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

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

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SUSTAINED AND TARGETED DELIVERY OF RAPAMYCIN
USING FKBP-ELASTIN LIKE POLYPEPTIDE FUSION PROTEINS

by
Jingmei Yu

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

August 2021

Copyright 2021 Jingmei Yu
ii
Acknowledgements
I would like to thank my advisor Dr. J. Andrew MacKay for his excellent guidance and continuous
support during my two-years master study at University of Southern California. I am dedicated to
his patience, encouragement, and wonderful mentorship for me, which also enlighten my interest
in research area. I am also grateful to my committee members, Dr. Curtis Okamoto, and Dr. Ian
Haworth for their support of my Ph.D. application and valuable suggestions for my thesis.

I really appreciate my current and previous lab members in MacKay lab for their generous help,
especially my mentors Dr. Hao Guo and Dr. Santosh Peddi. Thank you for training me for several
scientific skills and providing useful guidance for my experiments. Without your help, I can’t make
my thesis possible.

iii
Table of Contents
Acknowledgements ......................................................................................................................... ii
List of Tables .................................................................................................................................. v
List of Figures ................................................................................................................................ vi
Abstract ......................................................................................................................................... vii
Chapter 1 Introduction .................................................................................................................... 1
1.1 Rapamycin: small molecular drugs ....................................................................................... 1
1.2 Rapamycin inhibit mTORC1 signaling pathway .................................................................. 2
1.3 Clinical limitations of rapamycin .......................................................................................... 3
1.4 Elastin like polypeptides (ELPs) is versatile platform for drug delivery .............................. 4
1.5 Cognate receptor fusion proteins for sustained delivery of rapamycin ................................ 6
Chapter 2: Elastin like polypeptides (ELPs) monoblock and di-block copolymers ..................... 10
Introduction ............................................................................................................................... 10
2.1 Material and Methods: ........................................................................................................ 12
2.1.1 Recombinant synthesis of ELP monoblock and diblock copolymers .......................... 12
2.1.2 Characterization of ELP transition temperatures ......................................................... 12
2.1.3 Rhodamine labeling, 5,6-carboxyfluorescein labeling and confocal imaging ............. 13
2.1.4 Circular dichroism ....................................................................................................... 14
2.2 Results ................................................................................................................................. 15
2.2.1 Physicochemical characterization of monoblock and diblock ELPs ........................... 15
2.2.2 Nanoparticles can encapsulate fluorescent dyes .......................................................... 18
2.2.3 Dynamic correlation of ELP secondary structures with temperature .......................... 19
2.3 Discussion and conclusion: ................................................................................................. 22
Chapter 3 FKBP-ELPs for the slow delivery of Rapamycin as immunosuppressant agent ......... 23
iv
Introduction ............................................................................................................................... 23
3.1 Material and Methods: ........................................................................................................ 25
3.1.1 FKBP-ELP purification, drug encapsulation and endotoxin removal ......................... 25
3.1.2 RP-HPLC to measure concentration of 5FA-Rapa and 5FV-Rapa ............................. 27
3.1.3 Induction of immune-mediated bone marrow failure in a mouse model and
immunosuppressive therapies ............................................................................................... 29
3.1.4 Flow cytometry analysis .............................................................................................. 31
3.2 Results ................................................................................................................................. 32
3.2.1 Physicochemical characterization of 5FA and 5FV ..................................................... 32
3.2.2 Bone marrow infiltration T cells analyses ................................................................... 34
3.2.3 5FA-Rapa and 5FV-Rapa suppress tumor growth in breast cancer ............................. 36
3.3 Discussion and conclusion .................................................................................................. 37
Chapter 4 GRP78-targeted FKBP-ELPs Rapamycin delivery for breast cancer .......................... 38
Introduction ............................................................................................................................... 38
4.1 Material and Methods: ........................................................................................................ 39
4.1.1 X-5FA plasmid molecular cloning, protein purification and drug encapsulation ....... 39
4.1.2 Mammalian Cell culture .............................................................................................. 39
4.1.3 Western blot assay ....................................................................................................... 40
4.2 Results ................................................................................................................................. 42
4.2.1 Physicochemical characterization of X-5FA ............................................................... 42
4.2.2 Western blot result ....................................................................................................... 43
4.3 Discussion and conclusion .................................................................................................. 46
References: .................................................................................................................................... 47

v
List of Tables
Table 1. Characterization of a library of purified ELPs ................................................................ 15
Table 2. Physiochemical characterization of 5FA and 5FV ......................................................... 33

vi
List of Figures
Figure 1. Chemical structure of rapamycin ..................................................................................... 1
Figure 2. mTOR signaling pathway ................................................................................................ 2
Figure 3. Reversible phase separation of Elastin-like polypeptides (ELPs) ................................... 4
Figure 4. Plasmid maps of 5FA and 5FV constructs in pET-25b (+) vector .................................. 7
Figure 5. Zr-5FV was retained longer at the subcutaneous administration site .............................. 8
Figure 6. PK analysis of IV 5FA, SC 5FA and SC 5FV ................................................................. 9
Figure 7. SDS-PAGE gel results high purify of ELPs with Cucl
2
staining .................................. 16
Figure 8. Optical density at 600 nm for (a) I48S48, (b) S48I48, (c) S96 and (d) I48 .................. 16
Figure 9. Concentration–temperature phase diagrams for ELP diblock copolymers I48S48,
S48I48 and monoblock S96 and I48 for CMT and Ttbulk .............................................................. 17
Figure 10. ELP microparticles can encapsulate hydrophilic cargo ............................................... 18
Figure 11. Circular dichroism was used to evaluate ELPs secondary structure changes versus
temperature ................................................................................................................................... 19
Figure 12. RP-HPLC to measure the concentration of rapamycin ............................................... 27
Figure 13. A schematic representation the whole experiment procedure ..................................... 29
Figure 14. Design of a depot forming ELP carrier subcutaneous delivery of Rapamycin ........... 30
Figure 15. Physicochemical characterization of 5FA and 5FV .................................................... 32
Figure 16. Bone marrow infiltrating T cells are effectively reduced by 5FA-Rapa and 5FV-Rapa
....................................................................................................................................................... 34
Figure 17. Frequency of both CD4+ and CD8+ T cells were reduced by 5FA-Rapa and 5FV-
Rapa treatments ............................................................................................................................. 35
Figure 18. Single dose of 5FV-Rapa outperformed repetitive doses of 5FA-Rapa against breast
cancer ............................................................................................................................................ 36
Figure 19. Physicochemical characterization of 5FA and X-5FA ................................................ 42
Figure 20. Western blot analysis of concentration-dependent effect on rpS6 .............................. 44
Figure 21. Western blot analysis of time-dependent effect on rpS6 ............................................. 45

vii
Abstract
Rapamycin is highly potent drug with cytostatic, anti-cancer and immunosuppressive properties.
However, the conventional methods for delivery of rapamycin in solvent or emulsions lead to drug
precipitation at injection site and cause toxic side effects like stomatitis, which limit the clinical
application of rapamycin. Elastin-like polypeptide (ELPs) are environmentally responsive protein
polymers with versatile applications as drug delivery vehicles. Being biocompatible, biodegradable,
and low-immunogenic, ELPs have intelligent phase behavior that can be tuned to remain soluble or
assemble viscous depots upon their transition temperature.

Thus, in this dissertation, (1). I present two generations of FKBP-ELP fusion-proteins of rapamycin
formulations with higher bioavailability, lower dose-limiting toxicity, and reasonable improvement of
pharmacokinetics. Specifically, the high-capacity carrier is designed to improve patience adherence
and compliance through forming subcutaneous depot and sustained release of rapamycin. To further
enhance the tumor targeting specificity, our lab fused a panel of csGRP78 targeting ligands and
evaluated the drug potency within HR+ breast cancer models. (2). I evaluated a library of ELP
monoblock and diblock copolymers to precisely engineer their properties for different therapeutic
platforms.

1
Chapter 1 Introduction
1.1 Rapamycin: small molecular drugs
Rapamycin (RAPAMUNE®), as known as sirolimus, is a natural hydrophobic macrolide drug
produced by the bacterium Streptomyces hygroscopicus (Fig. 1). It was first isolated in 1972 from
the soil samples on Easter Island
1
. Rapamycin was originally indicated as an antifungal agent, but
due to the discovery of its immunosuppressive and anti-proliferative properties, it was later
approved by the FDA in 1999 as an immunosuppressant to prevent rejection in organ
transplantation
2
. Specifically, in the situation of immunosuppression, rapamycin can inhibit the
activation and proliferation of T cells. Interestingly, rapamycin can also act as potent cytostatic
agent and have anti-cancer activities, which includes breast cancer
3
, prostate cancer
4
and kidney
cancer
5
.

Figure 1. Chemical structure of rapamycin

2
1.2 Rapamycin inhibit mTORC1 signaling pathway
In cells, rapamycin binds to its cognate receptor FK506 binding protein 12 (FKBP-12) with sub-
nanomolar affinity of 0.2 nM
6
. The FKBP-rapamycin complex then associates with the FKBP-
rapamycin binding domain (FRB) of the mammalian target of rapamycin (mTOR), inhibiting
mTORC1 pathway allosterically and leading to growth suppression
7,8
. mTOR forms two
functionally distinct macromolecular complexes mTORC1 and mTORC2 in the cells
31
. mTORC1
mediates immune cell growth, proliferation, and metabolism
9-11
(Fig. 2). The mechanism of action
is mTORC1 promotes the cell-cycle progression from G1 phase to S phase for cell growth and
proliferation through increased mRNA translation, protein synthesis and cytokine signaling like
IL-2
12
. Therefore, mTORC1 inhibitors like rapamycin are used as immunosuppressants through
blocking proliferation of T-cells. Additionally, mTORC1 is upregulated in multiple cancers and is
known to promote tumor growth and metastasis
12
. The activation of Akt, Rho or PKCα leads to
activation of mTORC1 and promotes cell survival, cell proliferation and cell metabolism
13
.

Figure 2. mTOR signaling pathway
13

3
1.3 Clinical limitations of rapamycin
Despite of the potent advantageous pharmacology, the clinical potential of Rapamycin is limited
by three major challenges
14-16
. Firstly, because of its low water solubility, rapamycin is only
available as an oral formulation like solution or tablet, so the bioavailability is poor, ~14%, in
humans after extensive first-pass metabolism. Secondly, rapamycin has undesirable clinical
pharmacokinetics because it has large intra- and inter- patient variability in PK parameters,
incomplete gut absorption and poor does to plasma concentration correlation
31
. The blood-to
plasma ratio is 36, which means most of the drug partition into red blood cells. Moreover, 92% of
drug that remains in the plasma is protein-bound. In addition, because of drug precipitation,
rapamycin also has prevalent dose-limited toxicity, such as stomatitis (incidence~40%), severe
anemia, pulmonary toxicity, infection, hyperlipidemia, acute kidney injury
17-19
. These painful side
effects lead to unplanned interruptions in treatment and reduce patient compliance. Therefore, it
may be necessary to develop novel formulations of rapamycin to enhance its therapeutic effects.
This thesis explores several ongoing strategies to do so.

4
1.4 Elastin like polypeptides (ELPs) is versatile platform for drug delivery
Elastin like polypeptides (ELPs) are a class of protein polymers derived from human tropoelastin
20
.
ELPs are repeats of pentameric amino acid sequence [Val-Pro-Gly-Xaa-Gly]n, where Xaa is any
amino acid and n is the number of repeated units
21
. ELPs can be precisely genetically engineered,
and it can be produced efficiently(~150mg/ml) by bacterial cells
22,23
. Given their human origin,
ELPs are potentially biocompatible, biodegradable, and non-immunogenic for sustained
circulation in the bloodstream
24
, which makes them great candidates as drug carriers
25-27
.

Many ELPs undergo thermo-responsive reversible phase transition (Fig. 3), whereby they remain
soluble as random coils below their transition temperature (Tt) and coacervate into β-sheet
structures when heated above Tt
26,28
. Taking advantage of this unique phase transition behavior,
ELPs can be easily purified through 3 rounds of heating and cooling inverse transition cycling
(ITC)
27
and be used as purification tag.

Figure 3. Reversible phase separation of Elastin-like polypeptides (ELPs)
27

5
The hydrophobicity, molecular weight, and concentration of ELPs are all essential elements to
determine their transition temperature (Tt). In addition, some external factors like salt
concentration, pH changes and light can be tuned to also trigger phase separation of ELPs
25,29,30

The transition temperature of ELP can be finely adjusted by choosing different hydrophobic guest
residue and by adjusting its molecular weight. Generally, ELPs with higher MW and more
hydrophobic guest residues exhibit lower transition temperatures. In contrast, lower ELP
concentration and salt concentration increase transition temperature
26
.

The nomenclature of ELPs used in this thesis is simple:
Monoblock ELPs are named as guest residue and repeat times. For example, I48 indicates an ELP
that contains 48 repeats of the VPGIG pentapeptide. Diblock ELPs are named as XnYm, where
the X and Y represent different guest residue, and n and m indicate repeat times. For example,
I48S18 indicates an ELP that contains 48 repeats of the VPGIG pentapeptide followed by 18
repeats of VPGSG.

6
1.5 Cognate receptor fusion proteins for sustained delivery of rapamycin
Our lab has developed two constructs of ELP-based rapamycin carriers called 5FV and 5FA with
cognate receptor technology to overcome the limitations of rapamycin including bioavailability,
toxicity, and a predictability of pharmacokinetics (PK). They are composed of multiple FKBP
domains and short ELP linker sequence
31
(Fig 4). FKBP protein is the cognate receptor of
rapamycin, which mediates hydrophobic rapamycin solubilization through high-affinity, high-
specificity non-covalent binding. Each molecule is designed to have 5 FKBPs that carry 5
rapamycin molecules. The drug loading capacity of the carriers is relatively high, around 4.4% by
mass as measured by reverse-phase high performance liquid chromatography (RP-HPLC).

Additionally, the ELP linker is designed to alter drug absorption rates by controlling their physical
state at body temperature in the subcutaneous administration sites
31,32
to reduce frequency of dose
and improve patient compliance. As for the 5FV, where valine is a very hydrophobic amino acid,
it is designed to phase separate below body temperature. The 5FV construct form a depot through
coacervation at the injection site and gradually releases the rapamycin. At the same time, a soluble
control 5FA, which has a less hydrophobic Alanine, was designed to have a higher transition
temperature than physiological temperature.

Moreover, the FKBP-ELP rapamycin carrier platform can release drug through integrin-mediated
cellular uptake
33
, reduce renal clearance through EPR effect, lower dose-limiting toxicity
34
, and
reduce the effective dose through RGD-peptide/integrin targeting
35
.

7

Figure 4. Plasmid maps of 5FA and 5FV constructs in pET-25b (+) vector
31
A. 5FA is composed of [FKBP-(VPGAG) 24] 4-FKBP
B. 5FV is composed of [FKBP-(VPGVG) 24] 4-FKBP

The binding affinity of rapamycin to FKBP-ELPs is evaluated through Isothermal Titration
Calorimetry (ITC), which was estimated to be 5, suggesting their correct folding of FKBP domain
in 5FA and 5FV and their ability to bind to rapamycin
31
. The equilibrium dissociation constant
was estimated to be low nanomolar range
31
.

8
Our lab conjugated a radioactive Zirconium (Zr89) to the carrier and used microPET imaging to
estimate the depot half-life of 5FV and 5FA
31
(Fig. 5). It is obvious that compared to Zr-5FA, Zr-
5FV was retained much longer at injection site. At 4 days post-injection, Zr-5FA is highly
eliminated but Zr-5FV still has high fraction of signal remaining at site of injection. Compared to
5FA for 8.9 hours, the half-life of the depot was 8.1days for 5FV.

Figure 5. Zr-5FV was retained longer at the subcutaneous administration site A. microPET images of Zr-5FV or Zr-5FA at mice
subcutaneous injection site B. Subcutaneous half-lives of Zr-5FA and Zr-5FV

9
To better understand the PK of 5FA and 5FV, Balb/C mouse blood samples were collected to
analyze the plasma concentration at multiple time point. 5FA with intravenous injection is used as
a control
31
(Fig. 6). Both 5FA and 5FV had similar bioavailability and apparent clearance; however,
the extended mean absorption time (MAT) lead to a longer terminal half-life for 5FV, which
indicates that 5FV-SC stays longer in the blood with slower absorption rate.

Figure 6. PK analysis of IV 5FA, SC 5FA and SC 5FV
31
A. Plasma concentration verses time profiles of IV 5FA, SC 5FA and
SC 5FV. B. Non-compartmental PK analysis of IV 5FA, SC 5FA and SC 5FV

10
Chapter 2: Elastin like polypeptides (ELPs) monoblock and di-block
copolymers
Introduction
As described above, ELPs is a class of protein polymers which can be used as a tunable drug
delivery platform. With high precision, recombinant DNA technology
36,37
, ELPs can be genetically
fused with biologically active proteins or drug payloads
38-40
. Unlike random and more polydisperse
synthetic polymers, genetic-engineered ELPs allow exquisite control over polymer composition,
molecular weight, and polydispersity
41
.

As for monoblock ELPs, the phase behavior is determined by guest residue hydrophobicity and
molecular weight. Diblock ELPs exhibit both critical micelle temperatures (CMTs) and bulk
transition temperatures (Tt, bulk). ELP diblock copolymers have emerged as attractive
biomaterials for drug delivery
42
. It assembles spherical nanoparticles and mediate cellular uptake
and in vivo biodistribution when heated above CMT
42
.

Di-block copolymers could self-assemble various structures like micelles, worms, and vesicles.
The ratio of hydrophilic polymer mass to total polymer mass, f, is one powerful determinant of
assembly of block copolymers
43-45
. Prior investigations with synthetic polymers suggest that
polymers with f > 45% often form micelles, whereas polymers with f ~ 25% are more likely to
form lamellar structures
46,47
. While ELP diblock copolymers with f > 45% have already been
described that assemble micelles
48
, there have been no reports of lamellar ELP structures capable
of encapsulating hydrophilic cargo.

11
To rational engineer ELP nanoparticles for different therapeutic applications, our lab synthesized
a library of genetically-encoded ELP diblock copolymers by joining hydrophobic (Val-Pro-Gly-
Ile-Gly)n and hydrophilic (Val-Pro-Gly-Ser-Gly)m ELP segments. ELP I48 is selected as
hydrophobic block because it undergoes assembly near room temperature (Table 1). As a control,
hydrophilic ELP S96 is selected because it does not undergo assembly until greater than 60 °C
(Table 1).

The polypeptide block copolymers were designed by systematically reducing the molecular weight
of the hydrophilic serine block, ranging in length from 18 to 48 pentameric unit. The phase
transition temperature is known to be length dependent
49
, and shorter (n = 18 to 48) serine ELPs
do not phase separate below 100 °C. Thus, we expect ELP copolymers disassemble below room
temperature and assemble into nanostructures at physiological temperatures, which may be useful
for encapsulating drugs inside the structures. Moreover, since there is limited information about
ELP phase changes on optical density (OD), circular dichroism is used to measure ELP peptide
secondary structure changes upon heating.

We aim to discover: (1). How diblock copolymers nanostructures relative with hydrophilic fraction
(f) (2) The capability of diblock ELP encapsulating hydrophilic cargo (2) The correlation of ELPs
secondary structure with temperature

12
2.1 Material and Methods:
2.1.1 Recombinant synthesis of ELP monoblock and diblock copolymers
ELP monoblock and diblock copolymers of specific chain length were generated using recursive
directional ligation method in a modified pET25b+ vector (Novagen, Madison, WI) in TOP10
cells (Invitrogen Corporation, Carlsbad, CA). Briefly, two cloning vectors containing different
ELP gene were digested with BssHII and AcuI or BssHII and BserI separately, which generating
compatible sticky ends. Then transformed the expression constructs into E. coli BLR(DE3) cells
(Novagen, Madison, WI) and proteins were purified by three times ITC. ELP molecular weight
and purity were determined by CuCl
2
SDS-PAGE staining with ＞95% purity. Molecular weights
of ELP polymers were further confirmed by using MALDI-TOF mass spectroscopy.

2.1.2 Characterization of ELP transition temperatures
The transition temperature of ELPs was obtained by measuring solution turbidity at 350nm optical
density. ELP transition temperatures were characterized over a range of concentrations (10, 25, 50,
and 100 μM ELP in phosphate buffer saline (PBS, Caisson, Labs, Smithfield, UT)) by raising the
temperature at 1 °C/min on a DU800 UV-vis spectrophotometer (Beckman Coulter, CA). The
transition temperature was defined at the maximum first derivative of the optical density at 350
nm. For block copolymers that undergo assembly nanostructures, the initial transition temperature
(Tt1) was defined as the critical micelle temperature (CMT) and the last major transition
temperature (Tt2) was defined as the bulk transition temperature (Tt, bulk).

13
2.1.3 Rhodamine labeling, 5,6-carboxyfluorescein labeling and confocal imaging
Dissolved NHS-Rhodamine (Thermo Fischer Scientific, Waltham, MA) in anhydrous DMSO
(Invitrogen, Carlsbad, CA) at 10 mg/mL and added 1.5x molar excess NHS-Rhodamine to di-
block copolymers. Incubated overnight at 4 °C and remove the untrapped free dye with Zeba
desalting columns (Thermo Fischer Scientific, Waltham, MA). Proteins eluted in phosphate buffer
saline. A Nanodrop spectrophotometer 2000 (Thermo Fischer Scientific, Waltham, MA) was used
to measure protein and rhodamine concentration by reading the absorbance at 280 nm and 555 nm
respectively. For diblock copolymers, the estimated molar extinction coefficient at 280 nm is 1285
M
-1
cm
-1
.

SDS-PAGE gel evaluated the purity of rhodamine labeled proteins and imaged on ChemiDocTM
(Bio-Rad, Hercules, CA) fluorescence imaging system. The diblock copolymers were observed
under LSM800 Confocal scanning laser microscopy (Carl Zeiss Microscopy, Thornwood, NY).
The labeling procedures for 5,6-carboxyfluorescein (Thermo Fischer Scientific, Waltham, MA) is
the same as NHS-Rhodamine as described above.

14
2.1.4 Circular dichroism
Circular dichroism measurements of ELP solutions were made on a Jasco CD spectrometer. The
path length for quartz cells is 0.1 cm and the measurement wavelength range is 190-240nm. ELP
samples were prepared in deionized water with a concentration range 10-15 μM. For temperature-
dependent CD scans, samples were first cooled at 15°C and then gradually increased to 95°C with
5°C intervals. At each temperature point, samples were equilibrated for 5 mins. Jasco software
processed data with simultaneous record of spectrums. Each spectrum was corrected by subtracting
the background spectrum recorded at 15°C. For data analysis, we converted the resulting spectra
into mean molar residue ellipticity (MRE) in mdeg cm
2
dmol
-1
. The data were fitted to spectra of
ideal secondary structures and analyzed using Dichroweb
50
software.

15
2.2 Results

2.2.1 Physicochemical characterization of monoblock and diblock ELPs

A library of ELPs with different arrangements and lengths of hydrophilic and hydrophobic blocks
was prepared (Table 1) and purified (Fig.7). As described above, isoleucine and serine ELPs are
a rational choice for the development of polypeptide block copolymers, which were combined to
cover a range of hydrophilic fractions, f. Hydrophobic I48 phase separates above 18 °C, whereas
hydrophilic S96 phase separates above 54 °C (Table 1). The concentration dependence of
transition temperature (Tt) was determined for the ELPs across a gradient from 10 to 100 μM (Fig.
8). Bulk Tt decreases for these ELP blocks upon increasing ELP concentration. I48 and S96
exhibits decrease in their Tt 21 °C to 18 °C and 60 °C to 54 °C respectively, as concentration is
increased from 10 to 100 μM (Fig 9). Both Tts were fit as a logarithmic function of concentration.

Table 1. Characterization of a library of purified ELPs

16

Figure 7. SDS-PAGE gel results high purify of ELPs with Cucl
2
staining A. Lane 1: MW marker, lane 2: I48, lane 3: S96, lane 4:
S48I48, lane 5: I48S48. B. I48S24 and I48S18.

Optical density (600 nm) was used to characterize phase behavior for ELP I48, S96, I48S48 and
S48I48 as a function of temperature and concentration (Fig. 8).

Figure 8. Optical density at 600 nm for (a) I48S48, (b) S48I48, (c) S96 and (d) I48
17
Based on optical density at 600nm, phase diagram for ELP block copolymers is a function of
concentration and temperature. Both CMT and Ttbulk are concentration dependent and can be fit to
a Log-linear model, Tt = b – m [Log10 (Concentration)]. Between CMT and Ttbulk, the diblock
copolymers assemble nanostructures; however, ELPs undergo bulk phase separation when
temperature above Ttbulk.

Figure 9. Concentration–temperature phase diagrams for ELP diblock copolymers I48S48, S48I48 and monoblock S96 and I48 for
CMT and T tbulk

18
2.2.2 Nanoparticles can encapsulate fluorescent dyes

Confocal laser scanning microscopy was used to characterize micron sized ELP nanostructures
(Fig 10). 5,6-carboxyfluorescein was used as a model drug for encapsulation experiments. NHS-
Rhodamine was conjugated to the amino terminus of di-block copolymers to visualize the structure
of nanoparticles. Particles were incubated over a period of 24 hours at 37°C prior to imaging.

Spherical structures (>1 micron) were observed with fluorescence in green (carboxy-fluorescein),
and it suggests the encapsulation of carboxy-fluorescein into ELP particles. (Fig. 10A). Confocal
microscopy analysis for I48S18 reveals that particles exhibit special morphology (Fig. 10B).

Figure 10. ELP microparticles can encapsulate hydrophilic cargo A. Unlabeled ELP block copolymers were assembled in the
presence of 50μM Carboxy-Fluorescein, free dye was removed, and the resulting particles retained green fluorescence. B. Covalent
modification of the terminal primary amine of I48S18 with rhodamine enabled visualization of microparticles with spherical
morphology

19
2.2.3 Dynamic correlation of ELP secondary structures with temperature

Figure 11. Circular dichroism was used to evaluate ELPs secondary structure changes versus temperature (a) I48S48, (c) S48I48,
(e) S96 and (g) I48 spectra were determined at 10-15 μM in DI water. Changes in percent secondary structure of (b) I48S48, (d)
S48I48, (f) S96 and (h) I48 were analyzed with Dichroweb.

20
Far UV Circular dichroism measurement is used to detect the change of ELP secondary structure
versus temperature
42
. CD spectra of I48S48, S48I48, S96 and I48 taken from 15°C to 95°C are
depicted (Fig. 11). The secondary structure content of ELPs was analyzed using the CDSSTR
method in Dichroweb software.

For each profile, five regimes were identified with different temperature. In regime 1, at
temperature below CMT, the spectra of I48, S96, I48S48 and S48I48 (Fig. 11a, c, e and g) consist
of a large negative band at ~195 nm and a smaller trough at ~220 nm, which are characteristic for
random coil and β-turns structures respectively. β-turns play an important role in protein folding
and stability
51
and ELP-mediated phase separation
52
. The β-turn structure is stabilized by hydrogen
bond and it gives the folded protein a globular shape by allowing the protein to reverse the direction
of the peptide chain
53
. A positive peak at ~ 210 nm is characteristic of type II β-turns
52
.

For diblock copolymers S48I48, with increasing temperature between 40°C and 45°C in regime 2,
the random coil signal becomes less pronounced, while type II β-turn content shows an increase,
indicating a transition toward a more ordered secondary structure
52,54
. The reversal of β-turn and
random coil between 65°C and 75°C indicates regime 4. However, with temperature increasing
from 45°C to 50°C or 75°C to 95°C, random coil increases while β-turn decreases, which is
indicates regime 3 and 5 respectively.

For I48S48, the total β-turn present almost remains the same level, while with an increase in type
II β-turns between 40°C and 45°C indicating regime 2, this could be attributed to the peptide-plane
flipping to a more stable secondary structure
55
. The type I β-turn and type II β-turn are completely
21
opposed with the essential difference being the orientation of the peptide bond residues at (i + 1)
and (i + 2)
42
. From 45°C and 50°C, there is an increase in random coil and a slightly decrease in
β-turn, which indicating regime 3. The reversal of β-turn and random coil between 75°C and 85 °C
indicates regime 4. From 85°C and 90°C, there is an increase in random coil and a decrease in β-
turn as regime 5.

ELP I48 provides hydrophobic core for diblock copolymers I48S48 and S48I4. Since the
precipitate hindered most of the light to catch signals between 20°C and 25°C (above Tt), the
spectra show no higher temperature transitions. However, we can still observe the conversion from
random coil to β-turn between 15°C to 20°C, which indicates regime 2.

ELP S96 was evaluated to determine the structure change of hydrophilic block as temperature
increase. The spectra show conversion of random coil to β-turn from 35°C to 45°C, which indicates
regime 2. From 45°C and 50°C, there is a slight increase in random coil and β-turn remains the
same, which may be regime 3. In addition, there is a bump backward at 70°C to 75°C, which
indicates regime 4. From 80°C and 85°C, there is an increase in random coil and a decrease in β-
turn as regime 5.

22
2.3 Discussion and conclusion:

Genetic engineering has emerged as a viable alternative to chemical synthesis of polymers, as it
allows precise control over composition, molecular weight, and polydispersity
41
. In controlling the
assembly of amphiphilic protein polymers, the relative hydrophilic fraction (f) of each block
determines the morphology of self-assembly
56,57
, including bilayer structures, cylindrical or
spherical morphologies.

ELP-based nanoparticles are considered as feasible biomaterials with long term biostability
58
,
enhanced targeting with fusion moieties
59
and conservative phase behavior with binding ligands
60
.
In this study, a library of ELP monoblock and diblock copolymers (I48, S96, I48S18, I48S24,
I48S48) was genetically engineered to discover protein polymers with different f that undergo
controlled assembly into potentially useful nanostructures.

These diblock copolymers encapsulated hydrophilic drug and allowed for precise control of
particle size through extrusion. ELP diblock copolymers I48S48 with f > 45% have already been
observed to form micelles
48
as synthetic polymers, and confocal microscopy revealed special
spherical morphology for I48S18. For the further direction, it is necessary to continue image other
diblock copolymers and observe their nanostructures.

Moreover, circulation dichroism measured ELPs peptide dynamics and inter-peptide interactions
changes under ELP coacervation phase except from Optical density, which may inspire future
studies on rationally engineering ELP therapeutics.

23
Chapter 3 FKBP-ELPs for the slow delivery of Rapamycin as
immunosuppressant agent
Introduction
Severe aplastic anemia (AA) is acquired bone marrow failure (BMF) disease caused by auto-
reactive T cells immune attack hematopoietic stem and progenitor cells (HSPCs)
61
, which is
characterized by pancytopenia and marrow hypocellularity
62
. As a result, patients with AA are
always at risk of bleeding episodes, infection, even uniformly fatal
63
. Most AA patient respond to
standard immunosuppressive therapy
64
with anti-thymocyte globulin (ATG) and cyclosporine A
(CsA) for 60%-70% effective, but the relapse on drug withdrawal or long-term administration
required alternative treatment
62
.

Rapamycin has been approved by FDA as immunosuppressant for transplantation as it inhibits
mTOR (Mechanistic Target of Rapamycin) pathway through arresting clonal proliferation of T-
cells in response to antigen stimulation. Moreover, rapamycin exhibits potent cytostatic and anti-
cancer properties. Given its advantageous pharmacology, rapamycin might be ideal
immunosuppressant agent for AA disease.

Previous research reported rapamycin is an effective therapy in BMF mice model with comparable
efficacy to CsA
62
. Rapamycin specifically expands regulatory T cells, suppress Th1 immune
response and eliminate infiltration CD8+ T cells in bone marrow, which support its potential
clinical usage in aplastic anemia treatment
62
.

24
On the other hand, patient non-adherence to prescriptions is an important contributor to long
duration, subsequent side effects like inflammation and complex dosing regimens of
immunosuppressive treatment
65
.

Thus, here we evaluate the sustained release of two novel rapamycin fusion carriers called 5FA
and 5FV in mice bone marrow failure (BMF) model. Bone marrow failure (BMF) has been
successfully modeled in animals by the infusion of allogeneic lymph node (LN) cells from donors
mismatched at major histocompatibility complex (MHC) antigens
66,67
. We expect solubilizing
rapamycin to improve bioavailability, lower toxicity, and an ensure predictable PK, we expect the
high-capacity FKBP-ELP rapamycin formulations to achieve the slow release of rapamycin at
subcutaneous injection site by forming coacervation depot at physiological temperature (37°C),
thereby reducing frequency of dose administration to once every 1-4 weeks. In this context, 5FA
remains soluble at injection point while 5FV forms a coacervate at 37°C (Fig.14).

25
3.1 Material and Methods:
3.1.1 FKBP-ELP purification, drug encapsulation and endotoxin removal
5FA and 5FV were generated by recursive ligation method as described above. Electrocompetent
ClearColi BL21 (DE3) cells (Lucigen, #60810-2) were transformed with 5FA or 5FV plasmid
DNA by electroporation. Between 50-200 µL of transformed cells were spread on agar plates
supplemented with 25 g/L Luria Broth (LB) and 1× carbenicillin (100 μg/mL) (Gold
Biotechnology, St. Louis, MO). Plates were incubated overnight at 37°C, and singular bacterial
colonies were picked for starter cultures in 50ml LB broth media (Sigma Aldrich, St. Louis, MO)
supplemented with 1× carbenicillin. Inoculated starter culture to 1L culture medium after overnight
shaking incubation at 37 °C. When the OD600 of culture reached 0.6-0.8, add 1 Molar IPTG (Gold
Biotechnology, St. Louis, MO) to a final concentration of 0.4 mM and further incubated for 24
hours at room temperature
31
.

Bacteria was pelleted by centrifugation, resuspended in PBS, and lysed by sonication. 0.5% (w/v)
polyethyleneimine solution (Sigma Aldrich, St. Louis, MO) was added to precipitate nucleic acids.
The lysate was clarified by centrifugation and proteins were purified by 3 rounds of ITC. 8M
Guanidine Hydrochloride (Thermo Fischer Scientific, Waltham, MA) was used to denature
proteins with 6:1 ratio and Nanodrop spectrophotometer was used to estimate protein
concentration follow Lambert Beer’s Law as described above.

26
For drug encapsulation, Rapamycin (LC Laboratories, Woburn, MA) dissolved in DMSO was
slowly added 10x stoichiometric excess Rapa dropwise to proteins with continuous stirring at 4 °C.
After 30 minutes, samples were centrifuged (13,000g, 15min, 4 °C) to remove insoluble drug
precipitate. The supernatant was subsequently dialyzed using X membranes with cold PBS at 4°C
in 1:300 ratio for at least 3 times buffer changes to remove free drug and residual DMSO. For
endotoxin removal, filtered 5FA-Rapa and 5FV-Rapa were passed through Acrodisc® Mustang E
syringe filters (Pall Corporation, Port Washington, NY) at 4 °C. LAL assay with recommended
manufacturer to measure the residual endotoxin burden to ensure formulation sterility.

27
3.1.2 RP-HPLC to measure concentration of 5FA-Rapa and 5FV-Rapa
To quantify hydrophobic rapamycin concentration, a standard curve was generated by C18
reverse-phase high-performance liquid chromatography (RT-HPLC) column column (Waters,
Milford, MA), which is feasible and robust analytical methods for rapamycin in pharmaceutical
dosage forms
68
. Methanol is used as organic solvent and water is used as aqueous solvent.

The HPLC method was developed by setting up three gradients: (1). 40% water and 60% methanol
wash column for 8mins (2). Gradually decrease water to 10% and increase methanol to 90% from
8min to 16mins (3). Gradually increase water to 40% and decrease methanol to 60% from 16mins
to 21mins. By adjusting the hydrophilicity and hydrophobicity of washing buffer, the more
hydrophilic ELP protein will be eluted first and then the hydrophobic rapamycin will be eluted.

Figure 12. RP-HPLC to measure the concentration of rapamycin A. Standard curve of free rapamycin B. Free rapamycin at
200μM C. a single aliquot of 5FA-Rapa D. a single aliquot of 5FV-Rapa

28
The column flow rate is 0.5ml/min and the temperature is set at 4°C. The standard curve of free
rapamycin is linear over a concentration range of 6.25 μM–200 μM. The formula of rapamycin
standard curve is y=0.9728X+2.067 with R square equal to 1 (Fig. 12A). After optimization of the
HPLC method, we obtained an efficient separation of ELP proteins and rapamycin with reasonable
retention times and good peak shapes (Fig. 12B).

Using a calibrated standard curve, both 5FA-Rapa and 5FV-Rapa concentration was quantified by
optical density at 210 nm. ELP proteins concentration was quantified by optical density at 280 nm.
Protein concentrations compared to rapamycin concentration (rapamycin µM/protein µM) were
used to evaluate drug loading efficiency for each sample.

29
3.1.3 Induction of immune-mediated bone marrow failure in a mouse model and
immunosuppressive therapies
Male CByB6F1 recipients were pre-irradiated with 5 Gy of total body irradiation (TBI) 4-6 hours
earlier by X-ray irradiator. To extract lymphocytes cells, C57BL/6 (B6) donor’s spleen was
crushed, filtered through 70um cell strainer and rinse with PBS. Incubate lymphocytes cells with
red blood cells (RBCs) lysis buffer, re-filter through 70um cell strainer and counted lymphocytes
cells in hemocytometer. Intravenously infused 5 million lymphocytes cells to CByB6F1recipients
to induce immune-mediate bone marrow failure. Then subcutaneously administrated recipient
mice with 5FA-Rapa or 5FV-Rapa for 12 days. Euthanized recipient mice and collect bone marrow
cells from bilateral femurs on day 12 post lymphocyte injection for flow cytometry analysis
(Fig.13).

Figure 13. A schematic representation the whole experiment procedure
62
CByB6F1 mice received 5 Gy total body irradiation (TBI)
4-6 hours earlier and intravenously infusion of 5 million B6 lymphocytes cell to induce BMF.

We set up four groups in total: (1). The total body irradiation (TBI) only group is a negative control,
we assume it as normal mice because it will recover if wasn’t infused the lymphocyte cells (2).
The bone marrow failure (BMF) group is a positive control group (3). The 5FA-rapa group is a
soluble control and we assume it works similarly to free rapamycin (4). The 5FV-rapa group is the
experiment group and we expect extended release of rapamycin.

30
5FA/5FV-Rapamycin was prepared as described above. 5FA-Rapa administered through
subcutaneous injection every other day for 12 days at 2 mg/kg, but 5FV-Rapa was only
administered as a single dose of 8mg/kg. 8-12 weeks of age mice were used in all experiments.

Figure 14. Design of a depot forming ELP carrier subcutaneous delivery of Rapamycin
31
. 5FA remains soluble while 5FV forms
a coacervate at 37°C.

31
3.1.4 Flow cytometry analysis
At 12 days, recipients CByB6F1mice were euthanasia by CO2 inhalation. Flush bone marrow (BM)
cells from bilateral femurs with PSB and filtered through 70um cell strainer. Incubated the BM
cells with Ammonium-Chloride-Potassium (ACK) lysis buffer for 5 min to lyse red blood cells
and resuspended the pellet in 1ml PBS. Count BM cells in hemocytometer and dilute/concentrate
the bone marrow cells samples into 2million/100μl.

To determine viability of live cells, 100 μl BM cells were first stained with an aqua antibody
(Thermo Fischer Scientific, #2240354). Then incubated BM cells with various antibodies CD3
(Biolegend, #100227), CD4 (Biolegend, #100512) and CD8 (Biolegend, #100712) for at least
30mins on ice and avoided light. Washed the BM cells for two times and resuspend the BM cells
in 500ul PBS. Transfer samples into FACS tube and analyze data on X 20 flow cytometer using
FlowJo software.

32
3.2 Results

3.2.1 Physicochemical characterization of 5FA and 5FV

5FA and 5FV (Figure 15A) were recombinantly expressed in Clear e coli, which are designed to
not trigger human endotoxic response in cells by genetically modify lipopolysaccharide. Protein
with >95% purity was achieved by 3 rounds of ITC. It resulted in yield of 50-100 mg/L bacterial
culture. The transition temperatures of 5FA and 5FV were obtained by measuring optical density
at 350nm on UV-Vis DU 800 spectrophotometers as described above. A rapid increase in solution
turbidity was performed by heating. 5FV undergo phase separated below physiological
temperature(37°C) at 25 μM, but 5FA remain soluble (Table 2). The transition temperature (Tt) of
5FA and 5FV were concentration dependent and can be fit to a Log-linear model, Tt = b – m [Log10
(Concentration)].

Figure 15. Physicochemical characterization of 5FA and 5FV
31
A. Coomassie SDS-PAGE gel staining verifies high purity and
confirm molecular weights of fusion proteins B. Optical density verses temperature profile for 5FA and 5FV at 25 μM C. The
transition temperature (Tt) of 5FA and 5FV

33
Table 2. Physiochemical characterization of 5FA and 5FV
31

After encapsulating rapamycin and dialysis to remove unbound drug and organic solvents,
endotoxin removal and LAL assay was performed as described. 5FA-Rapa and 5FV-Rapa
concentration measured through C-18 RP-HPLC column.

34
3.2.2 Bone marrow infiltration T cells analyses

To evaluate the sustained-release of our rapamycin formulation in immune-mediated Bone marrow
failure, we infused lymphocyte cells from B6 donors into MHC-mismatched, pre-irradiated
CByB6F1 recipients and induced severe Bone marrow failure in recipient animals with
dramatically increased proportion of total CD3 positive T cells on BMF group compared with TBI
group (Fig.16). Treatment with 5FV-Rapa at 2 mg/kg every other day and 5FA-Rapa at 8 mg/kg
for only single dose both can effectively reduce the infiltrating CD3+

T cells in the bone marrow.
(Fig. 16A) There is no significant difference observed among TBI only, 5FA-rapa and 5FV-rapa
groups (Fig. 16B).

Figure 16. Bone marrow infiltrating T cells are effectively reduced by 5FA-Rapa and 5FV-Rapa. 5 CByB6F1mice for each group;
Abbreviation: BMF (bone marrow failure), TBI (total body irradiation)

35
From the BMF group, it is clearly that the CD8 positive cells are the dominant population in bone
marrow T cells (Fig 17A). Relative to the TBI control, BM failure mice had marked expansion of
T cells in the bone marrow, and both 5FA-rapa and 5FV-rapa effectively eliminated most BM-
infiltrating T cells. (Figure 17 A and C). Both CD4+ and CD8+ T cells were significantly reduced
by our treatments, but stronger inhibitory effect was observed for CD8+ T cells, which reduced
from 67% to around 2%. This result is consistent with previous research
62
.

Figure 17. Frequency of both CD4+ and CD8+ T cells were reduced by 5FA-Rapa and 5FV-Rapa treatments A. CD8 positive cells
are the dominant population in BMF B. Stronger inhibitory effect was observed for CD8 positive cells.

36
3.2.3 5FA-Rapa and 5FV-Rapa suppress tumor growth in breast cancer

5FA-Rapa and 5FV-Rapa were tested against breast cancer and tumor volume were evaluated for
28 days (Fig. 18). 5FV-Rapa treatments and PBS groups were subcutaneous administrated for only
single dose. As control group, 1mg/kg 5FA-Rapa was given twice weekly, which received total 8
mg/kg over 4 weeks. Interestingly, with the equivalent dose of 8 mg/kg, single does 5FV-Rapa
outperformed repetitive doses of 5FA-Rapa against breast cancer with significantly decrease tumor
volume. Moreover, 2 mg/kg 5FV-Rpa is the maximum subtherapeutic dose. Therefore, our lab
will further be fused with targeting ligands to 5FV-Rapa at 2 mg/kg and expect it to achieve similar
efficacy as 4 mg/kg or even 8 mg/kg by concentrating at tumor site.

Figure 18. Single dose of 5FV-Rapa outperformed repetitive doses of 5FA-Rapa against breast cancer

37
3.3 Discussion and conclusion

In this study, we demonstrate that the administration of our two rapamycin formulations effectively
and reproducibly weakened immune-mediate BMF in AA mouse models. Both 5FA-Rapa and
5FV-Rapa showed high efficacy eradicating CD4 + and CD8+ T cells in recipients bone marrow,
especially for pathogenic CD8+ T cells. Single injection of 5FV-Rapa exerts comparable effect as
repetitive doses of 5FA-rapa in mitigating bone marrow failure.

For the future direction, it is necessary to compare single injection of 5FA-Rapa and 5FV-Rapa at
the same drug dose to directly evaluate the sustained release of 5FV-Rapa. As interferon γ (IFN-
γ) is typical Th1 immune response cytokine to mediate potent hematopoietic inhibition
69,70
in AA
disease, it is also necessary to collect blood plasma and measure proinflammatory cytokine IFN-γ
and hematopoietic stem and progenitor cells (HSPC) concentration.

38
Chapter 4 GRP78-targeted FKBP-ELPs Rapamycin delivery for breast cancer
Introduction
Breast cancer is the second most deadly cancer affecting American women which kills nearly 40
thousand women per year in United States. As mTORC1 inhibitor, rapamycin has potent cytostatic
pharmacology, and it combined with exemestane has been approved for hormone receptor positive
(HR+) breast cancer. However, patients often experience severe side-effects related to low
solubility and off-target disruption of mTOR pathway
71
.

As described above, 5FA and 5FV are high-capacity rapamycin formulations with diminishing
peaks and troughs increasing bioavailability, reducing renal toxicity and predicable PK profile.
Specifically, 5FA remains soluble at body temperature and it is absorbed with advanced
bioavailability around 60% within one day
72
. In this project, our lab developed next-generation
rapamycin carriers fused 5FA with a panel of GRP78-binding peptides (X=W-peptide, L, P-6, and
P-13
73
) to enhance tumor-specific targeting.

Cell-surface GRP78 (cs GRP78) is a 78 kDa glucose-regulated protein, which is preferentially
exposed on multiple cancers and tumor neovascular, but not on normal organs
74,75
. For cells under
ER stress, including multiple classes of breast cancer, GRP78 translocate from the cytosol to the
cell surface. Thus, we propose this new delivery method could improve therapeutic outcomes of
rapamycin. Additionally, we aim to identify the peptide ligands that enhance the cellular uptake of
5FA into BT474 cells.

39
4.1 Material and Methods:

4.1.1 X-5FA plasmid molecular cloning, protein purification and drug encapsulation

5FA was generated by recursive ligation method as described above and GRP78 targeting peptides
(X = W, L, P6, or P13) were synthesized by Genewiz cloning services and ligated into the N-
terminus of 5FA backbone through sticky ends. Electrocompetent ClearColi BL21 (DE3) cells
(Lucigen, #60810-2) were transformed with X-5FA plasmid DNA by electroporation. Between
50-200 µL of transformed cells were spread on agar plates supplemented with 25 g/L Luria Broth
(LB) and 100 μg/mL carbenicillin. Plates were incubated at 37°C overnight, and singular bacterial
colonies had picked us starter culture. The X-5FA protein purification, drug encapsulation and
endotoxin removal as described above.

4.1.2 Mammalian Cell culture

Mammary gland breast duct cells BT474 cell line (ATCC, Manassas, VA) was cultured in
complete Hybri-Care Medium supplemented with 10% heat inactivated fetal bovine serum and 1.5
g/L NaCHO3 (EMD, #SX0320-1) adjusted to a pH of 7.2 with NaOH (Corning Life Sciences,
Tewksbury, MA) at 37 °C in a humidified incubator with 5% CO2. Media was replenished every
2 days and sub-cultured after reaching 80 to 90% confluency; briefly, cells were detached in
minimal volume of pre-warmed 0.25% trypsin-EDTA (Gibco, #25200-056) for 5 minutes at 37 °C
and centrifuged to form a pellet. Cells were resuspended in fresh media and used for passaging to
a 75 cm
2
flask and 6-well plates for assaying. Cells line stocks were stored in a 5% anhydrous
DMSO (Invitrogen, #D12345) media solution submerged in liquid nitrogen (-170 °C). Fresh cell
stocks from the same lineage were used for each experiment.

40
4.1.3 Western blot assay

BT474 cells were sub-cultured on 6-well plates (Genesee Scientific, #25-105) and grown to 80-
90% confluency. For dose-response assay, incubated either 0, 0.1, 1, 10, 100, or 1000 nM
rapamycin, 5FA-Rapa, or X-5FA-Rapa (X = W, L, P6, or P13) for 2 hours at 37°C in a 5% CO2
incubator. For kinetic assay, incubated cells with 1 nM of rapamycin, 5FA-R, or X-5FA for either
0, 15, 30, 60, or 120minutes at 37°C in a 5% CO2 incubator. For both experiments, media was
discarded and washed with 4°C PBS and then incubated with 100 µL 1x protease phosphatase
inhibitor cocktail (Catalog no: 78440, Thermo Fischer Scientific, Waltham, MA) diluted in RIPA
buffer (Thermo Fischer Scientific, Waltham, MA) to lyse cells at 4°C. Cell monolayers were
manually removed using a cell scraper (VWR, #10062-904) and vortexed before deep freezing in
individual microcentrifuge tubes at -80°C. After freezing overnight, centrifuged cells at 13000g,
4°C for 15mins to remove cell debris. Protein concentration was quantified by PierceTM BCA
Protein Assay Kit (Thermofisher, #23227). 30 µg Protein was loaded on a 4-20% gradient Mini-
Protean TGX precast gel and transferred to a nitrocellulose membrane using an iBlot2 NC stack
(Invitrogen, #IB23001) on an iBlot 2 Dry Blotting System. Membranes were blocked in 5% BSA
(Sigma, #A9647) dissolved in 1x tris-buffered saline adjusted to pH 7.4 with 0.1% tween-20
(TBST) (Santa Cruz Biotechnology, #sc-29113) for 1 hour at room temperature. Then incubated
the membrane in Rabbit α-rpS6 (Ser235/236) (Cell Signaling Technology, #2211) diluted 1:1000
in 5% BSA overnight at 4°C with gentle shaking. After washing with 1x TBST in triplicate,
membranes were incubated with secondary α-Rabbit IgG HRP-linked antibody (Cell signaling,
#7074S) diluted to 1:5000 in 5% BSA for 1 hour at room temperature.

41
Blots were briefly incubated in a 1:1 luminol and peroxide solution (Prometheus Protein Biology,
#GSC-925-D10, #GSC-929-D10) and immediately imaged on a ChemiDoc® to detect
chemiluminescent signal. Membranes were stripped using RestoreTM Western Blot Stripping
Buffer (Thermofisher, #21059) for 15 minutes at room temperature and washed 3 times in 1x
TBST. Membranes were re-blocked in BSA and incubated in Mouse α-GAPDH (Cell Signaling
Technology, #97166) and α-Mouse IgM HRP (Cell Signaling, #7076S) chemiluminescence
repeating the protocol for rpS6 visualization. Raw data for rpS6 expression normalized to GAPDH
was extracted using FIJI and analyzed on GraphPad for comparisons.

42
4.2 Results

4.2.1 Physicochemical characterization of X-5FA

5FA was generated by multiple FKBP proteins linked with ELP A24 and X-5FA constructs was
designed to target cell surface GRP78 by fused ligands (X=W, L, P6, P13) on the N-terminus of
5FA backbone (Fig.19A). To demonstrate the carrier’s selectivity, the SubA protease cleaved
GRP78 from the cell surface, which prevents internalization of these drug carriers. The SubA
protease will be used to demonstrate the carrier’s selectivity as it cleaves GRP78 from the cell
surface and prevents internalization of these drug carriers. 5FA and X-5FA (Fig.19B) were
recombinantly expressed in Clear e coli with >95% protein purity by 3 rounds of ITC with the
yield about 80-200 mg/L (Fig.19C).

Figure 19. Physicochemical characterization of 5FA and X-5FA A. The schematic representation of X-5FA-Rapa target tumor cells.
B and C. SDS-PAGE show high purify with the correct MW of 5FA and X-5FA-Rapa constructs.

43
4.2.2 Western blot result

Western blots revealed potent mTOR inhibition of free rapamycin, 5FA-Rapa, or X-5FA-Rapa
treatment in BT474 cells line (Figure 20A, 21A). mTORC1 mediated the phosphorylation and
activation of p70S6 Kinase and its downstream effector ribosomal protein S6 (RPS6). Rapamycin
blocked mTORC1 pathway and thereby mediated rpS6 de-phosphorylation, which is parallel with
decreased ribosomal biogenesis, mRNA translation, and protein synthesis, partly accounting for
its anti-proliferative effect.

The dose-dependent phosphorylation inhibition of rpS6 was monitored (Fig 20A), where all
treatments showed significant inhibition effect (Fig 20B). Suppression of rpS6 phosphorylation
demonstrated successful Rapamycin delivery to the cytoplasm and subsequent inhibition of mTOR
kinase activity. GAPDH is used as total protein loading control. For quantitively comparison,
integrated volume for rpS6 bands were normalized to GAPDH (Figure 20B, 21B). Free rapamycin
had complete suppression of phosphorylation at 0.1nM, while P13-5FA-Rapa group had detectable
rpS6 signal until 100 nM. Compare with 5FA-Rapa treatment, L-5FA-Rapa and W-5FA-Rapa
treatments effectively inhibit phosphorylation of rpS6 at 1nM, which means peptide ligands can
concentrate better in BT474 cell line.

44

Figure 20.Western blot analysis of concentration-dependent effect on rpS6 A. rpS6 protein expression in free rapamycin/5FA/X-
5FA treatment over various concentration range from 0nM-1000nM B. quantification of rpS6 protein relative expression

Thus, 1nM was chosen to indicate the time-dependent effects of X-5FA treatments on rpS6 protein
expression in cultured BT474 cells (Fig 21A). Consistent with concentration-dependent results,
there was no significant differences in rpS6 expression of P13-5FA-Rapa detected over a range of
time. Compared to free rapamycin treatment, the inhibition effect of L-5FA-Rapa and L-5FA-Rapa
treatment were more pronounced at 120min, which indicating W and L might be potential ligands
to enhance internalization of BT474 cell (Fig 21B).

45

Figure. 21 Western blot analysis of time-dependent effect on rpS6 A. rpS6 protein expression in 1nM free rapamycin/5FA/X-5FA
treatment over various time periods from 0min-120min B. quantification of rpS6 protein relative expression

46
4.3 Discussion and conclusion

In this study, we demonstrate that free rapamycin, 5FA-Rapa, or X-5FA-Rapa treatment
significantly inhibit mTORC1 pathway in HR+ breast cancer models with dose-dependent effect.
1nM is the minimum concentration and 120min is the minimum time to effectively block rpS6
phosphorylation. These results suggest that X-5FA-Rapa formulation can successfully act on-
target in hormone receptor positive model, where L-5FA-Rapa and W-5FA-Rapa might be
promising carriers to concentrate better with better potency to enhance cellular uptake of 5FA in
BT474 cell lines, which should be further identified by flow cytometry and confocal microscopy.

For the future direction, the cytostatic activity of promising carrier and free rapamycin will be
evaluated using a MTT assay to statistically compare IC50 values. Stability will be assessed using
dynamic light scattering as a function of time and temperature. Further experiment to compare
efficacy and preclinical toxicology of promising rapamycin carrier and free rapamycin in murine
breast cancer models.

47
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Asset Metadata

Creator Yu, Jingmei (author)

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

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2021-08

Publication Date 07/14/2022

Defense Date 06/16/2021

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

Tag drug delivery,elastin like polypeptide,FKBP,fusion proteins,nanoparticle,OAI-PMH Harvest,rapamycin

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mackay, John Andrew (committee chair), Haworth, Ian (committee member), Okamoto, Curtis (committee member)

Creator Email estatejingmei@163.com,jingmeiy@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC15496662

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