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Comparing drug release kinetics of rapamycin bound FKBP-ELP fusion proteins
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Comparing drug release kinetics of rapamycin bound FKBP-ELP fusion proteins
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1
Comparing Drug Release Kinetics of
Rapamycin Bound FKBP-ELP Fusion Proteins
Shruti Singh Kakan
USCID: ██ ███ ██
A thesis presented to the Department of Pharmacology and Pharmaceutical
Sciences, School of Pharmacy at the University of Southern California in partial
fulfilment of the requirements for the degree MS in Pharmaceutical Sciences
August 2017
2
Acknowledgements
I extend my sincere gratitude to my advisor Dr. J. Andrew Mackay for his
continued support, encouragement, motivation and guidance throughout the
duration of my Master’s at USC. I would also like to thank my committee
members Dr. Ian Haworth and Dr. Curtis Okamoto, for their guidance and support
of my two-year education at USC. Their discussions in the courses Drug Discovery
and Drug Transport have significantly aided me in understanding my project
better. I am also grateful to Dr. Roger Duncan, for offering his critique on my
scientific writing.
I am immensely grateful to all my lab members for their constant support,
motivation and collaborative work that has immensely enriched my education in
research. I am particularly grateful to Aida Kouhi for training me initially and
helping me better navigate my master’s journey. I am also thankful to lab members
Santosh Peddi and Hao Guo for training me on several instruments, Hugo Avila
who has proved to be a valuable peer, and Jordan Despanie for his unconditional
support and encouragement in the past year.
3
Table of Contents
Abstract 4
Chapter 1: Elastin-Like Polypeptides for Delivery of anti- cancer
drugs 5
1.1 Challenges associated with existing small molecule anti-cancer drugs 5
1.2 How nanomedicine can overcome the challenges of conventional
chemotherapy 7
1.3 Elastin-like Polypeptides (ELP) for the delivery of anti-cancer drugs 9
Chapter 2: FKBP-ELP for the delivery of Rapamycin 10
2.1 Introduction 10
2.2 Material & Methods 14
2.3 Results and Discussion 19
2.4 Conclusion 39
References 40
4
Abstract
Traditional anti-cancer drugs are cytotoxic in nature and cause severe side-effects in
patients. Additionally, several of them lack tumor specificity, are difficult to formulate,
show high variability in pharmacokinetics among individuals, have shown to develop
drug resistance over time, and can even contribute to tumor recurrence. Many of these
challenged can be counteracted by nanomedicine, which can also have the added
benefit of tumor targeting.
Rapamycin is a cytostatic drug that arrests a dividing cell in its G1 phase and has
shown anti-cancer activity in several cancer cell lines. Being a cytostatic it is relatively
less toxic than conventional chemotherapy. Two of its analogues, Temsirolimus and
Everolimus, are in the market for the treatment of a range of tumors. However, the
extent of Rapamycin’s clinical potential (and that of its analogs) is marred by its lung
toxicity, adverse effects such as myalgia, sepsis, stomatitis, leukopenia, etc.,
unfavorable physicochemical properties, low oral bioavailability and inter-individual
variability in drug disposition.
Rapamycin’s aqueous solubility can be enhanced significantly by encapsulating it with
its cytosolic partner FKBP-12. As FKBP-Rapamycin would be cleared relatively quickly
through renal filtration owing to the 20kDa molecular weight of FKBP, we have
developed FKBP-ELP fusion proteins that would have longer circulation time in plasma
(for ELP fusion proteins with molecular weight > 45 kDa) or provide passive targeting
through the EPR effect (for nanoparticle forming ELPs). A high drug loading efficiency
was achieved irrespective of the ELP fused to FKBP. We have also studied how the
attachment of different ELPs affects the release rate of Rapamycin from an ELP fusion
protein. Of the five ELP fusion proteins studied all four of the monoblock ELPs show
extended rapamycin retention with little to no release over a time period of several days.
Drug release is also not affected by temperature or the phase of the thermo-responsive
ELPs as both coacervate and soluble fusion proteins displayed similar drug retention.
This indicates that rapamycin encapsulated fusion proteins may not release the drug at
all. Therefore, it remains to be investigated if Rapamycin encapsulated fusion proteins
can be internalized by a tumor cell and if that is the way this formulation exerts the anti-
cancer activity that has been reported previously
[1]
.
5
Chapter 1: Elastin-Like Polypeptides for delivery of anti-
cancer drugs
1.1 Challenges associated with existing small molecule anti-cancer
drugs
Conventional chemotherapeutics suffer from several problems. Poor tumor selectivity,
leads to many off target effects, which are quite severe due to the cytotoxic nature of
these drugs. These toxicities create an upper limit on the dose that can be administered
to the patient, which is often not sufficiently efficacious. These drugs also suffer from
poor penetration into solid tumor reducing their overall efficacy. There is also a growing
drug resistance due to cancer cells’ ability to express efflux transporters over time.
Many of these drugs face formulation challenges due to their hydrophobic nature and
poor aqueous solubility
[2]
. Due to rapid clearance (such as in the case of methotrexate)
there is low systemic exposure for each dose requiring relatively frequent dosing
[3]
.
1.1.1 Cytotoxicity, Poor Selectivity and Drug Resistance
Conventional chemotherapeutics kill tumor cells by inducing apoptosis by various
mechanisms such as inhibiting DNA polymerase, DHFR, topoisomerase I & II,
microtubule assembly, intercalating DNA base pairs, alkylating or cross-linking DNA,
etc.
[4][5]
. Inhibition of these processes in normal cells leads to severe side-effects such
as alopecia, myelosuppression, neurotoxicity, nephrotoxicity, GI bleeds, sarcopenia,
etc.
[4][5][6]
. These side effects occur because chemotherapeutics target all rapidly
dividing cells and have no specificity for tumor cells.
In a 2009 study it was reported that following treatment with letrozole (endocrine
therapy) and docetaxel (chemotherapy), the residual breast tumor tissue was found to
be enriched with tumor initiating cells, as a direct consequence of the toxicity of these
drugs. This suggests that chemotherapy may also be contributing or hastening tumor
recurrence
[7]
.
6
1.1.2 Low aqueous solubility
Several anti-cancer drugs such as Paclitaxel, Imatinib, docetaxel, cisplatin, etc.
[8]
are
hydrophobic in nature with poor aqueous solubility which presents a formulation
challenge. As a result, they often require solvents and/or surfactants to dissolve them.
For example, Paclitaxel’s current formula (taxol) is formulated in a 1:1 mixture of
Ethanol and Cremophore EL
[9]
. Cremophore by itself has been associated with
peripheral neuropathy, aggregation of erythrocytes, anaphalactoid hypersensitivity
reactions and hyperlipidaemia
[10]
. Similarly, docetaxel’s formulation with ethanol and
tween 80 is also undesirable as the surfactant can elicit severe hypersensitivity
reactions
[11]
.
1.1.3 Wide variation in PK
Chemotherapeutics display high PK variability among individuals and a same dose of a
drug can have widely varying levels of exposure between patients
[12]
. This can lead to
severe toxicities in some patients while at the same time leaving others with sub-optimal
dosage (as anti-cancer drugs often have a narrow therapeutic index). This makes it
harder to predict patient outcome for any given drug and warrants the need to tailor
make doses for every individual.
Where drug absorption is concerned, orally taken anticancer drugs tend to have higher
variability than intravenously administered. For e.g. Etoposide administered orally has a
58% coefficient of variation in area under the curve (AUC) as opposed to the 28% for
intravenous administration
[13]
. To an extent these variabilities occur due to variable
expression of efflux transporters in the gut and genetic polymorphism in metabolizing
enzymes in the liver
[14]
.
Several anti-cancer drugs have high protein binding. In case of cancer patients there is
often liver dysfunction and symptoms of hypoalbuminaemia that would reduce the
fraction of drug bound. On the other hand, conditions such as inflammation can
increase the levels of blood proteins such as alpha 1–acid glycoprotein leading to
7
increased drug binding
[15]
.
These situations can increase or decrease levels of drug in
plasma without warning.
Therefore, there is a need to develop therapeutics that are more specific in nature, with
lesser toxicity and a higher residence time, so that they are sufficiently efficacious and
require relatively less dosing.
1.2 How Nanomedicine can overcome the challenges of conventional
chemotherapy
Packaging of any given anti-cancer drug in a nano-formulation can help improve
bioavailability, reduce toxicity and allow for drug targeting. Nano drug delivery vehicles
can be made up of proteins, polymeric micelles, liposomes, lipoprotein based drug
carriers, dendrimers, carbon nano-materials, viral particles, etc. Ideally, such a vehicle
should biocompatible, non-immunogenic, non-toxic, biodegradable into non-toxic
molecules and should be able to avoid opsonization
[16]
.
Increase in Bioavailability and decrease in toxicity
Cancer drugs with poor solubility are able to overcome this challenge by piggybacking
on a soluble drug carrier. This improves bioavailability over oral formulation. Tumor
targeting, whether active or passive shields the healthy dividing cells from the cancer
drug. Liposomal formulations of Doxorubicin have been found to reduce drug related
toxicities such as alopecia, bone-marrow depression and cardiomyopathy. Studies
conducted during clinical trials show that liposomal formulation Myocet had twice fewer
cardiac events and 4 times less CHF than patients who were given doxorubicin
[17]
.
Stage I clinical trial of albumin bound Rapamycin (nab-Rapamycin) found that the
toxicities of stomatitis/mucositis were no longer dose-limiting, and pneumonitis (that can
be life-threatening) that is observed with mTOR inhibitors was not observed at all with
nab-Rapamycin
[18]
. Oral bioavailability of Doxorubicin is 5%, whereas that of
Rapamycin is 14%.
8
Tumor targeting
To an extent, nanoparticles have an inherent passive tumor targeting as they are able to
exploit the aberrant vasculature of the tumor micro-environment through the EPR effect.
Due to their size, when administered intravenously, they are not cleared by renal
filtration. They cannot move across tight endothelial junctions and circulate in blood
vessels for longer times but extravasation across extensively fenestrated vasculature
surrounding the tumor is possible
[19]
.
Active targeting can be achieved by targeting a cell surface receptor that is over
expressed by the tumor cell followed by internalization of the cargo. This type of
targeting is particularly important when the cargo is a macromolecule such as siRNA,
DNA or protein. Active targeting can also be achieved by targeting angiogenic receptors
that are overexpressed by endothelial cells surrounding the tumor
[20]
.
More favorable PK
Delivering drugs by means of a nano-carrier can help reduce inter and intra-patient
variability observed in PK parameters. For example, delivering a drug with a nano-
carrier can help reduce or prevent plasma protein binding and obviate the variations of
drug concentrations in plasma resulting from hyper or hypoalbuminaemia.
A longer plasma half-life is achieved by designing nanoparticles that are larger than the
fenestrations in blood vessels of nephrons. Proteins with a molecular weight greater
than 45 kDa
[21]
, and nanoparticles or liposomes > 6.2 nm
[22]
can escape renal filtration.
Hence, they would circulate for a relatively longer time. For example, Doxorubicin has
an elimination half-life of 0.2 h. Its liposomal formulation Doxil, however, has an
elimination half-life of 55h
[23]
.
On the other hand, for drugs that have a relatively high half-life owing to plasma protein
binding, nanomedicine can help improve variability in PK parameters. Stage I clinical
trial of albumin bound Rapamycin (or nab-Rapamycin) found that intravenous infusions
of the formulation improve C
max
and toxicity profile of the formulation while maintaining
9
efficacy and half-life of orally administered Rapamycin or intravenously administered
Temsirolimus. A 63-hour half-life was reported for the maximum tolerated dose of
100mg/m
2
. The C
max
at this dose was 3227.6 ng/mL which is significantly higher than
C
max
of 57.7 ng/mL for the 60 mg oral dose or the day-5 C
max
of 133 ng/mL after 5 days
of intravenous administration of Temsirolimus at a maximum dose of 19.1 mg/m
2 [18]
.
These values were consistent across patients.
1.3 Elastin Like Polypeptides (ELP) for the delivery of anti-cancer
drugs
Elastin Like Polypeptides (ELPs) derived from human tropoelastin, are peptidic repeats
of G(VPGXG)
n
Y. They are biodegradable, non-immunogenic, can be recombinantly
expressed and have the incredible ability to undergo a sharp reversibly phase change
going from soluble to coacervate at a unique transition temperature. By altering the
guest amino acid X and copy number n, we can engineer ELPs that phase separate at
physiologically relevant temperatures. ELPs retain this ability even when they are
conjugated to proteins of diverse physico-chemical properties or biological significance.
Such constructs have great potential as targeted drug delivery systems. Diblock ELPs
provide the advantage of nanoparticle drug delivery through EPR effect, while soluble
fusion proteins extend the half-life of the therapeutic they are appended to, as they
would have a higher circulation time
[24]
.
ELPs offer considerable advantage over synthetic dug carriers. They are peptides, and
hence are non-toxic and non-immunogenic in nature. They are completely
biodegradable into non-toxic amino acids. And they can be biosynthesized efficiently in
E.coli, without the use of toxic chemicals or organic solvents
[25]
. It is also quite easy to
covalently link molecules whether they be targeting moieties, drugs or investigative
molecules for research. Amphiphilicity allows them to interact well with both drug and
solvent.
10
Another advantage of ELPs could be the possibility of inherent targeting. When
Abraxane was formulated, its ability to be selectively internalized by cell surface
receptors such as SPARC, that is overexpressed on several cancers, was not known
[26]
. Hence, Abraxane not only solubilizes paclitaxel well, but also selectively delivers it
to the tumor. Investigations are underway to see if ELPs can also be selectively
internalized in a similar manner.
Chapter 2: FKBP-ELP for the delivery of Rapamycin
2.1 Introduction
Rapamycin is a macrolide molecule that was initially extracted from the bacterium
Streptomyces hygroscopicus in 1975
[27]
. In the beginning, it was being studied as an
anti-fungal agent but once its ability to cause immune-suppression was discovered,
those attempts were left behind. Rapamycin can inhibit T cell and B cell activation by
reducing production of IL-2 following inhibition of the Mechanistic Target of Rapamycin
(mTOR) downstream signaling
[28]
. As it is less toxic to kidneys than calcineurin
inhibitors such as Tacrolimus, Rapamycin was approved as an immunosuppressant in
1999 by the USFDA for prevention of organ rejection in kidney transplant.
Its potential as an anti-cancer drug was first reported by its discoverers in 1981
[27]
when
it was found to have anti-tumor activity against melanocarcinoma, ependymoblastoma,
and tumors of breast and colon
[29] [30]
. At that time, rapamycin’s binding to mTOR was
not known. mTOR was discovered in 1994-95 in the search for understanding the
mechanism of action of Rapamycin, and is therefore named after it.
Further research in the mTOR inhibition in breast cancer cells lines that were either
estrogen dependent, PTEN-deficient or had Her-2/neu overexpression were sensitive to
treatment with Rapamycin which exerted its cytostatic action via inhibition of mTOR
[31]
It was suggested that Rapamycin could be used as an adjuvant therapy for breast
11
cancer with the above-mentioned markers. Soon after Rapamycin was also found to be
effective in tumor xenografts of renal cell carcinoma and prostate cancer
[32] [33]
.
As of now several analogs of Rapamycin have been approved for treatment of various
cancers
[34]
.
Table 1. Analogues of Rapamycin and the type of cancers that they have been approved for or
the stage of development that they are in
Rapalogue FDA approved for/Stage of development
Rapamycin - Angiomyolipoma in adults with the tuberous sclerosis complex
or sporadic lymphangioleiomyomatosis (Phase I-II)
[35]
Everolimus - advanced neuro-endocrine tumors
[36]
- HR positive breast cancer (combination therapy with
exemestane)
[37] [38]
- renal cell carcinoma
[39]
- subependydymal giant cell astrocytoma
[40]
- renal angiomyolipoma
[41]
Temsirolimus - advanced renal cell carcinoma
[42]
- relapsed/and or refractory mantle cell lymphoma (under
investigation in US, approved in the European Union)
[43]
Ridaforolimus Phase III clinical trial for bone sarcoma
[44]
However, oral formulations of Rapamycin suffer from severe delivery complications
owing to the low solubility of Rapamycin (2.5 µg/mL) and high hydrophobicity, with a
logP of 4.3. As a result, the oral formulation of Rapamycin has a low oral bioavailability
of 14%. Owing to these physicochemical characteristics
[45]
, both oral and intravenous
formulations of Rapamycin is challenging. Currently Rapa is being formulated in a 40 to
75% solution of propylene glycol in water which gives a concentration of 0.25 mg/mL to
8 mg/mL [46]. Its oral solution is formulated in 50% phosphatidylcholine in propylene
glycol/ethanol carrier. This oily solution is inconvenient due to stringent storage
conditions. Sirolimus tablets are formed of nanocrystals of the drug. Even then with both
12
the oral formulations, a bioavailability of only 14 – 15 % can be reached
[45]
. Both
formulations however, show high inter-individual variability and fluctuations in serum
concentrations at any given time. This makes dosing quite challenging as the dose
would have to be personalized for every individual to achieve optimal efficacy.
To counteract these challenges, we propose to deliver Rapamycin with FK506 binding
protein- 12 (FKBP-12) which is one of its natural cytosolic partners.
Figure 1. Free Rapamycin binds cytosolic FKBP-12 forming a complex that then interacts with
the mTOR complex.
Rapamycin shares partial structural homology with FK506 (tacrolimus) which is also a
macrolide discovered from a different strain of Streptomyces. As it was already known
that FK506 binds the cytosolic protein FKBP, Rapamycin was also investigated for the
FKBP binding
[47]
and was found to have a k
d
of 0.2 nM for FKBP-12
[48]
. Resolution of
Rapa bound FKBP-12 showed that it forms H-bonds with side chains of Ile-56, Tyr-82,
Asp-37 and main-chain carbonyl of Gln 53
[49]
. Of these, interactions, Tyr-82 seems to
be the most crucial interaction as mutation of Tyr-82 in FKBP-12 can lead to 95% loss in
binding affinity
[50]
.
13
Rapamycin is a cytostatic drug, that can arrest cells in G1 phase but does not induce
apoptosis
[51]
. Therefore, it is relatively less toxic than conventional chemotherapeutics.
By encapsulating it with FKBP which has a high affinity for the drug, a water-soluble
formulation of rapamycin can be formed with a carrier that would be completely
biodegradable in the body.
Figure 2. Downstream signaling of Inhibition of mTOR by Rapamycin leading to G1 arrest
[52]
Molecular weight of FKBP at 20 kDa is lower than the cut-off of renal filtration. Rapa
encapsulated in FKBP would be cleared relatively quickly than if it was being carried
with a vehicle with a molecular weight >45 kDa or by itself (as Rapamycin has a half-life
of 54h due to high protein binding). To develop a formulation that improves on
Rapamycin’s solubility and also have a high plasma half-life, ELP of varying chain
length and characteristics can be attached. Similar to FKBP, ELPs are also expected to
be completely benign as they would be metabolized into amino acids.
In this manuscript, we have compared how attachment of different ELPs affects the
release of Rapamycin. Two of the FKBP-ELPs, FKBP-A192 (FA) and FKBP-A192-
FKBP (FAF), have molecular weights of 85.4 kDa and 97 kDa respectively, and
14
therefore may have a longer circulation time. FKBP-V48 (FV48) & FKBP-V72 (FV72)
would phase separate at body temperature and may find application as a depot forming
extended release formulation. FKBP-S48I48 (FSI) forms a micellar nano-particle of 27
nm and has been explored for passive tumor targeting [1].
2.2 Materials and Methods
Materials
The pET25b (+) vector was obtained from Novagen Inc. (Madison, WI). Top10 cells
were purchased from Invitrogen (Carlsbad, CA) and BLR(DE3) cells were purchased
from EMD Millipore (Billerica, MA). All E. coli cells were grown in TB dry growth media
bought from MO Bio Laboratories Inc. (Carlsbad, CA). Rapamycin amorphous powder
was obtained from LC Laboratories (Woburn, MA). Dulbecco’s PBS of 1X strength,
used for diluting & solubilizing proteins was purchased from Thermo Scientific, while 1x
PBS buffer for dialysis was prepared from 10x PBS powder obtained from Biopioneer
Inc. 1x Penicillin-Streptomycin was re-constituted from 100x Pen-Strep solution
obtained from Corning Inc. (Corning, NY). HPLC grade methanol and trifluoroacetic acid
were purchased from Fischer Scientific (Hampton, NH). 10 kDa Molecular weight cut off
(MWCO) Snakeskin dialysis tubing, and Dialysis cassettes of 10 kDa & 20 kDa
molecular weight cut off (MWCO), 3 mL in volume were purchased from Thermo
Fischer Scientific (Waltham, MA).
Preparation of Stock Solutions & Buffers
Rapamycin stock solutions were prepared in DMSO for Isothermal Titration Calorimetry
(ITC). DMSO stocks were first prepared at concentrations of 11 – 13 mM and frozen at -
20 ºC then diluted to 400, 500 or 750 µM to prepare 8, 10 and 15 µM of Rapamycin
solution in 2%DMSO/PBS, respectively. For encapsulating rapamycin in FKBP-ELPs,
Rapamycin stocks were prepared in 20% EtOH/Hexane and stored at -20 ºC. A fresh
stock was prepared every 3 months. All buffers were prepared with deionized Milli Q
water.
15
FKBP-ELP Expression & Purification
50 µL of BLR(DE3) E. coli cells were transformed with pET25b (+) vector carrying
FKBP-ELP or ELP genes by heat shocking at 41 ºC for 1 minute. Colonies carrying the
vector were then grown on Ampicillin positive agar plates for 18 h at 37 ºC. Following
this, a single colony was picked and grown for 12 hours in 50 mL of dry TB growth
media. BLR(DE3) Stocks were prepared by adding 1mL of 14% DMSO/PBS to 1 mL of
this culture and were stored at -80 ºC.
3 µL of the -80 ºC stock was transformed to 50 mL of TB dry growth media in the
presence of 100 µg/mL Carbenicillin for and allowed to grow for 12 h at 37 ºC. Following
which the culture was centrifuged and the bacterial pellet was then re-suspended in
double distilled water and transferred to 1L of dry TB growth media. The cells were
allowed to grow for 12 – 18 h at 37 ºC. The culture was centrifuged at 37 ºC, 4000 rpm
for 10 minutes. The supernatant was discarded, the pellet was re-suspended in DPBS
and sonicated to lyse the cells. The required FKBP-ELP or ELP was then purified by
inverse transition cycling
[53]
. The proteins were dialyzed in 1x PBS at 4 ºC for four hours
and then filtered using 0.2 µ syringe filters. Protein concentration was determined
following Beer Lambert’s law. UV-Vis measurements were taken using Nanodrop. Molar
extinction coefficients for ELPs, FKBP-ELP and FKBP-ELP-FKBP are 1285, 11585 and
20190 M
-1
cm
-1
, respectively.
FKBP-ELP Physicochemical Characterization
The purity of the constructs was determined by loading 20 µL of 5 – 10 µg of denatured
protein in SDS-PAGE gold gels. Gels stained with 10% w/v of Copper (II) Chloride were
then imaged in BioRad Gel Imager. Proteins were also run on an RP-HPLC to ensure
purity.
The ELP and the ELP fusion proteins were characterized for transition temperature (T
t
)
and critical micellar temperature (CMT, in case of FKBP-S48I48) by UV-Vis DU800
Spectrophotometer (Beckman Coulter, CA, USA). Briefly, 350 µL protein of
16
concentration ranging from 10 µM to 200 µM was loaded on Tm microcell cuvettes
(Beckman Coulter, Brea CA). The optical density (OD) was measured from 10 ºC to up
to 75 ºC at 350 nm, thrice a minute with temperature increasing 1 ºC per minute. The T
t
,
which is the first maximum derivative of OD with respect to. temperature, was reported
for the concentration of 25 µM of the protein.
The particle size of the fusion proteins was determined by Dynamic Light Scattering
which gives the hydrodynamic radius (R
h
). Briefly, 200 µL of 25 µM ELP fusion protein
was filtered using a 0.2 µ syringe filter and 60 µL of this was loaded in 3 wells of a 384
well plate (Griener Bio One). The wells were covered with 20 µL of mineral oil to prevent
evaporation during measurements. The well plate was centrifuged at 3000 rpm for 15
minutes to remove any air bubbles and the R
h
was measured using DynaPro Plate
reader (Wyatt, Santa Barbara CA). In the first characterization, particles size was
measured at temperatures ranging from 20 ºC to 50 ºC, whereas in the second
characterization, fusion proteins were tested for stability at 37 ºC, at time points ranging
from a few hours to several weeks following Rapamycin encapsulation. The data
reported is mean R
h
.
Encapsulation of Rapamycin
Rapamycin stock solution of 680 µM stored at -20 ºC was brought to room temperature.
In a 20 mL glass vial, 2 mL of 200 - 250 µM FKBP ELP was added to 2 mL of 680 µM
Rapamycin (nearly 3 eq.). As hexane is insoluble in PBS, two visible phases are
observed in the vial. The two phases are stirred at 200 rpm on a stir plate at room
temperature with the organic phase being evaporated under N
2
atmosphere. Following
20 minutes of stirring, when the organic phase was no longer visible, the contents of the
vial were centrifuged at 37 ºC thrice to remove any un-encapsulated Rapamycin. The
Rapa encapsulated FKBP ELP was then filtered using 0.2 µ syringe filter and dialyzed
at 4 ºC in 1xPBS for two hours using snakeskin dialysis tubing of 10 kD MWCO or
dialysis cassette of 10kD MWCO.
17
HPLC Determination of Rapamycin & FKBP-ELP
A validated RP-HPLC method was developed to measure concentration of Rapamycin
for plotting a standard curve and for determining the amount of Rapamycin retained by
FKBP-ELP at different time points for the dialysis experiment. A C18 XBridge column
from Waters Inc. (Millford, MA) was used as an analytical column. The temperature was
set to 20 ºC. The mobile phase constituted of 40% H
2
O / 0.1% TFA and 60% MeOH /
0.1% TFA at the beginning. The method consisted of alternating isocratic and gradient
flows, as follows:
Flow MeOH/0.1% TFA H
2
O/0.1% TFA Time (min)
isocratic 40 60 5
gradient 95 5 4.4
isocratic 95 5 5.5
gradient 40 60 4.5
The flow rate was set to 0.75 mL/min throughout. Rapamycin was detected by a UV
detector at 280 nm. The same method could also detect FKBP-ELP or ELP and was
used to plot standard curves and later determine the concentrations of fusion proteins
and Rapamycin in a sample. All samples were diluted 10-fold in 100% MeOH and 20 µL
of this was loaded on the column by the auto-sampler. The extent of encapsulation of
rapamycin in FKBP-ELP can be ascertained by -
Encapsulation efficiency =
!"#$%#&'(&)"# "+ ,(-(./$)# )# 01
!"#$%#&'(&)"# "+ 23456785 )# 01
∗100
Rapamycin Release Investigation by Dynamic Dialysis
2 mL of Rapamycin encapsulated fusion protein was loaded in 20 kD MWCO dialysis
cassettes and dialyzed into 1500 mL of PBS containing 1x Penicillin-Streptomycin to
prevent bacterial growth at 37 ºC. Buffer was changed twice on the first day and then
every alternate day from then on. 120 µL of sample was withdrawn at fixed times and
the concentrations of fusion protein and Rapamycin retained by them was determined
using RP-HPLC. As the detection was done at 280 nm, both Fusion protein and
rapamycin could be detected in the same run. Results were plotted as fraction of Rapa
18
retained with respect to time. Non-linear regression was used to determine the release
half-life of Rapamycin. The samples were withdrawn at the following time points for
each of the fusion proteins -
FKBP-S48I48 – 0h, 1h, 2h, 4h, 8h, 14h, 32h
Results for FKBP-S48I48 were not reported beyond 14h as the reported nano-particle
[1] size of 27 nm was not observed to be uniform after 14h.
FKBP-A192 – 0h, 1h, 2h, 4h, 8h, 24h, 48h, 96h, 192h
FKBP-A192-FKBP – 0h, 1h, 2h, 4h, 24h, 48h, 96h, 192h, 576h, 768h
In another series of dialysis experiments, release rate of rapamycin was compared
between soluble form of a fusion protein to that of its coacervate form. At 200 – 250 µM
Hence, two sets of dialysis experiments were conducted, in triplicates, with one at room
temperature (RT) and another at 37 ºC. The samples were withdrawn at the following
times –
FKBP-V72 – 0h, 1h, 2h, 4h, 8h, 24h, 48h, 96h, 144h
FKBP-V48 – 0h, 1h, 2h, 4h, 8h, 24h, 48h, 96h, 144h
In this case dialysis tubing was of 10 kD MWCO were used for dialysis. The dialysis
tubing was replaced every two days.
Determination of FKBP-ELP Rapamycin Binding Thermodynamics by Isothermal
Titration Calorimetry
Due to the limiting solubility of Rapamycin in PBS, for the experiment, both Rapamycin
and the ELP fusion proteins were solubilized in 2% DMSO/PBS. With this solvent Rapa
concentration of up to 15 µM could be reached. The binding affinity of Rapamycin to the
ELP fusion proteins was studied by Isothermal Titration Calorimetry. 280 µL of 8 µM, 10
µM or 15 µM of Rapamycin was loaded in the calorimeter sample cell using a Hamilton
syringe. 80 µL of 80 µM to 100 µM of ELP fusion protein was loaded on the syringe.
While normally, the drug is loaded on the syringe, in this case due to the poor solubility
of Rapamycin, the drug was loaded in the cell. The reference cell was filled with double
distilled H
2
O. For FA, FAF and FSI binding studies were conducted at 37 ºC. The speed
of the rotating syringe was set to 750 rpm and 3 µL of sample was injected second
19
injection onwards with time between two injections being 150s. Data was analyzed by
the MicroCal PEAQ software in the offset mode, which takes into account small heats of
dilution.
Results and Discussion
To address the delivery challenges faced by rapamycin, such as poor solubility and
variable PK, Rapamycin was encapsulated in FK506 binding protein-12 fused with
different ELPs. Five constructs were developed with different behavior at body
temperature: FKBP-S48I48 (FSI) forms nanoparticles, FKBP-A192 (FA) and FKBP-
A192-FKBP (FAF) are soluble, and FKBP-V48 (FV48) and FKBP-V72 (FV72) form
coacervates (depending on the concentration) as they phase separate close to body
temperature. Based on their behavior, they were expected to have different rates of
release for Rapamycin, which has been compared in this manuscript.
Recombinant expression of FKBP-ELP in E. coli delivers satisfactory yields
Following lysis of BLR(DE3) cells, FKBP-ELPs were purified by inverse transition
cycling. Typically, 2 to 3 rounds of ITC were employed for purification of FKBP-ELPs.
For ELPs, it was observed that four rounds of ITC were required in order to achieve
more than 99% purity. Following ITC, the FKBP-ELP and ELP constructs were run on
SDS-PAGE gel to ascertain purity. Later, an impurity was observed when FKBP-ELP
constructs were run on RP-HPLC, which could be removed by dialysis. Therefore, after
four hours of dialysis, concentrations were measured and yields were reported. Yield for
all constructs was between 20 – 36 mg/mL
20
Figure 3. a) SDS-PAGE gel run of FKBP-A192 (FA), FKBP-S48I48 (FSI), FKBP-A192-FKBP
(FAF), FKB-V48 (FV48), and FKBP-V72 (FV72). Dimers for FSI FV48 and FV72 are visible as
the 4x buffer used for staining samples was non-reducing. b) SDS-PAGE gel run of controls
A192, and S48I48 (SI). Gels were stained with Copper (II) Chloride
Table 2: ELP fusion proteins evaluated in this manuscript
ELP Construct Amino acid sequence
a
Tt
(ºC)
R
h
at
25 ºC
(nm)
R
h
at
37 ºC
(nm)
Slope,
m
b
Intercept
, b
b
(ºC)
Expected
MW
(kDa)
S48I48 (SI) G(VPGSG)
48
(VPGIG)
48
Y 27 39.8
FKBP-S48I48 (FSI) FKBP-G(VPGSG)
48
(VPGIG)
48
Y 24 5.6 23.1 3.88 28.4 51.6
A192 G(VPGAG)
192
Y 63.0 73.6
FKBP-A192 (FA) FKBP- G(VPGAG)
192
Y 58.3 8.1 7.9 6.26 66.6 85.4
FKBP-A192-FKBP (FAF) FKBP-G(VPGAG)
192
Y-FKBP 59.0 9.9 9.8 5.13 65.2 97.0
V48 G(VPGVG)
48
Y 43.0 19.7
FKBP-V48 (FV48) FKBP-G(VPGVG)
48
Y 40.9 3.9 3.2 8.50 52.7 31.5
V72 G(VPGVG)
72
Y 29.5
FKBP-V72 (FV72) FKBP- G(VPGVG)
72
Y 33.4 4.8 220 5.61 41.2 41.3
a
FKBP amino acid sequence:
“MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQ
RAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE”.
b
Phase diagrams were fit to T
t
= b – m[Log
10
(Concentration)]
21
Dialysis successfully removes impurity remaining after Inverse Transition Cycling
While characterizing FA peak on RP-HPLC, the chromatogram showed two peaks of
comparable areas under the curve (AUC), with retention times of 4 and 6 minutes. Upon
loading FSI and FAF, once again, two peaks were observed at 4 minutes and 8.5 and
6.5 minutes respectively. The 4-minute peak was observed each time irrespective of
whether the sample for RP-HPLC was diluted in MeOH or PBS (Figure 4a). As the 4-
minute peak remained constant in chromatograms of several FKBP-ELPs (with the later
peak changing retention time in a manner characteristic to each protein), it was
deduced, for the moment, that the later peak corresponded to FKBP-ELP. It was also
deduced that an impurity with significant UV absorbance near 280 nm remained in the
protein solution and could not be removed by inverse transition cycling.
a. b.
Figure 4. a) Chromatograms of FAF, FA and FSI. Two peaks are being detected by the UV
detector of RP-HPLC at 280 nm. The first peak with retention time of 4 minutes is common to
all. The 11-minute peak in the third chromatogram (with FSI) is an unknown trace impurity only
observed with FSI b) SDS-PAGE gel shows only a single band for FSI & FA. (The second faint
band in the three lanes with FSI is the FSI dimer, non-reducing 4x SDS-PAGE buffer was used
to stain the samples) Note: Copper (II) Chloride staining is sensitive for up to 10 ng of protein.
To determine which of the two peaks corresponded to the fusion protein, fractions
corresponding to the two peaks were collected manually during an RP-HPLC run. 5 - 10
µL of each of the fractions were loaded on an SDS-PAGE gel, with FA as a reference
22
(Figure 5a). The later peak was confirmed to correspond to FA. No band was observed
in the lane corresponding to the former peak.
Figure 5. a) Peaks 2 and 3 were collected manually in vials 5 and 7 respectively. b) 5 µL from
vial 7 was loaded on lane 4. Lane 6 was loaded with 10 µg of FA. A band matching the
molecular weight of FA was observed in lane 4.
Lane 7 was loaded with 10 µL of from vial 5. No band was observed on the gel.
Lane 8 was loaded with 10 µL from vial 6. Lane 9 was loaded with 10 µL from vial 8 and lane 10
was loaded with 10 µL from vial 9. No protein band was observed in any of these lanes.
Hence the second peak was confirmed as the FA peak.
The nature of the impurity is not known and it remains to be characterized. It is possible
that the concentration of the impurity is significantly lower than that of the protein.
However, it should be noted that the area under the curve (AUC) in an RP-HPLC
chromatogram is a function of the extent of the UV absorbance of a compound (in this
case, the RP-HPLC detector is a UV detector and samples are analyzed at 280 nm).
The two peaks observed upon loading any FKBP-ELP have comparable AUCs, and this
is a concern because concentration measurements are also made using UV-Vis
spectroscopy at 280 nm. As nearly 40% of the UV-Vis signal is coming from the impurity
(as in case of FKFSI), it is likely that absorbance measurements using UV-Vis for
FKBP-ELPs and subsequent concentration calculations have a maximum error of about
40%. Therefore, removal of this impurity was crucial.
23
Prior to characterization of FSI on RP-HPLC, an observation was made in
chromatograms from pilot dialysis experiments (studying rapamycin release from FSI).
At time t = 0 the 4-minute peak was observed in the chromatogram. This sample was
analyzed immediately before dialysis at 37 ºC. However, when the sample withdrawn at
t = 2 h after dialysis was analyzed, the 4-minute peak, was not observed. Similarly, the
4-minute peak was also not observed in Rapamycin release studies with FA and FAF in
samples analyzed at t = 2h. Based on these observations it seemed likely that the
impurity was being dialyzed out.
Figure 6. Chromatograms from FSI/Rapa drug release study, before and after 2 h of dialysis.
The peak with retention time of four minutes (shown with arrows) is not observed after 2 hours
of dialysis.
To confirm this possibility, 2mL of FSI containing the impurity was dialyzed in 1500 mL
PBS at 37 ºC using 10 kD MWCO dialysis tubing. After 1 hour of dialysis, 100 µL of the
sample was analyzed and it was observed that the 4-min peak had disappeared.
Dialysis was also conducted at 4 ºC for 2 mL FSI. Samples were analyzed at 1h, 2h and
3 h after dialysis was started. By the 3
rd
hour, there was no peak at 4 minutes in the
chromatogram. Concentration of FSI was measured using UV-Vis before dialysis and
3h after dialysis. At 0h concentration was 423 µM whereas after 3h, it decreased to 336
µM.
24
Therefore, it was concluded that presence of the impurity does skew concentration
measurements significantly. All proteins expressed following this finding were dialyzed
at 4 ºC in 1500 mL PBS after 3 rounds of ITC. Also, the purity was ascertained using
SDS-PAGE and RP-HPLC to ensure that the impurity peak was absent. Concentrations
were measured only after dialysis.
Figure 7. a) Chromatograms of FSI before and after 1h of dialysis at 37 ºC. b) Chromatograms
of FSI 1h, 2h and 3h after dialysis at 4 ºC
Thermo-responsive property of ELPs is retained by FKBP-ELP
The characteristic transition temperature of FKBP-ELPs was evaluated by measuring
OD at 350 nm and R
h
with increasing temperature. The behavior and architecture of
FKBP-ELPs was similar to their ELP counterparts. FSI forms nanoparticles similar to
S48I48 as reported previously above their CMT [1]. Therefore, FSI remains as a
nanoparticle at body temperature. As expected, the CMT of FSI (at 24.5 ºC) is slightly
lower than SI (at 27 ºC). The nanoparticle size was observed to be 27 nm consistently
for about 8 hours at 37 ºC, beyond which aggregation was observed. However, this
aggregation may have been precipitated by higher concentrations and presence of
penicillin-streptomycin. Previously, at 25 µM nanoparticles of 27 nm were observed with
greater than 90% intensity at 37 ºC for 24 hours.
25
Figure 8. a) Hydrodynamic radii of FKBP-ELPs with increasing temperature. FA & FAF (not
shown) maintain an average size of 8 nm throughout; FSI reaches critical micellar temperature
at 23 ºC; FV48 phase separates between 39 – 40 ºC; FV72 phase separates between 33 and
34 ºC. The results concur with the T
t
determined from UV-Vis spectroscopy. b) T
t
is directly
proportional to log
10
(concentration). Phase diagram was fit to the following linear regression:
T
t
= c - m[log
10
(concentration)]. r
2
for all evaluated constructs was > 0.99
FA and FAF have high transition temperatures (>60 ºC) and therefore remain soluble at
body temperature. Their molecular mass (85 and 97 kDa, respectively) is well above the
cut-off for renal filtration and are, therefore, expected to circulate in the body for longer
duration. Their particle size was observed to be between 7 – 10 nm consistently (Figure
8a).
FKBP-V48 and FKBP-V72 phase separate close to 37 ºC and can be expected to form
a coacervate at body temperature. Encapsulated with rapamycin, this formulation could
form a depot in the tissue. Hydrodynamic radius was close to 4 nm below transition
temperature for both the constructs (Figure 8a). FSI forms nanoparticles of about 25 nm
above 23 ºC. The CMT observed by DLS was in agreement with the CMT calculated
from UV-Vis temperature ramp (Figure 8a and 8b)
26
Figure 9. a) Transition behavior of FV48 was evaluated in presence of different concentrations
of DMSO. Presence of DMSO increases T
t
of FV48. Increase in Tt is proportional to
concentration of DMSO. b) T
t
of FV72 was determined in presence of 2% DMSO.
For designing Isothermal Titration Calorimetry experiments (that employ 2%
DMSO/PBS to enhance solubility of Rapamycin), it was necessary to know the precise
transition temperature of FV48 & FV72 with 2% DMSO at any given concentration.
These experiments aimed at studying Rapamycin binding coacervates of FV48 & FV72.
Also, the possibility of working with Rapamycin concentrations greater than 15 µM was
evaluated. Concentration greater that 15 µM in PBS could be achieved by increasing
the amount DMSO. As both ligand and the protein have to be dissolved in the same
buffer for ITC, the effect of working with greater than 2% DMSO in FV48 had to be
evaluated (Figure 9a).
27
Figure 10. Change in T
t
of a) FV48 and b) FV72 with respect to the amount of DMSO present in
the solution. There is a linear increase in T
t
of FV48 with increasing concentration of DMSO.
The data was fit to y = mx + c with c = 0.03, m = 1.45 and r
2
= 0.98.
Table 3. Effect of DMSO on T
t
of FV48 and FV72
FKBP-ELP % DMSO Slope, m Intercept, b(ºC)
Average ∆T
t
(ºC) ∆T
t
per 1%
DMSO (ºC)
FV48
0 8.58 52.83 - -
2 10.2 58.85 2.9 1.45
5 10.6 63.94 7.18 1.43
10 10.7 71.73 14.78 1.47
FV72
0 5.59 41.18 -
2 5.61 42.83 1.68 0.84
Phase diagrams were fit to T
t
= c - m[log
10
(concentration)]. r
2
for all evaluated constructs was >
0.99
Addition of DMSO increased transition temperature for FKBP-ELPs. With 2 % DMSO,
an increase in c of nearly 1.6 ºC was observed for FV72 (Figure 10b), i.e., roughly 0.8
ºC increase for every 1% of DMSO. While the slope, m, of the T
t
= c - m[log
10
(conc.)]
equation remains the same for FV72, there is an increase in the intercept c, proportional
to the increase in T
t
at any given concentration. For FV48, a slight increase in slope, m,
was observed. Compared to FV72, the increase in T
t
per 1% increase in DMSO is about
1.45 ºC, nearly twice that of FV72.
Rapamycin is encapsulated efficiently by FKBP-ELPs
The procedure for encapsulating Rapamycin in FKBP-ELPs is relatively straightforward
lasting only a few hours. Concentrations of both Rapamycin and FKBP-ELP can be
determined in a single run of an established RP-HPLC method. Encapsulation efficiency
was determined by measuring concentrations of Rapa and the fusion protein
immediately before beginning dialysis, i.e. at t = 0h. Encapsulation efficiency for the four
monovalent fusion proteins ranged from 75 % in case of FV48, to 85% in case of FSI, to
28
nearly 100% in cases of FV72 and FA (Figure 11). FAF has two FKBP domains capable
of encapsulating drug and should be capable of encapsulating twice more drug per
molecule than the other four constructs. As theorized, encapsulation efficiency of FAF
was found to be nearly 200%, (binding two Rapa molecules per 1 molecule of FAF) .
Figure 11. Fraction of Rapamycin encapsulated by various FKBP-ELPs and controls.
The high encapsulation efficiency can be attributed to the high affinity of Rapamycin to
FKBP (k
d
= 0.2 nM) [48] which has a specific binding site. ELP, by itself in not capable
of forming specific interactions with Rapamycin and therefore does encapsulate any
drug. An exception to this is S48I48, the di-block nanoparticle forming co-polymer,
which can encapsulate a small amount of drug at its hydrophobic core. In comparison,
BSA Fraction V did not encapsulate any detectable drug by the two-phase
encapsulation method.
ELP fusion does not affect the binding affinity of Rapamycin with FKBP-ELP
Previously, ITC studies conducted at 37 ºC showed low nanomolar k
d
for FA, FAF and
FSI (table 2). While the reported k
d
for binding of Rapamycin to FKBP is 0.2 nM, such a
low value of k
d
cannot be detected by ITC as the limit of detection is at 1 nM. Therefore,
the k
d
values observed here cannot be taken at face value. It is possible that with SPR,
we might see values that are closer to value reported in literature. In any case, it can be
concluded that FKBP-ELPs, irrespective of the nature of the ELP, bind Rapamycin with
high affinity (Table 4).
29
Comparing the binding isotherms of FA, FSI and FAF (Figure 12), one can clearly see
that in case of FAF/Rapa, saturation is reached at a molar ratio 0f 0.6, which is roughly
half of the molar ratio for FSI or FA for reaching saturation of binding sites. As FAF has
two FKBP domains, each molecule of FAF can bind two molecules of Rapamycin, and
the ITC data is indicative of this. The signature plots (Figure 13) for Rapamycin binding
FSI, FA and FAF indicate that Rapamycin binding FKBP-ELP is an exothermic event
driven by enthalpy.
Table 4. Binding parameters of FKBP-ELP to Rapamycin.
FKBP
-ELP
Binding
Stoichiometry
(n)
Dissociation
Constant (nM)
Enthalpy of
binding (KJ/mol)
Free Energy of
Binding (KJ/mol)
-T∆S
FSI 1.38 ± 0.01 11.9 ± 4.11 -40.8 ± 2.70 -47.1 -6.31
FA 1.16 ± 0.003 8.46 ± 2.17 -55.1 ± 0.72 -48.0 7.11
FAF 0.58 ± 0.002* 2.88 ± 0.4 -102 ± 0.786 -50.7 51.5
* 1 molecule of Rapa binds half a molecule of FAF, in other words, 2 molecules of Rapa will be
needed to bind 1 molecule of FAF
Figure 12. Raw data and biding isotherm for a) FSI/Rapa, b) FA/Rapa and c) FAF/Rapa. Once
again, the evidence of bivalency of FAF is visible. Experiment was carried out at 37 ºC.
30
Concentration of FSI and FA used was 80 µM, while for FAF, 40 µM of the protein was used.
Concentration of Rapamycin was at 8 µM in all the cases. Figures b) and c) are the work of
graduate student Santosh Peddi.
Figure 13. Signature plots of ∆G (in blue), ∆H (in green) and -T∆S (in red) for a) FSI/Rapa; b)
FA/Rapa; c) FAF/Rapa. The increase in Entropy for FSI may have to do with nanoparticle
formation inside the cell. For the three fusion proteins, rapamycin binding is enthalpy driven
which is indicative of formation of H-bonds or other polar interactions. Rapamycin is known to
form four H bonds with FKBP.
FKBP-ELPs display extended stability and drug retention
The release of rapamycin from FKBP-ELP was studied by measuring the concentrations
of Rapamycin bound to the fusion protein inside the dialysis cassette. Data was plotted
as the fraction of Rapamycin bound to FKBP-ELP (concentration of Rapamycin/
Concentration of the fusion protein) with respect to time. Only unbound Rapamycin that
had been released from the fusion protein can dialyze out of cassette. It was expected
that as rapamycin is released over time, this fraction would go down.
31
Figure 14. a) Non-linear One-phase decay of Rapamycin from FKBP-S48I48. b) Fraction of
Rapa bound to FKBP-A192 with respect to time. FA displayed extended drug retention, as no
significant release was observed for a period of two weeks. c) fraction of Rapamycin bound to
FAF. Similar to FA, FAF displayed extended drug retention. d) particle size analysis of FSI with
respect to time. Aggregation was observed beyond 14 hours. e & f) particle size analysis for FA
and FAF show that particle size remains under 10 nm for the duration of the experiment with no
sign of aggregation.
However, it was observed that the fraction of rapamycin bound remained more or less
unchanged for the entire duration of the experiment. In case of FSI, the experiment was
carried out for 14 h only, because beyond this time particle size analysis showed signs
of aggregation, with R
h
nearing 200 nm. For the duration of the experiment, the ratio of
concentration of Rapa to concentration of FSI varied from 0.85 at t = 0h to 0.76 at t =
14h (Figure 14a). When the data was plotted for non-linear one phase decay, a half-life
of 200 h was calculated. For the duration of the experiment, particle size of FSI
maintained a size of nearly 27 nm (Figure 14d).
In case of FAF, previous dialysis studies had indicated a half-life of nearly 2000 h.
Therefore, the experiment was designed to study drug release over a period of a month.
However, fraction of rapamycin bound to FAF remained nearly unchanged, going from
2.16 at t = 0h to 2.12 at t = 768 h (Figure 14c). A half-life could not be ascertained as
32
there was no decay. Particle analysis at each time interval gave an average particle size
of 6.4 nm indicating that there was no precipitation during the experiment (Figure 14e).
Similar results were obtained with FA, albeit with lower drug loading than FAF. The
experiment was carried out for two weeks as prior studies had indicated a release half-
life of 500 h. Once again, fraction of rapamycin bound varied between 0.97 and 0.81
over a period of two weeks (Figure 14b).
Phase of ELP has no effect on release rate of Rapamycin from FKBP-V48 & FKBP-
V72
FV48 and FV72 are expected to form a coacervate at body temperature. It was
theorized that at higher temperatures as FV48 and FV72 phase separate, they would
hold on to the drug for a relatively longer time and release drug slowly, that at lower
temperatures, when they are soluble. Experiments were designed to test this. Dynamic
dialysis experiments were conducted at RT and 37 ºC for FV72 and FV48.
Figure 15. a) Fraction of Rapamycin bound to FV48 with respect to time at 37 ºC. At
concentrations in the vicinity of 200 µM, FV48 forms a coacevate b) Fraction of Rapamycin
bound to FV48 with respect to time at Room temperature. Extended drug retention by FV48 was
observed irrespective of whether it was a coacervate or soluble.
At 37 degrees, ratio of drug to FKBP-V48 at t = 0h was 0.75, which goes down to about
0.65 by t = 156 h. The data was fit to non-linear regression one phase decay and a half-
33
life of about 1911 h was calculated (Figure 15a). At room temperature however, the
fraction of rapamycin bound varies from 0.77 at t = 0h to 168 h (Figure 15b). The data
could not be fit to any non-linear regression as no decay was observed, much like the
results from the Rapa release studies carried out with FA & FAF.
Similar experiments were conducted with FKBP-V72. The fraction of rapamycin bound
to FKBP-V72 prior to beginning dialysis was at an average of 1.18. After 144 h of
dialysis, FV72 was found to retain an average of 1.08 rapamycin molecule per molecule
of FV at 37 ºC and 0.99 molecule of rapamycin per molecule of FV at RT (Figure 16a
and 16b). It seems that release of rapamycin from various FKBP-ELPs (for monoblock
ELPs) is neither affected by the nature of the ELP or the temperature. This is in part due
to the high affinity of Rapamycin for FKBP and in part due to the poor solubility of
Rapamycin in PBS.
Figure 16. a) Fraction of Rapa bound to FV72 with respect to time at 37 ºC b) Fraction of Rapa
bound to FV72 at room temperature.
For each time interval, samples withdrawn were also evaluated for their particle size to
ensure that fusion proteins were stable for the entirety of the experiment and to also see
their behavior in the dialysis tubing. For the study being carried out at 37 ºC, the phase
separated FV72 had a particle size in the vicinity of 900 nm (Figure 17a). Whereas the
for the RT study, samples showed a mean particle size of 4.5 nm, as expected (Figure
17b).
34
Figure 17. a) Particle size analysis of FV72/Rapa at 37 ºC. Hydrodynamic radius was greater
than 600 nm at all times due to coacervation. b) Particle size analysis of FV72/Rapa at RT. In
this case particle size remains below 5 nm at all times. No signs of aggregation were observed.
Phase separation of FKBP-ELP reduces the binding affinity of Rapamycin to
FKBP
ITC was used to determine how the binding affinity would differ when Rapamycin binds
a phase separated fusion protein (i.e. in its coacervate form) as opposed to a soluble
fusion protein. The ITC cell contains 280 µL of one of the binding entities while the
syringe contains a maximum of 40 µL of the second binding entity. To achieve a binding
isotherm where saturation is reached halfway through the injections (assuming a
binding stoichiometry of 1:1), concentration of the molecule in the syringe has to be at
lease 10-fold higher than the concentration of the molecule in the cell. Due to the
limiting solubility of Rapamycin in PBS, 2% DMSO had to be used to achieve
Rapamycin concentrations in the range of 8 – 15 µM. Therefore, Rapamycin was taken
in the cell, and the fusion protein was taken in the syringe.
At room temperature, the binding parameters observed for both FV48 & FV72 were
similar to those of FSI, FA and FAF (Table 5). Binding stoichiometry remained close to
1, and the dissociation constant was in the low nanomolar range. Also, judging by the
signature plots of enthalpy and entropy, the binding was exothermic in nature (Figures
19 & 21). When a coacervate’s binding to Rapamycin had to be studied, presence of 2
35
% DMSO presented a complication, as it increased the transition temperature at any
given concentration by nearly 1.6 ºC.
Following the second injection of 3 µL of 80 µM FV48 or FV72, the concentration of the
fusion protein in cell (which has a volume of 280 µL) was close to 1 µM. Neither FV72
nor FV48 would form a coacervate at 1 µM at 37 ºC. This explains why k
d
values (or any
of the thermodynamic parameters) do not change appreciably when the experiment is
done at RT or at 37 ºC.
Therefore, several changes had to be made to ensure that the ELP fusion protein would
remain a coacervate following the second injection (because the first injection of 0.4 µL
is ignored in calculations). Concentration of the fusion protein in syringe was increased
to 300 µM; volume of fusion protein injected in the cell per injection was increased to 4
µL; Rapamycin’s concentration in the cell was increased to 15 µM. With these settings,
following the second injection, concentration of FV72 in cell would be about 4.4 µM. The
transition temperature at this concentration is 39.2 ºC. Therefore, run temperature of
ITC was increased to 40 ºC (to take into account errors in calculating concentration or
pipetting errors) (Figure 18b).
With these settings, a k
d
of nearly 300 nM was observed, consistently. For all the runs
binding stoichiometry remained close to 1. Also, judging by the signature plot (Figure
19b), binding was enthalpy driven. The data also shows that there is a significant loss of
entropy and binding is favorable due to the high negative ∆H.
Table 5. Binding Parameters for FKBP-V48 & FKBP-V72 at various temperature
FKBP
-ELP
T (ºC) Binding
Stoichiometry
(n)
Dissociation
Constant (nM)
Enthalpy of
binding
(KJ/mol)
Free Energy
of Binding
(KJ/mol)
-T∆S Phase
FV48 15 0.91 ± 0.003 3.14 ± 0.71 -70.6 ± 0.622 -48.6 22.0 Soluble
FV48 37 0.93 ± 0.003 4.4 ± 1.36 -65.0 ± 0.488 -49.6 15.4 Soluble
FV48 52 1.07 ± 0.02 109 ± 79.4 -63.4 ± 2.81 -43.3 20.9 coacervate
36
FV72 15 0.67 ± 0.005 2.38 ± 1.16 - 58.2 ± 0.95 -47.6 10.6 Soluble
FV72 37 1.16 ± 0.001 20.5 ± 7.09 -61.3 ± 1.9 -45.7 15.6 Soluble
FV72 40 0.953 ± 0.005 351 ± 27.8 -114 ± 1.08 -38.7 75.0 coacervate
Figure 18. Raw data & binding isotherm for FV72 at a) Room Temperature and b) 40 ºC. Due to
the limitations of total injection volume of the syringe, experiment had to be restricted to 10
injections of 4 µL each.
37
Figure 19. Signature plots of ∆G (in blue), ∆H (in green) and -T∆S (in red) for FV72 at a) RT and
b) at 40 ºC.
The same experimental conditions did not work well with FV48 (Figure 20b). For 4 µM,
the transition temperature of FV48 is above 50 degrees. Because there is an upper limit
on the total volume that the ITC injector can take in at once (40 µL), injection volume
could not be exceeded beyond 4µL per injection. While running the experiment at 52 ºC,
raw data read by the instrument could not be resolved, and it is possible that the
components in the cell had begun to degrade (Figure 20b). At room temperature
however, binding stoichiometry was close to 1 and the k
d
was close to 3nM (Table 5).
38
Figure 20. Raw data & binding isotherm for FV48 at a) Room Temperature and b) 52 ºC. It is
likely that at 52 ºC, the protein begins to unfold.
Figure 21. Signature plots of ∆G (in blue), ∆H (in green) and -T∆S (in red) for FV48 at a) room
temperature and b) 52 ºC. Rapamycin binding is an exothermic process in both instances.
39
Conclusion
ELPs are polypeptides of the general sequence G(VPGXG)
n
Y
derived from tropoelastin,
the soluble precursor of elastin. They have the incredible ability to undergo reversible
hydrophobic collapse upon reaching a temperature characteristic to their sequence. A
range of ELPs can be genetically engineered by varying the chain length n and/or the
guest residue Y to create a library of ELPs with varying characteristics.
In this manuscript, we have studied five FKBP-ELP fusion proteins for enhancing the
delivery of Rapamycin and compared how ELP architecture affects the rate at which
they release the drug. Fusing the FKBP-12 domain to ELPs does not alter their ability
to undergo reversible phase separation. At physiologically relevant temperatures, FA
and FAF remain soluble, FSI forms stable nanoparticles whereas FV48 and FV72 form
a coacervate. Rapamycin has a high binding affinity to FKBP-ELP fusion proteins,
irrespective of the nature of the ELP. The binding is exothermic and indicative of
formation of strong polar interactions such as hydrogen-bonds. The drug can be
encapsulated efficiently through a simple two-phase encapsulation method, with a high
encapsulation efficiency. Four of the five fusion proteins – FAF, FA, FV48 and FV72
displayed extended drug release, with little release being detected over several days.
Particle size analysis using DLS also showed extended stability for all four fusion
proteins. Drug release is not affected by the phase of the ELP. FV48 and FV72, whether
soluble or coacervate, retained the bound drug in a similar manner.
Initially it was assumed that Rapamycin would release from FKBP-ELPs and the free
drug would exert its effect. However, data from the release experiments indicate that
this may not be the case, because Rapamycin appears to remain bound almost
indefinitely. Anti-tumor efficacy has been reported with Rapamycin bound FSI in breast
cancer xenografts in mice
[1]
, meaning Rapamycin from the fusion protein is certainly
reaching its target inside the cell. Additionally, internalization of S48I48 in murine
hepatocytes followed by sequestration in lysosomes has been reported
[54]
. Therefore, it
40
is likely that Rapamycin bound FKBP-ELPs are being internalized by the cell. This
requires further investigation. If the fusion protein carrying rapamycin is, indeed, being
internalized, it would be interesting to determine whether FKBP-ELP-Rapamycin
escapes the lysosomal compartments and interacts with the FRB domain of mTOR
directly, or if it is degraded in the lysosomal compartments eventually releasing the free
drug. It would also be interesting to determine whether the internalization is receptor
mediated or simply fluid-phase endocytosis.
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Abstract (if available)
Abstract
Traditional anti-cancer drugs are cytotoxic in nature and cause severe side-effects in patients. Additionally, several of them lack tumor specificity, are difficult to formulate, show high variability in pharmacokinetics among individuals, have shown to develop drug resistance over time, and can even contribute to tumor recurrence. Many of these challenged can be counteracted by nanomedicine, which can also have the added benefit of tumor targeting. ❧ Rapamycin is a cytostatic drug that arrests a dividing cell in its G1 phase and has shown anti-cancer activity in several cancer cell lines. Being a cytostatic it is relatively less toxic than conventional chemotherapy. Two of its analogues, Temsirolimus and Everolimus, are in the market for the treatment of a range of tumors. However, the extent of Rapamycin’s clinical potential (and that of its analogs) is marred by its lung toxicity, adverse effects such as myalgia, sepsis, stomatitis, leukopenia, etc., unfavorable physicochemical properties, low oral bioavailability and inter-individual variability in drug disposition. ❧ Rapamycin’s aqueous solubility can be enhanced significantly by encapsulating it with its cytosolic partner FKBP-12. As FKBP-Rapamycin would be cleared relatively quickly through renal filtration owing to the 20kDa molecular weight of FKBP, we have developed FKBP-ELP fusion proteins that would have longer circulation time in plasma (for ELP fusion proteins with molecular weight > 45 kDa) or provide passive targeting through the EPR effect (for nanoparticle forming ELPs). A high drug loading efficiency was achieved irrespective of the ELP fused to FKBP. We have also studied how the attachment of different ELPs affects the release rate of Rapamycin from an ELP fusion protein. Of the five ELP fusion proteins studied all four of the monoblock ELPs show extended rapamycin retention with little to no release over a time period of several days. Drug release is also not affected by temperature or the phase of the thermo-responsive ELPs as both coacervate and soluble fusion proteins displayed similar drug retention. This indicates that rapamycin encapsulated fusion proteins may not release the drug at all. Therefore, it remains to be investigated if Rapamycin encapsulated fusion proteins can be internalized by a tumor cell and if that is the way this formulation exerts the anti-cancer activity that has been reported previously.
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Asset Metadata
Creator
Singh Kakan, Shruti
(author)
Core Title
Comparing drug release kinetics of rapamycin bound FKBP-ELP fusion proteins
School
School of Pharmacy
Degree
Master of Science
Degree Program
Pharmaceutical Sciences
Publication Date
08/08/2017
Defense Date
08/07/2017
Publisher
University of Southern California
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Tag
cancer,elastin like polypeptides,OAI-PMH Harvest
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English
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Electronically uploaded by the author
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Mackay, John Andrew (
committee chair
), Haworth, Ian S. (
committee member
), Okamoto, Curtis Toshio (
committee member
)
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shruti.singh.222@gmail.com,singhkak@usc.edu
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