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Development of positron emission tomography (PET) labeled polypeptide nanoparticles for tumor imaging and targeting

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Development of positron emission tomography (PET) labeled polypeptide nanoparticles for tumor imaging and targeting

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Content DEVELOPMENT OF POSITRON EMISSION TOMOGRAPHY (PET)
LABELED POLYPEPTIDE NANOPARTICLES FOR TUMOR
IMAGING AND TARGETING

by

Siti Najila Mohd Janib

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 2013

Copyright 2013 Siti Najila Mohd Janib
ii

Dedicated to my parents –
for their endless love, support and encouragement

iii

ACKNOWLEDGEMENTS
I would like to express my sincere gratitude and appreciation to my advisor Dr J. Andrew
MacKay for his continuous support, patience, motivation and enthusiasm throughout the course
of my graduate study. His mentoring and encouragement have been especially valuable and I
could not have imagined having a better advisor and mentor for my PhD study.
Besides my advisor, I would like to thank the other members of my committee – Dr
Walter Wolf, Dr Peter Conti and Dr Frank Markland for the assistance and guidance they
provided at all levels of the research project. Their encouragement and insightful comments are
most appreciated.
Appreciation also goes to Dr Zibo Li and Dr Shuanglong Liu of the USC Molecular
Imaging Centre and Dr Steve Swenson and Dr Radu Minea from the Markland Lab for their kind
assistance in lending me their expertise and time throughout the course of my study. .
I would also like to thank my friends in the MacKay lab – Martha Pastuszka, Suhaas
Aluri, Ara Moses, Pu Shi, Wan Wang, Jugal Dhandhukia, Dr Josh Gustafson and previous
members of the lab, for useful discussions, exchanges of knowledge and skills and venting of
frustration during my time in the lab. You all have enlivened the graduate student experience
with good humor and good camaraderie! My sincere gratitude also to friends and colleagues at
the Molecular Imaging Centre – Grant Dagliyan, Ryan Park, Dr Chiun Wei-Huang, Dr Li Peng-
Yap, Madlen Aldadyan, Lindsey Hughes and Joe Cook for their kindness, assistance and
friendship.
iv

I gratefully acknowledge the Malaysian Public Services Department for awarding me a
full scholarship to carry out my doctoral study at USC. In addition my thanks also go to the
Malaysian Nuclear Agency for giving me the opportunity to pursue a PhD.
And last but by no means least, my eternal gratitude to my parents Mohd Janib Johari and
Hasnah Jamaluddin, and my family for all their unequivocal support, unfailing patience and love
for which no words would ever come close to sufficiently express.

v

TABLE OF CONTENTS

LIST OF TABLES viii
LIST OF FIGURES ix
LIST OF ABBREVIATIONS xi

PROLOGUE 1

CHAPTER 1 Introduction

1.1 The cancer problem 4
1.2 Cancer nanotechnology 5
1.2.1 Drug conjugates and complexes 6
1.2.2 Micelles 7

1.3 Protein polymers 8
1.3.1 Elastin-like polypeptides 10

1.4 Angiogenesis and integrins 15
1.4.1 Disintegrin 20

1.5 Integrin imaging 22

CHAPTER 2 Biophysical characterization of protein polymer nanopaerticles
composed from elastin-like polypeptides

2.1 Abstract 26
2.2 Introduction 27
2.3 Materials and methods 32
2.3.1 Materials 32
2.3.2 Modification of pET 25b(+) vector 32
2.3.3 ELP gene insertion 33
2.3.4 Recombinant synthesis of ELPs by plasmid reconstruction recursive directional 35
ligation (pre-RDL)
2.3.5 Generation of ELP block copolymer library 34
2.3.6 Protein purification by inverse transition cycling 35
2.3.7 Optical density characterization of protein polymer phase diagrams 35
2.3.8 Secondary structure determination using circular dichroism (CD) 36
2.3.9 Temperature-dependent dynamic light scattering 36
2.3.10 Conjugation of ELP block copolymers to carboxyfluorescein (CF) 37

2.4 Results 38
2.4.1 Generation of ELP monoblock and diblock copolymer libraries 38
2.4.2 Thermal characterization of ELP block copolymers 42
2.4.3 Role of secondary-structure during assembly of ELP nanoparticles 48
vi

2.4.4 Cellular localization of the polymeric micelles 52

2.5 Discussion 53

2.6 Conclusion 54

CHAPTER 3 An assessment of the effects of molecular weight, amino acid
composition, and nanostructure on the biodistribution of ELP protein
polymers

3.1 Abstract 55

3.2 Introduction 57

3.3 Materials and methods 61
3.3.1 Recombinant synthesis of ELPs 61
3.3.2 Protein purification by inverse transition cycling 62
3.3.3 Dynamic light scattering of particle assembly 62
3.3.4 Transmission electron microscopy (TEM) sample preparation 63
3.3.5 Orthotopic xenograft of human breast cancer model 64
3.3.6 Preparation of AmBaSar-ELP conjugates 64
3.3.7 Radiolabeling 64
3.3.8 Stability of 64Cu-ELP-Sar constructs 65
3.3.9 MicroPET imaging study 65
3.3.10 Quantitative analysis of PET images 66

3.4 Results 67
3.4.1 Preparation and characterization of ELPs and ELP-Sar constructs 67
3.4.2 Stability of radiolabeled 64Cu-ELP-Sar constructs 70
3.4.3 In vivo microPET imaging of protein polymers 72
3.4.4 Quantitative analysis of PET images 73

3.5 Discussion 77

3.6 Conclusion 78

CHAPTER 4 Development of anti-angiogenic protein polymer nanoparticles and
their evaluation using positron emission tomography

4.1 Abstract 80

4.2 Introduction 82

4.3 Materials and methods 86
4.3.1 Cells 86
vii

4.3.2 Recombinant synthesis of ELPs 86
4.3.3 Protein purification by inverse transition cycling 87
4.3.4 LCST characterization of ELP and ELP-fusion protein 87
4.3.5 Dynamic light scattering of particle assembly 88
4.3.6 Transmission electron microscopy (TEM) sample preparation 88
4.3.7 Fluorescence activated cell sorter (FACS) analysis 88
4.3.8 Confocal microscopy 89
4.3.9 Cell integrin receptor binding assay 90
4.3.10 Orthotopic xenograft model of human breast cancer 90
4.3.11 Preparation of DOTA Conjugate 91
4.3.12 Preparation of AmBaSar-ELP conjugates 91
4.3.13 Radiolabeling 92
4.3.14 MicroPET imaging study 92
4.3.15 Quantitative analysis of PET images 92

4.4 Results 94
4.4.1 Characterization of ELP-VCN constructs 94
4.4.2 Evaluation of the expression of integrin αvβ3 receptor 96
4.4.3 Intracellular uptake of ELP-VCN by flow cytometry analysis 97
4.4.4 Quantitative evaluation of the cellular uptake of ELP-fusion proteins 98
4.4.5 Cell Integrin Receptor-Binding Assay 103
4.4.6 Small animal PET scan 104
4.4.7 Quantitative analysis of PET images 107

4.5 Discussion 113

4.6 Conclusion 114

CHAPTER 5 Conclusion and future directions

5.1 Conclusion 115

5.2 Future directions 119

References 124

viii

LIST OF TABLES
Table 1-1 Classification of disintegrins 20

Table 1-2 Library of ELP genes expressed and characterized for phase behavior of 34
monoblocks.

Table 2-2 Libraries of ELP diblock copolymers characterized 39

Table 1-3 Properties of ELP protein polymers evaluated in this study 67
ix

LIST OF FIGURES
Figure 1-1 Structural representations of nanoparticle classes functionalized for theranostics 6
Figure 2-1 Reversible phase transition of ELPs 11
Figure 3-1 ELP-based drug delivery strategies 13
Figure 4-1 The different steps of the angiogenic cascade 15
Figure 5-1 The Integrin Receptor Family 17
Figure 6-1 Integrin conformation-function relationships: a model 18
Figure 7-1 Structure of trimestatin 21
Figure 8-1 Bifunctional chelating for tagging a molecule with metallic radionuclide 24
Figure 1-2 Biosynthesis of ELP block copolymers that assemble micelles 41
Figure 2-2 Transition temperature for monoblock polypeptides depends on molecular 47
weight and hydrophobicity.
Figure 3-2 Particle diameter and micelle stability depend on molecular weight 44
Figure 4-2 Bulk phase transition temperature for stable ELP nanoparticles depends on the 52
hydrophilic block.
Figure 5-2 ELP assembly behavior and particle radius are independent of diblock 53
orientation
Figure 6-2 Nanoparticle assembly is accompanied by formation of secondary structure 51
Figure 7-2 ELP nanoparticles are internalized to low pH compartments in HeLa cells 52
Figure 1-3 Conjugation scheme of the bifunctional chelating agent AmBaSar and ELP 63
Figure 2-3 ELP diblock copolymers assemble nanoparticles at physiological temperatures 69
Figure 3-3
64
Cu-ELP constructs are stable in serum for 24 hours 71
Figure 4-3 Serial microPET imaging of protein polymer nanoparticles in an orthotopic 81
model of human breast cancer
Figure 5-3 Non-compartmental pharmacokinetics of
64
Cu-ELPs in the heart 74
Figure 6-3 Biodistribution of
64
Cu-ELP 76
x

Figure 1-4 Characterization of ELP-fusion proteins. 95
Figure 2-4 Relative expression of target integrin across primary and transformed 106
cell lines
Figure 3-4 Binding of integrin-targeted ELPs is receptor-mediated. 97
Figure 4-4 Specific uptake of protein polymers in HUVEC, MDA-MB-231 and 112
MDA-MB-435 cells observed by confocal laser scanning microscopy
Figure 5-4 Competition binding to integrin receptors expressed on MDA-MB-435 cells. 103
Figure 6-4 Serial microPET imaging of ELP and ELP-fusion protein in an orthotopic 116
model of human breast cancer
Figure 7-4 Circulation half-life of A192-DOTA, A192-Sar, A192-VCN-DOTA and 118
A192-VCN-Sar
Figure 8-4 Biodistribution of
64
Cu-ELP 111
Figure 9-4 Blocking study 112
Figure 1-5 Proposed formation of A192-VCN ‘nanostructure’. 120

xi

LIST OF ABBREVIATIONS
%ID/g percentage injected dose per gram
64
Cu copper-64
CD circular dichroism
CF carboxyfluoroscein
CMC critical micelle concentration
CMT critical micelle temperature
CN controtostatin
DLS dynamic light scattering
DOTA 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid
DTPA diethylene triamine pentaacetic acid
EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
ELP elastin-like polypeptide
EPR enhanced permeability and retention
FACS fluorescent activated cell sorter
FOV field of view
HUVEC Human umbilical vein endothelial cell
IC50 inhibitory concentration at 50%
ITC inverse transition cycling
LCST lower critical solution temperature
MALDI-TOF Matrix-assisted laser desorption/ionization- time of flight
MPS mononuclear phagocyte system
MW molecular weight
OD optical density
PBS phosphate buffered saline
PEG polyethylene glycol
PET positron emission tomography
PK pharmacokinetics
pre-RDL plasmid reconstruction recursive directional ligation
RDL recursive directional ligation
RES reticulo-endothelial system
RGD arginine, glycine, and aspartic acid
R
h
hydrodynamic radius
ROI region of interest
SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
SNHS N-hydroxysulfosuccinimide
SOD superoxide dismutase
TEM transmission electron microscope
T
t
transition temperature
VCN vicrostatin

1

PROLOGUE

The two main problems currently stalling the efficient treatment of cancer has been detecting
cancer early enough in the disease process for successful treatment, and treating cancer cells
while avoiding excessive toxicity to normal tissues. Arguably the most important factor in the
fight against cancer, besides prevention is early detection because the cancer will be easier to
treat and less likely to have drug resistance.

The work highlighted in this thesis attempts to address the issues related to the effective
treatment and management of cancer. The objective of this work is to develop new materials
and methods for co-assembly of drugs and imaging agents that permit quantitative imaging of
drug delivery and disease progression. By using molecular imaging technique to non-invasively
study and detect various molecular markers of diseases can allow for much earlier diagnosis,
earlier treatment, and better prognosis that will eventually lead to personalized medicine.
Exploration of particulates and polymeric carriers is gaining momentum in diagnostic imaging,
initiated by successful therapies using long circulating liposomes. However, liposomes are
challenging pharmaceuticals, which include many chemical components, require complex drug
encapsulation strategies, and must be physically sheared to control their particle diameter and
polydispersity. Polymeric nanocarriers have emerged as an alternative to liposomes as carriers
of drugs and imaging agents. Co-inclusion of therapeutic and imaging agents, into these carriers
might be advantageous because they increase solubility of hydrophobic agents, may enhance
permeability across physiological barriers, alter drug biodistribution, increase local
bioavailability and reduce of side effects.
2

Chapter 3 discusses the viability of using protein polymers as a basis for constructing micelle-
based nanocarriers. The hypothesis put forward was that amphiphilic block copolymers
composed of elastin-like polypeptide (ELP) would be able to form micelles upon the stimulation
of heat. To this end ELP block copolymers with different lengths and amino acid sequence was
recombinantly synthesized and characterized from which micelles of stable and monodisperse
particles were obtained. Identifying the minimum cut-off a point for such a formation aids in the
rational design of ELP micelles with a specific length, critical micelle temperature (CMT), and
size.

The physical properties of nanocarriers such as size, charge, shape and surface chemistry plays
an important role in its in vivo distribution. To facilitate biodistribution analyses, it would be
advantageous if the circulation time and organ accumulation of nanocarriers could be visualized
non-invasively in vivo in real time. Testing the hypothesis that circumventing renal filtration,
leads to prolonged blood circulation which results in an increase in tumor accumulation,
Chapter 4 explores the biodistribution of ELPs of different lengths, amino acid sequence and
nanostructure which has been radiolabeled with the positron emitter copper 64. Positron
emission tomography (PET) was chosen as the modality of choice as it is sensitive and
quantitative and the longer half-life of the copper, allows the biodistribution of the constructs to
be tracked for up to 48h. The outcome of this study reinforces the importance of size in
determining the blood half-life of nanocarriers used. Through the use of molecular imaging it is
thus possible to non-invasively optimize carrier characteristics for the delivery of therapeutic
drugs and proteins.
3

The success of molecular targeted agents has led to increasing interest in combining molecular
targeting and nanoparticle delivery. This spurs the hypothesis that nanoparticles with a targeting
domain will be taken up better into target cells than non-targeted nanoparticles. Chapter 5
highlights studies carried out with an ELP-fusion protein composing of a therapeutic peptide
(vicrostatin, VCN) that is appended to an ELP-based nanocarrier. Its targeting ability against the
integrin αvβ
3
, an angiogenic marker was evaluated. It was found that ligand targeted nanocarrier
was selectively taken up and accumulated in tumor tissues with greater efficiency than the
untargeted counterpart. Selective tumor accumulation was also observed in in vivo imaging
experiments. The effectiveness of using macromolecular nanocarriers such as ELPs to improve
and extend the biological half-life of a short therapeutic peptide was also demonstrated. This
work is timely because molecular imaging of receptors or markers expressed on the surface of
tumor cells is becoming a major field of investigation in clinical oncology, especially for the
detection of cancer at its earliest stages.
4

Chapter 1
Introduction
1.1 The cancer problem
Cancer remains one of the leading causes of death in the worldwide accounting for 7.6 million
deaths in 2008
1
. This number is projected to continue to rise over 13.1 million in 2030. Despite
advances in the understanding of molecular and cancer biology, discovery of cancer biomarkers
and conventional surgical procedures, radiotherapy and chemotherapy the overall survival rate
from cancer has not significantly improved in the past two decades
2
. The development of novel
approaches for early detection and cancer marker specific and eventually personalized treatment
of cancers is urgently needed to increase patient survival.

Human cancer is a complex disease caused by genetic instability and accumulation of multiple
molecular alterations
3, 4
. Current diagnostic and prognostic classifications more often than not
fail to accurately make prediction for treatment and patient outcome as it does not take into
account this heterogeneity. Furthermore most current anticancer agents are unable to
differentiate between cancerous and normal cells, leading to systemic toxicity and adverse
effects. In addition, cancer is often diagnosed and treated too late, when the cancer cells have
already invaded and metastasized into other parts of the body. At this stage, therapeutic
modalities are limited in their effectiveness. Due to these problems, cancer has overtaken heart
disease as the leading cause of death for adults in the United States [United States Cancer
Statistics, Centers for Disease Control and Prevention (CDC)
[http://www.cdc.gov/cancer/npcr/uscs]
3
.

5

1.2 Cancer nanotechnology
Nanotechnology is a multidisciplinary field involving disciplines in biology, chemistry,
engineering and medicine. A branch of nanotechnology, called nanomedicine is described as the
application of nanotechnology to medicine and healthcare thus allowing the development of
nanoparticle drug delivery system
5
. These nanoscale (1-100nm) therapeutic systems have
emerged as novel therapeutic modalities for cancer treatment and are expected to lead to major
advances in cancer detection, diagnosis and treatment. The progression of nanoparticle
technology toward solving these problems is mainly a result of the properties of these
compounds, which include:

i. Their diminutive size allows preferential accumulation in the tumors via the enhanced
permeability and retention effect (EPR). In addition the sizes also allow escape from
renal elimination for increase therapeutic efficacy.
ii. The ability to endow disease specific targeting agents to enhance of therapeutic delivery
in a tissue- or cell-specific manner.
iii. The ability to deliver a combination of imaging and therapeutic agents for real time-
monitoring of therapeutic efficacy.
iv. The high surface area to volume can allow increase loading therapeutics
v. Nanoparticle multivalency greatly enhance its ability to bind to targets with great affinity
and avidity

Nanoparticles can be made from a number of materials including proteins, peptides, polymers,
lipids, metals and metal oxides, and carbon. While other materials can form nanoparticles, these
6

are the predominant materials under development. The most relevant nanoparticle structures
include drug conjugates and complexes, dendrimers, vesicles, micelles, core–shell structures,
microbubbles, and carbon nanotubes, which can all be functionalized with a targeting moiety,
therapeutic, and contrast agent (Fig. 1-1).

Figure 1-1. Structural representations of nanoparticle classes functionalized for theranostics.
Schematics of a functionalized a) drug conjugate; b) dendrimer; c) vesicle; d) micelle; e) core–shell
nanoparticle; f) microbubble; and g) carbon nanotube
6
.

1.2.1 Drug conjugates and complexes
In contrast to self-assembled structures like vesicles and micelles, complexation and covalent
conjugation are other direct routes to prepare nanoparticles. Drug complexes rely on reversible
interactions between carrier and drug, whereas drug conjugates utilize covalent interactions.
Drug conjugates can be prepared using many chemical pathways, which often depends on the
chemistry of the drug as well as the carrier. The two major classes of drug conjugates currently
under development for theranostic use include protein and peptide-associated and polymer-
associated drugs. As with vesicular structures, the effectiveness of a drug conjugate is related to
7

its ability to improve therapeutic index relative to free drug, generally, by reducing toxicity
and/or improving efficacy.

Proteins and peptides are versatile materials that can form nanoparticles in several ways,
including complexation. The best example of a successful protein nanoparticle already in clinical
use is Abraxane
TM
, a formulation of paclitaxel reversibly bound to 130–150 nm albumin
nanoparticles via high pressure homogenization
7, 8
. Abraxane
TM
outperformed conventional
paclitaxel at an equitoxic dose while decreasing toxicity, due to longer circulation time and
decreased off-target activity
7, 9
.

Polymer-drug conjugation is a relatively reliable method to improve pharmacokinetics and
increase therapeutic index; non-covalent complexes have a similar effect. Albumin-bound
paclitaxel is routinely used in the clinic. The barriers to drug conjugate and complex
development into TNPs include the necessity for efficient drug loading and minimization of
complexity to control production cost and reliability. Both natural and synthetic polymers are
excellent platforms for overcoming these barriers
6
.

1.2.2 Micelles
Micellar nanoparticles are attractive structures for carriers of drug and contrast agents because
they can form relatively uniform size structures, be prepared from a variety of amphiphilic
materials, increase solubility of hydrophobic molecules, and incorporate multiple functionalities
into a single structure. Lacking an aqueous core, the drug and contrast agent must be bound to
the polymer prior to formation, conjugated to an anchor molecule, or entrapped and associated
8

within the dense, hydrophobic core of the micelle
10
. Micellar structures, including polymeric
micelles, have been extensively studied as drug carriers
11
.

Like polymersomes or polymer-drug conjugates — similar polymeric materials can be used to
form polymeric micelles. In general, a hydrophobic block of the copolymer forms the micellar
core, while a hydrophilic portion forms the corona. This corona (commonly consisting of PEG,
HPMA, or equivalent hydrophilic polymer) confers these micelles with biocompatibility, stealth-
like properties, and a platform for functionalization
12-14
. Genexol-PM
TM
is a formulation of
paclitaxel encapsulated in a polymeric micelle formed by the solid dispersion technique from the
biodegradable block copolymer, monomethoxy poly(ethylene glycol)-block-poly(D,L-lactide)
15
.
Several clinical trials have already been performed evaluating and validating the safety and
efficacy of Genexol-PM
TM
in metastatic breast cancer, solid tumors, and non-small-cell lung
cancer
16-18
.

Due to ease of formation, stability, ability to encapsulate hydrophobic molecules, and therapeutic
success in preclinical and clinical studies, polymeric micelles are widely accepted as viable drug
and imaging agent delivery systems
13, 19-23
. However, the stability of micelles depends on their
critical micelle concentration, which makes them prone to exchange their components with other
physiological membranes.

1.3 Protein polymers
Recent years have seen an increasing demand for technologically advanced smart polymers that
responds to small changes in their environment. Protein-based polymers, which are composed of
9

repeat units of natural or unnatural amino acids, have recently emerged as a promising new class
of stimulus-responsive materials. Biopolymers offer various advantages over other synthetic
polymer-based nanoparticles, such as good biocompatibility, biodegradability, and homogeneity.
These properties make them ideal for use as biomedical devices. Some examples of natural
biopolymers currently being investigated include collagen
24, 25
, elastin
26-28
, silk
29-31
and resilin
32,
33
.

The maturation of recombinant DNA technologies has allowed these protein based materials to
be synthesized in high yields, while retaining precise control over their molecular mass,
composition sequence and monodispersity. These physical properties have a profound effect on
the pharmacokinetics and cellular uptake of the biopolymers and as such complete sequence and
architecture control allows such materials to be designed with an extremely high degree of both
functional and structural complexity. It is now possible to encode functional peptide domains
that confer mechanical, structural or bioactive properties making it possible to fine-tune the
characteristics of the produced biomaterial, adapting it to its eventual use.

Bipolymeric materials can be constructed by using concatemerization, step by step directional
approach and recursive directional ligation (RDL). Concatemerization is a useful method when a
library of genes of different sizes is desired, but has limitations in the preparation of genes with
specific sizes
34
. To overcome this limitation step-by-step ligation and RDL can be used as an
alternative. RDL allows for facile modularity, where control over the size of the genetic
cassettes is achieved. Moreover RDL eliminates the restriction sites at the junctions between
10

monomeric genetic cassettes without interrupting key gene sequences with additional base pairs
that makes it different from the step by step approach
35
.

Conventional chemical synthesis could not even begin to offer the same level of complexity as
that provided by genetic engineering techniques. While solid state synthesis can be efficient for
small scale production and testing, scaling up can prove to be expensive and time consuming.
Whilst polymers that are monodisperse, ranging from a few hundred Daltons to more than
200kDa can be easily produced via biosynthetic processes, solid state synthesis struggles to
produce peptide of lengths over 35 residues in any acceptable yields
36
.

It is thus becoming increasingly clear that through the combination of advanced protein
engineering techniques and the advantages associated with biopolymers, this field will remain an
active area of research, possibly becoming a source for new and novel biomaterials for use in
medicine.

1.3.1 Elastin-like polypeptides
One of the protein polymers that are currently under investigation for its potential in biomedicine
is a class of proteins called the elastin like polypeptide (ELP). It is a biopolymer derived from
the structural motif found in mammalian elastin protein
37
and has a sequence dependent
transition temperature that can be used as nanocarriers to treat diseases. ELP is a protein
comprised of a five amino acid repeat (VPGXG, where X is any amino acid except proline).
ELPs are attractive as polymeric carriers for drug delivery because they undergo an inverse
temperature phase transition. Below a characteristic transition temperature (T
t
), ELPs are
11

structurally disordered. But, when the temperature is raised above their T
t
, they undergo a sharp
(2–3
o
C range) phase transition, leading to the aggregation of the biopolymer
37, 38
. This process is
reversible (Fig. 2-1). The T
t
is a function of the identity of the guest residue X, molecular weight
and the length of pentameric repeat. Phase transition can also be triggered by other external
stimuli such as changes in ionic strength, pH, solvent, magnetic fields etc.

Figure 2-1. Reversible phase transition of ELPs. ELPs are soluble below their transition
temperature (Tt) but aggregate above it. This process is reversible.

Being able to genetically engineer these polypeptides allow precise control over their length,
sequence and monodispersity. ELPs can be expressed at high yields of 50-500mg/L and can be
induced to self-assemble into nanoparticles. More importantly however it is both biocompatible
and biodegradable making it suitable for biomedical applications. A complete series of
American Society for Testing and Materials (ASTM) biological tests for materials and devices in
contact with tissues, tissue fluids and blood demonstrated their unmatched biocompatibility.
Tests such as Ames mutagenicity, cytotoxicity-agarose overlay, acute systemic toxicity,
intracutaneous toxicity, muscle implantation, acute intraperitoneal toxicity, and systemic
antigenicity were conducted and yielded acceptable values
39
. In addition tests for
immunogenicity revealed that it has not been possible to raise any antibodies against them. The
12

immune system seemingly ignores the polymer because it cannot distinguish from natural
elastin.
40

Two main fields of interest for ELPs have been as drug delivery systems and tissue engineering.
In the former, polymers of ELP can be used as a matrix, aggregate or devise which release drugs
upon stimulation. For applications in tissue engineering, the polymer, usually in the form of a
matrix works as temporary scaffold that is gradually replaced by endogenous growing tissue.
Hence the artificial tissue must possess the mechanical properties of the natural tissue for which
it is intended as a substitute while also forcing cell proliferation and gradual replacement by new
tissues
41
. The focus of this thesis will primarily be on the use of ELPs as a nanoparticulate
system for tumor imaging and targeting with a view for further development as a drug delivery
system.

Over the past decades, ELPs has been applied in drug delivery as both macromolecular carriers
42,
43
and nanocarriers
44-46
. In addition to these carrier systems, ELP-based drug depots for
controlled release have also been designed to enhance the local delivery of drugs (Fig. 3-1).

13

Figure 3-1. ELP-based drug delivery strategies. a) Therapeutic agents can be appended to either
the N- or C-terminus of a hydrophilic ELP. b) ELP block copolymers can be induced to form micelles
upon stimulations with heat. Hydrophobic drugs can be encapsulated within the core for delivery to
diseased sites. c) ELP-depots can be delivered locally via injection at the site of interest, where it will
form an insoluble coacervate at body temperature which forms a long lasting depot.

Macromolecular carriers are capable of overcoming transport barriers that limit drug delivery to
tumors. Typically therapeutic agents are attached to a polymeric compound, like ELPs, in order
to enhance the delivery of the active substance to the diseased tissue thereby reducing any toxic,
off target effects. Macromolecular carriers such as these are able to extravasate and accumulate
preferentially in tumor tissue relative to normal tissues via the enhanced permeability and
retention (EPR) effect. By appending these substances to a high molecular weight carrier, it also
has the added advantage of extending the circulation half-life of the therapeutic agent leading to
an enhanced therapeutic effect. Furthermore, the ability to functionalize the construct with
targeting moieties and triggered release mechanisms significantly improves its drug delivery
function. Using peptide based polymers as a carrier has additional advantages in that it’s PK and
biodistribution can be easily controlled due to its molecular weight and sequence uniformity.
Furthermore as the carrier is primarily composed of amino acids, it ensures its biocompatibility
as well as that it can be easily degraded and excreted through normal metabolic pathways.
14

Micellar nanocarriers have already been applied successful for delivery of hydrophobic drugs
10-
12
. A polymeric micelle usually consists of a hydrophobic and a hydrophilic block. These then
self-assembled in an aqueous environment giving rise to a hydrophobic core and a hydrophilic
corona. The core is usually formed by the hydrophobic block and is thus ideal for encapsulating
cancer drugs, which tends to be hydrophobic in nature, or imaging agents. The hydrophilic block
makes up the corona of the micelle. The corona forms this polymeric “brush” not unlike PEG,
which suppresses opsonization of blood components thereby prolonging circulation times. It can
also be functionalized by targeting ligands like peptides, antibodies and carbohydrates.

ELP –assisted local delivery of drugs is realized via several strategies. Here the objective is to
increase the local drug concentration by implanting a drug near the site of interest. This
enhances the diffusion into the desired site, overcoming the negative effects of systemic delivery.
In the first strategy soluble ELPs are injected and their coacervation is triggered by the body
temperature. Here, the drug can be covalently attached to the ELP or it can just be mixed with
the ELP. In another approach, cross-linked ELP depots containing a drug can be produced and
then implanted to generate a stable release of the drug
47
.

Exploiting the phase transition properties of ELPs three different types of drug delivery systems
can be defined
47
:
i. Macromolecular nanocarriers - Soluble ELPs with a Tt above the body temperature.
ii. Micelles - Stimulus responsive ELPs with a Tt in a region where transitions can be
triggered in vivo, Tt is thus at or slightly above the body temperature
iii. Depot - Insoluble ELPs with a Tt below body temperature.
15

With this in mind it is now possible to design an ELP-based carrier that is specific for a
particular function.

1.4 Angiogenesis and integrins
Angiogenesis, the formation of new blood vessels from pre-existing capillaries is a prominent
feature during pathogenesis of cancer. Without angiogenesis tumor cannot grow more than 2mm
in diameter. Once a tumor size has reached 1-2mm, simple diffusion of oxygen and nutrients
from pre-existing vasculature becomes insufficient, leading to the generation of its own blood
supply (Fig. 4-1). As such this process plays a pivotal role in the initiation, growth and
metastasis of tumor cells.

Figure 4-1. The different steps of the angiogenic cascade. I. Outgrowth of a tumor will involve
generation of hypoxia, leading to the onset of angiogenic genes, such as vascular endothelial growth
factor (VEGF) and fibroblast growth factor (FGF). II. The secretion of these factors activates endothelial
cells of preexisting nearby capillaries to produce matrix metalloproteinases to breakdown the extracellular
matrix. This will allow the endothelial cells to start migrating towards the stimulus. III. Endothelial cells
proliferate and form vascular sprouts that can transport blood but are initially very leaky. IV. Only after
the formation of a new extracellular matrix and basement membrane, a new blood vessel is available for
oxygenation of the tissue and removal of waste products. New vessels in tumors can be leaky and may
allow migration of tumor cells to distant sites
48
.

16

One of the major players that have been implicated in the angiogenic process is a class of
molecules called the integrins. Integrins are a family of transmembrane receptors, composed of
18 α and 8 β chains that can be combined to form 24 heterodimeric glycoproteins with distinct
cellular and adhesive specificities (Fig. 5-1a). Each integrin subunit contains a large extracellular
domain, a single transmembrane domain and a short cytoplasmic domain
49, 50
(Fig. 5-1b). They
play critical roles in cell-cell and cell-matrix interaction and are responsible for cellular
activation, migration, proliferation, survival, and differentiation. Studies over recent decades
have shown that they help mediate the transmission of extracellular cues across the plasma
membrane, linking up, via their short cytoplasmic tails, to the intracellular signaling machinery
— so-called ‘outside-in’ signaling. They also respond to intracellular stimuli, undergoing
conformational changes that influence how their extracellular heads interact with the nearby
extracellular environment — so-called ‘inside-out’ signaling
51
(Fig. 6-1).

17

Figure 5-1. The Integrin Receptor Family. a) Integrins are αβ heterodimers; each subunit
crosses the membrane once, with most of each polypeptide (>1600 amino acids in total) in the
extracellular space and two short cytoplasmic domains (20–50 amino acids). The figure depicts
the mammalian subunits and their αβ associations; 8 β subunits can assort with 18 α subunits to
form 24 distinct integrins. Cell 2002 110:673–687 b) Schematic diagram of integrin structure.
The overall structure is that of a head region [propeller and thigh domains of the α-subunit and
the βA (also known as βI), hybrid and PSI domains of the β-subunit] supported on two legs that
are made up of the calf1 and calf2 domains in the α-subunit and the EGF repeats and β-tail
domain in the β-subunit. The binding of ligands takes place at an interface between the propeller
domain and βA domain
52
.

To date 9 of the 24 known integrin heterodimers have been implicated in angiogenesis namely
α1β1, α2β1, α3β1, α4β1, α5β1, αvβ1, αvβ3, αvβ5, αvβ8, α6β4
53
. Historically, most of the data
have pointed to the role of αv integrins in angiogenesis in particular αvβ3 and αvβ5. However,
there is emerging evidence that the integrin α5β1 and its ligand fibronectin are also found to be
upregulated on blood vessels in human tumor biopsies to play critical roles in angiogenesis
54
.
Among the integrins αvβ3 has been the most studied as it has been well documented that is
upregulated and overexpressed on activated endothelial cells during the process of angiogenesis
while only having minimal level of expression on quiescent vessels. The expression level of
integrin αvβ3 is an important factor in determining the invasiveness and metastatic potential of
18

some malignant tumors such as melanoma, glioma, ovarian and breast cancer
55
, with the higher
the expression, the poorer the prognosis.

Figure 6-1. Integrin conformation-function relationships: a model. A five-component model
illustrating conformational changes that are associated with inside-out and outside-in integrin signaling.
The α-subunit is in red and the β-subunit in blue
52
.

Erkki Ruoslahti and coworkers discovered a common sequence shared by matrix proteins that
can bind the integrins that are generally overexpressed on angiogenic blood vessels
56, 57
. This
arginine‐ glycine‐ aspartic acid (or RGD) sequence is expressed on fibronectin, vitronectin,
osteopontin, collagens, thrombospondin, fibrinogen, and von Willebrand factor. Eight integrins
19

(α5β1, α8β1, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and αaIIbβ3), of which αvβ3 is one of them,
classically binds to extracellular matrix (ECM) proteins via the arginine-glycine-aspartic acid
(RGD) peptide sequence
56, 58, 59
, therefore RGD peptides and their derivatives can serve as
integrin inhibitors to induce endothelial cell apoptosis and inhibit angiogenesis or as integrin
specific probes for monitoring tumor angiogenic and metastatic activities. The RGD motif
served as a starting point for the rapid development of a variety of αvβ
3
-based small molecule
antagonists. The first iteration of such molecules is linear peptides containing the RGD
sequence. However linear RGD peptides proved highly susceptible to chemical degradation
which is due to the reaction of the aspartic acid residue with the peptide back bone
60
.
Cyclization is commonly employed to improve the binding properties of RGD peptides and since
cyclization confers rigidity to structure it greatly improves the selectivity of the RGD sequence
for a specific integrin subtype
61
. In cyclic peptides, the RGD peptide sequence is flanked by
other amino acids to build a ring system. These systems offer the possibility to present the RGD
sequence in a specific conformation for a selected integrin
62
. Since the rigidity conferred by
cyclization prevents this, cyclic peptides are more stable, more potent, and more specific. For
instance, high selectivity, and sufficient metabolic stability have been observed for the cyclic
pentapeptide cycloRGDfV which in turn strongly enhances its activity
63
.

Indeed of the 65 integrin-inhibiting compounds that is currently being developed in anti-cancer
treatment
64
, 12 targets RGD binding integrins. Of these only one compound has reached Phase
III, the cyclic RGD peptide inhibitor cilengitide (EMD 121974). Cilengitide has been reported to
inhibit both αvβ3 and αvβ5 integrins and is being investigated as anticancer treatment in high
20

grade gliomas and in other indications including non-small cell lung cancer (NSCLC) and head
and neck carcinoma
65
.

1.4.1 Disintegrin
A source of naturally occurring high affinity ligands for integrins is found in the family of
molecules known as disintegrin. Disintegrins are a family of small, molecular weight cysteine-
rich polypeptides found in various snake venoms. They were first described as inhibitors of
platelet aggregation by their binding to integrin αIIβ3 on the surface of platelets. This inhibitory
effect of disintegrin is due to the typical RGD sequence motif that binds with high affinity to
numerous integrins and consequently is the most potent known inhibitors of integrin function.
Currently disintegrins can be classified into four groups (Table 1-1)
66
.

Table 1-1. Classification of disintegrins

Type of disintegrin
No. of
residues
No. of disulfides Examples
Short 41 – 51 4
Echistatin (RGD);
obtustatin (KTS)
Medium 70 6
Barbourin (KGD);
flavovinudin (RGD);
atrolysin E (MVD)
Long 84 7

Bitistatin (RGD)

Homodimers/heterodimers -
Four intrachain
disulphides, a 2 interchain
cysteine linkages
EC3A-VGD

Correct folding of disintegrins is important for their biologic activity, to maintain the correct
conformation of the RGD receptor-binding site. This is facilitated by the appropriate pairing of 8
to 14 cysteines by disulphide bridges, which maintain the RGD-containing loop in its active
21

conformation. NMR studies confirmed that the RGD motif is located in a flexible loop joining 2
short β-strands, protruding 14 -17 A from the protein core, which plays an important role in
determining their 3D topology
67-69
(Fig. 7-1a). Structure function studies has also indicated that
the amino acid residues flanking the RGD sequence as well as the C-terminus region of the
peptide are additional determinants for integrin binding, affinity and selectivity. A docking
model of trimestatin and αvβ3 illustrated possible contact points between the RGD sequences
with the integrin subunits. Additionally the model also illustrates that the C-terminal region
could serve as another potential binding site with integrin receptors (Fig. 7-1b).

Figure 7-1. Structure of trimestatin. a) The RGD sequence, amino acid residues adjacent to the RGD
sequence and the C-terminal residues (considered important for receptor-binding) are labeled and their
side-chains are indicated in ball-and-stick presentation. The six disulfide bonds are depicted in ball-and-
stick presentation. b) Representation of trimestatin (green) docked on the surface of αvβ3 (PDB code
1L5G; orange, the propeller domain of αv subunit; yellow, the βA domain of β3 subunit; blue spheres,
manganese ions at MIDAS and ADMIDAS sites). The side-chains of the RGD sequence are labeled and
indicated in ball-and-stick presentation. The corresponding RGD sequence of the cyclic peptide ligand in
1L5G is superimposed in gray ball-and-stick presentation for comparison. The side-chains of the C-
terminal residues that interact with receptors are also labeled and indicated in ball-and-stick
presentation
70
.

22

1.5 Integrin imaging
Integrin ligands are also under investigation as tumor endothelium-targeted diagnostic agents and
integrin targeting is proving useful in tumor imaging. Tumor imaging plays a key role in clinical
oncology with radiological examination able to detect solid tumors, determine recurrence and
monitor therapeutic responses. The timing of that detection is important since it will have an
intense effect on a patient’s prognosis. Unfortunately many cancers do not have recognizable
symptoms until the disease has progressed beyond a local disease. Consequently the prognosis is
poor. Typically solid tumors must be larger than 10mm to be reliably detected by anatomic
imaging techniques such as computed tomography (CT). In a similar function, nuclear medicine
techniques are limited to detection of larger tumors, so many lesions are missed. Thus, there is a
need to develop new techniques for the early detection of tumors, so that treatment can be
administered earlier, improving the patients’ prognoses. Considering that the expression of αvβ3
is strongly correlated with tumor progression, it could serve as an ideal prognostic indicator of
tumor development and could be used as a marker for the early detection of cancer. In addition,
the ability to non-invasively visualize and quantify αvβ3 integrin expression level will provide
new opportunities to document tumor receptor expression, help identify appropriate patients for
integrin related therapy and monitor the therapeutic responses paving the way to a more
personalized form of medicine.

For integrin imaging radionuclide based imaging (SPECT and PET) have the highest clinical
impact, which has prompted intense efforts in the development of αvβ3 integrin
radiopharmaceuticals. Positron emission tomography (PET) is a powerful modality for
noninvasive imaging of different molecular targets and events thanks to its high sensitivity,
23

reasonable spatial resolution, and good quantification ability. Positron emitting isotopes
frequently used include C-11 and F-18. Recently non-traditional PET radionuclides particularly
those of the transition metals e.g. Y-90, Cu-64 have gained considerable interest because of
increased production and availability
71
. Significant research, however have been focused on the
copper radionuclides as they offer varying range of half-lives and positron energies. Their rich
coordination chemistry allows for its reaction with a wide variety of chelator systems that can
potentially be linked to protein or polymer and lipid-based nanoparticles.

The normal approach to tagging a biomolecule with a metallic radionuclide e.g. Cu-64 is first to
conjugate a suitable chelating agent to the protein or nanoparticle and then to form a complex
between the metal and the conjugated chelated molecule (Fig. 8-1). Bifunctional chelating
agents are compounds with a strong metal chelating group at one end and a reactive functional
group, capable of binding to proteins or nanoparticles at the other. An array of reactive
functional groups or conjugation of bifunctional chelating agents has been reported in literature.
Beyond just the simple amine or carboxylate for use in conjugation chemistry protocols,
haloacetamide or maleimide have been reported for reaction with sulfyhydryl moieties that are
either extant or introduced. Chelates that can hold radiometals with high stability under
physiological conditions is essential in achieving high uptake of the copper radionuclide in the
tissue or organ of interest while minimizing their non-selective binding or incorporation into
non-target organs or tissues. The consequences of loss or dissociation of the radionuclide are
associated with radiotoxicity in the case of therapeutics and poor image qualities for diagnostics.
24

Figure 8-1. Bifunctional chelating for tagging a molecule with metallic radionuclide
72
.

By far the most extensively used class of chelators for Cu-64 has been the macrocyclic poly-
amino carboxylates and two of the most important chelators in this class/family or DOTA and
TETA. Maeres and co-workers
73
reported the first use of macrocyclic chelators for Cu-67
labeling of monoclonal antibodies. Since then DOTA and TETA derivatives have been
successfully used for the Cu-64/67 labeling of biomolecules, including antibodies and small
peptides. While DOTA has been used as a bifunctional chelator for Cu-64, its promiscuity as a
chelating agent and its relative instability compared to TETA have made it only a marginal Cu-
64 chelator. Although Cu-TETA complexes are more stable than
64
Cu-DOTA demonstrated,
their instability in vivo has been well documented. Bass et al.
74
demonstrated that nearly 70% of
the Cu-64 from
64
Cu-TETA-OC was transchelated to superoxide dismustase (SOD) in the liver
20h post injection.

25

It is evident that the in vivo instability of these
64
Cu-complexes emphasizes the need for more
stable Cu-64 chelators. Metal chelators that can form complexes with high thermodynamics and
kinetic stability and be resistant to in vivo processes such as transchelation to binding proteins
and inert to metal dissociation are currently being developed. A class of ligands that has gained
attention as potential Cu-64 chelators are the hexa-azamacrobicyclic cage type ligands which are
based on the sarcophagine cage motifs first synthesized by Sargeson and co-workers
75
. These
ligands can be prepared in high yields and high purity at low cost. They can also be complexed
≤30min under aqueous conditions at pH 5.5 – 6.5 at room temperature and micromolar
concentrations of the ligand.

Voss et al.
76
has used this sarcophagine-based bifunctional chelate to develop a new class of
tumor-specific Cu-64 radiopharmaceuticals for imaging neuroblastoma and melanoma. The
novel chelating agent, SarAr, stably held the Cu ion in place. So much so that Cu
2+
cannot be
removed under physiological conditions and thus resists transfer to copper binding proteins such
as SOD.

26

Chapter 2
Biophysical characterization of protein polymer nanoparticles composed from elastin-like
polypeptides
2.1 Abstract
Genetically-engineered protein polymers can assemble switchable nanostructures with potential
applications as biomaterials and nanomedicines. For example, diblock copolymers composed of
elastin-like polypeptides (ELPs) can assemble proteins and peptides into nanoparticles. ELPs are
repetitive polypeptides that reversibly associate at tunable temperatures. Above a critical micelle
temperature (CMT) ELP diblock copolymers assemble nanostructures, which remain stable for
days. This report focuses on how the phase behavior of the monoblocks alone influences the
CMT and bulk phase transition temperature (T
t,bulk
) of diblock copolymers. We are the first to
observe that there is a minimum molecular weight required to form stable ELP nanoparticles.
The CMT was found to be mostly independent of the hydrophilic block, while T
t,bulk
depends
strongly on the hydrophilic block. For the first time, we report that altering the amino acid
sequence and molecular weight of the hydrophilic domain can control bulk phase separation of
these particles. To quantify this effect we developed a mathematical model to characterize four
ELP monoblock libraries and correlated their phase behavior to that of a library of block
copolymers. In addition, nanoparticle assembly was associated with the conversion in secondary
structure from a mixture of random coil and beta-sheet to type-2 β turns. The contents of this
report are intended to enable the rational design of genetically-engineered nanoparticles
functionalized with therapeutic peptides and proteins.
27

2.2 Introduction
Protein polymers are repetitive amino acid sequences that combine advantageous properties of
polymers with the ability to be genetically engineered. As such, they are emerging as a platform
for the assembly and controlled delivery of peptides and proteins with therapeutic activity.
Protein polymers derived from silk
29,30,31
, collagen
77
and elastin
26,27,28
each have unique stimulus-
responsive properties
78
and present opportunities to control biodegradation
79
. More recently, it
was discovered that protein polymers designed as block copolymers can assemble monodisperse
nanostructures
45, 80, 81
. These nanostructures have proposed applications in drug delivery, tissue
engineering and biosensors
82,83
. To this end recombinant DNA technology has been utilized to
synthesize various protein-based block copolymers that include silk-like
29,84
, resilin-like
85
and
elastin-like polypeptides
86,87,44
, coiled-coil
88
and leucine zipper domains
89
and various peptide
amphiphiles
90
. In comparison with synthetic polymer chemistry, genetic engineering offers
exquisite control of the composition, lengths, and molecular weight of the block copolymer.
Moreover, large repetitive sequences can be constructed with high fidelity using
concatamerization
91,92,93
, step by step directional approach (seamless cloning)
94
and recursive
directional ligation
35,95
.

Recursive directional ligation (RDL) is a step-by-step approach that enables absolute control
over the synthesis of repetitive polypeptides of specific and pre-determined chain length. RDL
involves oligemerization of the DNA fragment in a succession of single and uniform steps. Each
of these steps grows the polymer gene by one pentameric repeat. In this method a synthetic gene
containing the desired sequence is first inserted into a cloning vector. To further elongate the
pentameric repeats, two cloning vectors which contained the gene are cut with two different sets
28

of restriction enzymes to generate compatible sticky ends. When the two sets of cut vectors are
ligated together, the overhangs form the point of ligation, resulting in the extension of the
pentameric repeats. Additional RDL cycles proceed identically, using products from previous
rounds as starting materials, until a gene of the desired length or architecture is obtained.
Recently McDaniel and coworkers
95
reported an improvement to the RDL method, called pre-
RDL. preRDL dimerizes the two halves of a vector containing a copy of the ELP gene to
reconstruct a functional plasmid. This new technique solves two of the major drawbacks of its
predecessor, namely, self-ligation of the plasmid and the low efficiency caused by nonproductive
circularized forms of the gene fragment during ligation. In addition this process seamlessly joins
the two sets of sequences without interrupting key gene sequences with additional base pairs
between the start and stop codon, which may be expressed on the protein polymer.

Elastin like polypeptide (ELP) is a protein polymer inspired from a structural motif found in
human tropoelastin
96
. They are polymers composed of a five amino acid repeat (Val-Pro-Gly-
Xaa-Gly)
l
, where the identity of Xaa and length (l) determine their phase behavior. ELPs are
attractive as polymeric carriers for drug delivery partly because they phase separate at elevated
temperatures and concentrations. Below a characteristic transition temperature (T
t
), ELPs are
predominantly disordered. When the temperature is raised above T
t
, they undergo a sharp (2–3
o
C
range) phase separation, which results in the coacervation of the biopolymer
38, 37
. This process is
fully reversible. In addition to their potential environmentally responsive behavior, soluble ELPs
may be useful as high molecular weight carriers for peptide therapeutics. Hybrid block
copolymers of ELPs have also been reported; silk-elastin-like proteins
97,98
, ODN-ELP
99
, and
cartilage-oligomeric matrix protein
100
. Block copolymers with elastin-like sequences with blocks
29

of different hydrophobicity have also recently emerged as biomaterials for tissue engineering and
delivery of therapeutics
79, 80, 101
.

Amphiphilic block copolymers have attracted a great deal of attention in terms of their ability to
form various types of nanoparticles. These polymers usually consist of hydrophilic and
hydrophobic monomer units. These monomeric units can be arranged into 2, or more,
conjugated blocks of equal or differing monomer lengths. The relative hydrophobicity and
molecular weight of these two blocks determines their characteristic behavior, which sometimes
can lead to the formation of stable polymeric micelles. Generally when the hydrophilic segment
is longer than the core block, the shape of the resulting micelles is spherical. Conversely,
increasing the length of the core segment beyond that of the corona-forming chains may generate
various non-spherical structures such as rods or worms. In water, the hydrophobic portion

of the
block copolymer self-associates into a semi-solid core,

with the hydrophilic segment of the
copolymer forms a coronal

layer. Assembly of the hydrophobic micelle can be sterically
stabilized by a neutral hydrophilic corona or electrostatically stabilized by a charged hydrophilic
corona. An important advantage of micellar copolymer structure is the ability to customize both
their core and coronal properties. Alterations to the composition of the constituent copolymers
can influence important performance related parameters including micelle size, core-drug
compatibility, drug loading capacity, drug release kinetics, and stability. Many types of
copolymers have been used for micelle formation, but the requirements of biocompatibility and
oftentimes biodegradability have limited the choice of copolymers in clinical applications.
Protein based block copolymers have many advantages over conventional synthetic polymers
because they are able to assemble into stable, ordered conformations. This ability depends on
30

the structures of protein chains as well as the microenvironment. Protein-based block
copolymers have the ability to form varied nanostructures in aqueous solution with multiple
potential biomedical applications. For example, the hydrophobic core may encapsulate
hydrophobic compounds and increase their solubility in an aqueous environment. Polymeric
micelles typically have a critical micelle concentration (CMC) that is lower than for surfactant
micelles like SDS, allowing for self-assembly at concentrations relevant for drug delivery.
Micelles also confer the beneficial aspects of polymeric delivery, such as prolonged plasma half-
life, because the size of a typical micelle allows it to exceed the renal filtration cutoff (Rh >5nm)
while evading the reticulo-endothelial system (Rh <100nm). Furthermore, predictable self-
assembly behavior allows for the incorporation of functional groups in the corona that range
from passive components such as PEG to active groups such as targeting ligands, protein
therapeutics, or molecular imaging agents.

To develop a rational approach to engineer the chemico-physico properties of ELP nanoparticles,
our group engineered a library of ELP block copolymers of different molecular weight (MW),
orientations, and amino acid sequence. To date, no comprehensive studies have been made that
connect the behavior of ELP monoblocks with related ELP diblock copolymers, which has
presented challenges to the development of nanoparticles for therapeutic applications. Based on
the library of data included in this manuscript, we observe a number of novel findings, which
include: i) the assembly of ELP diblock copolymers requires a minimum length of ELPs; ii)
assembly of ELP nanoparticles is accompanied by a change in secondary structure to favor type-
2 β turns along the polypeptide backbone; and iii) the bulk phase transition temperature of ELP
31

nanoparticles can be tuned by changing the amino acid sequence of the hydrophilic block
without significantly influencing the CMT.
32

2.3 Materials and methods

2.3.1 Materials
Restriction enzymes and calf intestinal phosphatase (CIP) were purchased from New England
Biolabs (Ipswich, MA). T4 DNA ligase was purchased from Invitrogen (Carlsbad, CA). The
pET-25b+ cloning vector was obtained from Novagen Inc. (Madison, WI), and all custom
oligonucleotides were synthesized by Integrated DNA Technologies Inc. (Coralville, IA).
Top10
™
cells were purchased from Invitrogen (Carlsbad, CA) and BLR(DE3) competent cells
were purchased from Novagen (Madison, WI). All E. coli cultures were grown in TBDry
™

media purchased from MO BIO Laboratories, Inc (Carlsbad, CA). The DNA miniprep kit and
the Illustra GFX Gel Band Purification Kit were purchased from Qiagen Inc. (Germantown, MD)
and GE (GE Healthcare, Buckinghamshire, UK) respectively.

2.3.2 Modification of pET 25b(+) vector
1.5µg of the plasmid was digested with 1µL of XbaI and BamH1 in NEBuffer 3, for 3h at 37
o
C.
The linearized vector was then dephosphorylated with 1µL calf intestinal phosphate (CIP) for 1h
at 37
o
C. After the incubation period, the vector was then gel purify using the Qiagen Gel
Extraction Kit (Qiagen, Valencia, CA). At the same time, two chemically synthesized
oligonucleotides that encode for the sense and anti-sense of the DNA monomer containing the
new Acu1 cut site are annealed together. The two synthetic oligos were annealed together in the
presence of T4 DNA ligase buffer and were placed on a heating block at 95
o
C for 1min before
letting the annealing mixture cool down for 3h.
The oligomer was then ligated into the modified cloning vector for 1h using the T4 DNA ligase.
The ligated product was transformed with the Top10 cells (Novagen, Madison, WI, USA) and
33

plated onto an ampicillin-supplemented TB dry agar plate. Positive transformants were selected,
screened by diagnostic restriction enzyme and sent for sequencing for confirmation.

2.3.3 ELP gene insertion
The two strands of synthetic gene containing the ELP sequence were annealed together as
described above. The modified vector was digested with 1µL of Bser1 for 1h at 37
o
C and the
ELP encoding gene was ligated into the vector using the T4 DNA ligase (Invitrogen, Carlsbad,
CA) at room temperature for a further 1h. The ligated product was transformed, screened and
positive clones with the desired inserts were DNA sequenced to confirm in-frame insertion.

2.3.4 Recombinant synthesis of ELPs by plasmid reconstruction recursive directional ligation
(pre-RDL)
To generate ELPs of a specific and pre-determined chain lengths the following RDL strategy was
employed. Two cloning vectors which contained the ELP gene were cut with two separate sets
of restriction enzymes. One vector was cut with BssHII and AcuI, while the enzymes BssHII
and Bser1 was used to cut the second vector generating compatible sticky ends. Enzyme
digestion was performed using 1µL of enzymes (250 units) each, at 37
o
C for 3h. The two sets of
cut vectors were ligated together using the T4 DNA ligase (Invitrogen, Carlsbad, CA), resulting
in the extension of the pentameric repeats.

34

2.3.5 Generation of ELP block copolymer library
A similar strategy was employed for the generation of the ELP block copolymer, where the N-
terminal gene of one monoblock was ligated to a C-terminal ELP gene of another via RDL. The
length, amino acid sequence and orientation can be varied and synthesized easily.

Table 1-2. Library of ELP genes expressed and characterized for phase behavior of
monoblocks.

Nomenclature
*
Nucleotide sequence of open reading frame
**
Encoded
amino
acid sequence
ELP MW
(Da)
S72
atg gg(t gtt ccg ggc tct ggt gta cca ggt agc ggt gta ccg ggt tct ggc
gta cct ggc tcc ggt gtc ccg ggt tcc ggt gtt ccg ggt tct gg) 12 t tac tga
G(VPGSG) 72Y 28,853
S96
atg gg(t gtt ccg ggc tct ggt gta cca ggt agc ggt gta ccg ggt tct ggc
gta cct ggc tcc ggt gtc ccg ggt tcc ggt gtt ccg ggt tct gg) 16 t tac tga
G(VPGSG) 96Y 38,392
S144
atg gg(t gtt ccg ggc tct ggt gta cca ggt agc ggt gta ccg ggt tct ggc
gta cct ggc tcc ggt gtc ccg ggt tcc ggt gtt ccg ggt tct gg) 24 t tac tga
G(VPGSG) 144Y 57,468
S192
atg gg(t gtt ccg ggc tct ggt gta cca ggt agc ggt gta ccg ggt tct ggc
gta cct ggc tcc ggt gtc ccg ggt tcc ggt gtt ccg ggt tct gg) 32 t tac tga
G(VPGSG) 192Y 76,545
A96
atg gg(t gtt ccg ggc gct ggt gta cca ggt gca ggt gta ccg ggt gcc ggc
gta cct ggc gca ggt gtc ccg ggt gcc ggt gtt ccg ggt gct gg) 16t tac tga
G(VPGAG) 96Y 36,987
A144
atg gg(t gtt ccg ggc gct ggt gta cca ggt gca ggt gta ccg ggt gcc ggc
gta cct ggc gca ggt gtc ccg ggt gcc ggt gtt ccg ggt gct gg) 24t tac tga
G(VPGAG) 144Y 55,296
A192
atg gg(t gtt ccg ggc gct ggt gta cca ggt gca ggt gta ccg ggt gcc ggc
gta cct ggc gca ggt gtc ccg ggt gcc ggt gtt ccg ggt gct gg) 32t tac tga
G(VPGAG) 192Y 73,605
V24
atg gg(t gtt ccg ggc gtg ggt gta cca ggt gtc ggt gta ccg ggt gtc ggc
gta cct ggc gtc ggt gtc ccg ggt gtt ggt gtt ccg ggt gta gg) 4t tac tga
G(VPGVG) 24Y 10,197
V36
atg gg(t gtt ccg ggc gtg ggt gta cca ggt gtc ggt gta ccg ggt gtc ggc
gta cct ggc gtc ggt gtc ccg ggt gtt ggt gtt ccg ggt gta gg) 6t tac tga
G(VPGVG) 36Y 24,939
V48
atg gg(t gtt ccg ggc gtg ggt gta cca ggt gtc ggt gta ccg ggt gtc ggc
gta cct ggc gtc ggt gtc ccg ggt gtt ggt gtt ccg ggt gta gg) 8t tac tga
G(VPGVG) 48Y 20,025
V96
atg gg(t gtt ccg ggc gtg ggt gta cca ggt gtc ggt gta ccg ggt gtc ggc
gta cct ggc gtc ggt gtc ccg ggt gtt ggt gtt ccg ggt gta gg) 16t tac tga
G(VPGVG) 96Y 39,680
V144
atg gg(t gtt ccg ggc gtg ggt gta cca ggt gtc ggt gta ccg ggt gtc ggc
gta cct ggc gtc ggt gtc ccg ggt gtt ggt gtt ccg ggt gta gg) 24t tac tga
G(VPGVG) 144Y 59,336
V192
atg gg(t gtt ccg ggc gtg ggt gta cca ggt gtc ggt gta ccg ggt gtc ggc
gta cct ggc gtc ggt gtc ccg ggt gtt ggt gtt ccg ggt gta gg) 32t tac tga
G(VPGVG) 192Y 78,991
I18
atg gg(t gtt cct ggt atc ggt gtt ccg ggc atc ggt gta cct ggc att ggt gtc
cca ggt att ggc gtt cca ggt atc ggc gta cca ggt att gg) 3t tac tga
G(VPGIG) 18Y 7,861
I24
atg gg(t gtt cct ggt atc ggt gtt ccg ggc atc ggt gta cct ggc att ggt gtc
cca ggt att ggc gtt cca ggt atc ggc gta cca ggt att gg) 4t tac tga
G(VPGIG) 24Y 10,403
I48
atg gg(t gtt cct ggt atc ggt gtt ccg ggc atc ggt gta cct ggc att ggt gtc
cca ggt att ggc gtt cca ggt atc ggc gta cca ggt att gg) 8t tac tga
G(VPGIG) 48Y 20,557
I96
atg gg(t gtt cct ggt atc ggt gtt ccg ggc atc ggt gta cct ggc att ggt gtc
cca ggt att ggc gtt cca ggt atc ggc gta cca ggt att gg) 16t tac tga
G(VPGIG) 96Y 40,896
*
Gene sequence confirmed by N and C terminal DNA sequencing and diagnostic restriction digestions. Parentheses
indicate location of restriction cut sites (BserI, AcuI) used for recursive directional ligation. A BserI recognition site
was located to the 5’ direction of the start codon (atg). An AcuI recognition site was located to the 3’ direction of the
stop codon (tga).
**
Translation of open reading frame excluding start and stop codons. A carboxy terminal tyrosine was incorporated
for A
280 nm
quantification of ELP concentration (molar extinction coefficient 1,285 M
-1
cm
-1
)
35

2.3.6 Protein purification by inverse transition cycling
pET25b(+) expression vectors containing the desired constructs were transformed into E. coli
BLR (DE3) cells for protein hyperexpression and proteins were purified by inverse transition
cycling
102
. Briefly, overnight cultures were centrifuged and re-suspended in cold PBS. The
proteins were liberated from the bacteria by intermittent probe-tip sonication for a total of 3
minutes. The sonicated product is centrifuged for 15min at 4
o
C, 12000rpm and the supernatant,
which contains soluble ELPs, is transferred to another tube. Excess polyethylene imine was
added to precipitate any remaining nucleic acids and the solution was centrifuged again. The
supernatant was heated to 37°C until the induction of turbidity, characteristic of ELP
coacervation. This solution was centrifuged at 37°C to collect insoluble ELP coacervate. The
pellet was re-suspended in cold PBS and centrifuged at 4
o
C. 5-6 rounds of hot and cold
centrifugation steps were performed to increase the polymer purity.

2.3.7 Optical density characterization of protein polymer phase diagrams
The temperature-concentration phase behavior of each member of the ELP library was
determined by measuring optical density as a function of temperature. ELP solutions in
phosphate buffered saline (PBS) were observed at a constant heating rate of 1°C/min in a
temperature controlled multicell holder of a UV visible spectrophotometer (DU800
Spectrophotometer, Beckman Coulter, CA, USA). The transition temperature (T
t
) is defined as
the point of one half maximal turbidity
37
. For the block copolymer the critical micelle
temperature (CMT) is recorded as the temperature at which micelles begin to form, where the
OD first increased from baseline. The second T
t
(T
tbulk
) is defined as the temperature at which
optical density changed the fastest with respect to temperature.
36

2.3.8 Secondary structure determination using circular dichroism (CD)
Measurements were made on a Jasco CD spectrometer (JASCO, Japan) with a 0.1cm path length
quartz cell in a wavelength range between 190-240 nm. An ELP solution in phosphate buffer
(pH 7.4), at a concentration of 25-50µM was used for measurements. Data analysis was
performed using the Spectra Manager II software (JASCO, Japan). Each spectrum was corrected
by subtracting the corresponding background spectrum recorded at the same temperature. The
resulting spectra was smoothed out, and afterward converted into mean molar residue ellipticity
in mdeg cm
2
dmol
-1
.

All plots were exported using Jasco Spectra Manager II to excel and deconvoluted assuming that
the molar elipiticity [θ] observed is a weighted linear sum of secondary structure ellipticity (Eq.
7). The data was fit using nonlinear regression on Microsoft Excel.
[ 𝜃 ] = ∑ 𝐶 𝑠𝑡 𝑑 [ 𝜃 𝑠𝑡 𝑑 ] Eq. 7

2.3.9 Temperature-dependent dynamic light scattering
Determination of the hydrodynamic radius of the protein polymers was performed on a Dynapro
plate reader (Wyatt Technology Inc., Santa Barbara, CA, USA). 10-25µM of polypeptide in
phosphate buffered saline (PBS) pH 7.4 was subjected to a temperature ramp between 10
o
C –
60
o
C with 1
o
C increment. Before use, the solutions were filtered through Whatman filters with a
0.02µM pore size and centrifuged at 4
o
C, 1200rpm for 10min to remove air bubbles. Mineral oil
was applied to prevent evaporation and the preparation was centrifuged again before
measurement.

37

2.3.10 Conjugation of ELP block copolymers to carboxyfluorescein (CF)
The ELP block copolymer was conjugated to carboxyfluorescein succinimidyl ester (CFSE)
thorugh the N-terminal amine. Excess fluorophore was removed using a PD10 desalting column.
HeLa cells were plated at a density of approximately 20,000 cells per well and incubated
overnight in 10% FBS/DMEM at 37
o
C in a humidified 5% CO
2
atmosphere. After overnight
incubation media was removed and cells was washed with phosphate buffered saline (PBS).
Media was then replaced. Lysotracker, a fluorescent probe which labels and tracks lysosomes in
live cells, ELP-CF block copolymers at 2µM, 20µM and 200µM is added to each plate and
incubated for 3h at 37
o
C in a humidified 5% CO
2
atmosphere. Following incubation, the cells
were rinsed with PBS and media was replaced. Fluorescence images were acquired with a
scanning confocal microscope. The incubation temperature of 37
o
C is well above the CMT of
the respective block copolymers.
38

2.4 Results
2.4.1 Generation of ELP monoblock and diblock copolymer libraries
To explore the relationship between the behavior of monoblock and diblock ELPs, we expressed
and characterized a number of ELP monoblocks (Table 1-2) and diblock copolymers (Table 2-2).
This was achieved by genetically engineering libraries of genes encoding for a number of novel
ELPs (Table 1-2). Libraries with Xaa= Ser, Ala, Val, and Ile were constructed of various lengths,
l, using a modification of the plasmid reconstruction-recursive directional ligation (pre-RDL)
103
.
In pre-RDL, two cloning vectors that contain the desired oligomers are cut with 2 separate sets of
restriction enzymes (Fig. 1-2a). When the two fragments of digested vectors are ligated together,
the resulting oligomeric sequence is extended and reconstituted on an intact plasmid. Each of the
gene products resulting from pre-RDL was explored for expression and purification by phase
separation, a technique known as inverse phase transition cycling
104
. All ELP genes that could be
expressed and purified have been characterized here. Purification of the ELPs by inverse phase
transition cycling yielded proteins of high purity at their expected molecular weight (>95%)
(Fig.1-2b). The identity of each diblock copolymer was confirmed independently using MALDI
TOF (Table 2-2). When viewed under a transmission electron microscope, the ELP micelles was
found to be monodisperse, and of regular spherical shape (Fig. 1-2c and 1-2d) with a mean
diameter of 40±5nm and 48±4nm for I48S48 and I96S96 respectively. A similar spherical
morphology was also observed with cryo-TEM. The block copolymer I48S48 and I96S96
measured 31±3nm and 38±4nm in diameter respectively (Fig. 1-2e and 1-2f).

39

Table 2-2. Libraries of ELP diblock copolymers characterized

**
ELP diblock libraries
[Xaa:Yaa]
ELP
Nomenclature
*
Amino acid sequence
**
Calculated ELP
MW (Da)
***
Observed ELP
MW (Da)
Ile:Ser I18S18 G(VPGIG)
18
(VPGSG)
18
Y 15,015 14,599
I24S24 G(VPGIG)
24
(VPGSG)
24
Y 20,012 19,820
I36S36 G(VPGIG)
36
(VPGSG)
36
Y 29,572 29,826
I48S48 G(VPGIG)
48
(VPGSG)
48
Y 39,643 39,749
I96S96 G(VPGIG)
96
(VPGSG)
96
Y 79,049 79,216
Ser:Ile S48I48 G(VPGSG)
48
(VPGIG)
48
Y 39,643 39,670
Ile:Ala I48A48 G(VPGIG)
48
(VPGAG)
48
Y 38,946 38,940
I96A96 G(VPGIG)
96
(VPGAG)
96
Y 77,655 77,233
Ala:Ile A48I48 G(VPGAG)
48
(VPGIG)
48
Y 38,946 38,952
A96I96 G(VPGAG)
96
(VPGIG)
96
Y 77,655 77,840
*
Gene sequence confirmed by N and C terminal DNA sequencing and diagnostic digestion.
**
Estimated from open reading frame excluding methionine start codon
***
Results from matrix assisted laser desorption ion time of flight mass spectrometry

The phase diagram of each member of the monoblock library was studied by measuring solution
turbidity as a function of temperature and concentration. The temperature at which optical
density changed the fastest with respect to temperature was defined as the ELP transition
temperature, T
t
. ELP phase diagrams are strong functions of the hydrophobicity of Xaa and
length (or MW) (Fig 2-2). All ELPs followed a log-linear relationship between T
t
and
concentration. For all of the ELPs, except Xaa=Ser, there was a strong dependence on the
polymer length. As desired, T
t
is a strong function of the hydrophobicity of Xaa. Both of the
hydrophilic libraries (Xaa=Ser, Ala) phase separate well above physiological temperatures, thus
they were selected as the hydrophilic component of subsequent diblock copolymers. The
hydrophobic library with Xaa=Val, had T
t
near physiological temperature, which may lead to
unstable assembly in the body (Fig. 2-2c). At constant length and concentration, the most
hydrophobic ELP monoblock Xaa=Ile had a T
t
close to room temperature (Fig. 2-2d); therefore,
the library with Xaa=Ile was selected to generate a block copolymers assemble well below body
temperature and can easily be disassembled under refrigeration.
40

From this initial library of ELP block copolymers where an N-terminal ELP gene with a high T
t

(VPGSG or VPGAG) was ligated to a C-terminal ELP gene with a much lower T
t
(VPGIG). By
generating a construct consisting of a block with a high and low T
t
, it is possible to modulate the
formation of micelles at physiologically relevant temperature. A 1:1 ratio of hydrophobic to
hydrophilic monomer blocks is chosen as it is expected to produce spherical micelles with the
minimum needed number of ELP repeats. Conversely increasing the length of the hydrophobic
block, beyond that of the corona-forming block may generate various non-spherical structures
including, rods, vesicles or worms.

d)
41

Figure 1-2. Biosynthesis of ELP block copolymers that assemble micelles. a) ELP genes were
designed using a plasmid-reconstruction recursive directional ligation method
103
. Double endonuclease
digestion was used to cut plasmids at the BseR1/BssHII or AcuI/BssHII. This generates two base pair
sticky ends, which enables recursive joining of ELP genes. b) Copper-staining was used to enhance an
SDS PAGE gel and show a representative library of purified ELPs. The left lane contains a MW standard,
which has a mass indicated to the left. Lane 1: MW marker, lane 2: I18S18, lane 3: I24S24, lane 4:
I36S36, lane 5: I48S48, lane 6: I96S96 and lane 7: S96. The right column indicates the expected mass of
each ELP, which was independently confirmed using MALDI (Table 2-2). Transmission electron
microscopy of ELP block copolymers stained with 1% uranyl acetate. Micrographs of c) I48S48, mean
diameter 40 ± 5 nm and d) 196S96, mean diameter 48 ± 4 nm are shown. Cryo-TEM images were also
obtained for e) I48S48 micelles, mean diameter 31 ± 3 nm and, f) I96S96, mean diameter 38 ± 4 nm.
42

Figure 2-2. Transition temperature for monoblock polypeptides depends on molecular weight
and hydrophobicity. ELP phase diagrams were characterized using optical density (350 nm) in
phosphate buffered saline as a function of concentration, for which a best-fit line is indicated. a)
Hydrophilic ELPs with Xaa=Ser and l = 72 to 192. b) Hydrophilic ELPs with Xaa=Ala and l = 96 to 192.
c) Hydrophobic ELPs with Xaa=Val and l = 24 to 192. d) Hydrophobic ELPs with Xaa=Ile and l = 18 to
96. The complete library of genes encoding these ELPs can found in Table 1-2.

2.4.2 Thermal characterization of ELP block copolymers
As with the monoblocks the inverse temperature phase transition of ELP block copolymers was
characterized by monitoring the optical density of an aqueous ELP solution at 350nm as a
function of temperature. Examination of their thermal properties revealed that upon stimulation
with heat, a two-step thermal response was exhibited by the ELP block copolymer; at low
temperatures <24 – 32
o
C, the ELP block copolymers was in solution and transparent in
43

appearance, but as the temperature rises (26 – 66
o
C), the absorbance increased and persisted
above the baseline levels (Fig. 3-2a). Higher temperatures caused the turbidity to increase
sharply above an upper inflection. The first increase in optical density was correlates with the
phase transition of the isoleucine ELP block. Upon further heating, the higher T
t
serine ELP
block, underwent phase separation yielding highly turbid aggregates. The resulting polypeptide
nanoparticles are hypothesized to consist of a hydrophobic core [VPGIG]
l
that is stabilized by a
hydrophilic shell composed of ELP [VPGSG]
l
.

44

Figure 3-2 Particle diameter and micelle stability depend on molecular weight. ELP block
copolymers were characterized for assembly using optical density and dynamic light scattering in
phosphate buffered saline. a) Optical density (350 nm) for I48S48 as a function of temperature and
concentration. b) Concentration-temperature phase diagrams for ELP diblock copolymers I18S18,
I24S24, I36S36, I48S48, I96S96. Detectable critical micelle temperatures (CMT) and bulk inverse phase
transition temperatures (T
t,bulk
) are indicated. c) Longer ELP block copolymers (I48S48, I96S96) form
nanoparticles of stable hydrodynamic radius at 25 µM. A monoblock ELP, S96, does not assemble and is
included as a negative control. d) Shorter ELP diblock copolymers (I18S18, I24S24, I36S36) form larger
nanoparticles above their CMT with unstable hydrodynamic radius (25 µM).

Dependence of phase transition on the concentration and length of ELP block copolymers was
observed as determined by spectrophotometric readings (Fig. 3-2b). Both the CMT and T
t
bulk
exhibited a negative linear relationship with concentration that indicates that the difference
between the CMT and T
tbulk
at each concentration is about 50-60
o
C.
45

To confirm the assembly and stability of micelles, temperature-dependent assembly was
monitored with dynamic light scattering (DLS) to determine hydrodynamic radius,
polydispersity, the critical micelle temperature and thermal stability. Based on our isoleucine-
serine [1:1] library, we have identified a minimal molecular weight necessary for the self-
assembly of ELP-based micelles. I48-S48 and I96-S96 formed large stable micelles above a
critical micelle temperature (25, 20 °C respectively), with a hydrodynamic radius proportional to
their MW (R
h
= ~20, ~40 nm respectively) [Fig. 3-2c]. Formation of these stable micelles persists
up to 60
o
C. In contrast, above their CMT the block copolymers (I18-S18, I24-S24, I36-S36)
formed larger structures of unknown morphology (Fig. 3-2d). Despite this difference in
nanostructure stability, for all ELPs diblock copolymers evaluated the CMT primarily correlated
with the transition temperature expected for the isoleucine monoblock alone. In contrast, the
monomeric ELP serine-96 exhibited no micellar formation over the same temperature range.
Lastly, the orientation of the block copolymers in the reverse orientation to I48S48, S48I48
showed similar behavior (Fig. 5-2b).

Following an initial identification of the MW cutoff for the formation of stable micelles, a library
composed of the Ile-Ala was constructed and characterized. When compared to the thermal
behavior of the Ile-Ser library, similar profile was obtained especially for the onset of micelle
formation (CMT) However a difference in the bulk T
t
was observed, with the T
tbulk
for the
hydrophilic serine ELP blocks being higher than that of hydrophilic alanine ELP blocks (fig. 4-
2). This is perhaps unsurprising as it has been demonstrated that serine monoblock ELPs do
have a higher T
t
than alanine (Fig. 2-2). Despite differences in the identity and phase behavior
between these two hydrophilic blocks, both the serine and alanine ELP blocks yielded similar
46

CMTs and hydrodynamic radius (R
h
). However further investigation of the stability of the Rh at
37
o
C revealed slight differences between the two types of block copolymers. The alanine ELP
hydrophilic block yielded a more stable hydrodynamic radius, despite its relatively lower
transition temperature. Conversely stabilization of a micelle via the serine ELP hydrophilic block
permits the slow formation of larger radius nanoparticles of unknown morphology. Certainly this
will have implications for in vivo applications, as perhaps it would be more favorable to use a
construct that is kinetically stable at physiologically relevant temperatures.

Due to the flexible and adaptive nature of the pre-RDL method, a library consisting of block
copolymers in the reverse orientation can be easily synthesized. Here the Ile-Ala and Ile-Ser
blocks are reversed to give Ala-Ile and Ser-Ile blocks (Fig. 5-2). The ability to switch the blocks
orientation enables the presentation of different functional groups e.g COO
-
or NH
3+
at the
corona surface for bioconjugation purposes. Again the Ile-Ser and Ser-Ile and Ile-Ala and Ala-
Ile blocks exhibited near superimposable CMT values to each other and to some extent the T
tbulk

too (Fig. 5-2a). The R
h
of the reverse orientation blocks also does not differ significantly (Fig. 5-
2b and 5-2c).

47

Figure 4-2. Bulk phase transition temperature for stable ELP nanoparticles depends on the
hydrophilic block. ELP block copolymers with two different hydrophilic blocks (Xaa = Ser, Ala) and
two lengths (l = 48, 96) were characterized for their assembly properties using optical density and
dynamic light scattering in phosphate buffered saline. a) Concentration-temperature phase diagrams for
ELP diblock copolymers I48S48, I48A48, I96S96, I96A96. The critical micelle temperature (CMT) was
minimally influenced by the sequence of the more hydrophilic block. In contrast, the bulk phase transition
temperature (T
t,bulk
) was highly dependent on the sequence of the hydrophilic block. b) Above their CMT,
ELP diblock copolymers with Xaa = Ala (I48A48, I96A96) assemble micelles that are stable at
physiological temperatures and undergo bulk phase separation ~10 °C above body temperature (25 µM).
c) Higher molecular weight ELP diblock copolymers have a larger hydrodynamic radius than smaller
ELPs. The distribution of hydrodynamic radius for a soluble monoblock ELP, S96, is indicated as a
negative control.

48

Figure 5-2. ELP assembly behavior and particle radius are independent of diblock orientation.
ELP block copolymers were characterized for assembly using optical density and dynamic light scattering
in phosphate buffered saline. a) Concentration-temperature phase diagrams for ELP diblock copolymers
I48S48, S48I48, I48A48, A48I48, I96A96, A96I96. The reversal of orientation from hydrophobic-
hydrophilic did not appear to influence the Critical micelle temperature (CMT) and bulk inverse phase
transition temperature (Tt,bulk). b) Hydrodynamic radius for ELP block copolymers with the
hydrophobic block at the carboxy terminus (S48I48, A48I48, A96I96) also form nanoparticles of stable
radius at 25 µM. c) The distribution of hydrodynamic radii (37 °C) observed for S48I48, A48I48, and
A96I96 roughly matches the particle radii observed for the equivalent block copolymers with the
hydrophobic block at the amino terminus (Fig. 4-2).

2.4.3 Role of secondary-structure during assembly of ELP nanoparticles
To determine the dominant peptide secondary structure of the block copolymer, far UV circular
dichroism (CD) measurements were conducted. The CD spectra of S96, I48-S48 and I48-A48
taken at various temperatures are depicted (Fig. 6-2a, c, e and g). At low temperature, the CD
49

spectra of S96, I48 and I48-S48 consist of a large negative band at ~195nm and a smaller trough
at ~220nm, which are characteristic for random coil and β-turn structures respectively. With
increasing temperature, the random coil signal becomes less pronounced, while the β-turn
increases, indicating a transition of the peptide backbone from a less to more ordered secondary
structure
105,106
. β-turns are a type of secondary structure with important roles in protein folding
and stability
107
. In a β -turn, a tight loop is formed when the carbonyl oxygen of one residue
forms a hydrogen bond with the amide proton of an amino acid three residues down the chain.
This hydrogen bond stabilizes the β-bend structure. As it is a means by which the protein can
reverse the direction of its peptide chain it plays a role in defining the globularity of folded
proteins
108
; however, its role in the ELP-mediated assembly process remains undetermined.

Deconvolution, via non-linear regression, of each spectra yielded a more detailed breakdown of
the changes in secondary structure content of the construct while it undergoes phase transition
(Fig. 6-2b, d, f and h. The general trend is that ELPs show a decrease in random coil and β-sheet
content, while the the α-helical and β-turn 2 content increases. The fact that this transition is well
underway below the CMT or T
t
suggests that an increase in polypeptide secondary structure may
provide an additional entropic driving force that is ultimately relaxed when the ELP enters the
more disordered coacervate phase. No significant change in the β-turn 1 content was observed
for any of the polypeptides studied except for Ser-96. For reference, β-turn 1 and β-turn 2 are
diametrically opposed with the essential difference between them being the orientation of the
peptide bond residues at (i+1) and (i+2).

50

The changes in spectra of the diblock copolymers I48S48 and I48A48 are of particular note
because, at the examined 50 µM concentration, they have similar CMTs. I48S48 undergoes a
micelle phase transition at 27.5°C, while I48A48 has a transition at 26°C. While the spectra of
the two are remarkably similar at the first post-micellization temperature analyzed of 37 °C, the
two show differences in the extent of conformational change between 37 °C and 50°C. Between
37 and 50°C the random coil structure of I48S48 and I48A48 remains similar, with a decrease
from 53.4% content to 51.4% in I48A48, and a decrease from 52.3% to 50.8% in I48S48. The β-
turn 2 content changes slightly more, with an increase from 22.2% to 27.2% in I48A48, and from
22.5% to 24.6% in I48S48. The changes in α helix structure are similarly slight, with an increase
from 12.6% at 37°C to 17.5% at 50°C in I48A48, and an increase from 12.2% to 14.1% in
I48S48. The greatest change is evident in the β sheet content, which decreases from 11.8% to
4.0% in I48A48, while decreasing from 12.9% to 10.4% in I48S48. While the changes in
secondary structure content are slight, they are indicative of alterations in the composition of the
ELP solution. A possible explanation for the observed discrepancies is the bulk phase transition,
which occurs at 50.7°C for I48A48, and 84.2°C for I48S48. The final 50°C measurement may
display the greatest variation as it is quite close to the bulk phase transition temperature for
I48A48, so it is plausible to presume that the ELP solution will be primed for this transition, and
is therefore subject to conformation changes to a greater extent than I48S48. It is yet unclear how
these changes will affect the overall properties and behavior of the respective micelles but it
certainly warrants further investigation.

51

Figure 6-2. Nanoparticle assembly is accompanied by formation of secondary structure.
Circular dichroism for polypeptides at 50 µM in 10 mM phosphate buffer, pH 7.4 below, near, and above
the CMT. a) The monoblock ELP I48, Tt
bulk
= 20.1°C, b) The monoblock ELP S96, Tt
bulk
= 55.5°C, c) The
diblock copolymer I48S48, CMT = 25 °C, c) The diblock copolymer I48A48, CMT = 25 °C.
Deconvolution of spectra for b) Ile-48, d) S96, f) I48S48 and h) I48A48.
52

2.4.4 Cellular localization of the polymeric micelles
The subcellular localization of ELP nanoparticles was examined qualitatively by confocal
microscopy (Fig. 7-2). After 3 hours of incubation the ELP nanoparticles displayed distinct
localization in live HeLa cells. The combined images of the green carboxyfluorescein labeled
nanoparticles and the red lysotracker dye revealed that the nanoparticles were mainly located
within the lysosomes. The main function of lysosomes is normally to breakdown waste
materials and cellular debris. The implication of this sequestration is that the ELP polymeric
micelles can be easily degraded by proteases and cleared from the system. It is hypothesized that
this could also be a mechanism whereby drugs can be released from encapsulation within the
nanoparticle core.

Figure 7-2. ELP nanoparticles are internalized to low pH compartments in HeLa cells. Cell
cultures were treated with carboxyfluorescein-labeled ELP CF-I48-S48 (green) micelles at 37
o
C at 2, 20
and 200µm for 3 hour. Cells were counterstained with lysotracker red and imaged with a scanning
confocal fluorescence microscope.
53

2.5 Discussion
We have demonstrated that by modulating the length and amino acid composition of the block
copolymers, we can control the onset of nanoparticle self-assembly as well as their size. These
assembly properties primarily correlate with the transition temperature and molecular weight of
the hydrophobic isoleucine ELP core. In contrast, the identity of the hydrophilic ELP block
copolymer played a limited role in assembly, but a major role in the bulk phase separation of
these protein polymer nanoparticles. Both the assembly temperature and stability of nanoparticle
diameter are critical to their disposition in the body. Other factors, including the N to C vs. C to
N orientation of the hydrophilic to hydrophobic blocks played almost no role in the assembly
temperature or radius.

Based on this data, we surmise that hydrophilic portion/block makes up the outer portion or
corona of the ELP nanoparticles. As the corona is usually in contact with the cellular
environment its composition usually imparts the characteristics that allow for prolonged
circulation of the particle, by preventing the opsonization and removal by the reticuloendothelial
system (RES). Polyethylene glycol (PEG) has thus far been the polymer of choice for endowing
any nanoparticles/polymers with stealth properties to protect them from premature clearance.
This polymeric brush afforded by the PEG, is thought to adopt a splayed appearance, which
sterically suppresses the binding of opsonins. Similarly it may be possible that the hydrophilic
corona of the ELP micelle can serve role similar to that of PEG in ensuring extended circulation
time. As of now we remain unsure as to what type of stealth characteristics that can be attributed
with the changes in amino acid sequence as well as the different secondary structure
conformation that it adopts. Certainly, in vivo characterization of these constructs will give a
54

better indication/understanding of this correlation; furthermore, these are investigated in depth in
the following chapter.

2.6 Conclusion
A library of ELP protein polymers of various lengths and MW, have been recombinantly
prepared and demonstrated to assemble into stable micelle-like nanoparticles upon stimulation
with heat. This self-assembly is maintained at physiologically relevant temperatures.
Furthermore, the monomer length of the hydrophobic core had the most profound effect on the
ability of the block copolymers to form stable micelles. Previous in vivo studies have suggested
that ELP nanoparticles are potentially useful as macromolecular nanocarriers of
chemotherapeutics
46
. Pursuant to this concept, this library of biomaterials may now be utilized as
a platform for the controlled combination of a host of therapeutic and imaging modalities with
potential applications to treat a wide range of indications including, but not limited to, cancer.
55

Chapter 3
An assessment of the effects of molecular weight, amino acid composition, and
nanostructure on the biodistribution of ELP protein polymers
3.1 Abstract
Protein polymers are repetitive amino acid sequences that can assemble monodisperse
nanoparticles with potential applications as cancer nanomedicines. Of the currently available
molecular imaging methods, positron emission tomography (PET) is the most sensitive and
quantitative; therefore, this work explores microPET imaging to track protein polymer
nanoparticles over several days. To achieve reliable imaging, the polypeptides were modified by
site-specific conjugation using a heterobifunctional sarcophagine chelator, AmBaSar, which was
subsequently complexed with
64
Cu. AmBaSar/
64
Cu was selected because it can label particles in
vivo for days, consistent with the timescales required to follow long-circulating nanotherapeutics.
Using an orthotopic model of breast cancer, we observed four protein polymer elastin-like
polypeptides (ELPs) of varying molecular weight, amino acid sequence, and nanostructure. To
analyze this data, we developed a six-compartment image-driven pharmacokinetic model capable
of describing their distribution within individual subjects. Surprisingly, the assembly of an ELP
block copolymer (78 kD) into nanoparticles (R
h
= 37.5 nm) minimally influences
pharmacokinetics or tumor accumulation compared to a free ELP of similar length (74 kD).
Instead, ELP molecular weight is the most important factor controlling the fate of these
polymers, whereby long ELPs (74 kD) have a heart activity half-life of 8.7 hours and short ELPs
(37 kD) have a half-life of 2.1 hrs. These results suggest that ELP-based protein polymers may
56

be a viable platform for the development of multifunctional therapeutic nanoparticles that can be
imaged using clinical PET scanners.
57

3.2 Introduction
Protein polymers are genetically-engineered polypeptides with emerging applications as cancer
therapeutics
78, 109-113
. More recently, protein polymers constructed as block copolymers have
been developed as a platform for the direct assembly of biodegradable, multivalent
nanoparticles
79, 101, 114
. While protein polymer nanoparticles present unique opportunities to
assemble protein-based therapeutics, it remains unknown if assembly significantly alters their
pharmacokinetics and biodistribution. To address this issue, we describe a chelation approach
enabling serial positron emission tomography (PET) imaging of protein polymer nanoparticles in
vivo over a period of several days. Molecular imaging is a powerful tool for characterizing
biological processes at the cellular and sub-cellular level, both in vitro and in vivo
6, 115, 116
.
However, many experimental and clinically-approved PET contrast agents currently consist of
low molecular weight compounds with rapid clearance
117
. Unlike low molecular weight
diagnostics, therapeutic nanoparticles are intended to circulate for extended periods. To track
long-circulating therapeutics, it is advantageous to use chelation-based strategies to carry easy-
to-obtain positron emitters that retain activity over several days.

Various imaging modalities have been used to explore nanoparticulate-based contrast agents,
including ultrasound, magnetic resonance imaging, and PET
6
. Here we selected microPET
because it has high sensitivity, good resolution, no limitation caused by depth of penetration, and
can be calibrated for quantification. While PET radioisotopes such as
18
F,
11
C,
13
N, and
15
O
118,
119
have been the mainstay of clinical and molecular imaging, continuing development of large
biomolecules such as proteins, peptides, antibodies, and nanoparticles necessitates the
development of non-traditional PET radioisotopes
72
. In their application to protein nanoparticles,
58

the aforementioned non-metallic radioisotopes possess critical limitations. Chief among them
are their short radiological half-lives, which prohibit the investigation of biological processes
over several days. To overcome this limitation metallic radioisotopes of Zr, Y, In, Ga, and Cu
have been investigated as they provide a wide range of decay half-lives, which are compatible
with long biological pharmacokinetic half-lives
71
. In addition, metallic radioisotopes are
amenable to non-covalent chelation, which makes them simple to attach to biological molecules
immediately prior to administration. Of these radioisotopes,
64
Cu is advantageous due to its low
positron energy, high specific activity, availability, and reasonably long half-life (12.7 hrs)
120
.
These properties allow investigation of biological processes that take place over days
121-123
.

The standard approach to tagging a biomolecule with a metallic radionuclide such as
64
Cu is to
first conjugate a suitable chelating agent to the protein or nanoparticle and then to complex the
metal to the chelated biomolecule. Chelates that can hold radiometals with high-stability under
physiological conditions are essential in achieving high uptake of the copper radionuclide in the
tissue or organ of interest while minimizing their non-selective binding or incorporation into
non-target organs or tissues
124
. Unfortunately, the cuprous ion does not chelate as effectively
with the macrocyclic 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as do
other metals
71
. In view of this, and based on the comparative stability of the sarcophagine-based
chelator
125, 126, 127
,

the chelating agent AmBaSar (Fig. 1-3a) was selected for this study over a
traditional macrocyclic chelator.

Many nanoparticle platforms are under investigation for packaging, transport, and delivery of
imaging and therapeutic agents
6
. One example is a class of protein polymers derived from
59

human tropoelastin, called elastin-like polypeptides (ELPs)
27, 78, 96
. ELPs are composed of a five
amino acid repeat (Val-Pro-Gly-Xaa-Gly)
n
. ELPs undergo an inverse phase transition above a
transition temperature (T
t
), which is primarily a function of the guest residue Xaa, n, and
concentration
96, 128
. In solution, ELPs are structurally disordered. When the temperature is raised
above their T
t
, they undergo a sharp (2–3
o
C range) phase transition, leading to biopolymer
coacervation
96
. This process is fully reversible when the temperature is lowered below T
t
. Phase
separation can be triggered by other external stimuli such as changes in ionic strength, pH,
solvent, and magnetic fields
96, 129, 130
.

The in vitro and in vivo properties of nanoparticles depend on a number of key physicochemical
characteristics including size and size distribution, surface morphology, surface chemistry,
surface charge, surface adhesion, steric stabilization, drug loading efficiency, drug release
kinetics and hemodynamic properties of the nanoparticles. Particle size and size distribution is
one of the most widely accepted defining characteristics of NP-based medicines because size can
significantly impact the PK, biodistribution and safety. After administration particles less than
5 nm are rapidly cleared from the circulation through extravasation or renal clearance
131
, while
particles 200nm or greater in size are more efficiently taken up by the MPS, with cells in the
liver, spleen and bone marrow
132, 133
. In addition, work by Perrault et al.
134
demonstrated that
sub-20nm particles have rapid permeation into tumors but have poor retention/accumulation
compared to larger molecules (~100nm), but the larger molecules were retained much longer
within the tumor via the EPR effect.

60

Nanoparticle based formulations are designed to improve the biodistribution and the target
accumulation of systematically administered therapeutic agents. To facilitate biodistribution
analyses it would be advantageous if the circulation time and the organ accumulation of
nanoparticle systems could be visualized non-invasively in vivo in real time. To achieve this
goal, many different nanomedicines have been cofunctionalized with contrast agents in order to
track their PK and biodistribution. In the majority of cases radionuclides have been used for this
purpose. Several different types of radionuclide-labeled antibody, liposomes, polymers and
micelles have been subjected to biodistributional analyses over the years, both in animal models
and the patients and it has become clear that such studies substantially assist in improving our
understanding of the drug delivery process, as well as in predicting the therapeutic potential of
targeted in nanomedicine. In addition to monitoring the biodistribution of nanomedicine
formulations in real time, nanotheranostics can also be used to non-invasively assess target site
accumulation. Both radionuclides and MR contrast agent labeled nanomedicines are highly
suitable for monitoring the targeted drug delivery to the pathological sites. Here we report the
characterization of ELPs with various lengths and nanoparticle structure using microPET
imaging to track their pharmacokinetic and biodistribution properties.

61

3.3 Materials and methods
All reagents and solvents were purchased from Sigma Aldrich Chemicals (St. Louis, MO, USA)
and unless otherwise stated were used without further purification. PD10 desalting columns were
purchased from GE Healthcare (Piscataway, NJ). Female athymic nude mice, 5-6 weeks old
were supplied by Harlan Laboratories (Indianapolis, IN).
64
Cu was obtained from Washington
University (St. Louis, MO) and was produced by the
64
Ni (p,n)
64
Cu nuclear reaction.

3.3.1 Recombinant synthesis of ELPs
To generate ELPs of a specific and pre-determined chain lengths the following plasmid
reconstruction recursive directional ligation (preRDL) strategy was employed
103
. Two cloning
vectors, which contained the ELP gene were cut with two separate sets of restriction enzymes,
which was described previously by our group
101
. One vector was digested with BssHII and
AcuI, while BssHII and Bser1 was used to digest the second vector. Enzyme digestion was
performed using 1 µL of each enzyme, at 37
o
C for 3 h. The two sets of cut vectors were gel
purified and ligated together using the T4 DNA ligase (Invitrogen, Carlsbad, CA), resulting in
the recursive extension of the genes encoding for pentameric repeats. The same strategy was
employed to generate the ELP block copolymer, where the N-terminal gene of one monoblock
(Xaa = Ala) was ligated to a C-terminal ELP gene of another (Xaa=Ile) via preRDL. Gene
sequences encoding for the desired polypeptides (Table 1-3) were confirmed using diagnostic
DNA digestion and DNA sequencing from both N and C termini.

62

3.3.2 Protein purification by inverse transition cycling
pET25b(+) expression vectors containing the desired constructs were transformed into E. coli
BLR (DE3) cells for protein hyperexpression and proteins were purified by inverse transition
cycling (ITC)
102
. Briefly, overnight cultures were spun down and re-suspended in cold PBS.
The proteins were liberated from bacteria by periodic probe-tip sonication for a total of 3
minutes. Insoluble debris was collected by centrifugation for 15 min at 4
o
C, 12,000 rpm, and
the supernatant was transferred to another tube. Excess poly-ethylene imine (MW=3,000) was
added to precipitate nucleic acids and the solution was centrifuged. The supernatant, containing
soluble ELP, was heated to 37 °C to induce phase separation, and the coacervate was collected
by centrifugation. The ELP was then re-suspended in cold PBS and centrifuged at 4
o
C again,
completing one round of ITC. 4-6 rounds of ITC were completed, sufficient to ensure the purity
indicated (Fig. 1-3b).

3.3.3 Dynamic light scattering of particle assembly
Determination of the hydrodynamic radius of the free protein polymers and nanoparticles was
performed on a Dynapro plate reader (Wyatt Technology Inc., Santa Barbara, CA, USA). 10-25
µM of polypeptide in phosphate buffered saline (PBS) pH 7.4 was subjected to a temperature
ramp between 10 – 40
o
C with 1
o
C increments. Before analysis, the solutions were filtered
through Whatman Anotop
TM
filters with a 0.02 µM pore size and centrifuged to remove air
bubbles. Mineral oil was applied to prevent evaporation and the preparation was centrifuged
again before running the samples.

63

Figure 1-3. Conjugation scheme of the bifunctional chelating agent AmBaSar and ELP. a)
AmBaSar is chemically conjugated to the N-terminus of either linear ELPs or a block copolymer.
AmBaSar then chelates
64
Cu endowing the construct with radioactive properties. b) The purified
polymers were evaluated for identity and purity using SDS-PAGE and stained with copper chloride. Lane
1: Ladder; Lane 2: A96; Lane 3: A192; Lane 4: S192; Lane 5: A96I96.

3.3.4 Transmission electron microscopy (TEM) sample preparation
TEM measurements were obtained using a JEM 2100 LaB6 microscope (JEOL, Tokyo, Japan)
using an accelerating voltage of 200 kV. A small drop of heated ELP solution (37 °C) was
pipetted on a plasma-treated carbon/formvar-coated 200-mesh copper grid (Ted Pella, Redding,
CA, USA) and stained using heated 1 % uranyl acetate solution. The excess liquid was wicked
off using filter paper and the sample was dried at 37 °C. The images were processed and
analyzed using ImageJ (NIH, USA).

64

3.3.5 Orthotopic xenograft of human breast cancer model
All animal experiments were performed in compliance with the guidelines established by the
USC Institutional Animal Care and Use Committee. MDA-MB-231, a human breast cancer cell
line, was suspended in DMEM and matrigel, and injected into the right mammary fat pad
(2.5x10
6
cells per mouse) and allowed to grow for 2 weeks before imaging.

3.3.6 Preparation of AmBaSar-ELP conjugates
AmBaSar
127
was activated by 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
(EDC) and N-hydroxysulfosuccinimide (SNHS) at pH 4.0 for 30 min (4 °C), with molar ratio of
AmBaSar:EDC:SNHS of 10:9:8. 5 mg of AmBaSar (11.1 µmol) and 1.9 mg of EDC (10 µmol)
were dissolved in 100 µL of water. After mixing, 0.1 N NaOH (150 µL) was added to adjust the
pH to 4.0. SNHS (1.9 mg, 8.8 µmol) was then added to the stirring mixture on an ice-bath, and
pH was adjusted again to 4.0 by the addition of 0.1N NaOH. The reaction was allowed to
incubate for 30min at 4
o
C. The theoretical concentration of active ester AmBaSar-OSSu was
calculated to be 8.8 µmol. Then, 5–20 times AmBaSar-OSSu based on molar ratios was mixed
with the protein polymer. The pH was adjusted to 8.5 using borate buffer (1M, pH 8.5). The
reaction was kept at 4 °C overnight. Size-exclusion PD10 chromatography was used to remove
unreacted reagents from the protein polymer.

3.3.7 Radiolabeling
The AmBaSar conjugates were labeled with
64
Cu by addition of 1–5 mCi of
64
Cu (10–50 nmol
protein per mCi
64
Cu) in 0.1 N ammonium acetate (pH 5.5) buffer followed by 45 min incubation
65

at 40 °C. Before purifying the
64
Cu-proteins using a PD10 column, DTPA (3µL, 10mM, pH
6.02) was added to remove
64
Cu that weakly interacts with the peptide backbone.

3.3.8 Stability of
64
Cu-ELP-Sar constructs
Each of the constructs was incubated in fetal bovine serum (FBS, Life Technologies, Grand
Island, NY) and PBS (Cellgro, Manassas, VA) for up to 48 hrs to assess the stability of the
complexed copper. Aliquots were removed and centrifuged using concentrator tubes (Corning,
MWCO 10k) at 10,000 rpm for 10 min. After centrifugation the activity remaining on the filter
and in the eluent was measured using a gamma scintillation counter. The percentage of
radioisotope, F
%
, retained by the construct was calculated as follows:
Eq. 1
Where A
filter
and A
eluent
are the activities remaining in the filter or eluent.

3.3.9 MicroPET imaging study
Molecular imaging was performed using a microPET R4 rodent model scanner (Concorde
Microsystems, Knoxville, TN). Mice were injected with ~100-150 µCi
64
Cu-labeled ELP via the
tail vein. For imaging, the mice were anaesthetized with 2% isofluorane and placed near the
center of the field of view (FOV), where the highest resolution and sensitivity are obtained.
Static scans were obtained at 0.08, 0.75, 1.33, 2.5, 4, 24 and 48h post injection. The images were
reconstructed by a two dimensional ordered subsets expectation maximum (2D-OSEM)
algorithm.

66

3.3.10 Quantitative analysis of PET images
Time-activity biodistribution for selected tissues was obtained by drawing regions of interest
(ROI) over the tissue area. The counts per pixel/min obtained from the ROI were converted to
counts per ml/min by using a calibration constant obtained from scanning a cylinder phantom in
the microPET scanner. The ROI counts per mL/min were converted to counts per g/min,
assuming a tissue density of 1 g/mL, and divided by the injected dose to obtain an image ROI-
derived percent injected dose of
64
Cu tracer retained per gram of tissue (%ID/g). As a proxy for
the blood concentration, the time-activity curve data obtained from the heart was fit to a one-
phase exponential decay curve using Prism (GraphPad Sortware Inc., San Diego, CA).
67

3.4 Results
3.4.1 Preparation and characterization of ELPs and ELP-Sar constructs
As particle diameter significantly influences the nanoparticle fate in vivo
135, 136
, we extensively
characterized the assembly properties of the ELPs evaluated in this study (Table 1-3).

Table 1-3. Properties of ELP protein polymers evaluated in this study

Label Amino acid sequence
*

MW
(Da)
**

T
t
at 25 µM
(
o
C)
Construct
A96 G(VPGAG)
96
Y 36987.00 84.3

A192 G(VPGAG)
192
Y 73604.56 61.9

S192 G(VPGSG)
192
Y 76619.32 57.4

A96I96 G(VPGAG)
96
(VPGIG)
96
Y 77655.30 20.6
***

*Gene sequence confirmed by N and C terminal DNA sequences and diagnostic restriction digestion.
**Estimated from open reading frame excluding methionine start codon and confirmed using SDS-PAGE.
***Critical micelle temperature (CMT)

These four protein polymers were selected to determine if the effect of molecular weight (A96
vs. A192), guest residue (A192 vs. S192), and nanoparticle assembly (A192 vs. A96I96). To
promote effective blood circulation, all four ELPs were designed to be soluble at physiological
temperature; however, the diblock copolymer A96I96 was designed to assemble nanoparticles at
~20 °C. The behavior of each ELP was characterized using dynamic light scattering (DLS) to
identify the hydrodynamic radii, R
h
, (25 µM, 37 °C) of 5.2 ± 0.6 nm, 7.1 ± 0.5 nm, 7.4 ± 0.4 nm
and 37.4 ± 2.5 nm for A96, A192, S192 and A96I96 respectively (Fig. 2-3a). Modification of
these polymers at the amino terminus with the heterobifunctional chelator AmBaSar (Sar) had a
negligible effect on these radii (Fig. 2-3a). To determine if the conjugation altered the assembly
properties of the ELP block copolymer, A96I96, a temperature ramp was used to monitor the
thermal assembly of the nanoparticle forming construct A96I96 and A96I96-Sar (Fig. 2-3b).
While modification with Sar induced a minor depression in the critical micelle temperature
68

(CMT), both the labeled and unlabeled constructs form nanoparticles at physiological
temperature with a stable R
h
of ~40 nm. DLS was also used to characterize the distribution of
hydrodynamic radii for ELPs A96, A192, and S192 at physiological temperature (Fig. 2-3c) and
also for the block copolymer A96I96 at 10 and 37 °C (Fig. 2-3d), which indicates the ability of
this polymer to assemble from a monomer to nanoparticle. Based on previous characterization,
A96I96 will retain its self-assembling properties at a concentration far below than what is used in
this study. Thus, it is expected that its structure will remain micellar at the point of
administration and initial circulation.
69

Figure 2-3. ELP diblock copolymers assemble nanoparticles at physiological temperatures.
Dynamic light scattering (DLS) was used to characterize the hydrodynamic radius of the protein polymers
in phosphate buffered saline. a) Hydrodynamic radius (R
h
) of A96, A192, S192 and A96I96 at 37
o
C
before and after modification with AmBaSar (Sar). The ELP block copolymer A96I96 assembles
nanoparticles. Bars represent mean ± SD. b) Above 15-18
o
C, A96I96 forms nanoparticles of stable
hydrodynamic radii at 25 µM. c) Distribution of hydrodynamic radii for linear ELPs at 37 °C. d)
Distribution of hydrodynamic radii for ELP block copolymer A96I96 at 10 and 37 °C. e) Transmission
electron microscopy (TEM) of negatively stained A96I96 nanoparticles (white round objects) with an
average particle diameter of 33.3 ± 11.5 nm stained with uranyl acetate (black clusters). Scale bar 50 nm.
f) Histogram of A96I96 nanoparticles (n=141) was obtained using image analysis across 9 TEM images.
70

As independent confirmation of nanoparticle assembly, negative-stained transmission electron
microscopy (TEM) was used to observe contrast-excluding (light) nanoparticles of A96I96 (Fig.
2-3e). These particles appeared as round, monodisperse particles with a diameter of 33.3 ± 11.5
nm (Fig. 2-3f). In this case, the radius of the particles by TEM is approximately half of that
observed by DLS, which may result from several possible causes: i) the hydrophilic block
includes a significant fraction of water in solution, which increases the hydrodynamic radii
compared to a sample dried for TEM; and ii) the hydrophobic core of A96I96 nanoparticles
excludes uranyl acetate contrast, while the hydrophilic corona does not. Currently, we are unable
to distinguish between these possibilities; however, both DLS and TEM indicate that the diblock
copolymer A96I96 assembles homogenous particles at physiological temperature.

3.4.2 Stability of radiolabeled
64
Cu-ELP-Sar constructs
A stability assay was performed to confirm that
64
Cu remains associated with the ELP over a 48
h time period and that the biodistribution patterns observed are not from dissociated, free
64
Cu.
Due to the half-life of
64
Cu (12.7h) measurement beyond 48h time point is less useful as only
minimal counts will be detected due to radiological decay. The PBS solution tests the stability of
the chelator to retain the radioisotope, while the serum solution mimics the proteinacious
environment during circulation. Figure 3-3a and 3-3b show minimal loss of
64
Cu from A96 and
A192 until around 48 hours. The radiochemical retention of the constructs remained high (>98%)
for 48 h in phosphate buffered saline. However, a slightly greater reduction in percentage
retention for S192 and A96I96 was detected under the same conditions (Fig. 3-3c,d). A96 and
A192 also showed relatively high serum stability. Conversely the stability of the constructs
S192 and A96I96 suffers slightly more in serum (~83 and 75% respectively). A two-way
71

ANOVA (F
7,14
=43.1, R
2
=0.956, p=2x10
-8
) at the 48-hour time point showed that all ELPs lose
retention in serum compared to PBS (p=3x10
-6
). Loss of retention depended significantly on the
ELP identity (p=1x10
-8
), with S192 (p=5x10
-5
vs. A192) and A96I96 (p= 2x10
-8
vs. A192). A96
and A96I96 were statistically indistinguishable. This suggests that the polymer architecture may
play a role in the stability of the chelators strategy; however, all four polymers were stable for 24
hours in serum.

Figure 3-3. 64Cu-ELP constructs are stable in serum for 24 hours. Stability of radiolabeled ELPs
over 48 h in serum and PBS was measured using retention in a dialysis cassette. a) A96, b) A192, c)
S192, and d) A96I96. A two-way ANOVA at the 48-hour time point showed that all ELPs lose retention
in serum compared to PBS (p=3x10
-6
). Loss of retention depended significantly on the ELP identity
(p=1x10
-8
), with S192 and A96I96 losing significantly more than A192. Bars represent mean ± SD (n =
3/group).
72

3.4.3 In vivo microPET imaging of protein polymers
Using biodistribution and microPET imaging, the influence of macromolecular structure on
biodistribution and blood circulation was evaluated by examining the performance of
nanoparticles derived from different ELPs namely, A96, S192, A192 and A96I96 (Fig. 4-3). To
this end
64
Cu-labeled ELPs were administered intravenously to nude tumor-bearing mice, and the
blood retention, tumor accumulation, and sequestration in the major excretory organs (liver,
kidneys) was observed. Upon administration (5 min) high levels of activity were observed in the
heart, suggesting retention of nanoparticles in the circulation. To ensure the accuracy of the
analysis, the heart signal was traced through all the imaging slices so as to avoid any additive
signal from other overlapping tissues. Activity in the heart remained high even after 4h for
A192, S192 and A96I96; however, a significant reduction in signal was observable for A96. For
all constructs, the heart signal diminishes to approximately one tenth of its initial concentration
by 24 hours post injection. What was unexpected was that the amount of signal in the heart
remained relatively constant between 24 and 48 hours, which suggested that the remaining
isotope is no longer freely circulating in the blood. Uptake of
64
Cu-ELPs by the liver is apparent,
suggesting that this is the primary route of clearance for ELPs above the renal filtration cutoff for
A192, S192 and A96I96. In contrast, the liver accumulation of A96 was lower than for other
constructs.

73

Figure 4-3. Serial microPET imaging of protein polymer nanoparticles in an orthotopic model
of human breast cancer.
64
Cu-labelled ELPs were administered systemically to mice carrying MDA-
MB-231 tumors. Serial imaging was performed, and coronal images centered on the tumor for A96,
A192, S192 and A96I96 are depicted at 0.08, 0.75, 1.33, 2.5, 4, 24 and 48 h post injection. A
representative mouse is shown from each group (n=3/group). Within each 5 min panel, two major pools
of blood are present in the heart (top) and liver (middle). At later time points, the gastro-intestinal track
(lower) and the bladder (bottom) enhance in contrast. The tumor locations are indicated by arrows.

3.4.4 Quantitative analysis of PET images
To gain an estimate of the rate of blood clearance, the heart intensity was monitored over time.
The time-activity curve of the heart showed that A192, S192, A96I96 and to a lesser extent A96,
were still circulating in the blood stream after 4h, but all approached the background signal
found in the muscle by 24h (Fig. 5-3a). By fitting an exponential decay curve to the data, a
correlation coefficient r
2
> 0.97 was obtained for each protein polymer. The heart-activity half-
74

life for A96, A192, S192 and A96I96 was determined to be 2.1, 8.7, 8.3, and 7.3 h respectively,
where A96 was cleared significantly faster than the other constructs (Fig. 5-3b).

In addition to the heart half-life, serial microPET imaging was used to estimate the kinetics and
magnitude of accumulation in several other easily identifiable tissues (Fig. 6-3). Especially for
the earlier time points, the tissues in the chest and abdominal cavity seem to overlap, making
quantification difficult. However, since these tissues are easily discernible at later time points,
we used the locations determined here, to guide the positioning of the ROI that optimally
captures the different tissues with minimal overlap. Time-activity curves corresponding to the
kidneys, liver and muscle (Fig. 6a-c) are presented. From these results, no obvious differences
were observed between the constructs in terms of muscle accumulation (Fig. 6-3a). A96, the
shortest and lowest molecular weight construct, exhibited the highest kidney uptake (Fig 6-3c).
Renal clearance of A96 occurs rapidly until it plateaus at 4h post injection. Conversely the extent
of renal clearance of A192, S192 and A96I96 was relatively low over time (Fig. 6-3b)

Figure 5-3. Non-compartmental pharmacokinetics of 64Cu-ELPs in the heart. a) The time
activity curve of blood concentration can be estimated using the intensity in the heart as a surrogate
measure, whereby
64
Cu-ELPs (n=3/group) are expressed as %ID/g. Values indicate the mean ± 95%CI.
b) By fitting the initial rate of log-linear decay (0-4 hrs for A96; 0-24 hours for A192, S192 and A96I96),
the half-life of activity in the heart was indicated as the mean ± 95%CI.
75

All
64
Cu-ELP-Sar constructs exhibited hepatic clearance in varying degrees (Fig. 6-3c).
Considering that A192, S192 and A96I96 have a hydrodynamic radius that is larger than the
cutoff point for renal filtration; these constructs appear to be cleared primarily via the
immobilization in the liver. Notably, the liver accumulation for diblock copolymer A96I96 is
more prominent compared to the monoblock ELPs, which is consistent with expectations of a
nanoparticle. Hepatic concentrations for S192 reaches a maximum at 24h where they remained
constant until 48h post injection. In contrast there was a decrease in liver accumulation over time
for A192 and A96. A96 exhibited the lowest accumulation in the liver, perhaps due to its ability
to be cleared by the kidney.

Tumor uptake profiles (Fig. 6-3d) are slightly different for each construct during the first hour
post injection; A96 exhibits the earliest detectable tumor signal, which subsequently decreases,
perhaps due to its lower molecular weight and higher vascular permeability. For both S192 and
A192, the tumor signal can be easily detected at 45 and 80 min respectively and remained
constant for the duration of the study. In contrast, a steady increase in tumor uptake can be
observed with the nanoparticle-forming A96I96. Despite differences in the kinetics of uptake, all
four constructs achieved a similar tumor concentration in the range of 3-4 %ID/g body weight.

76

Figure 6-3. Biodistribution of 64Cu-ELP in athymic nude mice implanted with MDA-MB-231 cell
line (n=3) within a) muscle, b) kidneys, c) liver and d) tumor expressed as %ID/g calculated from ROI
image analysis. A96 accumulates over time in the kidneys, while A192 and S192 do not. A96I96
accumulates over time in the liver. Values indicate the mean ± 95%CI.
77

3.5 Discussion
Among the factors that affect pharmacokinetics and biodistribution are the architecture and
molecular weight of the nanocarriers
137
. Through genetic engineering we can easily manipulate
and control both factors when designing protein polymer-based nanocarriers. Here we present a
study to determine if ELP nanoparticles can be tracked using serial molecular imaging via
stable
138,126
chelation (AmBaSar) of a positron emitter (
64
Cu) with a sufficient radiological half-
life to image distribution over several days (Fig. 1-3a). We explored the hypothesis that protein
polymer architecture would influence the kinetics and magnitude of biodistribution. Long-
circulation is an important feature of nanomedicines; furthermore, we were encouraged to
observe that nanoparticle assembly of A96I96 has minimal effect on the half-life of activity in
the heart (Fig. 5-3b); however, assembly moderately redirects clearance to the liver (Fig 6-3c).
For soluble ELPs with Xaa=Ala or Xaa = Ser, there were minimal differences between route of
clearance or apparent kinetics of biodistribution. Our predominant finding was that the lower
molecular weight ELP, A96, clears via the kidney compared to the larger molecular weight
protein polymers, A192, S192, A96I96.

One of the challenges to the field of nanomedicine is the identification of patient-specific and
tissue-specific biodistribution patterns and the development of a platform for interpreting this
information
139
. Typical preclinical tumor models average data across multiple animals to
characterize biodistribution and pharmacokinetics, which are not translational approaches
140, 141
.
In contrast, the molecular imaging approach used here can deliver quantitative spatio-temporal
data within an individual. Armed with this information, clinicians and engineers can develop
personalized pharmacokinetic models that directly describe the fate of nanomedicines within
78

their patients. More importantly, this information may directly answer the question of whether or
not a given nanoparticle preferentially interacts with its target in a patient.

In this study, no active targeting moiety was appended to the carriers and tumor sequestration
was achieved only through passive or non-specific uptake mechanisms such as the enhance
permeability retention (EPR) effect
142
. While all of the constructs do exhibit tumor
accumulation, no significant difference in extent of the accumulation was observed (Fig. 6-3d).
Other than passive accumulation, no effort was made to characterize target-mediated delivery to
the tumor; however, this model may have potential applications in future studies of targeted
therapeutics. Active targeted delivery of therapeutic agents to tumor tissue remains a promising
approach to improve cancer treatment, as it may deliver higher doses to tumor sites while
minimizing exposure to normal tissues. This work shows that it is possible to tailor make specific
protein polymer nanocarriers that shift their clearance from renal to hepatic routes of elimination;
furthermore, both monomeric (A192) and nanoparticulate (A96I96) carriers appear to remain
viable platforms for delivery. Partnered with molecular imaging to quickly select drug carriers
with the most selective tumor accumulation, these protein polymer nanoparticles are an emerging
solution to nanotherapeutics engineering.

3.6 Conclusion
Using microPET imaging this thesis describes the effect of amino acid composition, molecular
weight, and nanostructure on the biodistribution and pharmacokinetics of several ELP protein
polymers. The ELP diblock copolymer (A96I96) that assembles into nanoparticles is cleared
more rapidly by the liver than a monoblock ELP (A192); however, the primary determinant of
79

the blood half-life appears to be the ELP molecular weight. Through molecular imaging of stable
AmBaSar/
64
Cu chelates, this library of protein polymers may now be optimized using non-
invasive imaging to carry therapeutic proteins and drugs.

80

Chapter 4
Development of anti-angiogenic protein polymer nanoparticles and their evaluation using
positron emission tomography
4.1 Abstract
Multiple nanocarriers are under investigation as multifunctional agents for packaging, transport
and delivery of imaging and therapeutic agents. The polymeric nanocarriers described here are
intended to visualize and measure functional characteristics of physiological
microenvironments. In this study several elastin-like polypeptide (ELP)-based PET imaging
agents were designed and evaluated for their potential use as diagnostic imaging and therapeutic
agents for breast cancer. To improve tumor accumulation and bioavailability a neovasculature-
targeting protein (7 kD) called disintegrin (VCN) was appended to ELPs. A series of control
ELPs and ELP-VCN fusion proteins of low and high molecular weight were
expressed. Biodistribution and microPET imaging studies with ELP alone (A192), VCN and
A192-VCN was performed in mice with palpable MDA-MB-231 tumors at 0.5, 2, 4, 18 and 48h
hours post administration. Image comparison between DOTA labeled and a novel sarcophagine-
based chelating agent was also performed.
Passive accumulation in tumors via the EPR effect (ELP alone) or through specific association of
VCN and A192-VCN with the integrin receptors upregulated on the tumor was observed. This
specific association was successfully blocked by prior injection with cold, unlabeled A192-
VCN. The combination of ELP and disintegrin (VCN) seem to prolong intratumoral contrast,
resulting in a higher accumulation of the peptide in the tumor when compared to the
controls. Increased tumor accumulation with the disintegrin-targeted ELP was observed when
81

compared to ELP or VCN by itself. The A192-VCN construct benefited from the longer
biological half-life resulting in tumor signal enhancement in PET.
82

4.2 Introduction

Targeting tumor angiogenesis for drug delivery has been identified as a promising approach for 3
main reasons
143
(i) angiogenesis is a common and genetically stable characteristic of most solid
tumors; (ii) it is readily accessible from the blood stream; (iii) it can be targeted by specific
RGD-containing peptides binding to integrins. The integrin αvβ3 is poorly expressed on
quiescent endothelium and is selectively overexpressed on activated endothelial cells of growing
vessels
144, 145
. Overexpression of αvβ3 has been shown to correlate with tumor progression and
poor prognosis in several malignancies
146-148
.

The RGD sequence is currently the model for a variety of RGD-containing peptides that display
preferential binding to either αvβ3-integrin or other types of integrins. As integrin inhibitors
these peptides are able to induce endothelial cell apoptosis and inhibit angiogenesis
145, 149
.

A source of naturally occurring high affinity ligands for integrins is found in the family of
molecules known as disintegrins. These small (40-100 amino acids), cysteine-rich polypeptides
were first isolated from viper venoms. Most disintegrins contain RGD/KGD sequence and they
bind with a high affinity to numerous integrins and are potent inhibitors of platelet
aggregation
150, 151
. Correct folding of disintegrins is important for their biologic activity and the
activity of disintegrins depends on the appropriate pairing of 8-14 cysteine residues by disulfide
bridges, which maintain the RGD containing loop in its active conformation
152
. The disulfide-
stabilized framework forms the correct conformational presentation of the integrin-binding
83

loops, and coupled with the C-terminal sequence of the disintegrin determines the integrin
binding specificity.

Although the pharmacological properties of the disintegrin contortrostatin (CN) have made it an
intriguing molecule for potential anticancer therapeutic strategies, it exists as a very small
fraction of the total venom protein (~0.01%). To overcome this obstacle, a novel recombinant
disintegrin was designed by the Markland laboratory, vicrostatin (VCN), whose structure is
based on the amino acid sequence of CN
153
. VCN has proven to be a powerful anti-angiogenic
agent with impressive anticancer activity, as shown in murine models of human breast and
prostate cancer
153
. VCN is as active as native CN both in vitro and in vivo and has a higher
affinity for integrin α5β1, which is overexpressed on angiogenic endothelial cells
153, 154
. With its
limited cellular access and poor PK parameters, short peptides, like disintegrin make poor drugs
because they do not circulate for significant periods in the bloodstream. As shown on previous
studies, cell permeability and PK characteristics of therapeutic peptides may be dramatically
improved by conjugating these peptides to macromolecular carriers
155-157
.

Many types of protein polymers are undergoing investigation as nanocarriers for transport and
delivery of imaging and therapeutic agents. These polymer conjugates are typically large
hydrophilic molecules linked to a therapeutic agent, which can target tumors either “passively”
through the EPR effect or “actively” through a triggered stimulus or affinity towards the site of
therapy. These macromolecular carriers have a longer plasma half-life, show reduced systemic
toxicity, retain activity against multiple drug resistant cell lines, and increase the solubility of
84

poorly soluble drugs. All of these attributes have led to higher anticancer efficiency of these
polymer conjugates compared to free drug.

Protein-based polymers, which are composed of repeat units of natural or unnatural amino acids,
have recently emerged as a promising new class of stimulus-responsive materials. Elastin like
polypeptide (ELP) is a biopolymer derived from the structural motif found in mammalian elastin
protein and has a sequence dependent transition temperature that can be used as nanocarriers to
treat diseases
37
. ELP is a protein comprised of a five amino acid repeat (VPGXG, where X is
any amino acid except proline). ELPs are attractive as polymeric carriers for drug delivery
because they undergo an inverse temperature phase transition. Below a characteristic transition
temperature (Tt), ELPs are structurally disordered. But, when the temperature is raised above
their Tt, they undergo a sharp (2–3
o
C range) phase transition, leading to the aggregation of the
biopolymer
37, 38
. This process is fully reversible when the temperature is lowered below Tt.
Phase transition can also be triggered by other external stimuli such as changes in ionic strength,
pH, solvent, and magnetic fields.

Here we have developed a fusion gene-product that can significantly alter the tumor
bioavailability of biopharmaceuticals such as disintegrins. Fusing the disintegrin, vicrostatin
(VCN) to a high molecular weight ELP substantially decreases its clearance and increases its
accumulation in a tumor. The resulting fusion protein still retains its integrin binding/targeting
properties albeit at a higher IC50 when compared to VCN by itself. Altering the length of the
ELP carrier used or the orientation of the VCN sequence may result in the improvement and
recovery of the VCN activity and will be further investigated. A control peptide with a short
85

RGD sequence was also appended to an ELP of the same length and would serve as a point of
comparison in terms of activity and behavior.
86

4.3 Materials and methods

4.3.1 Cells
Human umbilical vein endothelial cells (HUVEC) were purchased from PromoCell GmbH
(Heidelberg, Germany) and cultured in EC growth medium containing low serum (2%) EC
growth supplements (PromoCell). MDA-MB-231 and MDA-MB-435 cell line was purchased
from American Type Culture Collection and grown in Dulbecco's Modified Eagle Medium
medium (Gibco, NY) supplemented with 10% (v/v) fetal bovine serum. All cells were
maintained at 37°C under an atmosphere containing 5% CO
2
.

4.3.2 Recombinant synthesis of ELPs
A synthetic oligonucleotide containing the vicrostatin and RGD sequence was designed and
inserted via cassette mutagenesis into the pET25b(+) cloning vector. To facilitate insertion of
the ELP sequence the following plasmid reconstruction recursive directional ligation (preRDL)
strategy was employed
95
. Two cloning vectors, which contained the ELP gene and another which
contained the targeting sequence were cut with two separate sets of restriction enzymes. One
vector was digested with BssHII and AcuI, while BssHII and Bser1 were used to digest the
second vector. Enzyme digestion was performed using 1 µL of each enzyme, at 37
o
C for 3 h.
The two sets of cut vectors were gel purified and ligated together using the T4 DNA ligase
(Invitrogen, Carlsbad, CA), generating the ELP-fusion protein. Gene sequences encoding for the
desired polypeptides were confirmed using diagnostic DNA digestion and DNA sequencing from
both N and C termini.

87

4.3.3 Protein purification by inverse transition cycling
pET25b(+) expression vectors containing the desired constructs were transformed into E. coli
Origami B(DE3) competent cells for protein hyperexpression and proteins were purified by
inverse transition cycling (ITC)
102
. Briefly, overnight cultures were spun down and re-suspended
in cold PBS. The proteins were liberated from bacteria by periodic probe-tip sonication for a
total of 3 minutes. Insoluble debris was collected by centrifugation for 15 min at 4
o
C, 12,000
rpm, and the supernatant was transferred to another tube. Excess poly-ethylene imine
(MW=3,000) was added to precipitate nucleic acids and the solution was centrifuged. The
supernatant, containing soluble ELP, was heated to 37 °C to induce phase separation, and the
coacervate was collected by centrifugation. The ELP was then re-suspended in cold PBS and
centrifuged at 4
o
C again, completing one round of ITC. 4-6 rounds of ITC were completed,
sufficient to ensure the purity indicated (Fig. 1-4a).

4.3.4 LCST characterization of ELP and ELP-fusion protein
The LCST characterization of the ELP and ELP fusion protein was determined by measuring
solution turbidity as a function of temperature. Solutions of the polypeptide in phosphate
buffered saline (PBS) were at a constant rate of 1oc/min in a temperature controlled multicell
holder of a UV visible spectrophotometer (DU800 Spectrophotometer, Beckman Coulter, CA,
USA). The LCST or transition temperature (Tt) is defined as the point of one half maximal
turbidity.

88

4.3.5 Dynamic light scattering of particle assembly
Determination of the hydrodynamic radius of the free protein polymers and nanoparticles was
performed on a Dynapro plate reader (Wyatt Technology Inc., Santa Barbara, CA, USA). 25 µM
of polypeptide in phosphate buffered saline (PBS) pH 7.4 was subjected to a single time point
reading at 37
o
C. Before analysis, the solutions were filtered through Whatman Anotop
TM
filters
with a 0.02 µM pore size and centrifuged to remove air bubbles.

4.3.6 Transmission electron microscopy (TEM) sample preparation
TEM measurements were obtained using a JEM 2100 LaB6 microscope (JEOL, Tokyo, Japan)
using an accelerating voltage of 200 kV. A small drop of heated ELP solution (37 °C) was
pipetted on a plasma-treated carbon/formvar-coated 200-mesh copper grid (Ted Pella, Redding,
CA, USA) and stained using heated 1 % uranyl acetate solution. The excess liquid was wicked
off using filter paper and the sample was dried at 37 °C. The images were processed and
analyzed using ImageJ (NIH, USA).

4.3.7 Fluorescence activated cell sorter (FACS) analysis
Cellular binding of the ELP and ELP-fusion protein to cells were analyzed by FACS. For
quantification, ELP and ELP-fusion protein were labeled with rhodamine using NHS chemistry
(Thermo Scientific, Rockford, IL).

Cells (4x10
5
) were seeded on a 12 well plate and left overnight in a 37
o
C, 5% CO2 incubator.
After overnight attachment, cells were washed with DPBS and media was replaced. 25µM of
rhodamine labeled ELP and ELP-fusion protein was added to the cells and was left to incubate
89

for 2h. After the incubation period, cells were detached with Trypsin-EDTA (0.05% trypsin, 0.5
mM EDTA pH 8.0) and suspended in PBS containing 1%BSA. 1x10
4
cells were analyzed for
fluorescence using a FACScan (Becton Dickinson, San Jose, CA). Non-treated cells were used
as controls. For blocking studies 50 µL (10mg/mL) VCN was added to the cells and left for
30min. After the incubation period the VCN was removed and the cells were washed with
DPBS. Media was replaced and 25µM A192-VCN-rhodamine was added and left to incubate for
a further 1.5hrs.

4.3.8 Confocal microscopy
Cells (2x10
5
) were plated on coverslips coated with fibronectin in 12-well plates and cultured for
48h. Rhodamine labeled ELP and ELP-fusion protein (25µM) were added to the cells and left to
incubate for 1h at 37
o
C in a humidified 5% CO
2
atmosphere. For blocking study, a blocking
dose of unlabeled VCN (50µL 10mg/mL) was first added to the cell for 30min before addition of
the labeled ELP-fusion protein. Following incubation period, cells were washed with DPBS and
then fixed with 4% paraformaldehyde in DPBS at room temperature for 15min. Nuclear staining
was performed by adding Hoescht 33285. Fluorescence images were acquired with a scanning
confocal microscope (Zeiss LSM 510 Meta, Thornwood, NY) which is equipped with argon and
red and green HeNe lasers. A separate experiment in which the cells were incubated at
temperature which does not permit receptor mediated endocytosis (4
o
C) was conducted
concurrently.

90

4.3.9 Cell integrin receptor binding assay
In vitro integrin binding affinities and specificities of peptides were assessed via displacement
cell-binding assays using 125I-echistatin as the integrin specific radioligand
158
. Experiments
were performed on the a
v
β
3
positive MDA-MD-435 human breast cancer cells. Cells were grown
in Dulbecco’s modified Eagle medium (Gibco) supplemented with 10% FBS at 37
o
C in a
humidified atmosphere containing 5%CO
2
. During the cell-binding assay experiment, the cells
were harvested, washed twice with PBS, and resuspended (2 × 10
6
cells/mL) in binding buffer
(25mM Tris, pH 7.4, 150mM NaCl, 1mM CaCl2, 1mM MgCl2, 1mM MnCl2, 0.1% bovine
serum albumin). Filter multiscreen DV plates (96-well; pore size, 0.65 μm; Millipore) were
seeded with 10
5
cells and incubated with 125I-echistatin (~100pM/well) in the presence of
increasing concentrations of different RGD peptide analogs (0–10000nM). The total incubation
volume was adjusted to 200μL. After the cells were incubated for 2 h at room temperature, the
plates were filtered through a multiscreen vacuum manifold and washed twice with cold binding
buffer. The hydrophilic polyvinylidenedifluoride (PVDF) filters were collected and the
radioactivity was determined using a gamma counter. The best-fit 50% inhibitory concentration
(IC50) values for the 435 cells were calculated by fitting the data by nonlinear regression using
GraphPad Prism (GraphPad Software, Inc.). Experiments were performed with triplicate
samples.

4.3.10 Orthotopic xenograft model of human breast cancer
All animal experiments were performed in compliance with the guidelines established by the
USC Institutional Animal Care and Use Committee. MDA-MB-231, a human breast cancer cell
91

line, was suspended in DMEM and matrigel, and injected into the right mammary fat pad
(2.5x10
6
cells per mouse) and allowed to grow for 2 weeks before imaging.

4.3.11 Preparation of DOTA Conjugate
DOTA was activated by EDC at pH 5.5 for 30 min (4 °C), with molar ratio of
DOTA:EDC:SNHS ) 10:5:4. Typically, 12 mg of DOTA (24 µmol) dissolved in 500 µL of water
and 2.3 mg of EDC (12 µmol) dissolved in 130 µL of water were mixed, and 0.1 N NaOH (250
µL) was added to adjust the pH to 5. SNHS (2.1 mg, 9.6 µmol) was then added to the stirring
mixture on ice-bath, and 0.1 N NaOH (50 µL) was further added to finalize the pH to 5.5. The
reaction was allowed for 30 min at 4 °C. The theoretical concentration of active ester DOTA-
OSSu was calculated to be 9.6 µM. Then, 5–20 times DOTA-OSSu based on molar ratios was
added to the proteins of interest. The pH was adjusted to 8.5 using borate buffer (1M, pH 8.5).
The reaction was kept at 4 °C overnight. Size-exclusion PD10 chromatography was used to
remove unreacted reagents from the protein polymer.

4.3.12 Preparation of AmBaSar-ELP conjugates
AmBaSar
127
was activated by 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
(EDC) and N-hydroxysulfosuccinimide (SNHS) was added at pH 4.0 for 30 min (4 °C), with
molar ratio of AmBaSar:EDC:SNHS of 10:9:8. 5 mg of AmBaSar (11.1 µmol) and 1.9 mg of
EDC (10 µmol) were dissolved in 100 µL of water. After mixing, 0.1 N NaOH (150 µL) was
added to adjust the pH to 4.0. SNHS (1.9 mg, 8.8 µmol) was then added to the stirring mixture
on an ice-bath, andpH was adjusted again to 4.0 by the addition of 0.1N NaOH. The reaction was
allowed to incubate for 30min at 4
o
C. The theoretical concentration of active ester AmBaSar-
92

OSSu was calculated to be 8.8 µmol. Then, 5–20 times AmBaSar-OSSu based on molar ratios
was mixed with the protein polymer. The pH was adjusted to 8.5 using borate buffer (1M, pH
8.5). The reaction was kept at 4 °C overnight. Size-exclusion PD10 chromatography was used to
remove unreacted reagents from the protein polymer.

4.3.13 Radiolabeling
The DOTA and AmBaSar conjugates were labeled with
64
Cu by addition of 1–5 mCi of
64
Cu
(10–50 nmol protein per mCi
64
Cu) in 0.1 N ammonium acetate (pH 5.5) buffer followed by 45
min incubation at 40 °C. Before purifying the
64
Cu-proteins using a PD10 column, DTPA (3µL,
10mM, pH 6.02) was added to remove
64
Cu that weakly interacts with the peptide backbone.

4.3.14 MicroPET imaging study
Molecular imaging was performed using a microPET R4 rodent model scanner (Concorde
Microsystems, Knoxville, TN). Mice were injected with ~100-150 µCi
64
Cu-labeled ELP via the
tail vein. For imaging, the mice were anaesthetized with 2% isofluorane and placed near the
center of the field of view (FOV), where the highest resolution and sensitivity are obtained.
Static scans were obtained at 0.5, 2, 4, 24 and 48h post injection. The images were reconstructed
by a two dimensional ordered subsets expectation maximum (2D-OSEM) algorithm.

4.3.15 Quantitative analysis of PET images
Time-activity biodistribution for selected tissues was obtained by drawing regions of interest
(ROI) over the tissue area. The counts per pixel/min obtained from the ROI were converted to
counts per ml/min by using a calibration constant obtained from scanning a cylinder phantom in
93

the microPET scanner. The ROI counts per mL/min were converted to counts per g/min,
assuming a tissue density of 1 g/mL, and divided by the injected dose to obtain an image ROI-
derived percent injected dose of
64
Cu tracer retained per gram of tissue (%ID/g). As a proxy for
the blood concentration, the time-activity curve data obtained from the heart was fit to a one-
phase exponential decay curve using Prism (GraphPad Software Inc., San Diego, CA).

94

4.4 Results
4.4.1 Characterization of ELP-VCN constructs
A vector containing the VCN and RGD sequence was designed and using the preRDL method
appended to an ELP with 192 pentameric repeats (A192). Purification of the ELPs by inverse
transition cycling yielded proteins of high purity (>95%) (Fig. 1-4a). The thermal behavior was
studied by measuring solution turbidity as a function of temperature. Compared to a free A192,
A192-VCN has slightly lower phase transition temperatures (Fig. 1-4b). However, A192-RGD
exhibited a higher T
t
than both A192 and A192-VCN.

To promote effective blood circulation, the ELP fusion protein was designed to be soluble at
physiological temperature. The behavior of ELP carrier and the fusion protein was
characterized using dynamic light scattering (DLS) to identify the hydrodynamic radii, R
h
, (25
µM, 37 °C) of 7.8 ±1.2 nm, 7.1 ±0.8nm and 22.3 ±5.3 nm for A192, ARGD and A192-VCN
respectively (Fig. 1-4c). It seems in both the cumulants and regularization fits A192-VCN
exhibited an R
h
much bigger than the ELP carrier by itself. This is certainly curious as the
appended peptide is only 1/10
th
of the size of the carrier and it was not foreseen that it would
exert a significant influence on the overall size of the construct. However, upon viewing with
negative-stained transmission electron microscopy (TEM) dark colored spots of round,
monodisperse particles with a diameter of 23.1 ± 2.8 nm was clearly observed (Figs. 1-4d and 1-
4e). It is speculated that the VCN portion of the protein aggregates among itself, possibly due to
intra-disulfide linkages, forming a VCN core with an ELP brush.

95

Figure 9-4. Characterization of ELP-fusion proteins. a) The purified fusion proteins were
evaluated for identity and purity using SDS-PAGE and stained with copper chloride. Lane 1: ladder; lane
2: A192; lane 3: ARGD; lane 4: A192-VCN. b) The temperature-concentration phase diagram for ELP
and an ELPVCN fusion protein. Both proteins have a T
t
well above body temperature. A best-fit curve
and 95% CI is indicated; c) Hydrodynamic radius (R
h
) of A192, ARGD and A192-VCN at 37
o
C. Bars
represent mean ±SD. d) Transmission electron microscopy (TEM) of negatively stained A192-VCN with
an average particle diameter of 23.13nm±2.81 nm stained with uranyl acetate. Scale bar 200nm. e)
Histogram of A192-VCN (n=31) was obtained using image analysis across 3 TEM images.
96

4.4.2 Evaluation of the expression of integrin αvβ3 receptor
To evaluate the expression of α
v
β
3
integrins, flow cytometric analysis of MDA-MB-231, MDA-
MB-435 and HUVEC (Figs. 2-4a-c) was carried out using specific anti-integrin antibodies. The
number of αvβ3 integrin receptors in each cell line was obviously different with the pattern of
expression levels HUVEC>MDA-MB-435>MDA-MB-231. Binding to FITC-VCN was also
assessed using the same cell lines (Figs. 2-4d – f). Minimal binding of VCN to 231 and 435 cells
were exhibited. However, a ~6-fold increase in signal from the control was observed for
HUVEC cells.

Figure 2-4. Relative expression of target integrin across primary and transformed cell lines.
The expression of integrin αvβ3, as stained by anti-αvβ3 antibody on a) MDA-MB-231, b) MDA-MB-
435 and c) HUVEC cells was confirmed by flow cytometry. Representative histogram from flow
cytometry of d) 231, e) 435 and f) HUVEC cells showing association of FITC-VCN. Dotted lines
indicated non-treated control cells.

97

4.4.3 Intracellular uptake of ELP-VCN by flow cytometry analysis
Flow cytometry was used to determine the uptake of ELP-fusion protein by endothelial cells
(HUVEC) and 2 breast cancer cell lines (MDA-MB-231 and MDA-MB-435). Figure 3-4a – c
show the cellular uptake of fusion protein after cells were incubated with rhodamine labeled ELP
for 2h at 37
o
C. Binding within each cell line also showed minimal association with A192-rho.
However as perhaps is expected the fusion construct of ARGD and A192-VCN (AV) binds at a
much higher level than A192 with A192-VCN exhibiting the highest binding.

Figure 3-4 Binding of integrin-targeted ELPs is receptor-mediated. Cell- surface binding of
various ELP constructs on endothelial cells (HUVEC) and breast cancer cell lines (MDA-MB-231 and
MDA-MB-435). Cells were incubated for 2h at 37
o
C with 25µM A192-rho, AV-rho and ARGD-rho.
Representative histogram from a) 231, b) 435, and c) HUVEC showing the relative binding of A192,
ARGD and A192-VCN. Dotted lines indicated non-treated cells. d) Histogram shows median
fluorescence intensity detected by flow cytometry (one representative experiment). Result are mean ± SD,
n=3. e) Specific binding of ELP constructs to HUVEC, 231 and 435 cells. Cells were pre-incubated for
30min at 37
o
C with excess VCN before addition of AV-rho for 1.5h and analyzed by flow cytometry.
Median fluorescence intensity (MFI) of AV-rho bound to cells is represented (one representative
experiment). Results are mean ± SD, n=3.
98

The cell line which minimally expresses the integrin αvβ3 (231 cells) showed minimal binding
of the RGD containing fusion protein – ARGD and A192-VCN exhibiting only ~2 fold higher
than median fluorescence intensity (MFI) exhibited by the ELP carrier (A192) by itself (Figs. 3-
4d and 3-4e). Conversely in HUVECS the MFI of the fusion protein are 2.7- and 4.5-fold
higher for ARGD and A192-VCN respectively. ANOVA analysis revealed a statistically
significant difference between A192 vs. ARGD in the 231 cells and HUVECs (p<0.001).
Perhaps unsurprisingly there is a statistically significant difference in all the cells for A192 vs.
A192-VCN and ARGD vs. A192-VCN (p< 0.001). Further comparison yielded a significant
difference in the uptake of ARGD between the 231 and 435 cells (p<0.001). The ARGD and
A192-VCN uptake is also statistically different between the 231 cells and HUVECs (p<0.001).
The uptake of A192, ARGD and A192-VCN between 435 cells and HUVECs are similarly
significant (p<0.05, p<0.001 and p<0.001 respectively). In addition the MFI of ARGD and
A192-VCN showed an intensity of about 17.9% and 27.2% respectively after blocking with
unlabeled VCN (Fig. 3-4e). This blocking effect was found to be statistically significantly
different (p<0.001) for both the 435 cells and HUVECs.

4.4.4 Quantitative evaluation of the cellular uptake of ELP-fusion proteins
Cellular uptake and intracellular distribution of the various construct by 231, 435 and HUVECs
was further investigated by fluorescent microscopy as shown in Figure 4-4a - c. The confocal
study showed there was no binding of A192 in all cell lines. In comparison a high level of
association of fluorescent labeled A192-VCN, VCN and A192-RGD with the 435 and the
HUVEC was demonstrated (Figs. 4-4b and 4-4c). However, little internalization was observed
for all the constructs within the 231 cells. When excess VCN was added, it competitively
99

inhibits the binding of A192-RGD and A192-VCN to the 435 cell line and HUVEC. This seems
to be indicative that ARGD and AV specifically bind to αvβ3 integrin.

Overall the quantitative results from confocal imaging studies were in good agreement with the
results obtained from flow cytometry confirming again the decoration of the surface with the
RGD moiety could facilitate uptake and internalization in tumor cells.

100

101

102

Figure 4-4. Specific uptake of protein polymers in HUVEC, MDA-MB-231 and MDA-MB-435
cells observed by confocal laser scanning microscopy. Representative confocal microscopic images of
a) MDA-MB-231, b) MDA-MB-435 and c) HUVEC cells incubated with 25µM A192, ARGD and A192-
VCN labeled with rhodamine for 1h at 37
o
C. A blocking study where cells were pre-incubated with
excess VCN for 30min before adding AV-rho was also performed. Cells were fixed with 4%
paraformaldehyde and incubated with Hoescht for nuclear staining.
103

4.4.5 Cell Integrin Receptor-Binding Assay
The receptor-binding affinity studies of VCN, A192, A192-VCN and A192-RGD for αv integrin
were performed using αv integrin–positive MDA-MB-435 cells rather than isolated receptors
αvβ3. Binding on the cell membrane allows cross-linking and integrin receptor multimerization,
by which multivalent binding and clustering of receptor is studied in the natural context of the
integrin. All the peptides inhibited the binding of 125I-echistatin to 435 cells to varying degrees
(Fig. 5-4). The binding of 125I echistatin to cell surface integrin αvβ3 was competed off by the
polypeptides in a concentration dependent manner. The IC50 values for VCN, A192-VCN,
A192-RGD (ARGD) and A192 were 0.48±0.109 µM, 1.64±0.079 µM, 51.82±0.268 µM and
900±0.288 µM respectively.

Figure 5-4. Competition binding to integrin receptors expressed on MDA-MB-435 cells.
Varying concentrations of ELP-constructs were incubated with 125I-echistatin and allowed to compete
for binding to integrin receptors expressed on the surface of 435 cells. Percent of 125I-echistatin bound
to the cell surface is plotted versus the concentration of peptides. Data shown are the average of triplicate
values and error bars represent SD. The addition of the high MW carrier reduces the potency of VCN
from 0.48±0.109 µM to 1.64±0.079 µM. An IC50 of 51.82±0.268 µM and 900±0.288 µM for ARGD and
A192 were determined respectively.
104

4.4.6 Small animal PET scan
Using biodistribution and microPET imaging, the biodistribution and blood circulation of the
ELP-fusion protein was evaluated (Fig. 6-4). To this end
64
Cu-labeled ELPS were administered
intravenously to athymic nude tumor bearing mice, and the blood retention, tumor accumulation
and sequestration in the major excretory organs (liver, kidneys) were analyzed.

High levels of activity were observed in the heart at 1h post injection for A192 and A192-VCN,
indicating a large amount of nanocarrier was in circulation. Activity in the heart remained high
even after 4h for all the ELP constructs. However, no significant signal was observed for VCN
throughout the study period. The heart signal completely diminishes for A192-DOTA by 18h
where only a residual signal remains for A192-Sar, AV-DOTA and AV-Sar indicating that the
64Cu-ELPs have been cleared from the bloodstream.

Uptake of
64
Cu-ELPs by the liver is also clearly apparent, suggesting that significant amount of
VCN, AV-DOTA and AV-Sar were sequestered by the reticuloendothelial system (RES) which
persisted until 48h post administration. The liver accumulation of A192-DOTA however is
observed not to be as high as the other constructs, as confirmed by the qualitative ROI analysis in
Figure 8-4d. While the liver showed high uptake at 24h, widespread activity could be also
observed in GI tract. This activity indicated that
64
Cu-ELP (and their metabolic products)
present in the liver underwent biliary excretion into the GIT to be eliminated in the feces.

As is indicated in Figure 6-4a, a marked difference in tumor accumulation is observed with the
three preparations of peptide. As the ELP itself is of high molecular weight, it is not surprising to
105

see it accumulate in the tumor. Due to the EPR effect, the polypeptide is passively sequestered
within the tumor achieving maximum accumulation at ~18hrs. Slight tumor accumulation was
also observed with vicrostatin alone. However since VCN is a small peptide (7kDa) it suffers
from rapid and early clearance by the kidneys. The rapid systemic clearance and high kidney
uptake of VCN may also quickly deplete circulating trace concentrations and thus may lead to
the lower tumor uptake values. Conversely tumor uptake is detected much early on and more
intensely with ELP-VCN. This seems to indicate that some degree of active targeting via the
VCN moiety has occurred resulting in the higher accumulation of ELP-VCN in the tumor.
Significant liver and intestinal uptake was also observed with the ELP-VCN. It is thought that
this staining or accumulation is due to the in vivo instability of the DOTA chelator.

Representative coronal images of MDA-MB-231 tumor bearing mice with a blocking injection
of A192-VCN are shown in Figure 6-4b. Tracer uptake in the 231 tumor was reduced in the
presence of cold, unlabeled A192-VCN indicating the in vivo integrin binding specificity of
64
Cu-A192-VCN. The uptake of radiolabaled fusion protein in other organs was also lower,
similar to that observed for RGD-peptide-based tracers
159
.

106

Figure 6-4. Serial microPET imaging of ELP and ELP-fusion protein in an orthotopic model of
human breast cancer.
64
Cu-labelled ELPs were administered systemically to mice carrying MDA-MB-
231 tumors. Serial imaging was performed, and coronal images centered on the tumor for VCN, A192,
A192-VCN are depicted at 2, 4, 18 and 48 h post injection. A representative mouse is shown from each
group (n=3/group). Two major pools of blood are present in the heart (top) and liver (middle). At later
time points, the gastro-intestinal track (lower) and the bladder (bottom) enhance in contrast. The tumor
locations are indicated by arrows. b) Coronal images of MDA-MB-231 tumor bearing mice after
injection of 64Cu-A192-VCN with a blocking injection of unlabeled A192-VCN.
107

4.4.7 Quantitative analysis of PET images
Based on the promising activity exhibited by A192-VCN in vitro, its biodistribution and PK
parameters will be evaluated with PET. The polymeric carrier (A192) and therapeutic peptide
(VCN) is also evaluated. Two different chelating agents the traditional DOTA and a
sarcopagine-based chelator AmBaSar will also be assessed.

Blood clearance was assessed by monitoring signal form the heart over time. The time activity
curve of blood showed that A192-DOTA, A192-sar, A192-VCN-DOTA and A192-VCN-sar,
were still circulating in the blood stream after 4h, but all were almost entirely cleared from the
blood stream by 48h. Minimal heart signal was detected with the VCN-DOTA construct as
perhaps being the smallest construct investigated (1/10
th
of the other ELP construct) means that it
is cleared rather quickly from the circulation. The biological half-life for A192-DOTA, A192-
sar, A192-VCN-DOTA and A192-VCN-sar was determined to be 2.6h, 6.0h, 5.4h and 4.3h
respectively (Fig. 7-4).

108

Figure 7-4 Circulation half-life of A192-DOTA, A192-Sar, A192-VCN-DOTA and A192-VCN-
Sar. Heart %ID/g was fitted to a one-phase exponential decay curve. The half-life of activity in the
heart was indicated as the mean ± 95%CI.

Time activity curves corresponding to the muscle, kidneys, bladder, liver, intestine, tumor and
brain are shown in Figure 8-4a – g. From these results no significant difference was observed
between all the constructs in terms of muscle accumulation (Fig. 8-4a). Perhaps unsurprisingly
VCN, being the smallest construct, exhibited the highest kidney uptake. Renal clearance of
VCN occurs rapidly and continues to decline over the period of the study. Conversely the extent
of renal clearance of A192-sar, A192-VCN-DOTA and A192-VCN-sar were lower which
decreased over time. The A192-DOTA exhibited the lowest renal accumulation of all the
constructs (Fig. 8-4b). Subsequent transfer from the kidneys to the bladder resulted in a clear
signal in this tissue which indicated a reduction in the radioactivity concentration over time (Fig.
8-4c).
109

All
64
Cu-ELP construct exhibited some hepatic clearance in varying degrees (Fig. 8-4d).
Considering that A192 and A192-VCN have a hydrodynamic radius that is larger than the cutoff
point for renal filtration; these constructs are cleared primarily via the hepatobiliary pathway.
Liver accumulation for the fusion protein is more prominent compared to the others with the
%ID/g for these constructs decreasing over time. Similarly for A192-DOTA and A192-sar, at
varying levels both constructs also showed a pattern of reduction in signal over the period of the
study. In contrast a steady increase in liver signal is observed with the VCN-DOTA. The latter
also exhibited the lowest accumulation in the liver as a result of its extensive renal clearance.
For the constructs that ended up in the liver, after hepatobiliary processing they are excreted into
the biliary system and enter the gall bladder and intestines where minimal radioactivity is
observed (Fig. 8-4e).

231 tumor uptake profiles for each construct are shown in Figure 8-4f. Minimal tumor signal is
observed for VCN up until 4h p.i., with a slight increase in signal at 18h and 48h post injection.
A steady and sustained accumulation via the EPR effect was seen with A192-DOTA achieving a
maximum tumor accumulation of 1.4 %ID/g at 4h p.i. Conversely a much improved
accumulation profile is obtained with A192-sar construct with a nearly ~3-fold increase
observed. This increase could partly be due to the longer biological half-life for A192-sar when
compared to A192-DOTA. Extended circulation times would ensure increased availability of the
construct for sequestration in tumors. Furthermore the chelating efficiency of the respective
chelators could exert a profound effect on the resulting signal detected in tumors. For both AV-
DOTA and AV-sar a steady increase in accumulation over time is observed until 18h post
injection. ANOVA analysis revealed that there is statistically significantly difference between
110

the tumor uptake of VCN vs. AV-Sar at 2h and 4h p.i. (p<0.05 and p<0.001 respectively), A192-
DOTA vs. AV-Sar at 4h p.i (p<0.01), A192-Sar vs. AV-Sar at 4h p.i. (p<0.05) and AV-DOTA
vs. AV-Sar at 4h p.i. (p<0.01). This seems to indicate that adding a large hydrophilic polymer to
a small therapeutic peptide has a desirable effect on its increased sequestration in tumors.
Additionally using a high stability chelator yielded better distribution results.

All other tissues evaluated such as the brain showed almost negligible uptake (Fig.8-4g).

Tumor uptake was reduced by the prior injection of unlabeled, excess AV, indicating receptors
specific binding in the tumor tissues. In the blocking experiment, there is also an observable
reduction of tracer uptake in normal tissues (liver, kidneys, muscle) [Fig. 9-4]. This is in
agreement with published results, as it has been reported that αvβ3 integrin imaging probes
demonstrate low but blockable uptake in normal tissues. This may be because of the expression
of low levels of αvβ3 or related integrins in normal tissues.

111

Figure 8-4. Biodistribution of 64Cu-ELP in athymic nude mice implanted with MDA-MB-231 cells
(n=3) within a) muscle, b) kidneys, c) bladder, d) liver, e) intestine, f) tumor and g) brain expressed as
%ID/g calculated from ROI image analysis. Values indicate the mean ± 95%CI.
112

Figure 9-4. Blocking study. Biodistribution of MDA-MB-231 tumor bearing mice after injection of
64
Cu-A192-VCN with a blocking dose of A192-VCN. Values indicate the mean ± SD.

113

4.5 Discussion
Targeted delivery to tumor vasculature is considered a powerful strategy for cancer treatment
since angiogenesis is essential for tumor growth. α
v
β
3
integrins are minimally expressed on
normal quiescent endothelial cells, but significantly upregulated on proliferating endothelium.
Studies described that RGD-peptides have been introduced into proteins, polymers, liposomes,
micelles, viruses and gene delivery vehicles to improve diagnosis via imaging or to deliver
therapeutics to solid tumors.

Neovasculature-targeted disintegrins halt the growth of solid tumors by specifically targeting
integrins. An obstacle to their development, disintegrins are small peptides (7.1 kD) and are
readily cleared by the kidneys. Additionally these small peptides, as demonstrated by PET
imaging achieved only moderate target-to-background ratios, with a low percentage of injected
dose per gram tumor. This may be because rapid blood disappearance minimizes delivery of
radioligand to the target tissue, or because the radioligand is not retained well in target tissue
after binding. To overcome this problem, we have used genetic engineering to fuse a disintegrin
to a high molecular weight ELP-based ‘carrier’. This carrier has previously been demonstrated to
prevent renal elimination thereby prolonging its circulation in vivo. The slightly slower blood
clearance rate of this fusion protein was shown to increase bioavailability and the percent of dose
which is able to bind to tumors.

Any given class of macromolecules with characteristics size, shape and polarity will show tumor
uptake to a certain extent via the EPR effect. In molecular targeting, this lower limit has to be
exceeded to visualize specific binding of the radiotracer. Numerous nanoparticles based agents
114

such as liposomes
160
and polymeric micelles
11
, have been reported to accumulate in tumors.
However, the question of whether this uptake is specific for the intended target is not always
addressed. Thus Heneweer et al.,
161
proposed that the target tissue accumulation by EPR effect
should be determined as a minimum threshold value, with a non-targeted analog of the targeted
radiotracer in addition to competition studies to describe the uptake of a targeted macromolecular
as high or specific. Through biodistribution analysis of the constructs adding the targeting
domain to the ELP is able to significantly increase the uptake of drug into the tumor without
significantly altering the pharmacokinetics of the drug in the rest of the body. Based on these
findings, other methods to further increase the uptake in the tumor can be applied such as adding
multiple VCN binding domains or using a targeting agent that binds more strongly to tumor
cells.

4.6 Conclusion
Using both in vitro and in vivo evaluations, we demonstrated that the ELP nanocarrier grafted
with VCN was able to target the tumor endothelium and was shown to exhibit preferential
accumulation. Confocal microscopy and flow cytometric analysis showed that A192-VCN were
significantly associated to HUVEC and 435 cells more than A192 due to their ability to bind
specifically to the α
v
β
3
integrin.

115

Chapter 5
Conclusion and future directions
5.1 Conclusion
According to Nie et al
3
., the current problems and unmet needs in translational oncology are:
1. Advanced technologies for tumor imaging and early detection
2. New methods for accurate diagnosis and prognosis
3. Strategies to overcome the toxicity and adverse side effects of chemotherapy drugs
4. Basic discovery in cancer biology leading to new knowledge for treating aggressive and
lethal cancer phenotypes such as bone metastasis

Advances in these areas would go a long way in making the practice of personalized medicine in
oncology a reality. The ability to tailor an individual’s therapy as well as being able to predict
disease development, progression and clinical outcomes will certainly improve the management
of oncology patients and their long term prognosis. The concept of personalized medicine has
been espoused for several years now but it has gained momentum due to the recent developments
in the field of nanotechnology. Personalized medicine has been defined as giving the “right,
medicine at the right time at the right dose”
162
. Administration of such a treatment takes into
account the genomic, genetic, metabolic, environmental and other factors that influence response
to therapy
163
. Nanoscale constructs are particularly suited for tailoring patient specific therapy in
two different ways, namely through targeted delivery of therapeutics and molecular imaging
agents.
116

Conventional chemotherapy treatments are usually non-specific and highly toxic where only a
small percentage of administered drug reaches the intended target resulting in severe side effects.
To overcome this issue targeted therapeutics have been developed based on nanocarriers that
encapsulate drugs and are functionalized with affinity ligands such as antibodies, aptamers and
peptides. Furthermore by incorporating contrast agents, it allows real time monitoring of carrier
accumulation

Protein polymers have emerged as a new class of biomaterials that possess unique and often
superior properties when compared to conventional materials. Growth in this field is primarily
attributable to the great advancement in recombinant technologies. Protein engineering has
become a useful approach to engineer protein polymers as it provides advantages to the field of
biomaterials in terms of sequence control, composition and molecular orientation. So much so
that many peptide-based biomaterials form the basis of many biomedical and biotechnological
applications
113, 164, 165
.

Elastin-like polypeptides (ELPs) are biodegradable
166
and biocompatible
39
, which makes it
possible that they can be useful in a living system. Enhanced by genetic engineering techniques
ELPs can easily be designed/functionalized to give the desired structure, architecture and
function. It is this modularity that we have tried to exploit in this dissertation. Be it for drug
delivery and therapeutic application or for diagnostic imaging purposes, we have successfully
demonstrated ELPs versatility and adaptability to its many potential uses. The tunable property
of ELP makes it easy to tailor a peptide with the desirable characteristic for its eventual
application. We were also able to demonstrate that by modulating the length and amino acid
117

composition of the block copolymers, we can control the onset of their self-assembly and the size
of the nanoparticle structure formed. The latter is especially relevant for drug encapsulation.

Increasing numbers of therapeutic proteins are being developed for the treatment of cancer and
other diseases, as evidenced by the increasing numbers of pre-clinical and clinical studies of
protein therapeutics. The RGD-containing ligands have been in development for the past three
decades for the treatment of cancer. However, poor pharmacokinetics
60, 167
often limit the
efficacy of these ligands. The basis for this dissertation is that the PK properties of these
molecules can be improved by coupling them to a carrier system. Such a strategy would reduce
renal clearance, since the higher molecule size of the carrier prevents glomerular filtration which
may lead to prolonged blood circulation times and longer presentation to target receptors within
the tissue
168
. In addition the higher molecular weight of most carriers leads to passive
accumulation in some human and murine tumors via enhanced permeability and retention
effect
169
.

Exploiting the advantages of positron imaging, real-time biodistribution data in an intact living
system can be obtained and quantified. To this end we have used image-guided biodistribution
studies to aid in the determination of the ideal protein characteristics for the desired PK profile of
a particular ELP construct. It is evident by our findings that it is possible to tailor-make specific
ELP nanocarriers for various functions based on their pharmacokinetics. Polymeric carriers can
be used to carry drugs (therapeutic) and/or imaging agents (diagnostic). The requirement for
each differs and the nanocarriers used for both or either needs to be reconciled with their
eventual use. For use as diagnostic (or contrast) agents, they may be optimized to provide a
118

quick, high-fidelity snapshot of the living system. This requires rapid uptake, prolonged tumor
accumulation and fast clearance. Based on our results ELP carriers using A96 would probably
be more suitable for use as a diagnostic imaging agent. In contrast, therapeutic nanoparticle
formulations that need to be long-circulating for better efficacy would benefit from using the
larger construct like A192
170
.

This dissertation has attempted to merge the inherent advantages of PET imaging, new
biomaterials and potent therapeutic peptides to try and meet some of these unmet needs.
Molecular imaging for non-invasive assessment of angiogenesis is of potential interest for
clinicians as well as the pharmaceutical industry. Increased development of anti-angiogenic
agents spurred demand for imaging modalities that can determine optimum dosage and evaluate
treatment response. Since anti-angiogenic agents may stop tumor progression rather than cause
tumor shrinkage, measuring tumor size may not be applicable. Therefore there is great interest
in identifying and developing reliable biomarkers of early tumor response to non-cytotoxic
drugs. Ideally we would like to use A192-VCN as a biomarker for the process of angiogenesis.
And when it’s coupled to an imaging modality that is both sensitive and quantitative could
provide an early indicator of effectiveness at a functional and molecular level. However, as was
revealed by the PET imaging study, the signal to noise ratio of
64
Cu-A192-VCN is not as good as
that obtained for small, radiolabeled RGD ligands
171, 172
. Thus at this point we are only able to
demonstrate that A192-VCN is able to predict the efficiency of a carrier, via improved
circulation times and tumor targeting, that could in the future be armed with other therapeutics.
We have shown that A192-VCN is specific for αvβ3 (in vitro and in vivo), taken up extensively
by cells expressing αvβ3, preferentially accumulates in tumors in tumor bearing mice and
119

enhances the tumor targeting effect. Taken together, we have successfully developed a targeted
fusion protein strategy that is specific and selective, has improved pharmacokinetics and the
ability to monitor tumor-targeting efficacy.

5.2 Future directions
While the work presented in this dissertation has some promising results a few shortcomings
have been identified that needs to be addressed. Chief among them is the decrease in
affinity/potency of the ELP-VCN construct. As reported by Minea et al
153
. VCN alone has a
very high potency which has been reduced approximately 10 fold by appending it to a large
hydrophilic polymer. In addition dynamic light scattering data indicated that this fusion protein
pre-assembles into small nanostructures of ~45 nm even at low temperatures. This is surprising
as it was not expected that fusing a 7kDa protein to a 70kDa would have a profound effect on its
assembly properties. Nevertheless, this ‘nanoparticle’ like structure has been confirmed by TEM
imaging. It is thus hypothesized that this nanostructure is formed by the VCN forming the core
of the structure, with the ELP acting like a polymeric brush (Fig. 1-5). Considering that the
VCN itself is disulfide rich, it is speculated that it could associate among itself via intra-disulfide
bonds. This proposed nanostructure would perhaps explain the reduced binding and potency of
A192-VCN. As the figure indicates, if the active part of the construct is buried within the core of
the structure it is less accessible for making contacts with the integrin receptor. However, since
the ELP is somewhat flexible, this polymeric brush will intermittently exposes the core allowing
the VCN to interact with the integrin, hence exerting its effect.
120

Figure 1-5. Proposed formation of A192-VCN ‘nanostructure’.

In view of this, modifications to the original construct need to be made in order to improve its
activity. Some of the changes that could be made include:
1. Fusing the VCN to the C-terminus of the ELP construct instead of the N-terminus. It has
been reported by Christensen et al.,
173
that placing the ELP at the C-terminus of the target
protein not only increases the expression of the fusion protein but enhances its specific
activity. It was presumed that misfolded and less active conformers are preferentially
degraded when the ELP is at the N-terminus of the fusion protein.
2. Using a shorter ELP to reduce the thickness of the polymeric brush, increasing the
accessibility of the VCN core.
3. Enzyme-triggered release of VCN by incorporating a sequence that is recognized by enzymes
e.g. cathepsin B
174
, MMP-7
175
, ubiquitously expressed by tumors. Upon cleavage, the
therapeutic portion of the construct would be released allowing it to exert its action.
4. Employing polymer directed enzyme prodrug therapy (PDEPT)
176
. PDEPT is a 2-step anti-
tumor approach that uses polymeric prodrug and polymer enzyme conjugate to generate
cytotoxic drug at the tumor site. A polymeric prodrug containing a linker designed for
121

enzymatic linkage is first administered. The conjugate is usually composed of a polymer that
has a molecular weight of ~30kDa, and once the circulating polymer-drug conjugate has been
cleared, a polymer enzyme conjugate can then be administered as a second step. This second
conjugate usually has a higher MW (50-100kDa) and thus circulates longer. Both conjugates
would accumulate at the tumor site either passively, by the EPR effect or actively via specific
interaction of the VCN with the integrin receptors. When they both meet, activation of the
prodrug, released through enzymatic cleavage, leads to localized drug release at the tumor
site
177
.

Many investigators have reported enhanced tumor uptake when nanocarriers are endowed with a
targeting peptide
178-181
. The data presented in this thesis has also demonstrated a similar benefit
of such an approach. An increase in the flux into tumors by A192-VCN compared to A192 taken
up presumably by the EPR effect was observed. Further imaging studies could perhaps be
performed to determine the robustness of this targeting strategy. To further develop the concept
of personalized therapy a two tier study could perhaps be proposed whereby the first step would
involve determining whether
64
Cu-A192-VCN can differentiate between integrin expressing
versus non-expressing integrin cell lines. A blinded imaging study with the associated PK
analysis can be performed to assess the tumor uptake in these two types of cells to see whether
the construct would be able to distinguish between the two. This is especially relevant clinically
because molecular stratification of tumor lesions can be used to guide therapeutic decisions.
After selection of ‘positive responders’, the second part of the study would involve
administration of a therapeutic dose of anti-angiogenic agent.
122

Diagnostic tools that predict a patient’s response to chemotherapy could reduce the use of non-
effective drugs and enable physicians to explore additional therapeutic options rapidly. Thus to
expand on the previous study, a non-invasive prediction of tumor response could be performed.
Here either avastin or cilegitide is administered and the treatment progression followed with the
administration of
64
Cu-A192-VCN.

Being amenable to site specific conjugation by chelating agents not only allows the ability to
image targeted cells, but it would also afford the opportunity to carry a radiotherapeutic isotope.
Radiation therapy utilizes radiation energy to induce cell death. Systemic radiotherapy delivers
radiation energy from the radioisotopes that are conjugated to a suitable delivery carrier, such as
antibodies, liposome emulsions or nanoparticles with tumor targeting ligands, and transported to
the tumor site
182
. Radioisotopes commonly used in radiation therapy are
111
In,
90
Y and
177
Lu.

The assembly of A192-VCN into multivalent nanoparticles was entirely unexpected but its
formation could be exploited to improve and further increase the versatility of the construct. For
instance by appending a second fusion protein that targets other biomarkers of tumor
angiogenesis to the corona of the nanoparticle could result in the multiple targeting of the
angiogenic process. Apart from integrins, endoglins and VEGF-2 are also among the best
characterized molecular markers of tumor angiogenesis. By appending a targeting moiety
against these markers it could lead to an enhanced therapeutic effect.

Eventually as has been reported by the Markland group
153
, it would perhaps be of interest to
assess the in vivo anti-angiogenic activity of A192-VCN on integrin expressing cell lines,
123

especially if the issue of decreased potency is overcome. However, this is not to say that the
original construct will not exert a therapeutic effect. Any anti-tumor effect would perhaps be
achieved due to prolonged circulation (longer exposure of the peptide to the tumor) than to high
affinity/association with the integrin. This construct is particularly unique in that it serves both
as a targeting as well as a therapeutic agent thanks to the specificity of the VCN towards
integrins as well as its intrinsic anti-integrin properties that inhibits the process of angiogenesis.
Furthermore being able to label the construct with an imaging agent allows A192-VCN to
become a true theranostic agent that can potentially detect, image and treat tumors.

124

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Asset Metadata

Creator Janib, Siti Najila Mohd (author)

Core Title Development of positron emission tomography (PET) labeled polypeptide nanoparticles for tumor imaging and targeting

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmacy / Pharmaceutical Sciences

Publication Date 04/26/2013

Defense Date 01/29/2013

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

Tag block copolymer,elastin like polypeptides (ELP),micelles,nanoparticle,OAI-PMH Harvest,positron emission tomography (PET),thermal self-assembly,vicrostatin (VCN)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mackay, John Andrew (committee chair), Conti, Peter (committee member), Markland, Francis S., Jr. (committee member), Wolf, Walter (committee member)

Creator Email ceetee41@yahoo.com,mohdjani@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-244201

Unique identifier UC11288418

Identifier etd-JanibSitiN-1608.pdf (filename),usctheses-c3-244201 (legacy record id)

Legacy Identifier etd-JanibSitiN-1608.pdf

Dmrecord 244201

Document Type Dissertation

Rights Janib, Siti Najila Mohd

Type texts

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

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

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA