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Development of protein polymer therapeutics for the eye

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Development of protein polymer therapeutics for the eye

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Content i

DEVELOPMENT OF PROTEIN POLYMER THERAPEUTICS FOR THE EYE
By

Wan Wang

A Thesis presented to the
Department of Pharmacology and Pharmaceutical sciences
University of Southern California
In partial fulfillment of the
Requirements for the degree
DOCTOR OF PHILOSOPHY
PHARMACEUTICAL SCIENCES

June 2014

Copyright 2014 Wan Wang
ii

Dedication
This thesis is dedicated to all of my family, mentors, and friends.
iii

Acknowledgments
I would like to acknowledge my gratitude to my mentors Dr. J. Andrew MacKay, Dr.
Sarah F. Hamm-Alvarez, Dr. David R. Hinton and Dr. Gordon W. Laurie for their
guidance, support and patience. I would like to thank all of my committee members Dr.
Wei-chiang Shen, Dr. Curtis T. Okamoto and Dr. James C. H. Tan for their feedback
and time spent in reviewing my thesis. I would like to appreciate Dr. Ram Kannan, Dr.
Parameswaran G Sreekumar, Dr. Robert L. McKown, Dr. Elizabeth Fini, Dr. Martin
Heur, Dr. Gabriel Gordon, Dr. Honggang Cui for their important suggestions and help in
my research. I would like to thank all my lab member and friends for their support. I
would like to appreciate all the funding resources for the research: the
USAMRMC/TATRC grant W81XWH1210538, NIH EY011386, the National Institute of
Health R21EB012281, P30 CA014089, P30 RO1EY03040 and RO1EY01545, the
Translational Research Laboratory at the USC School of Pharmacy, the USC Whittier
Foundation, USC Clinical and Translational Science Institute SC CTSI
(NIH/NCRR/NCATS) Grant # UL1TR000130, and the Arnold and Mabel Beckman
Foundation.

iv

Table of Contents

Dedication ............................................................................................. ii
Acknowledgements .............................................................................. iii
List of Tables ........................................................................................ ix
List of Figures ....................................................................................... x
Abbreviations ........................................................................................ xiii
Introduction ........................................................................................... xiv
1.0. Chapter 1: A thermo-responsive protein treatment ................................
for Dry Eye Disease (DED)
1
1.1. Introduction .................................................................................. 1
1.2. Materials and methods................................................................. 6
1.2.1. Animals ..................................................................................... 6
1.2.2. Instruments and Reagents ........................................................ 6
1.2.3. Biosynthesis of Lacrt-ELP fusions ............................................. 6
1.2.4. Expression and purification of Lacrt ELP fusion protein ............ 7
1.2.5. Thermal characterization of Lacrt ELP fusion proteins .............. 8
1.2.6. Cell isolation, culture and treatments ........................................ 9
1.2.7. Secretion of β-hexosaminidase ................................................. 9
1.2.8. LGAC morphology changes upon stimulation ........................... 10
1.2.9. Rhodamine labeling and cell uptake of Lacrt ............................
and Lacrt ELP fusion proteins

10
1.2.10. Tear secretion and immunohistochemistry of the mouse LG ... 11
1.2.11. Stability of Lacrt ....................................................................... 11
v

1.2.12. Statistical Analysis ................................................................... 12
1.3. Result ........................................................................................... 12
1.3.1. Construction and Purification of a Lacrt ELP fusion protein ...... 12
1.3.2. LV96 stimulates β-hexosaminidase secretion from ...................
primary rabbit LGACs
19
1.3.3. Fusion with V96 influenced uptake of exogenous .....................
Lacrt into LGACs
25
1.3.4. LV96 stimulates tear secretion from ..........................................
non-obese diabetic (NOD) mice
25
1.4. Discussion ................................................................................... 31
1.5. Conclusion ................................................................................... 33
2.0. Chapter 2: Self-assembly of nanoparticles for corneal wound healing .. 35
2.1. Introduction .................................................................................. 35
2.2. Materials and methods................................................................. 41
2.2.1. Materials and equipment ........................................................... 41
2.2.2. Construction of LSI nanoparticles ............................................. 41
2.2.3. Characterization of LSI phase behavior ....................................
and nanoparticle formation
42
2.2.4. TEM imaging of LSI nanoparticles ............................................ 43
2.2.5. SV40-immortalized human ........................................................
corneal epithelial cell (HCE-T) culture
43
2.2.6. Ca
2+
imaging ............................................................................. 43
2.2.7. Scratch assay ........................................................................... 44
2.2.8. Exogenous cell uptake assay .................................................... 44
2.2.9. Murine corneal abrasion and recovery study ............................ 45
vi

2.2.10. Statistics .................................................................................. 45
2.3. Result ........................................................................................... 48
2.3.1. LSI forms thermo-responsive nanoparticles .............................. 48
2.3.2. LSI nanoparticles exhibit mitogenic activity using .....................
SV-40 transduced human corneal epithelial cell (HCE-T) model
50
2.3.3. LSI undergo uptake into HCE-Ts .............................................. 55
2.3.4. LSI nanoparticles heal abrasion wounds ................................... 55
2.4. Conclusion ................................................................................... 60
3.0. Chapter 3: Thermo-responsive loading and release ..............................
of protein polymers from contact lenses
61
3.1. Introduction .................................................................................. 61
3.2. Materials and methods................................................................. 64
3.2.1. Decoration of contact lenses with rhodamine labeled ELPs ...... 64
3.2.2. ELPs inverse phase transition characterization ........................ 64
3.2.3. Release of fluorescent ELPs from contact lenses ..................... 64
3.2.4. Spatiotemporal HCE-T cell uptake ............................................ 65
3.2.5. Statistical Analysis .................................................................... 65
3.3. Results and discussions .............................................................. 66
3.3.1. Discovery of ELPs specific attachment to .................................
Proclear Compatible
TM
contact lens
66
3.3.2. T
t
and temperature dependent attachment of ELPs to the lens 68
3.3.3. Contact lenses decorated with a ring of LV96 ...........................
mediated spatiotemporal HCE-T cell uptake
72
3.4. Conclusion ................................................................................... 74
vii

4.0. Chapter 4: sHSPs-ELPs as potential therapeutics ................................
for Age-related Macular Degeneration (AMD)
76
4.1. Introduction .................................................................................. 76
4.2. Materials and methods................................................................. 81
4.2.1. Materials and reagents .............................................................. 81
4.2.2. Construction of ELP genes ....................................................... 81
4.2.3. ELP expression and purification ................................................ 82
4.2.4. Transmission Electron Microscopy (TEM) imaging ................... 82
4.2.5. Characterization of ELP particle formation ................................
and phase transition temperature
83
4.2.6. Bis-ANS assay .......................................................................... 84
4.2.7. Quantification of chaperone activity .......................................... 84
4.2.8. NHS-Rhodamine labeling of recombinant ELP fusions ............. 84
4.2.9. Protection of RPE cells from H
2
O
2
induced cell death .............. 85
4.2.10. Intracellular uptake in RPE cells under H2O2 induced stress .. 86
4.2.11. Statistical analysis.................................................................... 86
4.3. Results ......................................................................................... 86
4.3.1. Purification of ELP fusion proteins that assemble nanoparticles 86
4.3.2. The mini-peptide from αB-crystallin shifts .................................
the phase diagram for ELP nanoparticles
91
4.3.3. The mini-peptide from αB-crystallin shifts .................................
the distribution of hydrodynamic radii
93
4.3.4. crySI and cryS96 behave as molecular chaperones ................. 98
4.3.5. Exogenous cryS96 and crySI protect ........................................
RPE cells from oxidative stress induced cell death
99
viii

4.3.6. Cell uptake and nuclear localization of crySI and cryS96 ..........
under oxidative stress is important for anti-apoptosis activity
101
4.4. Discussion ................................................................................... 106
4.5. Conclusion ................................................................................... 116
5.0. Chapter 5: Elastin-like polypeptides (ELPs) in .......................................
ocular drug delivery and tissue engineering
117
5.1. Introduction .................................................................................. 117
5.1.1. Ocular drug delivery and tissue engineering ............................. 117
5.1.2. ELPs in drug delivery and tissue engineering ........................... 118
5.2. ELPs in ocular drug delivery ........................................................ 119
5.2.1. Lacrimal gland ........................................................................... 119
5.2.2. Corneal epithelium .................................................................... 122
5.2.3. Intraocular delivery .................................................................... 124
5.2.4. Drug delivery via contact lenses ............................................... 127
5.3. Conclusion and future perspective ............................................... 129
6.0. References ............................................................................................ 130

ix

List of Tables
Table 1: Nomenclature, amino acid sequence, .................................................
and transition temperature of Lacrt-ELPs
14
Table 2: Representative Lacritin cleavage sequences identified by MALDI-TOF 17
Table 3: Nomenclature, amino acid sequence, and ..........................................
physicochemical property of LSI and LS96
46
Table 4: Modeling of ELP modified contact lens release kinetics ...................... 70
Table 5: Nomenclature, sequence and .............................................................
molecular weight of cryELP fusion proteins
88

x

List of Figures
Figure 1: Rationale of utilizing Lacrt-ELP drug depots for sustained ................
stimulation of tear secretion for dry eye disease (DED)

5
Figure 2: Purification and thermal characterization of LV96 .............................

15
Figure 3: Stepwise Lacrt ELP purification process ........................................... 16
Figure 4: Purified lacritin is susceptible to proteolysis ......................................
of an unidentified origin
18
Figure 5: Lacrt-ELP fusion proteins are prosecretory in LGACs ....................... 21
Figure 6: Fusion with V96 influenced uptake of exogenous Lacrt into LGACs .

23
Figure 7: Lacrt-ELP fusion proteins stimulate tear secretion in NOD mice .......

27
Figure 8: Intra-lacrimal injection of a labeled Lacrt-ELP ...................................
fusion protein produces a depot

28
Figure 9: Intra-lacrimal gland injection of Lacrt-ELPs in normal male ..............
C57BL/6 mice

29
Figure 10: Carbachol (CCh) triggered Ca
2+
scintillation in LGACs ................... 30
Figure 11: Rationale of using LSI nanoparticles to heal corneal ......................
wounds via topical application

40
Figure 12: Construction and thermal behavior of LSI fusion proteins ............... 47
Figure 13: Nanoparticle characterization of LSI and SI .................................... 49
Figure 14: LSI nanoparticles stimulate Ca
2+
wave propagation in HCE-Ts ...... 52
Figure 15: Lacrt heals scratch wound in HCE-Ts ............................................. 53
Figure 16: LSI nanoparticles undergo uptake in HCE-Ts ................................. 54
Figure 17: LSI nanoparticles heal abrasion wounds on ....................................
the ocular surface of female NOD mice

58
Figure 18: LSI nanoparticles exhibit faster wound ............................................
healing efficacy compared to a macromolecular control LS96

59
xi

Figure 19: ELP selectively phase separate onto ..............................................
Proclear compatibles
TM
contact lenses
67
Figure 20: T
t
and temperature dependent affinity of ELPs ...............................
towards ProclearCompatible
TM
contact lens

71
Figure 21: Spatiotemporal HCE-T cell uptake .................................................. 73
Figure 22: Rational bioengineering of cryELP nanoparticles ............................
and macromolecules to rescue retinal pigment
epithelial cells (RPEs) from oxidative stress

80
Figure 23: Purity of ELP fusions and morphology ............................................
of protein polymer nanoparticles

89
Figure 24: Fusion to the S96 protein polymer inhibits the ................................
fibril formation for the αB crystallin mini-peptide

90
Figure 25: Phase diagrams for mono and diblock ELP fusion proteins 92
Figure 26: The αB-crystallin mini-peptide influences the ..................................
assembly and radius of ELP nanoparticles

95
Figure 27: ELP fusion proteins have protective chaperone ..............................
activity on ADH and insulin

97
Figure 28: ELP fusion proteins protect RPE cells from.....................................
H
2
O
2
induced cell death

100
Figure 29: Uptake and nuclear translocation of ................................................
exogenous ELPs in RPE cells

103
Figure 30: Uptake and nuclear translocation plays...........................................
an important role incryS96’s anti-apoptotic activity

104
Figure 31: Intracellular trafficking of cryS96 and ..............................................
cry peptide under H
2
O
2
stress

105
Figure 32: cryS96 exhibits concentration dependent .......................................
chaperone activity against Alcohol Dehydrogenase (ADH)

111
Figure 33: Nuclear translocation of cryS96 is dynamin dependent .................. 112
Figure 34: A fluorescent probe mixed with CrySI reveals .................................
a temperature-dependent increase in hydrophobicity

113
xii

Figure 35: Proposed intracellular trafficking and protective ..............................
mechanism of cryS96 under oxidative stress

115
xiii

Abbreviations
ELP, Elastin like polypeptides, TEM, Transmission electron microscope, DED, dry eye
disease, Lacrt, lacritin, T
t
, transition temperature, LG, lacrimal gland, LGACs, lacrimal
gland acinar cells, NOD, non-obese diabetic, CCh, carbachol, CMT, critical micelle
temperature, HCE-T, SV40-Adeno vector transformed human corneal epithelial cell,
BPE, Bovine Pituitary Extract, EGF, Epidermal Growth Factor, cry, mini-αB-Crystallin,
CMT, critical micelle temperature, ITC, inverse transition cycling, DLS, dynamic light
scattering, R
h
, hydrodynamic radius, ADH, alcohol dehydrogenase, RPE, retinal
pigment epithelial, sHSPs, small heat shock proteins, MDC, Monodansylcadaverine,
CsA, cyclosporine A.
xiv

Introduction
Collecting visual information from the surroundings and transmitting the signals to
the brain, the eye plays a crucial role in our daily life. Visual impairment is one of the
most popular diseases worldwide and patients suffer from short-term eye discomfort to
irreversible blindness. Unique features of the eye, such as delicate and complex
anatomy, multiple blood-ocular and tissue barriers, Immune privilege, provide both
benefits and challenges for ocular drug discovery and delivery. At the same time,
knowledge of the pathogenesis underlying ophthalmic diseases has accumulated
substantially over the decades and clinical trials are underway to evaluate existing/new
drugs delivered to novel targets in both the anterior and posterior segments of the eye.
Thus so, designing sustained ocular drug delivery platforms with improved
biocompatibility, smarter environment responsiveness, precise spatiotemporal control
and better combination with classic medical devices to create novel functions remains
an important focus in the field. As peptides and proteins emerge as promising ocular
biopharmaceuticals in addition to small molecule therapies, the importance of finding
the proper delivery vehicle has attracted increased attention in both academia and
pharmaceutical industry. Herein, we explored the application of thermo-responsive
Elastin-like polypeptides (ELPs) as a novel protein polymer drug delivery system in
representative ocular compartments: the lacrimal gland, the corneal epithelium, the
retina and in combination with contact lens. Model protein/peptide biopharmaceuticals,
lacritin and mini-αB-crystallin, were selected for proof-of-concept demonstration.
Chapter 1: Millions of Americans suffer from dry eye disease, and there are few
effective therapies capable of treating these patients. A decade ago, an abundant
xv

protein component of human tears was discovered and named lacritin. Lacritin has
prosecretory activity in the lacrimal gland and mitogenic activity at the corneal
epithelium. Similar to other proteins placed on the ocular surface, the durability of its
effect is limited by rapid tear turnover. Motivated by the rationale that a thermo-
responsive coacervate containing lacritin would have better retention upon
administration, we have constructed and tested the activity of a thermo-responsive
lacritin fused to an Elastin-like polypeptide (ELP). Inspired from the human tropoelastin
protein, ELP protein polymers phase separate into viscous coacervates above a tunable
transition temperature. This fusion construct exhibited the prosecretory function of
native lacritin as illustrated by its ability to stimulate β-hexosaminidase secretion from
primary rabbit lacrimal gland acinar cells. It also increased tear secretion from non-
obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis, when administered
via intra-lacrimal injection. Lacritin ELP fusion proteins undergo temperature-mediated
assembly to form a depot inside the lacrimal gland. We propose that these lacritin ELP
fusion proteins represent a novel potential therapy for dry eye disease and the strategy
of ELP-mediated phase separation may have applicability to other diseases of the
ocular surface.
Chapter 2: The avascular corneal epithelium plays an important role in maintaining
normal vision and protecting the corneal interior from harmful environmental infections.
Delayed ocular wound recovery caused by clinical conditions and popular refractive
surgeries such as LASIK and PRK strengthen the need for accelerating corneal wound
healing to restore the normal functions of the ocular surface. In this study, we
bioengineered thermo-responsive elastin-like polypeptides (ELPs) based nanoparticles
xvi

with model mitogenic biopharmaceutical lacritin at the corona. The LSI fusion protein
self-assembled into stable nanoparticles at physiological relevant temperatures.
Calcium wave propagation and scratch wound healing assays illustrated LSI’s in vitro
mitogenic activity in transformed human corneal epithelial cells (HCE-Ts). Fluorescein
imaging and immunohistochemistry suggested that topical administration of LSI onto the
ocular surface significantly promoted corneal wound healing and epithelium proliferation
compared to plain ELP SI, epidermal growth factor (EGF) and bovine pituitary extract
(BPE) co-treatment, and no treatment groups using a non-obese diabetic (NOD) mouse
model. Exogenous LSI nanoparticles were taken up by HCE-Ts and similar intracellular
retention was observed in the murine corneal epithelium 12 h after treatment.
Collectively, self-assembly of ELP fusion protein nanoparticles present a novel strategy
to rationally bioengineer biopharmaceuticals as promising treatments for corneal wound
healing and other diseases of the ocular surface.
Chapter 3: Contact lenses are emerging as an alternative ophthalmic drug delivery
system to therapeutically manage ocular anterior segment disorders beyond their
traditional role as vision correction and cosmetic aid. In this chapter, we reported the
surprising discovery of ELPs’ thermally-reversible, spatiotemporal and sustained
attachment to Proclear Compatibles
TM
contact lens as an elastic bridge. Moreover, we
illustrated that attachment and release of ELPs to/from Proclear contact lens was a T
t

and temperature dependent process. As a proof of concept for targeted protein
therapeutic delivery, we modified the lens with prosecretory mitogenic fusion protein
Lac-V96 and demonstrated spatial cell uptake via contact lens using human corneal
epithelial cell model (HCE-Ts).
xvii

Chapter 4: αB-crystallin is a protein chaperone with anti-apoptotic and anti-
inflammatory activity that is apically secreted in exosomes by polarized human retinal
pigment epithelium. A 20 amino acid mini-peptide derived from residues 73-92 of αB-
crystallin protects human retinal pigment epithelial (RPE) cells from oxidative stress, a
process involved in the progression of age related macular degeneration (AMD).
Unfortunately, due to its small size, its development as a therapeutic requires a robust
controlled release system. To achieve this goal, the αB-crystallin peptide was re-
engineered into a protein polymer nanoparticle/macromolecule with the purpose of
increasing the hydrodynamic radius/molecular weight and enhancing potency via
multivalency or an extended retention time. The peptide was recombinantly fused with
two high molecular weight (~40 kD) protein polymers inspired by human tropoelastin.
These elastin-like-polypeptides (ELPs) include: i) a soluble peptide called S96; and ii) a
diblock copolymer called SI that assembles multivalent nanoparticles at physiological
temperature. Fusion proteins, cryS96 and crySI, were found to reduce aggregation of
alcohol dehydrogenase and insulin, which demonstrates that ELP fusion did not
diminish chaperone activity. Next their interaction with RPE cells was evaluated under
oxidative stress. Unexpectedly, H
2
O
2
-induced stress dramatically enhanced cellular
uptake and nuclear localization of both cryS96 and crySI ELPs. Accompanying uptake,
both fusion proteins protected RPE cells from apoptosis, as indicated by reduced
caspase 3 activation and TUNEL staining. This study demonstrates the in vitro
feasibility of modulating the hydrodynamic radius for small peptide chaperones by
seamless fusion with protein polymers; furthermore, they may have therapeutic
applications in diseases associated with oxidative stress, such as AMD.
xviii

Chapter 5: Biomaterials have been playing a vital role in developing biocompatible
medical devices, efficient drug delivery systems and tissue engineering. Different from
other parts of the body, ocular drug delivery and tissue engineering remains challenging
due to unique compartmentalized structures of the eye and multiple tissue barriers
against efficient drug uptake. In this chapter, we address several parts of the ocular
systems, namely the lacrimal gland, the anterior segment of the eye and the retinal
pigment epithelium. We begin with introducing their anatomical features; then
summarize traditional drug delivery approaches; followed by highlight some of the novel
delivery and tissue engineering strategies; and finally outlook the perspectives of ocular
drug delivery systems and tissue engineering in near future. Specifically, we review our
own exploration and achievements thus far using Elastin-like polypeptides (ELPs).
1

Chapter 1
A thermo-responsive protein treatment for Dry Eye Disease (DED)
1.1 Introduction
The lacrimal gland-corneal axis plays a critical role in maintaining ocular surface health.
While the avascular cornea serves as both a protective barrier and the main refractive
element of the visual system, the lacrimal gland is the major organ secreting key
proteins and electrolytes into the tear film that bathes the cornea and, through nutrient
and antimicrobial proteins, sustains its function (Dartt, 2009; Laurie et al., 2008). Dry
eye disease (DED) is a multifactorial disease of the ocular surface causing visual
disturbance and tear film instability (Friedman, 2010) and can be due to either aqueous
tear insufficiency originating with defects in aqueous tear production by the lacrimal
gland (LG) (Stern et al., 1998a) or evaporative dry eye associated with meibomian
gland insufficiency (Javadi and Feizi, 2011; Qiao and Yan, 2013). Accordingly to
reports, severe DED affects approximately 5 million Americans above age 50 and its
global prevalence ranges from 5 % to 35 % of the population (Friedman, 2010).
Traditional approaches to treatment of DED include topical administration of artificial
tears or the conservation of secreted tears using tear plugs (Kojima et al., 2014) and
eye-shields (Kaufman et al., 1994). Since many cases of DED are associated with
inflammation (Lu et al., 2014; Wei et al., 2013), some treatments for DED have been
proposed that inhibit inflammation of the lacrimal gland

(Akpek et al., 2011). None of
these methods are satisfactory in replacing the lost regulatory functions provided by the
many components found in normal tears. To better sustain the health and homeostasis
2

of the ocular surface there remains a need for efficient, sustained and targeted DED
therapy.
The discovery of the glycosylated human tear protein, lacritin (Lacrt), provided critical
insight into the potential use of regulatory tear proteins to treat DED (Karnati et al.,
2013; McKown et al., 2009a). Lacrt was found in a systematic oligonucleotide screen of
a human lacrimal gland (LG) cDNA library and exhibited LG specific-expression (Sanghi
et al., 2001a). Subsequent studies have proven its efficacy in stimulating peroxidase
secretion in cultured rat (Sanghi et al., 2001a), and both lactoferrin and lipocalin
secretion in cultured monkey lacrimal acinar cells (Fujii et al., 2013). Lacrt also
promotes constitutive tear secretion by New Zealand white rabbits via topical treatment
(Samudre et al., 2011b), proliferation of transformed human corneal epithelial cells
(Sanghi et al., 2001a; Wang et al., 2006), and restored health of transformed human
corneal epithelial cells, primary human corneal epithelial cells (Wang et al., 2013c) and
primary monkey lacrimal acinar cells (Fujii et al., 2013) that had been stressed with the
inflammatory cytokines interferon-γ and tumor necrosis factor. Interestingly, Lacrt
displays growth factor-like behavior; however, its specificity for target cells of the ocular
surface system results from a unique ‘off-on’ switch controlled by heparanase
deglycanation of the cell surface protein, syndecan-1 (Ma et al., 2006), which both
exposes and generates a Lacrt binding site (Zhang et al., 2013b) as a prerequisite for
mitogenic signaling. Confirmed by 2-D electrophoresis, mass spectrometry and surface-
enhanced laser desorption/ionization studies, Lacrt (Velez et al., 2013) is down
regulated in blepharitis (chronic inflammation of the eyelid) vs normal tears (Ma et al.,
2008), and most aqueous deficient dry eye (Aluru et al., 2012). Whether down
3

regulation of Lacrt provokes disease is a key unresolved question, but its prosecretory
and corneal mitogenic activity suggest it might have activity as a protein therapeutic for
ocular surface diseases.
Great strides have been made to improve the bioavailability and simplify the
administration of existing drugs, which include depot formulations that deliver short
peptides such as leuprolide and bioadhesive polymers used in buccal drug-delivery
systems (Rosen and Abribat, 2005). Recently, stimuli-responsive polypeptides have
emerged as an attractive controlled release strategy. One such type of biomaterial are
the elastin-like-polypeptides (ELPs) (Hubbell and Chilkoti, 2012a). Biologically inspired
from human tropoelastin, ELPs are composed of a pentapeptide repeat (Val-Pro-Gly-
Xaa-Gly)
n
, where the ‘guest residue’ X can be any amino acid and n determines
molecular weight. One unique property of ELPs is their inverse temperature phase
transition behavior. ELPs are soluble in aqueous solutions below their transition
temperature (T
t
) and self-assemble into various-sized particles above T
t
(Dhandhukia et
al., 2013).

T
t
can be precisely modulated by adjusting the number of pentapeptide
repeats, n, and the hydrophobicity of the guest residue, X, which can determine whether
the ELP remains a soluble macromolecular drug carrier (Aluri et al., 2014a), assembles
a nanoparticle (Shi et al., 2013a), or phase separates into micron-sized coacervates
(Amiram et al., 2013b) at physiological temperature. With their distinctive thermo-
responsive, elastic, and biocompatible properties, ELPs have impacted fields such as
protein purification

(Meyer and Chilkoti, 1999), stimuli responsive hydrogels (Wang et
al., 2013a), tissue engineering (Koria et al., 2011; Nettles et al., 2010a), and targeted
4

cancer treatment (Callahan et al., 2012a; MacKay et al., 2009a). Yet, the application of
ELPs in ophthalmology has just started (Shah et al., 2013a).
To explore the concept of a thermo-responsive reservoir drug as a potential novel
treatment for DED (Kojima et al., 2014), we generated a novel Lacrt-ELP fusion with T
t

below physiological temperature (Fig. 1). The construct exhibits the thermo-
responsiveness of the parent ELPs while retaining the prosecretory efficacy of native
Lacrt, as demonstrated by its ability to stimulate dose-dependent β-hexosaminidase
secretion from primary rabbit lacrimal gland acinar cells (LGACs). Moreover, the Lacrt-
ELP fusion enhanced tear secretion from the non-obese diabetic (NOD) mouse model
of autoimmune dacryoadenitis when given via intra-lacrimal injection. Formation of a
local depot inside the LG was confirmed by confocal laser scanning microscopy. Finally,
we captured the intracellular trafficking and transcytosis of exogenous Lacrt in LGACs
using time-lapse confocal fluorescence microscopy, which was prolonged by fusion to
the ELP. These findings support the potential enhancement of lacritin therapeutics via
the linkage to a thermo-responsive ELP, which may have broader implications in the
treatment of DED.

5

Figure 1. Rationale of utilizing Lacrt-ELP drug depot for sustained stimulation of
tear secretion for dry eye disease (DED).
6

1.2. Materials and methods
1.2.1. Animals
In vitro studies were conducted using LG from Female New Zealand White rabbits (2.2
–2.5 kg) obtained from Irish Farms (Norco, CA). In vivo studies were conducted using
LG isolated from 12-week old male/female C57BL/6 (Jackson Labs, Bar Harbor/ME,
USA) or non-obese diabetic (NOD) (Taconic Farms, Germantown/NY, USA) mice. All
procedures performed were in accordance to the university approved IACUC protocol.
1.2.2. Instruments and Reagents
Terrific broth dry powder growth medium was purchased from MO BIO Laboratories,
Inc. (Carlsbad, CA). Isopropyl β-D-1-thiogalactopyranoside, OmniPur*. 99.0 % min. was
purchased from VWR (Visalia, CA). Amicon Ultra concentrators were purchased from
Millipore (Billerica, MA). Thrombin CleanCleave™ Kit, carbachol (CCh) and insulin-
transferrin-sodium selenite media supplement were purchased from Sigma-Aldrich (St.
Louis, MO). 4-20 % Tris-Glycine PAGEr GELS were purchased from LONZA (Allendale,
NJ). Cell culture reagents were from Life-Technologies (Carlsbad, CA). Peter's
Complete Medium (PCM) Medium consisted of 50 % Ham’s F-12 plus 50 % DME (low
glucose) supplemented with penicillin (100 U/ml), streptomycin (0.1 mg/ml), glutamine
(4 mM), hydrocortisone (5 nM), transferring (5 μg/ml), insulin (5 μg/ml), butyrate (2 mM),
linoleic acid (0.084 mg/L), carbachol (1 μM), laminin (5 mg/L) and insulin-transferrin-
sodium selenite (ITS) media supplement (5 μg/ml).
1.2.3. Biosynthesis of Lacrt-ELP fusions
A sequence encoding human lacritin without a secretion signal peptide was designed
using the best E. coli codons in EditSeq (DNAStar Lasergene, WI). A thrombin cleavage
7

site was encoded between the Lacrt sequence and ELP tag via insertion at the BseRI
site. A custom gene flanked by NdeI and BamHI restriction digestions sites at the 5’ and
3’ ends was purchased in the pIDTSmart-KAN vector from Integrated DNA
Technologies (IDT) as follows:
5’-
CATATGGAAGACGCTTCTTCTGACTCTACCGGTGCTGACCCGGCTCAGGAAGCTG
GTACCTCTAAACCGAACGAAGAAATCTCTGGTCCGGCTGAACCGGCTTCTCCGCC
GGAAACCACCACCACCGCTCAGGAAACCTCTGCTGCTGCTGTTCAGGGTACCGCT
AAAGTTACCTCTTCTCGTCAGGAACTGAACCCGCTGAAATCTATCGTTGAAAAATCT
ATCCTGCTGACCGAACAGGCTCTGGCTAAAGCTGGTAAAGGTATGCACGGTGGTG
TTCCGGGTGGTAAACAGTTCATCGAAAACGGTTCTGAATTCGCTCAGAAACTGCTG
AAAAAATTCTCTCTGCTGAAACCGTGGGCTGGTCTGGTTCCGCGTGGTTCTGGTTA
CTGATCTCCTCGGATCC-3’.
The gene encoding for LV96 was synthesized by recursive directional ligation in a
modified pET25b(+) vector as previously reported (Aluri et al., 2014a). The Lacrt-
thrombin gene was subcloned into the pET25b(+) vector between the NdeI and BamHI
sites. LV96 genes were synthesized by ligation of a gene encoding for the ELP V96 via
the BseRI restriction site, resulting in placement of the thrombin cleavage site between
Lacrt and ELP. Correct cloning of the fusion protein gene was confirmed by DNA
sequencing. ELPs used in this study are described in Table 1.
1.2.4. Expression and purification of Lacrt ELP fusion protein
Plain ELP V96 and the Lacrt fusion LV96 were expressed in BLR (DE3) E. coli
(Novagen Inc., Milwaukee, WI). Briefly, V96 was expressed for 24 h in an orbital shaker
8

at 37 ° C at 250 rpm. For LV96, 500 μM IPTG was adde d to the culture when OD 600nm
reached 0.5 and the temperature was decreased to 25 ° C to optimize protein
expression for 3 h. Cell cultures were harvested and re-suspended in phosphate buffer
saline (PBS). Proteins were purified from clarified cell supernatant by inverse transition
cycling as previously reported until ELP purity was determined to be approximately 99%
by SDS-PAGE stained with CuCl
2
. Due to partial degradation of LV96 during
biosynthesis, fusion proteins were further purified to homogeneity using a Superose 6
(GE Healthcare, NJ) size exclusion column at 4 ° C. After equilibration with PBS (pH
7.4), 10 mg LV96 was loaded onto the column and washed out by isocratic flow of PBS
at 0.5 ml/min. P1, representing LV96 (supplementary Fig. 1), was collected and
concentrated using an Amicon Ultra concentrator (10 kD). When desired, free Lacrt was
released by thrombin cleavage of LV96 fusion protein. Briefly, 300 μl of thrombin bead
slurry (Sigma-Aldrich) was added to 200 mg purified LV96 and incubated at room
temperature for 3 h. After pelleting the thrombin beads at 250 rpm, the solution was
warmed up to 37 ° C and centrifuged at 4,000 rpm for 10 min to remove ELP
coacervates. The supernatant was then concentrated using an Amicon Ultra
concentrator with a 3 kD M.W. cut-off (MWCO). Protein concentrations were determined
by UV-VIS spectroscopy at 280 nm (ε
ELP
=1285 M
-1
cm
-1
, ε
LV96
=6990 M
-1
cm
-1
, ε
Lacrt
=5500
M
-1
cm
-1
). Protein molecular weight was further confirmed by MALDI-TOF mass
spectrometry (AXIMA Assurance, Shimadzu).
1.2.5. Thermal characterization of Lacrt ELP fusion proteins
Inverse phase transition and self-assembly behavior of purified V96 and LV96 fusion
proteins were characterized by turbidity analysis using a DU800 UV-VIS
9

Spectrophotometer (Beckman Coulter, Brea, CA). Optical density was measured at 350
nm as a function of solution temperature. ELPs were observed in PBS over a range of
concentrations from 5 to 100 μM under heating at 1 ° C/min between 10 ° C and 45 ° C. T
t

at each concentration was defined as the maximum first derivative of turbidity change.
1.2.6. Cell isolation, culture and treatments
Isolation of primary cultured LGAC from female New Zealand white rabbits was
performed in accordance with the Guiding Principles for Use of Animals in Research.
Specifically, LGAC were isolated from rabbit LGAC and cultured by the method of da
Costa (da Costa et al., 1998) in Peter's Complete Medium (PCM) medium for 2-3 days.
1.2.7. Secretion of β-hexosaminidase
Fresh PCM medium was added to wells containing LGAC and incubations were
continued for additional 2 h. Baseline samples were then taken from each well, and the
cells were stimulated with 100 μM carbachol (CCh), Lacrt, V96, or LV96 at various
concentrations as indicated for 1 h. After stimulation, the cell supernatant was collected
and β-hexosaminidase activity in each aliquot was measured against a model substrate,
methylumbelliferyl-N-acetyl-β-D-glucosaminide. Assays of catalytic activity were
performed in black 96-well plates, and reaction product absorbance was determined
with a plate reader at 460 nm (Tecan Genios Plus; Phenix Research Products, Candler,
NC); signals were analyzed with the manufacturer’s software package (Magellan v6.6;
Phenix Research Products). Medium was then aspirated from all wells and 500 μl 0.5 M
NaOH was added into each well and incubated at 4 ° C for overnight to lyse the acini
and solubilize all protein. Total protein in each well was measured by the bicinchoninic
acid assay (BCA) assay using a bovine serum albumin standard curve. Secreted β-
10

hexosaminidase level was expressed as ΔOD465nm(Post-Pre)/μg total protein. Each
treatment was performed in triplicate and whole β-hexosaminidase secretion assays
were repeated 3 times. The LGAC response was defined as:
β hexosaminidase Secretion % of control

!"
#
$$%&

$$%'
#
$$%&
( 100%.
1.2.8. LGAC morphology changes upon stimulation
LifeAct-RFP adenovirus was generated as described previously (Chiang et al., 2011).
For amplification, QB1 cells, a derivative of HEK293 cells, were infected with the virus
and grown at 37 ° C and 5 % CO
2
in Dulbecco's Modified Eagle's Medium (DMEM, high
glucose) containing 10 % fetal bovine serum for 66 hours until completely detached
from the flask surface. The Adeno-X™ virus purification kit (Clontech, CA) was used for
virus purification and the Adeno-X™ rapid titer kit for viral titration. LGAC cells were
transduced at a multiplicity of infection of 8-10 for 2 h at 37 ° C and then rinsed and
cultured in fresh medium overnight to allow for protein expression. Live cell images
upon Lacrt/LV96 stimulation were captured using a Zeiss LSM 510 Meta confocal
fluorescence microscopy system.
1.2.9. Rhodamine labeling and cell uptake of Lacrt and Lacrt ELP fusion proteins
Lacrt, V96 and LV96 were conjugated with NHS-Rhodamine (Thermo Fisher Scientific
Inc, Rockford, IL) via covalent modification of the amino terminus. Conjugation was
performed in 100 mM borate buffer (pH 8.0) for 2 h (LV96 and Lacrt) or overnight (V96)
at 4 ° C followed by desalting on a PD10 column (GE Healthcare, Piscataway, NJ) to
remove free dye. Cell uptake was studied on 35 mm glass coverslip-bottomed dishes.
Briefly, after washing with warm fresh medium, LGACs were cultured in medium
containing 10 μM of proteins conjugated with rhodamine. After incubation at 37 ° C for
11

different time points, the cells were rinsed with warm fresh medium and images were
acquired using confocal fluorescence microscopy.
1.2.10. Tear secretion and immunohistochemistry of the mouse LG
For intra-lacrimal injection, mice were anesthetized with an i.p. injection of
xylaxine/ketamine (60-70 mg+5 mg/kg) and placed on a heating pad. After removing fur
from the cheek and cleansing the area with alcohol, a small incision (5 mm) was made
to visualize the LG. 5 μl of 50 μM carbachol (CCh), 100 μM LV96, 100 μM V96 or 100
μM Lacrt was injected into the LG using a 33 gauge blunt needle. The mice were
monitored on the heating pad until fully recovered from anesthesia. For quantification of
tear secretion, a glass capillary (Microcaps Drummond disposable micropipettes 2 μl)
was placed on the lower eyelid of the mice to collect tears (2 LG/each mouse, 30
min/each gland). After sacrificing the mice following surgery, LG were dissected and
fixed in 4 % paraformaldehyde for histological analysis.
1.2.11. Stability of Lacrt
For determination of the half-life of Lacrt, the purified proteins (20 μg) were incubated in
PBS at 37 ° C for 72 h followed by SDS-PAGE analysis . Peptide sequence analysis was
performed using MALDI-TOF (AXIMA Assurance, Shimadzu). Cleavage products were
assigned by MALDI-TOF mass by comparison of measured with predicted mass to
charge ratios (m/z) with +1 charge ionization ([M+H]
+
). For Western blotting of purified
Lacrt, 50 μg purified protein was loaded onto 4-20 % Tris-HCl polyacrylamide gels; with
blocking buffer at room temperature for 1 h and blotted with rabbit anti-N-terminal or
anti-C-terminal (1:200) lacritin antibody (Laurie et al., 2012) overnight at 4 ° C followed
by blotting with IRDye800 Donkey anti-rabbit IgG (H+L) (Rockland) (1:3000) at room
12

temperature for 1 h. Images were taken using the Odyssey infrared imaging system (Li-
Cor, Lincoln, NE).
1.2.12. Statistical Analysis
All experiments were replicated at least three times. Values are expressed as the mean
± SD. Quantification of β-hexosaminidase secretion was analyzed using two-way
ANOVA followed by Bonferroni post-hoc analysis (GraphPad Prism). Tear secretion and
Lacrt degradation results were analyzed using one-way ANOVA followed by Tukey’s
post-hoc test (GraphPad Prism). A p value less than 0.05 was considered statistically
significant.
1.3. Results
1.3.1. Construction and Purification of a Lacrt ELP fusion protein
We designed the cDNA encoding LV96 in the pET25b(+) vector resulting in the amino
acid sequence shown in Table 1. V96 forms viscous coacervates with T
t
below
physiological temperatures of 37 ° C (Janib et al., 2014) and thus was chosen as the
ELP backbone for potential depot formation. LV96 was also used to generate free Lacrt
control protein utilizing selective cleavage through the thrombin cleavage site (Fig. 2A).
In contrast to the previously reported intein system for lacritin purification (Sanghi et al.,
2001a), we demonstrated that LV96 showed a robust yield of more than 40 mg/L using
the inverse transition cycling purification approach (Fig. 2B). MALDI-TOF analysis
(Table 1) and Western blotting with anti-C terminus lacritin antisera (N-65) (Fig. 4)
further confirmed the successful construction of both LV96 and Lacrt. Interestingly,
SDS-PAGE analysis of purified LV96 (Fig. 3) suggested the spontaneous cleavage of
ELP (V96) from the fusion construct, which yielded a combination of fusion protein and
13

the ELP tag after purification. After several attempts at chromatographic separation, a
Superose 6 size exclusion column was chosen to remove free ELP tags as a final
purification step (Fig. 3). The internal Lacrt control was liberated from the ELP tag via
thrombin cleavage. Similar to previously reports (Ma et al., 2008), Lacrt ran higher on
SDS-PAGE than the expected M.W. of 12 kDa (Fig. 2B), but was equal to the expected
by mass spectrometry (Table 1).
Optical density measurements were used to confirm the phase behavior for all three
constructs (Table 1), which revealed that only LV96 and V96 phase separate at
physiological temperatures (Fig. 2C). The LV96 phase transition curve at 25 μM (Fig.
2C) was consistent with the phase transition behavior of the parent V96 with a 5 ° C
decrease. Further characterization of the concentration-temperature phase diagrams for
V96 and LV96 suggest LV96 was slightly less dependent on concentration compared to
V96 as fit by a log-linear regression line (Fig. 2D).
At physiological temperatures, the half-life of disappearance for Lacrt is about a day
(23.7 h); however, at 4 ° C the Lacrt was stable for extended periods. Despite this
apparent biodegradation, by optimizing the purification strategy and maintaining
proteins on ice, both Lacrt and LV96 were available at high purity and yields necessary
for further study (Fig. 2B).
14

Table 1. Nomenclature, amino acid sequence, and transition temperature of Lacrt-
ELPs
Protein
Label
*Amino Acid Sequence
**Expected
M.W.(kDa)
***Observed
M.W. (kDa)
****T
t
(° C)
V96 G(VPGVG)
96
Y 39.55 39.50 31.6

LV96
GEDASSDSTGADPAQEAGTSKPNEEISGPAEPAS
PPETTTTAQETSAAAVQGTAKVTSSRQELNPLKSI
VEKSILLTEQALAKAGKGMHGGVPGGKQFIENGS
EFAQKLLKKFSLLKPWAGLVPRGSG(VPGVG)
96
Y
52.52 52.29 26.8

Lacrt
GEDASSDSTGADPAQEAGTSKPNEEISGPAEPAS
PPETTTTAQETSAAAVQGTAKVTSSRQELNPLKSI
VEKSILLTEQALAKAGKGMHGGVPGGKQFIENGS
EFAQKLLKKFSLLKPWAGLVPR
12.84 12.73 NA
*After the start codon, a glycine spacer was added during cloning which is not present
on human lacritin
**Expected M.W.(kDa) was calculated by DNAStar Lasergene Editseq
***Observed M.W. (kDa) was measured by MALDI-TOF
****T
t
(° C) was defined at the point of the maximum first derivative of 25 μM protein
solution turbidity change at 350 nm in Phosphate Buffer Saline (PBS)

15

Figure 2. Purification and thermal characterization of LV96. A) Cartoon of the LV96
fusion protein showing Lacrt at the N-terminus and an ELP tag at the C-terminus, with a
thrombin recognition site between the two moieties. B) SDS-PAGE of purified LV96,
ELP alone (V96) and free Lacrt. Gels were stained using CuCl
2
. C) Representative
optical density profiles for LV96, V96 and Lacrt at 25 μM as a function of temperature,
which indicate a phase separation at 26.8 (LV96) and 31.6° C (V96) respectively. Lacrt
alone remains soluble, and does not increase optical density. D) Concentration
temperature phase diagram for LV96 and V96. A best fit line is indicated that follows the
following relationship, T
t
= b – mLog
10
[Conc]. b = 28.56 and 36.06 ° C, m = 1.19 and
3.25 ° C (Log
10
[μM])
-1
for LV96 and V96 respectively.
16

Figure 3. Stepwise Lacrt ELP purification process. A) SDS-PAGE showing pre
cleavage of V96 from LV96 after ITC. The free ELP tag was removed using a Superose
6 size exclusion column. B). Peak 1 (P1) was collected and concentrated using Amicon
Ultra concentrator (10 kD cut-off).

17

Table 2. Representative Lacritin cleavage sequences identified by MALDI-TOF
Fragment *Amino Acid Sequence
**Expected
M.W. (kDa)
***Observed
M.W. (kDa)
1 GEDASSDSTGADPAQEAGTSKPNEEISGPAEPASP
PETTTTAQETSAAAVQGTAKVTSSRQELNPLKSIVE
KSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQ
KLLKKFSLLKPWAGLVPR|
12.84 12.96

2 GEDASSDSTGADPAQEAGTSKPNEEISGPAEPASP
PETTTTAQETSAAAVQGTAKVTSSRQELNPLKSIVE
KSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQ
KLLKKFSLL|K|
11.84/
11.97
12.00

3 GEDASSDSTGADPAQEAGTSKPNEEISGPAEPASP
PETTTTAQETSAAAVQGTAKVTSSRQELNPLKSIVE
KSILLTEQALAKAGKGMHGGVPGGKQFIENGSEFAQ
KLLK|K|
11.25/
11.38
11.35/
11.47/
11.50
4 |PNEEISGPAEPASPPETTTTAQETSAAAVQGTAKVT
SSRQELNPLKSIVEKSILLTEQALAK|
6.42 6.36

* Underlined sequence: Syndecan-1 binding site
**Expected M.W. (kDa) was calculated by DNAStar Lasergene Editseq
***Observed M.W. (kDa) was measured by MALDI-TOF

18

Figure 4. Purified lacritin is susceptible to proteolysis of an unidentified origin. A)
Western blot of purified Lacrt probed with an anti-lacritin antibody (raised against Lacrt
lacking 65 amino acids at the amino terminus) revealed multiple bands smaller than the
M.W. expected for Lacrt. B) MALDI-TOF analysis of Lacrt degradation products upon
incubation at 37 ° C. C-D) Time dependent degradation of purified Lacrt into fragments
(Table 2) C) SDS-PAGE stained with Coomassie blue showing disappearance of the
Lacrt band; D) Lacrt degradation was quantified by fitting to a single exponential decay
model, t
1/2
=23.7 h (R
2
=0.99).

19

1.3.2. LV96 stimulates β-hexosaminidase secretion from primary rabbit LGACs
The concentration-dependent prosecretory activity of human recombinant Lacrt was first
observed using freshly isolated rat lacrimal acinar cells with peroxidase as the marker of
secretory activity. Signaling was effective in wells exposed to 10-20 μM lacritin coating
solution and at 162 ng/ml (13 nM) or 10 ng/ml (0.8 nM) when presented as soluble
lacritin (Sanghi et al., 2001a). The latter was confirmed in assays of several cultured
human cell lines over a broad dose range (Wang et al., 2006). Yet, lacritin partially
purified from monkey tears is apparently optimal at 1 μM. Further in vivo study
indicated that 10-100 μg/ml (0.8 - 8 μM) lacritin topically administered at both a single
dose and chronically over two weeks elevated basal tearing of healthy New Zealand
White adult female rabbits (Kurzbach et al., 2013). Rabbit LGACs do not secrete
peroxidase; therefore, we monitored β-hexosaminidase secretion, a robust secretory
marker, from primary rabbit LGACs treated with Lacrt or LV96 (Fig. 5). Compared to
V96, the LV96 coacervate significantly stimulated secretion at a concentration of 10 μM
(p<0.01) and 20 μM (p<0.001) while significant Lacrt-triggered stimulation was observed
at 20 μM (p<0.05) (Fig. 5B); the effects of either Lacrt or LV96 at 1 μM or 0.1 μM were
not statistically significant. This data suggests that receptors exist on rabbit LGACs that
respond to human Lacrt delivered by an ELP fusion.
In response to secretagogues, LGACs exocytose mature secretory vesicles containing
tear proteins at their apical membranes for release into the acinar lumen, an event that
involves F-actin remodeling around fusing secretory vesicles at the luminal region

(Jerdeva et al., 2005). Motivated to understand the cellular mechanisms of LGAC
secretory activity in response to LV96, we tracked the morphology of live LGACs
20

transduced with adenovirus Ad-LifeAct-RFP to investigate changes in F-actin filament
rearrangement beneath the apical and basal membranes of the acini during exocytosis
evoked by 20 μM LV96 (Fig. 5C). While 100 μM CCh acutely increased significant F-
actin filament turnover and promoted transient actin coat assembly around apparent
fusion intermediates in 15 min as previously reported (Jerdeva et al., 2005); LV96
exhibited a slower and more sustained effect on F-actin remodeling, which triggered
increased irregularity in the actin filaments around the lumen and formation of actin-
coated structures beneath apical and basal membrane (white arrows) after 20 min. No
significant remodeling of actin filaments were observed in the negative V96 control
group.

21

Figure 5. Lacrt-ELP fusion proteins are prosecretory in LGACs. A) Cartoon
depicting structure for ex vivo clusters of LGACs obtained from rabbits. These primary
cultures form an apical lumen (L) that is bounded by a thick network of actin filaments.
SV: secretory vesicles. B) Rabbit secretory vesicles (SV) release β-hexosaminidase in a
dose dependent manner in response to secretagogues. The percentage of cell
response is plotted compared to a positive control, CCh-stimulation, which is defined as
100 %. LGACs were treated with 0.1 to 20 μM of LV96, Lacrt, V96, or no treatment for 1
h at 37 ° C. 10 μM and 20 μM LV96 significantly enha nced secretion compared to the
V96 group (p<0.01), and a similar effect was found with 20 μM Lacrt (p<0.05). Data
were shown as mean ± S.D. and analyzed by ANOVA followed by Bonferroni’s Post-
22

Hoc Test. C) Representative live-cell imaging of LGACs labeled for F-actin (red). Actin-
RFP is enriched beneath the apical membrane surrounding the lumen. Shortly after 10
min, increased irregularity of apical actin filaments and actin-coated secretory vesicle
(SV) formation beneath the apical membrane (white arrows) were observed in CCh
treated cells. 20 μM LV96 induced substantial time-dependent actin remodeling in
LGAC cultures after 20 min, which increased apical actin filaments irregularity and
secretory vesicle formation (white arrows). These were similar to the remodeling events
elicited acutely by CCh. No significant remodeling of actin filaments was observed in
V96 negative control group. White asterisk*: lumen. Scale bar: 10 μm.

23

24

Figure 6. Fusion with V96 influenced uptake of exogenous Lacrt into LGACs. A)
Time dependent uptake of rhodamine-labeled Lacrt into LGACs. At 10 min, most Lacrt
was bound to the basolateral membrane. After 30 min, significant fluorescent puncta
were detected in the cytosol and transcytosis were observed as evidenced by retention
of fainter rhodamine label within the lumen (marked by white *). After 2 h, basolateral
binding became less uniformly distributed. B) Examination of time-dependent uptake of
LV96 showed a more diffuse labeling pattern in the cytosol with some apparent
detection of accumulation in the lumen by 2 h, although this effect was less pronounced
than free Lacrt. C) Even after 2 hours, the negative control V96 did not show significant
uptake into LGACs. White *: Lumen; white arrow: basolateral membrane binding and
exogenous Lacrt/LV96 puncta in the cytosol. Scale bar: 10 μm.

25

1.3.3. Fusion with V96 influenced uptake of exogenous Lacrt into LGACs
Secreted by LGAC, transported via ducts and deposited onto rapidly renewing ocular
surface epithelia, Lacrt is thought to be preferentially mitogenic or prosecretory for the
cell types that it normally contacts during its glandular outward flow, such as the
corneal, limbal and conjunctival epithelial cells, meibomian and LG epithelium, retina,
and retinal pigmented epithelium/choroid (Zhang et al., 2011). Yet, no previous studies
have captured the real-time binding and transport of Lacrt in live cells. Herein, we
documented the uptake of exogenous Lacrt and LV96 in rabbit LGACs within a 2 h
timeframe (Fig. 6). Binding of native Lacrt to the basolateral membrane of LGAC was
recorded starting from 10 min of exposure (Fig. 6A), revealing detection of significant
fluorescent puncta in the cytosol after 30 min. Interestingly, we noticed an increase in
fluorophore retention in the LGAC luminal region, marked by white * in the lumen,
suggesting the possible transcytosis of exogenous Lacrt in LGACs. LV96 labeled the
surface and clearly reached the LGAC interior, as evidenced by the diffuse fluorescence
detected within cells at the same times as well as some fluorescent puncta (Fig. 6B).
The less abundant intracellular fluorescence accumulation relative to free Lacrt is
possibly caused by its phase separation, which could influence receptor binding affinity
and delay endocytosis (Ma et al., 2006).
1.3.4. LV96 stimulates tear secretion from non-obese diabetic (NOD) mice
To further explore LV96’s in vivo efficacy and influence of V96 coacervation on local
retention of Lacrt, we locally injected Lacrt and LV96 into the lacrimal gland of non-
obese diabetic (NOD) mice. The NOD mouse is one of the most utilized models for
study of DED symptomatic of Sjögren' syndrome (Robinson et al., 1997), which is
26

characterized by autoimmune infiltration of LG and reduced production of aqueous
tears. Although SjS is more prevalent in female patients, the 10-12 week male NOD
mice feature more symptoms of autoimmune inflammation in LG, including severe
lymphocytic infiltration, decreased production of lacrimal fluid, significant extracellular
matrix degradation and increased expression of matrix metalloproteinases (Schenke-
Layland et al., 2010). Surprisingly, LV96 and Lacrt significantly enhanced tear secretion
from both male and female NOD mice relative to the V96 negative control (Fig. 7).
Although no significant difference in secretion (Fig. 7C,D) was observed between LV96
and Lacrt, immunohistochemistry (Fig. 7B) and fluorescent imaging (Fig. 8) suggested
local depot formation of the viscous LV96 coacervates while Lacrt showed a more
diffuse distribution pattern in the LG. Compared to CCh stimulation (100 %), LV96’s
prosecretory effect was 40.9 % in male NOD mice and 50.0 % in female NOD mice,
both significantly higher than V96 treatment (p<0.01). Lacrt’s efficacy was 29.6 % in
male NOD mice and 42.9 % in female NOD mice accordingly.
Interestingly, no enhanced secretion was observed in age-matched male or female
C57BL/6 mice although a similar formation of an LV96 depot was confirmed (Fig. 9).
However, due to the invasiveness of the tear collection procedure, long term controlled
release and tear simulation of LV96 was not evaluated after 1h. These disparities
associated with the NOD disease model and the C57BL/6 mice suggest that features of
aqueous tear deficiency may sensitize the diseased LG to therapy with Lacrt.

27

Figure 7. Lacrt-ELP fusion proteins stimulate tear secretion in NOD mice. A)
Representative pictures showing tear secretion stimulated by 100 μM LV96 (5 μl) after
an intra-lacrimal injection in a male NOD mouse. Blue arrow: collected tear volume after
30 min. B) LG with LV96 administered by injection were visualized using
immunofluorescence to identify Lacrt by anti-C terminus lacritin antibody. Green: anti-
Lacritin antibody; Red: actin stained using Rho-phalloidin; Blue: nucleus stained by
DAPI. Scale bar: 10 μm. C-D) Tear volume quantification showing significantly
enhancement of tear secretion by LV96 and Lacrt compared to negative V96 controls
(**p<0.01, *p<0.05, n=9). Data were shown as mean ± S.D. and analyzed by ANOVA
followed by Tukey’s Post Hoc Test. C) Male NOD mice; D) Female NOD mice.

28

Figure 8. Intra-lacrimal injection of a labeled Lacrt-ELP fusion protein produces a
depot. Representative confocal images showing exogenous Rho-LV96 formed a local
drug depot in the LG of female NOD mice while Rho-Lacrt without ELP exhibited only
diffuse fluorescence 1 h after injection. Without the rhodamine label, the CCh treated
group did not show any fluorescent signal in the LG under the same imaging settings.
*LGs were harvested, fixed, frozen, sliced and directly imaged. Scale bar: 100 μm.

29

Figure 9. Intra-lacrimal gland injection of Lacrt-ELPs in normal male C57BL/6
mice. A) Immunohistochemistry figure showing possible enhanced
secretion/transcytosis of Lacrt treated mice and local LV96 depot formation near the
injection site. Green: anti-C terminus Lacritin Ab; Red: rhodamine or Rho-phalloidin;
White *: lumen; White arrow: local LV96 drug depot. B) Quantification of Lacrt-ELP
stimulated tear secretion showing tear secretion of LV96 compared to V96 vehicle
group (not significant, n=3). Data were shown as mean±S.D.
30

Figure 10. Carbachol (CCh) triggered Ca
2+
scintillation in LGACs. 10 μM CCh
triggered Ca
2+
scinlitation in LGACs using Fluo-4AM as Ca
2+
indicator. A) Before
stimulation; B) Increased intracellular fluorescence level after CCh stimulation. C)
Representative intracellular fluorescence change curve upon stimuli (n=10). No
significant effect was observed when cells were treated with lacritin. Scale bar: 20 μm.
31

1.4. Discussion
Hasn’t attracted much attention in drug industry, the eye is now a popular target for
development of new drugs, especially novel biological therapies (Garber, 2010) due to
the increased numbers of patients with ocular allergies and dry eye disease (Clark and
Yorio, 2003). A key element of drug development is optimization of the safety and
efficacy of drug candidates (Muller and Milton, 2012), and local ocular delivery provides
unique opportunities to enhance the therapeutic index of ophthalmic drugs by extending
local residence time while minimizing off-target effects and dose frequency (Novack,
2009a). Once rarely used for medical treatments, protein therapeutics have become
more popular because of their high target specificity, reduced interference with normal
biological processes and minimal immune responses to human self-proteins (Leader et
al., 2008a). The discovery of Lacritin offers a new therapeutic opportunity for DED and
an alternative to conventional approaches (McKown et al., 2009a). Its basic structural
features (McKown et al., 2009a), prosecretory and mitogenic significance (Sanghi et al.,
2001a), as well as associated downstream signaling transduction mechanisms (Ma et
al., 2006; Wang et al., 2006) have been gradually elucidated over the past decade. As a
proof of concept, in this study we aimed to generate a thermo-responsive Lacrt-ELP
fusion protein for extended release. The transition temperature

(Fig. 2C,D) supports our
hypothesis that Lacrt fused to an ELP exhibits similar phase separation and self-
assembly properties relative to the parent ELP. Significantly enhanced β-
hexosaminidase secretion from primary rabbit LGACs (Fig. 5) and increased tear
secretion from both male and female NOD mice via single-bolus intra-lacrimal injection
(Fig. 7) corroborated the prosecretory function of LV96 both in vitro and in vivo.
32

Although no significant prosecretory activity difference was observed between LV96 and
Lacrt during the time period tested, histological analysis did provide evidence for local
drug depot formation of LV96 (Fig. 7, 8). Moreover, we report uptake of exogenous
LV96 in LGACs (Fig. 6), with a slight decrease in cellular uptake for LV96.
We noted Lacrt’s susceptibility to degradation using a western blot (Fig. 4A), which
showed multiple bands below the expected Lacrt M.W. The blotting result was
augmented by MALDI-TOF analysis (Fig. 4B) and time-dependent analysis of
degradation by SDS-PAGE (Fig. 4C), suggesting a 24-hour half-life of native Lacrt (Fig.
4D), which is mediated by apparent cleavage among lysine residues in human Lacrt
(Table 2). Yet, we cannot rule out the possibility that Lacrt may exhibit autolysis similar
to trypsin, based on the potential His, Ser and Asp triad (Vestling et al., 1990). Whether
this is due to the absence of glycosylation or an auto-regulatory mechanism for ocular
Lacrt remains to be determined. Nevertheless, Lacrt and LV96 can be exploited in the
form of a depot whose release kinetics change in response to the local protease
environment (Law and Tung, 2009; Senter and Sievers, 2012). Published reports lay out
in remarkable detail how a crucial sequence at the C-terminus of Lacrt targets
heparanase-modified syndecan-1 and the sequential selective binding cascades (Wang
et al., 2013b; Zhang et al., 2013a). Our LGAC uptake experiments illustrate how the
fusion of a thermo-responsive ELP tag to the C-terminus of lacritin significantly
influences the transcytosis process (Fig. 6), supporting the importance of Lacrt in
receptor binding (Zhang et al., 2013a). Although signaling pathway of lacritin stimulating
LGAC secretion may differ from cholinergic agonist carbachol (CCh) (Fig. 10).
33

The concept of a ‘reservoir drug’ (Setton, 2008) is intended to tackle the problem of
inefficient target delivery and rapid payload clearance. By delicately modulating the
precise dose and location of the therapeutic agent reservoir, side effects can be
reduced and bioavailability may be enhanced (Langer, 2001). ELPs have shown
promise as reservoir scaffolds (Amiram et al., 2013b; Koria et al., 2011), as alternatives
to PLGA in situ (Parent et al., 2013) and in catechol-based adhesive gels (Kastrup et
al., 2012). In this study, the persistently high Lacrt signal for viscous coacervates
formed by LV96 shown in immunohistochemistry (Fig. 7) and fluorescence imaging (Fig.
8) illustrates the injected protein’s ability to be sequestered in the LG even after a period
of time that eliminates the Lacrt without an ELP tag. This suggests the formation of a
drug depot capable of releasing its contents in a sustained manner. Moreover, this
study confirms the ability of ELPs to absorb and release protein drugs (LaVan et al.,
2003). Further research unveiling the pharmacokinetics and underlying mechanism of
tear secretion from the murine model via Lacrt-ELP stimulation are of interest for future
studies.
1.5. Conclusion
Achieving sustained delivery of therapeutic proteins to treat ocular disease is one of the
major challenges of ophthalmology. In pursuing this goal, we fused ELPs with the model
dry eye disease therapeutic, Lacritin, to control local delivery. Lacrt-ELP fusion proteins
maintained thermo-responsive phase separation exhibited by the parent ELPs. They
also gained the prosecretory activity of native Lacrt. Detailed pharmacokinetics and
mechanistic studies of LV96 in the LG will require further investigation to translate this
34

discovery; furthermore, if successful, this approach may be useful to deliver a variety of
proteins to ocular targets.
35

Chapter 2
Self-assembly of nanoparticles for corneal wound healing
2.1. Introduction
Eye injury is reported to be the second most common cause of visual impairment in the
United States after cataracts (Haddadin et al., 2013). Back in 2006, emergency room
(ER) visits for eye injuries represented 1.4% of all ER visits (Haddadin et al., 2013). To
maintain corneal transparency and rigidity, the corneal epithelium serves as an
important barrier between the external environment and the delicate internal ocular
tissues (Kimura et al., 2008). Although the corneal epithelium normally recovers rapidly
from damage, certain clinical conditions including diabetic retinopathy, herpes simplex
virus infection, neurotrophic keratopathy, and corneal transplants result in delayed
wound healing, often precipitating sight-threatening complications (Suzuki et al., 2003).
Thus, there remains a need for more effective therapies to facilitate epithelial healing on
the ocular surface. Moreover, recovery times following popular refractive procedures,
such as photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK),
directly rely on the patients’ corneal wound healing response; however these
procedures may lead to haze, dry eye, nerve damage and Diffuse Lamellar Keratitis
(DLK) (Netto et al., 2005).
Corneal epithelium recovery after injury involves apoptosis, migration, proliferation and
differentiation of multiple cells in a cascade mediated by cytokines, growth factors and
chemokines (Suzuki et al., 2003). Naturally present in the anterior segment of the eye
and responsible for the migration and proliferation of corneal epithelial cells, growth
factors have become a class of promising therapeutic candidates in treating visual
36

impairments (Klenkler and Sheardown, 2004). An enhanced wound healing effect has
been observed in primate models and clinical trials via topical treatment with epidermal
growth factor (EGF) (Kitazawa et al., 1990; Scardovi et al., 1993), keratinocyte growth
factor (Sotozono et al., 1995), nerve growth factor (Lambiase et al., 1998), etc. As such,
it would be of great clinical value to rationally bioengineer growth factor-like proteins into
therapies using a robust formulation process. Successful earlier trials include increased
tensile strength of full thickness corneal wounds after topical epidermal growth factor
(EGF) treatment (Leibowitz et al., 1990) and better ulcer healing in diabetic patients
from platelet-derived growth factor-BB (PDGF-BB) in a topical gel formulation (Embil et
al., 2000). One novel candidate to stimulate wound healing on the ocular surface is the
mitogen known as Lacritin (Karnati et al., 2013). Lacritin is the most severely
downregulated protein in contact lens related dry eye and is similarly deficient in
blepharitis (a common inflammation of the eyelid) (Sanghi et al., 2001b). Previous in
vitro tests have shown that, on the ocular surface, Lacritin triggers Ca
2+
wave
propagation (Sanghi et al., 2001b), in addition to promoting the survival of primary and
cultured human corneal epithelial cells stressed with interferon-γ, tumor necrosis factor
(Wang et al., 2013c) and Benzalkonium chloride (BAK) (Feng et al., 2014), indicative of
both mitogenic and cytoprotective activity. This cell targeting specificity is triggered by a
unique ‘off-on’ switch controlled by heparanase deglycanation of the cell surface
protein, syndecan-1, which exposes a lacritin binding site as a prerequisite for its
downstream mitogenic signaling (Ma et al., 2006; Zhang et al., 2013b).
Although topical application of ophthalmic products has remained the most popular and
well-tolerated administration route for patient compliance, the bioavailability of eye
37

drops is severely hindered by blinking, baseline and reflex lachrymation, and
nasolacrimal drainage (Lang, 1995a). One solution to enhancing the therapeutic index
of topical treatments is through the application of polymeric nanoparticles as drug
carriers (Diebold et al., 2007; Nagarwal et al., 2009; Sahoo et al., 2008). Polymeric
nanoparticles displaying therapeutic ligands at the corona can interact with complex
biomolecular architectures through multiple simultaneous interactions (multivalency)
and exhibit the well-defined sizes required for efficient tissue penetration (Monopoli et
al., 2012). One such material capable of being employed as the scaffold are thermo-
responsive elastin-like polypeptides (ELPs) (Hubbell and Chilkoti, 2012a). ELPs are
composed of the repetitive pentapeptide motif (Val-Pro-Gly-Xaa-Gly) and exhibit unique
reversible inverse phase transition temperatures, T
t
, below which they solubilize and
above which they phase separate (Meyer and Chilkoti, 1999). T
t
can be modulated
through guest residue (Xaa) selection and changes in the number of pentameric
repeats, n (Chilkoti et al., 2006a; Nettles et al., 2010a). We have previously reported the
successful bioengineering of diblock ELP nanoparticles to suppress tumor growth with
rapamycin binding at both the corona as well as in the core (Shi et al., 2013b); uptake of
ELP nanoparticles displaying adenovirus knob domain into hepatocytes and acinar cells
has also been described (Sun et al., 2011). Moreover, Callahan et al. have
demonstrated enhanced intratumoral spatial distribution of ELP nanoparticles via triple
stimuli (Callahan et al., 2012b) while MacEwan et al. observed controlled cellular uptake
in HeLa, MCF7, and primary HUVEC cells using local Arg density modulation on ELP
nanoparticles (MacEwan and Chilkoti, 2012). Collectively, these studies illustrate the
38

potential of ELP nanoparticles to enhance both local and systemic therapeutic effects as
a drug carrier.
Inspired by the motivation to further explore lacritin’s function on the ocular surface,
enhance its bioavailability, and better target the corneal epithelium, we utilized a diblock
ELP (SI) micelle scaffold to bioengineer LSI nanoparticles with multivalent presentation
at the surface (Fig. 11). The thermo-responsiveness and self-assembly of LSI
nanoparticles was investigated by UV-Vis turbidity analysis, dynamic light scattering
(DLS), and transmission electron microscopy (TEM). LSI exhibited mitogenic activity in
vitro as confirmed by Ca
2+
wave propagation and scratch wound healing using SV40-
transduced human corneal epithelial cells (HCE-Ts). To further explore the in vivo
efficacy of LSI nanoparticles, we created abrasion wounds on the ocular surface of
female NOD mice mimicking the PRK procedure and topically treated the eye with two
doses of LSI nanoparticles within 12 h after the surgery. The LSI treated group exhibited
significant faster wound healing compared to SI, epidermal growth factor (EGF) and
bovine pituitary extract (BPE) co-treatment, and no treatment. To address the
importance of multivalency, we also included a simple macromolecule control LS96.
Murine corneal abrasion recovery study strongly supported enhanced healing efficacy of
LSI nanoparticles than LS96 in a 12 h timeframe. Histology analysis revealed that, after
LSI treatment, no significant corneal inflammation was observed and the reconstituted
ocular surface appeared as smooth as pre-procedure following 24 h. Collectively, we
have successfully bioengineered multivalent self-assembling LSI nanoparticles based
on the thermo-responsive SI micelle scaffold, confirmed its in vitro mitogenic activity
using HCE-Ts, and corroborated the efficacy using a novel murine corneal abrasion
39

model. Compared to macromolecular LS96, LSI nanoparticles exhibited faster in vivo
wound healing efficacy in 12 h. This study provides the first in vivo verification of
lacritin’s wound healing potential and can be further applied to rationally bioengineer
other therapeutics into self-assembling nanoparticles for treating visual impairments
using the ELP delivery system.
40

Figure 11. Rationale of using lacritin LSI nanoparticles to heal corneal wounds via
topical application.
41

2.2. Materials and Methods
2.2.1. Materials and equipment
TB DRY® Powder Growth Media was purchased from MO BIO Laboratories, Inc.
(Carlsbad, CA). NHS-Rhodamine was purchased from Thermo Fisher Scientific
(Rockford, IL). SV40-Adeno vector transformed cornea cells (RCB 2280, HCE-T) were
purchased from Riken Cell Bank, Japan. Keratinocyte-SFM medium supplied with
Bovine Pituitary Extract (BPE) and prequalified human recombinant Epidermal Growth
Factor 1-53 (EGF) was purchased from Gibco Invitrogen (Life Technologies, NY).
Calcium Indicator Fluo-4, AM, cell permeant was purchased from Life Technologies
(NY). Algerbrush II with 0.5 mm burr was purchased from The Alger Company, Inc., TX.
In vivo studies were conducted using 12-week female non-obese diabetic (NOD)
(Taconic Farms, Germantown/NY, USA) mice. All procedures performed were in
accordance to the university approved IACUC protocol.
2.2.2. Construction of LSI nanoparticles
Genes encoding for ELPs (SI) were synthesized by recursive directional ligation in
pET25b(+) vector as previous reported (Shi et al., 2013b). A sequence encoding human
lacritin without secretion signal peptide was designed using the best E. coli codons in
EditSeq (DNAStar Lasergene, WI). A thrombin cleavage site was designed between the
lacritin sequence and ELP tag via insertion at the BseRI site. Lacritin gene flanked by
NdeI and BamHI restriction digestions sites at the 5’ and 3’ ends was purchased in the
pIDTSmart-KAN vector from Integrated DNA Technologies (IDT) as follows:
5’-
CATATGGAAGACGCTTCTTCTGACTCTACCGGTGCTGACCCGGCTCAGGAAGCTG
42

GTACCTCTAAACCGAACGAAGAAATCTCTGGTCCGGCTGAACCGGCTTCTCCGCC
GGAAACCACCACCACCGCTCAGGAAACCTCTGCTGCTGCTGTTCAGGGTACCGCT
AAAGTTACCTCTTCTCGTCAGGAACTGAACCCGCTGAAATCTATCGTTGAAAAATCT
ATCCTGCTGACCGAACAGGCTCTGGCTAAAGCTGGTAAAGGTATGCACGGTGGTG
TTCCGGGTGGTAAACAGTTCATCGAAAACGGTTCTGAATTCGCTCAGAAACTGCTG
AAAAAATTCTCTCTGCTGAAACCGTGGGCTGGTCTGGTTCCGCGTGGTTCTGGTTA
CTGATCTCCTCGGATCC-3’.
The above gene was subcloned into the pET25b(+) vector and LSI gene was
synthesized by ligation of ELP SI gene via the BseRI restriction site. Correct cloning of
the fusion protein gene was confirmed by USC DNA core. LSI fusion protein was
expressed in BLR (DE3) E. coli (Novagen Inc., Milwaukee, WI) for 24 h in an orbital
shaker at 37 ° C at 250 rpm and purified via inverse phase transition cycling as
previously reported (Aluri et al., 2014b).
2.2.3. Characterization of LSI phase behavior and nanoparticle formation
The phase diagram for LSI fusion protein was characterized by optical density change
at 350 nm as a function of solution temperature using a DU800 UV-VIS
Spectrophotometer (Beckman Coulter, Brea, CA). T
t
was defined at the point of the
maximum first derivative. Self-assembly of nanoparticles was measured using dynamic
light scattering (DLS) using a DynaPro-LSR Plate Reader (Wyatt Technology, Santa
Barbara, CA). Light scattering data were collected at regular temperature intervals (1
° C) as solutions were heated from 5 to 50 ° C. The r esults were analyzed using a
Rayleigh sphere model and fitted into a cumulant algorithm based on the sum-of-
43

squares value. The critical micelle temperature (CMT) was defined as the lowest
temperature at which the R
h
is significantly greater than the average monomer R
h
.
2.2.4. TEM imaging of LSI nanoparticles
The TEM imaging was carried out on a FEI Tecnai 12 TWIN microscope (Hillsboro, OR)
at 100 kV. Briefly, a 100 μM solution (5 μL) was initially deposited on a copper grid with
carbon film (CF400-Cu, Election Microscopy Sciences, Hatfield, PA). After removing the
excess amount of solution with filter paper, the samples were negatively stained with
2% uranyl acetate, followed by removing excess uranyl acetate after 30 seconds. The
samples were then dried under room temperature for at least 3 hours before used for
imaging.
2.2.5. SV40-immortalized human corneal epithelial cell (HCE-T) culture
SV40-immortalized HCE-T cells (Riken Cell Bank, Japan) were grown in keratinocyte-
SFM media (KSFM, Life Technologies, Rockville, MD) containing bovine pituitary
extract (BPE, 50 μg/ml) and epidermal growth factor (EGF, 5 ng/ml). Cell passages 4-6
were used for Ca
2+
imaging, scratch and uptake assays in 35-mm coverslip-bottomed
dishes. To optimize responsiveness upon stimuli, cells were starved with EGF and BPE
free medium for 24 h before experimentation.
2.2.6. Ca
2+
imaging
HCE-Ts were rinsed twice with Ca
2+
and Mg
2+
free phosphate buffer saline (PBS) and
incubated at 37 ° C for 20 min in fresh KSFM medium containing 2.5 μM calcium probe
Fluo-4 AM (Invitrogen Life technologies, NY). The cells were then rinsed twice with
NaCl Ringer buffer (145 mM NaCl, 5 mM KCl, 1 mM CaCl
2
, 1 mM KH
2
PO
4
, 1 mM
MgCl
2
, 10 mM glucose, and 10 mM HEPES, osmolarity 300, pH 7.4) and kept in the
44

same buffer at room temperature for 30 min. For Ca
2+
free medium, 1 mM Ca
2+
was
replaced with 0.5 mM EGTA. The cells were illuminated at 488 nm, and their emission
was monitored every 3.15 seconds at 510 nm using Zeiss LSM 510 Meta confocal
microscope system. The field of interest contained 24 to 45 cells, and the fluorescent
intensity change was calculated for each region with image-analysis software. Ca
2+

dynamics were evaluated using the changes in fluorescence intensity of Fluo-4AM. The
data are presented as percentage change in fluorescence intensity at each time point
(F
t
) to the first time point (F
0
) reading: (F
t
-F
0
)/F
0
×100%.
2.2.7. Scratch assay
For scratch assay, confluent HCE-T monolayer was scraped in a straight line to create a
scratch wound with a p200 pipet tip (Liang et al., 2007). Cells were rinsed with KSFM
medium without BPE or EGF to remove debris and then incubated with fresh KSFM
medium containing BPE (50 μg/ml) and EGF (5 ng/ml), LS96, LSI or SI of different
concentrations. Images of wound at the beginning and after 24 h treatment were
captured using Zeiss LSM 510 Meta confocal microscope system.
2.2.8. Exogenous cell uptake assay
SI and LSI nanoparticles were conjugated with NHS-Rhodamine (Thermo Fisher
Scientific Inc, Rockford, IL) via covalent modification of the amino terminus. Conjugation
was performed in 100 mM borate buffer (pH 8.0) for 2 h (LSI) or overnight (SI) at 4 ° C
followed by desalting on a PD10 column (GE Healthcare, Piscataway, NJ) to remove
free dye. Briefly, after the cells were rinsed with fresh medium without BPE and EGF, 10
μM rhodamine labeled proteins were added into the dish. After incubation at 37 ° C for
45

different time points, the cells were rinsed and images were acquired using Zeiss LSM
510 Meta confocal microscope system.
2.2.9. Murine corneal abrasion and recovery study
Briefly, 12-week female NOD mice were anesthetized with an i.p. injection of
xylaxine/ketamine (60-70 mg+5 mg/kg) and placed on a heating pad. After cleaning the
ocular surface with eye wash (OCuSOFT, INC., TX), the corneal epithelium of the right
eye was removed down to the basement membrane using an algerbrush II (The Alger
Company, Inc., TX); the left eye was left intact as a contra lateral control. Mice were
allowed to heal for 24 h with 2 doses (5 μl) of 100 μM LSI, SI or no treatment at 12 h
intervals. After staining the ocular surface with 3 μl 0.6 mg/ml fluorescein (Akorn, IL),
images of the abrasion wound were captured using a Moticam 2300 camera after 12
and 24 h.
2.2.10. Statistics
All experiments were replicated at least three times. HCE-Ts scratch wound healing
was analyzed using one-way ANOVA followed by Tukey’s post-hoc test. HCE-T uptake
was analyzed using two-way ANOVA followed by Bonferroni post-test and murine
corneal epithelium recovery from abrasion wound were analyzed using Kruskal-Wallis
non-parametric ANOVA. Corneal wound healing comparison between LSI and LS96
after 12 h treatment was analyzed using Mann-Whitney U test. A P value less than 0.05
was considered statistically significant.
46

Table 3. Nomenclature, amino acid sequence, and physicochemical property of
LSI and LS96
Protein
Label
*Amino Acid Sequence
**Expected
M.W.(kDa)
***Observed
M.W. (kDa)
T
t1
(° C)
T
t2
(° C)
SI G(VPGSG)
48
(VPGIG)
48
Y 39.65 39.54 32.3 73.1
LSI
GEDASSDSTGADPAQEAGTSKP
NEEISGPAEPASPPETTTTAQET
SAAAVQGTAKVTSSRQELNPLK
SIVEKSILLTEQALAKAGKGMHG
GVPGGKQFIENGSEFAQKLLKK
FSLLKPWAGLVPRGSG(VPGSG)
48
(VPGIG)
48
Y
52.61 52.21 18.7

LS96
GEDASSDSTGADPAQEAGTSKP
NEEISGPAEPASPPETTTTAQET
SAAAVQGTAKVTSSRQELNPLK
SIVEKSILLTEQALAKAGKGMHG
GVPGGKQFIENGSEFAQKLLKK
FSLLKPWAGLVPRGSG(VPGSG)
96
Y
51.36 51.15 -

*After the start codon, a glycine spacer was added during cloning which is not present
on human lacritin
**Expected M.W.(kDa) was calculated by DNAStar Lasergene Editseq
***Observed M.W. (kDa) was measured by MALDI-TOF
****T
t
(° C) was defined at the point of the maximum first derivative of 25 μM protein
solution turbidity change at 350 nm in Phosphate Buffer Saline (PBS)

47

Figure 12. Construction and thermal behavior of LSI fusion proteins. (A) Cartoon
showing LSI nanoparticles with multivalent lacritin presenting at the corona above CMT.
(B) SDS-PAGE showing purified LSI and SI using inverse phase transition cycling (ITC).
(C) Representative phase transition curve of LSI and SI at 25 μM, which revealed a
single phase transition for LSI and two-stages of assembly for unmodified SI. (D)
Concentration-temperature phase diagram for LSI and SI. Dashed lines indicate the fit
of T
t
to the following equation T
t
=mlog[ELP]+b where [ELP] is the concentration, m is
the slope, and b is the transition temperature at 1 μM. b = 19.69 ° C, m = -0.64 ° C (Log
10[μM])
-1
for LSI. As of SI, for T
t1
, b = 65.06 ° C, m = -22.56 ° C (Log 10[ μM])
-1
. b = 77.98
° C, m = -3.72 ° C (Log 10[ μM])
-1
for bulk phase transition T
t2
.
48

2.3. Results and discussion
2.3.1. LSI forms thermo-responsive nanoparticles
LSI and SI (Table 3) were cloned into pET25(+) vector and purified using inverse phase
transition cycling (ITC) as previously reported (Fig. 12B). After confirmed the purity and
molecular weight of the proteins, we characterized the phase transition behavior of LSI
and SI based on optical density measurements (Fig. 12C,D). While monomeric ELPs
undergo single soluble monomers to coacervate phase transition when heated above
their T
t
, diblock ELP micelles exhibit two-step thermo-response: soluble monomer to
mono-dispersed micelles above T
t1
and micelle to coacervate above T
t2
. T
t1
is thus
defined as the critical micelle temperature (CMT) when stable mono-dispersed micelles
exist. T
t2
, the bulk phase transition temperature, represents the temperature at which
micelles further aggregate into coacervates. In contrast to its SI scaffold, which follows
this two-step phase separation with a CMT of 32.3 °C at 25 μM, LSI only showed one
phase transition at 18.4 ° C for 25 μM solution. Mor eover, LSI illustrated less
concentration dependent phase separation compared to the SI scaffold, as
demonstrated by a decreased slope when T
t
was fitted into the equation:
T
t
=mlog[ELP]+b, where [ELP] is the concentration, m is the slope, and b is the transition
temperature at 1 μM (Fig. 12D). To confirm the T
t
of LSI is the CMT instead of bulk
phase transition, we utilized dynamic light scattering (DLS) to analyze the temperature
dependent particle assembly process of the two constructs (Fig. 13A). Surprisingly, LSI
preassembled into 30-40 nm particles and formed 130-140nm nanoparticles when the
temperature was raised above its CMT. Consistent with our previous observation, SI
remained as 20-30nm micelles at physiologically relevant temperatures (Fig. 13A).
49

Figure 13. Nanoparticle characterization of LSI and SI. (A) Dynamic Light Scattering
(DLS) result showing SI existed as soluble monomers and aggregated into stable
monodispersed micelles with an Rh of 20-30 nm above its CMT (26.6 ° C). LSI
preassembled into 30-40 nm nanoparticles below T
t
and further reconstituted into 130-
140 nm nanoparticles above T
t
. (B&C) TEM images of SI and LSI nanoparticles, with
average diameter of 36.5±5.8 nm and 67.1±11.5 nm accordingly. Scale bar: 100nm.
50

Multivalent presentation of payload at the corona of self-assembling polymer
nanoparticles is important for downstream drug delivery efficiency. To further test
whether LSI was capable of forming nanoparticles with lacritin present at the corona
and not in the core, we observed its morphology at room temperature under
transmission electron microscopy (TEM). Consistent with DLS data, while SI formed a
mono-dispersed micelle structure with an average diameter of 36.5±5.8nm (Fig. 13B),
LSI formed larger nanoparticles that exhibit average diameters of 67.1±11.5nm (Fig.
13C). Interestingly, due to the fast degradation of lacritin (Fig. 4), there is a higher
variance in the observed nanoparticle size distribution of LSI compared to SI under
TEM, which suggested partially cleaved lacritin from the corona during sample
preparation.
2.3.2. LSI nanoparticles exhibit mitogenic activity using SV-40 transduced human
corneal epithelial cells (HCE-T)
Upon injury, one of the earliest reactions of many epithelial cells is a transient Ca
2+

wave spreading across the monolayer cell sheet (Berridge and Dupont, 1994). The Ca
2+

wave triggers downstream signaling pathways responsible for cell migration,
proliferation and other events associated with wound repair (Churchill et al., 1996; Sung
et al., 2003). Sanghi et al. first reported lacritin’s ability to stimulate Ca
2+
wave
propagation throughout HCE-Ts while initially exploring its efficacy on the ocular surface
(Sanghi et al., 2001b). Further studies have confirmed that this Ca
2+
signal leads to
lacritin’s protection of HCE cells stressed with benzalkonium chloride (BAK) (Feng et al.,
2014) and maintenance of cultured corneal epithelia homeostasis (Wang et al., 2013c).
To confirm whether LSI has inherited mitogenic activity from parent lacritin, we started
51

the efficacy testing with calcium transients and scratch wound healing assays based on
the reported HCE-T model (Arakisasaki et al., 1995). Whereas the signal triggered by SI
was negligible, the addition of LSI nanoparticles resulted in rapid calcium influx into the
cells (Fig. 14). Moreover, HCE-T cells appeared to have “memory” for exogenous LSI
treatment, as treating the same group of cells for the second time with the same amount
of proteins resulted in higher Ca
2+
influx (Fig. 14A). A similar Ca
2+
wave pattern was
observed in EGF positive control groups. To visualize the in vitro wound repair effect of
LSI, we applied a scratch wound in confluent HCE-Ts and captured the time-lapse
healing process (Fig. 15). As shown in the figure, after 24h treatment, LSI enriched
medium significantly accelerated scratch wound healing compared to plain medium
without growth-factors and SI control.
52

Figure 14. LSI nanoparticles stimulate Ca
2+
wave propagation in HCE-Ts. 40 μM
LSI nanoparticles (×2) triggered 3-6 fold intracellular Ca
2+
increase in HCE-T cells (A)
while same concentration of SI did not exhibit significant effect (B). Scale bar: 20 μm.
Fluorescence intensity change of Fluo-4AM in ten individual cells were chosen for
analysis purpose. The data are presented as percentage change in fluorescence
intensity at each time point (F
t
) to the first time point (F
0
) reading: (F
t
-F
0
)/F
0
×100%.

53

Figure 15. Lacrt heals scratch wound in HCE-Ts. Representative scratch assay
figures showing lacrt exhibited wound healing effect similar to EGF (5 ng/ml) and BPE
(50 μg/ml) co-treatment while plain medium did not heal the scratch wound significantly
after 24 h. Scale bar: 100 μm.

54

Figure 16. LSI nanoparticles undergo uptake in HCE-Ts. (A) Representative
confocal images showing time dependent uptake of LSI into HCE-Ts while SI control did
not exhibit uptake effect. Red: rhodamine labeled exogenous LSI and SI; Blue: DAPI
staining of nuclei. Scale bar: 10 μm. (B) Quantification of HCE-Ts uptake showing at 60
min, LSI exhibited significant higher cell uptake than SI (p<0.0001). Images were
quantified using ImageJ and analyzed via ANOVA followed by Bonferroni post-hoc
analysis.
55

2.3.3. LSI undergo uptake into HCE-Ts
Encouraged by LSI’s in vitro mitogenic activity, we further explored whether exogenous
LSI can enter the HCE-Ts. The cells were thus incubated with NHS-rhodamine labeled
LSI and SI nanoparticles for different time points and representative images were
shown in Fig. 16. Surprisingly, LSI undergoes cell uptake into HCE-Ts in a time
dependent manner (Fig. 16A). Significant cell entry was observed 10 min following
incubation, and after 1 h, LSI nanoparticles accumulated more within the peri-nucleus
sites of the cells, which exhibited significant higher cytosolic fluorescence than SI group
(p<0.0001, Fig. 16B). Nanomaterials of different sizes, shapes, and charges have been
widely used in biomedical imaging, tissue targeting, and cell uptake (Jiang et al., 2008;
Verma and Stellacci, 2010; Weissleder et al., 2005). More recently, using nanoparticle
based multivalent ligands to crosslink membrane receptors more efficiently and to
precisely regulate downstream signaling processes has attracted enormous attention,
especially in antibody mediated receptor crosslinking (Barua et al., 2013; Dubois et al.,
1992). Herein, we first demonstrated the uptake of exogenous LSI nanoparticles into
HCE-Ts in a controlled manner. Modulating the delivery of Lacritin-ELP and other
protein/peptide biopharmaceutical-ELP nanoparticles into the cells by engineering their
size and shape is of great interest in our future studies.
2.3.4. LSI nanoparticles heal abrasion wounds on non-obese diabetic (NOD) mice
ocular surface
The in vivo mitogenic activity of lacritin has not been previously reported. As such, our
in vitro results inspired us to apply LSI nanoparticles to the ocular surface and
investigate their in vivo efficacy via the easiest and most well-tolerated delivery
56

approach: eye drop. In this study, we utilized the corneal epithelial abrasion model on
female NOD mice to assess the wound-healing effect and in vivo uptake of LSI
nanoparticles. Non-obese diabetic (NOD) mice are frequently used as an animal model
for impaired wound healing in humans. Darby et al. have previously compared the skin
wound healing process in non-obese diabetic (NOD) mice and C57BL/6 mice. Reduced
cell proliferation, retarded onset of the myofibroblast phenotype, reduced procollagen I
mRNA expression and aberrant control of apoptotic cell death were observed in NOD
group (Darby et al., 1997). Rich et al. have used NOD mice as an impaired wound
healing model for studying the pathogenesis of Staphylococcus aureus infection (Rich
and Lee, 2005). Moreover, Lee et al. have implanted alginate hydrogels loaded with
model drug VEGF into NOD mice after femoral artery ligation to increase collateral
circulation under cyclic mechanical stimulation (Lee et al., 2000). Briefly, a circular
abrasion wound with a diameter of around 1.5 mm was created on the right eye of the
animal with an algerbrush II with 0.5 mm burr without hurting the limbal region.
Immediately after imaging, 5 μl of 100 μM LSI nanoparticles, SI micelles, or control
EGF+BPE were topically administered to the ocular surface, and the treatment was
repeated once 12 h after wound initiation. Images of abrasion wound were captured at
time 0, 12 h and 24h after fluorescein staining under cobalt blue light with internal ruler
scale (Fig. 17A). Wound area, average fluorescein intensity/area, total fluorescein
intensity, percentage of wound area, percentage of average fluorescein intensity/area,
and percentage of total fluorescein intensity were compared between groups at both 12
h and 24 h and analyzed using Kruskal-Wallis non-parametric ANOVA (Fig. 17B).
Notably, LSI at both 12 and 24 hours exhibited significant decreased percentage of
57

initial wound area (PctArea) compared to SI, EGF+BPE, and no treatment groups
(p=0.001), suggesting promising accelerated recovery efficacy. To corroborate the
fluorescein imaging result, we further processed the corneal epithelium after 24 h for
histology analysis using H&E staining (Fig. 17C). Remarkably, the LSI treatment group
showed complete corneal epithelium recovery with a smooth, reconstituted surface. The
untreated group did not exhibit complete new epithelium coverage over the wound area
due to keratocyte apoptosis (black arrow). Interestingly, although fluorescein test
revealed partial wound closure in the SI group, the new corneal epithelium did not
complete differentiation as illustrated by a rough ocular surface (black arrow). To
address the significance of multivalent presentation of lacritin payload, we further
compared the wound healing efficacy of LSI nanoparticles and macromolecule LS96 at
12 h (Fig. 18). Interestingly, LSI healed the abrasion wound faster than the non thermo-
responsive monomeric LS96 (p=0.028).
58

Figure 17. LSI nanoparticles heal abrasion wounds on the ocular surface of
female NOD mice. (A) Representative fluorescein staining images showing time-lapse
healing of abrasion wound on NOD mice ocular surface. (B) Analysis of abrasion wound
area change using Kruskal-Wallis non-parametric ANOVA showing LSI at both 12 and
24 h exhibited significant decreased percentage of initial wound area (PctArea)
compared to SI, EGF+BPE, and no treatment groups (p=0.001, n=4). *PctArea was
evaluated by a blind reviewer for objective justification of wound healing efficacy. (C)
H&E staining showing 24 h after wound initiation, corneal epithelium of LSI treating
group completely healed with no observed inflammation. Although reduced fluorescein
staining was observed in SI group, corneal epithelium did not recover fully with a
smooth monolayer surface (black arrow). EP: epithelium; BM: Bowman’s membrane;
ST: Stroma; DM: Descenet’s membrane; EN: Endothelium.
59

Figure 18. LSI nanoparticles exhibited faster wound healing efficacy compared to
macromolecule LS96. (A) Representative fluorescein staining images showing healing
of abrasion wound on NOD mice ocular surface after 12 h. (B) Analysis of abrasion
wound area change using Mann-Whitney U test showing LSI at 12 exhibited significant
decreased percentage of initial wound area (PctArea) compared to LS96 (p=0.028,
n=8). *PctArea was evaluated by a blind reviewer for objective justification of wound
healing efficacy.
60

2.4. Conclusions
To accelerate the corneal wound healing process, we developed a multivalent Lacritin-
ELP nanoparticle system as a means of delivering a model biopharmaceutical,
prosecretory mitogen lacritin, to the ocular surface. This fusion construct inherited
thermo-responsive self-assembly properties from the SI micelle scaffold and presented
payload lacritin at the corona at physiological relevant temperatures. LSI nanoparticles
triggered calcium wave propagation across SV40-Adeno vector transformed human
corneal epithelial cells (HCE-Ts) and healed scratch wounds in a dose dependent
manner using the same model. When topically applied on the ocular surface of female
NOD mice following abrasion, LSI nanoparticles promoted faster wound healing
compared to the SI and no treatment groups. Exogenous LSI underwent uptake into
HCE-Ts as confirmed by fluorescence imaging. Overall, this study highlights the
potential of ELPs as nanoparticle scaffolds to effectively deliver protein therapeutics to
the ocular surface and repair abrasion wounds. The application can be further extended
to other biopharmaceutical candidates. The mechanism underlying the LSI
nanoparticles’ mitogenic activity and their interaction with the corneal extracellular
matrix remains to be elucidated.
61

Chapter 3
Thermally-responsive loading and release of protein polymers from contact
lenses
3.1. Introduction
Despite the truth that millions of patients benefit annually from novel therapeutics in the
ophthalmic market, it is evident that lack of more efficacious, targeted, sustained
delivery of therapeutics to the ocular surface is a bottleneck that must be overcome in
the field (Urtti, 2006a). Accounting for approximately 90% of all ophthalmic medications,
topical ophthalmic solutions (eye drops) have long been the preferred and most widely
used administration route owing to its convenience and patient compliance (Lee and
Robinson, 1986). However, their main drawback is an inefficient pharmacokinetic
profile, featuring a transient overdose which may cause undesirable toxicity, followed by
a short period of therapeutic concentration, and then a prolonged period of insufficient
concentration (Urtti, 2006a). Moreover, bioavailability of eye drops is further lowered by
reflex tearing, blinking or nasolacrimal system drainage so that only 1 to 7% of an eye
drop is absorbed by the eye (Lang, 1995b). Thus multiple aspects of an improved ocular
drug delivery system have been addressed, including strategies such as ophthalmic
ointments, viscous polymer vehicles, nanoparticles, in situ gel-forming systems,
iontophoresis, and punctal plug (Gaudana et al., 2010; Gaudana et al., 2009; Novack,
2009b), etc. Yet, these systems are still not widely used because they are often not
optically clear, difficult and uncomfortable to self-insert or inherent instability (Sintzel et
al., 1996). Beyond their traditional role as vision correction and cosmetic aid, contact
lenses are emerging as an alternative ophthalmic drug delivery system to
62

therapeutically manage ocular anterior segment disorders (Guzman-Aranguez et al.,
2013a). From the industrial standpoint, it would be ideal for the contact lens drug
delivery device to be relatively simple in design; to not require complicated and
expensive manufacturing processes; to not significantly impair or interfere with the
patient's vision; and to not require a substantial change in the practice patterns of eye
physicians and surgeons (Ali and Lehmussaari, 2006). Reported efforts tend to mainly
focus on drug soaking (Kim and Chauhan, 2008; Li and Chauhan, 2007), drug-loaded
colloidal nanoparticles inclusion (Gulsen and Chauhan, 2005; Kapoor et al., 2009) and
molecular imprinting (Alvarez-Lorenzo et al., 2006). Although these approaches are
effective at extending the drug release duration time from contact lenses, most studies
only focused on delivery of small molecule therapeutics, such as cyclosporine A (Peng
and Chauhan, 2011a) and timolol (Hiratani and Alvarez-Lorenzo, 2004b; Jung et al.,
2013), rather than making extra effort to deliver protein therapeutics (Frokjaer and
Otzen, 2005; Leader et al., 2008b), which is more challenging due to steric hindrance
and complex template design. Furthermore, no previous studies have successfully
exerted spatiotemporal control over the delivery of therapeutic agent to desired site of
action while reducing side effects.
As development of growth factors and other intrinsic macromolecules in local ocular
microenvironment into novel therapies is garnering increased attention (Duncan, 2003),
it is worthwhile to search for novel drug delivery system which can be integrated with
contact lens and exhibit improved or new functionalities (Kearney and Mooney, 2013): i)
encapsulate small molecule therapeutics; ii) seamlessly fuse with protein
pharmaceuticals; iii) extend drug retention time on contact lens; iv) target deliver
63

therapeutics at required disease area on the ocular surface without interfering with
healthy region. One type of Elastin-based systems, Elastin-like polypeptides (ELPs),
has been of special interest in this regard (Hubbell and Chilkoti, 2012b). ELPs are
composed of repeated pentameric peptides, (Val-Pro-Gly-Xaa-Gly)n. They reversibly
phase separate from aqueous solution above a temperature (T
t
) which can be tuned by
adjusting the identity of X and the length n (MacEwan and Chilkoti, 2010). Fusion ELP
proteins or fluorescent labeled ELPs inherit the unique inverse phase transition behavior
(MacEwan and Chilkoti, 2010) and can thus be used as pharmacologically relevant and
biocompatible novel drug delivery vehicle (Chilkoti et al., 2006b) or imaging probe
(Janib et al., 2010). We have previously demonstrated the encapsulation (Shah et al.,
2013c) and fusion ability of thermo-responsive Elastin-like polypeptides (ELPs)
regarding delivering both small molecules and protein therapeutic treatments to the eye.
Herein, we reported the surprising discovery of ELPs’ thermally-reversible,
spatiotemporal and sustained attachment to Proclear Compatibles
TM
contact lens as an
elastic bridge. Moreover, we illustrated that attachment and release of ELPs to/from
Proclear contact lens was a T
t
and temperature dependent process using rhodamine as
a detection probe. Two types of ELPs, V96 (T
t
: ~30 ° C) and S96 ( T
t
: ~55 ° C) were
involved in the study. When attaching the lens with V96 at 37° C, around 80% of
fluorecence remained on the lens after one week incubation in PBS solution at 37 ° C,
while the plateau of fluorescence retention dropped down to below 10 % when releasing
at 4 ° C. Lenses modified with S96 did not exhibit s ignificant total fluorescence or
release profile differences at either 37 ° C or 4 ° C . Interestingly, lenses modified with
V96 at 4 ° C exhibited similar release pattern at 4 ° C compared to S96 group, both of
64

which can be described using a single two-phase decay model. As a proof of concept
for targeted protein therapeutic delivery, we modified the lens with prosecretory
mitogenic fusion protein Lac-V96 and demonstrated spatial cell uptake via contact lens
using human corneal epithelial cell model (HCE-Ts).
3.2. Materials and methods
3.2.1. Decoration of contact lenses with rhodamine labeled ELPs
Briefly, ELPs were covalently modified with NHS-Rhodamine (Thermo Fisher Scientific
Inc, Rockford, IL) via the primary amino terminus. The conjugation was performed in
100 mM borate buffer (pH 8.5) overnight at 4 ° C. E xcess fluorophore was removed
using a desalting PD-10 column (GE Healthcare, Piscataway, NJ) and overnight dialysis
against PBS at 4 ° C. Contact lenses were either inc ubated with 50μM labeled ELPs
overnight at 37° C in a 24-well plate or spot decora ted with concentrated labeled ELPs
using a 20 μl pipette at 37 ° C before being transfe rred to PBS solution.
3.2.2. ELPs inverse phase transition characterization
The temperature-concentration phase diagrams for rhodamine labeled ELPs/ELP fusion
proteins were characterized by optical density observation using a DU800 UV-Vis
spectrophotometer at 350 nm as a function of solution temperature. Typically, ELPs (5 –
100 μM) were heated at 1 ° C/min from 10 to 85 ° C and sam pled every 0.3 ° C. T
t
was
defined at the point of the maximum first derivative.
3.2.3. Release of fluorescent ELPs from contact lenses
ELP modified contact lens were gently rinsed with PBS and placed in 4 ml of PBS at
37° C or 4° C for 1 week. Samples of the solution (10 0 μl) were withdrawn at regular
intervals and kept at -20° C. After one week, lenses were thoroughly washed in PBS at 4
65

° C for 24 hours to detach ELPs. Rhodamine intensity of collected samples was
measured spectrophotometrically (Ex: 525nm, Em: 575nm) using Synergy™ H1m
Monochromator-Based Multi-Mode Microplate Reader (BioTek) and analyzed using
Gen5 2.01 Data Analysis Software (BioTek). Total fluorescence on the lens was
calculated using Equation 1. Retention rate was calculated using Equation 2. Raw data
were then fitted into either a one phase decay model (Equation 3) or two phase decay
mode (Equation 4) using SPSS. Goodness of fit and predicted values were collected.
+,-./ 0
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19:95;9_=3>5:
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AB-BC-D,C %
=3>5: E
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#∑ E
FNPNJQN_R
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RST
=3>5: E
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( 100% (2)
AB-BC-D,C % A
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(4)
3.2.4. Spatiotemporal HCE-T cell uptake
HCE-T cell uptake study was conducted on 35mm glass coverslip-bottomed dishes.
Briefly, HCE-T cells were grown to 70-80 % confluence and gently rinsed with warm
fresh medium before changed to fresh medium containing either rhodamine labeled
lacritin, Lac-V96 or Proclear Compatible
TM
contact lens modified with rhodamine labeled
Lac-V96. After incubation at 37 ° C for 1 hour, the cells were rinsed with fresh medium
and images were immediately acquired using ZEISS 510 confocal microscope system.
For uptake quantification comparison, images were analyzed using ImageJ.
3.2.5. Statistical Analysis
Data presented are representative curves or mean ± S.D. All experiments were
repeated at least three times. Statistical analysis was performed by Student t-test or
one-way ANOVA by SPSS. Differences between treatments were established with
66

Tukey’s post-hoc test. A p value of less than 0.05 was considered statistically
significant.
3.3. Results and discussion
3.3.1. Discovery of ELPs specific attachment to Proclear Compatible
TM
contact
lens
Surprising discovery of ELPs’ attachment to Proclear Compatible
TM
contact lens came
from a quick screen of four types of market contact lenses, including Acuvue Oasys
®
,
Acuvue Advance Plus
®
, Dailies AquaComfort Plus
TM
and Proclear Compatibles
TM
.
Unexpectedly, rhodamine labeled V96 selectively attached to Proclear Compatibles
TM

contact lens at 37 ° C after overnight incubation in PBS solution (Fig. 19a) and the
attachment was stable at 37 ° C in PBS solution for more than 24 hours. Motivated by
the rationale that the delivery system itself should not interfere with normal vision, we
further investigated whether it was possible to spatially decorate the lens with ELPs.
Interestingly, we were able to modify the lens with various shapes of ELPs according to
the need, such as ring, dots, etc (Fig. 19b).
67

Figure 19. ELP selectively phase separate onto Proclear compatibles
TM
contact
lenses. a) Among four types of contact lens tested, rhodamine labeled V96 selectively
phase separated onto Proclear compatibles
TM
contact lens. 1: Proclear compatibles
TM
;
2: Dailies (AquaComfort Plus); 3: Acuvue OASYS; 4: Acuvue Advance Plus. b) Different
shapes of rhodamine labeled V96 on Proclear compatibles
TM
contact lens. Upper: white
light; lower: fluorescence.

68

3.3.2. T
t
and temperature dependent attachment of ELPs to the lens
Even though the mechanism underlying ELPs’ attachment to Proclear Compatibles
contact lens remains to be explored, we proposed the attachment was partially T
t
and
temperature dependent. In the pilot screen experiment, we used V96, which phase
separated around 30 ° C. To further test the T
t
and temperature dependence of ELPs’
affinity to Proclear Compatible
TM
contact lens, we chose two types of representative
ELPs: V96 (T
t
at around 30 ° C) and S96 ( T
t
at around 55 ° C) for a five group
comparison study: i) Group one: lens incubated with V96 at 37 ° C and release at 37 ° C
(closed circle); ii) Group two: lenses incubated with S96 at 37 ° C and release at 37 ° C
(closed square); iii) Group three: lenses incubated with V96 at 4 ° C and release at 4 ° C
(open circle); iv) Group four: lenses incubated with S96 at 4 ° C and release at 4 ° C
(open square); v) Group five: lenses incubated with V96 at 37 ° C and release at 4 ° C
(half closed circle). After 24 h incubation, total attachment of V96 at 37 ° C (Group one)
was about six fold of S96’s attachment at 37 ° C (Gr oup two) and sixty-nine fold of V96’s
attachment at 4 ° C (Group three) (Figure 2a&b). Int erestingly, S96 incubated at 37 ° C
(Group two), V96 incubated at 4 ° C (Group three) an d S96 incubated at 4 ° C (Group
four) did not exhibit significant different contact lens attachment affinity (p>0.50) (Figure
20a&b). After one week release in PBS, only Group one exhibited around 80% of
fluorescence retention on the lens while all the other groups released most of the
attached ELPs (Figure 20c-e). Total fluorescence intensity provided the first clue of the
association between contact lens affinity and T
t
. To thoroughly compare fluorescence
release kinetics of all five groups, we fitted the data using both one phase decay and
two phase decay models by SPSS (Table 4). Both Group one and Group five data can
69

be described using a one phase decay model, with R
2
of 0.916 and 0.953 accordingly;
while the other three groups did not fit the one phase decay model very well (R
2
=0.646).
Interestingly, release kinetics of Group two, Group three and Group four can be
described using the same two phase decay model (R
2
=0.847). The modeling result
highly supported our hypothesis about the link between ELPs’ attachment to Proclear
Compatible
TM
contact lens and T
t
/temperature. Most significant different release profile
comes from Group one, which exhibited a predicted plateau of more than 75 %
retention after one week’s incubation at 37 ° C. Ret ention of V96 (Group five) on the lens
was significant lowered when the incubation temperature was changed to 4 ° C, with a
predicted plateau of less than 10 % using either model and a longer half-life of release
(Table 4). The link between lens affinity and T
t
was further corroborated by Group two,
three and four. As when both incubation and release temperatures were below ELPs’ T
t
,
no significant difference was noticed in either total binding fluorescence intensity (Figure
20b) or release kinetic profiles (Figure 20d).

70

Table 4. Modeling of ELP modified contact lens release kinetics

Group 1 Group 2
*
Group 3
*
Group 4
*
Group 5
ELP Type V96 S96 V96 S96 V96
Label Temp (° C) 37 37 4 4 37
Release Temp (° C) 37 37 4 4 4

Model
*

(one phase decay)
R=(R
0
-Plateau)*exp(-k*t)+Plateau
Predicted R
0
(%) 100.05±1.72 74.32±4.78 85.59±4.10
Predicted Plateau (%) 82.22±0.58 29.73±3.40 9.28±3.96
k (h
-1
) 2.88±0.65 0.17±0.07 0.070±0.02
t
1/2
(h) 0.24 3.98 10.05
R
2
0.92 0.65 0.95

Model
*

(two phase decay)
R=Plateau+Span
fast
*exp(-k
fast
*t)+Span
slow
*exp(-k
slow
*t)
Predicted Plateau (%) 75.00±36.26 0.00±27.79 6.44±2.67
Predicted Span
fast
(%) 16.56±1.57 40.50±6.21 64.42±4.21
k
fast
(h
-1
) 3.36±0.74 3.30±1.18 0.04±0.01
t
1/2fast
(h) 0.21 0.21 17.33
Predicted Span
slow
(%) 8.53±35.89 54.23±26.59 30.40±5.48
k
slow
(h
-1
) 0.00±0.02 0.01±0.01 1.94±0.80
t
1/2slow
(h) 231.05 86.64 0.36
R
2
0.96 0.85 0.99
*
SPSS analysis of release kinetics for Group two, Group three and Group four illustrated
the same predicted parameters using both one phase decay and two phase decay
models.
71

Figure 20. T
t
and temperature dependent affinity of ELPs towards Proclear
Compatible
TM
contact lens. a) Representative picture showing different affinity of V96
and S96 to the lens at 37° C and 4° C after 24 h incu bation. b) Total fluorescence
intensity quantification result showing ELPs’ attachment to the lens was T
t
and
temperature dependent. c) Group one (V96, labeled at 37 ° C and release at 3 7 ° C)
exhibited high retention on the lens after one week incubation. d) Group two (S96,
labeled at 37 ° C and release at 37 ° C), three (V96, labeled at 4 ° C and release at 4 ° C)
and four (S96, labeled at 4 ° C and release at 4 ° C) showed similar release pattern and
can fit into same two-phase decay model. e) Group five (V96, labeled at 37 ° C and
release at 4 ° C) illustrated different release kine tics from group one, with significant
lower plateau. ***p<0.001; grey line: predicted values using one phase decay model;
grey dash line: predicted values using two phase decay model.
72

3.3.3. Contact lenses decorated with a ring of LV96 mediated spatiotemporal HCE-
T cell uptake
To explore the targeted delivery potential of ELP-contact lens system, we chose one of
the potential protein therapies for ocular disease, lacritin, which has shown prosecretory
mitogenic activities as dry eye disease and cornea wound healing treatment (McKown
et al., 2009b). We have previously proved that Lac-ELP fusion proteins imparted similar
prosecretory/mitogenic function of lacritin and thermo responsiveness of ELPs.
Moreover, by fused to different ELP tags, uptake level and speed of exogenous Lac-
ELPs into HCE-Ts could be modulated (Figure 21a-c). The spatiotemporal controlled
HCE-T cell uptake effect was enhanced when we ring decorated the lens with
rhodamine labeled Lac-V96 (Figure 21d). Three representative regions underneath the
lens were chosen to compare cell uptake level and distribution (Figure 3e-g). As
illustrated in figures, Lac-V96 ring decorated contact lens successfully executed its
targeted delivery task. Region 1 (Figure 21e) was fully covered by the lens, exhibiting
evenly distributed highest cell uptake level. Region 2 (Figure 21f) was partially covered
by the lens and thus only showed one section of cell uptake. Region 3 (Figure 21g) was
outside of the Lac-V96 ring area, which illustrated the lowest cell uptake level.

73

Figure 21. Spatiotemporal HCE-T cell uptake. a) Representative pictures showing
time dependent uptake of Lac and Lac-V96 into HCE-T cells. b-c) Quantification result
showing V96 tag modulated cell uptake speed and amount of exogenous Lac. d)
Cartoon showing rho-Lac-V96 “ring” modified contact lens with three representative
regions. 1: rho-Lac-V96 fully covered cell region; 2: cell region half covered by rho-Lac-
V96; 3: cell region not covered by the lens. e-g) Representative pictures showing HCE-
T cell uptake of rho-Lac-V96 in three regions delivered by contact lens. Red: rhodamine;
Blue: DAPI staining of nuclei. Scale bar: 10 μm.
74

3.4. Conclusion
To develop new treatments or delivery mechanisms for ocular diseases and improve the
bioavailability, new drug vehicles are required to be biocompatible, biodegradable,
easily modified with bioactive peptides, small molecules or antibodies and can work in
concert with existing medical devices to provide novel functionality (Langer and Tirrell,
2004). In this communication, we reported the surprising discovery of thermal
responsive ELPs’ selective reversible attachment to Proclear Compatibles
TM
contact
lens; we described the T
t
and temperature dependence of this attachment and we
provided the first proof of concept to spatiotemporally deliver model ocular protein drug
lacritin via contact lens. Different from reported contact lens mediated drug delivery
systems, the ELP modification on contact lens can be T
t
and spatiotemporally
modulated so that delivery is more targeted to the disease site and delivery rate can be
further fine-tuned using external stimuli such as local cooling for on demand dosing
(Jung and Chauhan, 2012; Stuart et al., 2010b). In this study, the monoblock ELP
modified contact lens was decorated with a fluorescently-labeled therapeutic agent for
visual detection of release and in vitro cell uptake. We have demonstrated diblock ELP
copolymers also exhibit similar contact lens affinity. And thus the system’s application
catalogue can be broadened to anti-inflammation agents, antibiotics, polypeptides and
diverse protein/antibody therapeutic libraries via encapsulation or recombinant protein
expression strategies. Although the first generation of ELP-contact lens delivery system
has not met the standard of highly desirable zero-order release (Ali et al., 2007) and its
underlying mechanism remains to be elucidated, our discovery may provide a promising
75

new avenue to circumvent challenges associated with effective delivery therapeutics to
the ocular surface.
76

Chapter 4
Protein polymer nanoparticles engineered as chaperones protect against
apoptosis in human retinal pigment epithelial cells
4.1. Introduction
Small heat-shock proteins (sHSPs) maintain protein homeostasis by binding substrate
proteins in non-native conformations and preventing irreversible aggregation (Haslbeck
et al., 2005). Partly mediated by oligomerization, they shift between an inactive low-
affinity state and active high-affinity oligomers. Stress, such as heat shock, shifts their
equilibrium towards formation of active oligomers. By forming intermediate complexes
with nonnative protein conformations, these active oligomers guard against protein
denaturation (Haslbeck et al., 2005). One member of the sHSPs family, αB-crystallin
has attracted attention due to its roles in neuroprotection, anti-inflammation, and
biophysics of assembly (Arrigo et al., 2007). Ongoing research by our group has
focused on its potential for treating age-related macular degeneration (AMD) via
protection against apoptosis induced by oxidative stress (Sreekumar et al., 2010a). It
has been reported that αB-crystallin expression increases in retinal pigment epithelial
(RPE) cells of patients with both late ‘dry’ and ‘wet’ AMD (De et al., 2007). We reported
that prominent expression of αB-crystallin correlates with the observation of drusen,
which are extracellular subretinal deposits associated with early AMD (Kannan et al.,
2012). In RPE cells, αB-crystallin has significant cytoprotective effects mediated by
suppression of protein aggregation and the proteolytic action of caspase 3 (Sreekumar
et al., 2010a).
77

αB-crystallin is composed of a conserved α-crystallin domain flanked by a variable N-
terminal domain and a C-terminal extension. Similar to other sHSPs, the α-crystallin
domain consists of 6-8 β-strands organized in two β-sheets (Kampinga et al., 2009).
Surprisingly, Bhattacharyya and coworkers reported that residues 73−92 of αB-crystallin
(DRFSVNLDVKHFSPEELKVK) are sufficient to prevent aggregation of denatured
substrate proteins in vitro, similar to the full length protein (Bhattacharyya et al., 2006).
The fact that this ‘mini-peptide’ retains full chaperone activity suggests that it too has
therapeutic potential to rescue RPE cells from oxidative stress. In contrast, an
overlapping (underlined amino acids) fragment of residues 90-100 of αB-crystallin
(KVKVLGDVIEV) forms oligomeric fibrils exhibiting β-sheet–rich structures similar to
other amyloid oligomers (Laganowsky et al., 2012). These oligomers exhibit cytotoxicity
and can be recognized by an oligomer-specific antibody (Laganowsky et al., 2012).
Thus, overlapping short peptides from αB-crystallin appear to have diametrically
opposing effects on cell viability. Although the correlation between mini-αB-crystallin’s
oligomeric flexibility and its cytoprotective/cytotoxic role is less clear, one postulation is
that the peptide’s quaternary dynamics (Stengel et al., 2010) underlie its chaperone
function both in vitro and in the crowded cellular environment. Unfortunately, as a small
peptide, the residence time near the retina following either systemic or intravitreal
administration is expected to be short (Kim and Csaky, 2010; Urtti, 2006a; Vadlapudi
and Mitra, 2013; Zheng et al., 2012). For this reason, we are exploring simple
approaches to engineer the mini-peptide (residues 73-92) onto a high molecular weight
carrier that has the potential to modulate local and systemic residence time, potentiate
binding and internalization, and enhance protection from oxidative stress.
78

An emerging method to bioengineer peptides with potent biological activity is to fuse
them to protein polymers. Protein polymers can provide a platform for controlling
release, multivalency, molecular weight, phase behavior, and even nanoparticle
assembly (Fleige et al., 2012; Kost and Langer, 2001; Stuart et al., 2010a; Yang et al.,
2001). One class of protein polymers known as elastin-like polypeptides (ELPs) are
composed of the repetitive pentapeptide motif (Val-Pro-Gly-Xaa-Gly)
n
(MacEwan and
Chilkoti, 2010).

ELPs have unique reversible inverse phase transition temperatures, T
t
,
below which they solubilize and above which they phase separate. T
t
can be tuned
through selection of guest residue identity (Xaa) and the number of pentameric repeats,
n. ELP fusion proteins are being evaluated for effects due to their hydrodynamic radius,
self-assembly of nanoparticles, or formation of thermo responsive deposits in multiple
disease models (Dreher et al., 2008; Janib et al., 2013; Liu et al., 2010a). Prior to this
report, no studies of protein polymers have explored; i) their fusion with chaperone
proteins; ii) their ability to modify oligomerization of peptide fibrils; or iii) their intracellular
behavior under oxidative stress, all to which are relevant to the treatment of AMD.
Herein, we report the rational bioengineering of the ‘mini-peptide’ from αB-crystallin into
two types of ELP fusion proteins (Table 5, Fig. 22). crySI has been engineered onto a
nanoparticle ELP scaffold called SI (Fig. 23). For comparison, the peptide has also been
fused to a soluble ELP of similar molecular weight called cryS96. Similar to their parent
ELPs, the fusion constructs have temperature-dependent assembly that interrupted the
native oligomerization of cry peptide as confirmed by TEM and DLS. Similar to the free
‘mini-peptide’, the ELP fusion proteins inhibit the aggregation of substrate proteins,
including alcohol dehydrogenase (ADH) and insulin. Furthermore, exogenous cryELPs
79

also protect RPE cells against hydrogen peroxide (H
2
O
2
) induced apoptosis and
undergo nuclear translocation in RPE cells under H
2
O
2
stress. Interestingly, cryS96
exhibited better protection against apoptosis than the free cry peptide at the same molar
concentration.

80

Figure 22. Rational bioengineering of cryELP nanoparticles and macromolecules
to rescue retinal pigment epithelial cells (RPEs) from oxidative stress.
81

4.2. Materials and methods
4.2.1. Materials and reagents
TB DRY
®
Powder Growth Media was purchased from MO BIO Laboratories, Inc.
(Carlsbad, CA). NHS-Rhodamine was purchased from Thermo Fisher Scientific
(Rockford, IL). Alcohol dehydrogenase (ADH) and Insulin were purchased from Sigma-
Aldrich (St. Louis, MO). αB-crystallin mini-chaperone (cry,
DRFSVNLDVKHFSPEELKVK) and a scrambled peptide control (Neg,
DLPLKKNVEDKFHRSFVESV) were synthesized by Neo-peptide (Cambridge, MA).
Anti-LAMP1 antibody was purchased from Abcam and Monodansylcadaverine (MDC)
was obtained from Sigma. The protocol for the preparation and use of cultured human
fetal RPE cells were approved by the University of Southern California Institutional
Review Board under protocol #HS-947005 and adhered to the tenets of the Declaration
of Helsinki. RPE cells were isolated from human fetal eyes (gestational age 16–18
weeks) obtained from Advanced Bioscience Resources, Inc. (ABR, Alameda, CA).
Primary cultures of RPE cells were established as described previously (Sonoda et al.,
2009) and used at passages 3 to 4.
4.2.2. Construction of ELP genes
Genes encoding for ELPs (S96 and SI) were synthesized by recursive directional
ligation in pET25b(+) vector as previous reported (Janib et al., 2013). Sense and
antisense DNA oligonucleotides encoding for the mini-peptide were synthesized with
NdeI (5’) and BamHI (3’) overhangs were synthesized at University of Southern
California DNA core facility. Forward sequence: 5’-
TATGGGTGATAGATTTAGCGTTAACCTGGACGTGAAACATTTCTCCCCAGAAGAAC
82

TGAAAGTGAAGGGTTATTAGACTCCTCG-3’. Reverse sequence: 5’-
ATCCGAGGAGTCTAATAACCCTTCACTTTCAGTTCTTCTGGGGAGAAATGTTTCAC
GTCCAGGTTAACGCTAAATCTATCACCCA-3’. Complementary oligonucleotides were
heated at 95° C for 5 minutes and annealed at room t emperature for 2h. Annealed oligos
were inserted into pET25b(+) vector, digested with NdeI and BamHI. A BseRI
recognition site was also incorporated to facilitate the insertion of ELP genes. Correct
ligation of the fusion protein gene was confirmed by restriction digestion and DNA
sequencing. Sequences were confirmed by DNA sequencing in the core facility of the
Norris Cancer Center of the University of Southern California.
4.2.3. ELP expression and purification
ELPs (Table 5) were expressed in BLR (DE3) E. coli competent cells (Novagen Inc.,
Milwaukee, WI). Cells were inoculated in ampicillin medium and grown for 24 h at 37 ° C.
The bacterial culture was centrifuged, disrupted by probe-tip sonicated in ice cold PBS
and centrifuged to remove insoluble cell debris. ELPs were purified from the cell
supernatant by inverse transition cycling (ITC) (Hassouneh et al., 2010). Purity of ELP
fusion proteins was determined by SDS-PAGE gels stained with coomassie blue.
Protein concentrations were determined by UV-visible spectroscopy of the carboxy
terminal tyrosine at 280 nm (ε=1285M
-1
cm
-1
). Protein molecular weight was further
confirmed by MALDI-TOF analysis.
4.2.4. Transmission Electron Microscopy (TEM) imaging
The TEM imaging was carried out on a FEI Tecnai 12 TWIN microscope (Hillsboro, OR)
at 100 kV. The samples were prepared by using the following protocol: A 100 μM
solution (5 μL) was initially deposited on a copper grid with carbon film (CF400-Cu,
83

Election Microscopy Sciences, Hatfield, PA). Excess amount of the solution was
removed by filter paper. The samples were then negatively stained with 2% uranyl
acetate, and the excess uranyl acetate solution was removed by filter paper after 30
seconds. The samples were dried under room temperature for at least 3 hours before
they were used for imaging.
4.2.5. Characterization of ELP particle formation and phase transition temperature
The temperature-concentration phase diagrams for ELP fusion proteins were
characterized by optical density, and the self-assembly of nanoparticles was confirmed
using dynamic light scattering (DLS). Optical density was recorded using a DU800 UV-
Vis spectrophotometer at 350 nm as a function of solution temperature. Typically, ELPs
(5 – 100 μM) were heated at 1 ° C/min from 10 to 85 ° C and sampled every 0.3 ° C. T
t

was defined at the point of the maximum first derivative. Similarly, hydrodynamic radius
(Rh) was recorded to characterize particle size distribution as a function of temperature.
Briefly, protein samples were prepared at 25 μM in PBS and filtered through a 20 nm
filter at 4 ° C. Autocorrelation functions were coll ected using a DynaPro-LSR Plate
Reader (Wyatt Technology, Santa Barbara, CA). Light scattering data were collected at
regular temperature intervals (1 ° C) as solutions w ere heated from 5 to 60 ° C. The
results were analyzed using a Rayleigh sphere model and fitted into either a
regularization or cumulant algorithm based on the sum-of-squares value. The critical
micelle temperature (CMT) for each protein construct was defined as the lowest
temperature at which the R
h
is significantly greater than the average monomer R
h
.

84

4.2.6. Bis-ANS assay
The hydrophobicity of cryELPs was evaluated using fluorescent probe 1,1*-bi(4
anilino)naphthalenesulfonic acid (bis-ANS) on a HORIBA Jobin Yvon Fluorolog-3
Spectrofluorometer. bis-ANS (Sigma-Aldrich) stock solution was prepared in 95%
alcohol and the concentration was determined by absorbance at 385 nm (^
_`a

bc, efg hi
#b
j
#b
). Samples with 5 μM bis-ANS were excited at 390 nm, and the
emission spectra were then recorded at 400-600 nm. The excitation and emission slits
were set at 5 nm.
4.2.7. Quantification of chaperone activity
Chaperone activity was measured using two protein substrates: ADH and insulin
(Bhattacharyya et al., 2006). The kinetics of aggregation was monitored by optical
density at 360 nm with a Beckman DU800 UV-Vis spectrophotometer equipped with a
temperature-controlled multi-cell transporter. Assays were done in the absence or
presence of ELP fusion peptides. Aggregation of ADH (100 μg /300 μl) was monitored
upon incubation at 48 ° C at a molar ratio of ADH to peptide of 1:250. For insulin
aggregation, 120 μg/300 μl insulin (bovine pancreas) were induced to aggregate by
addition of 80 mM Dithiothreitol (DTT) at 12 ° C at a molar ratio of insulin to peptide of
0.7:1. Heat capacity measurements were performed using a Perkin Elmer Differential
Scanning Calorimeter (DSC) 8500.
4.2.8. NHS-Rhodamine labeling of recombinant ELP fusions
For fluorescent visualization, ELPs were covalently modified with NHS-Rhodamine
(Thermo Fisher Scientific Inc, Rockford, IL) via the primary amino terminus. Briefly, the
conjugation was performed in 100 mM borate buffer (pH 8.5) overnight at 4 ° C. Excess
85

fluorophore was removed using a desalting PD-10 column (GE Healthcare, Piscataway,
NJ) and overnight dialysis against PBS at 4 ° C.
4.2.9. Protection of RPE cells from H
2
O
2
induced cell death
The anti-apoptotic effects of crySI and cryS96 were studied by TUNEL staining and
western blot detection of caspase 3 activation in confluent human RPE cells upon
challenge with H
2
O
2
. Briefly, human RPE cells grown on 4-well chamber slides were
starved overnight in FBS free medium and treated with 200 μM H
2
O
2
for an additional
24 h. For TUNEL staining, cell death was assessed by terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) following the manufacturer’s protocol
(Roche, IN). After treatment RPE cells were washed in PBS, air dried, and fixed with
freshly prepared 4 % paraformaldehyde. Cells were permeabilized with Triton X-100
and incubated with the TUNEL reaction mixture for 1 h in the dark at 37 ° C. Cells were
washed, stained with 4',6-diamidino-2-phenylindole (DAPI) and viewed under a spinning
disc laser scanning confocal microscope (PerkinElmer, MA). TUNEL positive cells were
counted and quantified as described (Sreekumar et al., 2010a). For immunoblot
analysis, protein was extracted from the RPE cells at the end of indicated experiments.
Equal amounts of protein (60 μg) were resolved on 15 % Tris-HCl polyacrylamide gels.
Membranes were probed with rabbit polyclonal anti-cleaved caspase-3 antibody
(1:1000, Cell Signaling, MA). After incubation with the corresponding secondary
antibodies, signals were detected using a chemiluminescence system. GAPDH was
used as a loading control.

86

4.2.10. Intracellular uptake in RPE cells under H
2
O
2
induced stress
Confluent human RPE cells cultured on 4 well chamber slides were co-incubated with
200 μM H
2
O
2
and 5 μM rhodamine labeled ELPs or cry peptide for indicated time
periods. For immunofluorescence, the cells after the experiments were fixed in 4 %
paraformaldehyde for 20 min, followed by permeabilization with 0.1 % triton-X for 15
min. Cells were blocked with 5 % goat serum and incubated with LAMP-1 (1:100) over
night at 4 ° C. After washing the cells were incuba ted with FITC labeled secondary
antibody for 30 min at RT, cells were mounted with DAPI and observed under a LSM
510 confocal microscope (Zeiss).
4.2.11. Statistical analysis
Data presented are representative curves or mean ± S.D. All experiments were
repeated at least three times. Statistical analysis was performed by Student t-test, one-
way or two-way ANOVA by GraphPad Prism 5.01. Differences between treatments
were established with Tukey’s or Bonferroni’s post-hoc test. A p value of less than 0.05
was considered statistically significant.
4.3. Results
4.3.1. Purification of ELP fusion proteins that assemble nanoparticles
To investigate the potential for assembling protein polymers with chaperone activity,
ELP genes with and without the chaperone mini-peptide from αB-crystallin were cloned
into a pET25b(+) vector (Table 5), expressed in E. coli cells, and purified using Inverse
Transition Cycling (ITC) as previously reported (Shah et al., 2013b; Sun et al., 2011).
After purification all four protein polymers yielded between 30-40 mg/L culture. Purified
proteins were confirmed to have the correct molecular weight and high purity when
87

assessed using SDS PAGE (Fig. 23A). MALDI-TOF was used to reconfirm that all
proteins purified were within 1.5 % of their expected molecular weight (Table 5). The
ELPs resulting from a diblock copolymer, SI and crySI were assessed for nanoparticle
morphology using TEM (Fig. 23B). The dominant species observed for both SI and
crySI formulations were spherical nanoparticles with diameters of 29.1 ± 3.4 nm and
28.7 ± 6.2 nm respectively. Using TEM, the free cry peptide was confirmed to mediate
the formation of oligomers and fibers; furthermore, fibril formation was disrupted by
scrambling the amino acid sequence (Fig. 24). Interestingly, cryS96 did not form either
fibril or oligomer structures (Fig. 24), which suggests that the ELP fusion may sterically
interfere with the oligomerization of the native cry peptide.
88

Table 5. Nomenclature, sequence and molecular weight of cryELP fusion proteins
Peptide
label
Amino Acid Sequence
*Expected
M.W. (kDa)
**Observed
M.W. (kDa)
SI G(VPGSG)
48
(VPGIG)
48
Y 39.65 39.54
crySI
GDRFSVNLDVKHFSPEELKVKG(VPGSG)
48
(VPGI
G)
48
Y
42.07 42.06
S96 G(VPGSG)
96
Y 38.39 38.22
cryS96 GDRFSVNLDVKHFSPEELKVKG(VPGSG)
96
Y 40.82 40.72
* Expected M.W.(m/z) was calculated by DNAStar Lasergene Editseq excluding the
methionine start codon
** Observed M.W. ([M+H]
+
) was measured by MALDI-TOF

89

Figure 23. Purity of ELP fusions and morphology of protein polymer
nanoparticles. A) SDS-PAGE was used to characterize the purity and molecular
weight for ELP fusion proteins (Table 1), which were stained using Coomassie blue.
The molecular weight for the ladder is indicated to the left. B) TEM image of SI
nanoparticles, with average diameters of 29.1±3.4nm. C) TEM image of crySI
nanoparticles, with average diameters of 28.7±6.2nm. Scale bar: 100nm.
90

Figure 24. Fusion to the S96 protein polymer inhibits the fibril formation for the
αB crystallin mini-peptide. Negative stained transmission electron microscopy was
used to evaluate the oligomerization and fibril formation of the cry peptide. A) The mini-
peptide cry alone assembles amyloid-like fibrils. B) The scrambled version of the mini-
peptide (Neg) formed irregular clusters, but does not form fibrils. C) No well-defined
oligomers or fibrils were observed for cryS96 alone. Scale bar: 100nm.
91

4.3.2. The mini-peptide from αB-crystallin shifts the phase diagram for ELP
nanoparticles
To characterize a concentration-temperature phase diagram for the ELP fusions, optical
density was monitored as a function of temperature to identify relevant transition
temperatures (T
t1
, T
t2
) (Fig. 25). SI exhibited a steep thermal response at 26 ° C, T
t1
,
which correlates with the phase transition temperature of the more hydrophobic
(Xaa=Ile) block. This lower transition temperature can be defined as the critical micelle
temperature (Janib et al., 2013). At a higher temperature, T
t2
, near 75 ° C, the more
hydrophilic block (Xaa=Ser) collapses and SI undergoes bulk phase separation (Fig.
25A). Similar to SI, crySI shows two obvious phase transitions, where T
t1
, T
t2
are 13, 30
° C respectively. In comparison to SI, the mini-pept ide on crySI has the effect of
depressing both transition temperatures across the experimental concentration range
(Fig. 25B). On the contrary, both S96 and cryS96 exhibit only one T
t
near 55 ° C, which
was unchanged by addition of the mini-peptide (Fig. 25C,D). Most notably,
concentration dependence of T
t1
for crySI (-0.4 ± 0.6 ° C / Log
10
[μM]) was significantly
suppressed compared to SI (-22.6 ± 16.0 ° C / Log
10
[μM]) as fit with a log-linear
regression line (mean slope ± 95% confidence interval) Despite this impact on the
phase diagram, crySI still forms a population of relatively monodisperse nanoparticles of
a similar diameter to SI at room temperature (Fig. 23C).
92

Figure 25. Phase diagrams for mono and diblock ELP fusion proteins. Optical
density (OD
350 nm
) was used to monitor temperature-dependent assembly of ELPs. A)
The SI and crySI diblocks (25 μM) undergo two obvious phase transitions. T
t1
is
associated with phase separation of the hydrophobic ELP (Xaa=Ile), which drives
nanoparticle assembly. Occurring at a higher temperature, T
t2
is attributed to bulk phase
separation into larger nanostructures. For SI, T
t1
and T
t2
are 26 and 70 ° C respectively.
Similarly, for crySI, T
t1
and T
t2
are 12 and 30 ° C respectively. B) Concentration-
temperature phase diagrams for SI and crySI diblock copolymers. C) In contrast, the
monoblocks protein polymers S96 and cryS96 (25 μM) demonstrate only one phase
separation near 56° C. D) Concentration-temperature phase diagram for S96 and
cryS96. Dashed lines indicate the fit of T
t
to the following equation T
t
=mlog[ELP]+b
where [ELP] is the concentration, m is the slope, and b is the transition temperature at
1 μM.
93

4.3.3. The mini-peptide from αB-crystallin shifts the distribution of hydrodynamic
radii
Due to the shift in the concentration-temperature phase diagram induced by fusion with
the mini-peptide, dynamic light scattering (DLS) was used to determine what species
exist above and below the boundaries identified by optical density (Fig. 26). Two fitting
models were used to interpret the DLS autocorrelation functions. First a cumulant fit
was performed, which assumes the existence of only a single population of particles.
This robust approach was adequate for fitting both SI and S96 (Fig. 26A,D); however,
fusion of the mini-peptide to either polymer resulted in a scattering dominated by a
particle of larger dimensions (Fig. 26B,E). Since SI and S96 do not assemble structures
until above 25 and 60 ° C respectively, the data sug gest that the mini-peptide alone
mediates nanoparticle assembly. Assuming that a smaller population of free crySI and
cryS96 exist in equilibrium with a larger assembled population, a second model called
regularization was used to interrogate the data (Fig. 26C,F). Regularization corrects for
the relative difference in scattering intensity from mixtures of particles with different
hydrodynamic radii. Using the regularization model, SI and S96 continued to behave as
single population of particles. This was not the case for crySI and cryS96, which were fit
best at the lowest temperatures as a mixture of a small (4 to 5 nm) and large (30 to 40
nm) population of particles, where the smaller population represents more than 95 % of
the polymer mass in solution (Fig. 26C,F). For cryS96, this distribution was roughly
constant over the entire temperature range (Fig. 26F). This suggests that even in the
absence of ELP-mediated assembly, the mini-peptide mediates relatively weak
assembly of nanoparticles. For crySI, regularization fitting identified two particle
94

populations over the observed temperature range. As observed using optical density
(Fig. 25B), crySI undergoes two thermally-induced changes in the distribution of
hydrodynamic radii. Below 13 ° C, the smaller popula tion of crySI particles accounts for
the majority of the sample monomers, which was similar to cryS96. Between 13 and 26
° C crySI increases in optical density (Fig. 25B), a nd the resulting mixture could not be fit
by either the cumulant or regularization models (Fig. 26B,C). There was a narrow
intermediate window (26 to 28 ° C) where crySI was d ominated (100 % by mass) by
nanoparticles similar to SI. Above 30 ° C, crySI nan oparticles proceeded to assemble
into larger nanoparticles (300 to 500 nm), which dominated the sample (99 % by mass).
Above 30 ° C, a small fraction of the sample remaine d in nanoparticles similar to SI;
however, this represents only 1 % of the mass of the sample. Thus, both crySI and
cryS96 have thermal assembly properties similar to their unmodified ELP; however, the
mini-peptide significantly impacts the distribution of particle sizes.
95

Figure 26. The αB-crystallin mini-peptide influences the assembly and radius of
ELP nanoparticles. The particle hydrodynamic radius (R
h
) was measured (25 μM) as a
function of temperature using dynamic light scattering. Panels A,B,D,E represent the
cumulant fit, which reflects the particle population that scatters the most light. Panels
C,F represent a regularization fit, which can separate populations of particles with
different size. A) SI diblocks form monodisperse nanoparticles above their critical
micelle temperature (26 ° C) that remain stable at p hysiologic temperatures (37 ° C). B)
In contrast, crySI shows an increased radius even at the lowest assayed temperature (7
° C). Extended autocorrelation times were observed i n the indicated grey region that
prevented the estimation of hydrodynamic radius using standard models. Above 30 ° C,
crySI forms ~400nm nanoparticles. C) Regularization analysis enabled two populations
of particles to be observed for crySI. Below 30 ° C the dominant population of small
particles behaved like SI, which accounted for about 95 % of the sample by mass.
96

Above 30 ° C a population of larger particles domina ted the sample. D) S96 remains
monomeric over 10 to 60 ° C. E) In contrast, cryS96 has a larger radius than S96 over
the entire temperature range. F) Regularization analysis enabled the observation of two
populations of particles in the sample of cryS96. The dominant species for cryS96 has a
radius similar to monomeric S96, which represented about 95 % of the sample by mass.
97

Figure 27. ELP fusion proteins have protective chaperone activity on ADH and
insulin. A) Representative chaperone activity of cryS96 and mini-peptide alone (cry)
against heat induced ADH aggregation at 48 ° C. B) Both reduced (p<0.001) the onset
of turbidity compared to a scrambled mini-peptide (Neg) at 1h (500 μM). C) DSC
measurement of ΔH change at 48 ° C showing cryS96 reduced the aggreg ation enthalpy
per mass of ADH in a dose dependent manner. (*p<0.05). D) An insulin aggregation
assay confirmed the chaperone activity of the ELP fusions with the mini-peptide (100
μM) at 12 ° C. Both cryS96 and crySI protect against DTT induced aggregation, while
unmodified S96 and SI did not. E) A quantitative comparison of insulin aggregation in
the presence of ELPs at 30 min. crySI and cryS96 significantly (p<0.001) inhibited
aggregation compared to SI and S96 respectively. Data indicate mean+S.D. (n=3),
which were analyzed using one-way ANOVA. (***p<0.001).
98

4.3.4. crySI and cryS96 behave as molecular chaperones
Substrate proteins alcohol dehydrogenase (ADH) and insulin were used to test
chaperone activity of ELPs with and without the αB-crystallin mini-peptide. Chaperone
activity against ADH was characterized at 48 ° C (Fi g. 27A,B). Both the mini-peptide
alone and cryS96 inhibited ADH aggregation in a concentration dependent manner, with
an IC
50
of 260 ± 99 μM and 205 ± 29 μM (Fig. 32). To confirm the importance of the
mini-peptide sequence, a scrambled mini-peptide (Neg) was also evaluated and
showed no chaperone activity. Due to the confounding optical densities of SI and crySI
at 48 ° C (Fig. 25B), heat induced ADH aggregation w as inconclusive and is not shown.
To further investigate the mechanism of chaperone activity, differential scanning
calorimetry was used to determine the enthalpy of ADH aggregation in the presence
and absence of cryS96 (Fig. 27C). This study revealed that cryS96 reduced the specific
enthalpy of ADH aggregation, while the ELP alone did not (Fig. 27C). To compare the
relative chaperone activity of crySI and cryS96, an insulin chaperone assay was
performed at 12 ° C upon the addition of DTT (Fig. 2 7D,E). At 30 minutes, both crySI
and cryS96 suppressed more optical density compared to controls SI and S96
(p<0.001). Noticeably, crySI inhibited insulin aggregation by 64.6 ± 5.4%, which was
greater than the protective effect of cryS96 (50.9 ± 4.2%) (p<0.05) (Fig. 27D,E). Control
ELPs SI and S96 as well as the scrambled peptide Neg lacked any chaperone activity
towards insulin.

99

4.3.5. Exogenous cryS96 and crySI protect RPE cells from oxidative stress
induced cell death
Protein chaperones protect RPE cells against apoptosis by interfering with caspase
activation and/or mitochondrial processes that lead to cell death (Sreekumar et al.,
2013). Having demonstrated that crySI and cryS96 both have chaperone activity, their
potential anti-apoptotic properties were evaluated on RPE cells co-incubated with 200
μM H
2
O
2
for 24 hours. To quantify apoptosis, the TUNEL assay was used to identify
apoptotic cells, which showed around 20 % cell death. Similar to the free cry peptide,
crySI and cryS96 treatment significantly reduced cell death (p<0.05) compared to
unmodified ELP controls (Fig. 28A,B). Unmodified ELPs did not produce apoptosis in
RPE; however, co-incubation of plain ELP with H
2
O
2
resulted in cell death. To confirm
that the TUNEL assay was detecting apoptosis, caspase 3 activation was also
characterized using a western blot. Activation of caspase 3 in RPE was reduced in
cryS96 and crySI compared to ELPs alone (Fig. 28C). Collectively, results from the
TUNEL and caspase 3 activation assays demonstrate that only the ELPs with the cry
peptide activity exhibit anti-apoptotic properties, and both crySI and cryS96 inhibited
caspase 3 activation to a similar degree.
100

Figure 28. ELP fusion proteins protect RPE cells from H
2
O
2
induced cell death.
Human RPE cells were treated with 200 μM H
2
O
2
for 24h with or without crySI or
cryS96. Cell death was assessed by the TUNEL assay and activation of cleaved
caspase-3 by immunoblot analysis. A) TUNEL assay showing co-treatment of H
2
O
2
and
unmodified ELPs did not inhibit apoptosis in RPE cells whereas crySI and cryS96
significantly reduced cell death. Confocal images of TUNEL-positive cells (red) and
nuclei (blue) are shown. B) Quantification of TUNEL-positive cells. Data are presented
as percent of TUNEL-positive cells. (*p<0.05, **p<0.01) C) Representative western blot
showing exogenous crySI and cryS96 protected RPE cells from oxidative stress by
inhibiting activation of caspase-3. Caspase-3 activation was prominent in control cells
treated with 200 μM H
2
O
2
. Unmodified ELPs SI and S96 failed to protect against
caspase-3 activation. Scale bar: 20 μm.
101

4.3.6. Cell uptake and nuclear localization of crySI and cryS96 under oxidative
stress is important for anti-apoptosis activity
We have previously shown that exogenous recombinant αB crystallin and the mini-αB
crystallin peptide (cry) can be taken up by RPE cells (Sreekumar et al., 2013), which
may play a role in the suppression of apoptosis. To determine if exogenous ELP fusions
to the mini-peptide would also have access to the cytosol, confocal microscopy was
used to track the internalization of rhodamine labeled material. In addition to incubation
with 5 μM of labeled ELPs, cells were also observed in the absence and presence of
oxidative stress from H
2
O
2
(200 μM for 24 h). All ELPs showed evidence of punctate
intracellular accumulation in the cytoplasm; however, to our surprise oxidative stress
significantly induced nuclear localization of SI, crySI, and cryS96 (Fig. 29). crySI
exhibited higher cell uptake than cryS96 (p<0.001).
Based on the combined observations that both cryS96 and crySI are protective
against apoptosis (Fig. 28) and translocate to the nucleus under oxidative stress (Fig.
29), we next investigated whether uptake was necessary for protection and dependent
on endocytosis. Since both cryS96 and crySI provide the same degree of apoptotic
protection, chaperone activity, and nuclear translocation, cryS96 alone was
subsequently characterized for its mechanism of internalization (Fig. 30). We
hypothesized that if cryS96 internalizes by clathrin-mediated endocytosis, then inhibitors
of this pathway should both block nuclear translocation and also abolish the anti-
apoptosis activity. Monodansylcadaverine (MDC) blocks clathrin-mediated endocytosis;
furthermore, it efficiently halted the nuclear translocation of cryS96 under oxidative
stress (Fig. 30A). A similar effect was observed when cells were treated with Dynasore,
102

which blocks dynamin-dependent internalization (Fig. 33). Remarkably, inhibition of
cryS96 endocytosis completely eliminated protection against TUNEL positive cells
under oxidative stress (Fig. 30B,C).
Based on the evidence that cryS96’s anti-apoptotic activity depends on cellular
uptake, we further hypothesized that the exogenous chaperone must partially escape
from the classical endosome-lysosome pathway in order to reach its intracellular
targets. We thus used the late endosome/lysosome marker LAMP1 to track localization
of cryS96 and cry peptide inside RPE cells at early time points (Fig. 31). Surprisingly, at
1h most cryS96 exhibited nuclear translocation while a significant amount of free cry
peptide was retained in the cytosol and partially colocalized with LAMP1. At 4 h, both
cryS96 and cry peptide had translocated to the nucleus and no longer showed co-
localization with LAMP 1. This study thus suggests that both the cry peptide alone and
cryS96 bypass the endo-lysosomal compartment of human RPE cells under oxidative
stress. This access to the cytosol results later in their translocation to the nucleus (Fig.
29) and correlates with their anti-apoptotic effect (Fig. 28,30).
103

Figure 29. Uptake and nuclear translocation of exogenous ELPs in RPE cells. A)
RPE cells were starved in serum free medium and treated with either 5 μM rhodamine
labeled ELPs alone or co-treated with 200μM H
2
O
2
for 24h. Oxidative stress induced
nuclear uptake of labeled crySI and cryS96. B) Quantification of RPE cell uptake of
ELPs. Both crySI and cryS96 exhibited significantly enhanced nuclear uptake level
under H
2
O
2
stress compared to ELP control groups (***p<0.001). Uptake level of crySI
nanoparticles was higher than cryS96 (***p<0.001). Scale bar: 20 μm.
104

Figure 30. Uptake and nuclear translocation plays an important role in cryS96’s
anti-apoptotic activity. Cells were pre-incubated with 100 μM MDC for 30 min followed
by co-incubation with 5 μM rhodamine labeled cryS96 for 24 h along with 200 μM H
2
O
2
.
Control cells represent no treatment with either MDC or H
2
O
2
. A) Representative
confocal images of cell uptake showing MDC can inhibit cell uptake of exogenous
cryS96. Red: rhodamine labeled cryS96; Blue: DAPI. B) Representative TUNEL images
showing MDC significantly (*p<0.05) reduced the anti-apoptosis efficacy of cryS96
under H
2
O
2
stress. Red: TUNEL+ cells (apoptosis), Blue: DAPI. C) Quantification of
TUNEL positive cells from B). (*p<0.05). Scale bar: 20 μm.
105

Figure 31. Intracellular trafficking of cryS96 and cry peptide under H
2
O
2
stress. A)
Representative images showing time dependent nuclear translocation of cryS96 (red)
under H
2
O
2
stress in human RPE cells. B) Intracellular trafficking of the free cry peptide
(red) under same condition. Red: fluorescence; Green: LAMP1; Blue: DAPI. Scale bar:
20 μm.
106

4.4. Discussion
Age-related macular degeneration (AMD) is the leading cause of blindness among older
individuals in the western world, with more than 13 million cases per year in the United
States alone (Augood et al., 2006). Currently there is no effective therapy for dry AMD;
however, we and others have proposed that exogenous sHSPs may be developed as
therapeutics for AMD (McClellan et al., 2005; Neef et al., 2011). The immunomodulatory
and anti-apoptotic function of molecular chaperones should be an important
consideration in the development of therapeutic interventions due to their ability to
enhance cell viability under deleterious conditions including oxidative stress (Morimoto
and Santoro, 1998; Mymrikov et al., 2011). As successful examples, Ousman et al.
have demonstrated the negative regulatory role of αB-crystallin on several inflammatory
pathways in the immune and central nervous systems (Ousman et al., 2007); Rothbard
and co-workers have illustrated the therapeutic effects of αB-crystallin associated with
specific proinflammatory plasma protein binding via systemic administration (Rothbard
et al., 2012). Moreover, Arac et al. have described effective treatment of experimental
stroke using αB-crystallin even twelve hours post-stroke onset (Arac et al., 2011).
Herein, we intended to utilize the intermolecular association/dissociation of a mini-
peptide derived from αB-crystallin (Knowles et al., 2007; Mornon et al., 1998), explore
its thermal and chaperone activities in fusion with ELPs (Fig. 23), and evaluate the
fusion protein’s cell-protective and uptake properties under oxidative stress in a human
RPE model. The resulting fusions may have applications as therapeutic chaperones for
AMD.
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A prevalent view regarding the mechanism of action for sHSPs is that they interact with
exposed hydrophobic patches on client proteins undergoing aggregation (Stengel et al.,
2010). Previous studies indicated that exposure of hydrophobic surface change and
reorganization of oligomer architecture are associated with αB-crystallin’s chaperone
function (Kumar et al., 2005; McHaourab et al., 2009; Reddy et al., 2006). TEM images
of all peptides and fusion proteins involved in the study provided the first clue of ELPs’
modulation of native cry peptide assembly (Fig. 23 & 24). To gain more insight into the
hydrophobicity change of cry peptide by fusion with ELPs, we analyzed the temperature
dependent hydrophobicity of cry, crySI and cryS96 via Bis-ANS assay (Bhattacharyya et
al., 2006) (Fig. 34). Noticeably, only crySI exhibited a significant temperature triggered
hydrophobicity change (****p<0.0001), which was consistent with its observed stepwise
aggregation via DLS (Fig. 26). Neither cry peptide nor cryS96 changed hydrophobicity
in response to temperature changes.
The TEM, DLS and Bis-ANS results raised the question as to how the assembly
influenced cry’s activity as a molecular chaperone both in vitro and in the crowded
cellular environment. We thus explored the thermodynamic mechanism underlying
cryELPs’ inhibition of ADH aggregation using differential scanning calorimetry (Fig.
27C). For protein systems, either a decrease in enthalpy decrease or an increase in
entropy is beneficial for maintaining stability. Although no direct studies have focused on
the thermodynamic mechanism underlying sHSPs’ chaperone activity towards ADH,
several studies suggest that stabilization of client proteins by sHSPs is dominated by
enthalpic changes (Kazlauskas et al., 2012; Kumar et al., 2005). The enthalpy increase
of both plain ELPs (Urry, 1997) and ADH when heated indicated that both the phase
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transition and aggregation are endothermic processes (data not shown). Most notable
at equal mass, cryS96 significantly reduced the enthalpy of ADH aggregation (p<0.05)
(Fig. 27C), while S96 did not exhibit any effect. Remarkably, the RPE cell uptake result
corroborated our interpretation of the in vitro chaperone function (Fig. 29). Without
oxidative stress, crystallin ELP fusions do not translocate into the nucleus. However,
when oxidative stress was applied, the highest uptake was observed for crySI (large
nanoparticles), followed by cryS96 (soluble macromolecule), followed by the SI ELP
(small nanoparticles) lacking the αB-crystallin minipeptide. This suggests that besides
cry peptide, the nanoparticle size can also influence cellular internalization in response
to oxidative stress (Geng et al., 2007; Nel et al., 2009; Petros and DeSimone, 2010).
Based on the summary of these findings, we have proposed the mechanism for
intracellular trafficking of cry96 under H
2
O
2
stress (Fig. 35).
Oxygen radicals that damage the sensitive cells in the retina are known to play a central
role in the causes of AMD (Sanvicens et al., 2004). We and others have reported that
the expression of αB-crystallin was increased with mild oxidative stress but decreased
when H
2
O
2
level reached a pharmacologically toxic dose (Shin et al., 2009; Yaung et al.,
2007). Moreover, our studies suggested secretion of αB-crystallin from RPE cells
involved participation of exosomes (Sreekumar et al., 2010a). The same cell model was
used in this study to evaluate the in vitro interaction of exogenous cry ELP fusion
proteins. Interestingly, the constructs have similar anti-apoptotic effects on human RPE
cells under H
2
O
2
stress (Fig. 28). Unexpectedly, oxidative stress in these primary cells
initiated a striking level of translocation from the cell-surface and cytoplasm into the
nucleus (Fig. 29), which roughly correlated with anti-apoptotic activity (Fig. 30). To the
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best of our knowledge, the nuclear translocation of a nanoparticle in response oxidative
stress in RPE has not been observed. Once internalized, these protein polymer fusions
would have direct access to misfolding client proteins inside the cytosol and the
nucleus. Jeong and coworkers have previously reported that αB-crystallin physically
interacts with caspase subtypes in the nucleus, which protects RPE from apoptosis
(Jeong et al., 2012). Similarly, Dastoor and coworkers reported that endogenous heat
shock cognate protein70 (Hsc70) translocates from the cytoplasm to the nucleus when
oxidative stress is applied (Dastoor and Dreyer, 2000). We have similarly reported the
translocation of fluorescein labeled human recombinant αB-crystallin into the nucleus
when compared with unstressed cells (Sreekumar et al., 2010a).
This manuscript shows that both cryS96 and cry escape from the endosome-lysosome
degradation pathway and reach the nucleus (Fig. 31). CryS96 shows less colocalization
with LAMP1 and faster trafficking to the nucleus. Del Pozo-Rodríguez and coworkers
have shown that in ARPE-19 cells, inclusion of proline rich Sweet Arrow Peptide (SAP)
in a delivery vesicle induced a change from clathrin dominant endocytosis to
caveolae/raft-dependent endocytosis, thereby decreasing lysosomal degradation of
DNA vectors (del Pozo-Rodriguez et al., 2009). Similarly, Fernández-Carneado et al.
addressed the importance of proline-rich peptides in facilitating lysosomal escape as
potential peptide carriers (Fernandez-Carneado et al., 2004). Both of these studies
utilized fusion with the endosomal membrane as vesicle escape mechanism (Varkouhi
et al., 2011). It is thus possible that the high proline content in S96, (Val-Pro-Gly-Ser-
Gly)
96
, protected cryS96 from lysosomal attack; however, more detailed intracellular
trafficking mechanisms remain to be investigated.
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One of the most effective drug delivery routes for treating dry AMD is direct intravitreal
administration, which is a skilled outpatient procedure associated with risks and
noncompliance (Evans and Syed, 2013). Extended release may reduce risks and costs
by reducing the frequency of procedures (Evans and Syed, 2013; Garber, 2010).
Biodegradable polymeric macromolecules may form a local depot in the vitreous
capsule and extend their effect over longer periods (Nagarwal et al., 2009). The ELP
system has been proposed as a potential platform for controlled release with orthopedic
applications (Adams et al., 2009), diabetes (Amiram et al., 2013a) and cancer treatment
(Liu et al., 2006). With this in mind, this manuscript describes the creation of new ELP
nanoparticles/macromolecules with chaperone activity and potential applications in the
treatment of AMD and thus may have wider applications for other therapeutic
approaches in the eye.
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Figure 32. cryS96 exhibits concentration dependent chaperone activity against
Alcohol Dehydrogenase (ADH). Optical density was used to evaluate the aggregation
of ADH (100 μg) at 360 nm after incubation at 48 ° C for 60 min vs. ADH alone (100%).
Inhibition of ADH aggregation for both cryS96 and cry was dose dependent. Raw data
points were independently fit to a sigmoidal curve:
% ,k lmn .oopBo.-D,C q,--,r ? +,Z q,--,r ( st,Cu v
w
/0t
yU
w
? st,Cu v
w

This predicted IC
50
values are displayed (mean ± SEM) for cry (top) and cryS96
(bottom). A
z1{|}~

0.9926; A
z1{

0.9871.
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Figure 33. Nuclear translocation of cryS96 is dynamin dependent. Human RPE
cells were incubated with rhodamine labeled cryS96 (red) and H
2
O
2
(200 μM) for 24 h in
the A) absence and B) presence of 50 μM dynasore. Nuclei were labeled with DAPI
(blue). Scale bar: 20 μm.
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Figure 34. A fluorescent probe mixed with CrySI reveals a temperature-dependent
increase in hydrophobicity. The Bis-ANS (5 μM) probe was used to explore shifts in
fusion peptide hydrophobicity for the cry mini-peptide and its ELP fusion proteins, crySI
and cryS96. Ex: 390nm; Em: 400-600nm; slit: 5nm. A-D) Peptides were incubated at 1
μM and evaluated over a range of temperatures. A) crySI. B) cryS96. C) The cry
peptide alone. D) CrySI displayed a temperature dependent intensity change, which
114

was significantly greater than for cryS96 or cry alone (****p<0.0001). Values indicate the
mean ± SD. (n=3, ANOVA, followed by the Bonferroni post-hoc test). E-F) When
peptides were incubated at 5 μM at a fixed temperature (25 ° C), both crySI (~10 fold)
and cryS96 (~1.7 fold) increased the fluorescence intensity compared to the cry peptide
alone (***p<0.001), which suggested enhanced hydrophobicity. Data were presented as
mean±S.D. (n=3, ANOVA, followed by the Tukey's post-hoc test).
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Figure 35. Proposed intracellular trafficking and protective mechanism of cryS96
under oxidative stress. When human RPE cells are incubated with cryS96 in the
absence of oxidative stress, the peptide partially traffics to lysosomes, as indicated by
colocalization with LAMP1 (Fig. 31). In contrast, under H
2
O
2
stress the cryS96
translocates to the nucleus (Fig. 29 and Fig. 30) as well as gains access to the cytosol.
Having access to the cytosol, the cryS96 peptide halts apoptosis, by blocking caspase 3
activation and inhibiting apoptosis (Fig. 28). Under oxidative stress, both nuclear
translocation of cryS96 and anti-apoptosis activity were blocked by a dynamin-
dependent mechanism (Fig. 33) or inhibited by monodansylcadaverine (Fig. 30). The
mechanism by which cryS96 binds to the cell surface, escapes from endosomes under
oxidative stress, or blocks the activation of caspase 3 remains unknown.
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4.5. Conclusion
This manuscript reports the modification of a chaperone peptide via fusion with two
protein polymers, one of which assembles polymeric micelles. Unlike full-length
chaperone proteins, the polymers are decorated with a sHSP ‘mini-peptide’ that gives
them chaperone and anti-apoptotic activity in human RPE cells. Unlike control ELPs,
both crySI and cryS96 exhibit comparable anti-apoptotic activity in a model of human
RPE cells, which are used to study the pathophysiology of AMD. ELP fusion modulated
the assembly process, chaperone activity and intracellular uptake pathway of the native
cry peptide. These findings point to the potential for intravitreal administration of these
nanoparticles/macromolecules to treat AMD; however, a more detailed understanding of
the mechanism underpinning oxidative stress induced nuclear translocation and in vivo
evaluation will now be required to assess the likelihood of clinical benefit.
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Chapter 5
Elastin-like polypeptides (ELPs) in ocular drug delivery and tissue engineering
5.1. Introduction
5.1.1. Overview of ocular drug delivery and tissue engineering
Biocompatible materials have been playing a vital role in developing advanced medical
devices, efficient drug delivery systems and smart engineering tissues (Langer and
Tirrell, 2004). During the past decades, progress in the understanding of the pathology
underlying ocular diseases and innovations in drug delivery systems have prompted
significant advances towards the regeneration of tissues such as bone (Puelacher et al.,
1996), heart valves (Shinoka et al., 1995), myocardial tissue (Leor et al., 2005) and
cartilage (Vacanti et al., 1991). Different from healing other parts of the body, ocular
drug delivery and tissue engineering remains challenging due to unique
compartmentalized structure of the eye and multiple tissue barriers against efficient
drug uptake (Ali and Lehmussaari, 2006). To achieve the ideal goal of delivering the
effective therapeutic agent at proper dose to the target ocular tissue with minimal off-
target side effects (Weiner and Gilger, 2010), one needs to consider three major
aspects: the bioavailability of the active drug, target property of the product and
patience compliance. In this chapter, we address several representative compartments
of the ocular system, namely the lacrimal gland, the corneal epithelium and intraocular
drug delivery (especially to the retina). For each section, we start with introducing the
basic anatomical features; then summarize traditional drug delivery approaches;
followed by highlight some of the novel delivery and tissue engineering strategies; and
finally outlook the perspectives of ocular drug delivery systems and tissue engineering
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in near future. Specifically, we focus on the exploration and achievements using protein
polymer Elastin-like polypeptides (ELPs) as novel ocular drug delivery and tissue
engineering system.
5.1.2. ELPs in drug delivery and tissue engineering
Elastin-like polypeptides (ELPs) are one class of polypeptides derived from natural
human tropoelastin. ELPs are composed of repetitive pentapeptide motif (Val-Pro-Gly-
Xaa-Gly)n where Xaa can be any amino acid (Nettles et al., 2010b). Human elastin itself
is a major protein component of extracellular matrix (ECM) and provides both strength
and extensibility to the connective tissue, such as skin, ligament, arteries, and
specialized cartilages. ELPs inherit multiple physical characteristics of natural elastin
and can undergo reversible thermo-responsive hydrophobic assembly from aqueous
solution (Daamen et al., 2007; Kim and Chaikof, 2010). Aggregation forms can be
varied from nanometer sized nanoparticles (Janib et al., 2013; Sallach et al., 2006; Shi
et al., 2013b) to extended fibril networks (Aluri et al., 2012) to micron-sized/gel-like
viscous coacervate (Amiram et al., 2013c; Liu et al., 2010b; Sinclair et al., 2013)
through appropriate design of ELPs’ block copolymer architecture. ELPs exhibit several
interesting features to be used as drug delivery and tissue engineering scaffold (Chilkoti
et al., 2006a; MacEwan and Chilkoti, 2010; Nettles et al., 2010b): i) Chemical
conjugation of small molecules and short peptides to ELPs provides a platform for fast
screening purpose; ii) recombinant technology secures the precise control over the
amino acid sequence, stereochemistry and molecular weight of ELPs and ELP-fusion
proteins, which is not easily achieved via chemical synthetic approach (Hassouneh et
al., 2012). And a well-defined molecular sequence can lead to a rich complexity of
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structure and function; iii) Expression of ELPs/ELP fusions from a heterologous host
(e.g., bacteria or eukaryotic cell) yields satisfactory quantities of target products and
most of the purification process can be simplified utilizing ELPs’ inverse phase transition
behavior without the need for chromatography, which meets the need of large scale
production; iv) composed of natural amino acids, ELPs are biocompatible,
biodegradable, and non-immunogenic. Thus, ever since the first characterization, the
application of ELPs have infiltrated into protein purification (Bellucci et al., 2013; Liu and
Chen, 2013), active/passive targeting cancer treatment (Aluri et al., 2014b; MacKay et
al., 2009b; Shi et al., 2013b), cartilage and intervertebral disc tissue engineering (Betre
et al., 2006; Betre et al., 2002), vascular graft tissue engineering (Lin et al., 1992; Nicol
et al., 1992), liver tissue engineering (Janorkar et al., 2008; Swierczewska et al., 2008),
cell penetrating (MacEwan and Chilkoti, 2012), intracellular microdomains sorting
(Pastuszka et al., 2012; Shi et al., 2014) and cell-sheet engineering (Mie et al., 2008),
etc. Reviews about ELPs’ application in other organs have been reported extensively
elsewhere, herein we focus on ELPs’ emerging role in ocular drug delivery and tissue
engineering.
5.2. ELPs in ocular drug delivery
5.2.1. Lacrimal gland
We started the exploration of ELPs’ application in the gland where human tear
originates. The lacrimal functional unit (LFU) is an integrated ocular functional defense
system against environmental stress via homeostatic regulation of tear flow and tear
film formation (Tiffany, 2003). It is composed of the cornea, conjunctiva, the main and
accessory lacrimal glands, the meibomian glands, mucin-producing epithelial cells and
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goblet cells, the blink mechanism, and closed eye condition events (VanderWerf et al.,
2003). In addition, homeostasis modulation involves a reflex arc between the ocular
surface and the brain (Stern et al., 1998a, b), as well as immunologic (Knop and Knop,
2005), inflammatory (Knop and Knop, 2005), and endocrine regulation (Sullivan et al.,
1996). The lacrimal gland is an important component of the LFU (Stern et al., 2004) and
secretes the tear film aqueous layer, which contains multiple growth factors (e.g.,
EGFs), antimicrobial factors (e.g., lactoferrin, defensins), anti-inflammatory factors (e.g.,
IL-1RA) and mucins (Pflugfelder, 2014).
Although debate exists about whether dry eye disease (DED) is mainly caused by
dysfuncion of lacrimal gland or meibomian gland, numerous studies suggested that
inflammation in the lacrimal gland contributes to less tear production, altered tear
composition and stability. Proposed underlying mechanisms include cholinergic
blockade from autoantibodies to muscarinic acetylcholine receptor 3 (Humphreys-Beher
et al., 1993; Kovacs et al., 2005), inhibition of acinar secretion by inflammatory
cytokines such as IL-1 (Zoukhri et al., 2007), epithelial cell death caused by cytokine
(Nguyen and Peck, 2009; Rahimy et al., 2010) and increased lymphocyte infiltration of
the acini (Rahimy et al., 2010). Thus, besides traditional topical lubrication, current
therapies targeting lacrimal gland dysfunction include ocular surface protection and anti-
inflammatory therapy such as cyclosporine A (CsA), which has been proved to inhibit a
variety of cytokines and significantly increase conjunctival goblet cell density
(Pflugfelder et al., 2008). Immunosuppressive effects of cyclosporin A (CsA) was
observed via systemic and topical application. To obtain effective drug concentration at
the target area and minimize various side effects, improved local administration
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formulations of CsA have been reported during the past years, including emulsion,
microspheres, implants, liposomes and contact lenses, etc (Yavuz et al., 2012).
To better deliver therapeutic agents to the lacrimal gland utilizing its unique anatomical
features and specifically expressed receptors, we started by thoroughly investigating
morphology and function of the acinar epithelial cells of the lacrimal gland and
underlying machinery that modulates its secretory activity, which suggested interesting
modulation efficacy of adenovirus capsid proteins (Hamm-Alvarez et al., 2003). To
bioengineer nanoparticles with multivalent adenovirus capsid protein presenting at the
corona and further improve their cellular internalization efficacy, we developed a fusion
protein that self assembles into nanoparticles decorated with the knob domain of
adenovirus serotype 5 fiber protein based on Elastin-like polypeptides (ELPs) (Sun et
al., 2011). Although no significant difference was observed when plain ELP and knob–
ELP were bound to the outside of hepatocytes that express adenovirus receptor (CAR),
the knob-ELP fusion protein exhibited more internalization and localization to lysosomes
of hepatocytes. In order to systemically treat the lacrimal gland inflammation caused by
lymphocyte infiltration, we tested FKBP12-ELP nanoparticles mediated delivery of
immunosuppressant rapamycin via intravenous injection into non-obese diabetic mouse
(NOD) and evaluated its efficacy in the inflamed lacrimal gland (Shah et al., 2013c).
Significantly suppressed lymphocytic infiltration in the lacrimal gland of 12-week male
NOD mice was noticed in the FSI rapamycin treatment group. Moreover, we further
tested locally delivering biopharmaceuticals to the lacrimal gland to minimize systemic
toxicity. We injected model biopharmaceutical Lacritin fused with a viscous coacervate
ELP tag (V96) into the lacrimal gland of NOD mice. The Lacrt-ELP fusion construct
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induced tear secretion from NOD mice via single bolus injection and formed local drug
depot inside the lacrimal gland (Fig. 1). Out of expectation, transcytosis of exogenous
ELP fusion proteins in polarized lacrimal gland acinar cells (LGACs) both ex vivo and in
vivo were noticed in some cases, such as Lacrt-ELPs (Fig. 6) and knob-ELPs (data not
shown).
5.2.2. Corneal epithelium
The rigid, transparent, avascular cornea is the anterior part of the corneoscleral
envelope that protects the eye from environmental stress (Efron et al., 2013). Major
layers of the cornea include an outermost stratified epithelium, a stroma composed of
collagen fibrils and an innermost monolayer of endothelial cells (Sommer et al., 2006).
Eye drops remain the most popular yet not efficient drug delivery approach to the ocular
surface due to the native anatomy of cornea and lacrimal fluid-eye barrier (Urtti, 2006a).
The most apical corneal epithelial cells form tight junctions and express glycocalyx that
confers wettability to the corneal surface. The glycocalyx and the tight junctions create a
relatively impermeable barrier that limits the paracellular drug permeation and the
passage of small, water-soluble molecules (Argueso et al., 2009; Argueso et al., 2006).
Drug bioavailability is further hampered by lacrimal fluid removal, nasolacrimal drainage,
and direct systemic absorption from the conjunctival sac via local blood capillaries (Urtti,
2006b). To prolong the duration of drug action, various formulation designs have been
proposed, such as gelifying formulations, ointments, and inserts. Take CsA as an
example, its high hydrophobicity causes variable and incomplete absorption from
conventional oral or topical formulations (Liu et al., 2007). Thus so, numerous groups
have been focusing on improving the solubility and enhancing drug absorption of CsA
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by changing formulations, such as α-cyclodextrins (Cheeks et al., 1992; Newton et al.,
1988), positive/negative charged emulsions (Milani et al., 1992; Sall et al., 2000), PLGA
and CD nanoparticles (Aksungur et al., 2011), Collagen shields (Pleyer et al., 1994;
Reidy et al., 1990), implants (Grisolano and Peyman, 1986; Kim et al., 2005), etc.
However, these approaches either cause poor patient tolerance or request complex
manufacturing or implanting techniques (Yavuz et al., 2012). To enhance the
biocompatibility and bioavailability of CsA, we have been focusing on re-formulating it
using Elastin-like polypeptides (ELPs) based nanoparticle formulations (data not
shown). At the same time, we are searching for novel therapeutic candidates to better
treat diseases on the ocular surface (e.g. wound healing, dryness, inflammation). Model
biopharmaceutical tested is lacritin, which stimulates human corneal epithelial cells
proliferation and protect them from proinflammatory cytokines’ stress (Sanghi et al.,
2001a). Topical lacritin administered to eyes of normal New Zealand White rabbits
either as a single dose or three times daily for 14 days have been proved to increased
basal tearing. Moreover, lacritin is well tolerated without triggering conjunctival swelling
or discharge, aqueous flare/anterior chamber reaction, loss of light reflex, iris
hyperemia, or corneal opacity or vascularization (Samudre et al., 2011a). Considering
the curvature of ocular surface and potential multiple binding sites via a multivalent
delivery approach, we bioengineered LSI nanoparticles based on ELP micelle SI
scaffold (Fig. 11). We have demonstrated the first proof of concept that these fusion
protein polymer nanoparticles heal scratch wound on cultured human corneal epithelial
cells (Fig. 15) and abrasion wound on non-obese diabetic (NOD) mice ocular surface
(Fig. 17). The murine corneal abrasion model mimics several aspects of photorefractive
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keratectomy (PRK) procedure and thus strongly supported therapeutic efficacy of
thermo-responsive LSI nanoparticles. Collectively, results from this study demonstrated
great potential for this strategy to transform other ocular protein therapeutics into protein
polymer nanoparticles.
5.2.3. Intraocular delivery
Pathologies affecting the posterior segment of the eye, such as diabetic retinopathy,
retinoblastoma, retinitis pigmentosa, choroidal neovascularization and Age-related
macular degeneration (AMD), are one of the major causes of blindness worldwide
(Herrero-Vanrell et al., 2014). However, compared to treating diseases on the anterior
segment of the eye, successful treatment of diseases at the back of the eye is more
challenging due to the anatomy and physiology of ocular barriers, and requires effective
concentrations of the active substance maintained during a long period of time in the
intraocular target site (Urtti, 2006a). Treating exudative AMD via an improved therapy is
one of the most popular topics in the field. To maximize drug concentration at the target
site and keep therapeutic drug concentration in the macula, most currently available
drugs such as pegaptanib, ranibizumab, and bavacizumab are administered directly into
the eye by intravitreal injection every 4 or 6 weeks (Augustin and Schmidt-Erfurth, 2006;
D'Amico and Grp, 2006; Martin et al., 2011). Yet this approach is invasive and due to
short half-life time of drug in the eye, repeated injections are required and can cause
clinical complications such as cataract formation, vitreous hemorrhage, retinal
detachment, and endophthalmitis (Bakri et al., 2007a; Bakri et al., 2007b; Cantrill et al.,
1989). Besides looking for novel treatment modalities, both academia and industry have
been focusing on improving efficacy and/or reducing injection numbers of currently
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available drugs using new formulations and drug delivery vehicles (Yasukawa et al.,
2011).
FDA approved and ongoing testing drug delivery systems are classified into 3 major
categories: (1) nonbiodegradable/biodegradable implants for intraocular controlled
release; (2) more targeted drug composition via systemic administration (e.g.
photodynamic therapy); and (3) drug penetration (e.g. gene transfer and iontophoresis)
(Yasukawa et al., 2011). Regarding the first two strategies, injectable Micro- and
nanoparticles have attracted numerous attention due to their small size and ability to be
modified with multivalent payload or tissue-specific uptake peptide (Moshfeghi and
Peyman, 2005). Some of the systems can also protect the therapeutic molecules from
degradation and thus enhance the duration of action (Fischer et al., 2011). As a
successful example, intravitreal and sub-Tenon’s (episcleral space) administrations of
triamcinolone acetonide have been widely used in the treatment of macular edema,
exudative AMD, and uveitis (Augustin and Schmidt-Erfurth, 2006). Moreover, dos
Santos et al. have demonstrated successful localized controlled delivery of anti-
transforming growth factor (TGF)-β2 phosphorothionate oligonucleotides (PS-ODN)
using PLGA microspheres in a rabbit model of glaucoma filtering surgery (dos Santos et
al., 2006). Bourges et al. have shown that the accumulation of PLA nanoparticles
loaded with Rh-6G and Nile red fluorochromes at the internal limiting membrane (ILM)
one hour after intravitreal injection in the rat retina, followed by a transretinal distribution
six hours after administration. Remarkably, the nanoparticles remain within the RPE
cells up to four months after injection (Bourges et al., 2003).
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We discovered the small heat shock protein αB-Crystallin exhibits anti-apoptotic and
anti-inflammatory functions and protects human retinal pigment epithelial (RPE) cells
from oxidative stress (Sreekumar et al., 2010b) and other group has previously reported
that the 73-92 (20-mer) mini-peptide of αB-crystallin (cry) exhibits chaperone activity
alone (Bhattacharyya et al., 2006). To provide a platform for enhancing mini-peptide’s
cellular internalization and activity, we generated crySI nanoparticles and cryS96
macromolecules based on elastin-like polypeptides (ELPs) (Fig. 22). Fusion
nanoparticles generated exhibited chaperone-like activity towards client proteins insulin
and ADH (Fig. 27, 28&30), underwent nuclear internalization into human RPE cells
when exposed to oxidative stress (Fig. 29) and inhibited apoptosis caused by oxidative
stress (Fig. 28). In vivo pharmacokinetic studies of the cryELP nanoparticles in murine
and rabbit models are undergoing in our group. These protective protein polymer
nanoparticles may have immediate applications in the treatment of dry AMD.
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5.2.4. Drug delivery via contact lenses
Beyond its function of providing millions of people with glasses-free vision correction,
contact lenses have been studied to serve as a way to therapeutically manage ocular
anterior segment disorders (Guzman-Aranguez et al., 2013b). Indications for using soft
contact lenses therapeutically include protecting a compromised ocular surface, pain
management, and promoting epithelialization or wound closure. Many studies have
investigated the ability of contact lenses to improve the corneal penetration and
bioavailability of topically applied pharmaceutical agents using different approaches
(Gulsen and Chauhan, 2004). In one method, lenses are simply soaked in the drug
solution and then placed on the eye and is commonly employed with antibiotics and
non-steroidal anti-inflammatory drugs (NSAIDs) postoperatively, and with antibiotics for
severe infections. However, this approach normally results in a high initial release,
followed by a slower, long-term release below therapeutic concentration. Alternatively, a
topical drug can be applied over the lens while the lens is in situ. This is often the
approach taken when a patient is wearing contact lens as a protective device (bandage
lens) following a corneal injury or serious infective complication, in which case a lens is
used as a shield or “bandage lens” to assist corneal repairing. The lens acts as a
reservoir, taking up drug from the tear film and then, slowly releasing it back into the
tears when the concentration of the drug over there declines. The rule of thumb is to
prolong the contact time of the drug with the cornea and thus improve its absorption.
Ongoing researches of drug-eluting contact lenses include copolymerizing the contact
lens’ hydrogel material (p-HEMA) with other monomer/polymer (dos Santos et al.,
2009), such as PLGA; releasing drug from microemulsions and nanoparticle contained
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in hydrogel prototype lenses (Jung et al., 2013); molecularly imprinted hydrogels
(Alvarez-Lorenzo et al., 2006; Cunliffe et al., 2005; Hiratani and Alvarez-Lorenzo,
2004a) and immobilizing drug-containing liposomes onto the surface of contact lenses.
As examples: Kapoor et al. have developed surfactant-laden poly-hydroxy ethyl
methacrylate (p-HEMA) contact lenses for releasing Cyclosporine A (CsA) at a
controlled rate for extended periods of time (Kapoor et al., 2009). Peng et al. have
demonstrated that Vitamin E incorporated silicone hydrogel (SiH) contact lens can
extend CsA release within the therapeutic window for a period of about a month (Peng
and Chauhan, 2011b). Ciolino et al. designed safe drug-eluting contact lens for
prolonged delivery of latanoprost for the treatment of glaucoma (Ciolino et al., 2014).
Their method is encapsulating latanoprostepoly(lactic-co-glycolic acid) films in
methafilcon by ultraviolet light polymerization. More recently, Kim et al. have
constructed a nanodiamond (ND)-embedded contact lens capable of lysozyme triggered
release of glaucoma drug timolol maleate (TM) for sustained therapy (Kim et al., 2014).
They have further confirmed the retention of drug activity in primary human trabecular
meshwork cells.
However, previously studies mainly focus on small molecule drugs other than
biopharmaceuticals. Achieving sustained, long-term drug delivery of emerging
therapeutic proteins and peptides at the normal physiological temperature, pH, and
salinity of human eye still remains a challenge. Furthermore, it would be desirable to
provide a contact lens drug delivery device which is relatively simple in design; which
does not require complicated and expensive manufacturing processes; which does not
significantly impair or interfere with the patient's vision; and which would not require a
129

substantial change in the practice patterns of eye physicians and surgeons. We
accidentally discovered ELPs’ thermo-reversible, spatiotemporal and sustained
attachment to Proclear Compatibles
TM
contact lens as a biodegradable bridge (Fig. 19).
Moreover, we illustrated that attachment and release of ELPs to/from Proclear
Compatibles
TM
contact lens was a T
t
and temperature dependent process using
rhodamine as a detection probe (Fig. 20). ELP fusion protein modified contact lens can
spatiotemporally mediate cell uptake (Fig. 21), which illustrated great potential as a
novel biopharmaceutical delivery system.
5.3. Conclusion and future perspectives
Efforts to design and develop better ophthalmic drug delivery systems and tissue
engineering devices in order to enhance bioavailability, improve patient compliance,
fine-tune targeting property and elongate duration of drug action time have been
accelerating within the past decade. Accumulated knowledge about the molecular and
cellular mechanism underpinning various ocular diseases, thorough investigation about
ocular kinetics and drug distribution, innovative exploration into new drug administration
routes and technologies all lead to the new generation of ophthalmological therapy. We
aim to apply thermo-responsive protein polymer elastin-like polypeptides (ELPs) in
ocular drug delivery and tissue engineering to improve the efficacy of available
therapeutics and possibly enable the creation of entirely new therapeutic entities. With
continued interest and research into the field, we believe that the proper utilization of
ELPs will not only improve conventional ophthalmic therapies, but also bridge the
translational gap between bench and bedside.

130

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Asset Metadata

Creator Wang, Wan (author)

Core Title Development of protein polymer therapeutics for the eye

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 07/02/2014

Defense Date 06/10/2014

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

Tag bioavailability,cell uptake,controlled release,drug delivery,elastin‐like polypeptides,OAI-PMH Harvest,ocular

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mackay, John Andrew (committee chair), Hamm-Alvarez, Sarah F. (committee member), Hinton, David R. (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email leilawangwan@gmail.com,wanwang@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-429068

Unique identifier UC11286606

Identifier etd-WangWan-2606.pdf (filename),usctheses-c3-429068 (legacy record id)

Legacy Identifier etd-WangWan-2606.pdf

Dmrecord 429068

Document Type Dissertation

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Rights Wang, Wan

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

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

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