shown to successfully deliver
therapeutics in vivo, such as insulin [213, 214], cyclosporin A, an immunosuppressant [215], and
enoxaparin, an anticoagulant [216], further supporting its clinical suitability.
Due to these benefits, in this study, we synthesized chitosan nanoparticles (CS-NP) to
test the hypothesis that CS-NP can be used as an oral delivery platform for ADPKD and other
chronic conditions. We investigated the synthesis parameters and its effect on the nanoparticle
size, polydispersity, loading efficiency, and degradation rate in pH ranges that are present in the
GI tract. We tested the ability of these nanoparticles to permeate an intestinal barrier model and
deliver the candidate ADPKD drug, metformin in vitro [217]. Finally, we evaluated the oral delivery
of chitosan nanoparticle met (CS-NP met) in a murine model of ADPKD, and demonstrate
enhanced therapeutic efficacy compared to the free drug upon oral delivery. These results lay the
groundwork for oral drug delivery applications using CS-NP, not only in the context of ADPKD
and chronic diseases as provided in this study, but treatments administered IV lacking the ability
to overcome enteric barriers.
61
Figure 5-1 Schematic of the physiological barriers for orally delivered nanoparticles. Adapted and
reprinted from Smart Servier Medical Art.
5.2 Methods and Materials
5.2.1 Synthesis of CS-NP
Chitosan with 95% degree of deacetylation and average molecular weight 150 kDa was
purchased from Heppe Medical (Germany). Mucin type II from porcine stomach, Rhodamine B,
pharmaceutical grade met, and poly-L-glutamic acid were purchased from Sigma-Aldrich (USA).
All other reagents were of analytical grade. Chitosan (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/ml) was
dissolved in MilliQ water containing 0.5% glacial acetic acid, sonicated and vortexed to obtain
homogenous mixtures. Similar concentrations (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/ml) of poly-L-
glutamic acid solutions were prepared in MilliQ water. Chitosan solution was added dropwise to
polyglutamic acid under constant stirring in a round bottom flask at 600 rpm; an opalescent
solution was seen upon successful formation of nanoscale particles. The final solution was
centrifuged at 14,000 rpm for 30 minutes at 14°C. The resulting pellet was serially washed with
20%, 75%, and 100% ethanol. The pellet of nanoparticles was resuspended in MilliQ water or
62
PBS and rapidly pipetted until a homogenous mixture was obtained and was used immediately
for further studies. To encapsulate a payload, met or rhodamine B was dissolved at desired
concentrations in poly-glutamic acid solution and used during nanoparticle synthesis. Loading
efficiency of the payload was calculated by quantifying the amount of unincorporated met or
rhodamine remaining in the supernatant. Rhodamine fluorescence was measured at λ excitation =
553 nm and λemission = 630 nm, while met absorbance was measured at 233 nm using a Varioskan
LUX plate reader [218, 219] (Thermo Fisher Scientific, Waltham, MA, USA). Loading capacity
was determined by dividing the weight of the known amount of encapsulated drug by the weight
of the total nanoparticle sample.
5.2.2 Nanoparticle Characterization
Dynamic light scattering (DLS) Nanoparticles derived from chitosan (0.5-3.0 mg/ml) and
poly-L-glutamic acid (0.5-3.0 mg/ml) concentrations were dispersed in 63 µL of MilliQ water and
measured by DLS to confirm size and polydispersity index (PDI). DLS measurements were
determined at 163.5° and 532 nm using a Wyatt Technology Möbiuζ system (Santa Barbara, CA,
USA, N ≥ 3). All measurements were carried out at 25°C after equilibrating for 5 minutes.
Zeta potential The zeta potential of chitosan nanoparticles was measured using the same
Möbiuζ system described above. Samples dissolved in MilliQ water were placed in a Quartz
cuvette with a polyether ether ketone (PEEK) and platinum dip probe (N ≥ 3) and measurements
were carried out at 25°C.
Transmission electron microscopy (TEM) TEM samples were prepared by placing 7.0 µL
of chitosan nanoparticles in MilliQ water on 400 mesh lacey carbon grids (Ted Pella, Redding,
CA, USA) for 5 minutes. Excess liquid was wicked away with filter paper and the grid was washed
with MilliQ water before placing 2 wt.% uranyl acetate solution for 2 minutes. After washing once
more with MilliQ water, samples were dried and immediately imaged on a JEOL JEM-2100F
(JEOL, Ltd., Tokyo, Japan).
63
5.2.3 Re-acetylation of Chitosan
Chitosan with ~95% deacetylation was re-acetylated to achieve varying degrees of N-
deacetylated chitosan. Chitosan (2 mg/ml) was mixed with 200 mM acetic anhydride in a 50:50
methanol/water mixture at 95°C and stirred for 1-12 hours. Confirmation of deacetylation degree
was measured from the first derivative of the UV-vis absorption spectra obtained from a Varioskan
LUX plate reader as specified by de Silva et al. [220]. Reaction times between 1-6 hours produced
chitosan with 95-85% degree of deacetylation, while 6-12 hours and >12 hours resulted in 70-85%
and 55-70% deacetylation, respectively.
5.2.4 Mucin-binding Assay
Porcine mucin (PM) in phosphate buffer (pH 7.4) was incubated with CS-NPs of varying
degrees of deacetylation verified by UV-vis, (90, 80, 70, 50% deacetylation, 50-300 nm diameter)
at room temperature (23°C) for 2 h (1:1, v/v), before centrifugation for 60 min at 14,000 rpm and
14°C. Absorbance of the remaining free PM in the supernatant was measured by UV
spectrophotometry at 251 nm. The mucoadhesiveness was expressed as PM binding efficiency
calculated by the following equation:
𝐵𝑖𝑛𝑑𝑖𝑛𝑔 𝑒 𝑓 𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 =
𝐶 0
−𝐶 𝑠 𝐶 0
× 100 (1)
where Co is the initial concentration of PM used for incubation (400 μg/mL) and C s is the measured
concentration of free PM in the supernatant after removal of chitosan-bound PM. The standard
curve was determined using 50, 100, 150, 200, 250, 300, 350 μg/mL PM solutions.
5.2.5 Drug Release and Morphological Response to pH
Drug release studies were performed on nanoparticles (2 mg/ml initial chitosan and 1
mg/ml poly-L-glutamic acid) suspended in PBS adjusted to pH 1.2, 2.5, 6.5, or 7.4 with the addition
of HCl or NaOH, simulated gastric fluid (SGF) composed of 2.0 g/L sodium chloride and 2.9 g/L
HCl (pH 1.3), or simulated intestinal fluid (SIF) composed of 0.62 g/L sodium hydroxide and 6.8
64
g/L potassium phosphate monobasic (pH 6.8) [221]. Free met released from nanoparticles was
quantified at 233 nm using a NanoDrop One microvolume UV-Vis spectrophotometer for up to 6
hours at room temperature (Thermofisher Scientific, Waltham, MA, USA).
To assess the morphology of nanoparticles in response to pH conditions, particles were
immersed in each pH condition for 6 hours, and imaged via TEM. All experiments were carried
out in triplicate.
5.2.6 Cell Culture
Human colon epithelial cells (Caco-2, ATCC HTB-37, ATCC, Manassas, VA, USA) were
cultured following the manufacturer’s recommendations. Cells were expanded in Dulbecco’s
Modified Eagle Medium (DMEM, ATCC-30-2003) supplemented with 10% fetal bovine serum
(FBS) and 1% penicillin-streptomycin. Cells were seeded at a density of 4.5 x 10
3
cells/cm
2
and
subcultured upon 50% confluence [222].
Mouse kidney cortical collecting duct (mpkCCDc14) cells were expanded in culture media
comprised of DMEM/F12 (11054-054, Waltham, MA, USA) supplemented with insulin,
dexamethasone, selenium, transferrin, triiodothyronine, glutamine, d-glucose, epidermal growth
factor (EGF), HEPES, sodium pyruvate as outlined by Bens et al. [223]. Complete media was
filtered before use, and media was changed every two days and subcultures were passaged every
7-8 days. Both cell lines were grown at 37°C in a humidified incubator under 5% CO 2.
5.2.7 In vitro Cell Compatibility
Biocompatibility was assessed with an MTS cell proliferation colorimetric assay following
the manufacturer’s instructions (BioVision Incorporated, San Francisco, CA, USA). MpkCCD c14
(5,000 cells/well) or Caco2 (5,000 cells/well) were incubated with either 10, 50, 100, or 500 μM of
CS-NP for 24 hours on a 96-well plate before the addition of MTS reagent. Assay fluorescence
was measured via a Varioskan LUX plate reader (Thermo Fisher Scientific, Waltham, MA, USA).
65
5.2.8 Transepithelial Resistance (TER) Surveillance
For transport and monolayer resistance experiments, Caco-2 cell monolayers were
seeded onto Transwell inserts (Corning, NY, USA; diameter 6.5 mm, growth area 0.33 cm
2
, pore
size 0.4 µm), at an initial density of 3 x
.
10
5
cell/cm
2
and maintained for 21 days in complete
medium to form a confluent monolayer. The change of TER, representing the tightness of the cell
monolayers, was measured by an EVOM2 Epithelial Voltohmmeter (World Precision Instruments,
USA). Monolayers reaching steady-state values in the range of 300-400 Ω-cm
2
were used for
studies [222].
5.2.9 Cellular Uptake and Transport of CS-NP
To assess the cellular uptake pathways of chitosan nanoparticles, Caco-2 cells were
pretreated with medium containing colchicine (transcytosis inhibitor, 10 μM, for 60 min) or
wortmannin (micropinocytosis inhibitor, 0.06 mM, for 3 hours) [224, 225] before 100 uL of 100 µM
rhodamine-loaded chitosan nanoparticles (CS-NP R) was administered in the apical chamber.
The amount of rhodamine fluorescence was measured in the basolateral chamber over the course
of 4 hours.
Additionally, the effect of paracellular transport through tight junctions was monitored via
TER of Caco-2 cells seeded on Transwell inserts. TER measurements were performed daily for
three days prior to treatment with CS-NP R, free Rhodamine (free R), or PBS control to establish
baseline measurements. TER was monitored every 6 hours, then again on day 2 and 3 post-
administration.
5.2.10 In Vitro Therapeutic Efficacy of CS-NP met Through ELISA and Epithelial Sodium
Channel (ENaC) Measurements
To assess therapeutic efficacy in vitro, the cellular levels of phosphor-AMPK (Kit #7959)
and total AMPK (Kit #7961) were measured via enzyme-linked immunosorbent assays (ELISA,
Cell Signaling Technologies, Danvers, MA, USA) according to the manufacturer's instructions.
66
mpkCCDc14 cells were treated for 12 hours with 300 µM of met in CS-NP met or free met, and
were compared to CS-NP or PBS treated controls. All standards and samples were measured on
a Varioskan LUX microplate reader at a wavelength of 450 nm.
To validate the therapeutic efficacy of met on the reduction of ENaC current, TER and
potential difference (PD) measurements were made on mpkCCD c14 cells seeded on Transwell
filters as described above [172]. ENaC-dependent equivalent short-circuit currents (I eq) was
estimated by Ohm’s law, dividing the measured PD by the TER value and the area of one
Transwell membrane (0.33 cm
2
). mpkCCDc14 cell cultures were treated with 100 uL CS-NP met
(300 µM met), CS-NP, or PBS at pH 7.4.
5.2.11 Ex vivo Imaging of Orally Administered CS-NP
To assess the biodistribution of CS-NP semi-quantitatively, 6-7 week old male and female
C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA) were orally gavaged with 10 mg/kg
of rhodamine loaded in 200 uL of 500 µM CS-NP R, 10 mg/kg of free R, or PBS control. Mice
were euthanized after 3, 24, or 48 hours post-injection and organs (e.g., brain, heart, lungs, liver,
kidneys, spleen, intestines, and bladder) were excised and imaged ex vivo on an AMI HTX in vivo
imaging system (Spectral Instruments Imaging, Tuscon, AZ, USA). The fluorescence signal was
quantified via Aura software (Spectral Instruments Imaging, Tuscon, AZ, USA, N ≥ 4), and
background was subtracted from the PBS-treated group. The mean radiance (photons/s/cm
2
/sr)
for each organ was quantified as a region of interest, and % of total organ fluorescence was
obtained by dividing each organ by the sum of all the organ regions. Urine and blood samples
were collected following organ harvest and stored at -20°C until further analysis. All animal
procedures followed NIH guidelines for the care and use of laboratory animals and were approved
by the University of Southern California’s Institutional Animal Care and Use Committee.
5.2.12 In vivo Half-life of CS-NP
67
To compare serum half- life, 200 uL of CS-NP R or free R was administrated at a dose of
10 mg/kg loaded rhodamine via oral gavage on 6-7-week-old male and female C57BL/6J mice (N
≥ 4). Blood draws were performed either retro-orbitally or via tail vein at 30 min, 3, 6, 12, 24, 36,
and 48 hours post-administration. Fluorescence was measured in serum and quantified using a
rhodamine calibration curve developed in mouse serum. Absolute bioavailability was calculated
for CS-NP R and free R relative to mice IV injected via tail vein with 10 mg/kg rhodamine dissolved
in 100 uL of PBS.
5.2.13 Histology and Immunohistochemistry
Following ex vivo imaging, the brain, heart, lungs, liver, kidneys, spleen, intestines, and
bladder were immediately frozen and embedded in OCT (Tissue Tek, Sakura Finetek, Torrance,
CA, USA). 10 μm sections were obtained (CM3050 S Cryostat, Leica, Nussloch, Germany) and
placed on Superfrost Plus slides (Fisherbrand, Waltham, MA, USA). Tissue sections were stained
with hematoxylin & eosin (H&E) and imaged (Leica DMi8, Leica, Wetzlar, Germany, N = 3). For
staining of intestinal mucin, tissue sample were processed with an alcian blue 1%, pH 2.5 stain
kit (Newcomer Supply, Middleton, WI, USA). Briefly, tissue sections on slides were washed in
acetic acid for 3 minutes, incubated in alcian blue for 30 minutes at 4°C in a humidified chamber,
and counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA, USA). Samples
were mounted using VectaMount™ (Vector Laboratories, Burlingame, CA, USA).
5.2.14 Therapeutic Efficacy in ADPKD Mice
To assess the ability CS-NP to enhance the therapeutic efficacy of orally administered
drugs in ADPKD, 300 mg/kg met-loaded CS-NP or free met was administered to Pkd1
fl/fl
;Pax8
rtTA;Tet-O-Cre mice. Pups were IP injected with doxycycline on postnatal day 10-11 (P10-11) to
induce a severe PKD phenotype as previously described [226]. Mice were orally gavaged every
two days starting on P12 and euthanized on P22 (N ≥ 4). Kidneys were excised to assess kidney
to body weight (KW/BW) ratio and stained with H&E to compare cystic index. Cystic index was
68
defined as the percentage of cystic area divided by total kidney area [227] and determined by
ImageJ.
5.2.15 Kidney Health in PKD Mice
Serum components, electrolytes, and kidney health markers including sodium (Na),
potassium (K), chloride (Cl), ionized calcium (iCa), total carbon dioxide (tCO 2), glucose (Glu),
blood urea nitrogen (BUN)/Urea, creatinine (Crea), hematocrit (Hct), hemoglobin (Hb), and anion
gap (AnGap) were assessed in diseased Pkd1
fl/fl
;Pax8 rtTA;Tet-O-Cre mice. 90 uL of blood taken
from the submandibular vein on the day of harvest was analyzed using Chem-8+ cartridges for
the i-Stat Handheld Blood Analyzer (Abbott, Chicago, IL, USA).
5.2.16 Statistical Analysis
A Student’s t-test was used to compare means of pairs. Analysis of variance (ANOVA)
with Tukey’s multiple comparison test post-hoc analysis was used to determine significant
differences among three or more means. A p-value of ≤ 0.05 was considered to be significant.
5.3 Results and Discussion
5.3.1. Fabrication and Characterization of Chitosan Nanoparticles
Chitosan is an easily procured biomaterial, and several synthesis methods have been
investigated to synthesize chitosan nanoparticles [228-231]. Ionic gelation was used in this study
as it has been reported to achieve high drug encapsulation and low polydispersity [210, 211], and
ionic gelation is based on electrostatic interaction between the amine group of chitosan and a
negatively-charged group of a polyanion such as poly-L-glutamic acid [207]. Due to this charge-
based interaction, negatively-charged payload drugs can also be easily incorporated during the
ionic gelation process [232].
In addition, one of the main advantages of using chitosan for oral delivery is its
mucoadhesive properties, as the mucous lining in the GI tract provides a significant barrier to
69
many drugs [233]. Mucous is composed of water (~90 to 98%), salts (~0.5 to 1.0% w/w), proteins
(~0.5% w/v), and mucins (0.2–5% w/w) [234] which are the glycoproteins responsible for
excluding large micrometer sized particulates by steric hinderance [235]. Without mucoadhesive
properties, oral delivery formulations lack the ability to withstand peristalsis movements [236] as
well as the extensive washing effect of body fluids, such as GI acids [237], which results in the
loss of drug payload available systemically.
In previous studies, chitosan polymers have been shown to bind to mucins more
effectively as the degree of deacetylation (DDA) increased [238], as the additional positively-
charged amino groups allow for increased interaction with negatively-charged sialic acid residues
of mucin [239]. Similarly, in this study, upon deacetylation of the starting chitosan (before
nanoparticle synthesis), the zeta potential of the chitosan solutions confirmed increasing %
deacetylation increased the positive charge from 15.8 ± 4.2 mV at 50% DDA to 27.3 ± 2.8 mV at
90% DDA. When chitosan polymers of varying DDA (90%, 80%, 70%, and 50% DDA) were tested
in a mucin binding assay, the highest DDA (90%) showed the greatest binding efficiency to mucin
(75.1 ± 5.0%) (Figure 5-2a).
70
Figure 5-2 Optimization of CS-NP synthesis parameters and mucin binding efficiency. (a) Binding
efficiency of chitosan to mucin increases upon chitosan DDA. (b) Nanoparticle diameter and (c)
polydispersity as a function of the starting chitosan and poly-glutamic acid crosslinker
concentration. The lowest polydispersity for CS-NP is seen at 2 mg/ml chitosan and 1 mg/ml
crosslinker concentrations. (d) Diameter of CS-NP with respect to mucin binding efficiency shows
highest binding at approximately 150 nm. (*p ≤ 0.05, **p ≤ 0.01, N ≥ 3) (e) TEM micrographs
confirm spherical morphology and a monodisperse population of CS-NP with 90% DDA chitosan
at 2 mg/ml and poly-L-glutamic acid crosslinker at 1 mg/ml synthesis conditions.
In order to identify the ideal nanoparticle diameter to adhere and diffuse through the
mucosal layer, 90% DDA chitosan was used to synthesize chitosan nanoparticles (CS-NP) of
various diameters (50, 100, 150, 200, 250, 300 nm), achieved by altering the starting
concentrations of chitosan and poly-L-glutamic acid crosslinker during ionic gelation. Nanoparticle
diameter has been found to affect the ability to bind and diffuse through mucus, as the mesh pore
size (10-200 nm) of mucus sterically limits nanoparticles larger than 200 nm [240]. Chitosan
concentrations were limited between 0.5 mg/ml and 3.0 mg/ml, as higher concentration ranges
resulted in immediate aggregation of particulates upon dropwise addition to polyglutamic acid,
and lower concentrations resulted in no nanoparticle formation. As shown in Figure 5-2, CS-NP
of approximately 150 nm in diameter demonstrated the highest mucin binding efficiency (74.8 ±
4.0%) (Figure 5-2d). We observe a decrease in binding for particles beyond 200 nm, which is
consistent with the study by S. Bandi et al. demonstrating nanoparticles ≥ 200 nm in diameter had
limited diffusivity through mucus due to mucin mesh steric hindrance [241]. Hence, we selected
90% DDA chitosan at 2 mg/ml and poly-L-glutamic acid crosslinker at 1 mg/ml synthesis
conditions, which resulted in nanoparticles with the desired diameter of 150 nm and a low PDI of
0.24, to proceed with further studies (Figure 5-2b,c,e). Increasing mucodiffusion has also been
a viable engineering strategy to access underlying enterocytes and increase systemic
bioavailability [242]. While not investigated in this study, the mucodiffusive properties of chitosan
may play a role in navigating the GI tract and will be evaluated in future studies.
71
5.3.2. Drug Release and Degradation Properties of CS-NP
To successfully deliver substances through the GI tract, enteric delivery systems must
protect the payload from degradation and premature release in the low pH environment of the
stomach [243]. The amino groups in chitosan (pK a = 6.5) are protonated to form NH 3
+
at low pH
(pH 1.2-2.5), providing strong electrostatic attractions to the oppositely-charged poly-glutamic
acid crosslinker, which allows the particles to remain compacted and remain at their original size
and retain the payload [244]. At higher pH values (pH = 6.5–7.4), the amine groups of chitosan
exist mostly in the NH2 form [245]. As a result, the electrostatic interactions between CS and
crosslinker are weakened, which favors dissolution [246].
To verify CS-NP have the ability to protect drugs past the gastric environment, we tested
met-loaded CS-NP (CS-NP met) in pH environments representative of a fasting stomach (pH 1.5),
fed stomach (pH 2.5), duodenum of the small intestine (pH 6.5), circulating blood (pH 7.4), SGF
(pH 1.3), and SIF (pH 6.8) [247, 248]. The loading efficiency and loading capacity of met into CS-
NP was determined to be 32.2 ± 2.8% and 37.3 ± 3.6%, respectively, likely due to the electrostatic
interaction of positively-charged met with the negatively-charged crosslinker during nanoparticle
synthesis [249]. To assess met release profiles, we quantified free met release from CS-NP met
in the various pH environments for up to 6 hours. We observed that payload release did not
exceed 25% at pH conditions 1.2, SGF (pH 1.3), and 2.5, while greater than 50% release was
found in pH 6.5, SIF (pH 6.8), and 7.4 at 3 hours (Figure 5-3a). Consistent with release studies,
TEM images showed minimal morphological change at pH 1.2 and 2.5, (Figure 5-3b), while an
increase in diameter (~250 nm) was seen in pH 6.5. At pH 7.4, CS-NP met lost their spherical
morphology and fused with adjacent particles. In agreement with observations in other chitosan
nanoparticle studies, water is able to intercalate through pores in the polymer matrix, expanding
the particle and causing swelling and degradation [250]. This further suggests that CS-NP can
72
remain stable and protect drugs under the low pH conditions found in the stomach but swell and
release drugs upon reaching neutral pH found in the small intestine and systemic circulation.
Figure 5-3 In vitro pH response of CS-NP. (a) In vitro release of met from CS-NP under various
pH conditions present in the GI tract (pH = 1.2, fasting stomach; 2.5, fed stomach; 6.5 intestines,
7.4 blood; 1.3, SGF; 6.8 SIF) (N ≥ 4). (b) TEM images of CS-NP confirm degradation at pH 6.5
and 7.4 after 6 hours.
5.3.3 In vitro Penetration Across Intestinal Epithelium
In addition to mucoadhesive properties and protection of payloads in the acidic
environment of the stomach, chitosan has been suggested to enhance the penetration of the
intestinal epithelial cell barrier by opening tight junctions and increase transport of luminal
peptides, nutrients, and nanoparticles [251]. To verify this, CS-NP rhodamine (CS-NP R), free
rhodamine, or PBS was tested in an intestinal epithelial barrier model consisting of human
colorectal Caco-2 cells, on Transwell membranes [252], and tight junction integrity was
determined via transepithelial resistance (TER) measurements [225]. TER measurements were
made three days before treatment, and again after administration of CS-NP R, free rhodamine,
or PBS for up to 3 days. No changes in TER occurred in the PBS or free rhodamine-treated
groups, while an 84.8% reduction in resistance to 53.1 ± 32.3 Ohm*cm
2
was observed for CS-NP
R 6 hours after administration (Figure 5-4a). A recovery to pre-treated baseline resistance levels
73
(355 Ohm*cm
2
) was seen after 3 days, suggesting the effects on tight junctions are reversible,
yet persist enough on the time scale that digestion occurs in the human gut [253].
In addition to paracellular transport, transcellular transport has been reported for
nanoparticles passage through the intestinal lining [102]. Previous studies have suggested
chitosan nanoparticles can undergo endocytosis (clathrin-mediated) and micropinocytosis in
intestinal cells [254]. Specifically, to determine if transcytosis or micropinocytosis is mainly
responsible for the transport of CS-NP, Caco-2 cell layers were treated with colchicine (10 μM,
for 60 min), wortmannin (0.06 mM, for 3 h), or no inhibitor before 100 µM of CS-NP R incubation,
and rhodamine fluorescence was measured in the basolateral chamber over the course of 4 hours.
A 27% reduction of transport was seen when the transcytosis inhibitor colchicine was
administered, while no reduction was seen when the micropinocytosis inhibitor wortmannin was
administered (Figure 5-4b). These initial findings suggest that transcytosis is a major pathway by
which CS-NP are transported across intestinal epithelial layers [225, 255, 256], in addition to
increasing paracellular permeation. Importantly, over 90% of cells were viable upon treatment
with CS-NP as indicated by an MTS assay, suggesting transport differences are not due to
cytotoxic effects of chitosan. The CS-NP R transport behavior is also distinct from free rhodamine
administered in the same conditions. No statistically significant reduction is observed in transport
of free R between the colchicine and wortmannin conditions, while colchicine reduces CS-NP R
transport.
5.3.4 In vitro Therapeutic Efficacy of Metformin-loaded CS-NP
Upon characterizing the biomaterial properties of CS-NP and verifying its potential to
deliver drugs through intestinal epithelia, met was loaded into CS-NP for ADPKD applications.
We selected met, a first-line therapy already approved for diabetes, due to its secondary benefits
in inhibiting ADPKD preclinically, resulting in several ongoing clinical trials repurposing met for
ADPKD including METROPOLIS (NCT03764605) and TAME (NCT02656017) [257, 258].
74
Specifically, met activates the 5' AMP-activated protein kinase (AMPK) pathway by
phosphorylating AMPK. This leads to inhibition of the mammalian target of rapamycin (mTOR)
pathway [259], responsible for the expansion of cysts due to the overproliferation of renal tubular
cells. Additionally, met inhibits intracellular generation of cAMP via inhibition of adenylyl cyclase
[260], a key signaling pathway that drives cystogenesis in ADPKD. In addition, AMPK activation
has been found to inhibit fluid secretion into cysts by inhibiting the cystic fibrosis transmembrane
receptor (CFTR) channel [261], the key apical membrane chloride secretory route in ADPKD [262].
In ADPKD preclinical murine studies, met was administered at a dose of 300 mg/kg/day which
exceeds the dose currently prescribed for patients with diabetes by ~38% when corrected for
volume of distribution and surface area-to-volume ratio (maximum 37.5 mg/kg/day). Even at low
doses, 25% of patients already suffer from GI discomfort and approximately 5% are unable to
tolerate met entirely due to these side effects [172-174]. Since the bioavailability of orally taken
met is only 40%, it is expected that high doses are needed for PKD efficacy, which may further
exacerbate the incidence of side effects [217]. To enhance the bioavailability of met for oral
delivery in ADPKD, met was loaded into chitosan nanoparticles (CS-NP met), and the size and
charge of CS-NP met was found to be unaltered compared to unloaded CS-NP (Table 5-1).
Table 5-1. Size and charge of unloaded CS-NP compared to CS-NP met.
To first test therapeutic potential in vitro, mpkCCDc14 cells, derived from the cortical
collecting duct, were treated with 300 µM met in CS-NP met, free met, unloaded CS-NP, or PBS
for 12 hours and phosphorylated (active) AMPK to total AMPK ratio was measured using an
75
ELISA assay. As shown in Figure 5-4c, an increase in phosphorylated AMPK to total AMPK ratio
in both met-containing groups was seen: 3.0 ± 0.1 for free met and 2.1 ± 0.1 for CS-NP met (p
< .005), while no change was found upon PBS and CS-NP blank treatment. While the free drug
showed higher therapeutic efficacy at the same dose, we believe this is due to the slow release
profile of met from CS-NP at 7.4 pH (Figure 5-3a), compared to the bolus effect of free met.
Additionally, the effect of CS-NP met on the reduction of ENaC current, a measure of the CFTR
activity, was analyzed. After 15 min, 3 hours, 24 hours, and 48 hours of CS-NP met, free met,
CS-NP blank, or PBS treatment, the CS-NP met and free met groups showed a marked decrease
in ENaC current in mpkCCDc14 cells. The largest change was seen after 48 hours, with CS-NP
met reaching 81.1 µA/cm
2
and free met at 79.2 µA/cm
2
, while unloaded CS-NP ENaC remained
at the 117.2 µA/cm
2
baseline (Figure 5-4d, p < 0.005). Taken together, these studies confirmed
the therapeutic efficacy of CS-NP met was not hindered, as it produced similar AMPK activity and
ENaC inhibition compared to free met. As over 90% of cells were viable upon treatment with CS-
NP when assayed by MTS, the observed ENaC and AMPK changes are not due to cytotoxic
effects of chitosan.
76
Figure 5-4 In vitro transport mechanisms and therapeutic efficacy of CS-NP (a) TER
measurements of a Caco-2 cell layer upon 100 µM CS-NP R, free R, or PBS treatment 3 days
before and after treatment show paracellular transport through tight junctions. (b) CS-NP R
permeation across a Caco-2 cell layer treated with 100 µM of CS-NP R after 4 hours, pretreated
with either colchicine (transcytosis inhibitor), wortmannin (micropinocytosis inhibitor) or no
inhibitor. (c) Phosphorylated AMPK to total AMPK obtained via ELISA; and (d) ENaC current
measurements of mpkCCDc14 cell monolayers treated for up to 48 h with CS-NP met (300 µM),
free met, CS-NP, and PBS show a significant decrease for the CS-NP met and free met groups,
compared to CS-NP, confirming therapeutic activity (****p ≤ 0.0001,***p ≤ 0.001, N ≥ 4).
5.3.5 Ex vivo imaging of CS-NP in vivo and Intestinal Localization
Next, to assess the ability of CS-NP to enhance drug bioavailability via oral delivery in vivo,
10 mg/kg rhodamine was first encapsulated into CS-NP (CS-NP R) and C57BL/6J mice were
orally gavaged with 200 uL CS-NP R or free R, and after 24 hours, ex vivo imaging was conducted.
As shown in Figure 5-5, ex vivo optical imaging demonstrated the majority of CS-NP R and free
R accumulated in the intestines, liver, kidneys, and bladder, and upon quantitative analysis, CS-
NP R showed 60.3 ± 11.0% of total organ fluorescence accumulation in the intestines vs. 37.4 ±
15.5% for free R (p<0.005, Figure 5-5a). A time course for ex vivo imaging using CS-NP R at 3
hours and 48 hours showed similar trends: at 3 hours, CS-NP R had 42 .1 ± 12.0% accumulation
while free R had 45.8 ± 14.0% (p < 0.005). At 48 hours, 52.3 ± 10.1% accumulation was found
for CS-NP R in the intestines whereas 38.5 ± 15.1% accumulation for free R (p < 0.005, Figure
S4). Notably, serum fluorescence showed a higher area under the curve (AUC) ratio of 1.3:1 for
CS-NP R compared to free R over the course of 7 days (Figure 5-5b), demonstrating enhanced
depot to systemic circulation.
77
Figure 5-5 Semi-quantitative biodistribution of mice treated with 10 mg/kg rhodamine in 200 uL
of CS-NP R and free R 24 hours after oral gavage. (a) Comparison of ex vivo imaging between
rhodamine fluorescence levels showed higher accumulation in the intestines for CS-NP R vs. free
R 24 hours post-oral gavage. (b) Serum fluorescence of CS-NP R shows a greater absolute
bioavailability and an extended release profile for the CS-NP formulation for up to 7 days (76.2%
for CS-NP R and 47.9% for free R; ***p ≤ 0.001, N ≥ 4). (c) Representative ex vivo images confirm
highest signal in the intestines in the CS-NP R condition 24 hours after oral gavage.
Upon further assessment of CS-NP R localization within the intestines via ex vivo imaging,
CS-NP R was found adhered to the jejunum of the intestines (Figure 5-6a,b). Fluorescence
microscopy of intestinal sections also confirmed higher rhodamine signal in the jejunum as well
as higher colocalization of CS-NP R with mucin vs. free R (Figure 5-6c). This is beneficial for oral
delivery as the Peyer’s patches located within the jejunum, as well as the larger surface area
compared to the duodenum and ileum, are responsible for the majority of nutrient uptake, as well
as facilitating nanoparticle transport into systemic circulation [263, 264]. Overall, these findings
suggest that through mucoadhesion, CS-NP is retained in the jejunum, which allows for sustained
drug release and bioavailability.
78
Figure 5-6 Quantification of intestinal localization of CS-NP R and free R 24 hours after oral
gavage. (a) Ex vivo fluorescence images and (b) quantitative comparison show the majority of
CS-NP R adhered to the jejunum, while free R treatment is localized to the duodenum and ilium.
(***p ≤ 0.001, N ≥ 4). (c) Alcian blue staining of mucus shows colocalization of the CS-NP R to
the intestinal mucosa, demonstrating mucoadhesion.
5.3.6 Therapeutic Efficacy of CS-NP met in PKD Mice
To confirm the viability of CS-NP to act as an oral delivery vehicle in chronic diseases,
CS-NP met was administered in the ADPKD murine model, Pkd1
fl/fl
;Pax8-rtTA;Tet-O cre [265]. In
this model, a rapidly progressing PKD phenotype can be developed by knockout of the PKD1
gene, induced by doxycycline injection on P10 and P11. Then starting on P12, mice were orally
gavaged with 300 mg/kg of met loaded in CS-NP met, control CS-NP, or free met every two days
and euthanized on P22 when a severe cystic phenotype is expected. Kidneys were excised to
assess kidney to body weight (KW/BW) ratio and stained with H&E to compare cystic index. In
79
CS-NP met-treated mice, a greater decrease in the KW/BW ratio was found compared to free met
(10.3 ± 1.1 vs. 13.1 ± 1.0, p ≤ 0.01), confirming enhanced therapeutic efficacy in slowing of
cystogenesis of met when delivered via CS-NP (Figure 5-7a). Moreover, cystic index was
statistically lower in CS-NP met-treated mice compared to mice treated with the free drug (57.6 ±
1.2% vs. 66.5 ± 0.8%, p ≤ 0.01, Figure 5-7b,c). A Cre- mouse serves as healthy control in which
the PKD1 gene knockout is not activated and Cre- kidneys represent normal kidney morphology.
Although a met dose of 300 mg/kg daily has been found to activate AMPK in previous
murine models [172], it is higher than what is currently prescribed for patients with diabetes
(maximum 37.5 mg/kg/day). Our study administered 300 mg/kg met every two days instead, and
confirmed efficacy via oral delivery that was comparable to previous IP delivery studies [172].
Future dose de-escalation studies will be conducted to examine the full benefits of CS-NP in
increasing therapeutic efficacy without compromising safety. Regarding renal biocompatibility,
kidney health markers including blood urea nitrogen, creatinine, and serum electrolytes were
found to remain similar between treatment groups, demonstrating CS-NP formulations do not
cause kidney damage (Table 5-2). The BUN levels correspond to mildly impaired renal function
expected in polycystic kidney mice, on the order of 40-80 mg/dL [266]. In sum, CS-NP
demonstrated a higher therapeutic efficacy when compared to free drug at the same dose, and is
a safe platform that can overcome the physiological barriers of oral delivery. Uniquely, this is the
first nanoparticle delivery platform for ADPKD, and our study highlights CS-NPs as a viable oral
delivery platform for chronic conditions.
80
Figure 5-7. In vivo therapeutic efficacy of CS-NP met. (a) A lower KW/BW ratio (10.3 ± 1.1 vs.
13.1 ± 1.0 (**p ≤ 0.01, N ≥ 4) and (b) cystic index (57.6 ± 1.2 vs. 66.5 ± 0.8; **p ≤ 0.01, N ≥ 4) was
seen in the CS-NP met group vs. free drug. (c) H&E staining of whole kidneys shows less severe
cystic phenotype in the CS-NP met group. A Cre-recombinase negative control is a non-diseased
kidney morphology.
Table 5-2. Serum components, electrolytes, and kidney health markers for CS-NP met, free met,
and CS-NP treated mice show no significant difference between groups. Measured values include
sodium (Na), potassium (K), chloride (Cl), ionized calcium (iCa), total carbon dioxide (tCO2),
glucose (Glu), blood urea nitrogen (BUN)/Urea, creatinine (Crea), hematocrit (Hct), hemoglobin
(Hb), and anion gap (AnGap).
81
5.4 Conclusion
Chitosan nanoparticles (CS-NP) were investigated as a promising drug delivery platform
for oral delivery in chronic kidney disease. CS-NPs were synthesized through ionic gelation and
their physiochemical properties were characterized. In vitro, CS-NPs demonstrated effective
mucoadhesion, while protecting premature release of the payload in the low pH environment of
the stomach. When met-loaded CS-NP were cultured with cells in vitro, therapeutic efficacy was
found via AMPK activation and ENaC current reduction. Moreover, upon oral gavage in a murine
model of PKD, disease burden was significantly reduced upon met delivery using CS-NP
compared to the free drug. While free met at the dosages used in the study did not cause
significant toxicities, future studies assessing high met dosages or increasing dose exposure in
the slowly developing mouse model of PKD that more closely mimics the chronic nature of the
human disease will more fully elucidate the benefits of delivering drugs in the CS-NP system.
Furthermore, the observed advantage of CS-NP in increasing systemic delivery may also be more
evident upon loading candidate ADPKD drugs with poor oral bioavailability, such as somatostatin
or bardoxolone methyl. Our study provides the framework to advance chitosan nanotechnology
for PKD; future studies will include additional animal models including slowly progressing PKD
models to further mimic the chronic nature of PKD, as well as large porcine animal models as we
look towards clinical translation.
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Chapter 6: Oral Delivery of Therapeutic Kidney Targeting Micelles
for ADPKD
6.1 Introduction, Objective, and Rationale
The past few decades have witnessed unprecedented endeavors for developing
nanomaterials as effective drug delivery vehicles [267]. As discussed in Chapter 4, peptide
amphiphile micelles have emerged as a promising class of nanoparticles with the ability to target
an organ of interest [14, 133]. However, these studies have relied on intravenous injection to
administer micelles and achieve 100% bioavailability [7]. Almost all FDA approved nanomedicines
rely on IV injection and are not suitable for chronic diseases such as autosomal dominant
polycystic kidney disease (ADPKD). Oral delivery is the delivery route of choice for patients, as it
achieves the greatest patient compliance, is pain free, and does not require a specialist to
administer [104, 109]. In Chapter 5, we demonstrated the results for a chitosan nanoparticle
system which can protect the payload from gastrointestinal (GI) tract degradation and bypass the
barriers inherent to oral delivery.
It is well recognized in the oral drug delivery industry that drug formulations consisting of
many small, discrete and individual drug delivery units, or multiparticles, have advantages over a
bulk solid or liquid formulation. These include pellets, beads, micro-granules, spheroids, or mini-
or micro-tablets. In 2015, multiparticle sales were in the order of $34 billion out of a $475 billion
total for oral solid dosage sales, or 7.15% of the market [268]. The advantages of multiparticle
formations include tunable drug release rates [269], ease of including combination therapy, and
reduced variation in gastric transit behavior, and multiparticle technology development is
expected to grow around 6% in the years to come. The reported size ranges of approved
multiparticle products vary, and may be as small as 150 μm, or as large as 2-3 mm [270]. Delivery
of constructs on the nanoscale are still mostly in preclinical and investigational applications.[271].
89
Some studies have taken to direct modification of micelles to resist the harsh GI tract,
usually composed of Pluronic or derivatives of poly(ethylene oxide) (PEO) with hydrophobic
poly(propylene oxide) (PPO) block copolymers [272]. However, modification of the amphiphile for
stability results in reduced ability to conjugate hydrophilic drugs which will be exposed on the
outer surface, and have mainly been limited to core loading hydrophobic drugs such as Paclitaxel
[273], Cyclosporin A [274], or Megestrol [275]. Therefore, the combination of our targeted KM
from Chapter 4 with our CS-NP platform from Chapter 5 can allow the delivery of intact micelles
with targeting ligands and a hydrophilic drug payload (metformin).
To our knowledge, the loading of supramolecular nanoparticles within chitosan has yet to
be studied and presents a novel methodology to improve poor bioavailability of nanoparticles
upon oral administration. In this final aim of this body of work, we encapsulate therapeutic kidney
targeting micelles within chitosan nanoparticles and investigate the in vitro permeation
enhancement across a Caco2 monolayer of this combinatorial system. To conclude, we
administer chitosan nanoparticles loaded with metformin kidney targeting micelles (CS-NP KM
met) into a slowly progressing ADPKD murine model. We pave the path to create an orally
deliverable, targeted micelle treatment for ADPKD (Figure 6-1).
90
Figure 6-1 Schematic of an orally delivered therapeutic kidney targeting micelle system. Kidney
targeting micelles are loading within chitosan nanoparticles (top). Loaded chitosan nanoparticles
are administered orally and deliver their payload across the GI tract upon contact with
physiological pH conditions (bottom).
6.2 Methods and Materials
6.2.1 Synthesis of Therapeutic KMs and Loaded Chitosan Nanoparticles
The (KKEEE)3K targeting peptide was again synthesized using standard Fmoc-mediated
solid phase peptide synthesis methods on rink Amide resin (Anaspec, Fremont, CA, USA) using
an automated benchtop peptide synthesizer (PS3, Protein Technologies, Tucson, AZ, USA). A
cysteine was added to the peptide sequence at the N-terminus to allow for a thioester linkage.
Peptides were cleaved from the resin and deprotected with 94:2.5:2.5:1 by volume trifluoroacetic
acid:1,2-ethanedithiol:H2O:triisopropylsilane and were precipitated and washed several times
with cold diethyl ether, dissolved in water, lyophilized, and stored as powders at 20°C. Crude,
peptide mixtures were purified by reverse-phase high performance liquid chromatography (HPLC)
91
(Prominence, Shimadzu, Columbia, MD, USA) on a C8 column (Phenomenex, Torrance, CA,
USA) at 50°C using 0.1% formic acid in acetonitrile/water mixtures and characterized by matrix-
assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectral analysis
(Autoflex Speed, Bruker, Billerica, MA, USA). Cysteine-containing peptides were conjugated to
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol 2000)
(DSPE-PEG(2000)-maleimide, Avanti Polar Lipids, Alabaster, AL, USA) by adding an equimolar
amount of the lipid to peptide in MilliQ water (pH 7.2). After gentle mixing for one week, the
resulting product was purified by HPLC on a C4 column as described above.
The fluorophore-conjugated monomer was synthesized by conjugating Cy7 via a peptide
bond to DSPE-PEG(2000)-amine (Avanti Polar Lipids, Alabaster, AL, USA) by adding an
equimolar amount of Cy7 mono-N-hydroxysuccinimide ester (GE Healthcare Life Sciences,
Pittsburgh, PA, USA) to the lipid dissolved in 10 mM aqueous sodium carbonate buffer (pH 8.5).
After reaction at room temperature for 24 hours, protected from ambient light, the mixture was
purified on a C4 column and characterized as described above.
The therapeutic monomer was synthesized by conjugating metformin hydrochloride via a
peptide bond to DSPE-PEG(2000)-NHS (Avanti Polar Lipids, Alabaster, AL, USA) by adding a 5x
molar excess of metformin to the lipid dissolved in 10 mM aqueous sodium carbonate buffer (pH
8.5). After reaction at room temperature for 24 hours, protected from ambient light, the mixture
was purified on a C4 column and characterized as described above.
Monomers were self-assembled into micelles via thin film evaporation. The appropriate
DSPE-PEG(2000) amphiphiles were dissolved in methanol or chloroform and evaporated under
a steady stream of air. The resulting film was dried under vacuum overnight and hydrated at 80˚C
with either MilliQ water or PBS, vortexed and sonicated as needed to obtain a clear solution and
allowed to cool to room temperature. Therapeutic KMs (metformin-KM) were composed of a
92
monomer molar ratio of 10:45:45 consisting of DSPE-PEG(2000)-Cy7:DSPE-PEG(2000)-
(KKEEE)3K:DSPE-PEG(2000)-metformin.
These assembled micelles were loaded into chitosan nanoparticles via ionic gelation.
Intact micelle solution was added to the anionic crosslinker solution. Chitosan (Heppe Medical
Chitosan GmbH, Halle, Germany) with 200 mPas viscosity and 85% degree of deacetylation was
dissolved at a 2.0 mg/mL concentration in a solution of 0.5% acetic acid in MilliQ water. Then a
solution of 1mg/ml anionic crosslinker of poly-L-glutamic acid sodium salt (Sigma Aldrich, St.
Louis, MO, USA) was prepared. This crosslinker solution was added as the solvent to a thin film
of metformin KM, hydrating them and forming micelles. The chitosan solution was added dropwise
under constant stirring to the crosslinker/micelle solution at a volume ratio of 5:2. An opalescent
suspension was formed spontaneously. Chitosan nanoparticles were separated by centrifuging
at 14,000 rpm at 14°C for 30 minutes and the pellet was washed with increasing grades of ethanol
in water and used immediate for studies, or frozen and lyophilized and stored at 4°–8°C. All other
chemicals used were reagent grade.
6.2.2 Characterization of Micelle Loading into Chitosan Nanoparticles
Dynamic Light Scattering (DLS): 100 μM of CS-NP KM solutions loaded with between 0-
3000 uM of KM were filtered through Puradisc 0.2-μm Polyvinylidene fluoride (PVDF) membrane
filters (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and measured immediately. DLS
measurements were determined at 163.5° and 532 nm using a Wyatt Technology Möbiuζ system
(Santa Barbara, CA, USA, N ≥ 3). All measurements were carried out at 25°C in MilliQ water after
equilibrating for 5 minutes.
Zeta Potential: Zeta potential of 100 μM solution of CS-NP KMs were dissolved in water
and filtered through Puradisc 0.2-μm PVDF membrane filters (GE Healthcare Life Sciences,
Pittsburgh, PA, USA) were measured using the same Möbiuζ system described above. Samples
93
were placed in a Quartz cuvette with a polyether ether ketone (PEEK) and platinum dip probe (N
≥ 3). All measurements were carried out at 25°C after equilibrating for 5 minutes.
Transmission Electron Microscopy (TEM): Negative stained samples for TEM were
prepared by placing 100 μM solution of CS-NP KM, loaded with between 0-3000 uM of KM in
MilliQ water on 400 mesh lacey carbon grids (Ted Pella, Redding, CA, USA) for 5 minutes. Excess
liquid was wicked away with filter paper and the grid was washed with MilliQ water before placing
2 wt.% uranyl acetate solution for 2 minutes, then washing with MilliQ water. Dried samples were
immediately imaged on a JEOL JEM-2100F TEM (JEOL, Ltd., Tokyo, Japan).
6.2.3 Drug Release of Metformin from Metformin-KMs
To assess how free metformin would be cleaved from KM-metformin, a drug release assay
was performed in the presence of protease. 1000 µM of metformin-KMs were synthesized and
treated with protease from streptomyces griseus (Sigma Aldrich, St. Louis, MO, USA) at 3.3 U/mL.
Samples of protease treated metformin-KM, free metformin, or untreated metformin-KM were
placed in Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific, Waltham, MA, USA) with a
molecular weight cutoff of 2000 Daltons in 10 mM sodium acetate buffer at pH 7.45.
Measurements of free metformin diffusing out of the dialysis chamber were made at select
timepoints (30 min, 1 hr, 2hr, hr, 3 hr, 4hr, 5hr, 6hr, 12hr). Measurements of the amount of
metformin released from the dialysis cassette was measured by UV-VIS spectrophotometer
(Nanodrop, Thermo Fisher Scientific, Waltham, MA, USA) at an absorbance of 233 nm.
6.2.4 Cell Culture
Cortical collecting duct (mpkCCDc14) cells were passaged between P10 and P20 for these
studies. Complete media was a 50% mixture of Dulbecco's Modified Eagle Medium (DMEM) and
HAMF-12 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Subculturing onto Transwell™
inserts (Thermo Fisher Scientific, Waltham, MA, USA) with 0.4 μm pore size in a 24-well plate
94
format preceded voltage measurements and western blot analysis. Media was exchanged every
3 days.
Human colon epithelial cells (Caco-2) were cultured following the manufacturer’s
recommendations. Cells were expanded in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were
seeded at a density of 4.5 x 103 cells/cm2 and subcultured upon 50% confluence. For transport
experiments, Caco-2 cell monolayers were seeded onto Transwell inserts (Corning; diameter 6.5
mm, growth area 0.33 cm2, pore size 0.4 μm), at an initial density of 3 x.10
5
cell/cm
2
and
maintained for 21 days in complete medium to form a confluent monolayer.
Mouse kidney cortical collecting duct (mpkCCDc14) cells were expanded in culture media
comprised of DMEM/F12 supplemented with insulin, dexamethasone, selenium, transferrin,
triiodothyronine, glutamine, d-glucose, epidermal growth factor (EGF), HEPES, sodium pyruvate.
Complete media was filtered before use, and media was changed every two days and subcultures
were passaged every 7-8 days. Both cell lines were grown at 37°C in a humidified incubator under
5% CO2. For transport experiments, Caco2 cells on Transwell inserts were placed in the apical
chamber, while mpkCCDc14 cells were seeded in the basolateral chamber. CS-NP, free
metformin, KM metformin, or CS-NP KM met was added to the apical chamber, and AMPK
activation of the basolateral mpkCCDc14 cells was measured by ELISA. Tight junctions in the
Caco2 monolayer were visualized in microscopy by a ZO-1 stain.
Clonal renal tubular epithelial cell lines generated from Pkd1flox/-:TSLargeT mice by in
vitro Cre recombinase transfection yielded (Pkd1 Null, Pkd1 Het) were cultured in DMEM/F12
media, 2% FBS, 1x ITSG, and ~2 nM of tri-ido-sodium salt. Cells were expanded at 37 °C in a
humidified incubator under 5% CO2. Cells at passage 3 were used for studies, and the media
were changed every 2–3 days.
95
6.2.5 3-D Matrigel Cell Culture of PKD1 Null and PKD1 Het Cells
50 μl of Matrigel from BD Biosciences is added to each well in a 96 well plate, and solidified
in a 37° incubator for 15 minutes. Pkd1flox/− or Pkd1−/− were trypsinized and resuspended with
150uL of 2% Matrigel™ in assay medium to achieve approximately 3000 cells/well, and the cells
were grown for 1–2 days. Then, treatment with either 300 µM metformin in KM met formulations,
and cells were treated from day 2-10 to assess effect on cyst growth. The cross-sectional areas
of the cell structures grown in 3-dimensional Matrigel culture were calculated using ImageJ
software. MTS cell proliferation colorimetric assays (BioVision) were also performed on the same
treatment groups following the manufacturer’s instructions.
6.2.6 Transepithelial Resistance (TER) Surveillance
For transport and monolayer resistance experiments, Caco-2 cell monolayers were
seeded onto Transwell inserts (Corning, NY, USA; diameter 6.5 mm, growth area 0.33 cm
2
, pore
size 0.4 µm), at an initial density of 3 x
.
10
5
cell/cm
2
and maintained for 21 days in complete
medium to form a confluent monolayer. The change of TER, representing the tightness of the cell
monolayers, was measured by an EVOM2 Epithelial Voltohmmeter (World Precision Instruments,
USA). Monolayers reaching steady-state values in the range of 300-400 Ω-cm
2
were used for
studies [222]. TER measurements were performed daily for three days prior to treatment with CS-
NP KM, CS-NP Blank, Free met, KM met, or PBS control to establish baseline measurements.
TER was monitored for baseline levels day 3, 2, and 1 before treatment, every 6 hours
immediately after treatment, then again on day 2 and 3 post-administration.
6.2.7 Drug Release and Morphological Response to pH
Drug release studies were performed on nanoparticles (2 mg/ml initial chitosan and 1
mg/ml poly-L-glutamic acid) suspended in simulated gastric fluid (SGF) composed of 2.0 g/L
sodium chloride and 2.9 g/L HCl (pH 1.3), or simulated intestinal fluid (SIF) composed of 0.62 g/L
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sodium hydroxide and 6.8 g/L potassium phosphate monobasic (pH 6.8) [221]. Free met released
from nanoparticles was quantified at 233 nm using a NanoDrop One microvolume UV-Vis
spectrophotometer for up to 6 hours at room temperature (Thermofisher Scientific, Waltham, MA,
USA). At the endpoint, the resulting degraded nanoparticles were spun down at 14,000 g for 10
minutes, and the supernatant was measured in DLS to verify intact micelles present.
6.2.8 In vitro Therapeutic Efficacy of CS-NP met Through ELISA and Epithelial Sodium
Channel (ENaC) Measurements
To assess therapeutic efficacy in vitro, the cellular levels of phospho-AMPK (Kit #7959)
and total AMPK (Kit #7961) were measured via enzyme-linked immunosorbent assays (ELISA,
Cell Signaling Technologies, Danvers, MA, USA) according to the manufacturer's instructions.
mpkCCDc14 cells seeded on the basolateral well of a Transwell with Caco2 cells seeded in the
apical chamber were treated for 12 hours with 300 µM of met in CS-NP KM met, CS-NP KM Blank,
KM met, free met, or PBS treated controls. All standards and samples were measured on a
Varioskan LUX microplate reader at a wavelength of 450 nm.
6.2.9 In vitro Therapeutic Effect of Metformin-KM and Western Blot
To validate the therapeutic effect of metformin-KMs, we treated polarized mice cortical
collecting cells (mpkCCDc14) on 0.4 µM pore Transwell membranes with 300 µM of metformin,
or an equivalent molar ratio of the therapeutic amphiphile within metformin-KMs for four hours.
Prior to harvest, the epithelial sodium channel (ENaC) current was measured by an epithelial
voltohmmeter (Millicell ERS; Millipore Co., Bedford, MA). Cultured cells were then lysed, and
protein was extracted for Western blotting using standard protocols. Briefly, AMPK lysis buffer
containing 1mM dithiothreitol (Goldbio Technology, St. Louis, MO, USA), 1mM
phenylmethylsulfonyl fluoride (Goldbio Technology, St. Louis, MO, USA) and 1X Complete™
Protease Inhibitor Cocktail (Sigma Aldrich, St. Louis, MO, USA) was used to lyse cells on ice with
50 uL of buffer per Transwell membrane. Cells were scraped with a cell scraper and kept on ice
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for 15 min. Samples were spun 15 min at 14,000 RPM. Samples were then incubated at 65˚C for
15 min and immediately run on NuPAGE Bis-Tris gels (Thermo Fisher Scientific, Waltham, MA,
USA) per the manufacturer’s instructions. Proteins were blotted onto nitrocellulose membranes
and probed with antibodies for Phospho-AMPKα (Thr172) and β-Actin (Cell Signaling Technology,
Danvers, MA, USA). The membrane was probed with appropriate secondary antibodies and
imaged on an Odyssey Imaging System (Li-Cor, Lincoln, NE, USA).
6.2.10 Ex vivo Imaging of Orally Administered CS-NP
To assess the biodistribution of CS-NP semi-quantitatively, 6-7 week old male and female
C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA) were orally gavaged with 200 uL
of 500 µM CS-NP KM Cy7 met, CS-NP NT Cy 7met, NT Cy7 met, KM Cy7 met, or free Cy7 met.
Mice were euthanized after 24 hours post-injection and organs (e.g., brain, heart, lungs, liver,
kidneys, spleen, intestines, and bladder) were excised and imaged ex vivo on an AMI HTX in vivo
imaging system (Spectral Instruments Imaging, Tuscon, AZ, USA). The fluorescence signal was
quantified via Aura software (Spectral Instruments Imaging, Tucson, AZ, USA, N ≥ 4), and
background was subtracted from the PBS-treated group. The mean radiance (photons/s/cm
2
/sr)
for each organ was quantified as a region of interest, and % of total organ fluorescence was
obtained by dividing each organ by the sum of all the organ regions. Blood draws were performed
either retro-orbitally or via tail vein at 30 min, 3, 6, 12, and 24 hours post-administration.
Fluorescence was measured in serum and quantified using a Cy7 met calibration curve developed
in mouse serum. All animal procedures followed NIH guidelines for the care and use of laboratory
animals and were approved by the University of Southern California’s Institutional Animal Care
and Use Committee.
6.2.11 Therapeutic Efficacy in ADPKD Mice
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To assess the ability CS-NP to enhance the therapeutic efficacy of orally administered
drugs in ADPKD, 300 mg/kg met-loaded CS-NP KM met, CS-NP NT met, KM met, or free met
was administered to Pkd1
fl/fl
;Pax8 rtTA;Tet-O-Cre mice. To induce a slowly developing ADPKD
model, mice were IP injected with doxycycline (50 mg/kg/day) on postnatal day 27-29 (P27-P29),
and again on P43 and P57. Mice were orally gavaged every three days starting on P30 and
euthanized on P150. Kidneys were excised to assess kidney to body weight (KW/BW) ratio and
stained with H&E to compare cystic index. Cystic index was defined as the percentage of cystic
area divided by total kidney area [227] and determined by ImageJ.
6.2.12 Histology and Immunohistochemistry
Following ex vivo imaging, the brain, heart, lungs, liver, kidneys, spleen, intestines, and
bladder were immediately frozen and embedded in OCT (Tissue Tek, Sakura Finetek, Torrance,
CA, USA). 10 μm sections were obtained (CM3050 S Cryostat, Leica, Nussloch, Germany) and
placed on Superfrost Plus slides (Fisherbrand, Waltham, MA, USA). Tissue sections were stained
with hematoxylin & eosin (H&E) and imaged (Leica DMi8, Leica, Wetzlar, Germany, N = 3).
6.3 Results and Discussion
6.3.1 Loading of Micelles within Chitosan Nanoparticles
Various methods of loading micelles into the chitosan nanoparticles based on motivations
from prior art were tested. These include encapsulating micelles into microparticles with layer by
layer assembly [276], incorporating the micelles into chitosan gel sheets [277], or hydrating the
micelle amphiphiles with the chitosan solution as demonstrated by Singh et al. [278]. Ultimately,
the chosen method of hydrating KM with the poly-glutamic crosslinker solution resulted in the
most robust method to achieve CS-NP KM diameters similar to that of unloaded CS-NP
developed in Chapter 5.
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Verification of loaded micelles within chitosan nanoparticles were performed with DLS
diameter measurements. The KM nanoparticles show a peak at 14 nm, while CS-NP have a peak
at ~160 nm (Figure 6-2a,b). When mixing 100 µM of micelles with 100 µM of chitosan
nanoparticles separately, we see two distinct size peaks corresponding to the expected micelle
size of 14 nm and the chitosan nanoparticles of ~160 nm. When micelles are loaded in chitosan,
an intact micelle peak is not observed (Figure 6-2c,d). Next, we probed the extent micellesthat
can be loaded within chitosan, ranging from 0 µM to 3000 µM. A similar diameter to that of
unloaded CS-NP is observed when the initial micelle concentration is under 1000 µM. Beyond
this concentration, the diameter of the CS-NP decreases and polydispersity increases, which is a
breakdown of ordered spherical morphology (Figure 6-2e). Thus, we concluded that a maximum
of 1000 µM micelle can be loaded inside CS-NP. Finally, we imaged the loaded CS-NP KM using
TEM to observe the morphology of the loaded chitosan constructs. The morphology is in
agreement with the DLS trend: When 500 or 1000 µM of micelle is loaded, we see individual
micelles encased within larger CS-NP nanoparticles. At concentrations higher than 1000 µM,
separation of the CS-NP and micelle populations occurs, with the majority of micelles existing
outside of CS-NP, and the CS-NP aggregating into disordered precipitates.
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Figure 6-2 KM loading into CS-NP. Representative DLS sizes of (a) KMs, (b) CS-NP, (c) KM
mixed with chitosan nanoparticles, and (d) KM loaded into CS-NP. Micelle peaks corresponding
to ~14 nm are not seen in the loaded condition, and are present in the mixed condition. (e) DLS
size of CS-NP loaded with various initial starting concentrations of micelles. (f) TEM images of
CS-NP KM loaded with varying initial starting concentrations of micelles between 0 uM and 3000
µM.
6.3.2 In Vitro Release of Metformin from KM met and Therapeutic Effect of KM met
Free metformin was confirmed to be cleaved from the amphiphile under intracellular
conditions. Release of free metformin from DSPE-PEG 2000 is triggered by proteases that cleave
the amine bond used for conjugation between the two molecules. Metformin has a molecular
weight of 129.16 g/mol, while DSPE PEG 2000-NHS and metformin-DSPE PEG 2000-NHS has
molecular weights of 2890 g/mol and 3055 g/mol, respectively, and are unable to escape the
dialysis compartment with a cutoff of 2000 g/mol. Measurement of metformin was correlated to %
cumulative drug release using a standard curve of metformin in 10 mM sodium acetate buffer at
pH 7.45. Results are consistent with other studies that demonstrate protease cleavage of the
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NHS-ester bond [279]. This shows that free metformin can be delivered to the cell target once the
micelle is endocytosed.
To evaluate the potential therapeutic efficacy of metformin KM, western blot was
performed on cortical collecting duct cells after 4 hours of treatment to confirm activation of the
AMPK pathway. Quantification by western blot shows roughly threefold higher levels of the
activated form of AMPK, pThr172 AMPK, in micelle formulated metformin compared to free
metformin or PBS at 4 hours (Figure 6-3).
Metformin is a known AMPK pathway activator, and recent literature suggests that
metformin’s activation of AMPK may be the result of its ability to prevent AMP breakdown,
although the exact mechanisms of action is still under investigation. Activation of AMPK also
causes an inhibition of the mTOR pathways, reducing proliferation. In the context of ADPKD, this
can help mitigate the expansion of cyst lining and over-proliferative phenotypes of tubule cells. In
transporting epithelial cells, AMPK activation also inhibits Cystic fibrosis transmembrane
conductance regulator (CFTR) and ENaC activity. This may reduce the rate of fluid secretion of
cystic cells [280]. This gives promise that the net effect of AMPK activation in ADPKD will have a
positive therapeutic response.
Figure 6-3 Drug release and in vitro therapeutic effect of KM met. (a) Drug release of metformin
over time from KM via protease cleavage. (b) Quantification by western blot shows higher levels
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of the activated form of AMPK, pThr172 AMPK, in metformin KM compared to free metformin or
PBS at 4 hours post treatment to mice cortical collecting cells on transwell membranes. Metformin
is a known AMP-activated protein kinase (AMPK) pathway activator. Expression level is quantified
by normalization to a housekeeping protein, B-actin. (c) Brightfield images of 3D Matrigel PKD1
null cells treated with PBS, NT Blank, KM Blank, NT met, KM met, or free met.
6.3.3 In Vitro Release and Therapeutic Efficacy of CS-NP KM
Upon confirming the therapeutic effectiveness of KM met and verifying its potential to
reduce cyst size in a 3D cyst Matrigel model, we wanted to confirm the release of therapeutically
potent met from CS-NP. Much like in Chapter 5, the release of KM met from CS-NP is pH
dependent. When we incubated CS-NP KM met in SGF, less than 25% total met release is
observed, signaling stability in low pH environments such as the stomach. When exposed to
higher pH in SIF, up to 74% of met was released from the nanoparticle, demonstrating a faster
release profile in the more neutral pH of the early intestines (Figure 6-4a). Furthermore, to
validate if the released payloads are indeed intact micelles, or simply disassembled monomers,
we spun down the samples at the final timepoint to collect chitosan debris. The supernatant was
then analyzed in DLS, and showed the characteristic ~10 nm size peak in both SIF and SGF
conditions (Figure 6-4b,c).
To test this full nanoparticle system intestinal permeation in a representative in vitro model,
we utilized a simple Transwell culture where we seeded Caco2 intestinal cells on the apical
membrane apical side to simulate the intestinal lining, while mpkCCDc14 cells, derived from the
cortical collecting duct, were seeded on the basolateral side. Transwells were treated with 300
µM met in CS-NP KM met, free met, unloaded CS-NP Blank, KM met, or PBS for 12 hours and
phosphorylated (active) AMPK to total AMPK ratio in the mpkCCDc14 was measured using an
ELISA assay. As shown in Figure 6-4c, an increase in phosphorylated AMPK to total AMPK ratio
in all met-containing groups was seen: Between 1.3 ± 0.8 for free met, 1.4 ± 0.3 for KM met, and
2.1 ± 0.1 for CS-NP KM met (p < .005), while no change was found upon PBS and CS-NP blank
treatment. The highest therapeutic effect was seen in CS-NP KM met, where the chitosan
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nanoparticles are able to deliver micelle payload through the Caco2 cells for an enhanced
therapeutic effect. While KM met and free met showed similar efficacy, we believe this is due to
the baseline uptake transport of micelles or small molecule across the Caco2 layer. Taken
together, these studies confirmed the therapeutic efficacy of CS-NP KM met is higher than the
constitution controls and can be a promising treatment for PKD.
6.3.4 Caco2 Permeation of CS-NP KM met
In addition to mucoadhesive properties and protection of payloads in the acidic
environment of the stomach, chitosan has been suggested to enhance the penetration of the
intestinal epithelial cell barrier by opening tight junctions and increase transport of luminal
peptides, nutrients, and nanoparticles [251]. To verify this, CS-NP KM met, CS-NP KM Blank, free
met, KM met, or PBS was tested in an intestinal epithelial barrier model consisting of human
colorectal Caco-2 cells, on Transwell membranes [252], and tight junction integrity was
determined via transepithelial resistance (TER) measurements [225]. TER measurements were
made three days before treatment, and again after administration nanoparticles or free drug for
up to 3 days. No changes in TER occurred in the PBS, free met, or KM met groups, while an 81.2%
reduction in resistance to 52.1 ± 31.3 Ohm*cm
2
was observed for CS-NP KM met and CS-NP
Blank 6 hours after administration (Figure 6-4f). A recovery to pre-treated baseline resistance
levels (335 Ohm*cm
2
) was seen after 3 days, suggesting the effects on tight junctions are
reversible, yet persist enough on the time scale that digestion occurs in the human gut [253].
Additionally, previous studies from Chapter 5 suggested that transcytosis is a major
pathway by which CS-NP are transported across intestinal epithelial layers [225, 255, 256], in
addition to increasing paracellular permeation. We performed tight junction staining with ZO-1
and observed a decrease in ZO-1 signal only in treatments groups that contained chitosan. The
CS-NP KM met and CS NP blank groups show marked decrease in ZO-1 signal 6 hours after
treatment (Figure 6-4e).
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Figure 6-4 In vitro release of KM from CS-NP. (a) In vitro release of KM from CS-NP under various
pH conditions present in the GI tract (pH = 1.3, SGF; 6.8 SIF, N ≥ 4). (b,c) DLS size measurements
of the supernatant from release studies at the final timepoint. (d) Phosphorylated AMPK to total
AMPK obtained via ELISA. (e) Caco2 monolayers stained with ZO-1 tight junction stain and DAPI.
Scale bar: 100 μm (f) TER measurements over time of Caco2 cell layers treated with CS-NP KM
met, CS-NP Blank, Free met, KM met, or PBS.
6.3.5 Ex vivo Imaging of CS-NP for Biodistribution
Next, to assess the ability of CS-NP to enhance drug bioavailability via oral delivery in vivo,
C57BL/6J mice were orally gavaged with 200 uL of 500 µM CS-NP KM Cy7 met, CS-NP NT Cy
7met, NT Cy7 met, KM Cy7 met, or free Cy7 met, and after 24 hours, ex vivo imaging was
conducted. As shown in Figure 6-5, ex vivo optical imaging demonstrated the majority of Cy7 met
accumulated in the intestines, liver, kidneys, and bladder. Upon quantitative analysis, a significant
difference in the intestines can be seen between the CS-NP KM Cy7 met and CS-NP NT Cy7 met
compared to the unmodified micelle KM Cy7 met and NT Cy7 met (p<0.005, Figure 6-5a). We
theorize that the bare KM and NT met Cy7 treatments are residing in the intestines and many not
be absorbed into systemic circulation to the degree of those with chitosan containing groups.
Notably, serum fluorescence showed consistently higher levels for CS-NP KM Cy7 met and CS-
NP NT Cy7 compared to free micelle or free Cy7 over the course of 24 hours (Figure 6-5b),
demonstrating enhanced depot to systemic circulation. Furthermore, the targeting ability of the
105
(KKEEE)3K peptide can be observed as there is a 9.2% and 8.1% increase, respectively, in kidney
accumulation when comparing the KM Cy7 met vs. NT Cy7 met and CS-NP KM Cy7 met vs. CS-
NP NT Cy7 met. The overall kidney accumulation levels of both CS-NP KM Cy7 met and CS-NP
NT Cy7 met have improved organ delivery compared to bare micelle KM and NT. This confirms
the overall hypothesis that delivery of targeted therapies investigated in Chapter 4 through
chitosan oral delivery platforms in Chapter 5 are indeed feasible and provide enhanced organ
accumulation.
Figure 6-5 Semi-quantitative biodistribution of mice treated with CS-NP KM Cy7 met, CS-NP NT
Cy 7met, NT Cy7 met, KM Cy7 met, or free Cy7 met, 24 hours after oral gavage. (a) Quantification
of ex vivo organ Cy7 fluorescence levels showed higher accumulation in the intestines for bare
KM Cy7 met and NT Cy7 met, as well as Cy7 met. (b) Serum fluorescence of chitosan containing
groups shows a signal and an extended-release profile compared to bare micelle and free Cy7
met.
6.3.6 Therapeutic Efficacy of CS-NP KM met in Slowly Progressing ADPKD Mice
To confirm the feasibility of CS-NP KM met to act as an oral delivery vehicle in chronic
diseases, CS-NP met was administered in the slowly progressing ADPKD murine model,
Pkd1
fl/fl
;Pax8-rtTA;Tet-O-Cre [281]. In this model, a slowly progressing PKD phenotype can be
developed by knockout of the PKD1 gene, induced by doxycycline injection on P27-29, and again
on P43 and P57. Starting on P30, mice were orally gavaged with 300 mg/kg of met loaded in CS-
NP KM met, KM met, or free met every two days and euthanized on P150 when a severe cystic
106
phenotype is expected. Kidneys were excised to assess kidney to body weight (KW/BW) ratio
and stained with H&E to compare cystic index. In CS-NP KM met-treated mice, a greater decrease
in the KW/BW ratio was found compared to bare micelle orally gavaged (KM met) (1.8 ± 0.4 vs.
7.65.1 ± 2.3, p ≤ 0.01), confirming enhanced therapeutic efficacy in slowing of cystogenesis of
met when delivered via CS-NP KM (Figure 6-6a). Moreover, cystic index was statistically lower
in CS-NP KM met-treated mice compared to mice treated with the bare micelle. (34.3 ± 3.6% vs.
53.4 ± 4.5%, p ≤ 0.01, Figure 6-6b). This shows the improved therapeutic effect of the protective
chitosan coating on micelles proved an enhanced therapeutic effect. In sum, CS-NP KM met
demonstrated a higher therapeutic efficacy when compared to bare micelle KM met at the same
dose that can overcome the physiological barriers of oral delivery. Uniquely, this is the first
nanoparticle delivery platform for ADPKD, and our study highlights CS-NP loaded with micelles
as a viable oral delivery platform for chronic conditions.
Figure 6-6 Therapeutic efficacy of orally gavaged nanoparticles in a slowly progressing ADPKD
murine model. (a) The total kidney weight to body weight ratio is dramatically reduced for CS-NP
KM met compared to KM met. (b) Cystic index of the CS-NP KM met group vs. KM met.
6.4 Conclusion and Future Directions
Chitosan nanoparticles (CS-NP) loaded with kidney targeting micelles (KM) were
investigated as a drug delivery platform for oral delivery in ADPKD. CS-NPs were synthesized
through ionic gelation and loaded with intact supramolecular micelles were characterized. To our
107
knowledge, this is the first development of a nanoscale multiarticulate delivery system for kidney
disease. In vitro, CS-NPs demonstrated effective release of the micelle payload in the high pH of
the intestines, while protected the micelles in low pH of the stomach. The chitosan containing
nanoformulations were also demonstrated to effectively deliver KM through a Caco2 intestinal
lining to collecting duct cells underneath. Moreover, upon oral gavage in a slowly progressing
PKD murine model, disease burden was significantly reduced upon met delivery using CS-NP
KM met compared to the KM only. While met at the dosages used in the study did not cause
significant toxicities, future studies assessing high met dosages or increasing dose exposure
more fully elucidate the benefits of delivering drugs in the CS-NP KM met system.
Additional follow up groups are required for the slow progressing PKD model, and due to
the long term dosing and breeding regiments will continue in the meantime. Groups containing
CS-NP NT, and free metformin controls are required to quantify the benefit of the targeting ligand
and the micelle system within the chitosan. Untreated PBS controls are also needed but have
been performed by other groups and expect to see a KW/BW ratio of ~20%.
Furthermore, finer probing of micelle uptake and processing by renal cells would allow
greater knowledge of the future design of nanomedicine. Initial intravital imaging studies, which
provide real-time imaging of glomerular filtration and renal cell uptake have begun to suggest the
benefits of a fully assembled micelle in accessing the glomerular space compared to free
monomer. Finally, the utilization of different targeting peptides can easily be investigated, as the
modular micelle platform can be of wide use to many different cell types in the kidney. The
preliminary findings in this chapter suggest CS-NP KMs can be a promising strategy for PKD
treatment and aid the development of precision nanomedicine.
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Chapter 7: Epigenetic Therapeutics for Treatment of PKD:
Preliminary Findings
7.1 Introduction, Objective, and Rationale
In addition to the main genetic aberrations in ADPKD, PKD1 or PKD2, discussed in
Chapter 3, recent work suggests that epigenetic modulation of gene expression may play a large
role in ADPKD cyst progression. The epigenome, which consists of heritable modifications to DNA
such as methylation and histone acylation, can also alter gene expression [282]. Epigenetic
modifications can also be modified through environmental stimuli as well as through therapeutics,
serving as an alternative way to correct cellular function. For example, it was shown by Fiedler et
al. that the AMPK pathway, the same pathway utilized by our chosen therapeutic metformin in
Chapters 5 and 6, can regulate the epigenome through phosphorylation of TET2 [283].
Additionally, it was discovered that PKD1 is hypermethylated in its gene-body regions, and
expression is subsequently downregulated in ADPKD patients [284].
These scientific insights bring to light some powerful epigenetic therapeutic potential for
PKD patients. For example, Li et al. demonstrated that targeting a histone deacetylase delayed
cyst growth in a PKD1 knockout mouse model [285]. Pharmacological inhibition of histone
deacetylases has also been shown to delay cyst growth [286, 287]. Pilot studies for combination
therapy of a potent epigenetic drug, 2-Deoxy-5-azacytidine (5 Aza), with metformin, and the only
FDA approved drug for PKD, tolvaptan, give us promising results for future clinical translation of
epigenetic modifiers. The motivation to use combination therapies is to reduce the needed dose
of each drug, thereby reducing side effects and potentially offering synergistic benefits [288].
Indeed, the potent 5 Aza drug has been utilized in many preclinical studies for cancer applications,
but was rejected by the FDA in 1967 due to high toxicity in bone marrow and lymphatic system
[289]. In this chapter, in order to investigate the potential of 5 Aza, or other epigenetic modifiers,
as a potential PKD treatment, we combine the DNA methyltransferase inhibitors 5 Aza and RG108,
111
and the histone deacetylase inhibitor Trichostatin A (TSA) with metformin and tolvaptan and probe
proliferation of PKD1 knockout cells, as a readout to limiting aberrant renal cell proliferation in
ADPKD.
7.2 Methods and Materials
7.2.1 Synthesis of Therapeutic Micelles
Metformin containing DSPE-PEG(2000) amphiphiles described in Chapter 6 amphiphiles
were dissolved in methanol or chloroform, along with the hydrophobic drug tolvaptan, and
evaporated under a steady stream of nitrogen. The resulting film was dried under vacuum
overnight, and then hydrated at 80 °C with either MilliQ water or PBS, vortexed and sonicated as
needed to obtain a clear solution and allowed to cool to room temperature. Amphiphile ratios
within the micelles consisted of a monomer molar ratio of 45:55 consisting of DSPE-PEG(2000)-
metformin : DSPE-PEG(2000) methoxy. Upon formation of micelles and sonicating for 15 minutes,
unincorporated free tolvaptan was filtered out using 0.2 µm PES syringe filters. Micelles were
formed in PBS at 10x the desired final concentration when added to complete media in future in
vitro experiments.
7.2.2 Cell Culture of PKD1 Null and Het Renal Cells for Cyst Studies
Clonal renal tubular epithelial cell lines generated from Pkd1flox/-:TSLargeT mice by in
vitro Cre recombinase transfection yielded (Pkd1 Null, Pkd1 Het) were cultured in DMEM/F12
media, 2% FBS, 1x ITSG, and ~2 nM of tri-iodo-sodium salt. Cells were expanded at 37 °C in a
humidified incubator under 5% CO2. Cells at passage 3 were used for studies, and the media
were changed every 2–3 days.
7.2.3 Biocompatibility for PKD1 Null and PKD1 Het cells
112
Biocompatibility was assessed with an MTS cell proliferation colorimetric assay (BioVision)
following the manufacturer’s instructions. PKD1 Null or PKD1 Het cells (2,000 cells/well) were
incubated with either free 50-1000 µM 5 Aza, 50-1000 µM RG 108, or 5 µM TSA (dissolved in
DMSO and added to complete media at 1% final vol/vol), along with either 300 µM metformin, 10
µM tolvaptan, or both 300 µM metformin and10 µM tolvaptan in micelle formulations for 24 hours
on a 96-well plate before the addition of MTS reagent.
7.3 Results and Discussion
7.3.1 Proliferation Dose Response of 5-Aza
To assess the ability of an epigenetic modifying drug, 5 Aza, to affect the proliferation and
therefore cyst progression of a PKD cell line, we initially performed a dose response. When
treated for 24 hours, the PKD1 het cells are observed to stay above 75% proliferation when 5 Aza
dose remains below 100 µM (Figure 7-1). Therefore, we proceeded with the next lowest
concentration of 50 µM in following studies utilizing combination therapy with the small molecule
drugs metformin and Tolvaptan, with the goal of utilizing the lowest effective dose of 5 Aza to
minimize toxicity in patients [289].
The PKD1 het cell line was utilized in this study, which possesses one functional copy of
PKD1, compared to the PKD1 null cells which have both alleles knocked out. We believe a more
significant effect can be observed while there is still one functional gene to undergo epigenetic
changes, although direct comparisons of these experiments with the PKD1 null line have yet to
be performed.
113
Figure 7-1 Proliferation dose response of PKD1 Het cells to 5 Aza between 0-1000 µM 24 hours
after treatment.
7.3.2 Proliferation Response of 5-Aza, Metformin, and Tolvaptan Combination Therapies
To assess the potential to reduce proliferation of diseased PKD cells in combination with
established small molecule drugs, 5 Aza, RG 108, and TSA were combined with either metformin,
tolvaptan, or metformin + tolvaptan dual therapy and incubated with PKD1 het cells. Utilizing
minimally effective concentrations suggested by prior art and our own dose response experiments,
50 µM 5 Aza, 50 µM RG 108, or 5 µM TSA was combined with either metformin at 300 µM,
Tolvaptan at 10 µM, or a dual treatment of 300 µM metformin and 10 µM Tolvaptan (Figure 7-2).
Looking across all treatment groups, when cells were treated with only epigenetic drug (5 Aza,
RG 108, or TSA), a reduction in proliferation to the 85% range was observed. When combining
each with a single small molecule drug, either metformin or tolvaptan, ranges of 75% proliferation
were observed, which is lower than the single small molecule drug control in either case,
suggesting an additive effect. The greatest reduction in PKD1 het proliferation was seen with a
combination of metformin and tolvaptan along with an epigenetic drug (Figure 7-2c), where a
reduction to 61% viability was observed.
114
Figure 7-1 Proliferation of PKD1 Het cells to 50 µM 5 Aza, 50 µM RG 108, or 5 µM TSA was
combined with either metformin at 300 µM, Tolvaptan at 10 µM, or a dual treatment of 300 µM
metformin and 10 µM Tolvaptan 24 hours after treatment.
7.4 Conclusions and Future Directions
Preliminary evidence suggests that epigenetic modifications, including DNA methylation
and histone deacetylation play an important role in ADPKD. Our pilot studies have explored the
potential of 5 Aza, RG 108, and TSA as beneficial treatments to limit aberrant proliferation in
ADPKD. Especially in conjunction with small molecule drugs such as metformin and tolvaptan,
synergistic or additive effects can be observed with the benefit of using lower individual drug
dosages.
However, as the specificity and the mechanisms of action of these inhibitors are not yet
fully clear, more work will be needed before these inhibitors can be evaluated in humans. The
exact DNA methylation levels need to be probed to further uncover the extent of epigenetic
modification or potential off target effects. Currently, collaboration with the Downing lab at UC
Irvine is ongoing with this goal. Additional avenues to explore would incorporating such inhibitors
into a micelle delivery system, which can offer the typical nanomedicine advantages of limiting
side effects in the bone marrow and lymph node, while increasing therapeutic potency at the
target organ. Epigenetic treatments may be more beneficial to a broader patient base than direct
genetic rescue approaches such as the use of miR or CRISPR, since there are hundreds of
recorded PKD1 and PKD2 mutations. Epigenetic modifiers “downstream” of the mutation thus
115
promise to be more efficacious in a larger percentage of the patient population. In summary,
epigenetics is clearly an emerging field in basic and clinical studies of ADPKD and can be married
with nanomedicine efforts to bring out their full potential.
7.5 References
282. Weinhold, B., Epigenetics: the science of change. Environmental health perspectives,
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283. Fiedler, E.C. and R.J. Shaw, AMPK Regulates the Epigenome through Phosphorylation
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284. Woo, Y.M., et al., Genome-wide methylation profiling of ADPKD identified epigenetically
regulated genes associated with renal cyst development. Human Genetics, 2014. 133(3):
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285. Li, L.X., et al., Lysine methyltransferase SMYD2 promotes cyst growth in autosomal
dominant polycystic kidney disease. The Journal of Clinical Investigation, 2017. 127(7): p.
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286. Zhou, X., et al., Sirtuin 1 inhibition delays cyst formation in autosomal-dominant polycystic
kidney disease. J Clin Invest, 2013. 123(7): p. 3084-98.
287. Fan, L.X., et al., Inhibition of histone deacetylases targets the transcription regulator Id2
to attenuate cystic epithelial cell proliferation. Kidney International, 2012. 81(1): p. 76-85.
288. Zhang, Y., et al., Chapter 8 - Nanoparticles as drug delivery systems of combination
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289. Ganesan, A., et al., The timeline of epigenetic drug discovery: from reality to dreams.
Clinical Epigenetics, 2019. 11(1): p. 174.
116
Chapter 8: Conclusion
8.1 Summary and Significance
Current treatments for ADPKD are inadequate. Symptomatic treatments only mend the
manifestations of destroyed kidney parenchyma such as hypertension and pain [70]. The only
FDA approved drug which can slow the progression of cyst growth and delay kidney failure,
tolvaptan, is difficult to tolerate and has reported cases liver damage requiring cessation of
treatment [76, 77]. To address this need, we have developed kidney targeting micelles that
increase accumulation of payloads to the kidney and reduce off-target side effects in addition to
increasing therapeutic potency of drugs [130]. In this thesis, we reviewed the advantages of
nanomedicines and the potential benefits of targeted drug delivery. Many concepts from the
background Chapter 3, including targeting peptides for kidney targeting [100] and kidney
therapeutics [67] have been published in Biomaterials Science, and Advanced Drug Delivery
Reviews, respectively.
In the first aim of this thesis, we synthesized and characterized a kidney targeting peptide
amphiphile micelle suitable to enhance the accumulation in the kidney. Micelles were found to be
biocompatible and localized to specific portions of the nephron based on the expected behavior
of our conjugated targeting peptide, (KKEEE)3K. Such particles can serve as a platform for
targeted delivery to renal tissue, not only for ADPKD, but potentially for many other chronic kidney
conditions. Results from this aim have been published in the Journal of Nano Research [7].
Additional probing into the mechanisms of micelle uptake would be informative, such as further
definition of renal tubular apical or basolateral surface preference. Currently, collaboration with
the Peti Peterdi Lab is utilizing intravital imaging to investigate this subject.
In the second aim of the proposal, we presented results towards the application of chitosan
nanoparticles for oral delivery. Successful delivery of nanoparticle metformin payload to murine
117
serum after oral gavage to a greater extent than free metformin gives promise to this strategy.
Ultimately, cystic burden was reduced to a greater extent with our CS-NP met compared to free
metformin, justifying chitosan’s effectiveness as an oral delivery enhancer. Results from this aim
have been published in the Journal of Controlled Release [156].
Finally, in aim 3, we combine the targeting properties of micelles with a therapeutic arm
and encapsulate the micelles within chitosan nanoparticles. Prior art for oral delivery of micelles
has focused on modification of the amphiphile blocks to remain stable at low pH, but this prevents
the use of hydrophilic conjugated targeting ligands or drugs exposed on the outer surface. With
our chitosan nanoparticles, we may open up the oral delivery route to wider range of
supramolecular micelles that may be loaded in the protective chitosan nanoparticle in their native
chemical composition. We observe the reduction of cyst burden in slowly progressing murine PKD
models to a greater extent in our fully assembled construct, CS-NP KM met, compared to bare
micelle KM when orally gavaged. Additional control groups are needed to compare to the standard
of care, tolvaptan, as well as investigation into the potent epigenetic drugs mentioned in Chapter
7. With these initial promising results in Aim 3, we believe these micelles within chitosan
nanoparticles provide a paradigm shift that differs from the use of bare nanoparticles through IV
injection, increasing patient compliance, while offering new gateway strategies to prevent kidney
failure and treat ADPKD.
8.2 References
7. Wang, J., et al., Design and in vivo characterization of kidney-targeting multimodal
micelles for renal drug delivery. Nano Research, 2018. 11(10): p. 5584-5595.
67. Wang, J., N. Tripathy, and E.J. Chung, Targeting and therapeutic peptide-based strategies
for polycystic kidney disease. Advanced Drug Delivery Reviews, 2020. 161-162: p. 176-
189.
70. Torra, R., Recent advances in the clinical management of autosomal dominant polycystic
kidney disease. F1000Research, 2019. 8: p. F1000 Faculty Rev-116.
76. Torres, V.E., et al., Tolvaptan in Later-Stage Autosomal Dominant Polycystic Kidney
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118
77. Patra, S., et al., Short term efficacy and safety of low dose tolvaptan in patients with acute
decompensated heart failure with hyponatremia: a prospective observational pilot study
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Association, 2014. 15(1): p. 1-5.
100. Wang, J., J.J. Masehi-Lano, and E.J. Chung, Peptide and antibody ligands for renal
targeting: nanomedicine strategies for kidney disease. Biomaterials Science, 2017. 5(8):
p. 1450-1459.
130. Black, M., et al., Self-Assembled Peptide Amphiphile Micelles Containing a Cytotoxic T-
Cell Epitope Promote a Protective Immune Response In Vivo. Advanced Materials, 2012.
24(28): p. 3845-3849.
156. Wang, J., et al., Oral delivery of metformin by chitosan nanoparticles for polycystic kidney
disease. Journal of Controlled Release, 2021. 329: p. 1198-1209.
119
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Abstract (if available)
Abstract
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited disorder of the kidneys and affects 12 million people worldwide. It is characterized by uncontrolled cyst growth in renal tissue, which destroys kidney filtration function and leads to kidney failure. Traditional clinical solutions have focused primarily on management of the symptoms, while cysts continued to grow. Only recently in 2018, Tolvaptan was approved by the Food and Drug Administration (FDA) as the first drug to slow cyst growth. However, Tolvaptan is difficult to tolerate, expensive, and may cause liver injury in patients, which has led to discontinuation of treatment by many patients. We hypothesize that the utilization of nanomedicine strategies, specifically a kidney targeting peptide amphiphile micelle (KM), can enhance accumulation of drugs like Tolvaptan to the kidneys, increasing therapeutic efficacy while reducing off-target side effects. Unfortunately, almost all FDA approved nanoparticles have relied on intravenous (IV) injection. However, this method of administration is not suitable for chronic diseases that require lifelong therapy like ADPKD. Oral delivery is the route associated with the highest patient compliance, but it also presents physiological barriers that may degrade or limit drugs and other payloads. Thus, we develop a biomaterials strategy using chitosan to deliver drugs and KMs into systemic circulation via oral delivery. Together, we encapsulate drug-loaded KMs into chitosan nanoparticles, which has promise in the treatment of ADPKD.
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Creator
Wang, Jonathan
(author)
Core Title
Chitosan nanoparticle mediated oral drug delivery of kidney targeting micelles for polycystic kidney disease
School
Viterbi School of Engineering
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Doctor of Philosophy
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Biomedical Engineering
Degree Conferral Date
2021-12
Publication Date
11/17/2021
Defense Date
07/15/2021
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ADPKD,autosomal dominant polycystic kidney disease,drug delivery,micelle,nanoparticle,OAI-PMH Harvest
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Chung, Eun Ji (
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ADPKD
autosomal dominant polycystic kidney disease
drug delivery
micelle
nanoparticle