University of Southern California Dissertations and Theses

Cognate receptor fusion proteins enable parenteral delivery of challenging rapalogues

(USC Thesis Other)

Cognate receptor fusion proteins enable parenteral delivery of challenging rapalogues

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

COGNATE RECEPTOR FUSION PROTEINS ENABLE
PARENTERAL DELIVERY OF CHALLENGING RAPALOGUES

By

Santosh Peddi

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERISTY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirement of the degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)

August 2020

ii

Dedicated to Vijay Kumar, Sri kala and Swathi Peddi for
their infinite love, care, freedom, support, and encouragement

iii
ACKNOWLEDGEMENTS

This Thesis would not have been possible without the excellent mentorship of my PI Dr. Andrew
MacKay. His style of teaching was instrumental in developing skills necessary to be a well-
rounded scientist. His guidance over the past few years instilled patience, motivation, perseverance
and the scientific freedom he allowed in the lab made my Ph.D. journey truly memorable. I would
also like to thank my committee members, Dr. Yong Zhang and Dr. Bangyan Stiles for their
valuable suggestions and support.

Many thanks to all the members of the Mackay lab: both past and present, especially Dr. Jugal
Dhandhukia for being my first mentor. Thank You Dr. Zhe Li for tagging along to every seminar
and always making time to discuss experiments. Many parts of this Thesis would not have been
possible without the selfless help offered by colleagues and friends Hao Guo, Yaping Ju and
Changrim Lee. I would also like to thank Xiaoli Pan for all her hard work in the lab which proved
essential for my projects.

Lastly, crossing the finish line would have been impossible without the constant belief and support
from my parents: Vijay Kumar and Srikala Peddi, and my friends, especially Pooja, Anupam and
Akhilesh.

iv
TABLE OF CONTENTS

Dedications ii
Acknowledgements iii
List of Tables ix
List of Figures x
Abstract xii

Chapter 1 Introduction 1
1.1 Current status of cancer treatment 1
1.2 Drug delivery strategies for chemotherapeutics 2
1.3 mTOR signaling in cancer 3
1.4 Rapalogues in breast cancer 4
1.5 Rapalogues in preventing transplant rejection 6
1.6 Cognate receptor fusion proteins for rapalogue delivery 7
1.7 Other applications of ELPs in drug delivery 11

Chapter 2 First generation FKBP-ELP carriers for delivery of Rapamycin
2.1 Abstract 15
2.2 Introduction 16
2.3 Materials and Methods 20
2.3.1 Recombinant protein purification and CMT measurements 20
2.3.2 Rhodamine labeling 21
2..3.3 Dynamic Light Scattering 22
v
2.3.4 Rapa encapsulation, concentration measurements and endotoxin removal 22
2.3.5 Measurement of residual endotoxins 23
2.3.6 Cell culture 24
2.3.7 Cellular uptake and co-localization analysis 24
2.3.8 Reactive Oxygen Species (ROS) assay 25
2.3.9 In vitro cytotoxicity assay 25
2.3.10 In vitro plasma coagulation time assay 26
2.3.11 BT-474 Xenograft model and drug treatment 26
2.3.12 Complement activation assay by Enzyme Immunoassay (EIA) 27
2.4 Results 28
2.4.1 Physicochemical characterization of ISR/FSI-Rapa nanoparticles 28
2.4.2 Estimation of HED, cellular uptake in HepG2 cell line 31
2.4.3 ROS generation assay 34
2.4.4 Hemolysis and complement activation assays 35
2.4.5 Plasma coagulation time and platelet aggregation assays 36
2.4.6 Efficacy in BT-474 xenograft mouse model 38
2.4.7 Changes in liver protein expression and serum biomarkers 41
2.5 Discussion 44
2.6 Conclusion 49

Chapter 3 Second generation FKBP-ELPs for the delivery of Rapa and Eve
3.1 Abstract 50
3.2 Introduction 52
vi
3.3 Materials and Methods 55
3.3.1 Recombinant protein purification in E.coli, drug encapsulation 55
and endotoxin testing
3.3.2 Dynamic Light Scattering 57
3.3.3 Rhodamine labeling 57
3.3.4 Cold competition binding assay and live cell imaging 58
3.3.5 Concentration-dependence of cellular association 58
3.3.6 Cellular uptake and co-localization analysis 59
3.3.7 Kinetics of cellular uptake and degradation 59
3.3.8 Split luciferase assay 60
3.3.9 Cell culture and in vitro cytotoxicity assay 61
3.3.10 Mouse xenograft model and drug treatment 61
3.3.11 Isothermal Titration Calorimetry (ITC) 62
3.3.12 Western blotting 62
3.3.13 Drug quantification by LC-MS/MS 63
3.4 Results 64
3.4.1 Physicochemical characterization of FAF-Rapa and FAF-Eve 64
3.4.2 Rhodamine labeling and Rapa encapsulation 69
3.4.3 Cellular uptake of Rho-FAF is receptor independent 70
3.4.4 Cellular uptake by macropinocytosis and translocation to low 72
pH compartments
3.4.5 Time-dependent cellular uptake and cellular degradation 75
3.4.6 Split luciferase assay 77
vii
3.4.7 In vitro cytotoxicity and BT-474 xenograft study 79
3.4.8 Assessing mTORC1 inhibition in off-target organs 84
3.5 Discussion 86
3.6 Conclusions 96

Chapter 4 Third generation FKBP-ELPs for the delivery of Rapamycin
for immunosuppression
4.1 Introduction 98
4.2 Materials and Methods 101
4.2.1 Recombinant protein purification, drug encapsulation and 101
endotoxin removal
4.2.2 Dynamic Light Scattering 102
4.2.3 Cell culture and in vitro cytotoxicity assay 103
4.2.4 Isothermal Titration Calorimetry (ITC) 103
4.2.5 Protease-coupled prolyl isomerase assay 104
4.2.6 Zirconium-89 labeling and in vivo microPET imaging 105
4.2.7 Rhodamine labeling and PK assessment in Sprague Dawley (SD) rats 106
4.2.8 Compartmental and non-compartmental PK analysis 108
4.3 Results 110
4.3.1 Physicochemical characterization of 5FA and 5FV 110
4.3.2 Mechanisms of inhibition and in vitro cytotoxicity 114
4.3.3 Zr-89 labeling, in vivo PET imaging, and mouse PK characterization 116
4.4 Conclusions 122
viii
4.5 Cognate receptor fusion technology: Summary and future 123

References 126

ix
List of Tables

Table 2.1: Physiochemical characterization of ELP di-block copolymers 29
evaluated in this chapter

Table 2.2: Toxicity assessment of ISR/FSI-Rapa via serum chemistry profiles 43

Table 3.1. Physiochemical characterization of ELP polymers evaluated in this 67
chapter

Table 4.1. Physiochemical characterization of ELP polymers evaluated in this 113

x
List of Figures
Figure 2.1: Physicochemical characterization of ISR/FSI-Rapa nanoparticles 31
Figure 2.2: Internalized ISR/FSI-Rapa nanoparticles traffic to acidic organelles 33
and induce oxidative stress in HepG2 cells

Figure 2.3: Neither ISR/FSI-Rapa nor control nanoparticles trigger measurable 36
hemolysis or complement activation

Figure 2.4: Plasma coagulation and platelet function are unaffected by the 38
presence of ISR/FSI-Rapa and control nanoparticles

Figure 2.5: ISR/FSI-Rapa nanoparticles potently inhibit the growth of BT-474 cell line 40
Figure 2.6: ISR/FSI-Rapa nanoparticles potently suppress tumor growth in a BT-474 41
xenograft mouse model

Figure 2.7: A month long exposure to ISR/FSI-Rapa nanoparticles does not induce 43
hepatic stress responses in treated mice

Figure 2.8: Presence of serum albumin does not impact the hydrodynamic radius (Rh) 45
and stability of ISR/FSI-Rapa nanoparticles

Figure 3.1: Physicochemical characterization of FAF-Rapa and FAF-Eve 66
Figure 3.2: FAF binds both Rapa and Eve at a stoichiometric ratio as determined by 68
isothermal titration calorimetry

Figure 3.3: Cellular uptake of Rho-FAF is receptor independent 70
Figure 3.4: Cellular association of Rho-FAF is non-saturable 71
Figure 3.5: Rho-FAF and FL-dextran display equivalent distribution after 73
cellular uptake

Figure 3.6: Both Rho-FAF and Rho-dextran localize to low pH compartments upon 74
cellular uptake

Figure 3.7: The kinetics of cellular association of Rho-FAF is minimally affected by 76
addition of Rapa

Figure 3.8: Loss of cellular Rho-FAF is minimally affected by addition of Rapa 77
Figure 3.9: FAF delays the access of Rapa to the cytosol in a manner consistent with 79
macropinocytosis
xi

Figure 3.10: Rapalogues bound to FAF retain cytostatic activity against BT-474 cells 80
Figure 3.11: FAF-Rapa significantly arrests tumor growth compared to oral Eve 83
Figure 3.12: FAF carrying rapalogues do not affect S6RP phosphorylation in liver and 85
spleen

Figure 3.13: Additional characterization of cell uptake mechanism of FAF-Rapa 91
Figure 3.14: Anti-mTOR immunoprecipitation of cell lysates treated with Rapa and 93
FAF-Rapa fail to detect FKBP12-Rapa-mTOR interaction.

Figure 3.15: Co-localization of FAF-Rapa and mTOR using immunofluorescence 95
Figure 4.1: Plasmid map depicting pET-25b(+) vector encoding high capacity 111
ELP fusions, which each contain 5 FKBP domains that are linked by elastin-like
polypeptide

Figure 4.2: Physicochemical characterization of 5FA and 5FV 112
Figure 4.3: 5FA binds to Rapa at an expected stoichiometric ratio as determined by 114
isothermal titration calorimetry

Figure 4.4: 5FA-Rapa displays equipotent mTOR inhibition as free Rapa 115
Figure 4.5: Molecular imaging reveals longer retention of 5FV at the injection site 118
Figure 4.6: Non-compartmental PK analysis of IV Zr89-5FA, SC Zr89-5FA and 120
SC Zr89-5FV

Figure 4.7: Compartmental PK modeling of IV Zr89-5FA, SC Zr89-5FA and SC 120
Zr89-5FV

Figure 4.8: Compartmental PK analysis of IV Zr89-5FA, SC Zr89-5FA and SC 121
Zr89-5FV

xii
Abstract

Rapalogues are a unique class of drugs with both cytostatic and immunosuppressive properties.
Two founding members, Rapamycin (Rapa) and its chemical derivative Everolimus (Eve) are
extremely potent, but their clinical use presents multiple challenges. Being water insoluble,
administration is restricted to the oral route, which results in a low bioavailability of <10%. Human
studies of rapalogues are reported to yield a high blood to plasma ratio and poor correlation
between blood concentration and dose. Moreover, treatment results in dose-limiting toxicities like
stomatitis and pneumonitis, which often leads to discontinuation of therapy. In this dissertation, I
present three generations of fusion-proteins as drug carriers for rapalogues. These proteins are
fabricated using elastin-like polypeptide (ELP) technology and aim to address the current clinical
limitations associated with rapalogue treatment.
1
Chapter 1: Introduction

1.1 Current status of cancer treatment

The failure of a significant number of tumors to respond to drug and/or radiotherapy is a serious
problem in the treatment of cancer. Currently, cancer is treated with one or a combination of
surgery, radiotherapy and chemotherapy. Surgery is an effective treatment strategy only when the
tumor site is accessible to surgeons, for example, in the breast, skin etc. It is generally effective in
only treating early stages of cancers and is not an option in case of blood based cancers and
metastasized tumors. At the time of diagnosis, more than 50% of patients are generally not good
candidates for surgery, and moreover the procedure can promote metastasis due to blood
circulation during surgery. Radiation therapy too is useful only when the tumor tissue is
anatomically localized and is not an option for late stages of cancer with metastasis. Despite recent
advances in radiotherapy, it is nearly impossible to concentrate high dose radiation completely to
abnormal tissue. Efforts to do so often destroy nearby healthy tissues and unwanted side effects.
Chemotherapy involves administration of one or more chemotherapeutic agents to disrupt cell
proliferation or metabolism. Although usually effective in all stages of cancer, chemotherapy
suffers from poor patient compliance due to highly toxic side effects including vomiting, loss of
blood cells, loss of weight, hair etc. Chemotherapy induced side effects are a result of poor target
specificity of the drugs as they circulate through most of the healthy normal tissues as well as
cancerous tissues. These side effects impact the quality of patient’s life and can be severe enough
to require hospitalization and in some cases cause death. Due to limitations of currently available
cancer treatments and the severity, heterogeneity and spread of cancer, it is essential to develop
alternative therapies to stabilize, treat, prevent or delay cancer. Novel therapies are required to
address these existing shortcomings of chemo, radiation and surgical therapy.
2
1.2 Drug delivery strategies for chemotherapeutics
The majority of chemotherapeutics are small organic molecules. They routinely suffer from poor
physico-chemico properties including water solubility, poor bioavailability and sub-optimal
pharmacokinetics (PK). To facilitate delivery of these extremely hydrophobic drugs, organic and
lipid based solvents are routinely employed; however, these solvents are also associated with
serious and dose-limiting toxicities that compound side-effects inherent to the drug. For example,
polyoxyethylated castor oil, a major component of the Taxol formulation (Paclitaxel) is known to
be pharmacologically active, leaches plasticizers from standard i.v tubing, and its infusion
produces histamine release in 20-40% of patients with well-documented hypersensitivity
reactions
1
, including anaphylaxis. It has also been associated with hyperlipidemia, abnormal
lipoprotein patterns, aggregation of erythrocytes, and prolonged, sometimes irreversible sensory
neuropathy
2-4
. Hypersensitivity reactions and neuropathies can also occur with Polysorbate 80
5
, a
solubilizing agent in Taxotere (docetaxel), Torisel (Temsirolimus) and many other clinical
formulations. Nanoparticles in the size range of 1-100 nm can potentially solubilize hydrophobic
drugs and address some issues presented by traditional chemotherapy and organic solubilizers. The
most studied materials for nanoparticle fabrication and synthesis are metals like gold and silver,
lipids, silicon and silica, carbon, and various synthetic polymers and proteins. Nanoparticle
technology is advantageous because it can: i) solubilize hydrophobic drugs; ii) specifically
concentrate the drug in tumor sites by enhanced permeation and retention (EPR) effect
6, 7
, thereby
reduce toxic drug effects; iii) improve drug mean plasma residence time by preventing renal
filtration and clearance pathways; and iv) are amenable to “active targeting” to further improve
nanoparticle specificity
8
by grafting tumor antigens on their surface.

3
1.3 mTOR signaling in cancer
The conserved serine/threonine kinase mTOR (the mechanistic target of rapamycin), a
downstream effector of the PI3K/AKT pathway, forms two distinct multiprotein complexes:
mTORC1
9, 10
and mTORC2. mTORC1 activates S6K1 and 4EBP1, which are involved in mRNA
translation. It is activated by diverse stimuli, such as growth factors, nutrients, energy and stress
signals, and essential signaling pathways, such as PI3K, MAPK and AMPK, in order to control
cell growth, proliferation and survival
11-13
. mTORC2 is considered resistant to rapalogues and is
generally insensitive to nutrients and energy signals. It activates PKC-α and AKT and regulates
the actin cytoskeleton. Deregulation of multiple elements of the mTOR pathway
(PI3K amplification/mutation, PTEN loss of function, AKT overexpression, and S6K1, 4EBP1
and eIF4E overexpression) have been reported in many types of cancers, including melanoma,
breast cancer, and renal cell carcinoma. Unfortunately, the clinical outcomes with mTOR
inhibitors are only modest, primarily because it is not cytotoxic, partially inhibits mTOR activities,
and is overcome by several feedback loops involved in cell survival. For example, under normal
circumstances, mTORC1-mediated activation of S6K1 promotes degradation of insulin receptor
substrate (IRS) and subsequent downregulation of PI3K signaling. Inhibition of mTORC1 by
rapamycin relieves this negative feedback loop, thereby increasing PI3K signaling and mTORC2
mediated AKT activation
14-17
. Nonetheless, mTOR inhibitors in combination with a wide variety
of chemotherapeutics remain an actively pursed area of research with numerous clinical trials
underway.

4
1.4 Rapalogues in breast cancer
The incidence of breast cancer remains significant with nearly a quarter of a million new cases of
invasive breast cancer diagnosed annually
18
. While the survival rates of early stage breast cancer
are relatively high, advanced disease results in nearly 40,000 deaths per year. As such breast cancer
remains the second most deadly cancer affecting American women. When a woman is diagnosed
with metastatic breast cancer, her prognoses ranges in median survival time between 18 and 24
months
19
. Until a decade ago, cytotoxic chemotherapy was the primary treatment option
20
.
Chemotherapeutics present a balance between dose-limiting side effects and the opportunity to act
on a target to halt or reverse tumor growth. It has become clear that validated drugs, such as
paclitaxel and doxorubicin, benefit from well-designed drug carriers that improve the balance
between side effects and efficacy
21-23
. One of the most successful demonstrations is paclitaxel pre-
loaded onto albumin, called Abraxane
TM
. Abraxane
TM
was approved for breast cancer in 2005 after
a randomized clinical trial demonstrated that it doubled the objective response rate and halved the
incidence of severe (grades 3-4) neutropenia among patients who failed chemotherapy
(www.cancer.gov). Beyond Abraxane
TM
, next generation drug carriers offer the opportunity to: i)
sequester drug, thereby reducing side effects; ii) utilize target-mediated delivery and release; iii)
direct multiple modalities to tumors (drugs, proteins, imaging agents); and iv) facilitate
combination drug trials by mitigating dose-limiting toxicity for one agent. There is a strong clinical
precedent for use of drug carriers to facilitate combination therapies, most notably the use of
docetaxel and trastuzumab in HER2+ breast cancer was limited by the side-effects of the free
taxane. When albumin-bound paclitaxel replaced docetaxel, the side effects were reduced and this
enabled a more potent combination therapy
24, 25
. When fully realized, these drug carrier strategies
have the potential to revolutionize small-molecule chemotherapy.
5

Since Abraxane
TM
, fundamental improvements in the treatment of breast cancer have been
achieved through the classification of patients into molecular subtypes. A significant proportion
(20-30%) of breast cancer is positive for the HER2 receptor, for which biologics (trastuzumab,
approved 2006) and receptor tyrosine kinase inhibitors (lapatinib, approved 2007) were
developed
26
. Alternatively, more than half of breast cancers are hormone receptor positive (HR+)
for the estrogen and/or progesterone receptors
27
, which respond to aromatase therapy (exemestane,
approved 2005). In 2012 the FDA approved the rapalogue known as everolimus to treat
HR+/HER2- breast cancer in combination with exemestane after it tripled progression free
survival
28
. Despite this clinical success, everolimus is recognized as having two important
limitations: i) inhibition of MTOR complex 1 (with raptor) produces a negative feedback that
selects for activation of AKT, allowing the mTOR complex 2 (with rictor) to facilitate rapalogue
resistance; and ii) its toxicity profile prevents patients from remaining on therapy
29
. To overcome
these limitations, the goal of this project is to design a rapalogue formulation to replace free
everolimus in combination with exemestane, which are indicated in HR+/HER2- breast cancer
patients.

6
1.5 Rapalogues in preventing transplant rejection
Every year about 32,000 transplantations are performed in the United States, with the majority of
them (~18,000) being kidneys[10]. Of these, 97% of kidney transplants are working at the end of
a month; 93% are working at the end of a year; 83% are working at the end of 3 years and only a
mere 50% are functional at 10 years post-transplant [10]. While 12 people die every day awaiting
a kidney transplant, 20% of kidney transplants every year are re-transplants, with choric
inflammation being the main cause for rejection. Along with multiple other factors, two reasons
underlying rejection include side effects of medications and patient non-compliance to
immunosuppressive prescriptions
30
. Currently, >90% of transplant patients receive calcineurin
inhibitor tacrolimus[10] and the anti-metabolite mycophenolate. The usage of mTOR inhibitors
and steroids is limited to <5 % and <70% of immunosuppressive regimens. Unfortunately, a major
side-effect of tacrolimus is kidney damage, along with high blood pressure, high blood potassium
and drug-induced diabetes. Non-adherence to prescriptions is reported in 23 cases per 100
recipients per year (PPY) and 36 PPY kidney recipients per year
31
, even when the lifelong need to
take immunosuppressants is routinely emphasized in clinical follow-up.

7
1.6 Cognate receptor fusion proteins for delivery of rapalogues

Elastin like polypeptides (ELPs) comprise a near-ideal protein-based material for drug delivery.
Derived from human tropoelastin
25
, ELPs are polypeptides comprised of a repeated pentameric
amino acid sequence [Val-Pro-Gly-Xaa-Gly]n, where Xaa can be any amino acid, and n is the
number of times the pentamer is repeated
26
. As a function of X aa and n, ELPs display inverse phase
transition temperatures (Tt) above which they phase separate to form a coacervate from an aqueous
solution
27-28
. ELPs are genetically encodable and are suitable for heterologous production in
various expression hosts. This unique feature results in homogenous, monodisperse products, a
feature not exhibited by synthetic polymers as their synthesis often results in heterogeneous,
polydisperse material. Moreover, ELPs are biocompatible and biodegradable
29
, a questionable
feature in case of metallic and polymeric nanoparticles as their biological accumulation,
immunogenicity and associated long term toxicity require continuous investigation
32
.

Similar to other drugs, rapalogues accumulate in normal tissue, which results in an often-untenable
toxicity profile. One way to rectify this is through drug carriers. By harnessing the emerging field
of protein polymer carrier technology
33-38
, I have synthesized high molecular weight ELPs that
combine the precision of recombinant protein expression with properties of polymers. When
heated above sequence-determined transition temperature (Tt), ELPs phase separate to form a
secondary liquid phase called a coacervate. Phase separation can even assemble nanoparticles
39, 40
.
An example of this phenomenon is the FKBP-ELP fusion called FSI, which includes a hydrophilic
ELP (Val-Pro-Gly-Ser-Gly)48 connected to a hydrophobic ELP (Val-Pro-Gly-Ile-Gly)48. At body
temperatures, the FSI di-block assembles ~22 nm radius nanoparticles decorated with FKBP
35, 36
.
8
In a mouse model of triple negative breast cancer (ER-, PR-, Her2-), intravenous FSI/Rapa
nanoparticles potently arrested tumor growth at 0.2 mg/kg BW compared to free Rapa. By
imparting an ability to specifically recognize integrin receptors overexpressed on tumor and
endothelial cells in the tumor, the effective dose was further reduced to 0.07 mg/kg BW.
Additionally, the MacKay group evaluated multiple FKBP-ELP architectures in the same model
using a more patient friendly subcutaneous route of delivery
41
and identified a carrier called FAF
(FKBP-[VPGAG]192-FKBP). FAF/Rapa suppressed tumor growth at 0.75 mg/kg BW dose while
preventing skin necrosis and injury at the injection site associated with free Rapa treatment. I now
propose to expand the existing technology by increasing the drug carrying capacity of our carriers
(5% by mass compared to 2% with FAF) and their potential for long-term self-administration of
rapalogues.

The success of liposome and albumin based drug carriers suggests that better carriers may improve
existing therapeutics; however, novel strategies are required to address clinical limitations of
potent drugs like the rapalogues. Technology developed in this dissertation innovates in four ways:
Innovation 1) FKBP-ELP nanocarriers are produced entirely through biological synthesis,
which is expected to provide a straightforward route to scaleup. Potential advantages of biological
synthesis include: i) control over the peptide-polymer primary structure, which is determined at
high fidelity by the gene sequence; ii) homogeneity-- protein translation delivers a monodisperse
product at a targeted molecular weight, which depends on the length of the ELP gene; iii)
immunogenicity-- humanized sources were selected for the ELP and FKBP; iv) nanoassembly of
particles with controlled hydrodynamic radius and density for drug-binding (FKBP). Control over
nanoassembly has been challenging to scale using other bioconjugation/nanoparticle synthetic
9
strategies. In contrast, the scaleup for biological therapeutics has been demonstrated for dozens of
drugs (insulin, growth hormone, monoclonal antibodies, etc.). Thus, biological synthesis offers
potential opportunities to take complex formulations through scale-up to clinical trials.
Innovation 2) Drug encapsulation is based on a non-covalent -- yet highly specific -- interaction
between the FKBP domain and the drug. Prior to work by Dr. MacKay’s research group
35, 36
,
recombinant FKBPs have never been explored for the delivery of rapalogues. The closest
analogous technology in the clinic, albumin offers a low affinity and low specificity interaction
with many drugs. Low affinity interactions allow rapid exchange to other hydrophobic lipid or
albumin species in the body. This may mask any beneficial sequestration offered by the drug carrier.
Alternatively, agents such as polymer or antibody drug conjugates rely on the formation and
reversible cleavage of a covalent bond. While covalent linkages are of high affinity and specificity,
optimization of release from covalent linkages remains challenging. Thus, the drug encapsulation
achieved by the FKBP-ELP/Rapalogue system is innovative in both its high specificity and non-
covalent nature.
Innovation 3) Biodegradation of the carrier and intracellular accessibility are made possible
because FKBP-ELPs are composed entirely of natural amino acids. Biodegradation of protein-
based polymers enables the direct exploration of large polymers and nanoparticles, because they
have a route of clearance from the body. In addition, proteolytic biodegradation of the FKBP-ELP
carriers is important because it provides a potential mechanism for intracellular drug release. Our
previous work shows that protein polymer nanoparticles traffic to low pH intracellular
compartments that are enriched in lysosomal proteases
42
. Using internalization as a trigger, this
provides an opportunity to bring the drug into close proximity with mTOR, which strongly
associates with lysosomes
43
, prior to release within the cell. This strategy also has the potential to
10
overcome multidrug resistance mutations
44
. Thus, the peptide nature of these nanoparticles is
important for their activity, because it provides an efficient means of triggered biodegradation.
Innovation 4) FKBP-ELPs do not employ surfactants for solubilization. Currently used drug
solubilizers can be associated with serious toxicities adding to the side-effects inherent to the drug.
For example, Polysorbate 80
5
, a solubilizing agent in intravenous temsirolimus (Torisel
â
) and
many other clinical formulations causes hypersensitivity reactions and neuropathies. Also,
polyoxyethylated castor oil, a major component of Taxol formulation (Paclitaxel) is known to be
pharmacologically active, leaches plasticizers from standard i.v tubing, and its infusion produces
histamine release in 20-40% of the patients with consequent well documented hypersensitivity
reactions, including anaphylaxis
1, 3, 4
. The unconventional use of humanized proteins as delivery
and solubilizing agents eliminates surfactant-related complications.

11
1.7 Other applications of ELPs in drug delivery
The versatility and above mentioned advantages of the ELP platform resulted in their application
in diverse fields of medicine with oncology, cardiovascular and diabetes being the most prominent.
Some of these applications are discussed below.
Dr. Chilkoti and colleagues were one of the first to report oncology applications for ELPs. They
demonstrated that local hyperthermia can increase localization of ELPs in the tumor environment
by inducing their phase separation. By conjugating small molecule chemotherapeutics to ELPs
with an optimized transition temperature (slightly above 37
o
C), such technology can be used to
concentrate drugs in target tissues, thereby improving efficacy and reducing off-target toxicities.
For example, a recombinantly synthesized ELP with a Tt of 41
o
C was found to localize ~2-fold
higher in the tumor when mice bearing human derived SKOV-3 and D-54MG xenografts were
exposed to local hyperthermia for 1-hour
45
. Using rhodamine labeled ELPs and fluorescence
video-microscopy, they demonstrated ELP coacervation in the tumor in response to hyperthermia,
but no aggregation was observed in control tumors which were not heated. Hence, local
coacervation was responsible for increased tumor localization. Another approach conjugated ELPs
to hydrophobic drugs via cleavable linkers. These ELP-drug conjugates assembled nanoparticles
with the drug at the core and ELPs on the corona
46, 47
. In one example, a highly water-soluble ELP
was fused to a short peptide sequence containing eight cysteine residues. The -SH side groups in
cysteines served as sites to conjugate paclitaxel by cysteine-maleimide chemistry. Using chemistry,
activated paclitaxel was fused to an acid-labile hydrazone linker that terminated with a maleimide
moiety. The reaction efficiency for ELP-paclitaxel conjugation was modest with each ELP
harboring ~2 molecules of paclitaxel. ELP-paclitaxel conjugates spontaneously assembled ~60 nm
micelles. Following endosomal uptake in tumor cells, the pH responsive hydrazone linker is
12
cleaved in acidic lysosomes, allowing free drug to reach its molecular target. ELP-paclitaxel
nanoparticles increased the systemic exposure of paclitaxel by seven-fold compared with free drug
and two-fold compared to Abraxane. The tumor uptake of these nanoparticles was two-fold greater
than Abraxane and their in vivo efficacy outperformed both free drug and Abraxane in mouse
xenograft models of triple-negative breast and prostate cancer.
To facilitate active targeting of ELP based therapeutics to tumors, the Mackay lab explored RGD
containing peptides/proteins that have affinity for integrin αV heterodimers. These integrins signal
angiogenesis, which in the tumor represents unregulated blood vessel growth to access nutrients
in the bloodstream. Tumor neovasculature overexpresses integrin αV heterodimers and can thus be
targeted by RGD-containing peptide
48, 49
. In one example, the disintegrin protein vicrostatin was
fused to an ELP
50
. These fusion proteins self-assembled into multimers with a hydrodynamic
radius of 15.9 nm and specifically interacted with cell-surface integrins on human umbilical vein
endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435.
Furthermore, the vicrostatin-ELP fusion proteins were internalized rapidly by cells. When
characterized using small animal positron emission tomography (microPET), the tumor
accumulation of vicrostatin-ELP was saturable which is consistent with integrin-mediated binding.
The high molecular weight of ELP tag improved the issue of rapid clearance of free vicrostatin
peptide via renal filtration. Such approaches constitute a new source of biomaterials with potential
diagnostic and therapeutic applications.
The ability of ELP fusion tag to improve the pharmacokinetics of small active peptides was also
harnessed to develop novel therapies for cardiovascular diseases, specifically hypertensive heart
disease. PhaseBio Inc. has developed an ELP-based treatment known as Vasomera™ for essential
hypertension. Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide that binds to
13
receptors VPAC1 and VPAC2
51
and promotes heart contractility and induces coronary
vasodilation, thereby relieving hypertension. Despite favorable pharmacology, VIP’s therapeutic
potential is limited by an extremely short half-life (<2 min). By fusing a high molecular weight
ELP to a VPAC2 specific VIP, scientists at PhaseBio Inc. were able to engineer a therapeutically
viable VPAC2 agonist
52
with an ~60 hr half-life in humans. Specificity for VAPC2 ensures
modulation of activity in lung and cardiac tissues while minimizing gastrointestinal abnormalities
associated with VPAC1 activation. When tested in a pulmonary arterial hypertension (PAH) rat
model, VIP-ELP (3-6 mg/kg) delivered via single bolus doses intravenously or intratracheally led
to rapid reductions in pulmonary artery pressure (−24 ± 3%) and sustained (>5 min) vasorelaxation
independent of administrate route (Intravenous: −17 ± 4% versus IT: −28 ± 4%). Additionally, in
conscious rats, VIP-ELP mediated dose-dependent blood pressure decreases that was sustainable
for about 12 hours post-dosing. These results were supported by promising efficacy data obtained
in other animal models of cardiovascular disease and hypertension
53, 54
. Supported by strong
clinical data, increasing doses of VIP-ELP were evaluated in a phase 1 human study with
pulmonary arterial hypertension patients
55
. Weekly dosing was found to be safe and well-tolerated
by both subcutaneous and intravenous routes, and produced a prolonged, dose-dependent reduction
in blood pressure. VIP-ELP was given orphan designation status by the FDA and is currently being
evaluated in Phase 2 studies, results of which are expected in 2021.
In the field of diabetes, an important application of ELPs is the development of glucagon-like
peptide 1 (GLP-1)-ELPs. GLP-1 is a 31 amino acid peptide incretin hormone secreted in the lower
intestine and colon post-prandially
56
. GLP1 induces satiety
57
, stimulates insulin secretion and
prevents β-cell apoptosis while enhancing their proliferation
58
. These properties make GLP1 an
attractive approach to maintain glucose levels in type 2 diabetes patients by regulating insulin
14
secretion. To address the short 2-min half-life of GLP1
59
, two 50 kDa GLP1-ELPs were designed.
By tuning the ELP sequence to achieve a Tt between room temp. and 37
o
C, a GLP1-ELP with
room temp. solubility with an ability to form a stable, coacervate-based drug depot upon
subcutaneous injection was realized
60
. The second GLP1-ELP served a non-transitioning control
and remained soluble at the site of subcutaneous injection. GLP1-ELPs increased the stability of
GLP1 against enzymatic degradation by neutral endopeptidase and no degradation was observed
even after 18 hr incubation with the enzyme. However, the bioconjugation to ELP polymer reduced
the potency of GLP1 ~ 50 times. When analyzed by NIR imaging in laboratory mice, depot
forming GLP1-ELP was retained at the injection site for over 120 hours, while the soluble GLP-1
formulation disappeared 24 hours post-injection. Moreover, efficacy data demonstrated that depot
forming GLP1-ELP formulation steadily reduced blood glucose levels up to ~30% in a dose-
dependent manner (e.g. 175, 350, and 700 nmol/kg over 24, 72, and 144 hrs respectively).
Alternatively, the soluble GLP1-ELP rapidly precipitated a 60% glucose level decline at a dose of
175 nmol/kg, causing a peak and valley response indicative of more rapid onset and loss of activity.
These results affirm that GLP-1 ELP fusions can reduce blood glucose levels across a range of
doses at intervals that depend on the phase behavior of the ELP in vivo. Based on these exciting
findings, depot forming GLP1-ELP underwent Phase I/IIA study under the trade name Glymera™
with 80 subjects. Four weeks of once weekly SC doses ranging from 0.6 to 1.35 mg/kg displayed
a clinically significant dose dependent effect on reduction in fasting plasma glucose and was able
to significantly reduce mean average daily glucose over the 7 day dosing interval with minimal
loss of glycemic control
61
. Moreover, the formulation was well tolerated with no significant safety
hurdles. Later, Glymera™ also underwent Phase IIB trials to evaluate response and safety of 3
dose levels following 20 weeks of weekly drug administration.
15
Chapter 2: First generation FKBP-ELP carriers for delivery of
Rapamycin: Efficacy and Toxicology

2.1 Abstract
The clinical utility of Rapamycin (Rapa) is limited by solubility, bioavailability, and side effects.
To overcome this, I report an elastin-like polypeptide (ELP) nanoparticle with high-affinity, non-
covalent drug-binding as well as integrin-mediated cellular uptake. Given the scarcity of
pharmacology/toxicology studies of ELP-based drug carriers, this chapter explores safety and
efficacy of ELP-Rapa. ELP-Rapa nanoparticles tested negative for hemolysis, did not interfere in
plasma coagulation nor platelet function, and did not activate the complement. Upon incubation
with HepG2 cells, ELP-Rapa revealed significant cellular uptake and trafficking to acidic
organelles, consistent with lysosomes. Internalized ELP-Rapa nanoparticles increased oxidative
stress 4-fold compared to free drug or free ELP controls. However, mice bearing orthotopic
hormone receptor positive BT-474 breast tumors, given a high dose (~10-fold above the
therapeutic dose), one-month administration of ELP-Rapa did not induce hepatotoxicity. On the
other hand, tumor growth and mTOR-signaling was suppressed without affecting body weight.
Nanoparticles assembled using ELP technology appear to be a safe and efficient strategy for
delivering Rapa.

16
2.2 Introduction

Elastin-like polypeptides (ELPs) are emerging biomaterials with numerous proposed applications
in drug delivery
47, 53, 62-67
, tissue engineering
68-71
, gene delivery
72-74
, and regenerative medicine
65,
75
. When heated above Tt, ELP diblock copolymers can self-assemble through hydrophobic
interactions into viral-size nanoparticles
76-78
. Nanoparticle assembly occurs rapidly over a narrow
range of temperature
79
, is reversible, and is remarkably conserved when ELPs are fused to
functional proteins or bioactive peptides
78, 80, 81
. Having been derived from a short, repetitive
peptide in human tropoelastin, they are reasonable candidates for biocompatible, biodegradable
drug carriers. Despite this, few toxicological studies have been reported to support or refute the
potential for these nanomaterials. While no ELPs are approved for human use, two ELP-containing
investigational drugs remain under evaluation for safety and efficacy in clinical trials
53, 82
.

Our group recently employed ELPs to fabricate nanoparticles that deliver rapamycin (Rapa)
80
.
Rapa, also called Sirolimus, is a potent anti-rejection drug approved for prevention of transplant
rejection. Through inhibition of the mammalian target of rapamycin complex 1 (mTORC1)
83
, it
and related drugs exhibit potent cytostatic activity and are under evaluation in multiple cancer
clinical trials. Although potent, Rapa’s clinical utility is limited by sub-optimal properties,
especially water insolubility and low oral bio-availability (10-15%)
84
. This results in poor
pharmacokinetics (PK) with wide inter and intra-patient variability in PK parameters
85, 86
.
Additionally, the drug’s multiple side effects include painful oral ulcers (incidence >30%) and
severe anemia (incidence >20%), which results in poor patient compliance and adherence to
treatment
87-91
.
17
Engineered nanoparticles of varying shapes and sizes, surface properties and materials have been
explored to aid delivery of chemotherapeutics. Most clinically successful nanoparticles are based
on liposomes; however, a number of other nanomaterials are under investigation. Well-designed
drug vehicles can improve the balance between adverse effects and anti-tumor efficacy, thereby
resulting in better patient outcomes. One such example is albumin loaded paclitaxel, called
Abraxane
TM92-94
. Abraxane
TM
was approved for breast cancer in 2005 after a randomized clinical
trial demonstrated that it doubled the objective response rate and halved the incidence of severe
(grades 3-4) neutropenia among patients who failed traditional chemotherapy. While the
mechanisms underlying superior performance of carrier-assisted delivery remain varied, several
principles may be conserved across systems: i) drug carriers can provide the hydrophobic
environment required to solubilize water-insoluble drugs; ii) drugs loaded in carriers (10-100 nm
in size) may shift their biodistribution away from non-target organs thereby changing toxicity
profiles; iii) carrier-associated drugs benefit from improved plasma residence times by escaping
renal filtration and other clearance pathways; iv) high nanoparticle surface area provides
opportunities to graft molecules that recognize tumor-associated receptors and selectively restore
cellular uptake and drug efficacy
8, 95
.

To explore the above criteria, the Mackay group developed ELP-Rapa nanoparticles comprising
two fusion proteins, FSI and ISR in an equimolar ratio, which successfully reduced the dose at
which tumor growth was suppressed with respect to either free drug or untargeted nanoparticles
80
.
FKBP12, the cognate receptor for Rapa was fused to the N-terminus of a di-block ELP polymer
called SI [(VPGSG) 48(VPGIG) 48] to generate FSI. Similarly, the tri-peptide RGD was fused to the
C-terminus of a di-block ELP called IS [(VPGIG)48(VPGSG)48] to generate ISR (Figure 2.1A).
18
Above room temperature, phase transition of the hydrophobic block (VPGIG)48 in FSI and ISR
triggers their co-assembly into colloidally stable bi-functional nanoparticles
80
. While FSI
solubilizes hydrophobic Rapa through high-affinity FKBP/Rapa interaction
96
, ISR targets these
nanostructures to cells by RGD-mediated recognition of integrins avb3 and avb5, which are
commonly overexpressed in tumors
97, 98
. The MacKay group extensively characterized the
therapeutic potential and targeting efficiency of ISR/FSI-Rapa nanoparticles both in vitro and in
vivo
80
. In mice bearing triple-negative MDA-MB-468 orthotopic tumors, a single administration
of
64
Cu labeled nanoparticles resulted in a modest two-fold higher accumulation in the tumor
compared to untargeted controls. Additionally, pronounced uptake by the liver was observed,
suggesting its role in nanoparticle clearance. Nonetheless, in long-term MDA-MB-468 mouse
xenograft studies, ISR/FSI-Rapa completely suppressed tumor growth at a 3-fold lower Rapa dose
compared to the untargeted FSI-Rapa control
80
. At an equivalent low dose (0.075 mg/kg), carrier-
free Rapa did not suppress tumor growth compared to untreated control mice.

To build on these findings, this chapter aims to further characterize aspects of the ISR/FSI-Rapa
formulation necessary for preclinical development. Questions to be addressed by this report
include mechanisms of cell uptake, toxicological consequences of liver accumulation, particle
interference in hemostasis and therapeutic efficacy of the formulation in other breast cancer models.
Breast cancer is a heterogenous disease and is classified into at least 4 subtypes based on tumor
expression of hormone receptor (HR), HER2 receptor and the protein Ki-67
99
. While previous
efficacy studies evaluated the triple negative (HR-/HER2-) subtype, this report investigates a HR+
breast cancer model. An HR+ model is representative of nearly 80% of breast cancer cases and
reflects the USFDA-approved indication for everolimus in HR+ breast cancer. Identifying broad
19
spectrum activity may strengthen the utility of ISR/FSI-Rapa nanoparticles and also aid in
choosing physiologically relevant concentrations for toxicity studies.

20
2.3 Materials and Methods

2.3.1 Recombinant protein purification and Critical Micelle Temperature (CMT)
measurements
Molecular cloning of FSI and ISR has been described elsewhere
50, 100
. For expression, BLR(DE3)
E.coli competent cells (Novagen, Madison, WI) were transformed with pET25b(+) vector
encoding either FSI or ISR. Cells were spread on agar plates with 100 µg/mL carbenicillin and
incubated overnight at 37 °C. A single colony was inoculated to 50 mL autoclaved Terrific Broth
(TB) medium (Mo Bio Laboratories, Carlsbad, CA) supplemented with 100 µg/mL carbenicillin
and incubated overnight at 37 °C in an orbital shaker. Starter cultures were transferred to 1L TB
medium supplemented with 100 µg/mL carbenicillin and allowed to grow for 24 hours at 37 °C in
an orbital shaker. Bacteria were collected by centrifugation at 5000g for 15 minutes, and the pellet
was re-suspended in phosphate buffered saline (PBS) (Caisson labs, Smithfield, UT). Bacterial
lysis and protein purification have been described elsewhere
101
. The purified proteins were
dialyzed overnight against PBS and sterile filtered using 200 nm Acrodisc
®
filters (Pall
Corporation, Port Washington, NY). Protein concentration was measured after dilution in 8M
Guanidine Hydrochloride (Thermo Fischer Scientific, Waltham, MA) on a Nanodrop 2000
spectrophotometer (Thermo Fischer Scientific, Waltham, MA) using Beer-Lambert’s law, with a
background correction:
𝐶 =

($
%&'
( $
)*'
)
,
-

where C is the solution concentration (M), A280 and A 350 are absorbances at 280 nm and 350
21
nm respectively, l is the path length (cm) and e is the estimated molar extinction coefficient at 280
nm: 11,585 M
-1
cm
-1
for FSI and 1,490 M
-1
cm
-1
1 for ISR
102
. The purity and expected molecular
weight of proteins were confirmed by SDS-PAGE gel stained with Coomassie blue dye.

To characterize phase transition behavior, varying concentrations of FSI and ISR (5-100 µM) were
heated from 15 to 50 °C at 1 °C/min on DU800 spectrophotometer (Beckman Coulter, Brea, CA)
and optical density at 350 nm (OD 350) was measured. CMT is defined as the temperature at which
OD350 (vs) Temperature plot has maximum slope. CMT vs. Conc. profiles were fit to a Log-linear
model:
𝐶𝑀𝑇=𝑏−𝑚[𝐿𝑜𝑔
89
(Concentration)]
where b represents the CMT at 1 µM and m represents change in CMT for a 10-fold change in
concentration.

2.3.2 Rhodamine labeling
NHS-Rhodamine (Thermo Fischer Scientific, Waltham, MA) was dissolved in anhydrous DMSO
(Invitrogen, Carlsbad, CA) at 10 mg/mL and frozen as single use aliquots. To 200 µM ISR in PBS,
2x molar excess NHS-Rhodamine was added and incubated overnight at 4 °C. Zeba desalting
columns (Thermo Fischer Scientific, Waltham, MA) were used according to manufacturer’s
protocol to remove unreacted free dye and elute Rho-ISR in PBS. Rhodamine concentration was
calculated using Nanodrop spectrophotometer as follows while ISR concentration was assumed
unchanged.
𝐶
:;<
=
$
***
,
-

22
where A555 is the absorbance at 555 nm, l is the path length (cm) and ε is the estimated molar
extinction coefficient: 80,000 M
-1
cm
-1
. Labeling efficiency, N, was calculated as:
𝑁 =
>
?@A
>
BC?

×100

The purity of Rhodamine-ISR (Rho-ISR) was evaluated by SDS-PAGE electrophoresis followed
by fluorescence imaging on ChemiDoc
TM
(Bio-Rad, Hercules, CA) imaging system. The
concentration of Rho-ISR throughout the chapter refers to ISR concentration unless otherwise
specified.

2.3.3 Dynamic Light Scattering
Hydrodynamic radius (Rh) measurements were obtained from a DynaPro plate reader II (Wyatt
Technologies, Santa Barbara, CA). Briefly, 20 µM protein solutions were passed through a 200
nm filter, and 60 µL sample was added in triplicate to a 384-well clear bottom plate (Greiner Bio
One, Monroe, NC). The wells were capped with 15 µL mineral oil to prevent evaporation during
measurements. Data was analyzed using Dynamics V7 software (Wyatt, Santa Barbara, CA)

2.3.4 Rapa encapsulation, concentration measurements and endotoxin removal
To prepare Rapa-loaded FSI, a two-phase encapsulation method was employed. To 10 mL 300 µM
FSI in PBS, 3x molar excess Rapa (LC Laboratories, Woburn, MA) in hexane/EtOH mixture (7:3
v/v) was added. After evaporation of the organic phase at 4 °C using a rotary evaporator, the
aqueous suspension was centrifuged at 13,000 g to pellet unbound Rapa precipitate. The
supernatant was subjected to additional rounds of centrifugation until no pellet was observed. FSI-
Rapa was added to a 10 kDa MWCO dialysis bag (Thermo Fischer Scientific, Waltham, MA) and
23
dialyzed against PBS (1:750 sample: dialysate) for 12 hours to remove free Rapa and residual
solvent. After mixing FSI-Rapa and ISR in an equimolar ratio, the working formulation was
subjected to endotoxin removal by poly-lysine chromatography using Pierce
TM
endotoxin removal
columns (Thermo Fischer Scientific, Waltham, MA) following manufacturer’s protocol. An
aliquot of the final material was injected onto a C-18 RP HPLC column (Waters, Milford, MA)
and Rapa was quantified at 280 nm using a calibrated standard curve. Concentration of ISR/FSI-
Rapa throughout the chapter refers to Rapa concentration unless otherwise noted.

2.3.5 Measurement of residual endotoxins
Endotoxin burden in the formulation was quantified using QCL-1000
™
endpoint chromogenic
LAL assay kit (Lonza Inc., Walkersville, MD). The human equivalent dose (HED) for ISR/FSI-
Rapa nanoparticles was estimated as
103

𝐻𝐸𝐷
J
KL
ML
N=𝑀𝑜𝑢𝑠𝑒
𝑑𝑜𝑠𝑒
J
KL
ML
N×
𝐾
K
𝑟𝑎𝑡𝑖𝑜

where mouse dose = 0.08 mg/kg Rapa and Km ratio = 0.081. The theoretical human plasma
concentration at HED (Cp) was estimated as
𝐶
X
=

YZ[
×
\
]

where W is reference human body weight = 60 kg and V is the volume of blood in an average adult
= 4.7 L. The endotoxin limit (EL) for the formulation was calculated as
104

𝐸𝐿 =
^
_

24
where K is the maximum endotoxin level allowed per dose = 5 EU/kg and M is the maximum dose
to be administered per kg of body weight per single hour = HED. Additionally, the maximum valid
sample dilution (MVD) for endotoxin estimation was calculated as
𝑀𝑉𝐷 =

Za
×
>
b

where C is the sample Rapa concentration and 𝜆 is the assay sensitivity (0.1 EU/ml for
chromogenic and turbidity LAL, and 3 EU/ml for gel-clot assay). All endotoxin measurements
were made at MVD. LAL reagent water served as the negative control.

2.3.6 Cell culture
HepG2 and BT-474 cell lines (HB-8065, ATCC, Manassas, VA) were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Thermo Fischer Scientific, Waltham, MA) and Hybri-Care
medium (ATCC, Manassas, VA) respectively. Media was supplemented with 10% fetal bovine
serum (FBS, Corning, NY) and cells were grown in a humidified incubator with 5% CO2 at 37 °C.

2.3.7 Cellular uptake and co-localization analysis
3x10
5
HepG2 cells were seeded on a 35 mm glass bottom dish (MatTek Corporation, Ashland,
MA) and allowed to attach overnight. The next morning, the culture medium was replaced with
pre-warmed complete medium containing 10 µΜ Rho-ISR/FSI. After 6 hours of treatment, cells
were washed three times with PBS and 1 mL of live cell imaging solution (Life Technologies,
Carlsbad, CA) was added to the dish with 2 drops of NucBlu
TM
reagent (Life Technologies,
Carlsbad, CA). When applicable, 1 µL LysoTracker
TM
Green DND-26 (LTG, Life Technologies,
Carlsbad, CA) was added. Images were captured using LSM800 confocal microscope (Carl Zeiss
25
Microscopy, Thornwood, NY) mounted on a vibration-free table with a Plan-Apochromat 63x oil
objective. For co-localization analysis, Mander’s Co-localization Coefficient (MCC) was
calculated for both red and green channel using ZEN2009 software (Carl Ziess Microscopy,
Thornwood, NY) using the following equations
𝑀
:de
=
∑ :
g,iAjAi g
∑ :
g g
𝑀
klddm
=
∑ k
g,iAjAi g
∑ k
g g

2.3.8 Reactive Oxygen Species (ROS) assay
HepG2 cells were seeded at a density of 3x10
4
cells/well in a black 96-well clear bottom plate
(Greiner Bio One, Monroe, NC). The next morning, media was aspirated, and cells were washed
twice with Hank’s Balanced Salt solution HBSS (Thermo Fischer Scientific, Waltham, MA). 25
µM 100 µL 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) dye in HBSS was added to each
well. After a 45-minute incubation, DCFDA was removed, cells were washed 1x with HBSS and
the appropriate treatment diluted in complete medium without phenol red was added. Fluorescence
intensity at Ex/Em 485/535 nm was measured using a Synergy H1 plate reader (BioTek, Winooski,
VT).

2.3.9 In vitro cytotoxicity assay
BT-474 cells were seeded at 3x10
3
cells/well density and allowed to attach overnight. Next
morning, media was exchanged for multiple dilutions of ISR/FSI-Rapa or free Rapa in complete
medium. After treatment for 4 days, 10 µL WST-1 reagent (Sigma Aldrich, St. Louis, MO) was
added per well and incubated for 2 hours before measuring formazan absorbance at 440 nm. A 440
was background corrected by subtracting absorbance at 690 nm reference wavelength. The %
26
maximum proliferation (%P) was calculated and plotted against varying Rapa concentrations using
the following equation:
%𝑃 =

($
pqrsprt
( $
'
)
($
uvpqrsprt
( $
'
)

where Atreated, Auntreated, and Ao refer to corrected absorbances of drug treated, untreated and blank
wells containing only media respectively. The IC 50 was determined by non-linear regression in 3
independent assays and reported as Mean ± SD. A student t-test was used to compare differences
between groups.

2.3.10 In vitro plasma coagulation time assay
Freshly collected human blood was spun at 2,500 g for 10 min at room temperature to separate
plasma. In a microcentrifuge tube, 900 µL plasma was mixed with 10x 100 µL ISR/FSI-Rapa and
incubated at 37 °C for 30 minutes. Coagulation tests were performed using a STart4 hemostasis
analyzer (Diagnostica Stago, Parsipanny, NJ) according to the manufacturer’s protocol.

2.3.11 BT-474 Xenograft model and drug treatment
All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC)
at Washington Biotech Inc. (Simpsonville, MD). One day before tumor implantation, 7-8 weeks
old female athymic nude mice (Envigo, Fredrick, MD) were ear tagged and received a 17-β-
estradiol pellet (Innovative Research of America, Sarasota, FL) administered subcutaneously using
a trocar. The next day, BT-474 cells were harvested and re-suspended in PBS with 20% matrigel
at the density of 20 X 10
7
cells/mL. 50 µL of the cell suspension was injected subcutaneously to
27
the mammary fat pad of each mouse. Tumor volumes were measured using calipers using the
formula:
𝑉 =

\
%
×
a
w

where V, W, and L denote the volume, width, and length of the tumor respectively. Two weeks
after implantation, mice were randomized into two groups (8/group) with average tumor volume
of 75 mm
3
. Mice received either PBS or 1 mg/kg ISR/FSI-Rapa subcutaneously on Monday,
Wednesday and Friday for 4 weeks. Two days after the last treatment, mice were euthanized and
organs (liver, kidney, spleen, and tumor) were harvested. Organs were either flash frozen or fixed
in 10% neutral buffered formalin for 2 days before being moved to 70% ethanol for long-term
storage.

2.3.12 Complement activation assay by Enzyme Immunoassay (EIA)
Human blood containing sodium citrate as the anti-coagulant was subjected to centrifugation at
2500g for 10 minutes and plasma was collected. In a microcentrifuge tube, 100 µL each of Veronal
buffer (Boston Bioproducts, Ashland, MA), plasma and test sample were mixed and incubated at
37 °C for 30 minutes. Appearance of iC3b was measured using the MicroVue iC3b EIA kit (Quidel
Corp., San Diego, CA) following manufacturer’s instructions. As a positive control, the vehicular
composition of Taxol® was prepared by mixing Cremophor-EL (Milliore Sigma, Burlington, MA)
1:1 with ethanol containing 2 mg/mL citric acid.

28
2.4 Results

2.4.1 Physicochemical characterization of ISR/FSI-Rapa nanoparticles
Both FSI and ISR were recombinantly expressed in E.coli. For purification, the bacterial lysates
were subjected to inverse transition cycling, a non-chromatographic strategy utilizing the stimulus
responsive properties of ELPs and ELP fusion proteins, specifically heat and solution ionic
strength
105
. By performing three rounds of centrifugation above and below their inverse phase
transition temperatures, FSI and ISR of >95% purity and a yield of 75 and 120 mg/L culture were
obtained (Figure 2.1B). The molecular weight estimated by SDS-PAGE electrophoresis were
comparable to the theoretical molecular weights of 51 and 40 kDa for FSI and ISR respectively
(Table 2.1).

Additional characterization included constructing Concentration vs. CMT phase diagrams. Since
the Tt of ELP containing proteins decreases with increasing solution concentration and vice versa,
phase diagrams provide a convenient means to determine their physical state at a given temperature
and concentration. Similar to previous observations, a drop in concentration from 100 to 5 µM
resulted in an increase in CMT from 21 to 26 °C for FSI, and from 22.5 to 28 °C for ISR. CMT vs.
log(Concentration) plots followed a straight line across the range of concentrations evaluated
(Figure 2.1C). For FSI, b and m were 30.0 ± 1.0
o
C and 4.0 ± 0.7
o
C respectively and for ISR, b
and m were 32.0 ± 0.8
o
C and 4.1 ± 0.6
o
C respectively (Mean ± 95% CI). By extrapolating the
line of best fit, at physiological temperature of 37 °C, both FSI and ISR are expected to remain
assembled across all the concentrations relevant to this chapter.

29
Table 2.1: Physiochemical characterization of ELP di-block polymers evaluated in this
chapter

Label Amino acid sequence MW
(kDa)
a

Purity
(%)
b
CMT
(°C)
c

Rh 15
°C
(nm)
d
Rh
37
°C
(nm)
FSI GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDS
SRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI
SPDYAYGATGHPGIIPPHATLVFDVELLKLEG(VPGSG)48
(VPGIG)48Y

51.4 99.1 25.0 4.9 ±
0.3
23.7
±
0.2
ISR G(VPGIG)48(VPGSG)48Y−GRGDGG 40.1 95.6 26.5 4.7 ±
0.1
24.3
±
0.3

a
Expected molecular weight based on amino acid sequence
b
Purity was determined by densitometry analysis of stained SDS-PAGE gel
c
CMT here is reported for 25 µM samples suspended in phosphate buffered saline (PBS)
d
Rh of 25 µM samples in PBS measured by DLS (Mean ± SD, n=12)

Rapa encapsulation was performed by a two-phase solvent evaporation technique. After removal
of unbound Rapa and traces of carryover solvents, drug loading ratio defined as CRapa/C FSI was
found to be 0.8, the theoretical maximum being 1. FSI-Rapa and ISR were mixed in an equimolar
ratio below their CMT and subjected to endotoxin removal by poly-lysine chromatography. To
measure residual endotoxins, two versions of the FDA approved Limulus Amoebocyte Lysate
(LAL) assay were compared. Assay inhibition or enhancement was tested using spike recovery
measurements. A small amount of known endotoxin was spiked in the presence of ISR/FSI-Rapa
and quantified using the assay protocols. Percent spike recovery for turbidity and chromogenic
LAL were 147 and 61 respectively, both within the acceptable range of 50-200%. FDA
recommended maximum endotoxin load[49] for the formulation was calculated to be 769 EU/mg
of Rapa. When measured by turbidity LAL assay, ISR/FSI-Rapa was found to have 218 EU/mg
Rapa while chromogenic LAL resulted in <7.5 EU/mg Rapa. Despite the discrepancy, both assays
30
measured residual endotoxins below the recommended limit. Additionally, gel-clot LAL assay
with a sensitivity of 750 EU/mg Rapa tested negative (no clot formed) for endotoxins.

At 37 °C, ISR/FSI-Rapa assembled homogenous particles with a hydrodynamic radius (Rh) of 22.7
± 0.2 nm (Mean ± SD, n=12) and a low polydispersity index (PDI) of 0.03 (Figure 2.1D). The Rh
remained constant even after 18 hr of incubation at 37 °C. On the contrary, Rh of 5.1 ± 0.3 nm
(Mean ± SD, n=12) at 15 °C (below the CMT) represents an unassembled state wherein FSI-Rapa
and ISR are soluble and monomeric. Similar behavior was observed for solutions containing only
FSI-Rapa and only ISR. Additionally, the size and stability of ISR/FSI-Rapa nanoparticles was not
affected by the presence of serum albumin (Figure 2.8). At native pH, ISR/FSI-Rapa
nanoparticles were charge neutral with a zeta potential of -1.1 ± 0.4 mV (Mean ± SD, n=3).
Although neutral particles are generally considered to be colloidally unstable and prone to
aggregation, no such effects were observed for ISR/FSI-Rapa.
31

Figure 2.1: Physicochemical characterization of ISR/FSI-Rapa nanoparticles. A) A schematic
representation of bi-functional ELP-based Rapa carriers. The two components FKBP12 (FSI) and
RGD (ISR) co-assemble above a critical micelle temperature (CMT) to form stable micelles that
solubilize Rapa. B) SDS-PAGE gel was stained with Coomassie stain to verify purity and identity
of fusion proteins. C) The CMT of fusion proteins was dependent on their concentration and can
be fit to a Log-linear model. At 37
o
C, both FSI and ISR exist in an assembled state at all
concentrations relevant to this chapter. D) DLS was used to measure hydrodynamic radii (Rh) of
the particles below and above CMT at 25 µM concentrations. At 15
o
C, the Rh was 4.9 ± 0.3 nm,
4.7 ± 0.1, and 5.1 ± 0.3 nm (Mean ± SD, n=12) for FSI, ISR and ISR/FSI-Rapa respectively. Above
CMT, monomeric FSI, ISR and ISR/FSI-Rapa assembled to form particles of radii 23.7 ± 0.2 nm,
24.3 ± 0.3 nm, and 22.7 ± 0.2 nm respectively. These results suggest that FSI retains its phase
behavior in a Rapa-bound state and that the drug itself does not precipitate into larger aggregates.

2.4.2 Estimation of HED, cellular uptake in HepG2 cell line
As previously reported, the efficacious dose for ISR/FSI-Rapa nanoparticles in mice was 0.08
mg/kg Rapa in a triple negative breast cancer (TNBC) model
80
. This translates to a human
equivalent dose (HED) of 6.4 µg/kg using the FDA recommended body surface area normalization
32
method. Furthermore, HED was used to estimate the theoretical human plasma concentration at
HED to be 0.1 µg/mL, or about 0.1 µM. Although efficacious concentrations are extremely low,
toxicological measurements are typically performed at concentrations orders of magnitude
higher
106
. For example, this is especially necessary when the plasma concentration does not reflect
the local concentration, in organs of accumulation. ISR/FSI-Rapa nanoparticles are known to
accumulate in the liver, but the biological consequences of this accumulation are unknown. Using
a hepatocellular carcinoma cell line called HepG2, I first studied their cellular internalization by
confocal microscopy. The N-terminus of ISR was labeled with Rhodamine (Rho) dye by NHS
chemistry to facilitate particle tracking. Rho-ISR/FSI-Rapa retained phase behavior and assembled
particles of the same size and physical stability as unlabeled equivalent. Upon incubation with 10
µM Rho-ISR/FSI-Rapa for 6 hours, significant uptake was observed in HepG2 cells (Figure 2.2A).
Additionally, the particles co-localized with Lyso Tracker Green (LTG), a fluorescent dye used to
label low pH organelles including lysosomes. The MCC for Rho-ISR/FSI-Rapa and LTG was 0.82
± 0.20 (n=10 cells), suggesting most of the internalized nanoparticles are trafficked to acidic
organelles, including lysosomes. These organelles may play a role in biodegradation or drug
release from the particles.

33

Figure 2.2: Internalized ISR/FSI-Rapa nanoparticles traffic to acidic organelles and induce
oxidative stress in HepG2 cells. A) Rho-ISR and Rapa loaded FSI were mixed in 1:1 ratio and
added to HepG2 cells at 25 µM. After a 6 hour incubation at 37 °C, cells were washed 3x and
labeled with Lysotracker green (LTG) before confocal imaging. Significant nanoparticle uptake
was observed and the internalized particles co-localized with LTG, a marker for acidic cellular
compartments including lysosomes. The Mander’s colocalization coefficient obtained in
individual cells averaged to 0.82 ± 0.20 for Rho-ISR/FSI-Rapa and LTG, thereby suggesting a
high degree of co-localization (Mean ± SD, n=10 cells). B) HepG2 cells were labeled with
DCFDA, a fluorescent probe for intracellular ROS and incubated with 25 µM ISR/FSI-Rapa for 1
hr and 16 hrs. Significantly higher fluorescence intensity (approximately 4-fold higher) was
measured in the presence of nanoparticles compared to untreated controls (Mean ± SD, n=3, One-
way ANOVA, Tukey Post-hoc ****p<0.0001). In contrast, cells treated with FSI, ISR, ISR/FSI,
or Rapa alone do not trigger ROS generation. C) A serial dilution DCFDA assay reveals weak
dose dependence for ROS induction. ISR/FSI-Rapa ranging from 25 µM to 300 nM of Rapa were
added to HepG2 cells for 1 or 16 hrs. Only concentrations above 8 µM induced significantly higher
34
ROS in a dose dependent fashion (Mean ± SD, n=3, One-way ANOVA, Tukey Post-hoc,
***p<0.001).

2.4.3 ROS generation assay
Internalized nanoparticles are known to induce cellular oxidative stress through ROS production
107,
108
. This triggers multiple cell signaling pathways that cause pathological effects like genotoxicity,
inflammation, and fibrosis. Various factors like particle composition, size and charge, and the
presence of metals dictate ROS generating potential of nanoparticles
109
. HepG2 cells were labeled
with the intracellular probe DCFDA which covalently reacts with ROS generated in the cell and
forms stable fluorescent molecules. Incubation of labeled cells with 25 µM ISR/FSI-Rapa
nanoparticles for 1 hour resulted in a nearly 5-fold higher cell associated fluorescence compared
to untreated control cells (Figure 2.2B). Surprisingly, neither 25 µM carrier (ISR/FSI) nor 25 µM
Rapa alone triggered ROS generation higher than untreated cells. Next, concentration dependence
of ROS generation was probed using a serial dilution of ISR/FSI-Rapa nanoparticles (Figure 2.2C).
Incubation of HepG2 cells with <3 µM ISR/FSI-Rapa did not generate measurable fluorescence
above background while concentrations >8 µM induced ROS in a dose dependent manner. Hence,
there appears to be a critical concentration, above which ROS induction becomes detectable.
Additionally, incubation of cells with the particles for longer time periods (16 hrs) did not cause a
proportional increase in cellular fluorescence (Figure 2.2C). This could be a result of probe
saturation causing additionally generated ROS to escape undetected. Alternatively, a cellular
adaptive mechanism (activation of stress responses) to rapidly increasing oxidative stress may
dampen ROS generation.

35
2.4.4 Hemolysis and complement activation assays
Damage to erythrocytes leads to release of intracellular material into blood plasma and is
associated with life-threatening pathological conditions. Since ISR/FSI-Rapa nanoparticles
circulate through the blood compartment, their hemolytic potential was evaluated using an in vitro
hemolysis assay
110
. The assay is based on quantitative colorimetric determination of total
hemoglobin in whole blood (TBH) and plasma-free hemoglobin (PFH). Hemolytic conditions are
expected to increase PFH. While incubation of whole blood with Triton X-100 positive control
caused 92 ± 0.7% (Mean ± SD, n=2) hemolysis (Figure 2.3A), ISR/FSI-Rapa nanoparticles did
not produce any detectable hemolysis across a range of concentrations (0.5 nM - 1 µM) greater
than expected for the HED (0.1 µM). Alternately, nanoparticles can cause infusion related pseudo-
allergies through activation of the complement system of proteins
111-113
, a component of the innate
immune system. The complement system consists of over thirty proteins and can be activated by
three different pathways termed classical, alternative and lectin pathways. When triggered, all
three proteolytic cascades converge to generate the same effector molecules C3b and iC3b through
protein C3 activation. Several effector molecules of this system are potent activators of immune
cells and cause hypersensitivity reactions. When human plasma was incubated with ISR/FSI-Rapa
nanoparticles at concentrations up to 1 µM, no measurable iC3b was detected by ELISA (Figure
2.3B). On the contrary, the positive control Cremophor activated the complement cascade and
produced about 75 µg/ml iC3b. The apparent absence of complement activation may be an
attractive aspect of ELP-based nanomaterials with respect to other nanoformulations and
emulsions.

36

Figure 2.3: Neither ISR/FSI-Rapa nor control nanoparticles trigger measurable hemolysis
or complement activation. A) Pooled human blood was incubated with 1 µM test nanoparticles
and 0.1 mg/ml Triton X-100 for 3 hours at 37
o
C. Total blood hemoglobin (TBH) and plasma free
hemoglobin (PFH) were determined by colorimetry and % hemolysis was calculated. While Triton
X-100 caused >90% hemolysis, none was detected with the test nanoparticles (n=2). B) Freshly
prepared human plasma was incubated with 1 µM test nanoparticles or CrEL 30 minutes at 37
o
C.
Generation of iC3b was quantified by ELISA as a measure of complement activation. The widely
used drug solvent CrEL activated complement system as a positive control and produced 79.5 ±
2.1 µg/ml (Mean ± SD, n=2) iC3b, but no detectable iC3b production was triggered by incubation
of test nanoparticles (n=2). This experiment was performed by the National Cancer Institute’s
Nanoparticle Characterization Laboratory.

2.4.5 Plasma coagulation time and platelet aggregation assays
Impairment of normal processes regulating blood coagulation and clotting (hemostasis) may result
in serious conditions like disseminated intravascular coagulation. Clot formation is complex, is
regulated by 13 factors termed factor I-XIII and can proceed via the intrinsic or extrinsic pathways
based on the trigger. Both pathways converge to a final common pathway. Functionality of these
processes can be assessed by measuring three different coagulation times using a coagulometer.
Activated thromboplastin time (APTT) is used to assess the intrinsic pathway, prothrombin time
37
(PT) measures extrinsic pathway and thrombin time (TT) assays the activity of the common
pathway. Nanoparticles may trigger coagulation by providing a surface to initiate clot formation
or impede clotting by adsorbing and depleting clotting factors from plasma. The PT, APTT, and
TT for untreated human plasma were found to be 10.0 ± 0.1 sec (Mean ± SD, n=3), 30.9 ± 0.1 sec
(Mean ± SD, n=3), and 16.2 ± 0.6 sec (Mean ± SD, n=3) respectively. In the presence of 1 µM
ISR/FSI-Rapa nanoparticles, the coagulation times were 10.1 ± 0.1 sec (Mean ± SD, n=3), 32.1 ±
0.4 sec (Mean ± SD, n=3), and 15.9 ± 0.2 sec (Mean ± SD, n=3) for PT, APTT and TT respectively
(Figure 2.4A). No significant differences in coagulation times were detected compared to
untreated controls, thereby suggesting no interference in clotting process. Using platelet rich
plasma (PRP), an independent assay studied potential thrombogenic or anticoagulant properties of
the particles through direct effects on platelets. When platelets aggregate, they reduce sample
turbidity and increase light transmission. Area under curve (AUC) for %transmission vs. time plots
can be compared across conditions. When PRP was spiked with ISR/FSI-Rapa nanoparticles to a
final concentration of 1 µM, the AUC recorded by the aggregometer was only 1.45 ± 1 units (Mean
± SD, n=3) (Figure 2.4B). Addition of collagen to PRP on the other hand triggered platelet
aggregation and produced an AUC of 366 ± 49 units (Mean ± SD, n=3). When collagen was added
to PRP containing 1 µM ISR/FSI-Rapa nanoparticles, the AUC was restored to 331 ± 14 units
(Mean ± SD, n=3). Taken together, ISR/FSI-Rapa nanoparticles appear to neither induce platelet
aggregation nor interfere with platelet aggregation.

38

Figure 2.4: Plasma coagulation and platelet function are unaffected by the presence of
ISR/FSI-Rapa and control nanoparticles. A) Freshly prepared human plasma was incubated
with 1 µM FSI, ISR and ISR/FSI-Rapa nanoparticles at 37 °C for 30 minutes. In vitro coagulation
times were measured by a coagulometer to assess the integrity of multiple coagulation pathways.
PT, ATPP and TT for untreated plasma were 10.0 ± 0.1 sec (Mean ± SD, n=3), 30.9 ± 0.1 sec
(Mean ± SD, n=3), and 16.2 ± 0.6 sec (Mean ± SD, n=3) respectively. No significant differences
in PT, ATPP nor TT were observed when plasma was treated with nanoparticles (One-way
ANOVA, α = 0.05, n=3) compared to untreated plasma. On the other hand, the positive control
Triton X-100 delayed the PT, ATPP and TT to 23.1, 74.0 and 41.1 sec respectively (n=1). B) When
platelet rich plasma (PRP) was spiked with FSI, ISR and ISR/FSI-Rapa nanoparticles, the AUC
for %transmission (vs) time profiles measured by the aggregometer were 2.7 ± 0.6 units (Mean ±
SD, n=3), 1.4 ± 1.1 units (Mean ± SD, n=3), 1.4 ± 1.0 units (Mean ± SD, n=3) respectively. On
the contrary, addition of the positive control collagen triggered platelet aggregation, increased light
transmission and produced an AUC of 366 ± 49 units (Mean ± SD, n=3). Additionally, addition of
collagen in the presence of FSI, ISR and ISR/FSI-Rapa nanoparticles resulted in platelet
aggregation to the same extent as with untreated PRP. The AUCs measured were not significantly
different between groups (One-way ANOVA, α = 0.05, n=3). These results suggest that the test
particles neither induce platelet aggregation nor interfere with collagen induced platelet
aggregation. This experiment was performed by the National Cancer Institute’s Nanoparticle
Characterization Laboratory.

2.4.6 Efficacy in BT-474 xenograft mouse model
BT-474 is a human breast epithelial cell line with ER+ HER2+ status, which contrasts with
previous studies of ISR/FSI-Rapa using triple negative breast cancers (TNBC). Because it is ER+,
BT-474 more closely reflects the hormone receptor positive status for which a closely related the
rapalogue known as everolimus is approved for clinical use by the US FDA. In an in vitro cell
proliferation assay, ISR/FSI-Rapa nanoparticles potently inhibited growth with an IC50 of 1.6 ±
0.3 nM (Mean ± SD, n=3) (Figure 2.5A). This is comparable to the potency of free Rapa which
39
has an IC50 of 1.3 ± 0.4 nM (Mean ± SD, n=3) in the same cell line. When mice bearing BT-474
xenografts were treated with ISR/FSI-Rapa nanoparticles (1 mg/kg Rapa dose) 3 times a week for
4 weeks, significant suppression of tumor growth was observed compared to PBS-treated mice
(Figure 2.6A). The dosing schedule adopted was based on our previous experience in TNBC
mouse model. On the last day of therapy, tumor volumes measured were 725 ± 358 mm
3
(Mean ±
SD, n=7) in PBS group and 175 ± 64 mm
3
(Mean ± SD, n=8) in ISR/FSI-Rapa group (Figure
2.6B). The difference was statistically significant (t-test with Welch’s correction, α = 0.05, p =
0.008). The treatment was well tolerated with no significant difference in body weights between
groups. Our efficacy study design lacks a free Rapa control because the goal was to evaluate the
formulation nanotoxicology at a standard dose. A dose of free Rapa (1 mg/kg) equivalent to
ISR/FSI-Rapa evaluated here has been reported to suppress tumor growth in the identical mouse
model
114
.

At the end of the study, tumors were excised and in vivo modulation of mTOR related signaling
was probed by western blotting. mTOR is a serine/threonine kinase and forms two distinct
multidomain complexes: mTORC1 and mTORC2. mTORC1
11, 13, 17
phosphorylates and activates
proteins involved in mRNA translation, S6K1 and 4EBP1. S6K1 is a kinase and phosphorylation
of its target substrate S6 ribosomal protein (S6RP) induces ribosomal protein synthesis. mTORC1
is sensitive to rapamycin and the loss of S6RP phosphorylation is a widely used marker of
mTORC1 inhibition. When 5 tumors from each group were randomly chosen and probed by a
phospho specific S6RP antibody, all tumors of ISR/FSI-Rapa group had complete suppression of
phosphorylation while all the tumors in PBS group had detectable P-S6RP signal (Figure 2.6C).
GAPDH served as a total protein loading control. This study demonstrates that this
40
nanoformulation is able to successfully act on-target in a hormone receptor positive orthotopic
model, while remaining tolerated.

Figure 2.5: ISR/FSI-Rapa nanoparticles potently inhibit the growth of BT-474 cell line. A)
BT-474 cells were treated with 150 nM – 10 pM ISR/FSI-Rapa and free rapa control for 4 days.
% Maximum proliferation determined by WST assay was plotted against Rapa concentration. Non-
linear regression analysis revealed an IC50 of 1.3 ± 0.4 nM (Mean ± SD, n=3) and 1.6 ± 0.3 nM
(Mean ± SD, n=3) for free Rapa and ISR/FSI-Rapa respectively. No significant differences in IC 50
were observed B) Carrier only controls did not affect cellular viability or proliferation. Hence,
these nano-carriers are biocompatible and not toxic to cells.
41

Figure 2.6: ISR/FSI-Rapa nanoparticles potently suppress tumor growth in a BT-474
xenograft mouse model. A) Female athymic nude mice (8/group) received subcutaneous
implantation of BT-474 cells to the mammary fat pad. When the average tumor volume reached
75 mm
3
, mice received either PBS or 1 mg/kg ISR/FSI-Rapa subcutaneously on Monday,
Wednesday and Friday for 4 weeks. Tumor volumes (Mean + SD, n=8) in the PBS group
progressively increased as opposed to ISR/FSI-Rapa treated group. B) On the last day of treatment,
tumor volumes were 725 ± 358 mm
3
(Mean + SD, n=7) in PBS group and 175 ± 64 mm
3
(Mean +
SD, n=8) in ISR/FSI-Rapa group. The difference was statistically significant (t-test with Welch’s
correction, p = 0.008). C) Body weights of mice were tracked to assess treatment associated
toxicity. ISR/FSI-Rapa nanoparticles were well tolerated with no significant difference in body
weights between groups. D) At the time of euthanasia, tumors were collected, protein extracted
and probed by western blotting. Phosphorylation of S6RP, a downstream target of rapamycin
sensitive mTOR signaling was completely suppressed in the ISR/FSI-Rapa group (n=5), while all
the tumors in the PBS group had detectable P-S6RP (n=5). GAPDH served as a total protein
loading control. Images are representative of 3 independent repeats. This experiment was
performed by Washington Biotechnology Inc.

2.4.7 Changes in liver protein expression and serum biomarkers
At the end of the one-month, multi-dose tumor study, livers from both the groups of mice were
harvested and total protein was extracted. Changes in gene expression of a targeted panel due to
exposure to nanoparticles were measured by western blotting (Figure 2.7A). Integrated volumes
42
for bands of interest were normalized to GAPDH for quantitative comparison (Figure 2.7B). Stress
response proteins of the heat-shock family HSP70 and HSP90α/ß were not affected by treatment.
Additionally, multiple proteins that cells upregulate to prevent oxidative stress were evaluated. No
significant differences in cellular levels of Glutathione S reductase (GSR), Thioredoxin (TRX),
Thioredoxin reductase (TrxR1), Peroxiredoxin (PRX), or γ-glutamylcysteine synthetase heavy
subunit (γGCSc) were detected between groups. Although ISR/FSI-Rapa nanoparticles at high
concentrations induced ROS generation in vitro, it appears like these effects are not recapitulated
in vivo. Additionally, serum biomarkers of organ toxicity were measured at the end of the tumor
study (Table 2.2). No statistical differences in markers of liver function (ALT, AST, Alkaline
phosphatase and GGT), nor kidney function (Creatinine, blood urea nitrogen) were detected
between PBS treated and ISR/FSI-Rapa treated groups. Hence, chronic exposure to ISR/FSI-Rapa
nanoparticles was found to be safe in organs responsible for clearing this nanoformulation.

43

Figure 2.7: A month long exposure to ISR/FSI-Rapa nanoparticles does not induce hepatic
stress responses in treated mice. At the time of euthanasia, livers of mice (4/group) from tumor
xenograft study were collected, total protein was extracted and probed by western blotting. A)
Expression of a panel of biomarkers comprising oxidative stress response proteins GSR, gGCSC,
PRX2/4, TRX, TRXr1 and heat shock proteins HSP70, HSP90α/ß were assessed. GAPDH was
used as a total protein loading control. Images are representative of 3 independent experiments. B)
Integrated band volumes from (A) were normalized to GAPDH and plotted as Mean ± SD,
n=4/group. No significant differences in expression levels of any of the markers tested were
detected between PBS and ISR/FSI-Rapa treated groups. These results support biocompatibility
and a lack of stress response to liver accumulation of ISR/FSI-Rapa nanoparticles.

Table 2.2: Toxicity assessment of ISR/FSI-Rapa via serum chemistry profiles (Mean ± SD,
n= 4/group)

Biomarker PBS ISR/FSI-Rapa
ALT (IU/L) 121.5 ± 80.2 180.8 ± 144.2
AST (IU/L) 43.1 ± 5.7 48.0 ± 9.6
BUN (mg/dL)
17.2 ± 2.9 20.2 ± 6.6
GGT (IU/L) 3.0 ± 0.0 3.0 ± 0.0
Alk. Phosphatase (IU/L) 109.5 ± 38.1 138.0 ± 46.0
Creatinine (mg/dL) 0.5 ± 0.0 0.5 ± 0.0
44
2.5 Discussion
This chapter reports pre-clinical safety of recently described ISR/FSI-Rapa nanoparticles, a novel
delivery strategy to repurpose the potent drug, Rapa. Clinically used oral formulations of Rapa
suffers from low bioavailability, poor and variable PK properties. To address this, multiple
intravenous Rapa formulations were developed using co-solvents like dimethyl acetamide and
propylene glycol
115, 116
, and Cremophor EL among others. However, none of these formulations
were tested in humans, possibly due to solvent-associated toxicities
2
. Alternatively, analogs of
rapamycin with improved solubility and drug-like properties were developed. One such analog
called Temsirolimus is clinically used and is available as an intravenous formulation. Although
temsirolimus is widely considered to be water soluble, its IV injection contains high proportions
of polysorbate 80, polyethylene glycol and alcohol for solubility enhancement. Both Cremophor
EL and polysorbate 80 activate the complement system of proteins
4, 113, 117
, and trigger infusion
related hypersensitivity, or anaphylactic reactions. On the contrary, this unconventional ELP
nanoformulation is free of organic solubilizers and leverages the natural affinity of Rapa for
FKBP12 for solubilization and delivery.

45

Figure 2.8: Presence of serum albumin does not impact the hydrodynamic radius (Rh) and
stability of ISR/FSI-Rapa nanoparticles. A) 100 µM ISR/FSI-Rapa containing 0.5 mM bovine
serum albumin (BSA) was heated to 37 °C. The Rh of assembled nanoparticles in this mixture was
estimated at 27.3 ± 0.6 nm (Mean ± SD, n=3) by regularization analysis of DLS. B) After 1 day of
incubation at 37 °C, the Rh of the nanoparticle peak appeared to remain constant at 28.7 ± 1.3 nm
(Mean ± SD, n=3) C) 100 µM ISR/FSI-Rapa nanoparticles were analyzed at 37 °C in PBS to
evaluate the population of nanoparticles alone. D) As a negative control, 0.5 mM BSA was
evaluated at 37 °C in PBS, which showed a single protein population (Rh < 10 nm) E) 10% heat
inactivated fetal bovine serum (FBS) in PBS was also evaluated at 37 °C. The presence of
particulate species that overlap in size with ISR/FSI-Rapa nanoparticles precluded its use in
analyzing particle serum stability.

In all the tests evaluated, ISR/FSI-Rapa nanoparticles performed as well as the negative controls
with the exception of in vitro ROS generation assay. None of the test particles evaluated were
found to be hemolytic. This is in accordance with previous observations of Tat-ELPs
118, 119
and
poly-(VPGVG)
120
being non-reactive towards erythrocytes. Plasma coagulation times (PT, ATPP
and TT) remained unchanged in the presence of ISR/FSI-Rapa nanoparticles indicating no
interference in coagulation pathways. Nonetheless, given the low sensitivity of the assay, surface
adsorption and depletion of plasma proteins cannot be excluded. It is known that even 50%
depletion of a given plasma factor will not result in prolonged coagulation time
121
. In fact, ELP
46
nanoparticles of composition (VPGLG)n and (VPGVG)n suspended in plasma assembled a protein
corona partly consisting of fibrinogen, plasminogen, antithrombin, and prothrombin, all of which
play a role in coagulation
122
. However, the effect on clotting time was not found to be
physiologically significant (prolongation > 2-fold versus untreated control is considered
physiologically significant).
Several clinically approved formulations, namely liposomal doxorubicin (Doxil)
111
, liposomal
amphotericin (AmBisome)
123
, and intravenous paclitaxel (Taxol)
1, 3
are inflammatory through
activation of the complement system of proteins
124
. Such immune activation may lead to a severe,
or even fatal condition called complement activation related pseudo-allergy (CARPA)
125, 126
.
CARPA is used to describe hypersensitivity reactions to nanomedicines occurring in up to 45% of
individuals[75]. Hence, its detection at an early stage of nanoparticle research is essential for their
successful development. ISR/FSI-Rapa nanoparticles did not activate the complement system of
proteins even at concentrations as high as 10 times the estimated human plasma concentration at
HED, thereby mitigating risk of CARPA. Equally positive results were observed in the platelet
function assay. As such, ELPs are known for their thromboresistant properties and are being
explored as a blood compatible coating material for vascular grafts
127-129
. Along similar lines,
ISR/FSI-Rapa and related control nanoparticles did not exhibit anticoagulant or thermogenic
properties, nor did they interfere with induced platelet aggregation. Given the good in vitro-in vivo
correlation for all the assays discussed so far
130
, the ELP Rapa nanoparticles are considered less
likely to induce these acute toxicities.
Cellular internalization often provides an opportunity for nanoparticle biodegradation. Compared
to some polymeric and inorganic materials, ELPs are biodegradable and can be cleared from the
47
body. Proteases trypsin
71
, elastase and collagenase
131
can act both intracellularly or extracellularly
to process ELPs. Upon internalization in HepG2 cells, ISR/FSI-Rapa nanoparticles are sequestered
in acidic endosomes and lysosomes which are rich in proteolytic enzymes, including elastase
132

and collagenase
133
. These enzymes may provide an efficient means for their triggered
biodegradation and drug release. Alternatively, high concentration of the nanoparticles in
lysosomes may cause their damage or dysfunction. Additional experiments are required to clarify
these possibilities. An unexplored avenue is the endocytic mechanism for ISR/FSI-Rapa
internalization. Although multiple internalization mechanisms have been reported for ELP based
materials
134-136
, the flexibility of the platform in fabricating particles of varying charge, size and
hydrophobicity makes it difficult to predict uptake mechanisms for a new ELP system. In MDA-
MB-468 cells, an untargeted FKBP-ELP called FAF was found to be internalized by
macropinocytosis
137
. Exploring such mechanisms for ISR/FSI-Rapa may provide additional
insights into possible toxicities.
As previously mentioned, ISR/FSI-Rapa nanoparticles triggered ROS generation in the DCFDA
assay while FSI, ISR and Rapa alone were inert. Induction was rapid (within 1 hour) and appeared
to be associated with a critical incubation concentration, above which ROS production is
detectable. While the underlying mechanisms remain unexplored, lysosomal dysfunction due to
nanoparticle accumulation can trigger ROS generation
138
. Since in vivo models have a higher
likelihood of identifying nanoparticle toxicities, ISR/FSI-Rapa was evaluated in a mouse model of
breast cancer. A month-long administration of nanoparticles was performed at an elevated dose,
which was 10 times higher than an effective dose in our prior report
80
. The effective dose did not
induce any hepatic anti-oxidant defense mechanisms. One possible explanation is that the
nanoparticles do not reach the critical concentration in the liver required for ROS induction.
48
Moreover, no significant immunology findings were noted with respect to hemolysis, complement
activation, plasma coagulation times, or platelet aggregation. No organ level toxicities were
observed either.

49
2.6 Conclusion
This chapter evaluates a panel of safety and efficacy studies that support the continued
development of ELP-based drug carriers. For the first time, this data confirms that ELP-Rapa is
effective at suppressing the mTOR signaling pathway in an orthotopic murine model of HR+ breast
cancer. The data mitigates concerns about complement activation, hemolysis, liver and kidney
toxicity. ELP-based drug carriers may have diverse applications; furthermore, this report suggests
that additional investigation is warranted. However, Chapter 2 (and the nanoparticles described
within) suffers from 3 major limitations: i) ELP-Rapa nanoparticles lack physical stability over
few days. With long-term incubation (>1 day), they are prone to aggregation. This is a concern
with regards to formulation stability and also raises concerns of in vivo spontaneous aggregation
ii) The drug-loading capacity is low (~0.8%), meaning 100 times more carrier (by weight) is
required for drug delivery which is inefficient iii) The in vivo xenograft study lacks a clinically
relevant control. Hence, effective conclusions cannot be made about the relative clinical
significance of these protein-polymer nanoparticles.

50
Chapter 3: Second generation FKBP-ELPs for the delivery of Rapa
and Eve
3.1 Abstract
Rapalogues are a unique class of drugs with both cytostatic and immunosuppressive properties.
Two founding members, Rapamycin (Rapa) and its chemical derivative Everolimus (Eve) are
extremely potent, but their clinical use presents multiple challenges. Being water insoluble,
administration is restricted to the oral route, which results in a low bioavailability of <10%. Human
studies of rapalogues are reported to yield a high blood to plasma ratio and poor correlation
between blood concentration and dose. Moreover, treatment results in dose-limiting toxicities like
stomatitis and pneumonitis, which often leads to discontinuation of therapy. I report an elastin-like
polypeptide (ELP) decorated with two-headed FKBP rapalogue-binding domains. Called ‘FAF,’
this biomacromolecular drug-carrier solubilizes, retargets, and releases rapalogues within disease
sites. FAF-rapalogue formulations are free of co-solvents or surfactants, which promotes their
parenteral administration. Unlike the first-generation FKBP-ELP carriers described in Chapter 1
(ISR/FSI-Rapa nanoparticles), FAF doesn’t assemble nanoparticles at physiological temperature
and hence expected to display high physical stability (over multiple days and weeks). The drug
loading capacity is twice (~1.6%) compared to first generation carriers. When given
subcutaneously (SC) in a mouse model of hormone receptor positive (HR+) breast cancer, FAF-
Rapa significantly suppressed tumor growth compared to an oral formulation of Eve (Affinitor
®
).
Additionally, mTOR, the pharmacological target of rapalogues was inhibited to a greater extent in
tumors of FAF-Rapa and FAF-Eve groups compared to mice that received oral Eve. No signaling
51
suppression was detected in the liver and spleen, which were evaluated to represent off-target
organs exposed to the circulating formulation.

52
3.2 Introduction
The mechanistic Target of Rapamycin (mTOR) is a serine/threonine protein kinase, and a key
downstream effector of the PI3K/AKT pathway
139
. It forms the catalytic subunit of two distinct
multiprotein complexes in the cell: mTORC1 and mTORC2. Upon activation by diverse stimuli,
including growth factors
140
, nutrients
141, 142
, and stress signals
143
, mTORC1 promotes cell growth,
proliferation, and survival by upregulating protein synthesis
144, 145
, lipid
146
, nucleotide
147
, and
glucose metabolism
148
, and suppressing pathways of protein catabolism, primarily autophagy
149
.
In contrast, mTORC2 is insensitive to nutrient and energy signals but instead promotes
proliferation and survival through phosphorylation and activation of AKT, a key effector of
Insulin/PI3K signaling pathway
12
. Targeting a master regulator of cellular physiology, mTOR
inhibitors are relevant to multiple human diseases, including cancer.

In 1964, a team of scientists collected soil samples from the isolated Easter island (also called Rapa
Nui) to find new antimicrobial agents. This resulted in the discovery of Rapamycin (Rapa), a
macrocyclic compound with potent antifungal
150
, immunosuppressive
151
and antitumor
activities
152
. Nearly three decades later, biochemical studies identified mTOR as Rapa’s
pharmacological target
153
. Rapa binds to cytosolic peptidyl-prolyl isomerase FKBP12 with sub-
nanomolar affinity, and this FKBP12-Rapa complex inhibits mTOR allosterically
154
. In cancer
cells, such inhibition arrests their proliferation by delaying G1 to S phase transition
155
, and in T-
cells, mTOR inhibition blocks activation and proliferation in response to antigen stimulation
156
.
Thus, Rapa and its chemical analogs (Rapalogues) are approved for human use as cytostatic agents
and immunosuppressants.

53
Of the several rapalogues currently in clinic and development, Rapa and its 40-O-(2-hydroxyethyl)
derivative Everolimus (Eve) offer the most clinical benefit. Rapa (marketed as Rapamune®) is
approved to prevent acute rejection of kidney transplants and is under evaluation in multiple
clinical trials for cancer and proliferative diseases like tuberous sclerosis. On the other hand, Eve
(marketed as Affinitor®) is approved for eight different conditions but is primarily used to treat
advanced hormone-receptor positive (HR+), HER2- breast cancer
157
. Despite being widely
prescribed, rapalogues present challenges that limit their utility. Firstly, orally administered Rapa
and Eve have poor bioavailability (<10%)
84
due to extensive first-pass metabolism. To address
this, attempts at a safe non-oral formulation have been made but were unsuccessful due to their
water insolubility
4, 115
. Secondly, both Rapa and Eve exhibit sub-optimal pharmacokinetic (PK)
properties, including incomplete absorption, preferential accumulation in red blood cells (blood :
plasma ratio of 5-35), and a large intra- and inter-patient variability in PK parameters
85, 86
. Such
variability coupled with poor correlation between blood concentration and drug dose as well as a
narrow therapeutic index necessitates therapeutic drug monitoring. Thirdly, treatment is associated
with dose-limiting adverse events, the most common being stomatitis with a >40% incidence
90, 91
.
These oral lesions are usually painful, harm quality of life, and lead to interruptions in treatment,
dose reduction and sometimes discontinuation of therapy.

To address some of the above limitations, the Mackay group previously designed a protein-based
drug carrier for rapalogues called ‘FAF’
41
. FAF was generated by fusing both the N- and C- termini
of an elastin-like polypeptide (ELP) linker with FKBP12. The two-headed ‘Berunda polypeptide,’
FAF (Table 3.1) is a 97 kDa protein that remains soluble at physiological temperature (Tt >37
o
C).
FKBP domains in FAF solubilize rapalogues through high-affinity binding (Kd <5 nM) and prevent
54
their precipitation in aqueous solutions. On the other hand, a high molecular weight ELP linker
potentially improves carrier half-life by delaying clearance by the glomerulus. FAF also sequesters
free circulating drug, thereby altering its bio-distribution and reducing toxicity
41, 158
. FAF-
rapalogue formulations are free of organic co-solvents, surfactants and can be safely administered
via non-oral routes. Our group previously reported potent inhibition of tumor growth and an
improved toxicity profile with FAF-Rapa in a mouse xenograft model of triple-negative breast
cancer
75
(HR- HER2-). To build upon these findings, this chapter evaluates two rapalogue
formulations (FAF-Rapa and FAF-Eve) in a mouse model of HR+ breast cancer. This subtype
reflects an FDA approved use of rapalogues in cancer and also accounts for nearly 80% of all
breast cancer cases. This chapter presents the first evidence that FAF binds everolimus;
furthermore, it shows how this formulation compares oral administration of Eve, which is a clinical
standard of care.

55
3.3 Materials and Methods

3.3.1 Recombinant protein purification in E.coli, drug encapsulation and endotoxin testing
Molecular cloning of FAF has been previously described
41, 159
. For recombinant expression,
chemically competent BLR(DE3) cells (Novagen, Madison, WI) were transformed with 50 ng of
pET25b(+) vector encoding FAF following manufacturer’s protocol. Cells were spread on agar
plate containing 100 µg/mL carbenicillin (Gold Biotechnology, St. Louis, MO) and incubated
overnight at 37
o
C. A single colony was aseptically transferred to a 50 mL starter culture, which
was later moved to 1L culture medium. All cultures were grown at 37
o
C in autoclaved Terrific
Broth (TB) medium (Mo Bio Laboratories, Carlsbad, CA) supplemented with 100 µg/mL
carbenicillin. Bacteria was pelleted by centrifugation, resuspended in phosphate buffered saline
(PBS, Caisson, Labs, Smithfield, UT), and lysed by sonication. To precipitate nucleic acids, 50%
(w/v) solution of polyethyleneimine (Sigma Aldrich, St. Louis, MO) was added to a final
concentration of 0.5% (v/v). The lysate was clarified by centrifugation and FAF was purified by
three rounds of inverse transition cycling
101
. To measure its concentration an aliquot of protein
was diluted in 8M Guanidine Hydrochloride (Thermo Fischer Scientific, Waltham, MA) and
loaded on a Nanodrop 2000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA). The
concentration was estimated as follows:
𝐶 =

($
%&'
( $
)*'
)
,
-

where C is the solution concentration (M), A 280 and A350 are absorbances at 280 and 350 nm
respectively, l is the path length (cm) and 𝜀 is the molar absorption coefficient at 280 nm (20,190
cm
-1
for FAF
41
). Protein purity and identity was assessed by SDS-PAGE followed by Coomassie
blue staining (Catalog no: LC6065, Thermo Fischer Scientific, Waltham, MA). Purified FAF was
56
subjected to endotoxin removal using Pierce
TM
endotoxin removal columns (Thermo Fischer
Scientific, Waltham, MA) following the manufacturer’s method for batches.

To characterize phase transition behavior, varying concentrations of FAF (5-100 µM) were heated
from 25 to 75 °C at 1 °C/min on DU800 spectrophotometer (Beckman Coulter, Brea, CA) and
optical density at 350 nm (OD350) was measured. Tt is defined as the temperature at which OD350
(vs) Temperature plot has maximum slope.

For drug encapsulation, 10 mL 150 µM FAF (in PBS) was placed in a round bottom flask and 3x
stoichiometric excess of Rapa (LC Laboratories, Woburn, MA) or Eve (LC Laboratories, Woburn,
MA) was added in a hexane/ethanol mixture (7:3 v/v). The organic phase was removed using a
rotary evaporator and the resulting aqueous suspension was centrifuged at 13,000g to pellet
unbound drug precipitate. The supernatant was dialyzed against PBS (1:300 sample: dialysate) for
12 hours to remove free drug and residual solvent. After another round of endotoxin removal, an
aliquot of FAF-Rapa and FAF-Eve was injected onto a C-18 RP-HPLC column (Waters, Milford,
MA) and drug concentration was quantified at by optical density at 280 nm using a calibrated
standard curve. FAF-Rapa and FAF-Eve were diluted to 100 µM working concentration, sterile
filtered using 200 nm Acrodisc® filters (Pall Corporation, Port Washington, NY) and frozen at -
80
o
C until needed. The concentration of FAF-Rapa and FAF-Eve throughout the chapter refers to
Rapa and Eve concentrations respectively, unless otherwise noted.

57
3.3.2 Dynamic Light Scattering (DLS)
Hydrodynamic radius (Rh) was measured using a DynaPro plate reader (Wyatt Technologies,
Santa Barbara, CA). Briefly, 60 µL samples at 25 µM concentration were added in triplicate to a
384-well clear bottom plate (Greiner Bio One, Monroe, NC). The wells were capped with 15 µL
mineral oil to prevent evaporation during long incubations. Data was analyzed using Dynamics
V7 (Wyatt Technologies, Santa Barbara, CA).

3.3.3 Rhodamine labeling
NHS-Rhodamine (Thermo Fischer Scientific, Waltham, MA) was dissolved in anhydrous DMSO
(Invitrogen, Carlsbad, CA) at 10 mg/ml and frozen as single use aliquots. To 200 µM FAF in PBS,
2x molar excess NHS-Rhodamine was incubated at room temperature for 1 hour. Zeba desalting
columns (Thermo Fischer Scientific, Waltham, MA) were used according to manufacturer’s
protocol to remove unreacted free dye and elute Rho-FAF in PBS. Concentrations of rhodamine
and FAF were calculated using Nanodrop spectrophotometer as follows:

𝐶
:;<
=
$
***
,
-

𝐶
y$y
=
($
%&'
( 9.{|
$
***
)
,
-

where A280 and A555 are absorbance at 280 nm and 555 nm respectively, l is the path length (cm)
and ε is the estimated molar extinction coefficient at 280 nm: 20,190 M-1cm-1 for FAF and 80,000
M-1cm-1 for rhodamine. Labeling efficiency, N, was calculated as:

58
𝑁 =
>
?@A
>
}~}

×100

The purity of Rhodamine-FAF (Rho-FAF) was evaluated by SDS-PAGE electrophoresis followed
by fluorescence imaging on ChemiDocTM (Bio-Rad, Hercules, CA) imaging system. The
concentration of Rho-FAF throughout the chapter refers to rhodamine concentration unless
otherwise specified.

3.3.4 Cold competition binding assay and live cell imaging
3x105 MDA-MB-468 cells were seeded on a 35 mm glass bottom dish (MatTek Corporation,
Ashland, MA) and allowed to attach overnight. After 24 hours, culture medium was replaced with
750 µL fresh medium supplemented with 25 mM HEPES. The dish was placed on ice and pre-
chilled Rho-FAF was added to a final concentration of 20 µΜ. After a 2-hour incubation, a 10-
fold excess of unlabeled FAF or an equal volume of cold PBS was added and incubated for another
2 hours. Cells were washed three times with PBS and 1 mL live cell imaging solution (Life
Technologies, Carlsbad, CA) was added to the dish with 2 drops of NucBluTM reagent (Life
Technologies, Carlsbad, CA). Images were captured using LSM800 confocal microscope (Carl
Zeiss Microscopy, Thornwood, NY) mounted on a vibration-free table with a Plan-Apochromat
63x oil objective. Integrated fluorescence intensities were measured by drawing regions of interest
(ROI) on ImageJ software (NIH, Bethesda, MD).

3.3.5 Concentration-dependence of cellular association
MDA-MB-468 cells were seeded in triplicate on a black bottom 96-well plate (Greiner Bio One,
Monroe, NC) at a density of 10,000 cells/well and allowed to attach overnight. Next, culture
59
medium was replaced with 100 µL fresh medium containing 1-100 µM Rho-FAF. After 16 hours
incubation at 37 °C and 5% CO2, medium was aspirated, and cells were washed three times with
PBS. 100 µL live cell imaging solution was added to each well and total fluorescence intensity
was measured using a Synergy H1 plate reader (BioTek, Winooski, VT).

3.3.6 Cellular uptake and co-localization analysis
3x10
5
MDA-MB-468 cells were seeded in a 35 mm glass bottom dish and allowed to attach
overnight. The next morning, culture medium was replaced with 1 mL fresh medium containing
either 30 µM Rho-FAF, 20 µM Fluorescein-dextran (70 kDa MW, Life Technologies, Carlsbad,
CA), 20 µM Rhodamine B-dextran (70 kDa MW, Life Technologies, Carlsbad, CA), or both 30
µM Rho-FAF and 20 µM Fluorescein-dextran (FL-dextran). After an 8-hour treatment, medium
was aspirated, and cells were washed three times with PBS. 1 mL live cell imaging solution was
added to each dish with 2 drops of NucBluTM reagent. When applicable, 1 µL LysoTrackerTM
Green DND-26 (LTG, Life Technologies, Carlsbad, CA) was added. Images were captured as
described above. For co-localization analysis, Mander’s Co-localization Coefficient (MCC) was
calculated for both red and green channel using ZEN2009 software (Carl Ziess Microscopy,
Thornwood, NY) using following equations

𝑀
:de
=
∑ :
g,iAjAi g
∑ :
g g
𝑀
klddm
=
∑ k
g,iAjAi g
∑ k
g g

3.3.7 Kinetics of cellular uptake and degradation
Cells were seeded in 35 mm dishes as previously described and treated with 30 µM Rho-FAF or
30 µM Rho-FAF-Rapa for 1, 4, 8, 16 and 24 hr at 37 ºC. At each time point, cells were washed,
60
imaged, and analyzed as described. To assess cellular degradation following pulsed incubation,
cells treated with 30 µM Rho-FAF or 30 µM Rho-FAF-Rapa for 16 hours at 37 ºC. After that,
culture medium was replaced with fresh medium and cells were imaged at 0, 1, 8, 24, 48 and 72
hrs as described above. After imaging, cells were washed with PBS and lysed using 120 µL RIPA
buffer (Thermo Fisher Scientific, Waltham, MA) containing 1x protease/phosphatase inhibitor
cocktail (Cell Signaling Technology, Danvers, MA). Total protein concentration in each cell lysate
was determined by BCA assay (Thermo Fisher Scientific, Waltham, MA) following
manufacturer’s protocol and 22.5 µg total protein was loaded in each well of a 4-20% Tris-Glycine
gel. Following SDS-PAGE, the gel was imaged on ChemiDocTM imaging system.

3.3.8 Split luciferase assay
30,000 cells/well were seeded in triplicate in a white opaque flat bottom 96-well plate (Greiner
Bio One, Monroe, NC) and allowed to attach overnight. The next morning, medium was aspirated
and 90 µL/well Opti-MEM medium (Thermo Fisher Scientific, Waltham, MA) was added. Each
well was transfected with 50 ng FKBP-SmBit and 50 ng FRB-LgBit (Promega, Madison, WI)
using 0.3 µL/well Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA) transfection
reagent according to manufacturer’s protocol. One-day post transfection, cells were treated with 1
mM amiloride (Sigma-Aldrich, St. Louis, MO) for 30 minutes. Following the above treatment, 25
µL/well Nano-GloÒ luciferase substrate (Promega, Madison, WI) was added and the plate was
equilibrated to room temperature. Cells were treated with either 30 nM Rapa/DMSO or 30 nM
FAF-Rapa and immediately placed in a plate reader to measure luminescence with 1 sec integration
time. Measurements were taken in kinetic mode for 2 hours at 30-second intervals.

61
3.3.9 Cell culture and in vitro cytotoxicity assay
The BT-474 cell line (ATCC, Manassas, VA) was cultured in Hybri-Care medium (ATCC,
Manassas, VA) supplemented with 10% heat inactivated fetal bovine serum (Corning Life
Sciences, Tewksbury, MA) at 37
o
C in a humidified incubator with 5% CO2. For formazan viability
assays, 3x10
3
cells/well were added to a 96-well tissue culture plate. Following cell attachment
overnight, the media was exchanged for multiple dilutions of Rapa, Eve, FAF-Rapa or FAF-Eve
in complete medium. After 4 days of drug treatment, 10 µL/well WST-1 (4-[3-(4-Iodophenyl)-2-
(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) reagent (Sigma Aldrich, St. Louis, MO)
was added and incubated for 3 hours. Absorbance at 440 and 690 nm was measured using a
Synergy H1 plate reader (BioTek, Winooski, VT) and % maximum proliferation (%P) was
calculated as follows:
%𝑃 =

($
pqrsprt
( $
'
)
($
uvpqrsprt
( $
'
)

where Atreated, Auntreated and A0 are background corrected absorbances of drug treated, untreated and
media only wells respectively. %P was normalized to extend from 0 to 100% using normalize
function of GraphPad Prism. Dose response curves were fit by non-linear regression and IC 50 was
estimated in 3 independent assays. One-way ANOVA followed by the Tukey post-hoc test was
used to test differences between groups.

3.3.10 Mouse xenograft model and drug treatment
All animal procedures were performed by Washington Biotechnology Inc. (Baltimore, MD)
following approval by their Institutional Animal Care and Use Committee. Eight week old female
athymic nude mice (Envigo, Fredrick, MD) received a 17-β-estradiol pellet (Innovative Research
of America, Sarasota, FL) subcutaneously (SC). The next day, BT-474 cells were harvested and
62
reconstituted in PBS with 20% Matrigel®(Corning Life Sciences, Tewksbury, MA) at 2 x 10
8

cells/mL density. 50 µL of the cell suspension was injected SC to the mammary fat pad of each
mouse. Tumor volumes were measured using calipers as:

𝑉 =

\
%
×
a
w

where V, W and L are the volume, width, and length of the tumor respectively. When the tumors
reached an average size of 75 mm
3
, mice were randomized into 3 groups of 8 mice each. To prepare
an oral Eve formulation, Affinitor® tablets were crushed in a ceramic grinder to a fine powder,
suspended in sterile water, and aliquoted while vortexing vigorously. Mice were treated on
Monday, Wednesday and Friday for 4 weeks at 1 mg rapalogue / kg body weight. FAF-Rapa and
FAF-Eve were administered subcutaneously. Oral Eve was administered by oral gavage. Two days
after the last treatment, mice were euthanized, organs were harvested and flash frozen.

3.3.11 Isothermal Titration Calorimetry (ITC)
ITC measurements were performed on a MicroCal PEAQ ITC (Malvern Instruments,
Northampton, MA) at 37
o
C. The calorimeter cell was filled with 8 µM Rapa or Eve dissolved in
PBS with 1% DMSO. The titration syringe was filled with 50 µM FAF in PBS with 1% DMSO.
Twelve 3 µL injections ensured complete saturation of binding sites. The resulting isotherm was
fit to a ‘one set of sites’ binding model using MicroCal ITC analysis software (Malvern
Instruments, Northampton, MA) and various thermodynamic parameters were estimated.

3.3.12 Western blotting
Approximately 25 mg of frozen mouse tissue was moved to tubes pre-filled with zirconium beads
(Catalog no: D1032, Benchmark Scientific, Sayreville, NJ). To each tube, 500 µL RIPA buffer
63
(Thermo Fischer Scientific, Waltham, MA) supplemented with 1x protease phosphatase inhibitor
cocktail (Catalog no: 78440, Thermo Fischer Scientific, Waltham, MA) was added and tissue was
homogenized using a bead homogenizer. Tissue debris was discarded by centrifugation and protein
concentration in lysate was measured by BCA assay (Catalog no: 23225, Thermo Fischer
Scientific, Waltham, MA). Samples (20 µg each) were resolved by SDS-PAGE and transferred to
nitrocellulose membrane (Catalog no: IB23001, Thermo Fischer Scientific, Waltham, MA) by
iBlot® dry blotting system. Membrane was blocked using 5% milk in tris-buffered saline with 0.1%
tween-20 pH 7.4 (TBST) and primary antibodies for P-S6RP (Catalog no: 2211, Cell Signaling
Technology, Danvers, MA) and GAPDH (Catalog no: 2118, Cell Signaling Technology, Danvers,
MA) were used as recommended. Imaging was performed using a ChemiDoc® (Biorad, Hercules,
CA) after exposing the blot to ECL substrate (Catalog no: 20-301, Genesee Scientific, San Diego,
CA).

3.3.13 Drug quantification by LC-MS/MS
LC-MS/MS based drug quantification was performed by the Scripps Center for Metabolomics and
Mass Spectrometry (La Jolla, CA). To 15-25 mg tumor tissue, 1 ml 4:1 (v/v) methanol/water
mixture was added and homogenized to facilitate drug extraction. The homogenate was
centrifuged, the organic extract was evaporated and reconstituted with 100 µL methanol containing
500 pg/µL tacrolimus, which served as an internal standard. Analysis was carried out on an Agilent
6495 triple quadrupole mass spectrometer. Standard curves were found to be linear from 5 pg/µL
to 500 pg/µL for both Rapa and Eve.

64
3.4 Results

3.4.1 Physicochemical characterization of FAF-Rapa and FAF-Eve
FAF was purified from E.coli by 3 rounds of inverse transition cycling, a non-chromatographic
protein purification technique utilizing responsiveness of ELPs to heat and salt concentration.
Protein with >95% purity (Figure 3.1B) and a yield of 80-100 mg/L bacterial culture was obtained
this way. The SDS-PAGE band for FAF matched its theoretical molecular weight of 97 kDa.

To confirm that FAF remains soluble at all temperatures evaluated herein, the optical density of
FAF solutions was measured as a function of temperature at various concentrations (Figure 3.1D).
The lower critical solution temperature (LCST) phase separation of ELPs is well studied
160, 161
.
As temperature increases, ELPs lose the water network solvating the polypeptide backbone
through entropy driven processes thereby increasing their hydrophobicity. This is accompanied by
a gradual conformational change towards more ordered secondary structures, predominantly type-
2 b spirals. Together, they result in ELP assembly and coacervation over a narrow temperature
range, usually 1-2 ºC, which can be detected as a sharp increase in solution turbidity. FAF exhibits
concentration dependent phase transition properties with the Tt dropping slightly from 60 ºC to 54
ºC as concentration increases from 5 µM to 100 µM (Figure 3.1E). Hence, at experimental
temperature of 37 ºC, FAF remains soluble across all the concentrations relevant to this chapter.

Endotoxin removal was performed prior to drug encapsulation using a two-phase evaporation
method as described above. Unbound drug and trace organic solvents were removed by
centrifugation, filtration and extensive dialysis. The drug loading ratio observed for FAF-Rapa
65
(CRapa/C FAF) and FAF-Eve (CEve/C FAF) were 1.8 and 1.7 respectively, which suggests most of the
FKBP domains in the formulation are bound to rapalogues. This translates to a drug loading
capacity of ~1.7% for both FAF-Rapa and FAF-Eve. After encapsulation, another round of
endotoxin removal was performed by poly-lysine chromatography and the residual endotoxin
burden was measured by FDA recommended chromogenic LAL assay. When samples were spiked
with 1.56 EU/ml endotoxin and tested, spike recovery was within the acceptable 50-200% range
thereby indicating no assay interference from FAF. At the injection concentration (100 µM
rapalogue), the endotoxin load was 6.6 and 29 EU/mL for FAF-Rapa and FAF-Eve respectively.
Since protein formulations are susceptible to misfolding and aggregation, assessing their physical
stability is crucial. When measured by DLS, FAF-Rapa and FAF-Eve retained a stable Rh of ~8
nm over a period of 2 days at 37
o
C (Figure 3.1C). No signs of higher order structures were
detected. This also confirmed that FAF-Rapa and FAF-Eve remain soluble at physiological
temperature since the coacervate phase typically contains particles of few hundreds of nanometers
in size.
66

Figure 3.1: Physicochemical characterization of FAF-Rapa and FAF-Eve. A) A schematic
representation of FAF loaded with Rapa and Eve. FKBP12 is fused to N- and C- termini of a linker
peptide (VPGAG)192. FAF is a monomeric protein that can solubilize rapalogues through high-
affinity binding with its FKBP domains. B) SDS-PAGE gel was stained with Coomassie to verify
purity and identity of fusion proteins. FAF remained intact without loss in purity through drug
encapsulation. C) DLS was used to measure hydrodynamic radii (Rh) and physical stability of
formulations. After 0 or 2 days of incubation at 37
o
C at 25 µM, the Rh for both FAF-Rapa and
FAF-Eve remained approximately 8 nm, which corresponds to that of a high molecular weight
monomeric protein. This size also confirms that these ELPs do not coacervate at 37
o
C, nor does
insoluble drug precipitate over this periods relevant to their dosing interval via SC administration.
(Mean ± SD. n=3) D) Optical density vs. temperature profile for 25 µM FAF and control ELP
A192 E) The transition temperature (Tt) of FAF was found to be concentration dependent and can
be fit to a Log-linear model, Tt = b – m [Log10 (Concentration)] where b represents the Tt at 1
µM, which is 63.8 ± 2.1 ºC, m represents the Tt change for a 10-fold change in concentration which
is 4.7 ± 1.5 ºC. These data show that FAF remains soluble at 37 °C. b, m and error bands in the
figure represent the Mean ± 95% CI.

67

Table 3.1: Physiochemical characterization of ELP polymers evaluated in this chapter

Label Amino acid sequence MW
(kDa)
a

Purity
(%)
b
Tt
(°C)
c

FAF MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRD
RNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYG
ATGHPGIIPPHATLVFDVELLKLEG(VPGAG)192VQVETISPGDG
RTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQE
VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATL
VFDVELLKLEG
97.0 98.5 57.8
A192 MG(VPGAG)192Y 73.6 95.6 62.1

a
Expected molecular weight based on amino acid sequence
b
Purity was determined by densitometry analysis of stained SDS-PAGE gel
c
Tt here is reported for 25 µM samples suspended in phosphate buffered saline (PBS)

Additional characterization included the use of calorimetry to study binding of rapalogues to FAF
(Figure 3.2). Due to their limited aqueous solubility, Rapa or Eve were added to the calorimeter
cell and titrated against FAF. For model fitting, it was assumed that both the FKBP domains in
FAF are equivalent. Saturation of FAF binding occurred at [FAF]/[drug] of ~0.5 which translates
to a binding stoichiometry of ~2. Hence, both the FKBP domains in FAF are functional and can
bind to rapalogues, which is consistent with the observed drug loading ratios, also ~2. Additionally,
FAF exhibited equivalent affinity for both Rapa and Eve since the estimated equilibrium binding
constants (Kd) estimated were of similar magnitude (4.7 nM for FAF-Rapa vs. 2.5 nM for FAF-
Eve). In terms of thermodynamics, FAF-rapalogue binding was exothermic with an enthalpy of
binding (ΔH) of -119 kJ/mol and -114 kJ/mol for FAF-Rapa and FAF-Eve respectively. The best
estimate for the entropy component (-TΔS) of binding was 69 kJ/mol and 63 kJ/mol for FAF-Rapa
and FAF-Eve respectively. Since –TΔS is positive, the overall entropy of the system reduces upon
rapalogue binding. This is expected since Rapa and Eve transition from a free to FKBP-bound
68
form. The spontaneity of binding is dictated by change in Gibbs free energy (ΔG), which in turn
is the sum of change in enthalpy (ΔH) and change in entropy (–TΔS). For both FAF-Rapa and
FAF-Eve, ΔG is negative (-50 and -51 kJ/mol for FAF-Rapa and FAF-Eve respectively) indicating
thermodynamically favorable processes.

Figure 3.2: FAF binds both Rapa and Eve at a stoichiometric ratio as determined by
isothermal titration calorimetry. A,B) 8 µM Rapa or Eve in the calorimeter cell was titrated
against 12 injections of 50 µM FAF in the syringe. The titration curves (upper panel) clearly reveal
saturation of binding sites towards the end of the titration. The binding isotherms (lower panel)
were fit assuming both the FKBP domains in FAF are equivalent. The best-fit values for
stoichiometry of binding (n) and various thermodynamic parameters are reported on the isotherm
and discussed in section 3.1.

69
3.4.2 Rhodamine labeling and Rapa encapsulation
To enable tracking by fluorescence microscopy and study cellular uptake, I labeled FAF with
rhodamine fluorophore. NHS activated rhodamine tagged lysine side chains in FAF through an
amide linkage. Incubation with two-fold excess dye yielded an efficiency of 160%, indicating an
average of 1.6 rhodamine molecules in each FAF molecule. Labeling strategies generally impact
substrate properties, thereby rendering good quality control practices essential. Since trace
amounts of unreacted dye in purified Rho-FAF can cause experimental artifacts, I tested our
purification technique using SDS-PAGE followed by fluorescence imaging (Figure 3.3A). Rho-
FAF was completely free of unreacted dye and any other impurities. To ensure labeling did not
induce any physical instability or aggregation, hydrodynamic radii (Rh) were measured using DLS.
At 37 ºC, FAF and Rho-FAF displayed a 7.8 nm radius with no significant differences in size
(Figure 3.3B). A working formulation of Rapa bound Rho-FAF (Rho-FAF-Rapa) was prepared
by two-phase encapsulation as described earlier. After removal of unbound Rapa, HPLC was used
to quantify solution concentrations of Rho-FAF and Rapa. Encapsulation ratio (ER), defined as
the ratio of Rapa to Rho-FAF concentration was found to be 1.8 ± 0.2 (n = 3, Mean ± SD). Since
ER is close to 2, I can conclude that both the FKBPs in Rho-FAF can bind and solubilize Rapa.
This further suggests the structure of FAF remains properly folded after rhodamine labeling.

70

Figure 3.3: Rhodamine-labeled FAF with and without Rapamycin is pure and stable: FAF
was labelled with NHS-Rhodamine and then used to encapsulate rapamycin. A) SDS-PAGE
imaged by fluorescence indicates successful labeling and complete removal of unreacted dye. B)
Dynamic light scattering was used to estimate the hydrodynamic radii of FAF, Rho-FAF and Rho-
FAF-Rapa. No significant differences between groups was observed, which suggests that neither
rhodamine labeling nor drug encapsulation induced aggregation or physical instability. (Mean ±
SD)

3.4.3 Cellular uptake of Rho-FAF is receptor independent
When MDA-MB-468 cells were incubated with Rho-FAF at 4 ºC, a bright ring staining pattern on
the cell surface was observed (Figure 3.4A). At 4 ºC, all endocytic pathways are arrested with
minimal disruption of affinity driven binding, thereby allowing detection of binding events at the
plasma membrane. When competed with ten-fold molar excess unlabeled FAF, there was no
significant decrease in cell surface rhodamine staining and the integrated fluorescence intensity
per cell remained constant (Figure 3.4B). This suggests absence of a specific receptor that can
bind to and mediate FAF internalization. Concentration-dependent cellular uptake of Rho-FAF
was next determined. A defining feature of receptor-mediated endocytosis is saturability at high
ligand concentrations. When cells were incubated with 1-100 µM Rho-FAF for a constant time
period, the total amount of internalized protein increased linearly with concentration (R2 = 0.9)
71
with no evidence of saturation (Figure 3.4C). This further supports a receptor-independent cellular
uptake mechanism for FAF.

Figure 3.4: Cellular association of Rho-FAF is non-saturable: A,B) MDA-MB-468 cells were
pre-incubated in complete media with Rho-FAF (20 µM) on ice for 2 hours, supplemented with
either unlabeled FAF (125 µM) or an equal volume of PBS, and incubated again for 2-hour on ice.
Cells were washed and live cell imaging was then performed using laser scanning confocal
microscopy. Red: Rho-FAF; Blue: Hoechst 33342; Scale bar: 20 µm. The integrated fluorescence
intensity per cell was determined using ImageJ (n=32, Mean ± SD). Excess unlabeled FAF was
unable to displace Rho-FAF. C) MDA-MB-468 cells in complete media were treated with
increasing concentrations of Rho-FAF at 37 °C for 16 hr, washed, and the total fluorescence
intensity was quantified using a plate reader. The cell-associated fluorescence was linearly related
to the incubation concentration, r
2
= 0.9. The apparent non-saturation of cellular association is
consistent with macropinocytosis. (n=4-6, Mean ± SD). These images were acquired by Xiaoli
Pan.

72
3.4.4 Cellular uptake by macropinocytosis and translocation to low pH compartments
Macropinocytosis is an ATP dependent, non-specific uptake of extracellular fluid and solutes
through membrane protrusions that collapse onto and fuse with the cell membrane thereby
generating large endocytic vesicles called macropinosomes
162
. FITC-dextran is a widely used
marker for macropinocytosis. When co-incubated with Rho-FAF and imaged using confocal
microscopy, equivalent cellular distribution of both Rho-FAF and FITC-dextran was observed
(Figure 3.5A). Consequently, superimposed images had abundant yellow pixels, consistent with
colocalization. To quantify colocalization, the MCC for Rho-FAF (red channel) and FITC-dextran
(green channel) were estimated as 0.77 ± 0.12 and 0.76 ± 0.11 respectively, which reflect a high
degree of cellular co-localization (Figure 3.5B). No leakage of FITC fluorescence into rhodamine
channel was detected when cells incubated with FITC-dextran only were imaged. These results
strongly support macropinocytosis as the mechanism for FAF internalization. To study the
intracellular fate of FAF, Lyso Tracker Green (LTG) was used to label low pH organelles including
lysosomes (Figure 3.6A). The MCC for Rho-FAF and LTG were 0.78 ± 0.22 and 0.24 ± 0.13
respectively (Figure 3.6B), suggesting a large proportion of cellular Rho-FAF is associated with
a small subset of acidic organelles. These organelles may play a role in FAF degradation or drug
release from FAF-Rapa complexes. Similar cellular distribution and co-localization coefficients
were obtained with the positive control Rho-dextran (Figure 3.6A,B), which is known to
translocate to lysosomes after macropinocytosis.
73

Figure 3.5: Rho-FAF and FL-dextran display equivalent distribution after cellular uptake.
A) MDA-MB-468 cells were incubated with FL-dextran at 37 °C in complete media, to mark
macropinocytotic uptake for 8 hours, washed with PBS, and imaged by laser scanning confocal
microscopy. When cells were co-incubated with Rho-FAF (30 µM) and FL-dextran (20 µM),
images showed high co-localization between red and green channels, which is consistent with
macropinocytosis as the mechanism for Rho-FAF internalization. Red: Rho-FAF; Green: FL-
dextran; Blue: Hoechst 33342. B) Mander’s Correlation Coefficient (MCC) for obtained in
individual cells were averaged to 0.77 ± 0.12, 0.76 ± 0.11 for Rho-FAF, FL-dextran respectively
(n=16, Mean ± SD). These images were acquired by Xiaoli Pan.

74

Figure 3.6: Both Rho-FAF and Rho-dextran localize to low pH compartments upon cellular
uptake. A) MDA-MB-468 cells were either incubated in complete media with Rho-FAF or RhoB-
dextran for 8 hours followed by addition of Lyso-Tracker Green (LTG) before laser scanning
confocal imaging. Images show high overlap of the red channel within pixels positive in the green
channel, which indicates that both Rho-FAF and Rho B-dextran accumulation in low pH
compartments. While a high degree of Rho-FAF and Rho B-dextran colocalized with LTG, a
significant amount of low pH compartments do not contain Rho-FAF or RhoB-dextran. Both
observations are consistent with macropinocytic vesicles. Red: Rho-FAF or RhoB-dextran; Green:
LTG; Blue: Hoechst 33342. B) Co-localization analysis in individual cells revealed a shared
pattern wherein a large fraction of intracellular FAF and dextran were associated with a small
fraction of acidic organelles. (n=12, Mean ± SD). These images were acquired by Xiaoli Pan.

75
3.4.5 Time-dependent cellular uptake and cellular degradation
To evaluate the kinetics of uptake, cells incubated with Rho-FAF or Rho-FAF-Rapa for 1 to 24
hours were imaged (Figure 3.7A). Increasing incubation time consistently resulted in higher
uptake in both the groups indicating faster internalization than degradation throughout the
timescale measured (Figure 3.7B). To study degradation kinetics, cells were pulsed with Rho-
FAF or Rho-FAF-Rapa and imaged up to 3 days after withdrawal of treatment. Confocal images
clearly show decreasing cell associated fluorescence with time (Figure 3.8A). The plot of
fluorescence intensity/cell against time followed a one-phase decay and the cellular half-lives of
Rho-FAF and Rho-FAF-Rapa were estimated to be 18 and 21 hours respectively (Figure 3.8B).
After imaging, cells were lysed, and the lysate was resolved using SDS-PAGE followed by
fluorescence imaging. Consistent with microscopy data, Rho-FAF band intensity diminished with
time with concomitant appearance of some low molecular weight bands (Figure 3.8C),
presumably intermediate degradation products. Lysate of untreated cells served as a negative
control and did not show any detectable bands.

76

Figure 3.7: The kinetics of cellular association of Rho-FAF is minimally affected by addition
of Rapa. A) MDA-MB-468 cells were incubated with Rho-FAF (30 µM) in complete media at
37 °C, washed at the indicated time points, and imaged using laser scanning confocal microscopy.
Increased fluorescence signal was observed with longer incubation time points; however, the
addition of Rapa had no dramatic effect on the kinetics of cellular uptake. B) At least 3 images of
each time point were analyzed for fluorescence intensity and cell number. Fluorescence
intensity/cell against time profile showed time-dependent uptake. (n=3-7, Mean ± SD). These
images were acquired by Xiaoli Pan.

77

Figure 3.8: Loss of cellular Rho-FAF is minimally affected by addition of Rapa. A) MDA-
MB-468 cells were pulse incubated with either Rho-FAF or Rho-FAF-Rapa for 16 hours at 37 °C.
After multiple washes, cells were incubated in complete media for the indicated time points and
imaged using laser scanning confocal microscopy. A decrease in fluorescence was observed with
time consistent with the degradation, dilution, or cellular export of FAF. B) Fluorescence intensity
per cell was measured using image analysis and plotted against time (n= 3-7, Mean ± SD).
Degradation followed one-phase decay with a half-life of 17.7 hours (15.6 - 20.4 hours, 95% CI)
and 21.3 hours (17.7 - 26.5 hours, 95% CI) for Rho-FAF and Rho-FAF-Rapa respectively. C) Cell-
associated fluorescence was recovered from cells and observed by SDS-PAGE. The band for Rho-
FAF diminished with time, along with multiple low molecular weight species. The estimated half-
life for Rho-FAF using densitometry was 19.0 hours (15.4-21.6 hours, 95% CI), which is in close
agreement with confocal images. These images were acquired by Xiaoli Pan.

3.4.6 Split luciferase assay
To study the release mechanisms of Rapa from FAF-Rapa, cells were co-transfected with plasmids
encoding FKBP-SmBit and FRB-LgBit. These fusion proteins together function as a sensor for
cytoplasmic Rapa. Upon access to the cytoplasm, free Rapa induces dimerization of FKBP and
78
FRB domains, thereby constituting a functional luciferase enzyme. In the presence of furimazine,
luciferase catalyzes its conversion to furimamide with concomitant emission of luminescence.
Treatment of cells with free Rapa resulted in a rapid luciferase activity with Rapa diffusing across
the plasma membrane even prior to obtaining the first measurement. On the other hand, cells
treated with FAF-Rapa produced luminescence with a very clear lag time of approximately 30
minutes, which suggests a different, slower mechanism of drug release to the cytoplasm compared
to free Rapa (Figure 3.9A). To better understand the underlying mechanisms, I repeated the assay
using cells pre-treated with various inhibitors. Amiloride, a selective inhibitor of macropinocytosis
163
completely arrested Rapa release from FAF with no significant effect on uptake of free Rapa
(Figure 3.9B). Considered together, these results provide three conclusions i) FAF-Rapa
complexes retain high affinity binding in media and do not undergo burst extracellular drug release;
ii) FAF traffics to low pH compartments, which may include lysosomes; and iii) macropinocytosis
of FAF-Rapa complexes is required for Rapa release to the cytoplasm.

79

Figure 3.9: FAF delays the access of Rapa to the cytosol in a manner consistent with
macropinocytosis. A) MDA-MB-468 cells were transfected with a split luciferase reporter that
enables the specific detection of Rapa within the cytosol. When incubated with cells, free drug
resulted in rapid luciferase activity consistent with diffusion across the plasma membrane as the
mechanism of cellular entry. In contrast, FAF-Rapa produced luminescence only after 30-minutes,
a period of time consistent with cellular uptake. B) Addition of the macropinocytosis inhibitor
amiloride completely blocks cytosolic detection of Rapa from FAF-Rapa. Free Rapa luminescence
kinetics were unaffected by amiloride. (Mean ± 95% CI, n=6)

3.4.7 In vitro cytotoxicity and BT-474 xenograft study
In a proliferation assay with the BT-474 cell line (Figure 3.10A), both FAF-Rapa and FAF-Eve
potently inhibited cell division in a dose-dependent fashion with an IC50 of 0.13 ± 0.05 nM (Mean
± SD, n=3) and 0.18 ± 0.06 nM (Mean ± SD, n=3) respectively. In the same model, free Eve
displayed a significantly higher IC50 of 2.2 ± 0.9 nM (Mean ± SD, n=3). This is in agreement with
previous observations of FAF-Rapa being ~6 times more potent than free Rapa in MDA-MB-468
cells
41
. In complete medium, free rapalogues are highly bound to serum albumin which reduces
their effective concentration available for pharmacological inhibition. FAF on the other hand
arrests albumin partitioning by binding to rapalogues with low nanomolar affinity. This likely
improves drug cellular bioavailability, which results in lower IC50s. In conclusion, both Rapa and
Eve retained their pharmacological activity when complexed with FAF without loss in potency.
As expected, FAF and A192 by itself were inactive (Figure 3.10B)
80

Figure 3.10: Rapalogues bound to FAF retain cytostatic activity against BT-474 cells: A) BT-
474 cells were treated with varying concentrations (100 nM – 6 pM) of Eve, FAF-Eve and FAF-
Rapa. After 4 days the percent of maximum proliferation was determined using the WST assay,
which was plotted against drug concentration. Non-linear regression analysis revealed an IC50 of
0.13 ± 0.05 nM (Mean ± SD, n=3) and 0.18 ± 0.06 nM (Mean ± SD, n=3) respectively for FAF-
Rapa and FAF-Eve respectively. Free Eve on the other hand had a significantly higher IC50 of 2.2
± 0.9 nM (Mean ± SD, n=3) compared to FAF-Eve (Global ANOVA/Tukey post-hoc test, α = 0.05,
p = 0.02). B) FAF carrier only and ELP only A192 controls did not affect cellular proliferation,
which supports their biocompatibility.

BT-474 is an ER+ HER2+ human breast epithelial cell line. Being ER+, these cells were selected
for study because they represent the HR+ subtype of breast cancer for which Eve is currently an
approved treatment. The goal of this study was to determine if at an equivalent dose, if either oral
Eve or FAF complexed to Eve have better pharmacological activity than FAF complexed with
81
Rapa at an equivalent dose. In the xenograft study (Figure 3.11A), mice with BT-474 tumors were
treated 3 times a week for 4 weeks with either oral Eve, or SC FAF-Eve, or SC FAF-Rapa (1mg/kg
body weight drug dose in all cases). On the last day of therapy, tumor volumes measured were 349
± 305 mm
3
(Mean ± SD, n=8), 186 ± 92 mm
3
(Mean ± SD, n=8), and 96 ± 56 mm
3
(Mean ± SD,
n=8) in the Oral Eve, FAF-Eve and FAF-Rapa groups respectively. Tumors in FAF-Rapa group
were significantly smaller (Figure 3.11B) than oral Eve group (p=0.03, One-way ANOVA with
Tukey’s multiple comparison test, α = 0.05). No other comparisons were statistically significant.
No significant loss of body weight was observed compared to at the start of treatment suggesting
all treatments were reasonably well tolerated (Figure 3.11C). This study design lacks a PBS (non-
treated) group since the goal was to evaluate two novel rapalogue formulations compared to a
clinically relevant control, oral Eve in this case.

A well-established downstream marker for mTOR kinase activity is the phosphorylation status of
S6 ribosomal protein (S6RP). To activate proteins involved in RNA translation and protein
synthesis, mTORC1 phosphorylates S6 kinase1 (S6K1) which in turn phosphorylates its substrate
S6RP. Hence, loss of S6RP phosphorylation indicates inhibition of mTORC1 activity. At the end
of the xenograft study, total protein from tumors was extracted and probed for Phospho-S6RP by
western blotting (Figure 3.11D). In the oral Eve group, 4 out of 5 randomly chosen tumors had
detectable levels of P-S6RP while none in the FAF-Eve, nor FAF-Rapa groups were positive for
P-S6RP. GAPDH served as a total protein loading control. These results suggest that rapalogues
are able to concentrate better in tumor tissues when delivered SC with FAF, as opposed to oral
administration. In fact, when drug concentration was quantified by LC-MS/MS two days after the
last day of treatment (Figure 3.11E), no Eve could be detected in tumors of 5 mice that received
82
oral Eve (Limit of detection = 5 pg/µL). In the FAF-Eve group, 2/5 mice had 32 and 36 pg/mg Eve
in their tumors while the other 3 remained undetectable. On the contrary, Rapa concentration in
FAF-Rapa group was 96 ± 40 pg/mg tissue (Mean ± SD, n=5), with all the tumors accumulating
detectable Rapa. These results are consistent with low oral bioavailability of Eve and can also
explain the relative potency of the SC FAF-Rapa formulation in suppressing tumor growth.
Although drug in the range of 30 - 100 pg/mg appears insignificant, it translates to ~ 30 -100 nM
for rapalogues (assuming a tissue density of 1 g/mL), which is orders of magnitude higher than
their IC 50 (Figure 3.10A)

83

Figure 3.11: FAF-Rapa significantly arrests tumor growth compared to oral Eve. A) Female
athymic nude mice (8/group) received orthotopic implantation of BT-474 cells to the mammary
fat pad. When the average tumor volume reached 75 mm
3
, mice received either oral Eve, SC FAF-
Eve, or SC FAF-Rapa at 1 mg rapalogue /kg body weight on Monday, Wednesday and Friday for
4 weeks. Tumor volumes (Mean + SD, n=8 per group) were estimated by calipers. B) On the last
day of treatment, tumor volumes were 349 ± 305 mm
3
, 186 ± 92 mm
3
, and 96 ± 56 mm
3
in the oral
Eve, FAF-Eve and FAF-Rapa groups respectively. The difference was statistically significant for
FAF-Rapa compared to oral Eve (p=0.03, One-way ANOVA with Tukey’s multiple comparison
test, α = 0.05). C) Body weights of mice were tracked to assess treatment associated toxicity. All
treatments were well tolerated with no significant difference in body weights between groups. D)
Two days after the last treatment, tumors were collected and signaling related to mTOR was
assessed by western blotting. In the oral Eve group, 4 out of 5 randomly chosen tumors were
positive for P-S6RP, while all the tumors in the FAF-Eve and FAF-Rapa groups had undetectable
P-S6RP (n=5). GAPDH served as a total protein loading control. The same oral Eve tumor lysates
were assayed in the top and bottom blots, and images are representative of 3 independent repeats.
E) Tumor rapalogue concentration was determined by LC-MS/MS. For statistical comparison,
samples with a concentration below the limit of detection (25 pg/mg) were assumed to have 25
pg/mg tissue drug. No Eve could be detected in tumors of 5 mice that received oral Eve. In the
FAF-Eve group, 2/5 mice had 32 and 36 pg/mg Eve in their tumors while the other 3 remained
undetectable. On the contrary, Rapa concentration in FAF-Rapa group was 96 ± 40 pg/mg tissue
(Mean ± SD, n=5), which was significantly higher than oral Eve and FAF-Eve groups (One-way
ANOVA, Tukey’s post hoc test, α = 0.05, **p < 0.005). The xenograft experiment was performed
by Washington Biotech Inc. and drug quantification by LC-MS/MS was performed by Scripps
Center for Metabolomics and Mass Spectrometry.

84
3.4.8 Assessing mTORC1 inhibition in off-target organs
Given the complexity of cellular signaling regulated by mTOR and its essential role in virtually
all cell types, it is not surprising mTOR inhibition causes a range of toxic effects
164
. The use of
rapalogues is associated with metabolic (hyperglycemia, dyslipidemia, hypercholesterolemia),
hematological (anemia, thrombocytopenia, neutropenia), respiratory (pneumonitis), renal and
dermatological toxicities (skin rash, oral stomatitis). Although these side effects are common with
incidences as high as 50% (stomatitis
165
and hyperglycemia for example), their underlying
molecular mechanisms and pathophysiology are not always clearly understood, and may partly be
occurring through mTOR-independent pathways. However, these toxicities are a result of drug
exposure to healthy tissues/organs. In a previous bio-distribution study
41
, Cy5.5 labeled FAF was
found to concentrate in the liver and spleen of mice where they are presumably processed and
cleared. This raised the possibility of FAF-Eve and FAF-Rapa causing off-target mTOR
suppression in these tissues. When organs of mice from the tumor xenograft study were assessed
by western blotting, levels of phosphorylated S6RP in the liver and spleen of treated mice (both
FAF-Eve and FAF-Rapa) were comparable to healthy controls (Figure 3.12 A-C). Hence,
although preliminary at this stage, FAF-rapalogue formulations appear to arrest off-target mTOR
inhibition, presumably through drug sequestration.

85

Figure 3.12: FAF carrying rapalogues do not affect S6RP phosphorylation in liver and spleen.
A) At the end of xenograft study, liver (4/group) and spleen (4/group) of mice treated with FAF-
Eve and FAF-Rapa were harvested and the phosphorylation status of S6RP was assessed by
western blotting. Comparisons were based on age, sex and strain matched untreated mice, with
GAPDH as a loading control. B,C) Integrated band volumes from (A) were normalized to GAPDH
and plotted as Mean ± SD, n=4/group. No significant differences in mTOR activity in the liver and
spleen of mice that received FAF-Eve and FAF-Rapa was observed, compared to untreated mice
(ANOVA, α = 0.05).

86
3.5 Discussion
This chapter provides additional evidence to support preclinical development of a rationally
designed two-headed protein drug carrier for rapalogues called FAF. Extensive first-pass and
intestinal wall metabolism restrict rapalogue bioavailability to ~10%, thereby suggesting possible
opportunities for parenteral dosage forms. Earlier approaches to an intravenous (IV) formulations
required high proportions of surfactants or organic solvents to stabilize hydrophobic rapalogues in
solution, which prevented their translation to patients. In fact, in 1982, Ayerst Pharmaceuticals
(later known as Ayerst-Wyeth Pharmaceuticals) shut down their Rapa project because formulating
IV Rapa for clinical trials seemed impossible
166
. A decade later, they abandoned efforts at a
parenteral formulation and started developing an oral form which gained FDA approval in 1999
as an anti-rejection agent for organ transplantation. However, years of poor clinical experience
sparked a renewed interest in this space and multiple drug delivery technologies are being tested
for their ability to deliver rapalogues. Among these, nanoparticle albumin-bound (nab) platform is
particularly noteworthy
167
. Nab-rapamycin (ABI-009) is Rapa bound to ~100 nm albumin
nanoparticles through hydrophobic interactions. It is a surfactant-free injectable suspension that
leverages natural affinity of albumin to hydrophobic drugs and its ability to selectively accumulate
(via gp60 and caveolae-mediated transcytosis)
168
and reside longer (via association with SPARC
protein) in tumor interstitium
169, 170
. ABI-009 showed good anti-tumor activity and safety profile
in preclinical models and is currently being evaluated in multiple phase 1/2 clinical trials
171, 172
in
combination with other chemotherapeutics.

The FKBP-ELP technology presented here is distinct from nab technology. While both
formulations are protein-based and rely on non-covalent drug-carrier binding, FKBP binds to
87
rapalogues with high affinity and specificity as opposed to albumin which offers a low affinity and
low specificity interaction with many drugs. Low affinity interactions allow rapid exchange to
other hydrophobic lipid, drug or albumin species in the body. This may mask beneficial
sequestration offered by the drug carrier. Additionally, due to its comparatively low hydrodynamic
radius, FAF is engineered to promote SC bioavailability of rapalogues
173
, while ABI-009 is
designed for direct IV administration. SC administration is advantageous for patient convenience
since less chair time is required to receive chemotherapy. SC or intramuscular FAF-Rapa could
be administered in an ambulatory setting, offers an opportunity for self-administration in educated
patients, and mitigates risk of bloodstream infections associated with IV injection. Despite these
advantages, only 8 chemotherapeutics (methotrexate, cytarabine, azacitidine, cladribine,
bortezomib, omacetaxine, bleomycin and trastuzumab) are approved for SC use. This is due to
irritation and inflammation caused by anticancer agents at the site of injection, also called vesicant
activity
174
. In this regard, skin histology could not detect any injection site abnormalities with SC
FAF-Rapa previously
41
. This is reasonable since FAF’s long retention may arrest free rapalogue
infiltration to surrounding dermal tissue.

This chapter also describes mechanisms of uptake and drug release from FAF-Rapa. Mammalian
target of Rapamycin complex 1 (mTORC1) is a nutrient sensing multi-protein complex
9
that
strongly promotes cell growth and proliferation. Rapa, through inhibition of mTORC1 kinase
activity, arrests cell cycle progression from G1 to S phase thereby exerting a cytostatic effect.
Deregulation of multiple elements of mTORC1 signaling have been reported in many types of
cancers
11
, including melanoma, breast cancer, and renal cell carcinoma, which renders the
pathway an attractive therapeutic target. Unfortunately, the clinical outcomes with mTOR
88
inhibitors have been modest, primarily because of their cytostatic and not cytotoxic effects, partial
inhibition of mTOR activities, and existence of several feedback loops involved in cell survival
responses
15-17
. Nonetheless, mTOR inhibitors in combination with a wide variety of
chemotherapeutics are an active area of research and a number of clinical trials are underway. An
additional factor hampering their clinical efficacy is poor drug-like properties, a problem that may
possibly be improved using FAF-Rapa formulation.

A variety of internalization mechanisms have been reported for ELP biomaterials. The uptake of
Cell penetrating peptide-ELP (CPP-ELP) fusion proteins
134
was driven by caveolae-independent
mechanisms. ATP depletion and inhibition of clathrin-mediated endocytosis had no significant
effect on CPP-ELP internalization. On the other hand, the uptake of GFP-K72 in A549 cells
135

was found to be primarily through caveolae-mediated endocytosis with no contribution from other
known mechanisms. Similarly, based on the size of endocytic vesicles, ELP[Val5Ala3Gly2]150
was proposed to be internalized by macropinocytosis
136
with enhanced uptake upon coacervation.
Taken together, it is difficult to predict or compare uptake mechanisms across ELPs or ELP fusion
proteins since they differ in their components and properties like charge, size, hydrophobicity etc.
In the present study, FAF co-localized with dextran, a marker for macropinocytosis and its cellular
association was not saturable across a range of achievable concentrations. Moreover, amiloride, a
specific macropinocytosis inhibitor completely blocked the cellular entry of Rapa upon FAF-Rapa
treatment. These results together support macropinocytosis as the mechanism for FAF-Rapa
uptake. Although no evidence for a specific receptor for FAF was observed, the presence of bright
surface staining in the cold binding experiment suggests FAF may weakly and non-specifically
bind to molecules of the plasma membrane, presumably lipids. Termed ‘adsorptive endocytosis’
89
175
, such behavior has been reported with ELPs
136
and may be advantageous as high surface
concentrations can drive their uptake.

A carrier system that utilizes macropinocytosis for drug delivery poses both advantages and
disadvantages. Efficiency of receptor-mediated endocytosis is dependent on expression levels of
the internalizing receptor, rendering the process heterogeneous across cell types. On the contrary,
macropinocytosis is a generally accepted hallmark of cancer with multiple cancer types exploiting
the process in various ways to establish and maintain their oncogenic phenotype
176, 177
. This
suggests a broad-spectrum cytostatic activity for FAF-Rapa against mTOR driven cancers. On the
downside, a carrier system utilizing macropinocytosis for drug delivery can exhibit off target side
effects since most normal cells, especially macrophages and dendritic cells depend on either ligand
induced or constitutive macropinocytosis for normal physiological function
178, 179
. This may result
in cellular entry of FAF-Rapa to non-cancerous cells, thereby causing off-target effects.

The ability to undergo biodegradation make ELPs excellent materials compared to some polymeric,
inorganic, and metallic nanoparticles. The MacKay group and other groups previously reported
enzymatic degradation of ELPs by trypsin
71
, elastase and collagenase
131
. In a physiological
system, these proteases could act intra or extracellularly, or both. In cells, Rho-FAF disappeared
with a half-life of 18 hours, during which time it significantly colocalizes with low pH
compartments, including lysosomes. Interestingly, both elastase
132
and collagenase
133
have been
identified in the lysosomes, which suggests they might be involved in FAF degradation. While
plausible, additional experiments are needed for definitive proof. It must be noted that the drop in
cell-associated fluorescence in the biodegradation experiment is a combined effect of
90
biodegradation, cellular recycling/export, and dilution due to cell division. In the Rho-FAF group,
there was a 15-fold drop in fluorescence after 72 hours relative to the start of experiment. With the
doubling time of MDA-MB-468 cells being about 48 hours and the experiment lasting for 72 hours,
only a maximum of 3 fold drop in fluorescence can be explained by cell division alone. Rho-FAF-
Rapa on the other hand may be less prone to dilution due to cell-division, because of Rapa’s
cytostatic effect
41
. An additional limitation of this study, the biodegradation experiment can be
strengthened by complementing SDS-PAGE with a western blot using anti-FKBP and anti-ELP
antibodies. Fluorophores, especially during long incubation times are prone to oxidation and other
chemical reactions that can cause photobleaching. In such cases, western blots may be reliable
alternative to quantify FAF. Nevertheless, with a cellular half-life of 21 hrs, the persistence of
Rho-FAF-Rapa greatly exceeds the approximately 0.5 hr time-scale required for intracellular
delivery of Rapa.

When added to cells, Rapa rapidly diffused across the membrane resulting in spontaneous
luciferase activity. This is in accordance with a previous report showing near quantitative
accumulation in human smooth muscle cells within few minutes of Rapa treatment
180
. On the
contrary, FAF-Rapa required 30 minutes before measurable luciferase activity was achieved
suggesting mechanisms other than trans-membrane diffusion mediate drug release. If FAF-Rapa
complexes caused burst release extracellularly, FAF-Rapa would have produced a similarly rapid
luciferase profile as free Rapa. More importantly, amiloride completely blocked cytoplasmic entry
of Rapa from FAF-Rapa, thus establishing the requirement for internalization in general, by
macropinocytosis in particular. This is in agreement with microscopy experiments that showed
Rho-FAF highly co-localized with the macropinocytosis marker dextran. Although proven to
91
selectively inhibit macropinocytosis without any effects on coat dependent endocytosis
163, 181, 182
,
amiloride may inhibit clathrin
183
and lipid raft mediated internalization
184
. Hence, the use of
multiple inhibitors can strengthen the proposed role of macropinocytosis. I used dynasore to
evaluate the dynamin dependence of FAF-Rapa uptake. In the split luciferase assay, pretreatment
of cells with dynasore completely suppressed luciferase signals triggered by both free Rapa and
FAF-Rapa (Figure 3.13 B). Since dynamin inhibition cannot arrest diffusion of free Rapa across
the cell membrane, the observed effect is likely an assay interference caused by dynasore by
unknown mechanisms.

Figure 3.13: Additional characterization of cell uptake mechanism of FAF-Rapa A) Inhibition
of lysosomal acidification does not inhibit Rapa release from FAF-Rapa. MDA-MB-468 cells were
transfected with a split luciferase reporter that enables the specific detection of Rapa within the
cytosol. When incubated with cells, FAF-Rapa (30 nM Rapa) resulted in delayed luciferase activity
consistent with endocytosis across the plasma membrane. Two inhibitors of lysosomal
acidification were added to determine if they affect the kinetics of Rapa delivery. Neither
Chloroquine (80 µM) nor Ammonium Chloride (25 mM) prevented the appearance of a luciferase
signal. (Mean ± SD, n=3) B) Dynasore mediated inhibition of dynamic dependent endocytosis
resulted in suppression of luciferase activity for both free rapa and FAF-Rapa. Since dynamin
inhibition cannot arrest diffusion of free Rapa across the cell membrane, the observed effect is
likely an assay interference caused by dynasore by unknown mechanisms.
92
Similar to the luciferase assay, microscopy experiments do not support a requirement for carrier
biodegradation in drug release. The half-life for Rho-FAF-Rapa degradation was found to be 21
hours, which is much longer than the time scale for triggering luciferase activity. To test the
possible role of endosomal/lysosomal pH in drug release through rapid disruption of FAF-Rapa
binding, the luciferase assay was performed using cells pretreated with lysosomal acidification
inhibitors chloroquine and ammonium chloride. This did not result in any measurable change in
carrier performance (Figure 3.13A). Although these findings point towards low pH of acidic
organelles not being necessary in Rapa release from FAF-Rapa, such conclusions can be made
only after confirming lysosomal basification under the treatment conditions employed.

Another possible mechanism that hasn’t been explored is a release-independent pathway wherein
FAF-Rapa directly binds to mTOR and inhibits its kinase activity. When free Rapa is added to
cells, it diffuses across the membrane, binds to endogenous FKBPs with high affinity and
FKBP/Rapa complexes target mTOR. Since FAF-Rapa structurally resembles naturally occurring
FKBP/Rapa, the possibility exists that it may directly bind to and inhibit mTOR. Such a process
would not require drug exchange from FAF-Rapa to cellular FKBPs for mTOR inhibition. This
can be envisioned as FAF-Rapa may escape endosomes and target cytoplasmic mTORC1. It is
well known that active mTORC1 attaches to the surface of lysosomes
185, 186
, and the microscopy
experiments suggest Rho-FAF-Rapa accumulates in the lysosomes. Such close proximity due to
translocation to the same cellular compartment might conceivably facilitate FAF-Rapa-mTOR
interaction across disruptions in the lysosomal membrane. This would require FAF-Rapa to
withstand affinity in the luminal milieu with a pH<5 and about a hundred potent hydrolases. To
verify a direct FAF-Rapa-mTOR interaction, anti-mTOR co-immunoprecipitation (co-IP) was
93
performed after treating cells with FAF-Rapa. Unfortunately, previously reported cell lysis
conditions for anti-mTOR co-IP preserved the mTORC1 complex but disrupted FKBP/Rapa-
mTOR interaction (Figure 3.14), thereby rendering the experiment inconclusive. As an alternative
to co-IP, I used immunofluorescence to detect mTOR Rho-FAF-Rapa co-localization. While
microscopy is limited by resolution in identifying direct molecular interactions, a strong co-
localization would be supportive of binding. Upon incubating cells with Rho-FAF-Rapa for 1, 8
and 16 hr, co-localization was observed (Figure 3.15) in a small population of cells (<20%) but
the average co-localization coefficient was a mere 0.13. Hence, while it cannot be ruled out, a
direct FAF-Rapa-mTOR interaction is unlikely.

Figure 3.14: Anti-mTOR immunoprecipitation of cell lysates treated with Rapa and FAF-Rapa
fail to detect FKBP12-Rapa-mTOR interaction. Cells were treated with Rapa (100 nM) or FAF-
Rapa (100 nM) for 30 minutes and lysed in CHAPS lysis buffer. Following anti-mTOR IP, the
immunoprecipitated and the supernatant fraction were resolved by SDS-PAGE and the indicated
markers were detected by western blotting. Lysis conditions preserved mTORC1 complex as
indicated by presence of Raptor in the IP product. On the other hand, the IP conditions interfered
with FKBP12/Rapa-mTOR binding resulting in detection of FKBP12 in the supernatant rather
than IP fraction. Suppression of p70S6K phosphorylation is indicative of successful Rapa delivery
to the cytoplasm and subsequent inhibition of mTOR kinase activity.
94
A key parameter to gauge the efficiency of a dosage form is its bioavailability. The mechanism
and extent of absorption of therapeutics from SC site is dictated by multiple factors. Among these,
biophysical properties of the molecule (molecular size, charge), formulation attributes
(concentration, buffer, excipients) and the biology of injection site play a key role
187
. Given the
high molecular weight of FAF, it likely reaches systemic circulation following uptake by the
lymphatic system. Our group previously characterized PK of subcutaneous FAF-Rapa extensively
in mice, and the bioavailability was found to be ~ 60%
173
. Additionally, FAF retained binding to
Rapa in circulation. These results are consistent with the superior in vivo mTOR inhibition of FAF-
Eve and FAF-Rapa compared to oral Eve. Despite this, only FAF-Rapa, but not FAF-Eve,
suppressed tumor growth significantly compared to oral Eve. To the best of our knowledge, no
published literature compared the potency of rapalogues in the same tumor model, and it remains
unknown if Eve and Rapa are equipotent. The observed difference in tumor inhibition here is likely
a complex outcome of differences in drug PK, metabolism, tumor clearance, mTOR independent
pharmacology and differential behavior of FAF when bound to Eve or Rapa.
95

Figure 3.15: Co-localization of FAF-Rapa and mTOR using immunofluorescence. Secondary
immunofluorescence was used to assess the intracellular distribution over time for mTOR in
relation to Rho-FAF. When MDA-MB-468 cells were treated with FAF-Rapa for 1, 8 and 16 hr,
a few areas of co-localization were seen as indicated by arrows. While some cells after 8 hours
show evidence of co-localization, the average co-localization co-efficient across many cells was
only 0.13. This suggests an unlikely direct interaction between FAF-Rapa and mTOR on time-
scales relevant to intracellular delivery of Rapa.

96
3.6 Conclusions
This chapter evaluates solvent-free formulations of two rapalogues Rapa and Eve that utilize a
biomacromolecular drug carrier called FAF. Unlike conventional drug carriers, the FAF-Rapa
formulation does not require toxic co-solvents or surfactants and instead utilizes Rapa’s cognate
receptor FKBP12 for high-affinity binding-mediated drug delivery. Compared to the first
generation FKBP-ELPs, FAF offers two advantages i) long-term physical stability by virtue of not
involving self-assembly ii) higher drug loading capacity (~1.6%). In MDA-MB-468 cells, FAF
was internalized by macropinocytosis and accumulated in low pH organelles, which include
lysosomes. Cellular uptake was dose and time-dependent but was not saturable. FAF-Rapa is a
biodegradable carrier with a cellular half-life of 21 hrs; however, it appears to deliver its Rapa
cargo to the cytosol within a time scale of 0.5 hrs. Drug release from FAF-Rapa was found to be
sensitive to amiloride, a macropinocytic inhibitor. While future studies must be performed to
determine the relative importance of other mechanisms of endocytosis, the findings in this
manuscript are consistent with macropinocytosis. When compared to clinically approved oral Eve
(Affinitor
®
), subcutaneous FAF-Rapa, but not FAF-Eve significantly suppressed tumor growth in
a mouse model of HR+ breast cancer. Both formulations resulted in higher drug accumulation in
the tumor and robust mTOR inhibition as compared to oral Eve. SC FAF-rapalogue formulations
achieve higher tumor concentrations compared with an orally-administered control, appear safe in
vivo, and are promising candidates for further preclinical development. However, 3x/week dosing
was used in the xenograft study. A lower dosing frequency is expected to be more patiently-friendly
and improve adherence. To achieve this, the next chapter describes third generation FKBP-ELP-
Rapa formulations that utilize body heat and phase separate at the site of subcutaneous injection.
97
This facilitates higher retention at the injection site and resembles plasma PK of a sustained release
formulation.

98
Chapter 4: Third generation FKBP-ELPs for the delivery of
Rapamycin for immunosuppression
4.1 Introduction
Every year nearly 32,000 transplantations are performed in the United States, with the majority of
them (~18,000) being kidneys
188
. Of these, 93% are functional at the end of a year; 83% are
working at the end of 3 years and only a mere 50% remain functional at 10 years post-transplant
188
.
While 12 people die every day awaiting a kidney transplant, 20% of kidney transplants are re-
transplants
188
, with choric inflammation being the primary cause for rejection. Along with other
factors, patient non-adherence to the prescribed immunosuppressive treatment plan
30
is an
important contributor to inflammation and subsequent organ rejection, as evidenced by multiple
well-designed studies
189-191
. Non-adherence to prescriptions is reported in 23 cases per 100
transplant recipients per year
31
, even when the lifelong need to take immunosuppressants is
routinely emphasized in clinical follow-up. This is likely an outcome of significant side effects,
long duration and complex dosing regimens of currently used immunosuppressive protocols
192
.
Currently, >90% of the patients receive the calcineurin inhibitor tacrolimus
188
and the anti-
metabolite mycophenolate, and ~60-70% of patients are prescribed steroids. The usage of mTOR
(Mechanistic Target of Rapamycin) inhibitors steadily decreased to <5% of current
immunosuppressive regimens
188
. These facts also hold true for other solid organ transplantations
including heart, liver, lungs, pancreas and skin.

mTOR is a serine/threonine protein kinase with a key role in activation and regulation of both
innate and adaptive immunity
193
. It forms the catalytic domain of two protein complexes in the
99
cell: mTORC1 and mTORC2. Through complex signaling involving the PI3-AKT, WNT-GSK3
and AMPK pathways
13, 194, 195
, mTORC1 acts as a master regulator of immune cell growth,
proliferation and metabolism. Upon activation of T-cells through the T-cell receptor (TCR) and
co-stimulatory molecule CD28, mTORC1 promotes cell progression from G0 to G1 phase
196

through increased mRNA translation and protein synthesis, increased glycolysis and ATP
accumulation, and increased cytokine signaling (especially by overexpressing IL-2 and IL-2Rα).
Consequently, mTORC1 inhibitors like rapamycin (Rapa) arrest clonal proliferation of T-cells in
response to antigen stimulation and are FDA approved for use as immunosuppressants.
Interestingly, mTORC1 is also deregulated in multiple cancers and is known to promote
tumorigenesis, tumor growth and metastasis
11
.

In cells, Rapa binds to cytosolic peptidyl-prolyl isomerase FKBP12 with sub-nanomolar affinity
and this FKBP12-Rapa complex inhibits mTORC1 allosterically
197
. In contrast, tacrolimus, the
most commonly used drug to prevent transplant rejection
198
works by inhibiting calcineurin and
its downstream transcription of cytokines important for immune response (IL-2, IL-10, IL-17 etc.).
Given mTOR’s oncogenic signaling, Rapa, but not tacrolimus additionally exhibits potent
cytostatic and anti-cancer properties. By virtue of dual pharmacology, Rapa is likely a better drug
for organ transplantations since transplant recipients are at an increased overall risk (between 3-4
fold) of cancer
199
compared to general population of the same age and sex. As such, malignancy
is the primary cause of mortality following transplantation
200, 201
. While Rapa’s ability to lower the
incidence of de novo cancers in transplant patients is being clinically tested, several clinical trials
show positive data
202-205
.

100
Despite being very potent
206, 207
with advantageous pharmacology, the clinical usage of Rapa is
limited by three major challenges: i) Rapa is only available as an oral formulation which has a
poor bioavailability (~ 10% in humans) due to extensive first-pass metabolism ii) Clinical
pharmacokinetics (PK) is sub-optimal with a large intra- and inter- patient variability in PK
parameters, incomplete gut absorption, poor correlation between blood concentration and
administered dose, and preferential partitioning to blood cells (blood : plasma ratio of 5-35) iii)
Dose-limiting toxicities including stomatitis (incidence ~40%) are prevalent. These oral lesions
are quite painful and lead to unplanned interruptions in treatment and reduce patient compliance.
To overcome these shortcomings, this chapter aims to develop novel protein-based formulations
of Rapa with higher bioavailability, lower toxicity and a predictable PK profile. Additionally, the
formulation is designed for low-frequency dosing via the subcutaneous route to improve patient
adherence and compliance. This is essential in increasing transplant longevity as discussed above.

Here I evaluate two new fusion proteins as drug carriers for Rapa: 5FA and 5FV (Figure 1A)
which comprise of multiple FKBP12 domains stitched together by ELP linker sequences (Figure
4.2A). The FKBP domains in 5FA and 5FV solubilize hydrophobic Rapa through high-affinity,
high-specificity non-covalent binding and prevent its precipitation in aqueous buffers. On the other
hand, the ELP linker can be designed to alter drug absorption rates from subcutaneous depots by
controlling their physical state at physiological temperature. In the context of this chapter, at 37
o
C, 5FA remains soluble while 5FV forms a coacervate.

101
4.2 Materials and Methods

4.2.1 Recombinant protein purification, drug encapsulation and endotoxin removal
For recombinant expression, electrocompetent ClearColi
®
BL21(DE3) cells (Lucigen, Middleton,
WI) were electroporated with 1 ng of pET25b(+) vector encoding 5FA or 5FV following
manufacturer’s protocol. Cells were spread on agar plate containing 100 µg/mL carbenicillin (Gold
Biotechnology, St. Louis, MO) and incubated for 30 hrs at 37
o
C. A single colony was aseptically
transferred to autoclaved 50 mL Miller LB broth media (Sigma Aldrich, St. Louis, MO)
supplemented with 100 µg/mL carbenicillin. Following overnight incubation at 37
o
C, the starter
was inoculated to 1L culture medium to a final OD600 of 0.1. When the OD600 of culture reached
0.6 – 0.8, IPTG (Gold Biotechnology, St. Louis, MO) was added to a final concentration of 0.4
mM and further incubated for 24 hrs at 37
o
C. Bacteria was pelleted by centrifugation, resuspended
in phosphate buffered saline (PBS, Caisson, Labs, Smithfield, UT), and lysed by sonication. To
precipitate nucleic acids, 50% (w/v) solution of polyethyleneimine (Sigma Aldrich, St. Louis, MO)
was added to a final concentration of 0.5% (v/v). The lysate was clarified by centrifugation and
proteins were purified by three rounds of inverse transition cycling
101
. To measure concentration,
an aliquot of protein was diluted in 8M Guanidine Hydrochloride (Thermo Fischer Scientific,
Waltham, MA) and loaded on a Nanodrop 2000 spectrophotometer (Thermo Fischer Scientific,
Waltham, MA). The concentration was estimated as follows:
𝐶 =

($
%&'
( $
)*'
)
,
-

where C is the solution concentration (M), A 280 and A350 are absorbances at 280 and 350 nm
respectively, l is the path length (cm) and 𝜀 is the molar absorption coefficient at 280 nm (50,475
102
cm
-1
for both 5FA and 5FV). Protein purity and identity was assessed by SDS-PAGE followed by
Coomassie blue staining (Catalog no: LC6065, Thermo Fischer Scientific, Waltham, MA).

For drug encapsulation, 10 mL 100 µM 5FA/5FV (in PBS) was equilibrated to 4
o
C. 10x
stoichiometric excess of Rapa (LC Laboratories, Woburn, MA) in ethanol was added to proteins
dropwise with continuous stirring. The final % ethanol was <5% (v/v). After 30 minutes of stirring
at 4
o
C, the resulting aqueous suspension was centrifuged at 13,000g to pellet insoluble drug
precipitate. The supernatant was dialyzed extensively against PBS (1:300 sample: dialysate) with
at least 3 buffer changes to remove free drug and residual ethanol. Drug loaded 5FA and 5FV were
subjected to endotoxin removal using Pierce
TM
endotoxin removal columns (Thermo Fischer
Scientific, Waltham, MA) following the manufacturer’s method for batches. Next, 5FA-Rapa and
5FV-Rapa were filtered using Acrodisc
®
Mustang E syringe filters (Pall Corporation, Port
Washington, NY) to ensure formulation sterility and further remove residual endotoxins. An
aliquot of 5FA-Rapa and 5FV-Rapa was injected onto a C-18 RP-HPLC column (Waters, Milford,
MA) and both drug and carrier concentration was quantified by optical density at 280 nm using a
calibrated standard curve. The formulations were diluted to 100 µM working concentration and
frozen at -80
o
C until needed. The concentration of 5FA-Rapa and 5FV-Rapa throughout the
chapter refers to Rapa concentration unless otherwise noted.

4.2.2 Dynamic Light Scattering (DLS)
Hydrodynamic radius (Rh) was measured using a DynaPro plate reader (Wyatt Technologies, Santa
Barbara, CA). Briefly, 60 µL samples at 25 µM concentration were added in triplicate to a 384-
well clear bottom plate (Greiner Bio One, Monroe, NC). The wells were capped with 15 µL mineral
103
oil to prevent evaporation during long incubations. Data was analyzed using Dynamics V7 (Wyatt
Technologies, Santa Barbara, CA).

4.2.3 Cell culture and in vitro cytotoxicity assay
MDA-MB-468 cell line (ATCC, Manassas, VA) was cultured in DMEM/F-12 medium
supplemented with 10% heat inactivated fetal bovine serum (Corning Life Sciences, Tewksbury,
MA) at 37
o
C in a humidified incubator with 5% CO2. For formazan viability assays, 3x10
3

cells/well were added to a 96-well tissue culture plate. Following cell attachment overnight, the
media was exchanged for multiple dilutions of Rapa or 5FA-Rapa in complete medium. After 3
days of drug treatment, 10 µL/well WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-
tetrazolio]-1,3-benzene disulfonate) reagent (Sigma Aldrich, St. Louis, MO) was added and
incubated for 3 hours. Absorbance at 440 and 690 nm was measured using a Synergy H1 plate
reader (BioTek, Winooski, VT) and % maximum proliferation (%P) was calculated as follows:
%𝑃 =

($
pqrsprt
( $
'
)
($
uvpqrsprt
( $
'
)

where Atreated, Auntreated and A0 are background corrected absorbances of drug treated, untreated and
media only wells respectively. %P was normalized to extend from 0 to 100% using normalize
function of GraphPad Prism. Dose response curves were fit by non-linear regression and IC 50 was
estimated in 3 independent assays. One-way ANOVA followed by the Tukey post-hoc test was
used to test differences between groups.

4.2.4 Isothermal Titration Calorimetry (ITC)
ITC measurements were performed on a MicroCal PEAQ ITC (Malvern Instruments,
Northampton, MA) at 37
o
C. The calorimeter cell was filled with 8 µM Rapa dissolved in PBS
104
with 1% DMSO. The titration syringe was filled with 20 µM 5FA in PBS with 1% DMSO. Twelve
3 µL injections ensured complete saturation of binding sites. The resulting isotherm was fit to a
‘one set of sites’ binding model using MicroCal ITC analysis software (Malvern Instruments,
Northampton, MA) and various thermodynamic parameters were estimated.

4.2.5 Protease-coupled prolyl isomerase assay
Prolyl isomerase enzymatic activity of 5FA and 5FV were measured using a chymotrypsin coupled
assay. The substrate peptide (Suc-AAPF-pNA) was dissolved in 20 mg/ml LiCl in trifluoroethanol.
First, the rate of thermal isomerization of substrate peptide was measured. To 170 µL assay buffer
(50 mM HEPES, 100 mM NaCl, pH 8), 20 µL chymotrypsin (60 mg/ml in 1 mM HCl) was added
in a chilled cuvette. After 5 min equilibration on ice, the solution was used as a blank for UV-Vis
spectrophotometer (DU-800, Beckman Coulter, Brea, CA) cooled to 10
o
C. 10 µL of 3 mM
substrate peptide was rapidly added, mixed and absorbance at 405 nm (A405) was measured in
kinetic mode (1.5 sec intervals). To measure enzymatic activity, 160 µL assay buffer, 20 µL
chymotrypsin and 10 µL enzyme (1 µg FKBP12 in the form of recombinant FKBP12, 5FA or 5FV)
was equilibrated on ice and used as the blank solution. 10 µL of 3 mM substrate peptide was rapidly
added, mixed and absorbance at 405 nm (A405) was measured in kinetic mode (1.5 sec intervals).
The kinetic curve from thermal isomerization (substrate only) was subtracted from (substrate +
enzyme) curves and slope of the curve in the linear part (first 30 sec) was calculated. Specific
activity was defined as moles of substrate isomerized/ min/ µg FKBP12 and was calculated using
the equation below:
𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐
𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦=

J
ΔOD
Δt
N𝑋
𝑉
𝜀
𝑙
𝑀

105
where (𝛥𝑂𝐷 /𝛥𝑡) is the slope of the linear portion of kinetic plot (corrected for thermal
isomerization), V is the volume of reaction, 𝜀 is the molar absorption coefficient of FKBP12 (9300
M
-1
cm
-1
), l is the path length of the cuvette and M is the mass of FKBP12 in the reaction.

4.2.6 Zirconium-89 labeling and in vivo microPET imaging
Zr-89 labeling was achieved using a 2-step protocol: DFO conjugation and Zr-89 chelation. First,
the pH of 225 µM 1.5 mL solutions of 5FA and 5FV (in PBS) was adjusted to 8.5-9 using 0.1 M
sodium bicarbonate. After dilution to 3 mL, 1.5x stoichiometric excess p-SCN-Bn-deferoxamine
(NCS-DFO, Catalog no: B-705, Macrocyclics, Plano, TX) was added from a DMSO stock and the
reaction was incubated at 37
o
C for one hour (for 5FA) or at 4
o
C overnight (for 5FV). Zeba
desalting columns (Thermo Fischer Scientific, Waltham, MA) were used following manufacturer’s
protocol to purify and elute DFO labeled conjugates in PBS, and their concentrations were
measured using Eq 1. The pH of zirconium oxalate (Washington University School of Medicine,
St. Louis, MO) solution was adjusted to 7.2 using 1 M sodium carbonate. To 1.5 mL of 110 µM
5FA-DFO and 5FV-DFO, 3.5 mCi of pH adjusted Zr-89 was added and incubated for one hour at
room temperature (for 5FA) or 4
o
C (for 5FV). Following chelation, free Zr-89 was removed using
zeba desalting columns and Zr-89 labeled proteins were eluted in PBS, and stored at 4
o
C until
injection. The protein recovery was assumed to be quantitative. Radiochemical purity was assessed
by radio TLC. Briefly, 1 µCi of the purified conjugate was spotted on a silica coated TLC sheet
(Sigma Aldrich, St. Louis, MO) and developed in 10 mM pentetic acid (DTPA, Sigma Aldrich, St.
Louis, MO). The plate was allowed to air-dry and was scanned using a radio-TLC scanner at 0.5
mm/sec.
106
For in vivo administration, three groups of mice (n=5/group) received 200 µCi of IV (via tail vein)
Zr89-DFO-5FA, or SC Zr89-DFO-5FA, or SC Zr89-DFO-5FV. At multiple time points following
injection, mice were imaged using a Siemens Inveon microPET scanner while anesthetized under
2% isoflurane in oxygen. Simultaneously, 20 µL blood was sampled via tail vein puncture, added
to heparinized PBS and Zr89 activity was measured by one minute scans using a gamma counter
(Wizard 2, Perkin Elmer, Waltham, MA) in the 350-650 keV energy window. All gamma counts
were corrected for isotope decay and also to account for day to day variability in counter efficiency.
At the end of the study, mice were euthanized, organs were harvested, weighed and total
accumulated activity was measured using a gamma counter and reported as %ID/g tissue.
The PET images obtained were reconstructed by a two dimensional ordered subsets expectation
maximum (2D-OSEM) algorithm and analyzed using AMIDE (UCLA School of Medicine, CA).
Activity at the site of injection was obtained by drawing regions of interest (ROI) over the SC
depot area. The counts per pixel per min obtained from the ROI were converted to counts per ml
per min by using a calibration constant obtained from scanning a cylinder phantom in the
microPET scanner. By using the volume of ROI estimated by AMIDE, total activity was calculated
and divided by injected activity to obtain %ID.

4.2.7 Rhodamine labeling and PK assessment in Sprague Dawley (SD) rats
NHS-Rhodamine (Thermo Fischer Scientific, Waltham, MA) was dissolved in anhydrous DMSO
(Invitrogen, Carlsbad, CA) at 10 mg/mL and frozen as single use aliquots. To 200 µM 5FA or 5FV
in PBS, 1.5x molar excess NHS-Rhodamine was added and incubated overnight at 4 °C. Zeba
desalting columns (Thermo Fischer Scientific, Waltham, MA) were used according to
107
manufacturer’s protocol to remove unreacted free dye and elute the proteins in PBS.
Concentrations of rhodamine and protein were calculated using Nanodrop spectrophotometer as
follows:

𝐶
:;<
=
$
***
,
-

𝐶
Xl<dm
=
($
%&'
( 9.{|
$
***
)
,
-

where A280 and A555 are absorbance at 280 nm and 555 nm respectively, l is the path length (cm)
and ε is the estimated molar extinction coefficient at 280 nm: 50,475 M-1cm-1 for 5FAand 5FV,
and 80,000 M-1cm-1 for rhodamine. Labeling efficiency, N, was calculated as:

𝑁 =
>
?@A
>
qAprgv

×100
The purity of rhodamine labeled proteins was evaluated by SDS-PAGE electrophoresis followed
by fluorescence imaging on ChemiDoc
TM
(Bio-Rad, Hercules, CA) imaging system. Rapa
encapsulation was performed as described above. Three groups of rats (n=3/group) received 1
mg/kg Rapa injected either IV (via tail vein) or SC. At various time-points ranging up to 2 weeks,
300 µL blood was collected using a blunt needle via jugular vein catheter and added to collection
tubes coated with EDTA (BD, Franklin Lakes, NJ). Plasma was separated by centrifugation at
1800xg and a calibrated plate reader was used to measure plasma fluorescence intensity. Using a
standard curve for fluorescence intensity vs. protein concentration, plasma protein concentrations
were calculated at all the timepoints.

108
4.2.8 Compartmental and non-compartmental PK analysis
For non-compartmental PK analysis, the area under the curve plasma conc. vs time profile (AUC)
and area under moments curve (AUMC) were calculated by trapezoidal rule. The mean residence
time (MRT) for both IV and SC and mean absorption time (MAT) for SC administration were
calculated as below
𝑀𝑅𝑇 =

𝐴𝑈𝑀𝐶
𝐴𝑈𝐶

𝑀𝐴𝑇=𝑀𝑅𝑇
(𝑆𝐶)−𝑀𝑅𝑇
(𝐼𝑉)
Next, bioavailability for SC administration was estimated as
𝐹 =

𝐷𝑜𝑠𝑒
(𝐼𝑉)
𝐴𝑈𝐶
(𝐼𝑉)
𝐴𝑈𝐶
(𝑆𝐶)
𝐷𝑜𝑠𝑒
(𝑆𝐶)

Next, plasma clearance (CL) for both IV and SC were calculated as
𝐶𝐿=

𝐹×𝐷𝑜𝑠𝑒
𝐴𝑈𝐶

Kelimination and Terminal half-life (T1/2, terminal) wers estimated by fitting the last two data points to
the following equation

Cp
=
Ae
-‐‑k
elimination
t

T1/2,terminal
=
0.693/kelimination

To obtain PK parameters for the compartmental analysis, the data set of each individual mouse
was fit to either a two-compartment (IV) or four-compartment (SC) PK model using SAAM II
(University of Washington, WA). It was not possible to account for the late peak times observed
109
using a one-compartment model of absorption from the injection site; however, the data was well
fit by two-step absorption from the SC injection site to an intermediate interstitial compartment
and then to the plasma compartment. To estimate the magnitude of the absorption parameters in
the SC data set, it was necessary to assume that the kinetic rate constant kdepot -> lymph is equal to
klymph→blood, which is denoted as k absorption (k abs). The dose and the plasma concentrations were fit
to these models to determine the volume of distribution (Vd), elimination rate constant ( kelimination),
and kabs. On the basis of these fit parameters, the maximum plasma concentration (Cmax),
clearance (CL), and elimination half-life (T1/2,elimination) were estimated for individual mice.
Average values for Vd and kelimination from the IV data set were adopted to perform fits for the SC
data sets. Equations used for compartmental analyses are as follows

CL
=
kelimination*Vd

AUC/F
=
Dose/CL

T1/2,absorption
=
0.693/kabsorption

F
=
kabsorption/(kabsorption
+
kdegradation)

Vd
=
Dose/Co

110
4.3 Results
4.3.1 Physicochemical characterization of 5FA and 5FV

5FA and 5FV (Figure 4.1) were recombinantly expressed in Clear coli, an engineered strain of
E.coli
208
. By introducing eight different mutations in parent BL21 cells, clear coli cells are
designed to utilize a genetically modified lipopolysaccharide (LPS) that does not trigger endotoxic
response in human cells. The six acyl chains of LPS that trigger the endotoxic response are
recognized by Toll-like receptor 4 (TLR4) in complex with myeloid differentiation factor 2 (MD-
2), causing NF-κB activation and production of proinflammatory cytokines. Modified LPS lacks
the two secondary acyl chains and does not induce formation of the activated hTLR4/MD-2
complex, thus evading the endotoxic response. Low endotoxicity of proteins purified from clear
coli is desirable. Protein purification was achieved by 3 rounds of inverse transition cycling, a non-
chromatographic protein purification technique utilizing responsiveness of ELPs to heat and salt
concentration. Protein with >95% purity (Figure 4.2B) and a yield of 50-100 mg/L bacterial
culture was obtained this way. Drug encapsulation was performed as described above. Unbound
drug and trace organic solvents were removed by centrifugation, filtration and extensive dialysis.
The drug loading ratio observed for 5FA-Rapa and 5FV-Rapa (CRapa/C Protein) was 4.4, which
suggests most of the FKBP domains in the formulation are bound to Rapa. This translates to a drug
loading capacity of ~4.5% for both 5FA-Rapa and 5FV-Eve. Endotoxin removal was performed
as described and residual endotoxin burden was measured by FDA recommended chromogenic
LAL assay. At the injection concentration (300 µM Rapa), the endotoxin load was <10 EU/mL for
both 5FA-Rapa and 5FV-Rapa.

111
A B

Figure 4.1 Plasmid map depicting pET-25b(+) vector encoding high capacity ELP fusions,
which each contain 5 FKBP domains that are linked by elastin-like polypeptide. A) 5FA
contains 5 FKBP domains linked by an ELP known as A24, which remains soluble at physiological
temperatures. B) 5FV contains 5 FKBP domains linked by an ELP known as V24, which is
expected to phase separate at physiological temperatures.

To verify proper folding of FKBP domains in 5FA and 5FV, their prolyl isomerase activity was
measured using a chymotrypsin-coupled isomerization assay (Figure 4.2C). The specific activity
for recombinant FKBP was found to be 414 ± 65 pmol/min/µg. The activity for 5FA and 5FV were
comparable at 474 ± 9 pmol/min/µg and 334 ± 41 pmol/min/µg respectively. This suggests
retention of FKBP enzymatic activity in recombinantly expressed 5FA and 5FV. Next,
concentration dependent phase transition property of 5FA and 5FV was evaluated. It is well studied
and established that the phase transition temperature (Tt) of ELPs and their fusion proteins
decreases with an increase in temperature, and Tt vs. log(concentration) assumes a linear
relationship. Such reliable behavior provides an opportunity to predict their physical state over a
wide range of concentrations. When concentration was varied from 100 to 6 µM (Figure 4.2E),
the Tt increased from 26 to 39
o
C for 5FV and from 49 to 56
o
C for 5FA. Using the linear fit
112
described in Table, 5FV is expected to form a coacervate at concentration above 8 µM while 5FA
remains soluble over a wide range of relevant concentrations.

Figure 4.2: Physicochemical characterization of 5FA and 5FV. A) A schematic representation
of high capacity drug carriers loaded with Rapa. Multiple FKBP12 domains are fused with a linker
peptide (VPGAG) 24 or (VPGVG)24. At the site of injection, 5FA is a soluble monomeric protein
while 5FV utilizes body heat to transition to a coacervate, thereby forming a drug depot. B) SDS-
PAGE gel was stained with Coomassie to verify purity and identity of fusion proteins. C) Prolyl
isomerase activity of 5FA and 5FV was measured to confirm proper structural folding of FKBP
domains. The specific activity of proteins was determined in the linear phase of the kinetic curve
and was found to be 414 ± 65 pmol/min/µg, 474 ± 9 pmol/min/µg and 334 ± 41 pmol/min/µg for
recombinant FKBP12, 5FA and 5FV respectively. D) Optical density vs. temperature profile for
25 µM 5FA and 5FV. Phase transition can be seen as a rapid increase in solution turbidity, as
measured by absorbance at 350 nm. 5FV, but not 5FA phase separated below physiological
temperature. E) The transition temperature (Tt) of 5FA and 5FV were found to be concentration
dependent and can be fit to a Log-linear model, Tt = b – m [Log10 (Concentration)] where b
represents the Tt at 1 µM and m represents the Tt change for a 10-fold change in concentration.

113
Table 4.1. Physiochemical characterization of ELP polymers evaluated in this chapter
Label Amino acid sequence MW
(kDa)
a

Purity
(%)
b
Tt
(°C)
c

b (°C)
d
m
d
5FA MG-[FKBP-(VPGAG)24]4-FKBP 99.5 97.5 53.2 59.5 5.2
5FV MG-[FKBP-(VPGVG)24]4-FKBP 102.0 96.6 27.7 46.8 11.0
FKBP amino acid sequence:
VQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGW
EEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEG
a
Expected molecular weight based on amino acid sequence
b
Purity was determined by densitometry analysis of stained SDS-PAGE gel
c
Tt here is reported for 25 μM samples suspended in phosphate buffered saline (PBS)
d
Parameter estimates for b and m for Tt = b – m [Log10 (Concentration)] fit curves

Additional characterization included the use of calorimetry to study binding of Rapa to 5FA
(Figure 4.3). Using Isothermal Titration Calorimetry (ITC), the binding stoichiometry of
5FA/Rapa interaction was estimated to be 5. This suggests all the FKBPs in 5FA are properly
folded and retain their ability to bind to rapamycin. Though the equilibrium dissociation constant
(Kd) was estimated to be 1.5 nM (Fig. 7), I believe the true Kd is much lower and beyond the limit
of detection of ITC (low nM range). In terms of binding thermodynamics, a negative binding
enthalpy (-58 kJ/mol interactions) suggests an exothermic binding, and a negative T∆S (-48 kJ/mol
interactions) suggests an entropic cost associated with binding, which can be explained by
rapamycin transitioning from a free, unbound state in solution to a more ordered FKBP bound
state. An overall negative Gibbs free energy (-10 kJ/mol interactions) indicates FKBP/Rapa
binding is thermodynamically favorable.
114
Figure 4.3: 5FA binds to Rapa at an expected stoichiometric ratio as determined by
isothermal titration calorimetry. A) 8 µM Rapa or Eve in the calorimeter cell was titrated against
12 injections of 20 µM 5FA in the syringe. The titration curve clearly reveal saturation of binding
sites towards the end of the titration. B) Titration of a control ELP A192 against Rapa doesn’t
reveal any binding. Only heats of dilution that remained consistent with injections were observed.
C) The binding isotherm for 5FA vs. Rapa was fit assuming all the FKBP domains in 5FA are
equivalent. D) The best-fit values for stoichiometry of binding (n) and various thermodynamic
parameters are reported as the best fit ± 95% CI.

4.3.2 Mechanisms of inhibition and in vitro cytotoxicity
When evaluated for anti-proliferative properties, 5FA-Rapa formulation potently inhibited the
growth of MDA-MB-468 (Figure 4.4B) breast cancer cells with an IC50 of 0.5 ± 0.2 nM, which is
comparable to IC50 of free rapa (0.3 ± 0.1 nM). Western blots revealed potent mTOR inhibition
when MDA-MB-468 cells were treated with either 5FA-Rapa or free rapa. Rapamycin treatment
blocked mTORC1 mediated phosphorylation (Figure 4.4A) of p70S6 Kinase, thereby inhibiting
phosphorylation and activation of downstream ribosomal protein S6 (rpS6). Rapamycin treatment
also inhibited 4EBP1Ser65 phosphorylation but at concentrations relatively higher than required
for p70S6K inhibition. Phosphorylation of 4EBP1 by mTOR leads to its dissociation from eIF4E,
115
allowing binding of eIF4G at the same site, hence allowing translation initiation complex
formation at the 5` end of mRNAs. Rapamycin mediated rpS6 and 4EBP1 de-phosphorylation
leads to decrease in ribosomal biogenesis, cap-dependent translation and protein synthesis, partly
accounting for its anti-proliferative effect. I also observed an increase in P-AktSer473 upon
treatment, the effect being more pronounced with 5FA-Rapa compared to free rapa treatment. This
rapamycin induced Akt activation is a result of inhibition of the S6K/IRS-1 feedback loop, a
phenomenon well characterized in literature and is thought to be the reason for sub-optimal clinical
performance of rapalogs.

Figure 4.4: 5FA-Rapa displays equipotent mTOR inhibition as free Rapa. A) Dose-dependent
mTORC1 inhibition. In MDA-MB-468 cells, free rapa (left panel) and 5FA-Rapa (right panel)
inhibited phosphorylation related to mTORC1, hence proving on-target inhibition. B) In MDA-
MB-468 cells, 5FA-Rapa and free rapa arrested growth with an IC50 of 0.5 ± 0.2 nM and 0.3 ± 0.1
nM (Mean ± SD) respectively. Error bands in the figure represent the Mean ± SD (n=3).
116
4.3.3 Zr-89 labeling, in vivo PET imaging, and mouse PK characterization
5FA and 5FV were labeled with radioactive Zirconium (
89
Zr) and microPET imaging was used as
a non-invasive technique to estimate depot half-life and understand carrier bio-distribution. PET
imaging has good sensitivity, good resolution, signal is not attenuated by depth of penetration, and
can be calibrated for quantification. Of the various radiotracers available,
89
Zr was chosen because
of its reasonably long radiological half-life (t1/2 = 78.4h), thereby facilitating in-vivo study of
processes taking few weeks, emits relatively low energy positron that produces high resolution
images and the conjugation chemistry is well established.
89
Zr labeling was achieved in two steps
i) 5FA and 5FV were labeled with desferrioxamine (DFO), the most commonly used chelator for
Zr
+4
, ii) 5FA-DFO/5FV-DFO were incubated with radioactive
89
Zr to chelate DFO and complete
the process. DFO labeling of 5FA and 5FV did not result in any aggregation behavior and the
labeled proteins retained the stability and hydrodynamic radius of parent molecules. Moreover,
DFO labeled conjugates had the same phase transition properties and transition temperatures as
parent molecules. The yield of zirconium labeling was ~90% and the radiochemical purity of Zr-
89 labeled constructs was found to be 100% (as measured by Instant Thin Layer Chromatography).

Mice received either IV Zr-5FA, or SC Zr-5FA or SC Zr-5FV. I did not administer IV Zr-5FV
because of the risk for coacervation in the bloodstream that could prove fatal to mice. When
administered subcutaneously, Zr-5FV was retained much longer at the site of injection compared
to Zr-5FA (Figure 4.5A). The depot half-life was found to be 8.9 hr for 5FA as compared to 8.1
days for 5FV (Figure 4.5B). In terms of biodistribution, the heart signal for IV administered 5FA
decreased with time with falling blood concentration (Figure 4.5C). At the same time,
accumulation in the liver and kidneys of mice was observed at longer time periods. Unfortunately,
117
the PET images of mice that received SC carriers did not reveal any signal in peripheral organs,
but the injection depot could be reliably imaged and quantified. At the time of euthanasia, organs
of mice in IV Zr-5FA and SC Zr-5FA were collected and carrier bio-distribution was estimated by
measuring accumulated activity in the while organ. In the IV group, liver showed the highest
accumulation (~ 22% ID/g tissue), followed by the spleen and kidneys (Figure 4.5D). In the SC
group, liver showed the highest accumulation as well (~ 5% ID/g tissue). However, the fraction of
dose that accumulated in peripheral organs was lower for SC administration compared to IV, likely
because of the low bioavailability of SC administration. These organs of accumulation may play a
role in carrier clearance.

118

Figure 4.5: Molecular imaging reveals longer retention of 5FV at the injection site. A) Balb/C
mice were injected SC with 0.2 mCi Zr89-5FV or Zr89-5FA and imaged up to 25 days since
injection. Representative PET images showing higher retention of FKBP-Depot 4 days after
administration B) Quantification of images revealed subcutaneous half-lives of 8.1 ± 2.1 days and
0.37 ± 0.13 days for 5FV and 5FA respectively (Mean ± SD, n=5). C) Non-invasive quantification
of biodistribution. 0.2 mCi 5FA was injected IV and imaged 8hr later. Heart, liver and kidney
show significant intensity. D) At the time of euthanasia, organs from mice that received Zr89-5FA
IV and SC were collected and total accumulated activity was quantified by gamma counting. Liver
showed highest carrier accumulation followed by the spleen and kidneys. SC injection resulted in
lower fraction of injected dose in the organs of accumulation, likely because of the lower
bioavailability associated with SC administration.

In the same mice, plasma PK was simultaneously characterized by sampling blood at multiple time
points post-administration. Gamma counting was used to measure Zr-89 activity in the blood and
was converted to a plasma carrier concentration. Plasma conc. vs time profiles (Figure 4.6A) were
used for non-compartmental PK analysis as described above and multiple PK parameters were
119
deduced (Figure 4.6B). The peak concentration for 5FA was ~ 5 times higher than for 5FV.
However, after 1-week of injection, only 5FV but not 5FA could be detected circulating in the
blood. These features are reflective of a sustained release profile for 5FV. Both 5FA and 5FV had
a bioavailability of ~20%. This is lower than the second generation carrier ‘FAF’ described earlier
which displayed a bioavailability of ~ 60%. Given third generation carriers have lower ELP
contribution to their design, these results suggest ELP may promote absorption from subcutaneous
depots. The mean absorption time (MAT) for 5FV was ~ 6 days compared to 22 hrs for 5FA.
Additionally, the terminal half-life for 5FV was significantly higher for 5FV than 5FA (~ 5.5 days
vs. 2 days). These parameters reflect a more sustained release behavior for 5FV. It should be noted
that all the concentrations measured were higher than the IC50 for Rapa which is < 1nM. Hence,
therapeutic efficacy can be expected.

To better understand PK, a compartmental approach was taken. Since the plasma conc. vs time
profile for IV 5FA showed a clear 2 phase behavior, a 2-comaprtment model (Figure 4.7) was
adopted. For SC administration, a 4-compartment model (Figure 4.7) was able to explain the
observed plasms conc. vs. time profile. The equations used to deduce various PK parameters by
compartmental fitting are stated (Figure 4.7). The fit lines indicating the goodness of fit for the
compartmental models adopted are shown in Figure 4.8 (left). Most of the data points are
accounted for. The estimate for bioavailability was the same (~20%) by both compartmental and
non-compartmental methods. The absorption half-life was longer for SC 5FV compared to SC
5FA (23 vs. 6.4 hrs), thereby revealing slower absorption kinetics and a sustained release for 5FV.
Overall, PK analysis by both the methods were in agreement.
120

Figure 4.6: Non-compartmental PK analysis of IV Zr89-5FA, SC Zr89-5FA and SC Zr89-
5FV. Balb/C mice were injected with 0.2 mCi activity and blood was sampled at multiple time
points. Residual radioactivity was measured by gamma counting and converted to plasma carrier
concentration. A) Plasma concentration vs. time profiles reveal distinct PK profiles for IV 5FA,
SC 5FA and SC 5FV. Concentrations are plotted as Mean ± SD, n=5. B) Non-compartmental PK
analysis was performed using methods described above. PK parameters are reported as Mean ±
SD, n=5.

Figure 4.7: Compartmental PK modeling of IV Zr89-5FA, SC Zr89-5FA and SC Zr89-5FV.
For IV and SC administrations, a two- and four- compartment model described above could
explain (fit) the observed plasma conc. vs time data. Equations that define the models are stated.
121
Figure 4.8: Compartmental PK analysis of IV Zr89-5FA, SC Zr89-5FA and SC Zr89-5FV.
Balb/C mice were injected with 0.2 mCi activity and blood was sampled at multiple time points.
Residual radioactivity was measured by gamma counting and converted to plasma carrier
concentration. Left) Plasma concentration vs. time profiles reveal distinct PK profiles for IV 5FA,
SC 5FA and SC 5FV. Fit lines indicate goodness of fit for compartmental modeling.
Concentrations are plotted as Mean ± SD, n=5. Right) Compartmental PK analysis was performed
using methods described above. PK parameters are reported as Mean ± SD, n=5.

122
4.4 Conclusion
This chapter evaluates third generation FKBP-ELP carriers 5FA and 5FV that offer two significant
advantages i) 5FV enables sustained release from subcutaneous drug depot and hence may reduce
drug dosing frequency required to maintain efficacy ii) higher drug loading capacity (~4.5%)
compared to previous FKBP-ELP generations. How these advantages translate to convenient
dosing in animal models of disease (cancer or transplantation) needs exploration. Such studies are
currently underway.

123
4.5 Cognate receptor-ELP fusion protein technology: Summary and future
This dissertation describes the use of fusion proteins to deliver small molecule drugs through
affinity driven drug retention. Multiple configurations of FKBP-ELP fusion drug carriers that rely
on FKBP-rapalogue interaction were evaluated to improve the current status of rapalogue delivery.
Some features of cognate receptor-ELP technology are noteworthy in the context of its broader
application, beyond the system described here, i) Its applicability is restricted to drugs with well
characterized protein targets. For example, drugs targeting DNA/RNA (DNA alkylating agents
etc.) are beyond the scope of the methodology, ii) The molecular weight of the target protein is a
key factor that predicts success. Since the technology relies on stoichiometric drug-receptor
binding, its loading capacity is dictated by molecular weight of the receptor. For example, using a
~ 100 kDa kinase to deliver a 0.5 kDa inhibitor would not be feasible. This issue can be partly
addressed by employing a simpler system, using the drug binding domain instead of the whole
protein for example. However, this may not be always feasible. Working with smaller receptors
(< 25 kDa) also increases the probability of successful expression of active proteins from E.coli,
iii) Formulations devised using the technology are compatible with all forms of parenteral
administration (IV, SC, IP, IM), the choice of which depends on the target PK profile and
frequency of administration for the particular disease state, iv) Since drug retention by carrier is
mediated by non-covalent interactions, only drug-carrier pairs with strong binding affinity are
expected to provide measurable benefits of carrier assisted delivery. The limits of acceptable
binding affinities can be defined by evaluating in vivo performance of multiple drug- receptor pairs.
However, a reasonable estimation can be obtained using an in vitro release dialysis experiment
41
,
v) Multiple studies suggest that PK of subcutaneously administered depot-forming ELPs is partly
124
controlled by its transition temperature. Hence, Tt of fusion carrier may provide a handle over drug
PK.
For a new drug-carrier pair utilizing the cognate receptor fusion protein technology, the following
sequence of steps would be a reasonable starting point to identify an optimized drug carrier. This
flowchart is proposed partly based on the results presented in this dissertation.
Step 1: Identify the drug and its cognate receptor. When possible, use the smallest possible domain
that retains drug binding affinity in lieu of the full target receptor. Clone the drug binding domain
(DBD) fused to both N- and C- termini of A192 ELP sequence. Express these 2 proteins using 3
different E.coli strains: BLR, Shuffle express and ClearColi following manufacturer’s protocol.
Identify expression and purification conditions that result in pure, soluble carrier protein. If >1
expression system is successful, prefer using ClearColi due to its low endotoxin contamination.
Step 2: Measure the Rh of purified carriers using DLS at room temp. Any presence of preassembled
nanoparticles that formed through expression/purification process is not desirable. If a mixture of
monomeric protein and pre-assembled particles are observed, consider using SEC to discard the
nanoparticle fraction. Next, verify proper folding of DBD using either an enzymatic assay, drug
binding assay (SPR, ITC), circular dichroism etc.
Step 3: Clone and purify DBD fused to S48I48. If DBD-SI can form stable micelles (as measured
by DLS), consider evaluating a nanoparticle formulation, else, clone and optimize purification of
a high capacity drug carrier (similar to 5FA for example). The orientation of high-capacity carrier
will depend on the activity of DBD when fused to N- and C-termini of ELP. The length of ELP
sequence can be modified to meet a target molecular weight of 100 kDa for the fusion carrier. This
125
increases probability of straightforward bacterial purification while escaping glomerular filtration.
Verify all DBDs in these carriers can bind to the drug (using ITC for example).
Step 4: Optimize a drug encapsulation protocol: If the drug is water soluble, incubate excess drug
(from DMSO stock) with the carrier protein (2 – 16 hrs) and perform extensive dialysis to remove
the unbound free drug and residual DMSO. If the drug is hydrophobic, add the drug dropwise
(from ethanol stock for example) in excess to the carrier protein ensuring the ethanol is low enough
to not induce protein precipitation. Excess unbound drug precipitate can be removed by
centrifugation, filtration and extensive dialysis.
Step 5: If sustained drug release from SC/IM site is desirable, clone and optimize purification of a
high-capacity depot forming DBD-ELP carrier (similar to 5FV for example). Using turbidimetry,
ensure reversible phase separation at physiological temperature. Irreversibility in solution turbidity
with decrease in temperature is indicative of protein aggregation and not phase separation.
Step 6: Upon identification of lead and control formulations, PK (both carrier and drug), and
efficacy studies can be performed. In conclusion, this technology presents a unique opportunity to
optimize delivery of small molecule drugs with poor physicochemical properties.

126
References
1. Rowinsky, E. K.; Donehower, R. C., Paclitaxel (taxol). N. Engl. J. Med. 1995, 332, (15),
1004-‐1014.
2. Gelderblom, H.; Verweij, J.; Nooter, K.; Sparreboom, A., Cremophor EL: the drawbacks
and advantages of vehicle selection for drug formulation. Eur. J. Cancer 2001, 37, (13), 1590-‐
1598.
3. Weiss, R. B.; Donehower, R.; Wiernik, P.; Ohnuma, T.; Gralla, R.; Trump, D.; Baker Jr, J.;
Van Echo, D.; Von Hoff, D.; Leyland-‐Jones, B., Hypersensitivity reactions from taxol. J. Clin.
Oncol. 1990, 8, (7), 1263-‐1268.
4. Lorenz, W.; Reimann, H.-‐J.; Schmal, A.; Dormann, P.; Schwarz, B.; Neugebauer, E.;
Doenicke, A., Histamine release in dogs by Cremophor EL® and its derivatives: Oxethylated oleic
acid is the most effective constituent. Agents Actions 1977, 7, (1), 63-‐67.
5. van Zuylen, L.; Verweij, J.; Sparreboom, A., Role of formulation vehicles in taxane
pharmacology. Investigational new drugs 2001, 19, (2), 125-‐141.
6. Maeda, H.; Fang, J.; Inutsuka, T.; Kitamoto, Y., Vascular permeability enhancement in
solid tumor: various factors, mechanisms involved and its implications. Int. Immunopharmacol.
2003, 3, (3), 319-‐328.
7. Maeda, H., The enhanced permeability and retention (EPR) effect in tumor vasculature:
the key role of tumor-‐selective macromolecular drug targeting. Advances in enzyme regulation
2001.
8. Gabizon, A.; Shmeeda, H.; Horowitz, A. T.; Zalipsky, S., Tumor cell targeting of liposome-‐
entrapped drugs with phospholipid-‐anchored folic acid–PEG conjugates. Adv. Drug Delivery Rev.
2004, 56, (8), 1177-‐1192.
9. Hara, K.; Maruki, Y.; Long, X.; Yoshino, K.-‐i.; Oshiro, N.; Hidayat, S.; Tokunaga, C.; Avruch,
J.; Yonezawa, K., Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action.
Cell 2002, 110, (2), 177-‐189.
10. Sarbassov, D. D.; Ali, S. M.; Kim, D.-‐H.; Guertin, D. A.; Latek, R. R.; Erdjument-‐Bromage,
H.; Tempst, P.; Sabatini, D. M., Rictor, a novel binding partner of mTOR, defines a rapamycin-‐
insensitive and raptor-‐independent pathway that regulates the cytoskeleton. Curr. Biol. 2004,
14, (14), 1296-‐1302.
11. Guertin, D. A.; Sabatini, D. M., An expanding role for mTOR in cancer. Trends Mol. Med.
2005, 11, (8), 353-‐361.
12. Sarbassov, D. D.; Guertin, D. A.; Ali, S. M.; Sabatini, D. M., Phosphorylation and
regulation of Akt/PKB by the rictor-‐mTOR complex. Science 2005, 307, (5712), 1098-‐1101.
13. Wullschleger, S.; Loewith, R.; Hall, M. N., TOR signaling in growth and metabolism. Cell
2006, 124, (3), 471-‐484.
14. Tremblay, F. d. r.; Gagnon, A.; Veilleux, A.; Sorisky, A.; Marette, A., Activation of the
mammalian target of rapamycin pathway acutely inhibits insulin signaling to Akt and glucose
transport in 3T3-‐L1 and human adipocytes. Endocrinology 2005, 146, (3), 1328-‐1337.
15. Shi, Y.; Yan, H.; Frost, P.; Gera, J.; Lichtenstein, A., Mammalian target of rapamycin
inhibitors activate the AKT kinase in multiple myeloma cells by up-‐regulating the insulin-‐like
127
growth factor receptor/insulin receptor substrate-‐1/phosphatidylinositol 3-‐kinase cascade.
Molecular cancer therapeutics 2005, 4, (10), 1533-‐1540.
16. O'Reilly, K. E.; Rojo, F.; She, Q.-‐B.; Solit, D.; Mills, G. B.; Smith, D.; Lane, H.; Hofmann, F.;
Hicklin, D. J.; Ludwig, D. L., mTOR inhibition induces upstream receptor tyrosine kinase signaling
and activates Akt. Cancer Res. 2006, 66, (3), 1500-‐1508.
17. Shah, O. J.; Wang, Z.; Hunter, T., Inappropriate activation of the TSC/Rheb/mTOR/S6K
cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies. Curr. Biol.
2004, 14, (18), 1650-‐1656.
18. American Cancer Society. Cancer Facts & Figures 2011.; American Cancer Society:
Atlanta, 2011.
19. Harris, J. R., Diseases of the breast. 4th ed.; Lippincott Williams & Wilkins: Philadelphia,
2010; p xxvi, 1174 p.
20. Foulkes, W. D.; Smith, I. E.; Reis-‐Filho, J. S., Triple-‐negative breast cancer. The New
England journal of medicine 2010, 363, (20), 1938-‐48.
21. Hawkins, M. J.; Soon-‐Shiong, P.; Desai, N., Protein nanoparticles as drug carriers in
clinical medicine. Advanced drug delivery reviews 2008, 60, (8), 876-‐85.
22. Schroeder, A.; Heller, D. A.; Winslow, M. M.; Dahlman, J. E.; Pratt, G. W.; Langer, R.;
Jacks, T.; Anderson, D. G., Treating metastatic cancer with nanotechnology. Nature Reviews
Cancer 2012, 12, (1), 39-‐50.
23. Barenholz, Y., Doxil (R) -‐ The first FDA-‐approved nano-‐drug: Lessons learned. Journal of
Controlled Release 2012, 160, (2), 117-‐134.
24. Mirtsching, B.; Cosgriff, T.; Harker, G.; Keaton, M.; Chidiac, T.; Min, M., A phase II study
of weekly nanoparticle albumin-‐bound paclitaxel with or without trastuzumab in metastatic
breast cancer. Clinical breast cancer 2011, 11, (2), 121-‐8.
25. Conlin, A. K.; Seidman, A. D.; Bach, A.; Lake, D.; Dickler, M.; D'Andrea, G.; Traina, T.;
Danso, M.; Brufsky, A. M.; Saleh, M.; Clawson, A.; Hudis, C. A., Phase II trial of weekly
nanoparticle albumin-‐bound paclitaxel with carboplatin and trastuzumab as first-‐line therapy
for women with HER2-‐overexpressing metastatic breast cancer. Clinical breast cancer 2010, 10,
(4), 281-‐7.
26. Hurvitz, S. A.; Hu, Y.; O'Brien, N.; Finn, R. S., Current approaches and future directions in
the treatment of HER2-‐positive breast cancer. Cancer treatment reviews 2013, 39, (3), 219-‐29.
27. Arpino, G.; Weiss, H.; Lee, A. V.; Schiff, R.; De Placido, S.; Osborne, C. K.; Elledge, R. M.,
Estrogen receptor-‐positive, progesterone receptor-‐negative breast cancer: association with
growth factor receptor expression and tamoxifen resistance. Journal of the National Cancer
Institute 2005, 97, (17), 1254-‐61.
28. Beck, J. T.; Hortobagyi, G. N.; Campone, M.; Lebrun, F.; Deleu, I.; Rugo, H. S.; Pistilli, B.;
Masuda, N.; Hart, L.; Melichar, B.; Dakhil, S.; Geberth, M.; Nunzi, M.; Heng, D. Y. C.;
Brechenmacher, T.; El-‐Hashimy, M.; Douma, S.; Ringeisen, F.; Piccart, M., Everolimus plus
exemestane as first-‐line therapy in HR+, HER2(-‐) advanced breast cancer in BOLERO-‐2. Breast
Cancer Res Tr 2014, 143, (3), 459-‐467.
29. Chia, S.; Gandhi, S.; Joy, A. A.; Edwards, S.; Gorr, M.; Hopkins, S.; Kondejewski, J.; Ayoub,
J. P.; Califaretti, N.; Rayson, D.; Dent, S. F., Novel agents and associated toxicities of inhibitors of
the pi3k/Akt/mtor pathway for the treatment of breast cancer. Current oncology 2015, 22, (1),
33-‐48.
128
30. Siegal, B.; Greenstein, S., Compliance and noncompliance in kidney transplant patients:
cues for transplant coordinators. Journal of Transplant Coordination 1999, 9, (2), 104-‐108.
31. Dew, M. A.; DiMartini, A. F.; Dabbs, A. D. V.; Myaskovsky, L.; Steel, J.; Unruh, M.;
Switzer, G. E.; Zomak, R.; Kormos, R. L.; Greenhouse, J. B., Rates and risk factors for
nonadherence to the medical regimen after adult solid organ transplantation. Transplantation
2007, 83, (7), 858-‐873.
32. Singla, R.; Guliani, A.; Kumari, A.; Yadav, S. K., Metallic nanoparticles, toxicity issues and
applications in medicine. In Nanoscale materials in targeted drug delivery, theragnosis and
tissue regeneration, Springer: 2016; pp 41-‐80.
33. Aluri, S.; Janib, S. M.; Mackay, J. A., Environmentally responsive peptides as anticancer
drug carriers. Adv Drug Deliv Rev 2009, 61, (11), 940-‐52.
34. MacKay, J. A.; Chen, M.; McDaniel, J. R.; Liu, W.; Simnick, A. J.; Chilkoti, A., Self-‐
assembling chimeric polypeptide-‐doxorubicin conjugate nanoparticles that abolish tumours
after a single injection. Nature materials 2009, 8, (12), 993-‐9.
35. Shah, M.; Edman, M. C.; Janga, S. R.; Shi, P.; Dhandhukia, J.; Liu, S.; Louie, S. G.; Rodgers,
K.; Mackay, J. A.; Hamm-‐Alvarez, S. F., A rapamycin-‐binding protein polymer nanoparticle shows
potent therapeutic activity in suppressing autoimmune dacryoadenitis in a mouse model of
Sjogren's syndrome. J Control Release 2013, 171, (3), 269-‐79.
36. Shi, P.; Aluri, S.; Lin, Y. A.; Shah, M.; Edman, M.; Dhandhukia, J.; Cui, H.; MacKay, J. A.,
Elastin-‐based protein polymer nanoparticles carrying drug at both corona and core suppress
tumor growth in vivo. J Control Release 2013, 171, (3), 330-‐8.
37. Shi, P.; Gustafson, J. A.; MacKay, J. A., Genetically engineered nanocarriers for drug
delivery. International journal of nanomedicine 2014, 9, 1617-‐26.
38. Dhandhukia, J. P.; Li, Z.; Peddi, S.; Kakan, S.; Mehta, A.; Tyrpak, D.; Despanie, J.; MacKay,
J. A., Berunda polypeptides -‐ multi-‐headed fusion proteins promote subcutaneous
administration of rapamycin to breast cancer in vivo. Theranostics 2017, 7, (16), 3856-‐3872.
39. Dreher, M. R.; Simnick, A. J.; Fischer, K.; Smith, R. J.; Patel, A.; Schmidt, M.; Chilkoti, A.,
Temperature triggered self-‐assembly of polypeptides into multivalent spherical micelles. J Am
Chem Soc 2008, 130, (2), 687-‐94.
40. Janib, S. M.; Pastuszka, M.; Aluri, S.; Folchman-‐Wagner, Z.; Hsueh, P. Y.; Shi, P.; Yi, A.;
Cui, H.; Mackay, J. A., A quantitative recipe for engineering protein polymer nanoparticles.
Polymer chemistry 2014, 5, (5), 1614-‐1625.
41. Dhandhukia, J. P.; Li, Z.; Peddi, S.; Kakan, S.; Mehta, A.; Tyrpak, D.; Despanie, J.; MacKay,
J. A., Berunda Polypeptides: Multi-‐Headed Fusion Proteins Promote Subcutaneous
Administration of Rapamycin to Breast Cancer In Vivo. Theranostics 2017, 7, (16), 3856.
42. Shah, M.; Hsueh, P. Y.; Sun, G.; Chang, H. Y.; Janib, S. M.; MacKay, J. A., Biodegradation
of elastin-‐like polypeptide nanoparticles. Protein science : a publication of the Protein Society
2012, 21, (6), 743-‐50.
43. Wang, A.; Carraro-‐Lacroix, L. R.; Owen, C.; Gao, B.; Corey, P. N.; Tyrrell, P.; Brumell, J. H.;
Voronov, I., Activity-‐independent targeting of mTOR to lysosomes in primary osteoclasts.
Scientific reports 2017, 7, (1), 3005.
44. Alakhova, D. Y.; Kabanov, A. V., Pluronics and MDR Reversal: An Update. Molecular
pharmaceutics 2014.
129
45. Meyer, D. E.; Kong, G. A.; Dewhirst, M. W.; Zalutsky, M. R.; Chilkoti, A., Targeting a
genetically engineered elastin-‐like polypeptide to solid tumors by local hyperthermia. Cancer
Res. 2001, 61, (4), 1548-‐1554.
46. Bhattacharyya, J.; Bellucci, J. J.; Weitzhandler, I.; McDaniel, J. R.; Spasojevic, I.; Li, X.; Lin,
C.-‐C.; Chi, J.-‐T. A.; Chilkoti, A., A paclitaxel-‐loaded recombinant polypeptide nanoparticle
outperforms Abraxane in multiple murine cancer models. Nature communications 2015, 6, (1),
1-‐12.
47. MacKay, J. A.; Chen, M.; McDaniel, J. R.; Liu, W.; Simnick, A. J.; Chilkoti, A., Self-‐
assembling chimeric polypeptide–doxorubicin conjugate nanoparticles that abolish tumours
after a single injection. Nat. Mater. 2009, 8, (12), 993.
48. Boudreau, N. J.; Varner, J. A., The homeobox transcription factor Hox D3 promotes
integrin α5β1 expression and function during angiogenesis. J. Biol. Chem. 2004, 279, (6), 4862-‐
4868.
49. Reiss, S.; Sieber, M.; Reiss, S.; Sieber, M.; Oberle, V.; Wentzel, A.; Spangenberg, P.; Claus,
R.; Kolmar, H.; Lösche, W., Inhibition of platelet aggregation by grafting RGD and KGD
sequences on the structural scaffold of small disulfide-‐rich proteins. Platelets 2006, 17, (3), 153-‐
157.
50. Janib, S. M.; Gustafson, J. A.; Minea, R. O.; Swenson, S. D.; Liu, S.; Pastuszka, M. K.; Lock,
L. L.; Cui, H.; Markland, F. S.; Conti, P. S., Multimeric disintegrin protein polymer fusions that
target tumor vasculature. Biomacromolecules 2014, 15, (7), 2347-‐2358.
51. Dvorakova, M. C.; Kruzliak, P.; Rabkin, S. W., Role of neuropeptides in cardiomyopathies.
Peptides 2014, 61, 1-‐6.
52. Yeh, S. T.; Youngblood, B. L.; Georgopoulos, L.; Arnold, S.; Hamlin, R. L.; del Rio, C. L.,
Novel Vasoactive Intestinal Peptide-‐ELP Fusion Protein VPAC-‐Agonists Trigger Sustained
Pulmonary Artery Vaso-‐Relaxation in Rats with Acute Hypoxia-‐Induced Pulmonary
Hypertension. In Am Heart Assoc: 2012.
53. del Rio, C. L.; Youngblood, B.; Yeh, S. T.; Georgopoulos, L.; Arnold, S.; Wallery, J.; Hamlin,
R. L., Evaluation of Vasomera™, A Novel VPAC2-‐selective Vasoactive Intestinal Peptide Agonist,
in Rats with Doxorubicin-‐Induced Cardiomyopathy: Evidence for Chronic Cardio-‐Protection. In
Am Heart Assoc: 2012.
54. Carlos, L.; George, R.; Kloepfer, P.; Ueyama, Y.; Youngblood, B.; Georgopoulos, L.;
Arnold, S.; Hamlin, R. L., Vasomera™, a novel VPAC2-‐selective vasoactive intestinal peptide
agonist, enhances contractility and decreases myocardial demand in dogs with both normal
hearts and with pacing-‐induced dilated cardiomyopathy. J. Am. Coll. Cardiol. 2013, 61, (10
Supplement), E645.
55. Free, A.; Brazg, R.; Matson, M.; Smith, W.; Chuck, L.; Georgopoulos, L.; Malatesta, J.;
Arnold, S.; Kramer, W.; Strange, P., A phase 1, multi-‐center, randomized, double-‐blind, placebo
controlled study to evaluate the safety/tolerability, pharmacokinetic and hemodynamic
response following single ascending subcutaneous doses of PB1046 (vasomera™) in subjects
with essential hypertension. Circulation 2014, 130, (suppl_2), A19112-‐A19112.
56. Mojsov, S.; Heinrich, G.; Wilson, I. B.; Ravazzola, M.; Orci, L.; Habener, J. F.,
Preproglucagon gene expression in pancreas and intestine diversifies at the level of post-‐
translational processing. J. Biol. Chem. 1986, 261, (25), 11880-‐11889.
130
57. Flint, A.; Raben, A.; Astrup, A.; Holst, J. J., Glucagon-‐like peptide 1 promotes satiety and
suppresses energy intake in humans. The Journal of clinical investigation 1998, 101, (3), 515-‐
520.
58. Drucker, D. J., Glucagon-‐like peptide-‐1 and the islet β-‐cell: augmentation of cell
proliferation and inhibition of apoptosis. Endocrinology 2003, 144, (12), 5145-‐5148.
59. Vilsbøll, T.; Agersø, H.; Krarup, T.; Holst, J. J., Similar elimination rates of glucagon-‐like
peptide-‐1 in obese type 2 diabetic patients and healthy subjects. The Journal of Clinical
Endocrinology & Metabolism 2003, 88, (1), 220-‐224.
60. Luginbuhl, K. M.; Schaal, J. L.; Umstead, B.; Mastria, E. M.; Li, X.; Banskota, S.; Arnold, S.;
Feinglos, M.; D’Alessio, D.; Chilkoti, A., One-‐week glucose control via zero-‐order release kinetics
from an injectable depot of glucagon-‐like peptide-‐1 fused to a thermosensitive biopolymer.
Nature biomedical engineering 2017, 1, (6), 1-‐14.
61. Christiansen, M.; Matson, M.; Brazg, R.; Georgopoulos, L.; Arnold, S.; Kramer, W.; Shi, L.;
Strange, P. In Weekly subcutaneous doses of glymera (PB1023) a novel GLP-‐1 analogue reduce
glucose exposure dose-‐dependently, Diabetes, 2012; AMER DIABETES ASSOC 1701 N
BEAUREGARD ST, ALEXANDRIA, VA 22311-‐1717 USA: 2012; pp A241-‐A241.
62. Liu, W.; Dreher, M. R.; Furgeson, D. Y.; Peixoto, K. V.; Yuan, H.; Zalutsky, M. R.; Chilkoti,
A., Tumor accumulation, degradation and pharmacokinetics of elastin-‐like polypeptides in nude
mice. J. Controlled Release 2006, 116, (2), 170-‐178.
63. Dreher, M. R.; Raucher, D.; Balu, N.; Colvin, O. M.; Ludeman, S. M.; Chilkoti, A.,
Evaluation of an elastin-‐like polypeptide–doxorubicin conjugate for cancer therapy. J.
Controlled Release 2003, 91, (1-‐2), 31-‐43.
64. Amiram, M.; Luginbuhl, K.; Li, X.; Feinglos, M.; Chilkoti, A., A depot-‐forming glucagon-‐like
peptide-‐1 fusion protein reduces blood glucose for five days with a single injection. J. Controlled
Release 2013, 172, (1), 144-‐151.
65. Wang, W.; Despanie, J.; Shi, P.; Edman, M. C.; Lin, Y.-‐A.; Cui, H.; Heur, M.; Fini, M. E.;
Hamm-‐Alvarez, S. F.; MacKay, J. A., Lacritin-‐mediated regeneration of the corneal epithelia by
protein polymer nanoparticles. J. Mater. Chem. B 2014, 2, (46), 8131-‐8141.
66. Walker, L. R.; Ryu, J. S.; Perkins, E.; McNally, L. R.; Raucher, D., Fusion of cell-‐penetrating
peptides to thermally responsive biopolymer improves tumor accumulation of p21 peptide in a
mouse model of pancreatic cancer. Drug Des. Devel. Ther. 2014, 8, 1649.
67. Aluri, S. R.; Shi, P.; Gustafson, J. A.; Wang, W.; Lin, Y.-‐A.; Cui, H.; Liu, S.; Conti, P. S.; Li, Z.;
Hu, P., A hybrid protein–polymer nanoworm potentiates apoptosis better than a monoclonal
antibody. ACS nano 2014, 8, (3), 2064-‐2076.
68. Betre, H.; Setton, L. A.; Meyer, D. E.; Chilkoti, A., Characterization of a genetically
engineered elastin-‐like polypeptide for cartilaginous tissue repair. Biomacromolecules 2002, 3,
(5), 910-‐916.
69. Betre, H.; Ong, S. R.; Guilak, F.; Chilkoti, A.; Fermor, B.; Setton, L. A., Chondrocytic
differentiation of human adipose-‐derived adult stem cells in elastin-‐like polypeptide.
Biomaterials 2006, 27, (1), 91-‐99.
70. Nettles, D. L.; Kitaoka, K.; Hanson, N. A.; Flahiff, C. M.; Mata, B. A.; Hsu, E. W.; Chilkoti,
A.; Setton, L. A., In situ crosslinking elastin-‐like polypeptide gels for application to articular
cartilage repair in a goat osteochondral defect model. Tissue Eng., Part A 2008, 14, (7), 1133-‐
1140.
131
71. Nettles, D. L.; Chilkoti, A.; Setton, L. A., Applications of elastin-‐like polypeptides in tissue
engineering. Adv. Drug Delivery Rev. 2010, 62, (15), 1479-‐1485.
72. Dash, B. C.; Thomas, D.; Monaghan, M.; Carroll, O.; Chen, X.; Woodhouse, K.; O'Brien, T.;
Pandit, A., An injectable elastin-‐based gene delivery platform for dose-‐dependent modulation
of angiogenesis and inflammation for critical limb ischemia. Biomaterials 2015, 65, 126-‐139.
73. Dash, B. C.; Mahor, S.; Carroll, O.; Mathew, A.; Wang, W.; Woodhouse, K. A.; Pandit, A.,
Tunable elastin-‐like polypeptide hollow sphere as a high payload and controlled delivery gene
depot. J. Controlled Release 2011, 152, (3), 382-‐392.
74. Kim, J.; Chu, H.; Park, K.; Won, J.; Jang, J., Elastin-‐like polypeptide matrices for enhancing
adeno-‐associated virus-‐mediated gene delivery to human neural stem cells. Gene Ther. 2012,
19, (3), 329.
75. Despanie, J.; Dhandhukia, J. P.; Hamm-‐Alvarez, S. F.; MacKay, J. A., Elastin-‐like
polypeptides: therapeutic applications for an emerging class of nanomedicines. J. Controlled
Release 2016, 240, 93-‐108.
76. Dreher, M. R.; Simnick, A. J.; Fischer, K.; Smith, R. J.; Patel, A.; Schmidt, M.; Chilkoti, A.,
Temperature triggered self-‐assembly of polypeptides into multivalent spherical micelles. J. Am.
Chem. Soc. 2008, 130, (2), 687-‐694.
77. Janib, S. M.; Pastuszka, M.; Aluri, S.; Folchman-‐Wagner, Z.; Hsueh, P.; Shi, P.; Lin, Y.; Cui,
H.; Mackay, J., A quantitative recipe for engineering protein polymer nanoparticles. Polym.
Chem. 2014, 5, (5), 1614-‐1625.
78. Shi, P.; Aluri, S.; Lin, Y.-‐A.; Shah, M.; Edman, M.; Dhandhukia, J.; Cui, H.; MacKay, J. A.,
Elastin-‐based protein polymer nanoparticles carrying drug at both corona and core suppress
tumor growth in vivo. J. Controlled Release 2013, 171, (3), 330-‐338.
79. Urry, D. W.; Trapane, T.; Prasad, K., Phase-‐structure transitions of the elastin
polypentapeptide–water system within the framework of composition–temperature studies.
Biopolymers 1985, 24, (12), 2345-‐2356.
80. Dhandhukia, J. P.; Shi, P.; Peddi, S.; Li, Z.; Aluri, S.; Ju, Y.; Brill, D.; Wang, W.; Janib, S. M.;
Lin, Y.-‐A., Bifunctional elastin-‐like polypeptide nanoparticles bind rapamycin and integrins and
suppress tumor growth in Vivo. Bioconjugate Chem. 2017, 28, (11), 2715-‐2728.
81. Sun, G.; Hsueh, P.-‐Y.; Janib, S. M.; Hamm-‐Alvarez, S.; MacKay, J. A., Design and cellular
internalization of genetically engineered polypeptide nanoparticles displaying adenovirus knob
domain. J. Controlled Release 2011, 155, (2), 218-‐226.
82. Varanko, A. K.; Chilkoti, A., Molecular and Materials Engineering for Delivery of Peptide
Drugs to Treat Type 2 Diabetes. Adv. Healthcare Mater. 2019, 1801509.
83. Dumont, F. J.; Su, Q., Mechanism of action of the immunosuppressant rapamycin. Life
Sci. 1995, 58, (5), 373-‐395.
84. Augustine, J. J.; Bodziak, K. A.; Hricik, D. E., Use of sirolimus in solid organ
transplantation. Drugs 2007, 67, (3), 369-‐391.
85. Trepanier, D. J.; Gallant, H.; Legatt, D. F.; Yatscoff, R. W., Rapamycin: distribution,
pharmacokinetics and therapeutic range investigations: an update. Clin. Biochem. 1998, 31, (5),
345-‐351.
86. Ferron, G. M.; Mishina, E. V.; Zimmerman, J. J.; Jusko, W. J., Population
pharmacokinetics of sirolimus in kidney transplant patients. Clin. Pharmacol. Ther. 1997, 61, (4),
416-‐428.
132
87. Marti, H.-‐P.; Frey, F. J., Nephrotoxicity of rapamycin: an emerging problem in clinical
medicine. Nephrol. Dial. Transplant. 2005, 20, (1), 13-‐15.
88. Pham, P.-‐T. T.; Pham, P.-‐C. T.; Danovitch, G. M.; Ross, D. J.; Gritsch, H. A.; Kendrick, E. A.;
Singer, J.; Shah, T.; Wilkinson, A. H., Sirolimus-‐associated pulmonary toxicity. Transplantation
2004, 77, (8), 1215-‐1220.
89. Danesi, R.; Boni, J. P.; Ravaud, A., Oral and intravenously administered mTOR inhibitors
for metastatic renal cell carcinoma: pharmacokinetic considerations and clinical implications.
Cancer Treat. Rev. 2013, 39, (7), 784-‐792.
90. Gomez-‐Fernandez, C.; Garden, B. C.; Wu, S.; Feldman, D. R.; Lacouture, M. E., The risk of
skin rash and stomatitis with the mammalian target of rapamycin inhibitor temsirolimus: a
systematic review of the literature and meta-‐analysis. Eur. J. Cancer 2012, 48, (3), 340-‐346.
91. de Oliveira, M. A.; e Martins, F. M.; Wang, Q.; Sonis, S.; Demetri, G.; George, S.;
Butrynski, J.; Treister, N. S., Clinical presentation and management of mTOR inhibitor-‐
associated stomatitis. Oral Oncol. 2011, 47, (10), 998-‐1003.
92. Miele, E.; Spinelli, G. P.; Miele, E.; Tomao, F.; Tomao, S., Albumin-‐bound formulation of
paclitaxel (Abraxane® ABI-‐007) in the treatment of breast cancer. Int. J. Nanomed. 2009, 4, 99.
93. Green, M.; Manikhas, G.; Orlov, S.; Afanasyev, B.; Makhson, A.; Bhar, P.; Hawkins, M.,
Abraxane®, a novel Cremophor®-‐free, albumin-‐bound particle form of paclitaxel for the
treatment of advanced non-‐small-‐cell lung cancer. Ann. Oncol. 2006, 17, (8), 1263-‐1268.
94. Micha, J. P.; Goldstein, B. H.; Birk, C. L.; Rettenmaier, M. A.; Brown III, J. V., Abraxane in
the treatment of ovarian cancer: the absence of hypersensitivity reactions. Gynecol. Oncol.
2006, 100, (2), 437-‐438.
95. Farokhzad, O. C.; Cheng, J.; Teply, B. A.; Sherifi, I.; Jon, S.; Kantoff, P. W.; Richie, J. P.;
Langer, R., Targeted nanoparticle-‐aptamer bioconjugates for cancer chemotherapy in vivo.
Proc. Natl. Acad. Sci. U. S. A. 2006, 103, (16), 6315-‐6320.
96. Banaszynski, L. A.; Liu, C. W.; Wandless, T. J., Characterization of the FKBP⊙
Rapamycin⊙ FRB Ternary Complex. J. Am. Chem. Soc. 2005, 127, (13), 4715-‐4721.
97. Chen, K.; Chen, X., Integrin targeted delivery of chemotherapeutics. Theranostics 2011,
1, 189.
98. Desgrosellier, J. S.; Cheresh, D. A., Integrins in cancer: biological implications and
therapeutic opportunities. Nat. Rev. Cancer 2010, 10, (1), 9.
99. Subik, K.; Lee, J.-‐F.; Baxter, L.; Strzepek, T.; Costello, D.; Crowley, P.; Xing, L.; Hung, M.-‐
C.; Bonfiglio, T.; Hicks, D. G., The expression patterns of ER, PR, HER2, CK5/6, EGFR, Ki-‐67 and
AR by immunohistochemical analysis in breast cancer cell lines. Breast Cancer: Basic Clin. Res.
2010, 4, 117822341000400004.
100. Dhandhukia, J.; Weitzhandler, I.; Wang, W.; MacKay, J. A., Switchable elastin-‐like
polypeptides that respond to chemical inducers of dimerization. Biomacromolecules 2013, 14,
(4), 976-‐985.
101. Hassouneh, W.; Christensen, T.; Chilkoti, A., Elastin-‐like polypeptides as a purification
tag for recombinant proteins. Curr. Protoc. Protein Sci. 2010, 61, (1), 6.11. 1-‐6.11. 16.
102. Pace, C. N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T., How to measure and predict the
molar absorption coefficient of a protein. Protein Sci. 1995, 4, (11), 2411-‐2423.
133
103. Nair, A. B.; Jacob, S., A simple practice guide for dose conversion between animals and
human. J. Basic Clin. Pharm. 2016, 7, (2), 27.
104. Malyala, P.; Singh, M., Endotoxin limits in formulations for preclinical research. J. Pharm.
Sci. 2008, 97, (6), 2041-‐2044.
105. Meyer, D. E.; Chilkoti, A., 18 Protein Purification by Inverse Transition Cycling. Protein-‐
protein interactions: A molecular cloning manual 2002, 329-‐344.
106. Oberdörster, G., Safety assessment for nanotechnology and nanomedicine: concepts of
nanotoxicology. J. Intern. Med. 2010, 267, (1), 89-‐105.
107. Manke, A.; Wang, L.; Rojanasakul, Y., Mechanisms of nanoparticle-‐induced oxidative
stress and toxicity. BioMed Res. Int. 2013, 2013, 1-‐15.
108. Fu, P. P.; Xia, Q.; Hwang, H.-‐M.; Ray, P. C.; Yu, H., Mechanisms of nanotoxicity:
generation of reactive oxygen species. J. Food Drug Anal. 2014, 22, (1), 64-‐75.
109. Abdal Dayem, A.; Hossain, M.; Lee, S.; Kim, K.; Saha, S.; Yang, G.-‐M.; Choi, H.; Cho, S.-‐G.,
The role of reactive oxygen species (ROS) in the biological activities of metallic nanoparticles.
Int. J. Mol. Sci. 2017, 18, (1), 120.
110. Malinauskas, R. A., Plasma hemoglobin measurement techniques for the in vitro
evaluation of blood damage caused by medical devices. Artif. Organs 1997, 21, (12), 1255-‐1267.
111. Chanan-‐Khan, A.; Szebeni, J.; Savay, S.; Liebes, L.; Rafique, N.; Alving, C.; Muggia, F.,
Complement activation following first exposure to pegylated liposomal doxorubicin (Doxil®):
possible role in hypersensitivity reactions. Ann. Oncol. 2003, 14, (9), 1430-‐1437.
112. Szebeni, J.; Muggia, F.; Gabizon, A.; Barenholz, Y., Activation of complement by
therapeutic liposomes and other lipid excipient-‐based therapeutic products: prediction and
prevention. Adv. Drug Delivery Rev. 2011, 63, (12), 1020-‐1030.
113. Weiszhár, Z.; Czúcz, J.; Révész, C.; Rosivall, L.; Szebeni, J.; Rozsnyay, Z., Complement
activation by polyethoxylated pharmaceutical surfactants: Cremophor-‐EL, Tween-‐80 and
Tween-‐20. Eur. J. Pharm. Sci. 2012, 45, (4), 492-‐498.
114. Hassan, B.; Akcakanat, A.; Sangai, T.; Evans, K. W.; Adkins, F.; Eterovic, A. K.; Zhao, H.;
Chen, K.; Chen, H.; Do, K.-‐A., Catalytic mTOR inhibitors can overcome intrinsic and acquired
resistance to allosteric mTOR inhibitors. Oncotarget 2014, 5, (18), 8544.
115. Sun, M.; Si, L.; Zhai, X.; Fan, Z.; Ma, Y.; Zhang, R.; Yang, X., The influence of co-‐solvents
on the stability and bioavailability of rapamycin formulated in self-‐microemulsifying drug
delivery systems. Drug Dev. Ind. Pharm. 2011, 37, (8), 986-‐994.
116. Waranis, R. P.; Harrison, M. M.; Leonard, T. W.; Enever, R. P., Rapamycin formulation for
IV injection. In Google Patents: 1996.
117. Qiu, S.; Liu, Z.; Hou, L.; Li, Y.; Wang, J.; Wang, H.; Du, W.; Wang, W.; Qin, Y.; Liu, Z.,
Complement activation associated with polysorbate 80 in beagle dogs. Int. Immunopharmacol.
2013, 15, (1), 144-‐149.
118. Massodi, I.; Thomas, E.; Raucher, D., Application of thermally responsive elastin-‐like
polypeptide fused to a lactoferrin-‐derived peptide for treatment of pancreatic cancer.
Molecules 2009, 14, (6), 1999-‐2015.
119. Massodi, I.; Raucher, D., A thermally responsive Tat-‐elastin-‐like polypeptide fusion
protein induces membrane leakage, apoptosis, and cell death in human breast cancer cells. J.
Drug Targeting 2007, 15, (9), 611-‐622.
134
120. Urry, D. W.; Parker, T. M.; Reid, M. C.; Gowda, D. C., Biocompatibility of the bioelastic
materials, poly (GVGVP) and its γ-‐irradiation cross-‐linked matrix: summary of generic biological
test results. J. Bioact. Compat. Polym. 1991, 6, (3), 263-‐282.
121. Roberts, H. R.; Monroe, D.; Hoffman, M., Molecular biology and biochemistry of the
coagulation factors and pathways of hemostasis. Williams hematology 2001, 6, 1409-‐34.
122. Bahniuk, M. S., Human Plasma Adsorption to Biomaterials: Fundamental Level Chemical
Modifications and Their Effects on Biocompatibility. 2017.
123. Bates, C.; Carey, P.; Hind, C., Anaphylaxis due to liposomal amphotericin (AmBisome).
Genitourin. Med. 1995, 71, (6), 414.
124. Szebeni, J.; Bedőcs, P.; Rozsnyay, Z.; Weiszhár, Z.; Urbanics, R.; Rosivall, L.; Cohen, R.;
Garbuzenko, O.; Báthori, G.; Tóth, M., Liposome-‐induced complement activation and related
cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and AmBisome.
Nanomedicine: Nanotechnology, Biology and Medicine 2012, 8, (2), 176-‐184.
125. Moghimi, S. M.; Farhangrazi, Z. S., Nanomedicine and the complement paradigm.
Nanomedicine: Nanotechnology, Biology and Medicine 2013, 9, (4), 458-‐460.
126. Szebeni, J., Complement activation-‐related pseudoallergy: a stress reaction in blood
triggered by nanomedicines and biologicals. Mol. Immunol. 2014, 61, (2), 163-‐173.
127. Srokowski, E. M.; Woodhouse, K. A., Evaluation of the bulk platelet response and
fibrinogen interaction to elastin-‐like polypeptide coatings. J. Biomed. Mater. Res., Part A 2014,
102, (2), 540-‐551.
128. Srokowski, E.; Blit, P.; McClung, W.; Brash, J.; Santerre, J.; Woodhouse, K., Platelet
adhesion and fibrinogen accretion on a family of elastin-‐like polypeptides. J. Biomater. Sci.,
Polym. Ed. 2011, 22, (1-‐3), 41-‐57.
129. Blit, P. H.; McClung, W. G.; Brash, J. L.; Woodhouse, K. A.; Santerre, J. P., Platelet
inhibition and endothelial cell adhesion on elastin-‐like polypeptide surface modified materials.
Biomaterials 2011, 32, (25), 5790-‐5800.
130. Dobrovolskaia, M. A.; McNeil, S. E., Understanding the correlation between in vitro and
in vivo immunotoxicity tests for nanomedicines. J. Controlled Release 2013, 172, (2), 456-‐466.
131. Shah, M.; Hsueh, P. Y.; Sun, G.; Chang, H. Y.; Janib, S. M.; MacKay, J. A., Biodegradation
of elastin-‐like polypeptide nanoparticles. Protein Sci. 2012, 21, (6), 743-‐750.
132. Menninger, H.; Burkhardt, H.; Röske, W.; Ehlebracht, W.; Hering, B.; Gurr, E.; Mohr, W.;
Mierau, H., Lysosomal elastase: effect on mechanical and biochemical properties of normal
cartilage, inhibition by polysulfonated glycosaminoglycan, and binding to chondrocytes.
Rheumatol. Int. 1981, 1, (2), 73-‐81.
133. Everts, V.; Korper, W.; Niehof, A.; Jansen, I.; Beertsen, W., Type VI collagen is
phagocytosed by fibroblasts and digested in the lysosomal apparatus: involvement of
collagenase, serine proteinases and lysosomal enzymes. Matrix Biol. 1995, 14, (8), 665-‐676.
134. Bidwell III, G. L.; Raucher, D., Cell penetrating elastin-‐like polypeptides for therapeutic
peptide delivery. Adv. Drug Delivery Rev. 2010, 62, (15), 1486-‐1496.
135. Pesce, D., Thermotropic liquid crystals from engineered polypeptides. University of
Groningen: 2015.
136. Raucher, D.; Chilkoti, A., Enhanced uptake of a thermally responsive polypeptide by
tumor cells in response to its hyperthermia-‐mediated phase transition. Cancer Res. 2001, 61,
(19), 7163-‐7170.
135
137. Peddi, S.; Pan, X.; MacKay, J. A., Intracellular delivery of Rapamycin from FKBP elastin-‐
like polypeptides is consistent with macropinocytosis. Front. Pharmacol. 2018, 9, (1184).
138. Marano, F.; Rodrigues-‐Lima, F.; Dupret, J.-‐M.; Baeza-‐Squiban, A.; Boland, S., Cellular
Mechanisms of Nanoparticle Toxicity. In Encyclopedia of Nanotechnology, Bhushan, B., Ed.
Springer Netherlands: Dordrecht, 2016; pp 498-‐505.
139. Saxton, R. A.; Sabatini, D. M., mTOR signaling in growth, metabolism, and disease. Cell
2017, 168, (6), 960-‐976.
140. Huang, J.; Manning, B. D., The TSC1–TSC2 complex: a molecular switchboard controlling
cell growth. Biochem. J. 2008, 412, (2), 179-‐190.
141. Gwinn, D. M.; Shackelford, D. B.; Egan, D. F.; Mihaylova, M. M.; Mery, A.; Vasquez, D. S.;
Turk, B. E.; Shaw, R. J., AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol.
Cell 2008, 30, (2), 214-‐226.
142. Kim, E.; Goraksha-‐Hicks, P.; Li, L.; Neufeld, T. P.; Guan, K.-‐L., Regulation of TORC1 by Rag
GTPases in nutrient response. Nat. Cell Biol. 2008, 10, (8), 935-‐945.
143. Feng, Z.; Hu, W.; De Stanchina, E.; Teresky, A. K.; Jin, S.; Lowe, S.; Levine, A. J., The
regulation of AMPK β1, TSC2, and PTEN expression by p53: stress, cell and tissue specificity, and
the role of these gene products in modulating the IGF-‐1-‐AKT-‐mTOR pathways. Cancer Res.
2007, 67, (7), 3043-‐3053.
144. Holz, M. K.; Ballif, B. A.; Gygi, S. P.; Blenis, J., mTOR and S6K1 mediate assembly of the
translation preinitiation complex through dynamic protein interchange and ordered
phosphorylation events. Cell 2005, 123, (4), 569-‐580.
145. Brunn, G. J.; Hudson, C. C.; Sekulić, A.; Williams, J. M.; Hosoi, H.; Houghton, P. J.;
Lawrence, J. C.; Abraham, R. T., Phosphorylation of the translational repressor PHAS-‐I by the
mammalian target of rapamycin. Science 1997, 277, (5322), 99-‐101.
146. Porstmann, T.; Santos, C. R.; Griffiths, B.; Cully, M.; Wu, M.; Leevers, S.; Griffiths, J. R.;
Chung, Y.-‐L.; Schulze, A., SREBP activity is regulated by mTORC1 and contributes to Akt-‐
dependent cell growth. Cell Metab. 2008, 8, (3), 224-‐236.
147. Ben-‐Sahra, I.; Hoxhaj, G.; Ricoult, S. J.; Asara, J. M.; Manning, B. D., mTORC1 induces
purine synthesis through control of the mitochondrial tetrahydrofolate cycle. Science 2016,
351, (6274), 728-‐733.
148. Düvel, K.; Yecies, J. L.; Menon, S.; Raman, P.; Lipovsky, A. I.; Souza, A. L.; Triantafellow,
E.; Ma, Q.; Gorski, R.; Cleaver, S., Activation of a metabolic gene regulatory network
downstream of mTOR complex 1. Mol. Cell 2010, 39, (2), 171-‐183.
149. Kim, J.; Kundu, M.; Viollet, B.; Guan, K.-‐L., AMPK and mTOR regulate autophagy through
direct phosphorylation of Ulk1. Nat. Cell Biol. 2011, 13, (2), 132-‐141.
150. Vezina, C.; Kudelski, A.; Sehgal, S., Rapamycin (AY-‐22, 989), a new antifungal antibiotic.
The Journal of antibiotics 1975, 28, (10), 721-‐726.
151. Martel, R.; Klicius, J.; Galet, S., Inhibition of the immune response by rapamycin, a new
antifungal antibiotic. Can. J. Physiol. Pharmacol. 1977, 55, (1), 48-‐51.
152. Eng, C.; Sehgal, S.; Vézina, C., Activity of rapamycin (AY-‐22, 989) against transplanted
tumors. The Journal of antibiotics 1984, 37, (10), 1231-‐1237.
153. Brown, E. J.; Albers, M. W.; Shin, T. B.; Keith, C. T.; Lane, W. S.; Schreiber, S. L., A
mammalian protein targeted by G1-‐arresting rapamycin–receptor complex. Nature 1994, 369,
(6483), 756-‐758.
136
154. Sabers, C. J.; Martin, M. M.; Brunn, G. J.; Williams, J. M.; Dumont, F. J.; Wiederrecht, G.;
Abraham, R. T., Isolation of a protein target of the FKBP12-‐rapamycin complex in mammalian
cells. J. Biol. Chem. 1995, 270, (2), 815-‐822.
155. Heitman, J.; Movva, N. R.; Hall, M. N., Targets for cell cycle arrest by the
immunosuppressant rapamycin in yeast. Science 1991, 253, (5022), 905-‐909.
156. Powell, J. D.; Pollizzi, K. N.; Heikamp, E. B.; Horton, M. R., Regulation of immune
responses by mTOR. Annu. Rev. Immunol. 2012, 30, 39-‐68.
157. Beaver, J. A.; Park, B. H., The BOLERO-‐2 trial: the addition of everolimus to exemestane
in the treatment of postmenopausal hormone receptor-‐positive advanced breast cancer. Future
Oncol. 2012, 8, (6), 651-‐657.
158. Shah, M.; Edman, M. C.; Janga, S. R.; Shi, P.; Dhandhukia, J.; Liu, S.; Louie, S. G.; Rodgers,
K.; MacKay, J. A.; Hamm-‐Alvarez, S. F., A rapamycin-‐binding protein polymer nanoparticle
shows potent therapeutic activity in suppressing autoimmune dacryoadenitis in a mouse model
of Sjögren's syndrome. J. Controlled Release 2013, 171, (3), 269-‐279.
159. Peddi, S.; Pan, X.; MacKay, J. A., Intracellular Delivery of Rapamycin From FKBP Elastin-‐
Like Polypeptides Is Consistent With Macropinocytosis. Front. Pharmacol. 2018, 9, 1184-‐1197.
160. Urry, D. W., Free energy transduction in polypeptides and proteins based on inverse
temperature transitions. Progress in biophysics and molecular biology 1992, 57, (1), 23-‐57.
161. Urry, D. W., Entropic elastic processes in protein mechanisms. I. Elastic structure due to
an inverse temperature transition and elasticity due to internal chain dynamics. Journal of
protein chemistry 1988, 7, (1), 1-‐34.
162. Lim, J. P.; Gleeson, P. A., Macropinocytosis: an endocytic pathway for internalising large
gulps. Immunology and cell biology 2011, 89, (8), 836.
163. Koivusalo, M.; Welch, C.; Hayashi, H.; Scott, C. C.; Kim, M.; Alexander, T.; Touret, N.;
Hahn, K. M.; Grinstein, S., Amiloride inhibits macropinocytosis by lowering submembranous pH
and preventing Rac1 and Cdc42 signaling. The Journal of cell biology 2010, 188, (4), 547-‐563.
164. Zhang, Y.; Yan, H.; Xu, Z.; Yang, B.; Luo, P.; He, Q., Molecular basis for class side effects
associated with PI3K/AKT/mTOR pathway inhibitors. Expert Opin. Drug Metab. Toxicol. 2019,
15, (9), 767-‐774.
165. Sonis, S.; Treister, N.; Chawla, S.; Demetri, G.; Haluska, F., Preliminary characterization of
oral lesions associated with inhibitors of mammalian target of rapamycin in cancer patients.
Cancer: Interdisciplinary International Journal of the American Cancer Society 2010, 116, (1),
210-‐215.
166. Halford, B., Rapamycin’s secrets unearthed. In Chem. Eng. News, ACS: 2016; Vol. 94, pp
26-‐30.
167. Desai, N.; Trieu, V.; Yao, Z.; Louie, L.; Ci, S.; Yang, A.; Tao, C.; De, T.; Beals, B.; Dykes, D.,
Increased antitumor activity, intratumor paclitaxel concentrations, and endothelial cell
transport of cremophor-‐free, albumin-‐bound paclitaxel, ABI-‐007, compared with cremophor-‐
based paclitaxel. Clin. Cancer Res. 2006, 12, (4), 1317-‐1324.
168. Schnitzer, J., gp60 is an albumin-‐binding glycoprotein expressed by continuous
endothelium involved in albumin transcytosis. American Journal of Physiology-‐Heart and
Circulatory Physiology 1992, 262, (1), H246-‐H254.
137
169. Trieu, V.; Hwang, J.; Desai, N., Nanoparticle Albumin-‐bound (nab) technology may
enhance antitumor activity via targeting of SPARC protein. New targets and delivery system for
cancer diagnosis and treatment (SKCC). San Diego, Abstract 2007, 53.
170. Trieu, V.; Damascelli, B.; Soon-‐Shiong, P.; Desai, N., SPARC expression in head and neck
cancer correlates with tumor response to nanoparticle albumin-‐bound paclitaxel (nab-‐
paclitaxel, ABI-‐007, Abraxane). In AACR: 2006.
171. Gonzalez-‐Angulo, A. M.; Meric-‐Bernstam, F.; Chawla, S.; Falchook, G.; Hong, D.;
Akcakanat, A.; Chen, H.; Naing, A.; Fu, S.; Wheler, J., Weekly nab-‐rapamycin in patients with
advanced nonhematologic malignancies: final results of a phase I trial. Clin. Cancer Res. 2013,
19, (19), 5474-‐5484.
172. Cirstea, D.; Hideshima, T.; Rodig, S.; Santo, L.; Pozzi, S.; Vallet, S.; Ikeda, H.; Perrone, G.;
Patel, K.; Desai, N., Dual inhibition of Akt/mTOR pathway by nab-‐rapamycin and perifosine
induces anti-‐tumor activity in multiple myeloma. Molecular cancer therapeutics 2010, 9, (4),
963.
173. Lee, C.; Guo, H.; Klinngam, W.; Janga, S. R.; Yarber, F.; Peddi, S.; Edman, M. C.; Tiwari, N.;
Liu, S.; Louie, S. G., Berunda Polypeptides: Biheaded Rapamycin Carriers for Subcutaneous
Treatment of Autoimmune Dry Eye Disease. Molecular pharmaceutics 2019, 16, (7), 3024-‐3039.
174. Fidalgo, J. P.; Fabregat, L. G.; Cervantes, A.; Margulies, A.; Vidall, C.; Roila, F.,
Management of chemotherapy extravasation: ESMO–EONS clinical practice guidelines. Ann.
Oncol. 2012, 23, vii167-‐vii173.
175. LLOYD, J. B.; WILLIAMS, K. E., Non-‐specific adsorptive pinocytosis. In Portland Press
Limited: 1984.
176. Ha, K. D.; Bidlingmaier, S. M.; Liu, B., Macropinocytosis exploitation by cancers and
cancer therapeutics. Front. Physiol. 2016, 7, 381.
177. White, E., Exploiting the bad eating habits of Ras-‐driven cancers. Genes & development
2013, 27, (19), 2065-‐2071.
178. Norbury, C. C., Drinking a lot is good for dendritic cells. Immunology 2006, 117, (4), 443-‐
451.
179. Liu, Z.; Roche, P. A., Macropinocytosis in phagocytes: regulation of MHC class-‐II-‐
restricted antigen presentation in dendritic cells. Front. Physiol. 2015, 6, 1.
180. Zhu, W.; Masaki, T.; Cheung, A. K.; Kern, S. E., In-‐vitro release of rapamycin from a
thermosensitive polymer for the inhibition of vascular smooth muscle cell proliferation. Journal
of bioequivalence & bioavailability 2009, 1, 3.
181. Dowrick, P.; Kenworthy, P.; McCann, B.; Warn, R., Circular ruffle formation and closure
lead to macropinocytosis in hepatocyte growth factor/scatter factor-‐treated cells. European
journal of cell biology 1993, 61, (1), 44-‐53.
182. West, M. A.; Bretscher, M. S.; Watts, C., Distinct endocytotic pathways in epidermal
growth factor-‐stimulated human carcinoma A431 cells. The Journal of cell biology 1989, 109,
(6), 2731-‐2739.
183. Meier, O.; Boucke, K.; Hammer, S. V.; Keller, S.; Stidwill, R. P.; Hemmi, S.; Greber, U. F.,
Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-‐
mediated uptake. The Journal of cell biology 2002, 158, (6), 1119-‐1131.
184. Wadia, J. S.; Stan, R. V.; Dowdy, S. F., Transducible TAT-‐HA fusogenic peptide enhances
escape of TAT-‐fusion proteins after lipid raft macropinocytosis. Nat. Med. 2004, 10, (3), 310.
138
185. Sancak, Y.; Peterson, T. R.; Shaul, Y. D.; Lindquist, R. A.; Thoreen, C. C.; Bar-‐Peled, L.;
Sabatini, D. M., The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1.
Science 2008, 320, (5882), 1496-‐1501.
186. Sancak, Y.; Bar-‐Peled, L.; Zoncu, R.; Markhard, A. L.; Nada, S.; Sabatini, D. M., Ragulator-‐
Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by
amino acids. Cell 2010, 141, (2), 290-‐303.
187. Turner, M. R.; Balu-‐Iyer, S. V., Challenges and opportunities for the subcutaneous
delivery of therapeutic proteins. J. Pharm. Sci. 2018, 107, (5), 1247-‐1260.
188. Hart, A.; Smith, J.; Skeans, M.; Gustafson, S.; Wilk, A.; Robinson, A.; Wainright, J.;
Haynes, C.; Snyder, J.; Kasiske, B., OPTN/SRTR 2016 annual data report: kidney. Am. J.
Transplant. 2018, 18, 18-‐113.
189. Gaynor, J. J.; Ciancio, G.; Guerra, G.; Sageshima, J.; Hanson, L.; Roth, D.; Chen, L.; Kupin,
W.; Mattiazzi, A.; Tueros, L., Graft failure due to noncompliance among 628 kidney transplant
recipients with long-‐term follow-‐up: a single-‐center observational study. Transplantation 2014,
97, (9), 925-‐933.
190. Sellares, J.; De Freitas, D.; Mengel, M.; Reeve, J.; Einecke, G.; Sis, B.; Hidalgo, L.;
Famulski, K.; Matas, A.; Halloran, P., Understanding the causes of kidney transplant failure: the
dominant role of antibody-‐mediated rejection and nonadherence. Am. J. Transplant. 2012, 12,
(2), 388-‐399.
191. Dobbels, F.; Ruppar, T.; De Geest, S.; Decorte, A.; Van Damme-‐Lombaerts, R.; Fine, R.,
Adherence to the immunosuppressive regimen in pediatric kidney transplant recipients: a
systematic review. Pediatr. Transplant. 2010, 14, (5), 603-‐613.
192. Bethany, J. F.; Ahna, L., Adherence in adolescent and young adult kidney transplant
recipients. The Open Urology & Nephrology Journal 2014, 7, (1).
193. Thomson, A. W.; Turnquist, H. R.; Raimondi, G., Immunoregulatory functions of mTOR
inhibition. Nature Reviews Immunology 2009, 9, (5), 324-‐337.
194. Sarbassov, D. D.; Ali, S. M.; Sabatini, D. M., Growing roles for the mTOR pathway. Curr.
Opin. Cell Biol. 2005, 17, (6), 596-‐603.
195. Yang, Q.; Guan, K.-‐L., Expanding mTOR signaling. Cell Res. 2007, 17, (8), 666-‐681.
196. Mondino, A.; Mueller, D. L. In mTOR at the crossroads of T cell proliferation and
tolerance, Semin. Immunol., 2007; Elsevier: 2007; pp 162-‐172.
197. Sehgal, S. In Sirolimus: its discovery, biological properties, and mechanism of action,
Transplant. Proc., 2003; Elsevier: 2003; pp S7-‐S14.
198. Bowman, L. J.; Brennan, D. C., The role of tacrolimus in renal transplantation. Expert
Opin. Pharmacother. 2008, 9, (4), 635-‐643.
199. de Fijter, J. W., Cancer and mTOR inhibitors in transplant recipients. Transplantation
2017, 101, (1), 45-‐55.
200. van de Wetering, J.; Roodnat, J. I.; Hemke, A. C.; Hoitsma, A. J.; Weimar, W., Patient
survival after the diagnosis of cancer in renal transplant recipients: a nested case-‐control study.
Transplantation 2010, 90, (12), 1542-‐1546.
201. Acuna, S. A.; Fernandes, K. A.; Daly, C.; Hicks, L. K.; Sutradhar, R.; Kim, S. J.; Baxter, N. N.,
Cancer mortality among recipients of solid-‐organ transplantation in Ontario, Canada. JAMA
oncology 2016, 2, (4), 463-‐469.
139
202. Alberú, J.; Pascoe, M. D.; Campistol, J. M.; Schena, F. P.; del Carmen Rial, M.; Polinsky,
M.; Neylan, J. F.; Korth-‐Bradley, J.; Goldberg-‐Alberts, R.; Maller, E. S., Lower malignancy rates in
renal allograft recipients converted to sirolimus-‐based, calcineurin inhibitor-‐free
immunotherapy: 24-‐month results from the CONVERT trial. Transplantation 2011, 92, (3), 303-‐
310.
203. Campistol, J. M.; Eris, J.; Oberbauer, R.; Friend, P.; Hutchison, B.; Morales, J. M.;
Claesson, K.; Stallone, G.; Russ, G.; Rostaing, L., Sirolimus therapy after early cyclosporine
withdrawal reduces the risk for cancer in adult renal transplantation. J. Am. Soc. Nephrol. 2006,
17, (2), 581-‐589.
204. Euvrard, S.; Morelon, E.; Rostaing, L.; Goffin, E.; Brocard, A.; Tromme, I.; Broeders, N.;
Del Marmol, V.; Chatelet, V.; Dompmartin, A., Sirolimus and secondary skin-‐cancer prevention
in kidney transplantation. N. Engl. J. Med. 2012, 367, (4), 329-‐339.
205. Hoogendijk-‐van den Akker, J. M.; Harden, P. N.; Hoitsma, A. J.; Proby, C. M.; Wolterbeek,
R.; Bouwes Bavinck, J. N.; de Fijter, J. W., Two-‐year randomized controlled prospective trial
converting treatment of stable renal transplant recipients with cutaneous invasive squamous
cell carcinomas to sirolimus. J. Clin. Oncol. 2013, 31, (10), 1317-‐1323.
206. Tedesco, J. S. H.; Rosso, C. F.; Medina, J. P., Reviewing 15 years of experience with
sirolimus. Transplantation research 2015, 4, (Suppl 1), 6-‐6.
207. Saunders, R. N.; Metcalfe, M. S.; Nicholson, M. L., Rapamycin in transplantation: a
review of the evidence. Kidney Int. 2001, 59, (1), 3-‐16.
208. Mamat, U.; Woodard, R. W.; Wilke, K.; Souvignier, C.; Mead, D.; Steinmetz, E.; Terry, K.;
Kovacich, C.; Zegers, A.; Knox, C., Endotoxin-‐free protein production—ClearColi™ technology.
Nat. Methods 2013, 10, (9), 916.

Abstract (if available)

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Effects of particle architecture on in-vivo pharmacokinetics and bio-distribution of therapeutic nanostructures

PDF

Sustained and targeted delivery of rapamycin using FKBP-elastin like polypeptide fusion proteins

PDF

Development of novel immunosuppressant-based therapies to treat dacryoadenitis in a Sjögren’s syndrome mouse model

PDF

Cellular uptake mechanism of elastin-like polypeptide fusion proteins

PDF

Tubulin-based fusion proteins as multifunctional tools

PDF

Temperature triggered protein assembly enables signaling switching and peptide drug delivery

PDF

Multivalent smart elastin-like polypeptide therapeutics with drug delivery and biosensing applications.

PDF

Comparing drug release kinetics of rapamycin bound FKBP-ELP fusion proteins

PDF

Temperature-mediated induction of caveolin-mediated endocytosis via elastin-like polypeptides

PDF

Development and therapeutic assessment of multivalent protein polymers for cancer and eye diseases

PDF

The modulation of dynamin and receptor endocytosis machinery using elastin-like polypeptides

PDF

Development of protein polymer therapeutics for the eye

PDF

Targeting LFA-1/ICAM-1 interaction using ICAM-1 binding elastin-like polypeptide

PDF

Synthetic biopolymers modulate cell signaling

PDF

Integrin-mediated targeting of protein polymer nanoparticles carrying a cytostatic macrolide

PDF

Methods and protocols for detecting the intracellular assembly of elastin-like polypeptides

PDF

Characterization of the transferrin oligomer and its potential application in drug delivery

PDF

Reconstruction of a proinsulin-transferrin fusion protein

PDF

Antibodies and elastin-like polypeptides: cellular and biophysical characterization of an anti-ELP monoclonal and an anti-CD3 single-chain-ELP fusion

PDF

Application of elastin-like polypeptides to therapeutics in leukemia

Asset Metadata

Creator Peddi, Santosh (author)

Core Title Cognate receptor fusion proteins enable parenteral delivery of challenging rapalogues

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 08/01/2020

Defense Date 05/06/2020

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

Tag drug delivery,elastin-like polypeptide,everolimus,nanoparticle,OAI-PMH Harvest,rapalogue,rapamycin

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor MacKay, Andrew (committee chair), Stiles, Bangyan (committee member), Zhang, Yong (committee member)

Creator Email santosh.peddi1992@gmail.com,speddi@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-356109

Unique identifier UC11664356

Identifier etd-PeddiSanto-8842.pdf (filename),usctheses-c89-356109 (legacy record id)

Legacy Identifier etd-PeddiSanto-8842.pdf

Dmrecord 356109

Document Type Dissertation

Rights Peddi, Santosh

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