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Analysis of endocytic and trafficking pathways of potential candidates for drug delivery in HeLa and lacrimal gland acinar cells

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Analysis of endocytic and trafficking pathways of potential candidates for drug delivery in HeLa and lacrimal gland acinar cells

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ANALYSIS OF ENDOCYTIC AND TRAFFICKING PATHWAYS OF POTENTIAL
CANDIDATES FOR DRUG DELIVERY IN HELA AND LACRIMAL GLAND
ACINAR CELLS

by

Janette Contreras

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

May 2012

Copyright 2012 Janette Contreras

ii

Dedication

This work is dedicated to my parents, Rafael and Evelia, who have always supported my
aspirations and decisions, and have showered me with more love than I could ever ask
for.

iii

Acknowledgements

With the utmost respect, I would first like to thank my advisor, Dr. Sarah F. Hamm-
Alvarez, for all her guidance and advice throughout the years. Her confidence in me has
helped me grow not only as a scientist, but also as a person. I truly admire the ease with
which she carries out her various roles as a scientist, leader, wife, and mother. She is, and
will remain, an inspiration to me.
I would also like to thank my friends and labmates in the Hamm-Alvarez laboratory,
especially Dr. Eunbyul Evans, Dr. Maria C. Edman, and Dr. Lilian Chiang, for their
friendship and support. The difficult times would have been much harder without you,
and the fun times were better because of you.
I would also like to acknowledge Francie Yarber for her all her hard work, especially in
preparing viral stocks. Her friendship and kindness made the lab feel like a home away
from home. A special thanks to Dr. Jiansong Xie and Dr. Kaijin Wu for their extensive
help in the early stages of my work, and their continued advice throughout.
This achievement would have been impossible without the loving support of my
wonderful family: my parents, Rafael and Evelia, my sister Evelyn, my brother Rafael,
and my fiancé, Mauricio A. De Lama. Their encouragement, love, and support have
helped me every step of the way on this long journey. I cannot thank them enough for
believing in me and instilling in me a genuine sense of happiness.
iv

Table of Contents

Dedication ii

Acknowledgements iii

List of Figures vi

Abbreviations viii

Abstract x

Chapter 1. Introduction
Foreword 1
The origin and scientific importance of HeLa cells 1
The lacrimal gland in health and as a therapeutic target 3
Brief review of endocytosis 7
Drug and delivery constructs
Difluoroboron dibenzoylmethane-polylactide nanoparticles 11
The Momordica cochinchinensis trypsin inhibitor: MCoTI-I 12
Adenovirus serotype 5 14

Chapter 2. Materials and Methods
Reagents 17
Antibodies 18
Difluoroboron dibenzoylmethane-polylactide nanoparticles 18
AlexaFluor 488-labeled trypsin inhibitor cyclotide 19
HeLa cell culture 19
Lacrimal gland acinar cell isolation and primary culture ` 19
Generation of recombinant proteins and Ad5 vectors 20
Cell-binding biochemical assays 20
Confocal fluorescence microscopy 21
BNP photostability assays 24
BNP uptake assays 26
Cytotoxicity assays 26
Transfection of rabbit lacrimal gland acinar cells 27
Analysis of plasmid transfection efficiency 28
Delivery of siRNA duplexes 28
Analysis of siRNA knockdown efficiency 29

v

Chapter 3. Intracellular localization and trafficking of BNPs in HeLa cells
In vitro and in vivo photostability of BNPs 30
Intracellular localization of BNP12 and the role of the cytoskeleton 33
Discussion 38

Chapter 4. Endocytosis and intracellular trafficking of MCoTI-I in HeLa and
LGAC
Endocytosis of MCoTI-I in HeLa cells 41
Intracellular trafficking of MCoTI-I in HeLa cells 51
Endocytosis and intracellular trafficking of MCoTI-I in LGAC 55
Discussion 61

Chapter 5. Endocytosis and intracellular localization of Ad5 in LGAC
Evidence of variations in the Adenovirus infection pathway 63
The importance of fiber for Ad5 internalization 64
Elimination of macropinocytosis as the prevalent endocytic route of Ad5 69
Effect of inhibitors on Ad5 endocytosis 71
Intracellular localization of Ad5 78
Discussion 82

Chapter 6. Optimization of nucleofection for efficient transfection of LGAC
Rationale 85
Transfection of LGAC for DNA plasmid delivery 85
Transfection of LGAC for siRNA delivery 88
Evaluation of nucleofection cytotoxicity 91
Discussion 92

Chapter 7. Conclusions and Perspectives 93

References 98

vi

List of Figures

Figure 1 Lacrimal gland acinar cell morphology 4
Figure 2 Schematic diagram of endocytic pathways 10
Figure 3 Adenovirus 5 structure 15
Figure 4 BNP photostability 32
Figure 5 BNP12 is not markedly colocalized with lysosomal compartments 34
Figure 6 Disruption of the cytoskeleton affects BNP12 uptake 36
Figure 7 MCoTI-I distribution in HeLa cells 42
Figure 8 Endocytosis of MCoTI-I in HeLa is temperature-dependent 44
Figure 9 Colocalization of MCoTI-I with markers of endocytosis in HeLa 46
Figure 10 Disruption of actin does not inhibit MCoTI-I uptake 48
Figure 11 Effect of EIPA treatment on MCoTI-I uptake 50
Figure 12 MCoTI-I is colocalized with lysosomal compartments 53
Figure 13 MCoTI-I-containing vesicles are in microtubule-associated motion 55
Figure 14 MCoTI-I distribution in LGAC 56
Figure 15 Endocytosis of MCoTI-I in LGAC is temperature-dependent 58
Figure 16 Colocalization of MCoTI-I with marker of endocytosis in LGAC 60
Figure 17 Uptake of penton base protein and Ad5 65
Figure 18 Penton base remains surface-bound in LGAC 67
Figure 19 LGAC express α
v
integrins 68
Figure 20 Fiber protein acquires trypsin resistance in LGAC 69
Figure 21 Inhibitors of macropinocytosis do not prevent fiber entry 70
Figure 22 Effect of Bafilomycin A1 on Ad5 uptake and binding 72
vii

Figure 23 Effect of Chlorpromazine on Ad5 uptake and binding 73
Figure 24 Effect of dynamin inhibitors on Ad uptake 75
Figure 25 Effect of Methyl- β-cyclodextrin on Ad5 uptake and binding 76
Figure 26 Effect of Nocodazole on Ad5 uptake and binding 77
Figure 27 Ad5 is not colocalized with EGF 79
Figure 28 Effect of Nocodazole on intracellular Ad5 distribution 81
Figure 29 Nucleofection of LGAC 86
Figure 30 Nucleofected LAC are able to accurately localize a vesicular 87
compartment protein

Figure 31 Transfection of LGAC 88
Figure 32 Effective gene expression knockdown 90
Figure 33 Nucleofection does not affect cell viability 91

viii

Abbreviations

HPV Human Papilloma Virus
CPP Cell-penetrating peptide
BF
2
dbmPLA Difloroboron dibenzoylmethane-polylactide
BNPs Difloroboron dibenzoylmethane-polylactide nanoparticles
CHO Chinese hamster ovary
CCK Cyclic cystine knot
EGF Epidermal growth factor
LGAC Lacrimal gland acinar cells
Ad Adenovirus
Ad5 Adenovirus serotype 5
CAR Coxsackievirus and adenovirus receptor
CME Clathrin-mediated endocytosis
Ad2 Adenovirus serotype 2
AP2 Adaptor protein complex 2
ER Endoplasmic reticulum
MW Molecular weight
CTX-B Cholera toxin B
GPI Glycophsophatidylinositol
MBCD Methyl- β-cyclodextrin
EIPA 5-(N-ethyl-N-isopropyl)amiloride
TR-EGF Texas Red-EGF
LysoRed LysoTracker Red
ix

LysoBlue LysoTracker Blue
AF594-CTX-B AlexaFluor 594-CTX-B
TR-10K Dex Texas Red-10,000 MW dextran
FITC Isothyocyanate
FITC-10K Dex Isothyocyanate-10,000 MW dextran
AF488-EGF AlexaFluor 488-EGF
RFP-Lamp1 Red fluorescent protein-lysosomal-associated membrane protein 1
NOC Nocodazole
Lat B Latrunculin B
CPZ Chlorpromazine
Baf A1 Bafilomycin A1
MCoTI-I Momordica cochinchinensis trypsin inhibitor I
MCoTI-II Momordica cochinchinensis trypsin inhibitor II
AF488-MCoTI-I AlexaFluor 488-MCoTI-I
ATCC American Type Culture Collection
PLA Polylactide
PEG Poly(ethylene glycol)
PLGA Polylactide-polyglycolide
RIPA Radioimmunoprecipitation
ROI Region of interest
Eth-D1 Ethidium homodimer-1
HPRT1 hypoxanthine phosphoribosyltransferase 1

x

Abstract

When identifying potential candidates for drug delivery or biomedical diagnostic tools, it
is of utmost importance to delineate, as best as possible, their uptake and intracellular
trafficking pathways. Here we investigated these pathways, in a simple cell model first,
followed by a more complex and physiologically-relevant cell model, for three potential
theranostic and drug delivery candidates. The first is a nanoparticle based on
difluoroboron dibenzoylmethane-poly(lactic acid), that exhibits unique molecular-weight
dependent emission properties. We showed that these nanoparticles are highly
photostable, in vitro and in situ, resisting laser-induced photobleaching under conditions
that destroy the fluorescence associated with a common photostable probe, LysoTracker
Blue. Also, we showed that the internalized nanoparticles do not accumulate in acidic
compartments such as late endosomes and lysosomes, but rather in a non-lysosomal
perinuclear compartment. Additionally, we demonstrated that their uptake utilizes actin
filaments and microtubules. These findings demonstrate the feasibility of using these
nanoparticles with unique emission properties for in situ, live cell imaging.
The second candidate examined is a cyclotide, MCoTI-I. Cyclotides are plant-derived
proteins that naturally exhibit various biological activities and whose cyclic structure
makes them remarkably stable and resistant to denaturation and degradation. Using real-
time confocal microscopy imaging, we showed that MCoTI-I is readily internalized in
HeLa and lacrimal gland acinar cells and that its endocytosis is temperature-dependent.
Endocytosis of MCoTI-I is achieved primarily through fluid-phase endocytosis in HeLa
xi

as evidenced by its significant colocalization with 10,000 MW dextran, but also through
other pathways as well as evidenced by its colocalization with cholera toxin-B and
epidermal growth factor. Uptake does not appear to occur only via macropinocytosis as
inhibition of this pathway did not affect MCoTI-I uptake. In lacrimal gland acinar cells,
endocytosis is also achieved primarily through fluid-phase endocytosis, albeit to a lesser
extent. In HeLa, a significant amount of MCoTI-I accumulates in late endosomal and
lysosomal compartments where MCoTI-I-containing vesicles continue to exhibit
microtubule-associated movements. In contrast, almost no MCoTI-I reaches acidic
compartments in lacrimal gland acinar cells in the same time frame. These findings
demonstrate internalization of MCoTI-I through multiple pathways that may be dominant
in the cell type investigated, and suggest that this cyclotide has ready access to general
endosomal pathways.
The third and final candidate investigated is Adenovirus 5. The established method of
Adenovirus 5 infection in most cells is by a penton-dependent mechanism. Here, we
demonstrated that while the penton base remains on the surface of lacrimal gland acinar
cells, fiber is readily internalized, suggestive of a fiber-dependent entry mechanism. We
also determined that macropinocytosis is not the prevalent route of uptake. Data from
studies involving various endocytosis inhibitors suggest that uptake of Adenovirus 5 in
lacrimal gland acinar cells may rely on multiple pathways other than primarily clathrin-
mediated endocytosis. Additionally, binding of Adenovirus 5 to its surface receptor
appears to be cholesterol-dependent. Also, the inward movement of internalized
Adenovirus 5 seems to rely on the microtubule network. Taken together, these studies
xii

indicate that the internalization and intracellular trafficking of Adenovirus 5 in lacrimal
gland acinar cells follow a different pathway than what is reported in the literature, and
may involve very complex and compensatory mechanisms.
Together, these findings demonstrate the unique challenges encountered on the path to
assessing the utility and feasibility of potential theranostics and drug delivery candidates.
They also highlight the importance of using physiologically- and disease-relevant cell
models, especially since information obtained from one cell type is likely not applicable
in another.

1

Chapter 1. Introduction

Foreword
Great scientific discoveries often begin with small observations. These, in turn, lead to a
great number of questions. As with all other things, one must address the simpler
questions first, preferably in a system that is relatively easy to decipher and explain. Once
we possess this information, we can pursue more complex questions in a correspondingly
more complex model. The work presented here attempts to accomplish precisely this.
Herein, we take a series of drug and delivery constructs and investigate their endocytosis
and intracellular trafficking patterns in a simple cell model first, followed by exploration
of processes deemed potentially valuable in a more complex and physiologically-relevant
cell model. Our goal is to understand these processes for identification of viable pathways
for uptake of potential candidates for theranostics and drug delivery.

The origin and scientific importance of HeLa cells
Cell models have long been a cornerstone for scientific discoveries. Perhaps the most
important cells to date have been those derived from the cervical adenocarcinoma of Mrs.
Henrietta Lacks in 1951. Known as HeLa cells, they became the first immortalized
mammalian cells capable of growing in culture. While there has been a long standing
debate over patient rights, informed consent, and whether these cells were obtained in an
ethical manner, the truth remains that these cells have contributed significantly to
scientific advancements made in the last 50 years. Among these include identification of
2

essential amino acids necessary for cell culturing (45), determination of the number of
human chromosomes (96), development of the polio vaccine (164), and elucidation of the
link between Human Papilloma Virus (HPV) and cancer (14, 90, 145, 175). Today, these
cells continue to be essential in a range of research fields including Acquired
Immunodeficiency Syndrome (AIDS) (4, 52, 126, 134), Parkinson’s Disease (5, 48, 57,
193), and of course, cancer (51, 91, 124, 136, 204). Their utility, however, has
transcended merely research of disease states. HeLa cells are routinely and extensively
used in basic research and have been especially valuable in studies of drug targeting and
delivery. They have been used in characterizations of viral (9, 12, 22, 82, 167),
nanoparticle (20, 38, 125, 168, 192), cell-penetrating peptide (CPP) (44, 53, 86, 211), and
quantum dot (2, 112, 205-206) endocytosis and trafficking. Because of their versatility,
HeLa cells have essentially become the ―go-to‖ cell type for initial investigations of
endocytosis and intracellular trafficking in mammalian cells. They are inexpensive, easy
to grow, and because of their long history, very well-studied. They serve as an important
first step from which pertinent observations can be made, and information gathered,
before progressing to a more relevant cell type or model. As useful as HeLa cells are, the
scope of information that can be attained from their use is limited by the inherent
characteristics of a transformed cell line. Although significant information can be
extracted, this information will not necessarily be indicative of the in vivo, or even in
vitro, behavior of a particular cell or tissue type. It is imperative that studies involving
more complex physiological systems, or cells representative of those systems, follow
those completed in simple cell models.
3

The lacrimal gland in health and as a therapeutic target
Maintenance of the tear film is crucial for ocular surface homeostasis, protection against
bacterial and viral infections, and provision of a smooth optical surface for light
refraction. It consists of three layers: the mucin, aqueous, and lipid layers. Goblet cells
present in the conjunctiva contribute the mucins that comprise the innermost, mucin
layer. The outermost lipid layer consists of a mixture of lipids secreted by the meibomian
glands. It is to the middle, aqueous layer that the lacrimal gland is the major contributor
(157). The lacrimal gland, along with accessory glands, is the tissue responsible for
maintaining the healthy integrity of the ocular surface. This is accomplished by regulated
secretion of tear proteins and fluid by the secretory lacrimal gland acinar cells, which
comprise over 80% of the gland. In addition to secreting water and electrolytes, these
cells also secrete lactoferrin, secretory IgA, lysozymes, hydrolases, and epidermal growth
factor (EGF) (39, 55, 116, 188) into the tear fluid. Lacrimal gland acinar cells (LGAC),
when isolated and reconstituted in culture, re-form their acinar-like structure and
maintain their polarity, as evidenced by the enrichment of actin filaments at the apical
plasma membrane (Figure 1).
4

Figure 1. Lacrimal gland acinar cell morphology. LGAC were processed to fluorescently label
actin (red) and nuclei (blue). A schematic is included to indicate the location of lumena (L) which
are bounded by the apical plasma membrane of these epithelial cells. The basolateral membranes
are also indicated. Bar = 10 µm.

The most common cause of dry eye disease is associated with deficiencies in the aqueous
layer of the tear film. This is most often attributed to decreased tear secretion by the
lacrimal gland (176-177) , often in combination with increased tear evaporation. A few of
the causes for reduced secretion include age (116, 187), contact lens wear (49), and
Sjögren’s Syndrome, a chronic autoimmune disease that affects most notably the lacrimal
and salivary glands, leading to severe dry eye and mouth (131, 157). Additionally,
changes in the composition of the aqueous layer combined with slow tear turnover can
lead to damage of the ocular surface (157). Current treatments for dry eye offer little in
5

terms of sustained relief. Among them are eye drops, including artificial tears, punctal
plugs, which serve to retain tears on the ocular surface for a longer period of time, and
prescription drugs, such as Restasis
®
, that suppress inflammation which disrupts proper
tear secretion. These options are all symptom-treating and do not address the underlying
cause of the disease. Manipulation of the lacrimal gland, to restore or repair function,
could potentially help treat not only dry-eye diseases, but also chronic diseases of the
cornea and front of the eye.
One such manipulation option is through modulation of gene expression, either through
gene therapy or siRNA. While a number of viral vectors have been tried for delivery to
the lacrimal gland (8), the most successful has been the use of Adenovirus (Ad)-mediated
gene delivery (165). Despite the successes of Ad-mediated delivery to the lacrimal gland,
major drawbacks impair its potential clinical use. One is the unstable transgene
expression attributed to a lack of integration into the host cell genome (68), and the other,
perhaps more serious, is the strong immune response elicited by these vectors (19, 150).
Such a response could pose a serious threat to the safety of patients. One way to
overcome this might be the use of viral capsid proteins, which coat the surface of the
virus itself. While these proteins, in their native form, can also elicit an immune response,
engineering of capsid proteins that maintain their ability to gain cellular entry, but do not
evoke an immune response might help circumvent the problem. It will be necessary,
however, to have a thorough understanding of the endocytosis and intracellular
trafficking mechanism(s) of the capsid protein(s), along with knowledge of the minimum
protein sequence necessary to elicit that mechanism, to ensure a successful approach.
6

Non-viral gene delivery is likewise a viable approach. Lipid-based methods are well-
established and commonly used in numerous cell types. Historically, however, primary
cultured LGAC have exhibited very low transfection efficiency using non-viral, lipid-
based methods. The difficulty with gene expression using typical transfection reagents
extends beyond plasmid DNA delivery to siRNA. Previous attempts at siRNA delivery in
LGAC using a cationic lipid reagent resulted at best in a 30% knockdown of the target
protein (202). It has also been shown that gene delivery using non-viral vectors, while
simple to use and inexpensive, is often dependent on a number of factors for
effectiveness, many of which are difficult to control in primary cultures and in terminally
differentiated cells (185). For these reasons, alternative non-viral methods for DNA and
siRNA delivery to LGAC must be explored. One possibility is the use of nucleofection, a
transfection method based on the physical method of electroporation. It uses a
combination of electrical parameters with cell-type specific reagents to enable transfer of
nucleic acids directly into the nucleus, and because it does not rely on cell division,
allows for transfection of non-dividing cells. It has recently been shown to be successful
in a number of cell types that have previously proven difficult to transfect (41-42, 171,
180, 207), and may offer an alternative approach for successful transfection of LGAC.
Additional possibilities for treatment of disease in lacrimal gland acinar cells include
delivery of macromolecular drugs for uptake into cellular compartments, such as
lysosomes. The protease, Cathepsin S, is known to be upregulated and implicated in the
disease development of Sjögren’s syndrome (74, 107). Introduction of potential inhibitors
of Cathepsin S selectively to lacrimal gland acinar compartments, while evading delivery
7

to regions of the body where Cathepsin S is physiologically necessary, is a desirable goal.
Some peptide scaffolds constitute new potential inhibitors for Cathepsin S and thus, their
internalization into acinar cells is likewise of interest.

Brief review of endocytosis
Endocytosis is the process by which internal membranes are produced by, and detached
from, the plasma membrane lipid bilayer. The process, which can occur through a
multitude of ways, is vital for the cell’s ability to communicate with, and sample, the
extracellular environment, as well as take up a variety of essential materials necessary for
proper functioning. Additionally, the process is often exploited by pathogens to gain
cellular entry. The following list is in no way complete as there are a number of
additional pathways described in the current literature. Some are better understood than
others with new ones being noted every day. Perhaps the most investigated and best
understood mechanism is the clathrin-mediated endocytosis (CME) pathway. This
pathway often involves uptake of high-affinity receptors and their bound ligands into
clathrin-coated pits on the plasma membrane. The pits are formed by assembly of
cytosolic coat proteins, mainly clathrin and the adaptor protein complex 2 (AP2) (26).
The other component of the machinery driving CME is dynamin, a multidomain GTPase
that is recruited to the necks of coated pits, where it assembles into a spiral ―collar‖ that
upon GTP hydrolysis, mediates release of clathrin-coated vesicles (147). Once inside the
cell, the pits form part of the classic endosome netwok, beginning with early endosomes
8

and ending with lysosomes or recycling endosomes, acquiring and losing cellular factors
along the way (123). CME is the most commonly observed pathway in virus entry.
Among the cargo known to pass through this route are Ad, G-protein-coupled receptors,
and transferrin receptors (43). Chemical approaches that have been used to inhibit this
pathway include potassium depletion (99), hypertonic sucrose (67), cytosolic
acidification (28, 163), and use of chlorpromazine (85). The first two function by
removing or dispersing the clathrin lattices associated with the plasma membrane.
Cytosolic acidification inhibits the budding off of the coated pit, while chlorpromazine is
a cationic amphipathic drug that inhibits CME of various plasma membrane proteins.
A second endocytic mechanism is the caveolae/caveolin-1-dependent pathway. Caveolae
are flask-shaped invaginations that are present in cholesterol and sphingolipid-rich
domains of the plasma membrane. The shape and structure of caveolae are given by
caveolin-1, a dimeric protein that binds to cholesterol, inserts into the inner leaflet of the
plasma membrane, and self-associates to form a caveolin coat on the surface of the
membrane invaginations (26, 43). Dynamin recruitment has been implicated in this
pathway as well (75, 135), and the actin cytoskeleton is believed to play a significant role
in the initial uptake and inward transport (140). Endocytosis via caveolae leads to
formation of cytoplasmic organelles known as caveosomes. These organelles are stable,
cholesterol-enriched structures that are devoid of markers of the classical endocytic and
biosynthetic organelles, including the known endocytic Rab GTPases (139). Additionally,
caveosomes do not accumulate ligands endocytosed via CME, nor do they accumulate
detectable amounts of fluid phase markers (140). Although the intracellular fate of
9

caveosomes has not been entirely elucidated, data suggests that ligands entering via
caveolae can be directed to the endoplasmic reticulum (ER), the Golgi apparatus, or can
transiently interact with early endosomes (94, 100). Among the cargoes using this
pathway are Cholera toxin B (CTX-B), SV40 virus, and glycophosphatidylinositol (GPI)-
linked proteins (43). Some of the chemical approaches used to inhibit this pathway
include the use of statins, which inhibit an enzyme important in cholesterol biosynthesis
(108, 170), use of methyl-β-cyclodextrin (MBCD), which depletes membrane cholesterol
(92), and use of filipin and nystatin, both of which sequester membrane cholesterol (85).
Macropinocytosis is a signal-triggered endocytic mechanism used by cells to internalize
larger amounts of fluid and membrane into large vacuoles called macropinosomes. These
organelles are formed by closure of characteristic membrane ruffles triggered by
activation of receptor tyrosine kinases and subsequent downstream changes in actin
dynamics (101, 109, 122). Once inside the cell, macropinosomes either recycle back to
the cell surface or acquire early endosome proteins required for homotypic fusion (64, 77,
149). While Na
+
/H
+
exchangers are important for this endocytic process, dynamin does
not appear to be (122, 133). Interestingly, it has been shown that this pathway is induced
in the presence of Ad, not for the purpose of gaining cellular entry, but rather to enhance
its acid-stimulated endosomal escape (120). Fluid phase markers and receptor tyrosine
kinases are among the cargoes implicated in this pathway. Because macropinosomes lack
a specific protein or lipid coat, it has been difficult to identify targets for pharmacological
inhibition of this pathway. Still, some of the chemical approaches used thus far include
use of sodium-proton exchanger inhibitors such as amiloride and its derivative 5-(N-
10

ethyl-N-isopropyl)amiloride (EIPA) (85), and F-actin depolymerizing drugs such as
cytochalasin D or latrunculin (142).

Figure 2. Schematic diagram of endocytic pathways. The three pathways discussed, and a
representative of their corresponding characteristic cargo, are depicted.

Drug and delivery constructs
The candidates examined in this work represent 3 very distinct approaches to the
development of theranostics, therapeutics, and delivery devices. They include a
nanoparticle composed of a polymer-dye conjugate, a cyclic micro-protein, and a virus.
The nanoparticles have potential in theranostic applications including sensing and
11

imaging. The cyclic micro-protein, also known as a cyclotide, could potentially be useful
in both therapeutic applications and drug targeting and delivery applications. Finally, the
viral candidate has the most potential in targeting and delivery applications. Each of these
candidates is discussed in further detail below.

Difloroboron dibenzoylmethane-polylactide nanoparticles
Light-emitting materials are very useful for biological imaging and sensing. Especially
desirable are systems in which the emission wavelengths are easily controllable.
Nanoparticles comprised of a fluorescent dye conjugated to a polymer, such as
polyethylene glycol, have been used to track endocytosis and intracellular localization
(184). Phosphorescence is also a very useful tool for imaging and sensing. It is especially
useful for oxygen sensing and imaging, including imaging of oxygen distribution in
tissue (6, 158, 190, 201). Additionally, nanosensors made from boron biomaterials, with
dual-emissive properties, have been shown to enable tumor hypoxia imaging with good
spatial and temporal resolution (209). Difloroboron dibenzoylmethane-polylactide
(BF
2
dbmPLA) is comprised of two parts: polylactic acid and a boron dye. Polylactic acid
or polylactide is a degradable polymer that is commonly used as a key component of
diverse biomedical devices such as bioresorbable sutures and implants, and drug delivery
systems (62). The boron dye component exhibits a large extinction coefficient, making
photon absorption in a given wavelength more likely, high emission quantum yield,
indicating high efficiency of the fluorescence process, two-photon absorption, allowing
12

high-depth tissue imaging, and sensitivity to the surrounding environment (21, 24).
Additionally, BF
2
dbmPLA has been shown to exhibit molecular weight-dependent
luminescence, as a single-component, when formulated into films and bulk materials
(208). Nanoparticles made from BF
2
dbmPLA, difloroboron dibenzoylmethane-
polylactide nanoparticles (BNPs) have been shown to retain their color-tunable
fluorescence in aqueous environments as well as the within the cellular milieu (143).
These were also shown to be internalized, and accumulate in an unknown perinuclear
compartment, in Chinese hamster ovary (CHO) cells (143). To expand upon our
understanding of the intracellular endosomal environment, and explore their potential as
possible theranostic tools, we explored the internalization behavior of these nanoparticles
in HeLa and LGAC.

The Momordica cochinchinensis trypsin inhibitor: MCoTI-I
Cyclotides are micro-proteins naturally expressed in plants ranging from 28 to 37 amino
acids. They exhibit various biological activities such as insecticidal, anti-microbial,
antiviral, and protease inhibitory (31-32, 56, 87). They share a unique head-to-tail cyclic
structure, called the cyclic cystine knot (CCK), that renders them remarkably stable as
well as resistant to chemical and thermal denaturation and enzymatic degradation (37,
56). In fact, these proteins were first described in a medicinal purpose where dry leaves
from the African plant Oldenlandia affins DC were boiled and given to women to
accelerate contractions and childbirth (161). While initial chemical investigations
13

indicated the presence of serotonin, the primary protein component was found to be a
uteroactive cyclotide (161). Cyclotides have since been isolated from a variety of plants
(18, 37, 146, 161) and while sequences have been reported for about 160 different
cyclotides (128, 195), it is estimated that approximately 50,000 might exist (63, 127).
They have been classified into three main families: the Möbius, the bracelet, and the
trypsin inhibitors (35, 76). The trypsin inhibitors, Momordica cochinchinensis trypsin
inhibitor I (MCoTI-I) and Momordica cochinchinensis trypsin inhibitor II (MCoTI-II),
were isolated from the seeds of Momordica cochinchinensis, a member of the
cucurbitaceae plant family. While MCoTI cyclotides do not share significant sequence
homology with other cyclotides beside the characteristic knot, they do share high
sequence homology with related linear cytine-knot squash trypsin inhibitors (76). These
squash trypsin inhibitors have recently been used as scaffolds for the incorporation of
novel bioactive peptides (95, 151), offering prospect for the use of MCoTI cyclotides as
molecular scaffolds for drug design (23, 31). Earlier we discussed the implication of
Cathepsin S in the development of Sjögren’s syndrome, and the use of inhibitors to
modulate its function. Cyclotides, some of which naturally exhibit protease inhibitory
activity, could potentially offer a sensible option for this therapeutic approach.
All cyclotides share the characteristic CCK motif despite their sequence diversity, and
with the exception of the conserved cysteine residues that comprise the knot, tolerate
mutation of all other residues. Cyclotides are naturally produced in plants, but can also be
produced chemically (36, 181-182) or recombinantly in bacteria (17, 93, 191), the latter
of which allows for production of folded cyclotides in vivo or in vitro using bacterial
14

expression systems (7, 17). The ease of production allows for the generation of large
libraries of these cyclotides that could be screened for selection of sequences that bind to
specific targets. Cyclotides offer many features that render them ideal drug development
tools. They are relatively small, remarkably stable, amenable to sequence variation, and
relatively easy to produce. More importantly, they were recently shown to gain cellular
entry in mammalian cells (61).

Adenovirus serotype 5
Adenovirus serotype 5 (Ad5) is a class C, non-enveloped, double-stranded DNA virus
with a 36KBP genome. The viral particle is composed of a protein shell, known as the
capsid, surrounding the DNA-protein core complex. Its icosahedral shape is conferred in
part by the 240 hexon capsomeres forming the 20 triangular facets (186). At each of the
twelve vertices, the trimeric fiber and pentameric penton capsid proteins form complexes
(15, 160) (Figure 3A). The fiber protein consists of two regions separated by an elongated
shaft (Figure 3B) containing repeating motifs of approximately 15 residues (40) . At the
proximal end, the tail region is bound to the penton base. The globular ―knob‖ region is
located on the distal end (40) and serves to mediate host cell attachment by noncovalent
binding to the extracellular D1 domain of the coxsackievirus and adenovirus receptor
(CAR) (10-11, 156, 183). The penton capsid protein participates in viral host cell
internalization by interacting with surface α
v
integrins to trigger endocytosis (114, 199).
15

Figure 3. Adenovirus 5 structure. (A). A stylized section of the virus particle showing the
relative orientation of the capsid proteins and viral DNA. (B). A representation of the fiber
protein domains.

The commonly accepted mode of Ad5 internalization begins with the host cell attachment
mediated by the binding of fiber knob to CAR on the cell surface. CAR, as mentioned
above, is the receptor that binds both Ad and group B coxsackievirus. The 46 kDa protein
is a member of the immunoglobulin (Ig) superfamily and has two Ig-like extracellular
domains (10). Its in vivo function is believed to be as a cell adhesion molecule mediating
homotypic cell adhesion as a component of tight junctions in polarized epithelial cells
(25, 29, 144). Following the initial fiber knob-CAR interactions, penton interactions with
surface integrins, mediated by a conserved Arg-Gly-Asp (RGD) sequence on the exterior
of the penton base, trigger virus internalization via CME (114, 121, 130). Penton is also
believed to play a role in subsequent endosomal escape of the virus (60, 130, 196),
16

although acid-stimulation has also been suggested to play a role (60, 155, 166). Once in
the cytosol, the virus travels along the microtubule network (106, 178) to the nuclear
membrane where it is imported via the nuclear pore complex (59).
Adenoviruses most often cause mild infections of the upper and lower respiratory tract
(104). While CAR is present in the airway epithelium, in the intercellular junctions and
the basolateral membrane, it is not readily accessible on the apical side which would be
the most opportunistic site for infection. For this reason, it has been suggested that more
virulent infections may occur in one of two possible ways: The first is through the release
of soluble fiber, by an infected cell, which in turn disrupts CAR-mediated cell-cell
adhesion (159, 194) . The other is via viral triggering of a cytokine response which in
turn would affect the integrity of the tight junctions (30). In any case, the result would be
greater accessibility of the receptor to the virus. Because human adenoviruses do not
replicate in rodent animal models, any clinical information regarding the native tropism
comes from experimental data involving gene therapy experiments in which a high viral
dose is administered intravenously (210). In these cases, the majority of replication –
defective Ad5 or Adenovirus serotype 2 (Ad2), another class C virus with high homology
to Ad5, is taken up by the liver (1, 103, 173). In only one study was the hepatotropism of
the virus greatly reduced and it necessitated removal of both the CAR and integrin
interactions (46). Given this strong in vivo tropism, it would be imperative to identify
specific modifications that would effectively aid in de-targeting of the liver to allow for
successful transduction of targeted tissues.
17

Chapter 2: Materials and Methods

Reagents: Texas Red-EGF (TR-EGF), LysoTracker
™
Red DN-99 (LysoRed),
LysoTracker
™
Blue (LysoBlue), AlexaFluor 594 CTX-B (AF594-CTX-B), Texas Red
10,000 MW dextran (TR-10K Dex), isothyocyanate (FITC) 10,000 MW dextran (FITC-
10K Dex), AlexaFluor 488 EGF (AF488-EGF), the LIVE/DEAD viability/cytotoxicity
kit for mammalian cells, CellLight
™
Lysosomes-RFP lysosomal-associated membrane
protein 1 (RFP-Lamp1), Lipofectamine
™
2000, DAPI, rhodamine-phalloidin, Alexa Fluor
647-phalloidin, and ProLong antifade mounting media were purchased from Invitrogen
(Carlsbad, CA). Latrunculin B (Lat B) was purchased from EMD Biosciences (San
Diego, CA). 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) was purchased from Alexis
Biochemicals (San Diego, CA) or Sigma-Aldrich (St. Louis, MO). Methyl-β-cyclodextrin
(MBCD), nocodazole (NOC), chlorpromazine (CPZ), and bafilomycin A1 (BafA1) were
purchased from Sigma-Aldrich (St. Louis, MO). The Dynamin Inhibitors Toolbox was
purchased from Ascent Scientific (Princeton, NJ). The Basic Nucleofector Kit for
Primary Epithelial Cells and the pmaxGFP plasmid were purchased from Lonza
(Gaithersburg, MD). GeneSilencer siRNA transfection reagent was purchased from
Genlantis (San Diego, CA). Fugene 6 was purchased from Roche (Rotkereuz,
Switzerland). The siControl non-targeting siRNA was obtained from Dharmacon RNA
Technologies (Lafayette, CO). CAR siRNA duplex (sense sequence: 5’-
GGUCAGAAGAAAUUGGAAATT-3’; antisense sequence: 5’-
UUUCCAAUUUCUUCUGACCTT-3’) was ordered from the USC/ Norris Cancer
18

Center DNA Core (Los Angeles, CA). The Cathepsin S-GFP plasmid was constructed
using the full-length cDNA of mouse cathepsin S in plasmid pCMV-SPORT6 (Open
Biosystems, Huntsville, AL). Matrigel was obtained from Collaborative Biochemicals
(Bedford, MA).

Antibodies: Rabbit polyclonal antibody to Ad5 was purchased from Access Biomedical
(San Diego, CA) or Abcam (Cambridge, MA). Mouse monoclonal antibody to Ad5 fiber
protein was obtained from Novus Biologicals (Littleton, CO). Mouse monoclonal
antibody to Cathespin S protein was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Goat anti-rabbit and anti-mouse IRDye 800-conjugated and IRDYE700-
conjugated secondary antibodies were purchased from Rockland Immunochemicals, Inc.
(Gilbertsville, PA). DyLight 488-conjugated donkey anti-goat secondary antibody was
purchased from Jackson ImmunoResearch (West Grove, PA). Mouse monoclonal
antibody to α-tubulin was purchased from Sigma-Aldrich (St. Louis, MO).

Difluoroboron dibenzoylmethane-polylactide nanoparticles: BNPs of various
molecular weights were fabricated, characterized, and kindly provided by the laboratory
of Dr. Cassandra L. Fraser (University of Virginia, Charlottesville, VA).

19

Alexa Fluor 488-labeled trypsin inhibitor cyclotide: The Alexa Fluor 488-labeled
Momordica cochinchinensis trypsin inhibitor I (AF488-MCoTI-I) cyclotide was
synthesized, characterized, and kindly provided by the laboratory of Dr. Julio A.
Camarero (University of Southern California, Los Angeles, CA).

HeLa cell culture: HeLa cells were obtained from the American Type Culture Collection
(ATCC) and were cultured in a humidified incubator at 37°C in 95% air/5% CO
2
in
phenol-red-free Dulbecoo’s modified essential medium (DMEM) (4.5 g/L glucose with
10% FBS, 1% glutamine, and 1% nonessential amino acids) and split with trypsin/EDTA
as recommended by the manufacturer. Cells were routinely analyzed at low passage in
the subconfluent state, although they were not synchronized.

Lacrimal gland acinar cell isolation and primary culture: All animal work was
performed in accordance with all institutional guidelines. Female New Zealand White
rabbits weighing between 1.8 and 2.2 kg were obtained from Irish Farms (Norco, CA).
LGAC were isolated and maintained in a laminin-based primary culture system for 2-3
days as described previously (34). These culture conditions result in reconstitution of
polarity, establishment of lumena, and formation of secretory vesicles (34, 58, 65, 88,
203).

20

Generation of recombinant proteins and Ad5 vectors
Recombinant knob protein, green fluorescent protein (GFP)-knob protein, and penton
base protein were produced from Escherichia coli as His
6
-tagged fusion proteins and
purified from bacterial lysates using Ni-NTA resin as described previously (66, 118-120,
153). Recombinant fiber protein was produced from a baculovirus expression system as
described previously (152). These recombinant proteins were kindly made by Hua Pei.
Replication-defective Ad5 containing the β-galactosidase reporter gene (AdLacZ), a
construct previously generated and described by our lab (198), was amplified kindly by
Francie Yarber. Briefly, the virus was propagated in QBI cells, grown in DMEM
containing 10% FBS at 37°C and 5% CO
2
, until the cells showed the typical
cytophathological effect. The cells were then harvested and purified on CsCl gradients by
ultracentrifugation. Viral titers were measured by plaque assay.

Cell-binding biochemical assays
To asses surface binding and subsequent uptake of recombinant penton protein and Ad5,
control HeLa or LGAC treated with or without recombinant penton (20 µg/ml) or Ad5
(MOI=15) for 0 min and 60 min at 37°C were then exposed to 0.2 mg/ml trypsin-EDTA
at 4°C for 1 h. The cells were lysed in radioimmunoprecipitation (RIPA) buffer by 15
passages through a 23-gauge syringe needle followed by two freeze-thaw cycles. The
lysate was centrifuged for 5 min at 5,000 rpm at 4°C and the supernatant collected. The
cell lysates (100-150 ug) were blotted with appropriate primary antibodies (rabbit
21

polyclonal anti-Ad5 antibody for penton detection and mouse monoclonal anti-fiber
antibody for Ad5 detection) and IRDye800 or IRDye700-conjuated secondary antibodies.
A similar protocol was used to evaluate Ad5 uptake (MOI=15) in LGAC following
various treatments. Cells treated with either EIPA (500 µM) or Lat B (10 µM) were
pretreated for 1 h at 37°C prior to addition of AdLacZ. For nocodazole (33 µM), cells
were first pre-cooled and following addition of NOC, were incubated for 30 min at 4°C
prior to addition of AdLacZ. For CPZ (10 µg/ml) and Baf A1 (50 nM), cells were
pretreated for 2 hrs at 37°C prior to addition of AdLacZ. For MBCD (5 mM), cells were
pretreated for 30 min at 37°C. For the dynamin inhibitors (30 µM) cells were pretreated
for 15 min at 37°C prior to addition of AdLacZ.

Confocal fluorescence microscopy
For BNP uptake studies, HeLa cells were seeded on 35 glass-bottom culture dishes at a
density of 8.5 x 10
4
cells/dish. On day 2 of culture, the cells were rinsed with phosphate-
buffered saline (PBS) and the media replaced with incubation buffer (phenol-red-free,
serum-free DMEM with 1% penicillin/streptomycin and 20 mM HEPES) prior to the
addition of either BNP (200 µL, dropwise) and incubation at 4°C for 1 hour. Unbound
BNPs were rinsed off with a gentle PBS wash, the incubation buffer replaced, and the
cells warmed to 37°C for 1 hour in the absence or presence of treatments. After
incubation, the cells were rinsed four times with PBS prior to imaging. For most studies,
BNPs were excited in intact cells using the 790 nm line of the multiphoton laser. For
22

assessment of colocalization of BNP and LysoTracker
™
Red, a Zeiss LSM 510 confocal
microscope equipped with UV, argon, and green HeNe lasers was used for confocal
fluorescence. For Lat.B pretreatment, 2 µM Lat B was added for 30 min at 37°C prior to
addition of BNPs. For NOC treatment, 33 µM NOC was added with the BNPs prior to
incubation at 4°C. For the colocalization study, LysoTracker
™
Red (50 nM) was added to
the cells prior to incubation at 37°C. For immunostaining, following treatment with either
33 µM NOC or 2 µM Lat B, the cells were fixed in 4% paraformaldehyde as previously
described (33-34, 88) prior to the addition of mouse monoclonal antibody to α-tubulin
and appropriate secondary fluorophore-conjugated antibody.
For MCoTI-I uptake studies in HeLa cells, these were seeded on 35 mm glass-bottom
dishes at a density of 8.5 x 10
4
cells/dish. As before, on day 2 of culture, the cells were
rinsed with PBS and the media replaced with incubation buffer prior to addition of
AF488-MCoTI-I (25 µM) and incubation at 37°C for 1 hour. Following this time, excess
MCoTI-I was rinsed off with a gentle PBS wash and the media replaced prior to imaging.
Intracellular distribution was analyzed at 1 hr and again at regular intervals for up to 10
hours. For analysis of the effects of Lat B or EIPA pre-treatment, 2 µM Lat.B or 50 µM
EIPA was added for 30 min at 37°C prior to addition of AF488-MCoTI-I. For analysis of
the effect of NOC, 33 µM was added to pre-cooled cells and incubated at 4°C for 30 min
prior to the addition of AF488-MCoTI-I. For colocalization studies, LysoTracker
™
Red
(50 nM), AF594-CTX -B (10 µg/ml), TR-10K Dex (1 mg/ml), or TR-EGF (400 ng/ml)
was added to cells simultaneously with AF488-MCoTI-I prior to incubation at 37°C.
Analysis of the extent of colocalization was done at 1 hr of uptake. For colocalization
23

with RFP-Lamp1, cells were treated with RFP-Lamp1-expressing BacMam (2 x 10
7

particles/plate) on the previous day. For temperature-dependent uptake studies, cells were
cooled on ice for 30 min prior to the addition of AF488-MCoTI-I in incubation buffer.
After incubation at 4°C for 30 min, the cells were imaged and subsequently incubated at
37°C for 1 hour before imaging again. For fixation and visualization of actin filaments or
microtubules following treatment with either 2 µM Lat B or 33 µM NOC, the cells were
fixed with 4% paraformaldehyde prior to the addition of rhodamine phalloidin, or mouse
monoclonal antibody to α-tubulin and appropriate fluorophore-conjugated secondary
antibody, and DAPI. For analysis of fluorescent pixel colocalization, cells from at least 3
different experiments were analyzed individually. Using the Zeiss LSM 510 software
colocalization tool, regions of interest (ROI) were selected and marked with an overlay to
encompass all pixels, following the Zeiss manual protocol. The threshold was
automatically set from these ROIs. For time-lapse imaging, cells were incubated with 25
µM AF488-MCoTI-I for 1 h at 37°C. Following this time, excess MCoTI-I was rinsed off
with a gentle PBS was and the media replaced prior to imaging. The time series image
capture was set to a 2.5 second delay between scans.
For MCoTI-I uptake studies in LGAC, these were grown on 35 mm glass bottom dishes
coated with matrigel. On day 2 of culture, the cells were transduced with Ad-Lifeact-RFP
virus (6 µl/dish, 1 x 10
9
pfu/ml) to allow visualization of actin and thereby, the acinar cell
morphology. On the following day, the cells were gently rinsed with PBS and the media
replaced with incubation buffer prior to the addition of AF488-MCoTI-I (40 µM) and
incubation at 37°C for 1 hour. For colocalization studies, LysoTracker
™
Red (50 nM) or
24

TR-EGF (400 ng/ml) was added to the cells simultaneously with AF488-MCoTI-I prior
to incubation at 37°C for 1 hour.
For detection of Ad5 or penton base in fixed cells, reconstituted rabbit LGAC cultured on
Matrigel-coated coverslips were incubated with replication-deficient AdLacZ (MOI=15)
or recombinant penton protein (20 µg/ml) for up to 2 hours at 4°C (0 min), then warmed
to 37°C for 1 h. After incubation, the cells were rinsed extensively in cold PBS and fixed
in 4% paraformaldehyde prior to addition of rabbit polyclonal antibody to Ad5 and
appropriate secondary fluorophore-conjugated antibodies. For comparison, HeLa cells
were seeded onto uncoated 12-well plates and treated in the same manner as the LGAC
above.
All confocal images, unless otherwise noted, were obtained using a Zeiss LSM 510 Meta
NLO imaging system equipped with Argon and red and green HeNe lasers mounted on a
vibration-free table. For live cell analysis, 5% CO
2
/95% hydrated air and a temperature of
37°C were maintained in an enclosed chamber. All image panels were compiled using
Adobe Photoshop 7.0 (Adobe Systems Inc, Mountain View, CA).

BNP photostability assays
For measurement of in vitro photostability, 12,000MW BNP (BNP12) was assayed using
a black, flat-bottomed 96-well plate. BNP12 was added dropwise (5 µL of a 1 mg/ml
stock) into 195 µL room-temperature incubation buffer (phenol-red-free, serum-free
25

DMEM with 1% penicillin/streptomycin and 20 mM HEPES). Serum was omitted from
the incubation buffer to prevent possible nanoparticle aggregation. LysoBlue was used as
a reference for a UV-excitable and photostable fluorophore (5 µL of a 1 mM stock).
Fluorescence intensity was measured at 0 h using an Envision 2103 Multilabel Reader
(Perkin-Elmer, excitation filter = 340 nm, emission filter = 460 nm). The plate was
exposed to direct UV light for 17 or 24 hours and the fluorescence intensity measured
again as before. For in vivo photostability studies, HeLa cells were seeded on 35 mm
glass-bottomed culture dishes at a density of 8.5 x 10
4
cells/dish. On day 2 of culture, the
cells were rinsed once with PBS and the medium replaced with cold incubation buffer
(800 µL). BNP12 was added dropwise (200 µL) and incubated at 4°C for 1 hour.
Unbound BNP12 was rinsed off and the cells were incubated at 37°C for 1 hour. For
reference, LysoBlue (2.5 µM final concentration) was added to cells after the initial rinse,
and the cells were incubated at 37°C for 1 hour. Following the incubation at 37°C, the
cells were rinsed with PBS and imaged. BNP12 or LysoBlue were imaged using time-
series sequential bleach settings. For each cycle, the image was acquired using a line 4
average, which was followed by 10 iterations of bleaching using the 790 nm line at 4%
power (74 mW). Bleaching was started after one scan and repeated after one scan. Time-
lapse imaging was performed using the Zeiss LSM Meta NLO imaging system equipped
with a Chameleon multiphoton laser mounted on a vibration-free table.

26

BNP uptake assays
HeLa cells were seeded on 6-well plates at a density of 1.5 x 10
5
cells/well. ON day 2 of
culture, cells were rinsed briefly with PBS containing 1 mM CaCl
2
and 0.5 mM MgCl
2

and the media replaced with incubation buffer (900 µL) with or without Lat B (2 µM)
and incubated at 37°C for 30 min. After cooling on ice for 10 min, BNPs (100 µL) were
added dropwise and incubated at 4°C for 1 h. For NOC pretreatment 33 µM NOC was
added prior to incubation at 4°C. Unbound BNPs were rinsed off with a gentle PBS
(+Ca
2+
, +Mg
2+
) wash, the incubation buffer was replaced, and the cells were warmed to
37°C for various times up to 1 h. After incubation, the cells were incubated with an ice-
cold mild acid wash buffer (0.1 M sodium acetate, 0.05 M NaCl, pH 5.5) for 10 min at
4°C. Following the acid wash, the cells were washed three times with ice-cold PBS
(+Ca
2+
, +Mg
2+
). To collect the cells, 700 µL of trypsin (37°C) was added to each well
and incubated at 37°C for 3 min. The cells were then transferred to 1.5 mL Eppendorf
tubes and centrifuged for 5 min at 800 rpm at 4°C. The cell pellet was resuspended in 0.5
mL of ice-cold PBS (without Ca
2+
or Mg
2+
) and filtered through a 70 µm nylon filter, nd
the fluorescence intensity was measured by flow cytometry.

Cytotoxicity assays
For analysis of cytotoxicity, LGAC were either mock-nucleofected, or nucleofected wit
either pmaxGFP or CAR-specific siRNA duplexes on day 2 of culture. Immediately
following nucleofection, the cells were transferred into 3 ml pre-warmed medium in a 6-
27

well plate and incubated at 37°C. After 24 hours, calcein (Ex 494/Em 517 nm) and
ethidium homodimer-1 (Eth-D1, Ex 528/EM 617 nm), both provided in the LIVE/DEAD
cell assay kit, were used to detect live/dead cells. Both were added to the cells (50 µl of
0.5 µM calcein AM and 3.0 µM Eth-D1) prior to incubating at 37°C for 15 minutes.
Fluorescence intensity was measured using an Envision 2103 Multilabel Reader. For
determining the cytotoxicity effect of MBCD on LGAC, cells were pre-treated with 5
mM MBCD for 30 min at 37°C prior to addition of calcein and Eth-D1.

Transfection of rabbit lacrimal gland acinar cells
Transfection was performed on LGAC on day 2 of culture. For transfection using Fugene
6, 1 x 10
6
cells were transfected with 3 or 6 µl of Fugene 6 reagent and either 1 or 2 µg
pmaxGFP to achieve the recommended 3:1, 3:2, and 6:1 Fugene 6 reagent:DNA ratios in
2 ml of culture medium in a 6-well plate, according to the manufacturer’s protocol. For
transfection using Lipofectamine™ 2000, 4 X 10
6
cells were transfected with 4 µg
pmaxGFP and either 4, 10 or 20 µl of Lipofectamine 2000 to achieve a 1:1, 1:2.5 and 1:5
DNA:Lipofectamine ratio in 2 ml of culture medium in a 6-well plate, according to the
manufacturer’s protocol. Nucleofection was performed on LGAC on day 1 or 2 of culture
using the Lonza Group nucleofector device and the Basic Nucleofector Kit for Primary
Mammalian Epithelial Cells. For the optimized reaction, 6 X 10
6
cells were resuspended
in 100 µl nucleofector solution with 3 µg either pmaxGFP or a cathepsin S-GFP.
Program Z-001 was used for the electrical settings. Immediately after nucleofection, the
28

cells were transferred into 3 ml pre-warmed medium in a 6-well plate. For imaging
experiments, the cells were subsequently plated onto Matrigel
™
-coated glass coverlips in
12-well plates (2 X 10
6
cells/well). Transfection efficiency was measured after 24 hours.

Analysis of plasmid transfection efficiency
For pmaxGFP, expression was detected by fluorescence microscopy and flow cytometry.
Immediately following nucleofection, the cells were transferred into 3 ml pre-warmed
media in 6-well plates and incubated at 37°C. After 24 hours, the cells were imaged and
then collected and processed for flow cytometry. For Cathepsin S-GFP, expression was
analyzed by immunofluorescence. As before, cells were transferred into 3 ml pre-warmed
media, but were immediately seeded onto Matrigel -coated coverslips in 12-well plates
at a density of 2 x 10
6
cells/well and incubated at 37°C. After 24 hours, the cells were
fixed in 100% ETOH (-20°C) for 5 minutes and rinsed extensively with PBS prior to the
addition of goat polyclonal antibody to Cathepsin S and appropriate fluorophore-
conjugated secondary antibody. Rhodamine-phalloidin or Alexa Fluor 647-phalloidin
was also used to label actin filaments. Confocal fluorescence images were obtained using
a Zeiss LSM 510 Meta NLO imaging system.

29

Delivery of siRNA duplexes
For delivery of siRNAs using nucleofection, 6 x 10
6
cells were resuspended in 100 µl
nucleofector solution with 2 µM siRNA duplexes directed against CAR or the siControl
non-targeting siRNA. Program Z-001 was used for the electrical settings. Immediately
after nucleofection, the cells were transferred into 3 ml pre-warmed medium in a 6-well
plate. For delivery of siRNAs using GeneSilencer® siRNA Transfection Reagent, 4 x 10
6

cells were transfected with 2 µg siRNA and 10 µl GeneSilencer reagent, according to
protocol. For delivery of siRNAs using Lipofectamine 2000, 4 x 10
6
cells were
transfected with 100 pmol siRNA duplexes and 5 µl Lipofectamine 2000 reagent,
according to protocol.

Analysis of siRNA knockdown efficiency
Uptake of CAR-specific siRNAs was evaluated by semi-quantitative real-time PCR
analysis of CAR mRNA. After nucleofection, cells were transferred into 3 ml pre-
warmed media in 6-well plates and incubated at 37°C. After 24 hours, the cells were
collected and the total RNA for both control and treated samples was isolated and used
for cDNA synthesis. Relative expression levels were detected by using TaqMan® gene
expression assays for the target, CAR, and the endogenous control, hypoxanthine
phosphoribosyltransferase 1 (HPRT1). Results were normalized to HPRT1 mRNA.

30

Chapter 3. Intracellular localization and trafficking of BNPs in HeLa cells

In vitro and in vivo photostability of BNPs
As mentioned earlier, BNPs possess several unique qualities that would make them quite
useful for imaging and sensing applications. To make them even more attractive for
biological applications, it would be important to investigate a) whether BNPs can
efficiently enter mammalian cells and b) whether they retain their unique properties
intracellularly. While they have been shown to enter CHO cells, and accumulate in a
perinuclear compartment (143), their optical properties and intracellular trafficking
mechanisms have not been investigated. These studies began in our lab, following the
kind donation of BNPs by Dr. Cassandra Fraser, in a HeLa cell model. As previously
mentioned, HeLa cells have been extensively used for studying internalization and
trafficking mechanisms of various entities. As such, we utilized this cell model hoping it
would provide us useful and applicable information for the study of BNPs in mammalian
cells. Initial studies indicated that BNPs could be internalized into HeLa cells and once
inside, could retain their unique MW-dependent optical fluorescence emission properties.
Their photostability, however, had not been investigated. Photostability is an important
characteristic to consider as it will often affect aspects of longevity, practicality, and even
storage conditions. To explore the in vitro and in vivo photostability, and for further
studies, we focused mainly on the 12,000MW BNP (BNP12) and compared it to a highly
photostable reference fluorophore, LysoBlue. This particular MW BNP was chosen
31

because initial screenings indicated that BNPs of all MW sizes appeared to behave
comparably, and BNP12 represented the middle of the MW range. For in vitro analysis,
BNP12 and LysoBlue were exposed to direct UV light for up to 24 hours and measured
fluorescence intensity before and after exposure. After 24 hours of direct exposure to UV
light over 50% of the fluorescence intensity of BNP12 remained, while less than 20%
was measured for LysoBlue (Figure 4A). To assess the in vivo, or intracellular
photostability of BNP12, HeLa cells were incubated with either BNP12 or LysoBlue for
1 hour and imaged over time during conditions of sequential bleaching. At the initial time
point, the fluorescence intensity of BNP12 exceeded that of LysoBlue, and after about 9.5
minutes of sequential bleaching, the BNP12 fluorescence signal was still readily detected
while the LysoBlue signal was almost completely photobleached (Figure 4B). This
experiment requires the use of strong laser power and as a result, cell damage and cell
death were detected at time points well preceding any loss in fluorescence signal of
BNP12. These studies indicate that BNPs are highly photostable, more so than a
commercially available, highly photostable probe, and under conditions that resulted in
cell damage and death due to high intensity illumination.
32

Figure 4. BNP photostability. (A) In vitro photostability. BNP12 (5 µL of a 1 mg/ml stock) was
added to a black, flat-bottomed 96-well plate, and the fluorescence intensity measured at 0 h.
Following exposure to direct UV light for 17 and 24 h, the fluorescence intensity was measured
again as before. LysoTracker Blue was used as a reference UV-excitable fluorophore (n = 3, * p ≤
0.05 relative to controls, and # p ≤ 0.05 relative to BNP12). (B) In vivo photostability. HeLa cells
seeded on glass-bottom culture dishes were incubated with either LysoTracker Blue or BNP12
and imaged over time as described in Materials and Methods. Bar = 5 µm.

33

Intracellular localization of BNP12 and the role of the cytoskeleton

To begin to decipher the internalization mechanisms and intracellular trafficking patterns
of BNP12, its intracellular localization once taken up into HeLa cells was investigated.
One way to accomplish this was to assess the extent of its colocalization with
intracellular membrane-bound compartments. LysoRed is a fluorescent probe that labels
acidic organelles, including late endosomes and lysosomes, in live cells. HeLa cells were
incubated with both BNP12 and LysoRed simultaneously for 1 hour and then imaged.
Little to no colocalization of BNP12 and LysoRed was detected (Figure 5). This suggests
that after 1 hour, BNP12 is not significantly localized to acidic organelles, but rather to
another perinuclear compartment. Interestingly, the intracellular distribution pattern
observed is consistent with an endocytic uptake mechanism. However, due to a lack of
adequate fixation and permeabilization techniques which could prevent significant
fluorescence loss, a problem that is often seen in nanoparticle fixation (16, 200), we were
unable to identify this compartment using traditional immunofluorescent compartment
markers.

34

Figure 5. BNP12 is not markedly colocalized with lysosomal compartments. HeLa cells were
incubated with BNP12 (200 µg/ml) and LysoTracker Red for 1 hour at 37°C as described in
Materials and Methods and then imaged. A UV laser was used for excitation of nanoparticles
(green) and a HeNe1 laser for the excitation of LysoTracker Red (red). Bar = 5 µm.

35

The cytoskeleton has been implicated in various ways in both endocytosis and subsequent
trafficking of endocytosed materials. Actin filaments have been shown to partake in
various endocytic mechanisms, including caveolar endocytosis (43), while the
microtubule network has been shown to be important in the sorting of internalized
materials within endosomal compartments (13, 34, 129). To assess the role of the
cytoskeleton in the uptake of BNP12, we examined the effects of nocodazole (NOC) and
latrunculin B (Lat B), inhibitors of the cytoskeleton. The first, NOC, is an inhibitor of
microtubule assembly (34). Lat B inhibits actin filament assembly by binding monomeric
actin, thereby preventing its incorporation into filaments (85). Both of these inhibitors
have previously been successfully used in HeLa cells (9, 50, 213). HeLa cells pretreated
with either NOC or Lat B showed significantly reduced uptake (approximately 40%) of
BNP12 with effects readily detectable by 20 min (Figure 6A & B). Similar results were
seen at 60 min, although the effect was less evident for NOC (data not shown).
Disruption of actin filaments and the microtubule network by their respective inhibitors
was confirmed by immunostaining (Figure 6C). These results suggest that the
cytoskeleton plays an important role in the endocytosis of BNP12.

36

Figure 6. Disruption of the cytoskeleton affects BNP12 uptake. (A) HeLa cells were untreated
(control) or pretreated with either 33 µM NOC or 2 µM Lat B prior to adition of BNP12 (200
µg/ml). Folowing uptake for 20 or 60 min at 37°C, remaining surface-bound BNPs were removed
by mild acid wash. The signal was collected by flow cytometry using appropriate laser and filter
settings (n = 8 for 20 min and n = 6 for 60 min NOC; n = 12 for 20 min and n = 14 for 60 min Lat
B. * p ≤ 0.05). (B) HeLa cells were untreated (control) or pretreated with either 33 µM NOC or 2
µM Lat B prior to addition of BNP12 (200 µg/ml). Following uptake for 20 min at 37°C, the cells
were imaged using confocal laser microscopy. Excitation = 790 nm. Bar = 5 µm. (C) HeLa cells
without any treatment (control) or treated with either 33 µM NOC or 2 µM Lat B for 60 min were
fixed and labeled with a primary antibody to α-tubulin (green) combined with an appropriate
secondary antibody in parallel with rhodamine-phalloidin to label actin (red). Bar = 5 µm.

37

As mentioned earlier, actin has been implicated in diverse endocytic pathways including
caveolar, clathrin-mediated, and CLIC/GEEC (clathrin-independent carrier/GPI-AP-
enriched early endosomal compartment) endocytosis (43). Because studies conducted by
a colleague in the lab indicated that BNP12 not only colocalized with CTX-B, a marker
of cholesterol/lipid-dependent endocytic pathways, but its uptake was significantly
inhibited by pre-treatment with MBCD, uptake of BNP12 is most likely dependent on
lipid rafts. Depletion of membrane cholesterol would likely affect multiple lipid-raft
dependent endocytic pathways including caveolar, CLIC/GEEC, and flotillin
endocytosis. We know that actin has been implicated in two of these pathways, caveolar
and CLIC/GEEC endocytosis, making them the most likely candidates for the
endocytosis of BNP12. Since it has been suggested that the CLIC/GEEC pathway may
play a role in transporting proteins and endocytosed materials to the Golgi network (113,
132), and caveolae-mediated endocytosis has been suggested to target the Golgi
apparatus and the Endoplasmic reticulum (100), entry via either pathway could lead to
the accumulation of BNP12 in perinuclear compartments.
The goals for this study included a comparison of uptake in HeLa and LGAC. As we
have shown, BNPs readily gain cellular entry through what appears to be a lipid-raft-
dependent pathway in HeLa. Initial studies seeking to examine BNP uptake in LGAC
proved unsuccessful as the BNP fluorescence signal could not be clearly detected
intracellularly (studies by Dr. Jiansong Xie, former lab colleague).

38

Discussion
The first steps in demonstrating the biological utility of a nanoparticle formulation are the
characterization of its properties in the biological environment of interest, as well as its
interactions with this biological environment. The unique optical properties of BNPs can
only be useful for biomedical diagnostic applications if they are retained within a
biological milieu. In this chapter, we demonstrated that the unique emission properties of
BNPs are retained and these nanoparticles are highly photostable, both in acqueous
solution and in situ, as our live cell photostability studies showed. In addition, the
demonstration that they are excitable by multiphoton extends their applicability into
animal models, and even into the clinical setting as this method allows an increase in
depth penetration.
Nanoparticle endocytosis can occur in a variety of ways. While many features, including
composition, formulation, size, and shape, may influence their uptake, two features that
have been found to have a major impact on the uptake of nanoparticles are size and
surface charge (20, 69, 71-72, 80). In fact, a single type of nanoparticle can employ
multiple routes simply by varying these factors. For example, carboxyl-modified
fluorescent polysterene nanoparticles that are 24 nm in diameter were able to enter HeLa
cells via a nonclassical (nonclathrin, noncaveolae, and noncholesterol dependent)
pathway while equivalently composed 43 nm nanoparticles entered cells predominantly
via CME (98). PLA and polylactide-polyglycolide (PLGA) nanoparticles have been
shown to enter vascular smooth muscle cells through a combination of fluid phase
39

pinocytosis and CME (69, 137-138). Cationic poly(ethylene-glycol)(PEG)-PLA
nanoparticles also use CME in HeLa cells whereas anionic PEG-PLA nanoparticles do
not (70). While the specific effects of size and charge are debatable, what is clear is that
nanoparticles with positive surface charge may be more readily endocytosed because of
their affinity for the negatively charged plasma membrane (80, 102). The wide use of
cationic liposomal formulations for cell transfection lends support to this notion (102,
110). It has been reported, however, that in cells enriched in lipid raft markers,
endocytosis of cationic liposomes is significantly reduced (97). This suggests that lipid
raft domains may exhibit a preference for negatively charged nanoparticles. The BNPs
investigated here appear to enter HeLa cells through a specific lipid-raft-mediated
pathway that relies on an intact cytoskeleton for proper uptake. These BNPs contain
PLA, which is known to form negatively charged nanoparticles (69), and therefore are
likely negatively charged. This may help explain their lipid-raft-mediated endocytosis.
Their accumulation in non-lysosomal perinuclear compartments confirms their
intracellular trafficking and suggests two possible, lipid-raft-dependent routes of entry:
caveolar endocytosis and/or the CLIC/GEEC pathway.
Although our studies focused on BNP12, earlier studies conducted in the lab by Dr.
Jiansong Xie indicated that BNPs of different molecular weights exhibited similar
intracellular distribution patterns. This suggests that BNPs of different sizes may utilize
similar trafficking pathways, indicating that small variations in their particle size may
play a secondary role to their surface characteristics. Lastly, the internalization
characteristics of BNPs will likely depend on the cell type investigated. Here, we used a
40

transformed, fibroblast-like, unsynchronized cell model (HeLa) to investigate the uptake
and intracellular localization of BNPs. However, when the same uptake experiments were
attempted in a primary epithelial secretory cell model (LGAC), the results were less than
favorable with almost no intracellular BNP fluorescence signal detected. For this reason,
future analyses which compare the pathways in different cell models and cell cycle states
may be of great interest.

41

Chapter 4. Endocytosis and intracellular trafficking of MCoTI-I in HeLa and
LGAC.

Endocytosis of MCoTI-I in HeLa cells
An integral step in assessing the utility of MCoTI-I as a drug development tool involves
the investigation of its cellular uptake and intracellular trafficking patterns. The first is
important to assess whether it can gain cellular entry, in a specific or non-specific
manner. The latter is important in beginning to evaluate the intracellular location where
its therapeutic effect might be exerted, be it in the cytoplasm or the nucleus. To begin to
study the cellular uptake of this cyclotide, we used AlexaFluor488-labeled MCoTI-I
(AF488-MCoTI-I) and HeLa cells. We first looked to see if we could detect any changes
in the distribution pattern of intracellular AF488-MCoTI-I over time. HeLa cells were
incubated with AF488-MCoTI-I for 1 hour and then imaged at various time points up to
10 hours. The internalized cyclotide was clearly visible within perinuclear punctate spots
after 1 hour (Figure 7). The observed distribution pattern remained largely perinuclear
and comparable at all time points examined. Although a similar distribution pattern has
been reported in macrophages for the uptake of the related cyclotide, MCoTI-II, this is
the first report of MCoTI-I uptake as visualized in live cells in real time.

42

Figure 7. MCoTI-I distribution in HeLa cells. HeLa cells were incubated with 25 µM MCoTI-I
for 1 h, followed by removal with gentle rinsing in PBS, and then monitored for distribution of
intracellular fluorescence at intervals from 1 to 10 h using confocal fluorescence microscopy.
Bar = 10 µm.
43

In order to determine whether internalization was via a temperature-dependent
mechanism, we explored the effect of temperature on the uptake process. Energy-
dependent and active endocytic mechanisms are inhibited at 4°C (172), although cell
surface binding can still occur. Incubation of AF488-MCoTI-I at 4°C for 1 hour resulted
in complete inhibition of uptake (Figure 8). This inhibition was completely reversible as
the same cells incubated again at 37°C for 1 hour showed the punctate intracellular
fluorescence pattern that was observed previously. These results confirmed that uptake of
AF488-MCoTI-I in HeLa cells is through an active endocytic internalization pathway. It
should be noted that no significant cell surface binding was observed following the
incubation at 4°C, suggesting that AF488-MCoTI-I does not bind any cell surface
receptors, even nonspecifically. Alternatively, the incubation at 4°C, which alters the
fluidity of the membrane, may affect the adequate distribution of surface proteins
necessary for proper MCoTI-I binding.

44

Figure 8. Endocytosis of MCoTI-I in HeLa is temperature-dependent. HeLa cells were
incubated with 25 µM MCoTI-I for 1 h at 4°C. After removal of the MCoTI-I-containing media,
and a gentle PBS wash, the cells were imaged. Following imaging, the MCoTI-I-containing
media were replaced and the cells were incubated at 37°C for 1 h and imaged again. Bar = 10 µm.

45

We next investigated the endocytic pathway used by AF488-MCoTI-I to enter HeLa
cells. Our approach was to examine its colocalization with various individual endocytic
markers. As shown in Figure 9, colocalization studies showed that after 1 hour,
significant colocalization was detected between the fluorescence associated with AF488-
MCoTI-I and that associated with the fluid phase marker, 10K-Dex (59 ± 4%). Less
colocalization was observed for studies with fluorescent CTX-B (39 ± 4%) and EGF (21
± 2%). This data suggests that the primary endocytic route utilized by AF488-MCoTI-I in
HeLa cells is fluid-phase endocytosis. The colocalization observed with CTX-B and EGF
suggests that AF488-MCoTI-I could potentially be utilizing additional pathways as well.
The observed colocalization, however, could also be attributed to the merging of
endosomal vesicles generated by different pathways, perhaps at the level of an early
endosome.

46

Figure 9. Colocalization of MCoTI-I with markers of endocytosis in HeLa. (A) HeLa cells
were incubated with 25 µM MCoTI-I and either 1 mg/ml TR-10K-Dex (10K-Dex), 10 µg/ml
AF594-CTX-B (CTX-B), or 400 ng/ml TR-EGF (EGF) for 1 h at 37°C as described in Materials
and Methods and then imaged. Bar = 10 µm. (B) Quantification of pixel colocalization was done
using the Zeiss LSM software for image analysis and measures the % of total fluorescent AF488-
MCoTI-I pixels in the ROI relative to red pixels associated with the different markers. (n = 13
cells for TR-10K-Dex, n = 11 cells for CTX-B, n = 10 cells for EGF, with cells assessed across 3
different experiments, * p ≤ 0.05 relative to 10K-Dex, # p ≤ 0.05 relative to CTX-B).
47

To explore whether the observed uptake and significant colocalization with 10K-Dex was
due to cointernalization by macropinocytosis, we examined the effect Lat B on the uptake
of AF488-MCoTI-I. Lat B is a potent inhibitor of actin polymerization, an essential
element in macropinocytosis. Treatment with Lat B did not significantly inhibit uptake of
AF488-MCoTI-I, although total disruption of the actin filament network was observed
(Figure 10).

48

Figure 10. Disruption of actin does not inhibit MCoTI-I uptake. (A) HeLa cells were
untreated (control) or treated with Lat B (2 µM) for 30 min at 37°C prior to addition of 25 µM
AF488-MCoTI-I. Following uptake for 1 h at 37°C, the cells were imaged using confocal
fluorescence microscopy. Bar = 10 µm. (B) HeLa cells without treatment (control) or treated with
2 µM Lat B for 30 min at 37°C were fixed and labeled with rhodamine-phalloidin to label actin
(red) and DAPI to label nuclei (blue). Bar = 10 µm.

49

In addition to Lat B, we also investigated the effect of EIPA on the uptake of AF488-
MCoTI-I. EIPA is a potent and specific inhibitor of the Na
+
/H
+
exchanger, whose activity
is important for macropinocytosis. Treatment of HeLa cells with 50 µM EIPA
significantly inhibited the uptake of both AF488-MCoTI-I and 10K-Dex (Figure 11). The
same treatment also significantly inhibited the uptake of CTX-B, suggesting that at this
concentration, the inhibitor is affecting multiple endocytic pathways in these HeLa cells
and not just macropinocytosis.
50

Figure 11. Effect of EIPA treatment on MCoTI-I uptake. HeLa cells were untreated (control)
or treated with EIPA (50 µM) for 30 min at 37°C prior to addition of either 10K-Dex (1 mg/ml),
MCoTI-I (25 µM), or CTX-B (10 µg/ml). Following uptake for 1 hr at 37°C, the cells were
imaged using confocal fluorescence microscopy. Bar = 10 µm.
51

Intracellular trafficking of MCoTI-I in HeLa cells
The first part of this study provided us valuable information. We determined that
endocytosis is by a temperature-dependent mechanism and identified that
macropinocytosis plays an important role, although other potential pathways may also be
involved. We next focused on the intracellular fate of the vesicles containing endocytosed
AF488-MCoTI-I. There are at least two pathways that involve the intracellular trafficking
of endosomal vesicles. The first is the recycling pathway, whereby recycling endosomes
sort material that will be effluxed and recycled back to the cell membrane. The second is
the degradative pathway which begins with routing of internalized materials to early
endosomes and continues through to late endosomes and lysosomes, where degradation
of materials occurs (174). Trafficking through the recycling pathway would result in a
decrease of intracellular signal over time, and based on the fact that we did not observe
this in the time-course experiment (see Figure 6), we eliminated the possibility of
MCoTI-I trafficking through this pathway. To explore the possibility that MCoTI-I was
trafficking through the degradative pathway, we first used LysoRed. This pH sensitive
probe labels acidic compartments including late endosomes and lysosomes. After a 1
hour incubation with AF488-MCoTI-I and LysoRed, significant colocalization (60 ± 4%)
was observed (Figure 12A). As an extension of these studies, we also investigated the
colocalization of AF488-MCoTI-I with lysosomal-associated membrane protein 1
(Lamp1), an established lysosomal marker (47, 54). For this experiment, live HeLa cells
were first infected with a Red Fluorescent Protein (RFP)-Lamp1-expressing BacMam
virus on the previous day before incubation with AF488-MCoTI-I for 1 hour and
52

subsequent imaging. As shown in Figure 12B, colocalization was also seen for AF488-
MCoTI-I and RFP-Lamp 1 (38 ± 5%), suggesting that after 1 hour, a significant amount
has already reached the lysosomal compartments. This data suggests that after 1 hour,
about 40% of the MCoTI-I has trafficked through the endosomal pathway to the
lysosomes and about 20% is already localized to late endosomes or other types of acidic
organelles.

53

Figure 12. MCoTI-I is colocalized with lysosomal compartments. Untreated (A) or BacMam-
RFP-Lamp1 treated (B) HeLa cells were incubated with 25 µM MCoTI-I and LysoRed, or
MCoTI-I alone, for 1 h at 37°C and then imaged. Bar = 10 µm. (C) Quantification of % of total
fluorescent AF488-MCoTI-I pixel colocalization with fluorescent pixels associated with both
markers was done using the Zeiss LSM software for image analysis. (n = 14 cells for LysoRed
and n = 11 cells for Lamp1 with cells selected from 3 separate experiments).

54

It has been reported that actin filaments and the microtubule network play an important
role in the perinuclear steady-state distribution of lysosomes (27, 115, 179). Microtubules
have also been shown to participate in the movement of early endosomes to late
endosomes and to lysosomes (3, 111). As an extension of these studies, we investigated
whether MCoTI-I containing vesicles were actively trafficking inside the cells by
capturing time-lapse video. Indeed, the captured video showed directed short- and long-
range movements of MCoTI-I-containing vesicles (Figure 13). This motility was readily
inhibited by treatment with the microtubule assembly inhibitor, NOC.

55

Figure 13. MCoTI-I-containing vesicles are in microtubule-associated motion. HeLa cells
were untreated (control) or treated with NOC (33 µM) for 30 min at 4°C prior to the addition of
MCoTI-I (25 µM). Following uptake for 1 h at 37°C the cells were imaged using time-lapse
microscopy with a 2.5 second delay between scans.

Because we know that a large portion of MCoTI-I has reached the lysosomal
compartment by 1 hour, these results suggest that while some of the movements may be
attributed to the steady-state distribution of lysosomes, the remaining MCoTI-I-
containing vesicles may still be trafficking through the cells from other membrane
compartments, most likely within late endosomes.

Endocytosis and intracellular trafficking of MCoTI-I in LGAC
Our studies in HeLa cells indicated that the MCoTI-I cyclotide has ready access to the
intracellular trafficking pathway without appearing to require binding to any specific
surface receptors. As indicated earlier, the lacrimal gland plays a vital role in the
56

maintenance of ocular health. Because its malfunction often leads to dry eye diseases,
which affect millions of people in the United States alone, the lacrimal gland is an
interesting therapeutic target. As an extension of the HeLa studies, we investigated the
uptake of MCoTI-I in a disease-relevant tissue cell type. The cell morphology of LGAC
is quite different than that of HeLa cells. Unlike the flat, transformed HeLa cells, LGAC
are primary polarized epithelial cells that reconstitute in culture to re-form their acinar-
like structure. We first looked at the intracellular distribution of MCoTI-I in LGAC. For
proper visualization of cell morphology, the cells were transduced on the previous day
with Ad-Lifeact-RFP virus, allowing the expression of red-colored actin. As shown in
Figure 14, AF488-MCoTI-I was readily detected within LGAC after 1 hour.

Figure 14. MCoTI-I distribution in LGAC. LGAC were transduced with Ad-Lifeact-RFP
virus (6 µl/dish, 1 x 10
9
pfu/ml) on day 2 of culture. On the following day, LGAC were uncubated
with MCoTI-I (40 µM) for 1 h at 37°C, gently rinsed with PBS and imaged using confocal
fluorescence microscopy. Bar = 10 µm.

57

Next we examined whether endocytosis in LGAC was also accomplished through a
temperature-dependent transport mechanism by looking again at the effect of
temperature. Uptake of AF488-MCoTI-I was completely inhibited after incubation at 4°C
for 1 hour (Figure 15). As before, the intracellular signal could be restored following
incubation for 1 hour at 37°C and once again, no significant cell surface binding was
observed. This suggests that uptake of MCoTI-I in LGAC is also by an active endocytic
mechanism, and reinforces the notion that MCoTI-I does not appear to preferentially bind
surface receptors despite the cell type.

58

Figure 15. Endocytosis of MCoTI-I in LGAC is temperature-dependent. LGAC were
transduced with live-act virus (6 µl/dish, 1 x 10
9
pfu/ml) on day 2 of culture. On the following
day, LGAC were uncubated with 40 µM MCoTI-I for 1 h at 4°C. After removal of the MCoTI-I-
containing media and a gentle PBS wash, the cells were imaged. Following imaging, the MCoTI-
I-containing media were replaced and the cells were incubated at 37°C for 1 h and imaged again.
Bar = 10 µm.

59

To gain a better understanding of the pathway utilized by MCoTI-I to enter LGAC, we
looked again at its individual colocalization with various fluorescent markers. As shown
in Figure 16, these studies showed that after 1 hour, a portion of AF488-MCoTI-I is
colocalized with 10K-Dex (33 ± 4%). Less colocalization was observed in studies with
EGF (21 ± 4%). It is interesting that while colocalization of MCoTI-I and 10K-Dex in
LGAC is nearly half of that in HeLa cells, the amount of colocalization between MCoTI-
I and EGF is comparable. This suggests that while fluid-phase endocytosis may very well
play an important role in the uptake of MCoTI-I in LGAC, it may be to a lesser extent.
Additionally, other endocytic mechanisms may participate as well. The possibility also
exists that the observed colocalization is again due to the merging of endosomal vesicles
from different pathways. Perhaps more interesting, very little colocalization was observed
for LysoRed (11 ± 3%), the marker of acidic vesicles including late endosomes and
lysosomes. This suggests that after 1 hour, very little MCoTI-I has trafficked through the
endosomal pathway to late endosomes and lysosomes. This is significantly different from
what we observed for the trafficking of MCoTI-I in HeLa cells. Due to the fact that very
little MCoTI-I was colocalized with LysoRed, we did not pursue this further with the
more specific lysosomal marker, Lamp1. Similarly, colocalization studies with CTX-B
were omitted since this marker is not well internalized in LGAC.

60

Figure 16. Colocalization of MCoTI-I with markers of endocytosis in LGAC. LGAC were
incubate with 40 µM MCoTI-I and either 1 mg/ml 10K-Dex, 400 ng/ml EGF, or 50 nM LysoRed
for 1 h at 37°C on day 3 of culture. Following the incubation, the cells were imaged using
confocal fluorescence microscopy. Bar = 10 µM. (n = 10 acini for 10K-Dex, n = 8 acini for EGF,
and n = 8 acini for LysoRed).

61

Discussion
Cyclotides represent a novel, attractive platform for drug development. Their unique,
stable structure offers a distinctive molecular scaffold for drug design. In this chapter, we
investigated the cellular uptake and intracellular localization of MCoTI-I in a relatively
simple cell model first (HeLa), and compared it to a physiologically- and disease-relevant
cell model (LGAC). We established that MCoTI-I gains cellular entry in both HeLa and
LGAC by a temperature-dependent endocytic mechanism. The cyclotide appears to lack
any specific affinity for proteins or lipids on the cell surface regardless of the cell type.
This is consistent with what has been reported for the related cyclotide, MCoTI-II, in
fixed-cell studies with macrophages (61). This lack of endogenous affinity for a specific
receptor or membrane constituent makes MCoTI-I ideal for engineering using more
specific, receptor-directed internalization motifs that might enable enhanced targeting to
a specific cell type.
In the previous chapter, we discussed in some detail the various features that affect
cellular uptake. While size and charge are important factors that help govern the uptake
of nanoparticles, cyclotides are small polypeptides and as such, will have a different set
of features that may affect their uptake. Our work demonstrated that MCoTI-I, a cationic
polypeptide, utilizes multiple endocytic pathways for internalization in HeLa, although
fluid-phase endocytosis appears to be the preferred route. The same is true in LGAC,
although uptake in these cells appears to be reduced in comparison to HeLa. This may be
due in part to the secretory nature of LGAC and their corresponding specialization in
62

exocytosis. Alternatively, the differences may be attributed to the endocytic preferences
of these very different cell types. A report on the uptake of MCoTI-II supports this idea.
In macrophages, uptake of MCoTI-II, which shares a high homology with MCoTI-I, was
shown to be mediated by macropinocytosis (61). However, these cells are known to be
specialized in large-scale sampling of extracellular fluid using macropinocytosis as the
dominant endocytic pathway.
Perhaps the most interesting finding here was the difference seen in the intracellular
localization of MCoTI-I in HeLa versus LGAC. In HeLa, we demonstrated the trafficking
of MCoTI-I through the endosomal pathway to late endosomes and lysosomes and
furthermore, revealed their continued movement along microtubules. In contrast, MCoTI-
I was not detected in acidic compartments in LGAC, suggesting that MCoTI-I is not
moving along the traditional endosomal network and may instead be moving through a
different intracellular trafficking pathway. Alternatively, MCoTI-I may be localized to
earlier endosomal vesicles, in which case, the use of fluorescent markers for detection of
earlier compartments, such as early endosomes, might help define the pathway. At this
point, we cannot ascertain whether some labeled MCoTI-I is able to escape from
endosomal compartments into the cytosol as these studies are hampered by the use of live
cell imaging due to the large dilution effect of moving from the highly confined volume
of a vesicle into the much larger cytosolic volume.

63

Chapter 5. Endocytosis and intracellular localization of Ad5 in LGAC

Evidence of variations in the Adenovirus infection pathway
Our studies with MCoTI-I in LGAC indicated that while the cyclotide gains cellular
entry, its intracellular trafficking is different than what was observed in HeLa cells. We
know that although Ad endocytosis and trafficking has been extensively studied, most of
these studies have been conducted primarily in HeLa cells and A549 cells, a cell line
derived from an airway epithelial carcinoma (60, 105, 117). Additionally, studies
investigating Ad endocytosis in different target cell types have yielded interesting results.
In fibroblasts and endothelial cells genetically modified to express CAR, Ad infection
results in accumulation of virus at the microtubule organizing center (105). Similarly, in
CAR-expressing macrophages, infection results in virus accumulation in what is believed
to be an acidic organelle (105). In either case, the virus fails to reach the nucleus. This
indicates that the classical intracellular trafficking pathway described for Ad does not
hold true for all cell types. Earlier we discussed the traditional route of entry for Ad5,
including the fact that the fiber capsid protein is important for binding, but uptake is
actually mediated by the penton protein. It has been reported that penton base interactions
with surface α
v
intergrins play a minimal role in Ad-mediated gene transfer in
hepatocytes (73). Instead, the interaction between the fiber protein and its receptor helps
mediate internalization. This fiber-dependent endocytosis opposes the established
paradigm. It should be noted that hepatocytes lack expression of α
v
β
3
and α
v
β
5
integrins
64

and accordingly may have mechanisms in place that prevent the need for the penton-
integrin interactions.

The importance of fiber for Ad5 internalization
Given the observations described above, we moved to investigate the pathways that
govern Ad uptake and trafficking in LGAC. Adenoviral vectors have already been found
to be the most effective gene transfer technique available for the transduction of lacrimal
epithelial cells (8, 189, 212). Also, previous studies in our lab have shown that LGAC are
transduced by replication-defective Ad5 with very high efficiency (88-89, 198). In a
comparison of transduction of LGAC with several other cell lines, LGAC were shown to
have comparable transduction efficiency to HeLa cells (202). Given that we previously
observed distinct trafficking patterns in HeLa and LGAC for the same cyclotide, we
investigated whether the uptake of Ad5 in LGAC follows the established, penton-
dependent internalization pathway. We first examined the intracellular distribution
pattern for penton base protein by confocal fluorescence microscopy. Uptake experiments
showed that after 1 hour, much of the membrane-associated penton protein could be
detected in the perinuclear region in HeLa cells (Figure 17A). In contrast, the penton
protein remained associated with the plasma membrane in LGAC, under the same
conditions, which did allow the internalization of intact virus (Figure 17B). This data
suggests that penton protein may not efficiently enter LGAC as would be expected, based
on the model described for HeLa cells.
65

Figure 17. Uptake of penton base protein and Ad5. (A) Penton base uptake in HeLa and
LGAC. HeLa cells or LGAC were seeded onto uncoated or Matrigel-coated glass coverslips in
12-well plates at 1 x 10
5
cells/well or 2 x 10
6
cells/well, respectively. On day 2 (HeLa) or day 3
(LGAC) of culture, cells exposed to penton base (20 µg/ml) for 0 min and 60 min at 37°C were
fixed and processed to fluorescence label penton base (green), actin filaments (red), and nuclei
(blue). Arrows, association of penton base with the plasma membrane; arrowheads, penton base
in preinuclear region. *, apical/luminal region. Bar = 10 µm. (B). Ad5 uptake in LGAC. Rabbit
LGAC exposed to replication-deficient Ad-LacZ (MOI = 15) for 0 min and 60 min were fixed
and processed to fluorescence label Ad5 proteins (green) and actin filaments (red). *,
apical/luminal region. Bar = 10 µm.
66

As a continuation of these studies, and to confirm whether the uptake pattern of penton
base protein was indeed different in LGAC, we used a trypsinization-based biochemical
assay to quantify the amount of penton base associated with the cell surface versus that
associated with intracellular membranes and the cytosol. The premise for this assay is
that penton base gaining cellular entry will acquire resistance to cell surface trypsin
exposure. On the other hand, penton base remaining on the cell surface will be cleaved
following exposure to trypsin. For this assay, the amount of penton base associated with
each cell type at 0 min in the absence of trypsin represents the control value. As shown in
Figure 18A, much of the penton base associated with HeLa cells after 60 min of
incubation at 37°C exhibited trypsin resistance, indicative of internalization. In contrast,
penton base associated with LGAC for either 0 min or 60 min exhibited a similar
sensitivity to trypsin, which resulted in cleavage of nearly all associated penton base
(Figure 18B). This response is consistent with a lack of internalization of the penton base
protein in LGAC. These biochemical studies confirmed the findings shown in the
microscopy studies, and suggest that penton base protein is not readily internalized in
LGAC. This finding was certainly unexpected given that penton base readily accumulates
in intracellular compartments of HeLa and other cells (81, 153).
67

Figure 18. Penton base remains surface-bound in LGAC. (A) Penton base acquires trypsin
resistance after incubation in HeLa cells. heLa cells were seeded on 12-well plates. When they
reached 80% confluence, they were exposed to penton base protein (10 to 50 µg/ml) for 0 or 60
min at 37°C or not exposed and then were treated with 0.2 ng/ml trypsin-EDTA for 1 hr at 4°C or
not treated and were lysed, using RIPA buffer. Cell lysates (150 µg/lane) were blotted with
polyclonal antibody to Ad5 proteins and IRDye800-conjugated secondary antibody. Data for each
preparation were normalized to that for penton base association at 0 min minus that for trypsin. *,
significant at p ≤ 0.05 based on results for cells at 0 min. Error bars represent SEM (n = 5). (B)
Penton base is vulnerable to trypsin at 60 min after exposure in LGAC. Rabbit LGAC were
seeded onto 150-mm petri dishes at 2 x 10
6
cells/ml. On day 2 of culture, LGAC with or without
exposure to penton base (20 µg/ml) for 0 min or 60 min at 37°C were treated with trypsin-EDTA
or not treated and processed as described above. Data were normalized in each preparation to that
for penton base association at 0 min minus that for trypsin. Error bars represent SEM. (n = 3).

Because a penton-independent pathway was previously described for hepatocytes, which
do not express α
v
integrins, we sought to confirm expression of these in LGAC. Western
blot analysis confirmed that α
v
integrin, the major receptor for penton base, is in fact
expressed in LGAC and at levels comparable to those for HeLa cells (Figure 19).
68

Figure 19. LGAC express α
v
integrins. Western blot of α
v
integrin expression in rabbit LGAC
and HeLa cells is shown. LGAC and HeLa cell lysates (150 µg/lane) prepared using RIPA buffer
were blotted with a rabbit polyclonal antibody to α
v
integrin and IRDye 800-conjugated
secondary antibody. The same lanes, reprobed for actin content, are shown below, while the
signal associated with the secondary antibody (2°) alone is shown in the panel to the right.

Using the same approach, we next assessed whether fiber protein acquired trypsin
resistance, indicative of internalization, or also remained surface bound in LGAC. As
shown in Figure 20A, much of the fiber protein associated with LGAC after 60 min of
incubation with intact Ad5 at 37°C exhibited trypsin resistance, indicative of
internalization. Intrigued by these results, we extended these studies to evaluate whether
trypsin resistance of fiber protein could be detected sooner than the 60 min time point.
Interestingly, as shown in Figure 20B, fiber protein acquires trypsin resistance as early as
69

15 minutes after incubation with Ad5 at 37°C. This implies that fiber is readily
internalized in LGAC and can still be detected in the cell after 1 hour.

Figure 20. Fiber protein acquires trypsin resistance in LGAC. Rabbit LGAC were seeded
onto 150-mm petri dishes at 2 x 10
6
cells/ml. On day 2 of culture, LGAC with or without
exposure to Ad-lacZ (MOI = 15) for 0 min or 60 min (A) or 0 min, 15 min, or 30 min (B) at 37°C
were treated with 0.2 mg/ml trypsin-EDTA for 1 h at 4 °C or not treated and lysed using RIPA
buffer. Cell lysates (150 µg/lane) were blotted with monoclonal antibody to fiber protein and
IRDye800-conjugated secondary antibody. Data were normalized in each preparation to that for
fiber association at 0 min minus that for trypsin. Error bars represent SEM. (n = 5).

Elimination of macropinocytosis as the prevalent endocytic route of Ad5
Macropinocytosis is one of the endocytic pathways used by viruses to gain cellular entry.
Adenovirus serotype 3, Herpes simplex virus-1, and Vaccinia virus all utilize
macropinocytosis as a direct means for internalization (123). Other viruses however, use
this pathway as an indirect approach to assist their uptake by a different mechanism of
endocytosis. For example, it has been reported that Ad2 and Ad5 stimulate this endocytic
70

mechanism not for host cell entry, but rather to enhance its acid-activated penetration
from endosomes (120-121). Although studies in our lab indicated that Ad5, fiber, and
knob protein could elicit macropinocytosis as well, it remained to be determined whether
this pathway played a significant role in the uptake of Ad5 in LGAC. To address the role
of macropinocytosis in the fiber-dependent internalization of Ad5 in LGAC, we
investigated the effects of EIPA and Lat B using the acquisition of trypsin resistance by
fiber protein as a marker of virus internalization. As shown in Figure 21, the amount of
Ad5 fiber protein internalized at 60 min with or without EIPA or Lat B treatment was
comparable. This data suggests that while Ad5 may trigger macropinocytosis in LGAC, it
does not utilize this pathway for its primary entry route.

Figure 21. Inhibitors of macropinocytosis do not prevent fiber entry. Rabbit LGAC were
seeded in 150-mm petri dishes at a density of 2 x 10
6
cells/ml. On day 2 of culture, vehicle-
treated rabbit LGAC or LGAC pretreated with either EIPA (500 µM) or Lat B (10 µM) for 1 h at
37°C were incubated with Ad5 (MOI = 15) for 0 or 60 min, followed by treatment with 0.2
mg/ml trypsin-EDTA for 1 h at 4°C and lysis using RIPA buffer. Cell lysates (150 µg/lane) were
blotted with monoclonal antibody to fiber protein and IRDye800-conjugated secondary antibody.
Error bars represent SEM. Values shown are fiber protein uptake at 60 min relative to that for the
control (0 min, incubated only with Ad5). (n = 3)
71

Effect of inhibitors on Ad5 endocytosis

The endocytosis pathway most commonly described for the penton-mediated uptake of
Ad5 is CME. Thus far, our studies in LGAC have indicated a very different endocytic
pattern than what has previously been described. Previous studies in our lab suggest that
Ad5 uptake in LGAC is also via CME (202), but this remains to be confirmed. In an
effort to verify the endocytic pathway, or begin to decipher the possible route(s) of entry,
we next investigated the effect of various endocytosis inhibitors on the uptake of Ad5.
We relied again on our established biochemical assay and used acquired trypsin
resistance as an indicator of cellular entry. The first inhibitor tested was Bafilomycin A1
(BafA1), a specific inhibitor of the vacuolar H
+
-ATPase. This pump has been implicated
in the endocytosis of certain viruses, specifically in the endosomal acidification
necessitated by their CME uptake (9, 12, 83, 141). Treatment of LGAC with BafA1 did
not significantly impair Ad5 uptake (Figure 22A), suggesting that if Ad5 uptake is indeed
via CME, the vacuolar H
+
-ATPase does not play a crucial role. Because in conducting
these biochemical experiments we collect data for the 0 min time point corresponding to
binding at 4°C prior to the warming step, we can also infer information regarding the
effect of these inhibitors on the cell surface binding of Ad5. Surface binding of Ad5 was
not significantly affected following treatment with BafA1 (Figure 22B).
72

Figure 22. Effect of Bafilomycin A1 on Ad5 uptake and binding. (A) BafA1 does not inhibit
uptake of Ad5 in LGAC. Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 x
10
6
cells/ml. On day 2 of culture, vehicle-treated rabbit LGAC or LGAC pretreated with BafA1
(50 nM) for 2 h at 37°C were incubated with Ad5 (MOI = 15) for 60 min, followed by treatment
with 0.2 mg/ml trypsin-EDTA for 1 h at 4°C and lysis using RIPA buffer. Cell lysates (150
µg/lane) were blotted with monoclonal antibody to fiber protein and IRDye800-conjugated
secondary antibody. Values shown represent fiber protein uptake at 60 min relative to that for the
control, 60 min incubated with Ad only. (n = 3) (B) BafA1 does not affect binding of Ad5 to
LGAC. Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 x 10
6
cells/ml. On
day 2 of culture, vehicle-treated rabbit LGAC or LGAC pretreated with BafA1 (50 nM) for 2 h at
37°C were incubated with Ad5 (MOI = 15) for 0 min, were treated or untreated with 0.2 mg/ml
and then processed as described above. Values shown represent fiber binding at 0 min relative to
that for control, 0 min incubated with Ad only. (n = 3) Error bars represent SEM.

We next examined the effect of chlorpromazine (CPZ), a cationic amphiphilic drug that is
believed to inhibit CME by a reversible translocation of clathrin to intracellular
endosomal membranes (12, 191, 197). As shown in Figure 23A, inhibition of Ad5 uptake
was not observed following treatment of LGAC with CPZ. Instead, a marked apparent
increase in fiber trypsin resistance was observed. Interestingly, CPZ treatment of LGAC
appeared to have an inhibitory effect on Ad5 surface binding (Figure 23B). Combined,
these data suggest that while treatment with CPZ, and by extension, partial removal of
73

plasma membrane clathrin, appears to negatively affect Ad5 binding to LGAC, it does
not inhibit its uptake and instead, appears to facilitate its internalization.

Figure 23. Effect of Chlorpromazine on Ad5 uptake and binding. (A) CPZ does not inhibit
uptake of Ad5 in LGAC. Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 x
10
6
cells/ml. On day 2 of culture, vehicle-treated rabbit LGAC or LGAC pretreated with CPZ
(100 µM) for 2 h at 37°C were incubated with Ad5 (MOI = 15) for 60 min, followed by treatment
with 0.2 mg/ml trypsin-EDTA for 1 h at 4°C and lysis using RIPA buffer. Cell lysates (150
µg/lane) were blotted with monoclonal antibody to fiber protein and IRDye800-conjugated
secondary antibody. Values shown represent fiber protein uptake at 60 min relative to that for the
control, 60 min incubated with Ad only. (n = 3) (B) CPZ inhibits binding of Ad5 to LGAC.
Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 x 10
6
cells/ml. On day 2 of
culture, vehicle-treated rabbit LGAC or LGAC pretreated with CPZ (100 µM) for 2 h at 37°C
were incubated with Ad5 (MOI = 15) for 0 min, were treated or untreated with 0.2 mg/ml and
then processed as described above. Values shown represent fiber binding at 0 min relative to that
for control, 0 min incubated with Ad only. (n = 3) Error bars represent SEM.

The literature indicates that while these pharmacological inhibitors are known to inhibit
CME, they do not possess absolute selectivity. For example, CPZ has also been shown to
block uptake of fluid-phase markers, making it quite difficult to distinguish between
74

CME and macropinocytosis. Additionally, these inhibitors may also have secondary
effects on the cytoskeleton and other biochemical cell processes, resulting in a possible
indirect effect on various clathrin-independent pathways as well. For these reasons, lack
of inhibition by these inhibitors does not permit elimination of this pathway for Ad5
uptake in LGAC.
We continued our uptake inhibition studies by looking next at the effects of dynamin
inhibitors. For these studies, we used a series of small molecule, cell permeable inhibitors
including MITMAB
™
, OctMAB
™
, Dynole-34-2
™
, and Iminodyn-22
™
. These molecules
exert their inhibitory effect on different sites of the GTPase. MITMAB
™
and OctMAB
™

target dynamin at the pleckstrin homology (lipid binding) domain, while Dynole-34-2
™

and Iminodyn-22
™
target different regions of the GTPase allosteric site. Recently, these
have all been shown to block dynamin-dependent endocytosis (78-79, 148). Treatment of
LGAC with MITMAB
™
, OctMAB
™
, or Iminodyn-22
™
, did not result in inhibition of
Ad5 uptake (Figure 24). Some reduced uptake was noted for LGAC treated with Dynole-
34-2
™
, however, the inhibition was not statistically significant.

75

Figure 24. Effect of dynamin inhibitors on Ad uptake. Rabbit LGAC were seeded in 150-mm
petri dishes at a density of 2 x 10
6
cells/ml. On day 2 of culture, vehicle-treated rabbit LGAC or
LGAC pretreated with MITMAB, OctMAB, Dynole-34-2, or Iminodyn-22 (30 µM for each) for
15 min at 37°C were incubated with Ad5 (MOI = 15) for 60 min, followed by treatment with 0.2
mg/ml trypsin-EDTA for 1 h at 4°C and lysis using RIPA buffer. Cell lysates (150 µg/lane) were
blotted with monoclonal antibody to fiber protein and IRDye800-conjugated secondary antibody.
Values shown represent fiber protein uptake at 60 min relative to that for the control, 60 min
incubated with Ad only. (n = 4) Error bars represent SEM.

These data suggest that Ad5 uptake in LGAC possibly occurs in a dynamin-independent
manner. We were unfortunately unable to confirm this as we encountered difficulties in
assessing the effects of these inhibitors on the dynamin-dependent uptake of EGF. While
transferrin is most often used for analysis of CME, this marker is not well internalized in
our LGAC system.
It was recently shown that cholesterol is required for the clathrin-dependent endocytosis
of Ad2 (84). Additionally, acute cholesterol depletion has been shown to block
76

internalization of tranferrin, a classical ligand of the CME pathway (154, 169). Because
we know that Ad2 has a high homology to Ad5, and both often exhibit similar behavior,
we investigated the role of cholesterol on the uptake of Ad5 in LGAC by looking at the
effect of MBCD. As shown in Figure 25A, treatment with MBCD did not affect uptake of
Ad5. Surprisingly, it did significantly inhibit Ad5 surface binding (Figure 25B),
suggesting that cholesterol in the plasma membrane is important for binding of Ad5 to its
surface receptor, but not for its internalization.

Figure 25. Effect of Methyl-β-cyclodextrin on Ad5 uptake and binding. (A) MBCD does not
inhibit uptake of Ad5 in LGAC. Rabbit LGAC were seeded in 150-mm petri dishes at a density of
2 x 10
6
cells/ml. On day 2 of culture, vehicle-treated rabbit LGAC or LGAC pretreated with
MBCD (5 mM) for 30 min at 37°C were incubated with Ad5 (MOI = 15) for 60 min, followed by
treatment with 0.2 mg/ml trypsin-EDTA for 1 h at 4°C and lysis using RIPA buffer. Cell lysates
(150 µg/lane) were blotted with monoclonal antibody to fiber protein and IRDye800-conjugated
secondary antibody. Values shown represent fiber protein uptake at 60 min relative to that for the
control, 60 min incubated with Ad only. (n = 4) (B) MBCD inhibits binding of Ad5 to LGAC.
Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 x 10
6
cells/ml. On day 2 of
culture, vehicle-treated rabbit LGAC or LGAC pretreated with MBCD (5 mM) for 30 min at
37°C were incubated with Ad5 (MOI = 15) for 0 min, were treated or untreated with 0.2 mg/ml
and then processed as described above. Values shown represent fiber binding at 0 min relative to
that for control, 0 min incubated with Ad only. (n = 3) *, significant at p ≤ 0.05. Error bars
represent SEM.
77

We mentioned earlier that the microtubule network is implicated in the intracellular
movement of virus particles. In our final inhibitor study, we looked at the effects of NOC
on the uptake of Ad5. Although an inhibitory trend in the uptake was observed for treated
cells, NOC did not significantly block Ad5 uptake or binding (Figure 26). This data
suggests that while microtubules may play a role in the initial inward movement of
endocytic vesicles, they are not vital for the internalization process itself in LGAC.

Figure 26. Effect of Nocodazole on Ad5 uptake and binding. (A) NOC does not significantly
inhibit uptake of Ad5 in LGAC. Rabbit LGAC were seeded in 150-mm petri dishes at a density of
2 x 10
6
cells/ml. On day 2 of culture, vehicle-treated rabbit LGAC or LGAC pretreated with NOC
(33 µM) for 30 min at 4°C were incubated with Ad5 (MOI = 15) for 60 min, followed by
treatment with 0.2 mg/ml trypsin-EDTA for 1 h at 4°C and lysis using RIPA buffer. Cell lysates
(150 µg/lane) were blotted with monoclonal antibody to fiber protein and IRDye800-conjugated
secondary antibody. Values shown represent fiber protein uptake at 60 min relative to that for the
control, 60 min incubated with Ad only. (n = 4) (B) NOC does not affect binding of Ad5 to
LGAC. Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 x 10
6
cells/ml. On
day 2 of culture, vehicle-treated rabbit LGAC or LGAC pretreated with NOC (33 µM) for 30 min
at 4°C were incubated with Ad5 (MOI = 15) for 0 min, were treated or untreated with 0.2 mg/ml
and then processed as described above. Values shown represent fiber binding at 0 min relative to
that for control, 0 min incubated with Ad only. (n = 4) Error bars represent SEM.

78

Intracellular localization of Ad5
To conclude or studies of Ad5 in LGAC, we investigated its intracellular localization.
Since based on our inhibitor data we could not rule out CME as the primary route of
entry, we investigated whether Ad5 colocalized with EGF, our marker for receptor-
mediated endocytosis, and by extension, CME. These colocalization studies indicated
that although Ad5 is in close proximity to EGF after 1 hour, it is not significantly
colocalized (Figure 28). Pixel colocalization analysis confirmed the minuscule amount of
colocalization (8 ± 1%). This suggests that after 1 hour, Ad5 and EGF are trafficking in
different vesicle populations. We cannot rule out the possibility that Ad5 and EGF may
have been colocalized at earlier time points and have since been sorted to different
compartments.

79

Figure 27. Ad5 is not colocalized with EGF. Rabbit LGAC were seeded onto Matrigel-coated
glass coverslips in 12-well plates at a density of 2 x 10
6
cells/well. On day 3 of culture, cells were
exposed to replication-deficient Ad-LacZ (MOI = 100) and EGF (400 ng/ml) for 60 min at 37°C.
These were subsequently fixed and processed to fluorescence label EGF (green), Ad5 proteins
(red), and actin (purple). Arrows indicate close proximity of Ad5 and EGF. Bar = 10 µm.

80

Next, we again looked at the effect of NOC, only this time we looked at its effect on the
intracellular distribution of Ad5. As shown in Figure 28, disruption of the microtubule
network did appear to affect the intracellular movement of Ad5 as evidenced by the
change in the distribution pattern following treatment with NOC. This is consistent with
the reported trafficking of Ad along microtubules to reach the nuclear membrane. Figure
28 also confirms the effective disruption of the microtubule network, thereby validating
the NOC data presented in the biochemical studies discussed earlier.

81

Figure 28. Effect of nocodazole on intracellular Ad5 distribution. Rabbit LGAC were seeded
onto Matrigel-coated glass coverslips in 12-well plates at a density of 2 x 10
6
cells/well. On day 3
of culture, control cells or cells treated with NOC (33 µM) for 30 min at 4°C were exposed to
replication-deficient Ad-LacZ (MOI = 100) for 60 min at 37°C. These were subsequently fixed
and processed to fluorescence label Ad5 proteins (red), microtubules (green), and actin (purple).
Bar = 10 µm.

82

Discussion
In this chapter we investigated the uptake mechanism used by LGAC to internalize Ad5.
In most cell types, uptake of Ad5 is mediated by the penton base protein, while the fiber
protein is responsible only for initial attachment to the surface receptor. In fact, it has
been reported that Ad2 sheds the fiber capsid protein in a very early step of the entry
process, following or coinciding with endocytosis (60). Surprisingly, our findings here
indicate that uptake of Ad5 in LGAC occurs by a very different, fiber-dependent
pathway. Our fluorescence microscopy and biochemical assays showed that while penton
protein associates with the plasma membrane, it does not appear to be internalized. This
is despite the fact that LGAC express high levels of α
v
integrins, the specific receptor for
penton base protein. They also showed that fiber protein is readily internalized in LGAC,
as early as 15 minutes after warming, and can still be detected intracellularly after 1 hour.
It should be noted that these cells express a high level of CAR (202), and may
correspondingly be more aptly suited for internalization of Ad5 through its high-affinity
receptor. Though this mechanism has not yet been described in the literature, it is
possible that these very specialized cells, with their extensive and complex trafficking
pathways, may very well internalize Ad5 as a ligand-receptor complex. Some evidence
exists for the internalization of CAR as HeLa cells have been shown to internalize, and
subsequently degrade CAR upon Coxsackievirus B3 uptake (22).
Although Ad5 has been shown to elicit macropinocytosis, our data suggest that this route
is unlikely to serve as the primary route of entry in these cells. Earlier studies in our lab
83

by Dr. Jiansong Xie showed evidence of Ad5 colocalization with early endosome antigen
1 (EEA1), a protein found in early endosomes, which receive traffic from CME (202).
Our efforts to investigate the role of this pathway in the uptake of Ad5 in LGAC yielded
interesting results. We found that pharmacological inhibition of CME did not inhibit Ad5
uptake, although the same treatment in these cells showed 80% inhibition in the uptake of
EGF, a marker for this pathway (data not shown). This alone would suggest that CME is
not the primary route of entry in these cells, however, since these pharmacological
inhibitors have been shown to lack specificity (85), we cannot rule out the possibility that
they are affecting multiple pathways. Additionally, the possibility exists that by blocking
one pathway, we may be enhancing a compensatory pathway. The CPZ data shown here
supports this idea as treatment with CPZ resulted in an apparent increase in Ad5 uptake.
In our studies, we did not observe colocalization of Ad5 with EGF, our marker for CME.
Because of the time point examined, we cannot rule out the possibility that at some
earlier time point they were localized to the same compartment. For this reason, it would
be interesting to investigate their colocalization at various earlier time points.
Unable to define the role of dynamin in the uptake of Ad5 (due to complications with the
assessment of the control protein, EGF), we moved to evaluate the role of cholesterol on
the uptake pathway. Although uptake was not significantly inhibited following removal
of plasma membrane cholesterol by MBCD, binding of Ad5 to the cell surface was
significantly reduced. This is not surprising given that CAR is an integral membrane
protein and likely requires adequate lipid composition for proper localization and
function. Similarly, the data resulting from our biochemical assessment of the role of
84

microtubules suggests that while they are not absolutely required for uptake, they do
appear to play a role in the efficiency of the process. This is substantiated by changes in
the intracellular distribution of Ad5 following NOC treatment, as shown in our
microscopy studies. This is consistent with the reported role of microtubules in the
inward motion, and intracellular trafficking of Ad5. Taken together, these data suggest
that the fiber-dependent uptake of Ad5 in LGAC is not only different from what is
reported for other cell types, but may involve several complex and compensatory
mechanisms.

85

Chapter 6. Optimization of nucleofection for efficient transfection of LGAC

Rationale
Earlier we discussed some of the limitations of transfection in LGAC, including the fact
that the only successful gene-delivery methodology is currently the use of Ad5. Although
effective, this approach relies on the customized production of viral constructs, which is
often time-consuming and expensive, and is not conducive to delivery of siRNAs. We
also previously mentioned that nucleofection has permitted transfection of various
previously-difficult to transfect cells. In hopes of adding LGAC to this list of transfection
successes, we explored and optimized transfection of LGAC using nucleofection
technology for DNA plasmid and siRNA delivery.

Transfection of LGAC for DNA plasmid delivery
We began our efforts by evaluating the nucleofection of LGAC using two genes encoding
cytosolic and membrane-encapsulated proteins, GFP and Cathepsin S-GFP, respectively.
The lysosomal protease, Cathepsin-S, has been implicated in the development of
Sjögren’s syndrome and therefore represents an interesting therapeutic target. After an
initial screening of several program settings, it was determined that program Z-001
yielded the best transfection efficiency and cell viability balance. Fluorescence
microscopy and flow cytometry analysis of cells nucleofected with pmax-GFP, a control,
86

GFP-encoding plasmid, indicated that 47 ± 2% of cells were GFP positive after 24 hours
(Figure 29).

Figure 29. Nucleofection of LGAC. Rabbit LGAC were seeded in 150-mm petri dishes at a
density of 2 x 10
6
cells/ml. On day 1 of culture, cells were nucleofected with pmax-GFP (3 µg).
Nucleofected cells were analyzed for expression of GFP by fluorescence microscopy 24 h post-
nucleofection (A). Bar = 50 µm. Following microscopy imaging, the cells were prepared for
FACS analysis and assayed by such (B). (n = 4)

To examine whether the nucleofection process affected LGAC morphology, we
nucleofected cells with a plasmid encoding Cathepsin S, a cysteine protease enriched in
lysosomes with traces in secretory vesicles. As shown in Figure 30, nucleofected cells
showed the normal organization of apical and basolateral membranes relative to the
control cells, despite the obviously increased expression of cathepsin S. Additionally, the
nucleofected cells were able to accurately localize the overexpressed protein to a series of
punctate intracellular compartments, consistent with targeting to lysosomes.
87

Figure 30. Nucleofected LGAC are able to accurately localize a vesicular compartment
protein. Rabbit LGAC were seeded in 150-mm petri dishes at a density of 2 x 10
6
cells/ml. On
day 2 of culture, cells were or were not nucleofected with a Cathepsin S-GFP plasmid (3 µg) and
seeded on Matrigel-coated glass coverslips in 12-well plates. After 24 h, the cells were fixed and
processed to fluorescently label Cathepsin S (green) and actin filaments (red). A schematic is also
included in which lumena are marked with an ―L‖. Note the expression of Cathepsin S-GFP
localized to punctate organelles. Bar = 10 µm.

Because there are a number of transfection reagents commercially available and widely in
use, we compared the optimized nucleofection method to two other non-viral techniques:
a lipid-based transfection reagent, Fugene 6, and a cationic lipid-mediated transfection
reagent, Lipofectamine
™
2000, using pmax-GFP. Transfection of LGAC using Fugene 6
yielded very poor efficiency across the suggested reagent-to-DNA ratios (Figure 31A),
making this reagent clearly unsuitable for use in LGAC. Similarly, transfection of LGAC
using Lipofectamine
™
2000 also resulted in poor transfection efficiency with the highest
88

efficiency reaching approximately 2% of total cells (Figure 31B). For comparison, HeLa
cells were transfected in parallel and resulted in a 17 ± 2% transfection efficiency with
the same reagent-to-DNA ratio, indicating that while it may be useful for some cells, it
would not be a suitable option for transfection of LGAC.

Figure 31. Transfection of LGAC. Rabbit LGAC were seeded in 150-mm petri dishes at a
density of 2 x 10
6
cells/ml. On day 2 of culture, cells were transfected with either Fugene 6 (A) or
Lipofectamine 2000 (B) and pmax-GFP as described in Materials and Methods. After 24 h,
control and transfected cells were collected and prepared for analysis by flow cytometry. For
comparison, HeLa cells were also transfected an analyzed in parallel. (n = 3 for HeLa cells and n
= 4 for LGAC)

Transfection of LGAC for siRNA delivery
We also investigated the efficiency of siRNA delivery via nucleofection. To assess the
efficiency, we performed knockdown of CAR, the Ad receptor, which is highly expressed
in LGAC, and evaluated the efficiency by real-time PCR analysis. Furthermore, we also
89

performed parallel experiments using GeneSilencer and Lipofectamine
™
2000 for delivery
of siRNA duplexes. A comparison of these methods indicated that the highest
knockdown efficiency was achieved using nucleofection (Figure 32). Surprisingly,
Lipofectamine
™
2000 was shown to be quite efficient for siRNA delivery despite its poor
efficiency in delivery of plasmid DNA. Interestingly, delivery of the siControl non-
targeting sequence by nucleofection resulted in an increase in CAR expression levels.
This may be attributed, at least in part, to what is believed to be the physiological
function of CAR, mainly as a cell adhesion molecule. It is possible that the nucleofection
process itself, in essence a type of electroporation, triggered increased expression of the
protein to aid in re-establishment of the cell—cell contacts necessary for proper acinar
structure of these cells. These cell-to-cell junctions have been shown to be important for
the establishment of proper epithelial cell polarity.

90

Figure 32. Effective gene expression knockdown. Rabbit LGAC were seeded in 150-mm petri
dishes at a density of 2 x 10
6
cells/ml. On day 2 of culture, cells were either transfected with 100
pmol siRNA and 5 µl Lipofectamine, 2 µg siRNA and 10 µl GeneSilencer, or nucleofected with 2
µM siRNA or a non-targeting sequence, siControl. After 24 h, the total RNA from control and
experimental samples was isolated, purified, and used for cDNA synthesis. Relative expression
levels were determined by quantitative real-time PCR. Results were normalized to HPRT1
mRNA and are expressed relative to control, untreated cells. *, significant at p ≤ 0.05. #,
significant relative to both control and Lipofectamine 2000 CAR-knockdown. (n = 4)

91

Evaluation of nucleofection cytotoxicity
To properly evaluate nucleofection as a viable transfection method in LGAC, it is
imperative to assess its effect on cell viability. To do so, we utilized a commercially
available viability/cytotoxicity kit. Analysis using this kit indicated that nucleofected
cells retained viability comparable to control cells and did not vary with cargo cargo
delivered (e.g. plasmid DNA or siRNA) (Figure 33), suggesting that the nucleofection
process does not affect cell viability. Viability of these cells is normally in the 60-70%
range as they are primary reconstituted cells that have undergone a substantial digestion
procedure during the cell culturing process.

Figure 33. Nucleofection does not affect cell viability. Rabbit LGAC were seeded in 150-mm
petri dishes at a density of 2 x 10
6
cells/ml. On day 2 of culture, cells were either mock-
nucleofected or nucleofected with either 3 µg pmaxGFP or 2 µM siRNA. After 24 h, the cells
were assayed using the LIVE/DEAD viability/cytotoxicity kit for mammalian cells. (n = 4)
92

Discussion
These data indicate that nucleofection offers a very efficient and useful technique for
transfection of LGAC. The level of transfection and knockdown reported here is the first
for this cell type. This optimized methodology presents a powerful, non-viral tool that
could help advance the understanding of the lacrimal gland and its associated diseases,
and may be relevant for use in other primary exocrine cells such as pancreatic acini.

93

Chapter 7. Conclusions and Perspectives
Understanding the endocytic mechanism of any potential drug and delivery constructs is
important to their applications as therapeutic and diagnostic tools. This often proves to be
quite a difficult task as there is such an abundance of possibilities in terms of
formulations, sizes, shapes, and compositions. As demonstrated here, even if one is able
to define the pathways that mediate cellular entry in one cell type, these will likely vary
considerably in a different cell line, model, or system. For this reason, studies that
graduate from a simple cell model, to a more complex, physiologically- and disease-
relevant one, are imperative. Of course, the transition from the simpler model to the more
complex one, introduces added difficulties. In our case, studies in LGAC are limited by
several factors. The first is their innate variability as primary cells. Unlike cell lines, these
cells are prepared from fresh tissue each time, and while consistency helps minimize
variability, from prep to prep there are slight differences. The second is the lack of
commercially available reagents, specifically antibodies and markers for specific
organelles. We found this to be especially limiting in our efforts to further characterize
the role of CAR in the uptake of Ad5. Although rabbit CAR shares a high homology to
human CAR, the commercially available anti-human CAR antibody was completely
useless in LGAC. One hurdle that our work here helped to overcome is their poor
transfection efficiency. In fact, the nucleofection methodology was optimized precisely to
further enable Ad uptake and binding studies.
94

Despite these limitations, LGAC offer the perfect investigative model for targeting of
diagnostics and therapeutics to the lacrimal gland. Due to their unique optical properties,
the BNPs studies here would be particularly useful as probes used to gather information,
such as oxygen concentrations, in microenvironments. Additionally, they would be ideal
for in vivo applications as they are two-photon excitable, bright, and degradable.
However, in their current, unmodified state, they are quite poorly internalized in LGAC.
An important question is whether these nanoparticles can be redirected from their default
pathway or modified through attachment of targeting ligands. Only after such a
modification, and re-evaluation in LGAC, could BNPs be considered possible probes for
use in the lacrimal gland.
Unlike BNPs, MCoTI-I was well internalized in LGAC and showed no specific affinity
for a surface receptor or protein. This suggests that MCoTI-I may be readily available for
uptake into many different cells. One of the advantages of this candidate is that it appears
to be highly amenable to sequence variation, making retargeting a likely possibility. This
would allow direction of the cyclotide to the dominant pathway or unique surface
receptor of the target cell. Another benefit is that MCoTI-I is a peptide. Although
peptides have been shown to be effective drug candidates (162), their utility has been
limited by their generally poor stability and limited bioavailability. With their remarkable
stability and demonstrated bioavailability(161), cyclotides such as MCoTI-I, overcome
these limitations. Because these cyclotides can serve as molecular scaffolds, they permit
the possibility of introducing biologically active therapeutic motifs. As proof of principle,
acyclic squash inhibitors, which show sequence homology to MCoTI cyclotides, have
95

already been used as scaffolds for the incorporation of novel bioactive peptides (95, 151).
It is of particular interest to further investigate the terminal location of MCoTI-I in
LGAC. Because some cyclotides naturally exhibit protease inhibitory activity, these
could be quite useful as inhibitors to modulate the function of Cathepsin S, implicated in
the development of Sjögren’s syndrome, thereby offering a novel therapeutic approach.
Ideally, if MCoTI-I is able to reach lysosomal compartments, it could efficiently target
Cathepsin S, a lysosomal protease. We clearly demonstrated the significant accumulation
of MCoTI-I in lysosomes in HeLa, but observed only a small amount (~10%) in LGAC at
an equivalent time point. Based on this observation, it is possible that given more time, a
larger percentage will accumulate in lysosomal compartments in LGAC. Additional
studies are necessary to confirm whether this is the case. Furthermore, studies looking at
the distribution of MCoTI-I over time, such as those conducted in HeLa, would provide
additional insights into the trafficking of MCoTI-I in LGAC. Overall, MCoTI-I offers an
attractive drug development tool that in a HeLa cell model, has ready access to general
endosomal and lysosomal pathways, but could readily be re-targeted to specific receptors
through addition of targeting ligands.
Our studies with Ad5 presented some of the more difficult challenges and left many
unanswered questions. The observation that LGAC are so effectively transduced
(routinely in the 90% range), initiated the query as to whether these cells might utilize a
unique endocytic mechanism. Our finding that uptake of Ad5 in LGAC was by a
different, fiber-dependent mechanism was surprising and quite interesting. Earlier data
suggested that CME may play a role in the uptake of Ad5 in these cells. However, this
96

still remains to be confirmed as our inhibitor studies did not yield conclusive results.
Instead, the picture that emerges for the uptake of Ad5 in LGAC is a complex one that
may involve compensatory mechanisms. This may be due in part to the fact that these
cells are themselves quite complex, and are specialized for highly regulated trafficking.
Although we know Ad5 has been successfully used for gene delivery in the lacrimal
gland, it is essentially useless for clinical approaches in its unmodified form since
immunogenicity is a major risk. For this reason, it is crucial to understand the
mechanisms that facilitate its uptake and trafficking in LGAC. With an understanding of
these pathways, as well as the minimum viral sequence necessary to elicit them, we could
begin to really evaluate potential drug delivery candidates without the current risks posed
to patient safety. Needless to say, there are still many questions to be answered with
regard to the uptake of Ad5 in LGAC. One of the major questions is what the exact role
of CAR is in these cells: whether it participates only as a binding receptor, or whether it
also directly mediates the uptake of Ad5. If it does facilitate uptake, the question then
arises as to whether the receptor itself is also internalized. If it only participates in
binding, and we know that penton does not enter the cells, then the question arises as to
what other surface receptor(s) may help mediate internalization. Although heparan
sulfate-glycosaminoglycans have been implicated in Ad5 infectivity (202), their precise
role remains unknown.
In summary, these findings demonstrate the unique challenges encountered on the path to
assessing the utility and feasibility of potential theranostics and drug delivery candidates.
They also highlight the importance of using physiologically- and disease-relevant cell
97

models, especially since information obtained from one cell type is likely not applicable
to another. Furthermore, the data obtained here serve as the foundation for the continued
investigation of these candidates as potential therapeutic tools for targeting to, as well as
treatment of diseases associated with, the lacrimal gland.

98

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Abstract (if available)

Abstract When identifying potential candidates for drug delivery or biomedical diagnostic tools, it is of utmost importance to delineate, as best as possible, their uptake and intracellular trafficking pathways. Here we investigated these pathways, in a simple cell model first, followed by a more complex and physiologically-relevant cell model, for three potential theranostic and drug delivery candidates. The first is a nanoparticle based on difluoroboron dibenzoylmethane-poly(lactic acid), that exhibits unique molecular-weight dependent emission properties. We showed that these nanoparticles are highly photostable, in vitro and in situ, resisting laser-induced photobleaching under conditions that destroy the fluorescence associated with a common photostable probe, LysoTracker Blue. Also, we showed that the internalized nanoparticles do not accumulate in acidic compartments such as late endosomes and lysosomes, but rather in a non-lysosomal perinuclear compartment. Additionally, we demonstrated that their uptake utilizes actin filaments and microtubules. These findings demonstrate the feasibility of using these nanoparticles with unique emission properties for in situ, live cell imaging. ❧ The second candidate examined is a cyclotide, MCoTI-I. Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose cyclic structure makes them remarkably stable and resistant to denaturation and degradation. Using real-time confocal microscopy imaging, we showed that MCoTI-I is readily internalized in HeLa and lacrimal gland acinar cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I is achieved primarily through fluid-phase endocytosis in HeLa as evidenced by its significant colocalization with 10,000 MW dextran, but also through other pathways as well as evidenced by its colocalization with cholera toxin-B and epidermal growth factor. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway did not affect MCoTI-I uptake. In lacrimal gland acinar cells, endocytosis is also achieved primarily through fluid-phase endocytosis, albeit to a lesser extent. In HeLa, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments where MCoTI-I-containing vesicles continue to exhibit microtubule-associated movements. In contrast, almost no MCoTI-I reaches acidic compartments in lacrimal gland acinar cells in the same time frame. These findings demonstrate internalization of MCoTI-I through multiple pathways that may be dominant in the cell type investigated, and suggest that this cyclotide has ready access to general endosomal pathways. ❧ The third and final candidate investigated is Adenovirus 5. The established method of Adenovirus 5 infection in most cells is by a penton-dependent mechanism. Here, we demonstrated that while the penton base remains on the surface of lacrimal gland acinar cells, fiber is readily internalized, suggestive of a fiber-dependent entry mechanism. We also determined that macropinocytosis is not the prevalent route of uptake. Data from studies involving various endocytosis inhibitors suggest that uptake of Adenovirus 5 in lacrimal gland acinar cells may rely on multiple pathways other than primarily clathrin-mediated endocytosis. Additionally, binding of Adenovirus 5 to its surface receptor appears to be cholesterol-dependent. Also, the inward movement of internalized Adenovirus 5 seems to rely on the microtubule network. Taken together, these studies indicate that the internalization and intracellular trafficking of Adenovirus 5 in lacrimal gland acinar cells follow a different pathway than what is reported in the literature, and may involve very complex and compensatory mechanisms. ❧ Together, these findings demonstrate the unique challenges encountered on the path to assessing the utility and feasibility of potential theranostics and drug delivery candidates. They also highlight the importance of using physiologically- and disease-relevant cell models, especially since information obtained from one cell type is likely not applicable in another.

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Creator Contreras, Janette (author)

Core Title Analysis of endocytic and trafficking pathways of potential candidates for drug delivery in HeLa and lacrimal gland acinar cells

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2012-05

Publication Date 02/21/2012

Defense Date 11/22/2011

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

Tag adenovirus,cyclotides,endocytosis,HeLa,lacrimal gland,nanoparticles,OAI-PMH Harvest,trafficking

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Language English

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Advisor Hamm-Alvarez, Sarah F. (committee chair), Garner, Judy A. (committee member), Okamoto, Curtis Toshio (committee member), Shen, Wei-Chiang (committee member)

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