University of Southern California Dissertations and Theses

Elastin like polypeptides as thermoresponsive modulators in reversible bioadhesives for fragile skin

(USC Thesis Other)

Elastin like polypeptides as thermoresponsive modulators in reversible bioadhesives for fragile skin

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

ELASTIN LIKE POLYPEPTIDES AS THERMORESPONSIVE MODULATORS IN
REVERSIBLE BIOADHESIVES
FOR FRAGILE SKIN
by
Vinit Gholap

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 2012

Copyright 2012 Vinit Gholap
ii

ACKNOWLEDGEMENTS
I would like to thank my mentor Dr. Andrew MacKay for such a wonderful opportunity to
work with Alfred Mann Institute at USC on such an innovative research project.
I am grateful to him for all his guidance and his encouragement throughout this project.
I would also like to thank Dr. Mark Thompson and Dr. Cesar Blanco for their valuable
advice and guidance on this project. My special thanks also go to Dr. Manuel Orosco,
Dr. Xiang Yu and Allan Kershaw for helping me out in learning various techniques.
Additionally, I would like to thank my colleagues from Dr. Andrew MacKay’s lab,
in particular Dr. Josh Gustafson, Martha Pastuszka, Suhaas Aluri, Siti Janib, Pu Shi, Mihir
Shah and Wan Wang for their cordial and generous support.
I would also like to thank my committee members Dr. Curtis Okamoto and Dr. Bogdan
Olenyuk for their advice and time they spent on reviewing my thesis.
Finally, I would like to thank USC School of Pharmacy as well as Alfred Mann Institute
for their support to this project.

iii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS
LIST OF TABLES
LIST OF FIGURES
ABBREVIATIONS
ABSTRACT
CHAPTER 1: INTRODUCTION AND BACKGROUND
1.1 Fragile Skin and Reversible Bio-adhesives
1.2 Pressure Sensitive Adhesives (PSA)
1.2.1 Acrylate Pressure Sensitive Adhesives
1.2.2 Polymerization Mechanism
1.2.3 Polymerization Techniques
1.3 Elastin Like Polypeptides (ELPs)
1.4 Characterization of Polymers: Thermal Analysis
1.5 Instron Material Testing
1.5.1 Tensile Test
1.5.2 T-Peel Test
1.5.3 Shear Test.
1.6 Specific Aims

ii
v
vi
viii
x

1
1
2
2
3
4
6
8
10
10
11
12
13

iv

CHAPTER 2: MATERIALS AND METHODS
2.1 Materials
2.2 Synthesis of Acrylate Pressure Sensitive Adhesives by Emulsion
Polymerization
2.3 ELPs Purification by ITC
2.4 ELPs Transition Temperature Determination by UV Spectrophotometer
2.5 SDS-PAGE
2.6 Matrix Assisted Laser Desorption/Ionization (MALDI) Mass
Spectrometry
2.7 PSA-ELP Conjugation
2.8 PSA-ELP Film Preparation
2.9 Differential Scanning Calorimetry
2.10 Instron Tensile Strength Testing

CHAPTER 3: RESULTS
3.1 Confirmation of Purity and Molecular Weight of ELPs and PSA using
SDS-PAGE and Mass Spectrometry.
3.2 Determination of T
t
of ELPs using UV-Spectrophotometer
3.4 PSA-ELP Conjugation Confirmation using FTIR-ATR
3.5 Thermal Analysis of PSA and ELP Films using Differential Scanning
Calorimetry
3.6 Measurement of Adhesive Strength as Function of Temperature using
Instron Tensile Strength Test
CHAPTER 4: DISCUSSION
CHAPTER 5: FUTURE DIRECTIONS
REFERENCES
14
14
15
16
17
17

18
18
19
20
21
23

23
26
28
29
34
38
42
44

v

LIST OF TABLES
Table 1: Formulation of pressure sensitive adhesive
Table 2: PSA-ELP film preparation
Table 3: ELP coacervate film preparation
Table 4: Summary of observed and calculated molecular weights of ELPs
Table 5: Summary of T
t
of ELP V
48
and ELP F
12
A
12

15
19
20
24
26

vi

LIST OF FIGURES
Figure 1: Acrylate monomers of pressure sensitive adhesives
Figure 2: Reaction mechanism of acrylate chain polymerization
Figure 3: Schematic representation of emulsion polymerization
Figure 4: The ELP phase transition
Figure 5: Reversible phase transitions of ELPs
Figure 6: Schematic representation of tensile strength test
Figure 7: Schematic representation of T-peel test
Figure 8: Schematic representation of lap shear test
Figure 9: Schematic representation of Instron tensile strength test
Figure 10: SDS-PAGE of ELP V
48
and F
12
A
12
Figure 11: Mass Spectrum of PSA polymer
Figure 12: Phase transition profile of ELP F
12
A
12
Figure 13: Phase transition profile of ELP V
48
Figure 14: FTIR spectrum of ELP F
12
A
12
Figure 15: FTIR spectrum of conjugated ELP F
12
A
12

3

4
5
7
8
11
12
13
22
24
25
26
27
28
29

vii

Figure 16: DSC thermogram of ‘PSA Only’ film
Figure 17: DSC thermogram of V48 films (unhydrated)
Figure 18: DSC thermogram of F
12
A
12
film (unhydrated)
Figure 19: DSC thermogram of V
48
film (0.5%w/w hydrated)
Figure 20: DSC thermogram of F
12
A
12
film (0.5% w/w hydrated)
Figure 21: Tensile strength of PSA+ELP films
Figure 22: Tensile strength of ‘PSA Only’ films
Figure 23: Tensile strength of Band Aid®
Figure 24: Tensile strength of F
12
A
12
coacervate films (0.5%w/w hydrated)
Figure 25: Tensile strength of V
48
coacervate films (0.5%w/w hydrated)
Figure 26: Proposed explanation

30
31
32
33
34
35
35
36
37
37
40

viii

ABBREVIATIONS
ASTM American Society for Testing and Materials
CHCA α-Cyano-4-hydroxycinnamic acid
DCC N,N’-Dicyclohexylcarbodiimide
DIPEA Di- isopropyl Ethyl Amine
DSC Differential Scanning Calorimetry
E.Coli Escherichia Coli
EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
EHA
2-ethyl hexyl acrylate
ELPs Elastin Like Polypeptides
FTIR-ATR
Fourier Transform Infrared Spectroscopy- Attenuated
Total Reflectance
ITC Inverse Transition Cycling
kDa Kilo Dalton
LCST Lower Critical Solution Temperature
MALDI Matrix Assisted Laser Desorption/Ionization
NHS N-hydroxy succinamide
PEI Polyethylenimine
PET Polyethylene terephthalate
PSA Pressure Sensitive Adhesive
SDS-PAGE
Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis
T
t
Transition Temperature

ix

T
g
Glass Transition Temperature
TB Terrific Broth
V
48
(VPGVG)
48
Y
F
12
A
12
(VPGFG)
12
(VPGAG)
12
Y

x

ABSTRACT
Adhesive bandages are widely used for wound healing treatment. However, use of these
bandages on fragile skin can aggravate healing problems as fragile skin is susceptible to
break while changing wound dressings. To overcome this difficulty, a new approach has
been proposed to develop temperature sensitive bioadhesive which will decrease in adhesion
below skin temperature (34
0
C). In the present study, acrylate pressure sensitive adhesive
(PSA) polymer has been used as a primary component for adhesives. To modulate its
adhesiveness with respect to temperature, LCST polymers of protein nature possessing
characteristic transition temperature (T
t
) have been used in conjunction to observe the
cumulative effect on adhesion below T
t
. Additionally, these protein biopolymers are also
studied without conjunction with PSA for their adhesive strength with respect to their T
t
.
The final section discusses the preliminary progress as well as provides future directions for
developing temperature sensitive bioadhesives for fragile skin.

1

CHAPTER 1
INTRODUCTION AND BACKGROUND
1.1 Fragile Skin and Reversible Bioadhesives:
Skin care treatment in patients with fragile skin has been a growing concern throughout
the world. There has been increasing demand among patients with fragile skin for
improved treatment to relieve pain. Such patients often include burn victims, patients
with pressure ulcers, deep surgical wounds and nevertheless patients with rare genetic
conditions such as epidermolysis bullosa caused by defective keratin KRT14/KRT5
genes(Coulombe et al 2009). One of the primary issues in such skin care treatment is
difficulties in use of adhesive dressings. Although most of the adhesive dressings or
patches are strong enough to rip off the fragile skin while changing the dressings, there
are very limited techniques currently being used to minimize such skin trauma. Some of
them include use of proxy adhesive surface and adhesive removal solvents such as
detachol(Winton & Salasche 1985). However, these procedures are time consuming,
inefficient and not patient friendly. Therefore a novel approach has been proposed to
develop reversible bioadhesive with temperature dependent adhesiveness. Such
bioadhesive would provide more effective and pain-free treatment for patients with
fragile skin. Here the initial progress of this project has been reported along with
discussion and future direction.

2

1.2 Pressure Sensitive Adhesives (PSA)(Brockmann & Huther 1996, Satas 1989):
Pressure sensitive adhesives as name suggests have a tendency to adhere to substrate
when pressure is applied. These pressure sensitive adhesives form weak bonds with
substrate which are primarily physical in nature. Therefore they can be easily removed
from the substrate without significant damage. These pressure sensitive adhesives are
classified on the basis of their physical form or their chemical composition. In the earlier
period most of the pressure sensitive adhesives are made of natural rubber. However,
today, these natural rubber polymers are have been replaced by synthetic polymers such
as polyacrylates, polyvinyl ethers and silicone based pressure sensitive adhesives. One of
such synthetic pressure sensitive adhesive which is widely used for skin care treatment is
acrylate pressure sensitive adhesive polymer. Acrylate pressure sensitive adhesives are
transparent, colorless, resistant to oxidation and do not change to yellow color in sunlight.
The following section describes their chemistry, mechanism of action and uses.

1.2.1 Acrylate Pressure Sensitive Adhesives:
The most commonly used pressure sensitive adhesives for skin care treatment are acrylate
pressure sensitive adhesives(Satas 1989). These synthetic adhesives can adhere to skin
instantaneously when pressure is applied. Therefore these adhesives are called as pressure
sensitive adhesives(Brockmann & Huther 1996). These acrylate adhesives are acrylic
esters which yield soft and tacky polymers possessing low glass transition temperature
(T
g
)(Satas 1989). The mechanism by which these polymers adhere to skin is by forming

3

physical bonds such as dipole induction, dipole-dipole forces, hydrogen bonds and Van
der Waal’s forces(Brockmann & Huther 1996, Gay 2002, Satas 1989). These adhesion
bonding and polymer flow properties depend upon type of monomer used for synthesis of
PSA. Three different types of monomers are generally used for acrylate pressure sensitive
adhesives for skin care treatment. They are 2-ethyl hexyl acrylate, butyl acrylate and
iso-octyl acrylate (Fig.1). However, the most frequently used monomers are 2-ethyl
hexyl acrylate and butyl acrylate due to its easy availability and better polymer
characteristics(Satas 1989). In the present study, 2-ethyl hexyl acrylate has been used as a
monomer of choice.

2-ethyl hexyl acrylate butyl acrylate iso-octyl acrylate

Fig.1: Acrylate monomers of pressure sensitive adhesives

1.2.2 Polymerization Mechanism(Odian 2004):
Polymerization in general can be classified in to step polymerization and chain
polymerization. Step polymerization, as name indicates, proceeds stepwise such as
monomer reacts monomer to form dimer. Dimer reacts with monomer or dimer to form

4

trimer or tetramer and so on. However, in case of chain polymerization, initiator is added
in to the reaction mixture which forms reactive species such as free radicals. Further
polymerization occurs by chain elongation of reactive species by addition of monomers
present in the mixture. This polymerization is also known as free radical polymerization.
Since acrylate monomers possess vinyl groups at one end, they follow free radical
polymerization by generating reactive α-carbons which further attack β-carbons of other
monomers and thus the chain polymerization propagates (Fig.2).

Fig.2: Reaction mechanism of acrylate chain polymerization(Odian 2004).

1.2.3 Polymerization Techniques(Odian 2004, Satas 1989):
Polymerization techniques for acrylate polymerization can be broadly classified as
solution polymerization and emulsion polymerization. Solution polymerization, as name
indicates, is a free radical polymerization in homogenous solution. If the polymer is
insoluble in the solvent, then such solution polymerization is termed as precipitation
polymerization(Satas 1989).

5

On the other hand, emulsion polymerization is a technique in which water insoluble
monomers are dispersed as small droplets. Emulsifiers are used to stabilize small droplets
of monomers as well as to form micelles. Some monomers tend to dissolve in micelles.
Almost 99% of particulate nucleation of chain polymerization occurs in micelles.
As chain polymerization continues, monomers from monomer droplets diffuse in to
micelles. (Fig.3)

Fig.3: Schematic representation of emulsion polymerization.

At the end of reaction, emulsion polymerization yields polymeric domains of high
molecular weight, low branching and with low residual monomer. In addition to that,
emulsion polymerization reaction, unlike solution polymerization, can be controlled by
changing its parameters.

6

Nevertheless, high polymer yield, low cost and high polymerization rate make emulsion
polymerization a preferred technique in the current study of reversible bioadhesives.
1.3 Elastin Like Polypeptides (ELPs):
Elastin Like Polypeptides are protein biopolymers with pentameric repeat sequence of
(VPG‘X’G)
n
where ‘X’ is any amino acid and ‘n’ denotes number of repeats. ELPs are
derived from human tropoelastin sequence. These ELPs have characteristic phase
transition temperature (T
t
) below which they are soluble in water and above which they
form insoluble amorphous state (Fig.4). Unlike other proteins, this transition is reversible.
It is also called as inverse transition temperature as unlike other proteins which lose their
structure at higher temperature, ELPs become more ordered above their T
t
resulting in
contraction of the polymer (Banta et al 2010, Urry 1997). On lowering the temperature
below T
t
, ELPs become less ordered resulting in relaxation of polymer. Such contraction
and relaxation of ELPs represent a mechanical model of energy transfer (Urry 1997). To
summarize, ELPs possess a characteristic inverse transition temperature which governs
their hydrophobic folding based on the principles of free energy transduction (Cirulis &
Keeley 2010, Urry 1997).

Transition temperature (T
t
) of ELPs can be modified by varying the guest residue ‘X’ in
the (VPG‘X’G) pentameric sequence. T
t
is also a function of concentration of ELPs,
length of ELPs ,pH of the medium and salt concentration(Dan E. Meyer 2002).

7

Considering this unique property, ELPs are used in this study to develop such
temperature dependent switch in PSA adhesiveness.

Fig.4: The ELP phase transition. (VPGIG)
48
Y, changes from a soluble state to an
insoluble state around a transition temperature (T
t
). (Image Courtesy: Siti M. Janib)

As mentioned earlier, ELPs undergo hydrophobic folding above T
t
excluding water inside
their pockets. If this insoluble amorphous state of ELPs is allowed to settle down slowly,
these ELPs form coacervate state (Fig.5). Coacervate state can be defined as a protein
enriched state with glue like consistency containing about 63% w/w of water(Urry 1997,
Urry et al 1985). Such coacervate state is reversible and the polymer returns to solution
phase when temperature is lowered below T
t
.

8

Fig.5: Reversible phase transitions of ELPs. As shown in the figure, ELPs when allowed
to settle down above T
t
, slowly goes in to coacervate state.

These ELP coacervates are tacky in nature and possess adhesive properties. The present
study also includes a determination of the adhesive strength of pure ELP coacervates as a
function of T
t
using tensile strength test.

These ELPs can be synthesized by either solid phase peptide synthesis or by genetic
recombination technique. Due to limits on chain elongation and stereospecificty, solid
phase peptide synthesis technique has been surpassed by genetic recombination technique
where ELPs are synthesized using Recursive Directional Ligation by (RDL) method in
E.coli bacteria(Chilkoti et al 2002). Plasmid constructs are transformed in E.coli cells and
ELPs are purified from bacterial culture using Inverse Transition Cycling (Dan E. Meyer
2002).

9

1.4 Characterization of Polymers: Thermal Analysis
Both PSA polymer and ELPs are characterized for their thermal transition behavior using
Differential Scanning Calorimetry (DSC). DSC is a widely used technique to study
thermally induced phase transitions in materials. With advances in DSC technologies,
precise information about transition temperature, enthalpy of transition and a type of
transition such as endothermic or exothermic reaction can be obtained. Additionally,
DSC is immensely useful in determining glass transition temperature (T
g
) of synthetic as
well as protein polymers.

Different phase transitions of ELPs are effectively studied using DSC. ELPs in water at a
temperature above T
t
, is an endothermic system. In addition to ‘soluble to insoluble’
phase transition, ELPs show crystalline phase at higher temperature. This phase is
exothermic in nature. Thermal degradation of peptides above crystallization phase can be
identified by distinct endothermic process (Hu et al 2007, Luan et al 1990, Rodriguez-
Cabello et al 2000).

In addition to that, ELPs in lyophilized condition can be studied using DSC to determine
their T
g
. However, in many cases, proteins possess weaker glass transition (T
g
) which
makes it difficult to detect by conventional DSC. Such weak transitions can be magnified
using low amount of sample and Hyper DSC technique where samples are subjected to
high scan rates maintaining precision(Katayama et al 2008).

10

In the present study, both conventional and hyper DSC techniques are used to study
different transitions of synthetic PSA polymer as well as ELPs.

1.5 Instron Material Testing (Kull et al 2009, Petrie 2000, Portelli 1986):
To evaluate strength and mechanical behavior of adhesive materials, mechanical testing
is considered as a primary criterion of their evaluation. In order to test the adhesion
strength of polymers, numerous standard tests have been developed. Evaluation and
material testing is very complex depending upon the condition in which tests are carried
out. In the present study, three major tests developed and published by American Society
for Testing and Materials (ASTM) have been used to test adhesion strength of PSA
polymers and ELPs. These tests are reliable and give important information about the
nature of adhesive joints, mechanism of adhesion and ways to modify adhesion strength
of material.

1.5.1: Tensile Strength Test:
Tensile strength test provides important information about the maximum load the
adhesive material can withstand without fracture or cohesive failure when being
stretched. By definition, tensile strength is a maximum load, P
c
,at fracture by cross
sectional area, A, of the bond(Petrie 2000).

11

The tensile strength is measured in pounds per square inch or newton per square
centimeter (N/cm
2
). The most commonly used model for tensile strength measurement is
a sandwich model where tensile strength of adhesive bonds between skin and adhesive
film is tested using T shaped metal surfaces as shown in Fig 6.

Fig.6: Schematic representation of tensile strength test.

1.5.2: T-Peel Test:
Peel test involves measurement of force required to peel or strip away flexible adherent
from either rigid or flexible surface such as skin. In other words it measures the resistance
of adhesive films to striping force. Most commonly used peel test is T-peel test (Fig.7).
Usually, adhesive films are peeled at an angle of 90 or 180 degrees(Petrie 2000).
Peel values are recorded in either pounds per inch of width (piw) or newton per
centimeter (N/cm). Although peel test is a good qualitative estimation of adhesive

12

strength, it gives the most fluctuating results out of rest of the adhesion tests due to very
small area on which stress is applied and due to angle of peeling if not maintained
precisely. Therefore identical test conditions and sample preparation techniques need to
be maintained for each T- peel test.

Fig.7: Schematic representation of T-Peel test.

1.5.3: Lap Shear Test:
Shear test involves application of force in the plane of adhesive layer on the adhesively
bonded structure. The applied force tends to slide two bonded surfaces resulting in shear
between two surfaces. Lap shear test is one of the shear tests which involves adhesively
bonded structure of two surfaces with certain length of overlap as shown in Fig.8. The
results are recorded in newton (N).

13

Fig.8: Schematic representation of lap shear test

1.6 Specific Aims:
Since the primary objective of this project is to develop reversible bioadhesive with
temperature dependent adhesiveness, ELPs were used as temperature dependent switch in
PSA adhesiveness. To conjugate ELPs to PSA, highly charged (carboxyl groups) PSA
was synthesized which would also possess high water holding capacity. The idea was
based on the hypothesis where based on the residual amount of water in PSA film, ELPs
in the film would go back to soluble state below T
t
which would eventually lead to
weakening of physical bonds causing decrease in adhesive strength of PSA(Urry 1997,
Guo et al 2007).
Additionally, since ELP coacervates have adhesive properties; a separate study was
carried out to test adhesive strength of pure ELP coacervates as a function of temperature.

14

CHAPTER 2
MATERIALS AND METHODS
2.1 Materials:
2- Ethyl hexyl acrylate (Sigma-Aldrich) was used as Pressure Sensitive Adhesive (PSA).
Acrylic acid and 2- carboxyethyl acrylate (Sigma-Aldrich) were used as polymerizable
surfactants in the polymerization reaction. Potassium persulphate (Fisher Scientific Inc.)
was used as initiator in emulsion polymerization. Potassium chloride was used as
emulsion stabilizer. Coupling agents required for protein conjugation reaction, viz.
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide
(DCC), N-Hydroxysuccinimide (NHS) were obtained from Thermo Scientific Inc.
N,N-Diisopropylethylamine (DIPEA) was used as non-nucleophilic base in conjugation
reaction. Porcine skin (Brennen Medical LLC) was used as substrate for tensile strength
tests. Polyethylene terephthalate (PET) was used as backing material for adhesive films.
Plasmid constructs of ELPs were selected from ELP library from Dr. Andrew MacKay’s
laboratory. ELPs were obtained and purified from BLR competent cells (EMD Millipore)
cultured in TB media (Mo-Bio Laboratories, Inc).

15

2.2 Synthesis of Acrylate Pressure Sensitive Adhesives by emulsion polymerization
(Janickova et al 2004, Reese & Asher 2002) :
Highly charged poly [2-ethyl hexyl acrylate (co-2-carboxy ethyl acrylate, co-acrylate)]
pressure sensitive adhesive was synthesized by emulsion polymerization in a triple neck
round bottom flask (RBF) immersed in pre-heated oil bath (70
0
C). While the magnetic
stirrer was set at 340 rpm, potassium persulphate solution (15mg/ml) was added as
initiator for polymerization. Emulsion was further stabilized by potassium chloride.
Reaction was allowed to take place for 45 min. Throughout the reaction; continuous cold
water flow was maintained in the condenser fitted on the top of RBF. Reaction was
completed under inert atmosphere and the milky product was collected in glass container.
Chemical %w/w of EHA Concentration Quantity
2-Ethyl Hexyl
Acrylate
- 9.7mmols 4ml
Potassium
Persulfate
0.86% of EHA 0.084mmols 3.00ml of stock
( 15mg/ml )
Potassium
Chloride
0.1 % of EHA 0.0097mmols 1.446mg
Acrylic Acid 1.5 % of EHA 0.305mmols 21ul
2-Carboxy
Ethyl Acrylate
1.5% of EHA 0.201mmols 24ul
DI Water - - 20ml

Table 1: Formulation of pressure sensitive adhesive: Pressure sensitive adhesive
polymer [2-ethyl hexyl acrylate (co-2-Carboxy ethyl acrylate, co-acrylate)] was
synthesized by emulsion polymerization.

16

2.3 ELP purification using ITC (Dan E. Meyer 2002):
Plasmid constructs of desired ELPs were selected from ELP library and transformed to
freshly thawed BLR competent cells. After overnight incubation at 37
0
C, on agar plate,
single bacterial colony was selected and inoculated in to 50 ml starter of TB media. After
overnight incubation at 37
0
C, bacterial culture was centrifuged at 4000 rpm for 10 min.
(Sorvall
®
RC 3C Plus). The pellets were re-suspended in TB media and inoculated in to 8
liters of culture media. After overnight incubation at 37
0
C, the culture was centrifuged at
4000 rpm for 10 min and pellets were resuspended in cold PBS. These resuspended
pellets were sonicated on ice.
Cold Spin: After sonication, lysed bacterial cells were subjected to cold centrifugation at
12000rpm for 15 min in Oakridge centrifuge tubes. Pellets were discarded and the
supernatant was collected for further treatment. Polyethylenimine (PEI) was added to
collected supernatant in the concentration of 0.25 % of final volume of supernatant. The
above mixture was again subjected to cold spin at 12000rpm for 15 min. Precipitated
DNA was discarded and supernatant was incubated at 37
0
C for 10 min.
Hot Spin: The above incubated supernatant was checked for turbidity indicating ELP
separation. 1-2 M of sodium chloride was added to supernatant, if necessary, to induce
transition. A turbid mixture was subjected to hot spin in at 4000 rpm for 10 min. After
hot spin, supernatant was discarded and a pellet containing ELPs was re-suspended in
cold PBS.

17

The above mixture was again subjected to cold spin to get rid of insoluble debris. This
cycle between cold and hot spins was repeated at least three times until high level of
purity is obtained in ELPs.

2.4 ELPs Transition Temperature Determination by UV-Vis Spectrophotometer:
ELPs transition temperature (T
t
) was determined over the range of concentrations from
5uM to 100uM (5, 10, 25, 50, 75, 100uM) in DI water using UV-Vis spectrophotometer
(Beckman Coulter). A temperature ramp was run from 10
0
C to 80
0
C at the scan rate of
1
0
C/min. Optical density was measured at 350nm. T
t
was measured as midpoint of
turbidity curve (Urry 1997). T
t
observed at 25uM concentration was considered as a
standard T
t
for a particular ELP.

2.5 SDS-PAGE:
Lonza PAGEr
®
Gold Precast 4-20% T-G gradient gels (Fisher Scientific Inc.) were used
to determine the purity of ELPs. 1X SDS solution was used as running buffer in
electrophoresis chamber. 5X SDS solution containing glycerol and bromophenol blue
was used as loading sample buffer. ELPs were mixed with loading sample buffer and
denatured at 95
0
C for 3 min. 20ul of denatured ELPs mixture was loaded in to wells.
Protein marker (New England Biolabs.) was loaded on both side wells of the sample.

18

Electrophoresis was run at 120mV for 1 ½ hour. Cupric chloride solution (5g/50ml) was
used to stain the ELP bands.

2.6 Matrix Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry:
Molecular weights of purified ELPs and molecular weight distribution of polymeric
particles of PSA were determined by MALDI mass spectrometry. For ELPs, sinapic acid
(Sigma Aldrich) matrix was prepared in 70% acetonitrile (ACN) containing 0.01% of tri-
fluoro acetic acid (TFA). Since, PSA polymeric domains are of low molecular weights as
compared to ELPs, a cold matrix of α-cyano-4-hydroxycinnamic acid (CHCA) was
prepared in 70% ACN containing 0.01% of tri-fluoro acetic acid (TFA)(Luo et al 2002).
Sample and matrix were mixed in 1:1 ratio. Bovine Serum Albumin (Sigma Aldrich) and
Apo myoglobin (Sigma Aldrich) were used as internal standards.

2.7 PSA-ELP Conjugation (Kim et al 2005, Melkoumian et al 2010, Nakajima & Ikada
1995):
Carboxyl groups of PSA polymeric domains were conjugated to amine groups of ELP
F
12
A
12
using coupling agents N,N'-dicyclohexylcarbodiimide (DCC) and
N-Hydroxysuccinimide (NHS). Coupling chemistry was carried out in anhydrous DMSO
under inert atmosphere. Final product was collected in DI water by dialyzing out DMSO
solvent and dicyclohexylurea (DCU) formed as a by-product. Conjugation reaction was

19

confirmed using FTIR-ATR technique which showed reduction in primary amine peak as
a result of conjugation(Coates 2000, Hsu 1997).

2.8 PSA-ELP Film Preparation:
PSA-ELP films were casted in two different ways. In the first case, PSA conjugated ELPs
were used to cast films however, since these conjugated nanoparticles could not form
films with optimum tackiness and surface characteristics, conjugation approach was
abandoned.
Further PSA-ELP films were prepared by pure blending of ELP F
12
A
12
with PSA in the
ratio as mentioned in Table 2.

Table 2: PSA-ELP film preparation: PSA-ELP films were prepared by blending ELP
F
12
A
12
with PSA.

Sample No.
ELP
F
12
A
12
wt.
( mg )
PSA
wt.
(mg)
PSA
(ul)
Total
wt. of
film
( mg)
%
w/w
ELP
Obs.
(Film
Formation)
Obs.
(Tackiness)
PSA+ELP 0.28 10 500 10.28 3.00 Yes Yes
PSA
Only
0 10 500 10 0 Yes
Yes

20

Additionally, films of pure ELP coacervate were also casted (Table 3) and studied for
their temperature dependent adhesiveness.
% hydration
ELP Total wt. of
film (mg)
Obs.(Film
Formation)
Obs.
( Tackiness)
Unhydrated
V
48
5 Yes No
F
12
A
12
5 Yes No
Hydrated
( 0.5%w/w)
V
48
5 Yes Yes
F
12
A
12
5 Yes Yes

Table 3: ELP coacervate film preparation: ELP coacervate films were prepared in
hydrated as well as unhydrated conditions.

2.9 Differential Scanning Calorimetry:
Thermal transitions of the films were studied using differential scanning calorimeter
(DSC 8000 Perkin Elmer Inc.). DSC 8000 was calibrated using indium as standard at the
scan rate of 5
0
C/min and 10
0
C/min. ‘PSA Only’ films and ELP coacervate films, each
weighing 1 mg, were casted in standard aluminum DSC pans (50ul). Films were studied
under unhydrated as well as hydrated (0.5%w/w) conditions. Standard crimper was used
to seal DSC pans. Using hyper DSC technique, each film was subjected to thermal
scanning from 5
0
C to 200
0
C at three different scan rates viz. 5
0
C/min. , 50
0
C/min. and
100
0
C/min. Transition temperatures corresponding to the centers of peaks were recorded.

21

2.10 Instron Tensile Strength Testing (ASTM F-2258-05):
Adhesive strengths of PSA and ELP films were tested by standard tensile strength test
using Instron Material Testing Machine. As mentioned in section 2.8, PSA and ELP films
were casted on polyethylene terephthalate (PET) pieces (2.5cm × 2.5 cm) which acted as
a backing material for films. Films were allowed to cure for 24 hours at room temperature
and humidity. Porcine skin (Brennen Medical LLC), wherever mentioned, was used as
substrate for testing adhesive films otherwise PET was used as substrate. Frozen porcine
skin sheet (12” × 12”) was thawed and cut in to pieces (2.5cm × 2.5 cm). Skin surface
with hair follicles was dehydrated by wiping out with 70% ethanol. Both porcine skin
piece and PET with adhesive film cured on it were fixed to ‘T’ shaped metal pieces using
Instant Krazy Glue® (2 full brush strokes). As depicted in Fig.9, T piece with Adhesive
film was pressed against another T piece with porcine skin/PET with a force in between
2-10N. Instron tensile strength test was carried out on each sample at two different
temperatures e.g. 15
0
C and 35
0
C in case of ‘PSA+ELP’ or ‘PSA only’ films. Each sample
was equilibrated in Instron environmental chamber at a respective temperature for 15
min. Tensile strength tests were carried out at the cross head speed of 2mm/min and load
at failure (maximum load sustained) and the type of failure was recorded. Adhesive
strength for each film was recorded in N/cm
2
.

22

Fig 9: Schematic representation of Intsron tensile strength Test. Substrate could be
either porcine skin or PET.

23

CHAPTER 3
RESULTS
3.1 Confirmation of purity and molecular weight of ELPs and PSA using
SDS-PAGE and Mass Spectrometry:
ELPs V
48
and F
12
A
12
selected from the ELP library were confirmed for their purity and
molecular weight using SDS-PAGE. Fig.10 shows molecular weight bands of V
48
and
F
12
A
12
around 20 KD and 10 KD respectively. Absence of any other band confirms purity
of ELPs. Further molecular weight confirmation of the ELPs was carried out using
MALDI mass spectrometry. Sinapic acid in 70% ACN containing 0.01% TFA was used
as matrix for MALDI mass spectrometry. Apo-myoglobin (Sigma Aldrich) used as
internal standard. Table 4 summarizes observed as well calculated molecular weights of
ELPs V
48
and F
12
A
12.

24

Fig.10: SDS-PAGE of ELP V
48
and F
12
A
12
. Absence of any other band confirms the
purity of these ELPs.

Table 4: Summary of observed and calculated molecular weights of ELPs

Sample
Molecular Weight (kDa)
Calculated Observed
V
48
19.835 19.780
F
12
A
12
10.248 10.320

25

Similarly, molecular weight distribution of PSA polymeric particles was determined
using MALDI mass spectrometry. Since PSA polymeric particles are of low molecular
weight compared to ELPs, CHCA matrix requiring low ionizing power was used. Fig.11
shows molecular weight distribution of PSA polymeric particles. Like a typical polymer
sample, PSA synthesized by emulsion polymerization gives following curve of its
molecular weight distribution.

Fig.11: Mass spectrum of PSA polymer. Figure shows molecular weight distribution of
PSA polymeric particles. The mean molecular weight is around 4kDa.

26

3.2 Determination of T
t
of ELPs using UV-Spectrophotometer:
ELPs V
48
and F
12
A
12
were characterized for their transition temperature (T
t
) at
concentrations 5uM, 10uM, 25uM, 50uM and 100uM in DI water using UV-Vis
Spectrophotometer (Beckman Coulter). T
t
was measured as midpoint of turbidity curve
(Urry 1997). T
t
observed at 25uM concentration was considered as a standard T
t
for a
particular ELP. Fig.12 and Fig.13 displays phase transition profile of ELPs V
48
and
F
12
A
12
with respect to their concentration. Increase in the concentration shows decrease
in T
t
of ELPs. Thus, from Fig. 12 and 13, T
t
for V
48
and F
12
A
12
at 25uM conc. are found
to be 40.1
0
C and 27.7
0
C respectively.

Fig.12: Phase transition profile of ELP F
12
A
12.
From the graph, T
t
of ELPs decreases as
their concentration increases.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 10 20 30 40 50 60 70 80
O D at 350nm
Temp (
0
C)
F
12
A
12

5uM
10uM
25uM
50uM
100uM

27

Fig.13: Phase transition profile of ELP V
48.
From the graph, T
t
of ELPs decreases as their
concentration increases.

Table 5: Summary of T
t
of

ELP V
48
and ELP F
12
A
12:
Experimental T
t
of ELP V
48
and
F
12
A
12
at various concentrations obtained using Beckman Coulter DU 800 UV-Vis
Spectrophotometer.

Conc.
(uM)
T
t
of V
48
(
0
C)
T
t
of F
12
A
12
(
0
C)
100 36.6 19.8
50 38.3 26.4
25 40.1 27.7
10 42.7 34.6
5 44.8 39.5
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
0 10 20 30 40 50 60 70 80
OD at 350nm
Temp (
0
C)
V
48
100uM
50uM
25uM
10uM
5uM

28

1646
1541
1453
1046
0
10
20
30
40
50
60
70
80
90
100
500 1000 1500
%
Absorption

Wavenumber (cm
-1
)
3.4 PSA-ELP conjugation confirmation using FTIR-ATR:
PSA and ELP F
12
A
12
chemical conjugation reaction was carried out using coupling agents
DCC and NHS. Conjugation was confirmed using FTIR-ATR technique using FTIR
Spectrometer (Bruker Vertex 80V). Briefly, 300uM of each of pure ELPs and conjugated
ELPs were used for such qualitative estimation of conjugation reaction. Fig.14 shows
FTIR spectrum of pure ELPs with distinct peak for primary amine C-N stretch(Coates
2000, Hsu 1997). On the other hand, Fig.15 depicts FTIR spectrum of conjugated ELPs
with marked reduction in the primary amine peak as a result of amide bond formation due
to conjugation of carboxyl group of PSA to amine group of ELPs.

Fig.14: FTIR Spectrum of ELP F
12
A
12
(300uM). Labeled peak depicts Primary amine
C-N stretch (1090-1020cm
-1
) and strong peak for primary amine N-H bend (1650-
1550cm
-1
).

Peak of primary
amine( C-N stretch)
Peak of primary
amine( N-H bend)

29

Fig.15: FTIR Spectrum of Conjugated ELP F
12
A
12
(300uM). Labeled peak shows
reduction in the primary amine C-N stretch peak (1090-1020cm
-1
) as well as peak for
N-H bend (1650-1590cm
-1
).

Additionally, peak labeled in green can be ascribed to methyl C-H asymmetric bend of
methyl groups of valine and alanine while peak labeled in yellow can be ascribed to
secondary amide N-H bend of amide bonds in ELPs.

1628
1540
1448
1046
0
10
20
30
40
50
60
70
80
90
100
500 1000 1500 2000
%
Absorption
Wavenumber ( cm
-1
)
Reduced peak of
primary
amine (C-N stretch)
Reduced peak of
primary
amine (N-H bend)

30

3.5 Thermal analysis of PSA and ELP films using Differential Scanning
Calorimetry:
PSA and ELP films were tested for their thermal transition using Differential Scanning
Calorimeter (DSC 8000 Perkin Elmer Inc.). DSC was calibrated at 5
0
C and 10
0
C /min
using Indium standard. PSA and ELP films were casted in aluminum pans (50ul) which
are then sealed using standard crimper. Different scans were run based on the type of
film. Fig.16 shows DSC thermogram of ‘PSA only’ film obtained at the scan rate of
5
0
C/min. No peak was observed in the thermogram.

Fig.16: DSC thermogram of PSA only film. From the above thermogram, it is evident
that ‘PSA only’ film does not show any transition in the given range of temperature.
Endo. Up
Temp. (
0
C)

31

Similarly, Fig.17 and Fig.18 show DSC thermograms of ELPs V
48
and F
12
A
12

respectively, obtained in unhydrated condition at scan rates of 5
0
C/min, 50
0
C/min and
100
0
C/min. To detect the weaker transitions, higher scan rates were used to magnify
transition peaks (Hyper DSC technique)(Katayama et al 2008). Such high scan rates
cause the heat flow to occur over the short period of time making the thermal transitions
more distinct. The broadening of the peak due to such high scan rates could be minimized
by proper calibration at each scan rate and by reducing the sample size(Katayama et al
2008).

Fig.17: DSC thermogram of V
48
films (unhydrated). As seen in the thermogram, higher
scan rates give magnified endothermic peaks around 60
0
C. Larger and broader peaks
around 100
0
C could be ascribed as endothermic peaks of vaporization of water.
Endo. Up
Temp. (
0
C)

32

Fig.18: DSC thermogram of F
12
A
12
film (unhydrated). As seen in the thermogram,
higher scan rates give magnified endothermic peaks around 60
0
C. Larger and broader
peaks around 100
0
C could be ascribed as endothermic peaks of vaporization of water.

Similarly, Fig.19 and Fig.20 show DSC thermograms of ELPs V
48
and F
12
A
12
respectively in 0.5%w/w hydrated condition. A distinct endothermic peak is observed at
around 35
0
C in DSC thermogram of V
48
. This peak could be ascribed to ELP V
48
transition from soluble to insoluble state in aqueous medium. No such peak is observed in
case of F
12
A
12
.

Endo. Up
Temp. (
0
C)

33

Fig.19: DSC thermogram of V
48
film (0.5%w/w hydrated). Transition peak at around
35
0
C are more distinct at higher scan rates. This peak could be ascribed to transition of
V
48
from soluble to insoluble amorphous state in aqueous medium.

Endo. Up
Temp. (
0
C)

34

Fig.20: DSC thermogram of F
12
A
12
film (0.5% w/w hydrated). No transition peak is
observed in case of ELP F
12
A
12
, which suggests a different mechanism for its onset of
turbidity in UV-Vis Spectrophotometer (Fig. 12).

3.6 Measurement of adhesive strength as function of temperature using Instron
Tensile Strength Test:
Adhesive strength of PSA and ELPs films were tested using Instron Machine. As
mentioned in Table 2, films were casted on PET and tested against porcine skin as
substrate. Fig.21 and Fig.22, describe tensile strength test results of the adhesive films at
15
0
C and 35
0
C. All the tests were carried out in triplicate. As seen the graphs, both
‘PSA+ELP’ and ‘PSA only’ films showed decreased adhesiveness with decrease in
temperature. Fig.23 shows tensile strength test results of Band Aid® used as standard
which, however, showed increased adhesiveness with decrease in temperature.
Endo. Up
Temp. (
0
C)

35

Fig.21: Tensile strength of PSA+ELP films. As seen the graph, adhesive strength
decreases with decrease in temperature. (n=3)

Fig.22: Tensile strength of ‘PSA Only’ films. From the graph, it is evident that adhesive
strength decreases with decrease in temperature. (n=3)

0
0.5
1
1.5
2
2.5
3
15 35
N/Cm
2
Temp. ( C
0
)
15
35
0
0.5
1
1.5
2
2.5
3
15 35
N/Cm
2
Temp (C
0
)
15
35

PSA+ELP films
PSA Only films

36

Fig.23: Tensile strength of Band Aid®. As seen in the graph, adhesive strength of Band
Aid® increases with decrease in temperature. (n=3)

Similarly, adhesive strength of ELP coacervate films (Table 3) was tested on Instron
machine. Both unhydrated and hydrated films were tested for their adhesive strength. It
was observed that unhydrated films of ELP coacervates do not have tackiness and hence
lack adhesiveness. However, when hydrated by 0.5%w/w of DI water, these films regain
their tackiness and show adhesive strength as function of the amount of ELPs per film.
Fig.24 display tensile strength of hydrated ELP F
12
A
12
coacervate films at two different
temperatures, 25
0
C and 40
0
C. It was observed that ELP F
12
A
12
coacervate films show
increase in adhesiveness above the transition temperature. Similar results were observed
in case of ELP V
48
coacervate films. (Fig.25)
0
0.5
1
1.5
2
2.5
3
15 35
N/Cm
2
Temp. ( C
0
)
15
35
Band Aid® films

37

Fig.24: Tensile strength of F
12
A
12
coacervate films (0.5%w/w hydrated). As shown in the
graph, tensile strength decreases with decrease in temperature. (n=2)

Fig.25: Tensile strength of V
48
coacervate films (0.5%w/w hydrated). As shown in the
graph, tensile strength decreases with decrease in temperature. (n=2)
F
12
A
12
coacervate films
V
48
coacervate films
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
25 35
N/cm
2
Temp. ( C )
25
35
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
25 40
N/cm
2
Temp. ( C )
25
40

38

CHAPTER 4
DISCUSSION
The primary aim of this project is to develop bioadhesive with temperature dependent
adhesiveness. One of the approaches is to develop ‘cool-off’ bioadhesive which will
reduce its adhesiveness at lower temperature than human skin temperature. In accordance
with this approach, an acrylate based pressure sensitive adhesive polymer was
synthesized by emulsion polymerization as mentioned in Table 1. In this formulation,
hydrophilic charged monomers were used as polymerizable surfactants(Janickova et al
2004, Reese & Asher 2002). On the other hand, protein polymers of Elastin Like
Polypeptides with characteristic transition temperature were obtained and purified from
bacterial culture. A particular ELP used for the conjugation study was F
12
A
12
. Initial
efforts were made to conjugate amine terminus of ELP to carboxyl groups of PSA.
However, this approach could not form adhesive films with optimum tackiness and
surface characteristics. Instead, it was found that films prepared using blending approach
possess better tackiness and film surface characteristics. Therefore, further films were
prepared by simple blending of ELP F
12
A
12
with PSA, as mentioned in Table 3.
Instron tensile strength tests were carried out using porcine skin as substrate. As shown in
Fig. 21 and Fig. 22, it was observed that PSA films were able to show decreased
adhesiveness on their own irrespective of addition of ELPs.

39

Since, PSA was synthesized by co-polymerizing with hydrophilic monomers, the initial
hypothesis to explain above phenomenon was that ‘co-polymerization with hydrophilic
monomers might have raised glass transition temperature (T
g
) of PSA causing cohesive
failure and decreased adhesiveness at low temperature’. To test this hypothesis, DSC
studies of PSA films were carried out. As shown in Fig. 16, DSC themogram of PSA
film, however, does not show any transition over the range of temperature. Therefore it
can be concluded that there is not significant increase in the T
g
of PSA due to
co-polymerization with hydrophilic monomers to cause decreased adhesiveness at low
temperature.
An alternative hypothesis to explain the decreased adhesiveness is that ‘hydrophilic
charged residues on PSA polymeric particles absorb water molecules from surrounding
atmosphere as temperature is lowered resulting in cohesive failure of PSA’. Since, PSA
is copolymerized with charged monomers; their unbound charged groups tend to reside in
the interparticulate spaces in the PSA film. When surrounding temperature is lowered
below room temperature (25
0
C), relative humidity increases i.e. water to air saturation
temperature gets closer to surrounding temperature. Therefore, relative hydrophilicity of
charged residues in PSA films increases to absorb water molecules from air. Such
absorbed water molecules saturate in interparticulate spaces of charged polymeric
domains(Houtman 2007). It is a well-studied phenomenon that water affects physical
bond strength of PSA leading to decrease in the adhesive strength of PSA polymers(Guo
et al 2007, Lai et al 1985). These water molecules cause swelling of adhesive films and
opens polymer network in the films. This process leads to destabilization of hydrogen

40

bonds within the films. Additionally, water has a plasticizing effect which makes
adhesive films softer. Cumulative effect of these processes results in decrease in adhesive
strength of adhesive films (Ryan Verhulst 2006).

Fig.26: Proposed explanation. Figure explaining proposed hypothesis of absorption of
water molecules in highly charged interparticulate space leading to adhesive failure of
PSA.

Since PSA was found to show decreased adhesiveness at lower temperature, it is being
proposed that ‘cool-off’ bioadhesive could be developed by optimizing the hydrophilicity
of the PSA polymer.
Polymer
particle

41

As mentioned earlier, ELPs also possess tackiness in their coacervate state. Therefore, a
separate study of ELP coacervate films was carried out to test their adhesiveness as a
function of temperature. Two different ELPs viz. V
48
and F
12
A
12
were selected from ELP
library and their films were casted as mentioned in Table 3. DSC studies were carried out
to identify transition temperature of ELP films (Fig.17-20). Hyper DSC technique was
utilized to magnify the transition peaks. It was observed that unhydrated films of ELP
coacervates do not have tackiness and hence lack adhesiveness. However, when hydrated
by 0.5%w/w of DI water, these films regain their tackiness and show adhesive strength as
function of the concentration of ELPs. Using DSC data (Fig.19-20), Instron tensile
strength tests were carried out at two different temperatures on hydrated films using PET
as substrate. As shown in Fig.24 and Fig.25, it was observed that above T
t
, ELP co-
acrevate films show moderate increase in the adhesive strength. Since, the amount of
ELP used to cast film was very low, the magnitude of adhesive strength is comparatively
less than PSA films.

.

42

CHAPTER 5
FUTURE DIRECTIONS
In the development of reversible bioadhesives, chemistry of PSA polymers plays an
important role. In this study, efforts were made to synthesize hydrophilic PSA using
highly charged monomers as polymerizable surfactants. From tensile strength results,
PSA showed decreased adhesiveness with decrease in temperature. Considering the
proposed hypothesis, this property of decreased adhesiveness could be ascribed to the
hydrophilicity of PSA. Since, the PSA showed decreased adhesiveness accompanied by
cohesive failure, current formulation of PSA needs to be optimized to achieve the desired
balance between hydrophilicity and adhesiveness of PSA film.
In the present study, emulsion polymerization technique was used to synthesize PSA
polymer. Since any emulsion polymerization process leaves small amount of monomer in
the final product, it needs to be studied for its residual monomer concentration to
determine the yield of polymerization process.
Any adhesive material is subjected to three standard tests for quantifying its adhesive
strength. These tests are tensile strength test, T-peel test and shear test. In the present
study, only tensile strength test was carried out. In future, other two adhesive strength
tests viz. T-peel test and shear test need to be carried out on PSA films.
Present study also includes measuring adhesive strength of ELP coacervate films as a
function of their transition temperature. Since magnitude of adhesive strength of ELP

43

coacervate films is directly related to amount and concentration of ELPs, further
readings with increasing amount of ELPs need to be taken for effective adhesive strength
measurement. Based on these readings, future study of ELPs needs to be carried out using
porcine skin as substrate.
In present study, DSC has been used to study the transition profile of polymers. In
particular, hyper DSC technique has been used to magnify weak transitions of protein
polymers i.e. ELPs. Since, DSC was not calibrated for high scan rates, transition curves
have been shifted to higher temperature range. Therefore, hyper DSC studies need to be
repeated with required calibration to maintain precision even at high scan rates.

44

REFERENCES
Banta S, Wheeldon IR, Blenner M. 2010. Protein engineering in the development of
functional hydrogels. Annual review of biomedical engineering 12: 167-86

Brockmann W, Huther R. 1996. Adhesion mechanisms of pressure sensitive adhesives.
International Journal of Adhesion and Adhesives 16: 81-86

Chilkoti A, Dreher MR, Meyer DE. 2002. Design of thermally responsive, recombinant
polypeptide carriers for targeted drug delivery. Advanced Drug Delivery Reviews
54: 1093-111

Cirulis JT, Keeley FW. 2010. Kinetics and Morphology of Self-Assembly of an Elastin-
like Polypeptide Based on the Alternating Domain Arrangement of Human
Tropoelastin. Biochemistry 49: 5726-33

Coates J. 2000. Interpretation of Infrared Spectra, A Practical Approach. In Encyclopedia
of Analytical Chemistry, ed. RA Meyers, pp. 10815-37. Chichester: John Wiley &
Sons Ltd

Coulombe PA, Kerns ML, Fuchs E. 2009. Epidermolysis bullosa simplex: a paradigm for
disorders of tissue fragility. J Clin Invest 119: 1784-93

Dan E. Meyer AC. 2002. Protein Purification by Inverse Transition Cycling. In Protein–
Protein Interactions: A Molecular Cloning Manual, pp. 329-43: Cold Spring
Harbor Laboratory Press

Gay C. 2002. Stickiness - Some fundamentals of adhesion. Integrative and Comparative
Biology 42: 1123-26

Guo J, Severtson SJ, Gwin LE. 2007. Optimizing the monomer composition of acrylic
water-based pressure-sensitive adhesives to minimize their impact on recycling
operations. Industrial & Engineering Chemistry Research 46: 2753-59
Houtman C. 2007. PROPERTIES OF WATER-BASED ACRYLIC PRESSURE
SENSITIVE ADHESIVE FILMS IN AQUEOUS ENVIRONMENTS. Presented at
8th TAPPI research forum on recycling, Ontario, Canada

45

Hsu CPS. 1997. Infrared Spectroscopy. In Handbook of Instrumental Techniques in
Analytical Chemistry, pp. 247-83: Prentice Hall

Hu X, Kaplan D, Cebe P. 2007. Effect of water on the thermal properties of silk fibroin.
Thermochimica Acta 461: 137-44

Janickova S, Capek I, Vasko P, Sedlak P, Capek P. 2004. Sterically stabilized emulsion
polymerization of 2-ethylhexyl acrylate. Des Monomers Polym 7: 541-51

Katayama DS, Carpenter JF, Manning MC, Randolph TW, Setlow P, Menard KP. 2008.
Characterization of amorphous solids with weak glass transitions using high ramp
rate differential scanning calorimetry. Journal of Pharmaceutical Sciences 97:
1013-24

Kim B, Kim SW, Bae YH. 2005. Synthesis, bioactivity and specificity of glucagon-like
peptide-1 (7-37)/polymer conjugate to isolated rat islets. Biomaterials 26: 3597-
606

Kull S, Martinelli I, Briganti E, Losi P, Spiller D, et al. 2009. Glubran2 Surgical Glue: In
Vitro Evaluation of Adhesive and Mechanical Properties. Journal of Surgical
Research 157: E15-E21

Lai JH, Knudson C, Hallgren L, Douglas RB, Griffin P. 1985. Effect of Humidity and
Temperature on the Adhesive Strength of Pressure Sensitive Adhesives. Polym
Eng Sci 25: 778-81

Luan CH, Harris RD, Prasad KU, Urry DW. 1990. DIFFERENTIAL SCANNING
CALORIMETRY STUDIES OF THE INVERSE TEMPERATURE
TRANSITION OF THE POLYPENTAPEPTIDE OF ELASTIN AND ITS
ANALOGS. Biopolymers 29: 1699-706

Luo GH, Marginean I, Vertes A. 2002. Internal energy of ions generated by matrix-
assisted laser desorption/ionization. Anal Chem 74: 6185-90
Melkoumian Z, Weber JL, Weber DM, Fadeev AG, Zhou Y, et al. 2010. Synthetic
peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte
differentiation of human embryonic stem cells. Nature biotechnology 28: 606-10

46

Nakajima N, Ikada Y. 1995. Mechanism of Amide Formation by Carbodiimide for
Bioconjugation in Aqueous-Media. Bioconjugate Chem 6: 123-30

Odian G. 2004. Introduction and Emulsion Polymerization. In Principles of
Polymerization, pp. 1-38, 350-71. New Jersey: Wiley Interscience

Petrie EM. 2000. Standard Test Methods In Handbook of Adhesivesand Sealants, pp.
145-51: McGraw-Hill

Portelli GB. 1986. Testing, Analysis and Design of Structural Adhesive Joints In
Structural Adhesives: Chemistry and Technology, ed. SR Hartshorn, pp. 409-19.
New York: Plenum Press

Reese CE, Asher SA. 2002. Emulsifier-free emulsion polymerization produces highly
charged, monodisperse particles for near infrared photonic crystals. Journal of
colloid and interface science 248: 41-6

Rodriguez-Cabello JC, Alonso M, Perez T, Herguedas MM. 2000. Differential scanning
calorimetry study of the hydrophobic hydration of the elastin-based
polypentapeptide, poly(VPGVG), from deficiency to excess of water.
Biopolymers 54: 282-88

Ryan Verhulst SS, Jihui Guo, Carl Houtman. TAAPI Engineering, Pulping and
Environmental Conference, Norcross, GA, 2006.

Satas D. 1989. Acrylic Adhesives In Handbook of Pressure Sensitive Adhesive
Technology, pp. 396-438. New York: Van Nostrand Reinhold

Urry DW. 1997. Physical chemistry of biological free energy transduction as
demonstrated by elastic protein-based polymers. J Phys Chem B 101: 11007-28

Urry DW, Trapane TL, Prasad KU. 1985. PHASE-STRUCTURE TRANSITIONS OF
THE ELASTIN POLYPENTAPEPTIDE WATER-SYSTEM WITHIN THE
FRAMEWORK OF COMPOSITION TEMPERATURE STUDIES. Biopolymers
24: 2345-56
Winton GB, Salasche SJ. 1985. WOUND DRESSINGS FOR DERMATOLOGIC
SURGERY. Journal of the American Academy of Dermatology 13: 1026-44

Abstract (if available)

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Physical chemistry and polymeric modification of elastin-like polypeptides

PDF

Flipping the switch on protein activity activity: elastin-like polypeptides assemble into cell switches and vesicles

PDF

Stable cell lines expressing elastin-like polypeptide fusions with epidermal growth factor receptor modulate gene expression in a heat dependent manner

PDF

Trafficking of targeted elastin‐like polypeptide nanoparticles in the lacrimal gland

PDF

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

PDF

Genetic engineering of thermally sensitive elastin-like polypeptide and its expression in HEK 293 cells

PDF

Tubulin-based fusion proteins as multifunctional tools

PDF

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

PDF

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

PDF

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

PDF

Cellular uptake mechanism of elastin-like polypeptide fusion proteins

PDF

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

PDF

Polymeric immunoglobulin receptor mediated drug carrier based on the genetically engineered temperature sensitive polypeptides

PDF

Expression and purification of different elastin like polypeptides (ELPs) constructs for therapeutic applications

PDF

Development of protein polymer therapeutics for the eye

PDF

Synthetic biopolymers modulate cell signaling

PDF

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

PDF

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

PDF

Characterization of IL-1β secretion by fusing elastin-like polypeptides to pro-caspase-1

PDF

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

Asset Metadata

Creator Gholap, Vinit (author)

Core Title Elastin like polypeptides as thermoresponsive modulators in reversible bioadhesives for fragile skin

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 07/31/2012

Defense Date 06/19/2012

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

Tag acrylate polymers,elastin like polypeptides,fragile skin,OAI-PMH Harvest,pressure sensitive adhesives,wound care.

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mackay, John Andrew (committee chair), Okamoto, Curtis Toshio (committee member), Olenyuk, Bogdan (committee member)

Creator Email gholap@usc.edu,vinitvgholap@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-78270

Unique identifier UC11290176

Identifier usctheses-c3-78270 (legacy record id)

Legacy Identifier etd-GholapVini-1071.pdf

Dmrecord 78270

Document Type Thesis

Rights Gholap, Vinit

Type texts

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

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

Repository Name University of Southern California Digital Library

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