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University of Southern California Dissertations and Theses
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Polymeric immunoglobulin receptor mediated drug carrier based on the genetically engineered temperature sensitive polypeptides
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Polymeric immunoglobulin receptor mediated drug carrier based on the genetically engineered temperature sensitive polypeptides
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Content
POLYMERIC IMMUNOGLOBULIN RECEPTOR MEDIATED DRUG
CARRIER BASED ON THE GENETICALLY ENGINEERED
TEMPERATURE SENSITIVE POLYPEPTIDES
by
Juhi Firdos
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)
May 2012
Copyright 2012 Juhi Firdos
ii
ACKNOWLEDGEMENTS
I am very grateful to my Principal Investigator, Dr Sarah Hamm Alvarez for her constant
encouragement, support and guidance. I acknowledge the valuable coaching and
mentorship of Dr Guoyong Sun, who has helped me immensely and provided me the
constructs of Hex-S48I48 and dmIgA-S48I48. He has patiently helped me with all my
queries. I am indebted to him for accelerating my learning process. I am thankful to my
committee members Dr. Andrew Mackay and Dr. Curtis Okamoto for their valuable
advice and time. I also appreciate my wonderful labmates, Frances Yarber, Hua Pei, Pang
Yu-Hsueh, Linlin Ma, Zheng Meng, Mihir Shah, Sri Kanth Janga, Maria Edman, Eun
Evans, Janette Contreras and Shi Xu , who made it a joy working in the lab even in tough
times. My family and friends have played a key role by instilling confidence and
encouraging me with my plans. Finally, I am thankful to USC School of Pharmacy for
providing me with this wonderful ecosystem and supporting my project.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ii
LIST OF FIGURES iv
ABBREVIATIONS v
ABSTRACT vii
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: BACKGROUND 3
2.1 Endocytosis and Transcytosis 3
2.2 Mucosal System 5
2.3 Polymeric Immunoglobulin Receptor 6
2.4 Nanoparticles 10
2.5 Elastin Like Polypeptides 11
2.6 Specific Aims 13
CHAPTER 3: MATERIALS AND METHODS 15
3.1 Materials 15
3.2 Design of the ELP and its synthesis 15
3.3 Protein Expression 20
3.4 Purification using ITC 20
3.5 SDS PAGE 22
3.6 ELP Characterization using UV-Vis Spectrophotometer 23
3.7 ELP Characterization using Dynamic Light Scattering (DLS) 23
3.8 ELP Characterization using MALDI-TOF 23
3.9 Cell Culture 23
3.10 Rhodamine Labelling of the ELP 24
3.11 Scanning with Confocal Microscopy 24
CHAPTER 4: RESULTS 25
4.1 Confirmation of the purity and molecular weight of ELPs 25
4.2 Characterization of Hex-S48I48 and dmIgA using
UV-Vis spectrophotometer 26
4.3 Characterization of ELP using DLS 29
4.4 Characterization of ELP using MALDI-TOF 31
4.5 Cellular Uptake of Hex-S48I48 and dmIgA 32
iv
CHAPTER 5: DISCUSSIONS 34
CHAPTER 6: CONCLUSIONS 36
CHAPTER 7: FUTURE DIRECTIONS 37
REFERENCES 38
v
LIST OF FIGURES
Figure 1: Structure of pIgR 8
Figure 2: pIgR Transcytosis 9
Figure 3: Phase Transition of ELPs 12
Figure 4: Plasmid Map 17
Figure 5: Purification of ELPs 22
Figure 6: SDS PAGE of S48I48Hex-S48I48, dmIgA-S48I48 25
Figure 7: Turbidity plot for Hex-S48I48 27
Figure 8: Turbidity plot for dmIgA-S48I48 27
Figure 9: Log –Plots of turbidity of Hex-S48I48 28
Figure 10: Log –Plots of turbidity of dmIgA-S48I48 28
Figure 11: Log –Plots of turbidity of S48I48 29
Figure 12: Hydrodynamic radius of Hex-S48I48 29
Figure 13: Hydrodynamic radius of dmIgA-S48I48 30
Figure 14: Hydrodynamic radius of S48I48 30
Figure 15: Mass Spectrometry data for Hex-SI 31
Figure 16: Mass Spectrometry data for dIgA-SI 31
Figure 17: Cellular Uptake data 32
vi
ABBREVIATIONS
DMSO Dimethyl sulfoxide (DMSO)
E. coli Escherichia coli
ELP Elastin-like polypeptides
ITC Inverse transition cycling
M.W Molecular weight
RDL Recursive directional ligation
PBS Phosphate buffer saline
PEI Poly (ethyleneimine)
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
TB Terrific broth
Tt Transition temperature
vii
ABSTRACT
The polymeric Immunoglobulin Receptor (pIgR) plays an important role in the mucosal
defense system. It transports immunoglobulins across many epithelial cells into their
secretions. It has domains which facilitate the binding and help in transport from one side
of the cell to another via transcytosis.
Elastin Like Polypeptides are peptapeptide repeats of (VPGXG) and are biocompatible.
Above their transition temperature, particular sequences of these peptides may undergo
phase transition and form micelles. They can be combined genetically with affinity
ligands to achieve targeted delivery. For my thesis, I focused on the ligands dmIgA and
Hexamer which bind to pIgR.
I constructed the diblock ELP-fusion proteins, Hex-S48I48 and dmIgA-S48I48, through
recursive directional ligation. I purified them using Inverse Transition Cycling which has
alternate rounds of hot spin and cold spin and their purity was confirmed through SDS-
PAGE.
They were characterized using UV-Vis Spectrophotometry to measure their transition
temperatures. Dynamic Light Scattering was used to measure their hydrodynamic radius
and their ability to form a nano-sized micelle. Mass spectrometry was used to measure
their molecular weight. These properties of these fusion proteins were then compared to
S48I48, the backbone without targeting ligands.
viii
To explore their trafficking in cells expressing pIgR, diblock ELP fusion proteins were
labeled with Rhodamine dye. From confocal fluorescence microscopy, it was observed
that Hex-S48I48, although it does not form nanoparticles, shows good cellular uptake.
Though dmIgA formed nanosized micelles, their cellular uptake was not optimum.
1
CHAPTER 1
INTRODUCTION
Elastin Like Polypeptides (ELPs) are pentapeptide repeats of VPGXG where X is the
guest amino acid. ELPs are denoted by the amino acid letter of the guest residue (X)
followed by the number of recurring units. ELPs respond to changes in temperature and
exhibit phase transitions. The phase behavior of the ELPs can be predicted by the
hydrophobicity of the guest amino acid. The ELPs are soluble in solution below their
transition temperature and they aggregate at higher temperatures. For the diblock ELP
like S48I48, it is possible to form a micelle as it has both hydrophilic and hydrophobic
blocks which have different phase transition temperatures. Above a Critical Micellar
Temperature (CMT), the hydrophobic part (Isoleucine) collapses to form the core of the
micelle and the hydrophilic block (Serine) forms the corona of the micelle. An affinity
ligand can be introduced to the ELP to achieve targeted drug delivery [36]. ELPs are
biocompatible and biodegradable.
The mucosal system participates in the body’s defense mechanism by transcytosing IgA
across the cell from the interstitial side to the mucosal side where IgA is released to the
external secretions. Polymeric Immunoglobulin Receptor (pIgR) is a transmembrane
spanning receptor with five domains. The extracellular region has five domains (D1-D5)
and is composed of 620 aminoacids and the transmembrane region has 23 amino acids.
2
The cytoplasmic tail with 103 aminoacids has the cues essential for its transport [9-11,
37].
The pIgR can bind to a ligand to form the pIgR-ligand complex which can be
transcytosed. Domain 1 binds to dmIgA and domain 3 and 4 bind to the Hexamer motif.
ELPs with the affinity ligand as either dmIgA or Hexamer can be used. The hexamer
motif is six amino acids sequence from SpsA (also known as CbpA), a surface displayed
protein virulence factor of Streptococcus pneumonia have been shown to bind human
pIgR [13, 37].
The pIgR mediated transcytotic pathway is considered a possible route to deliver drugs
from the plasma to mucosal surfaces. So, in my project, I focused on the ELP fusion
proteins: Hex-S48I48 and dmIgA-S48I 48 which bind to pIgR to see if they are
effectively transcytosed. This study has the possibility of targeting mucosal sites [13].
3
CHAPTER 2
BACKGROUND
2.1 Endocytosis and Transcytosis:
The extracellular cargo is transported into the cell through a process called endocytosis.
Cargo can be either fluid phase or associated with a receptor protein as a receptor-ligand
pair. Nanoscopic regions are formed on the cell membrane which deforms the local
membrane and forms curvatures. Bin/Amphiphysin/Rvs(BAR) domains mediate the
interaction between membrane and cytososolic proteins. BAR domains can also detect
and cause curvatures in the membrane. After the membrane invaginates to form a vesicle,
dynamin assembles at the neck of the vesicle. When GTP binds to dynamin, it helps in
release of the vesicle from the membrane. Early endosomes are rich in PI 3K and Rab5
GTPase which help in sorting and recycling the vesicles [3, 4, 34 and 39].
The entry into the cell can be mediated by any of the following: Clathrin, caveolin or
Rho-A. Clathrin mediated endocytosis, is important for nutrient uptake, receptor
signaling and recycling of the synaptic vesicles in neurons. Caveolin mediated
endocytosis participates in many biological functions like cell signaling, regulation of
lipids and vesicular transport. RhoA plays an important role in regulating the dynamics of
the actin cytoskeleton. Rho-A was found to play an important role in Baculo-virus entry
into non phagocytic human cells. [3, 4, 34 and 39].
4
Transcytosis is the transfer of cargo from one side of the cell to another with the help of a
vesicular carrier. It uses different carriers and different mechanisms to carry diverse types
of cargo between two different cellular environments without altering their composition.
In polarized cells like epithelial cells the transfer of cargo may be in either direction from
basolateral to apical or vice versa. In intestinal cells, cargo is internalized through clathrin
coated vesicles and transcytosis is a part of the endocytotic pathway. The cargo may be
ions, vitamins or macromolecules. In the process of transcytosis, the cargo can evade
degradation in lysosomes [5, 6].
As a part of immune response, trancytosis helps in the transport of antigens and
antibodies across epithelial barriers. The polymeric immunoglobulins, particularly the
dIgA are transcytosed across the cell to the apical side. The dIgA binds to the polymeric
immunoglobulin receptor on the basolateral side. On reaching the apical side the dIgA-
pIgR complex is proteolytically cleaved to give the secretory component. The secretory
component (SC) is the extracellular region of the pIgR bound to dIgA. SC protects the
IgA from degradation [37].
Transcytosis is helpful in delivering proteins to the apical surface by the virtue of which
polarity is maintained [5]. Transcytosis is also helpful in the passive immunity in the
transport of Ig G to the newborn [6].
5
2.2 Mucosal System:
The mucosal immune system is the first line of the immune system. It protects the tissue
from infectious agents and antigens [7]. It has the largest area of contact with the external
environment. Food ingestion and air inhalation expose the mucosal system to foreign
organisms. The mucosal system may also be invaded by toxins, bacteria and viruses and
has various mechanisms to prevent infection. Non specific mechanisms include mucosal
secretions like acid, lysozyme etc. Specific mechanisms include lymphocytes and
Immunoglobulins [5].
An important component of the mucosal immune system is secretory IgA. The secretory
antibodies help in non inflammatory protective function by trapping the antigens during
receptor mediated movement across the epithelial cell. In the mucosal secretions, IgA
restricts the entry of antigens. The IgA also preserves the integrity of the epithelial
barrier. The secretory antibodies can neutralize the viruses and transport pathogens to the
lumen and protect the epithelium. When this fails, the T-cells and other antibodies
induced by a pro inflammatory defense system come to the rescue. The secretory
immunity prevents the inflammation of the mucosal system [29].
The mucus membranes are equipped with broad range of the secretory antibodies. All
secretory effector tissues primarily contain IgA immunocytes which produce IgA. The
pIgR mediated transport across the epithelial cell depends upon the presence of a
polypeptide J- chain in Ig A and IgM. The J-chain helps in the dimerization of the
Immunoglobulin and plays an important role in binding the immunoglobulin to pIgR.
6
The pIgR is internalized and transferred to the basolateral early endosomes and then to
the common endosomes. From the common endosomes, it can be recycled to the
basolateral membrane. But, generally, it is transported to the apical membrane through
the apical recycling endosomes. The movement of polymeric IgA, especially the dimeric
IgA is preferred over Ig M as it is produced more in quantity [8, 30 and 31].
2.3 Polymeric Immunoglobulin Receptor (pIgR)
The Polymeric Immunoglobulin Receptor can be classified as a Type I transmembrane
protein which is synthesized by epithelial cells [6]. The pIgR is found on the basolateral
side of the cell membrane. It has 620 residue extracellular regions with five domains (D1-
D5), and their sequence is similar to Complementary determining loops (CDL) of Ig
variable regions. Domain 6 is the non- transmembrane part and has a non-homologous
region which is highly conserved.
The pIgR contains transmembrane region with 23 residues and a cytoplasmic tail with
103 residues. The cytoplasmic tail has most of the cues required for the transport. pIgR
expression is regulated by cytokines like INF-γ, TNF and IL-1. pIgR can also bind with
the bacterial components [9-11].
The domain 1 of pIgR has a folding topology similar to immunoglobulin variable
domains with differences in the complementary determining region (CDR) including the
helix in CDR1 and CDR3 which points away from the other CDR. It binds to pIgA and
pIgM which have a J-chain. The D1 is essential for binding and its CDR loops interact
7
with the antibody. Antibody variable region domains have three complementary
determining regions which together form a hetero-dimer [9-11].
The dimeric IgA is a major class of Immunoglobulins found in the secretions of the
mucosal and the exocrine glands. It is produced by B-lymphocytes mostly in the
intestinal tract [5]. It is formed by the monomeric units of Ig A joined by a polypeptide J-
chain. Ig A is composed of two heavy chains and two light chains which form same
antigen combining sites. The heavy chain has a variable region and 3 constant regions.
These regions have a β sheet. It also forms polymers through a certain constant region
and J chain. It protects against enzymatic action [9].
The pIgR is synthesized in respiratory, gastrointestinal and biliary regions. Domain 1 is
necessary and sufficient for binding to dIgA [10]. The D1 domain interacts noncovalently
with C α 3 and C α 2 domains of the Fc region of dimeric IgA. After that, a disulphide
bond is developed between Cys311 in the C-2 domain of the second IgA and Cys467 in
the C-terminal domain of the extracellular portion of pIgR. The dIgA binds to the pIgR
on the basolateral side of the membrane and is transcytosed to the apical side. Here the
complex undergoes proteolytic cleavage and secretory IgA is formed. The extracellular
region of pIgR bound to dIgA is called secretory component [11]. Each receptor can be
used for transport only once.
pIgR is considered a good model to study trafficking and post endocyting sorting [9].
Peptides which have the similar amino acid sequence as the binding site of dimeric IgA
were able to bind to the pIgR and be transcytosed. The residue 402-410 belonging to C
8
α3 region of IgA is the binding site for pIgR. This represents an ideal system to target the
therapeutics to the mucosal epithelial cells [13].
Figure 1: Structure of pIgR showing the five domains in the extracellular region, the
transmembrane region and the cytoplasmic tail. The extracellular region is cleaved to form the
secretory component [38].
9
Figure 2: pIgR Transcytosis pathway[41].The pIgR binds to the dIgA on the basolateral side. It is
transcytosed and released on the apical side by apical endosomes. On reaching the apical side, it
undergoes proteolytic cleavage to form Secretory Component
10
2.4 Nanoparticles:
Nanoparticles have revolutionized the field of drug delivery. They help in targeted drug
delivery to specific tissues, cells and cellular components like ribosomes, chromosomes,
DNA and even the nucleus. Targeted delivery has multifaceted advantages like reducing
the side effects to the rest of the body, using very less quantity of the drug, improving the
therapeutic index and infiltrating deep into the tissue. They can also exhibit sustained
release which may vary between few days to weeks [33].
A nanoparticle is generally between 1 nm to 100nm [14]. This size helps them easily
cross the potential barriers. They can be internalized through endocytosis and thus bypass
the receptors. Their surface to mass is relatively safer and biocompatible. Size of the
nanoparticle affects the biodistribution which in turn affects the bioavailabililty.
They can be of different size ranges and varieties. They belong to different categories like
polymers, viruses, lipids, organic and inorganic materials. Polymers may either be natural
like chitosan, albumin or synthetic. Liposomes consist of a central aqueous layer
surrounded by a lipid bilayer. Different viruses like bacteriophages are also used. Carbon
nanotubes are also used especially in diagnostics. They can be chemically modified to
make them water soluble [16, 17].
Of late, polymers which are biodegradable and release the drug after degradation are
hugely popular. The idea of biodegradable polymers originated from sutures.
Biopolymers are of various kinds like polylactic acid and polyglycolic acid. They can be
used in controlled release as the body can reabsorb them [18]. Stimuli in form of light or
11
heat may also be applied to the nanoparticle to exhibit its effect or for drug release [15].
Active targeting nanoparticles have an affinity ligand or antibody in the vehicle.
Targeting with the help of a ligand helps avoid drug resistance [17].
Thus nanoparticles help overcome the current obstacles in drug delivery. With the drug
delivery systems smaller than the size of their targets, there is an increase in treatment
efficacy. They can also be used in imaging, diagnosis and treatment [19].
2.5 Elastin Like Polypeptides:
Elastin Like Polypeptides mimic Elastin, an extracellular protein which gives elasticity to
arteries, skin, lungs etc. Its precursor Tropoelastin has structure which alternates between
both phobic and cross linking regions. Also, covalent crosslinks are formed which
stabilize the insoluble polymer matrix. The physical properties dictate its structure. It
undergoes a process called coarcervation, that is, with an increase in temperature, it
aggregates. This is a reversible process and the transition temperature depends on pH,
ionic strength of solution and concentration of polypeptide [20-22].
Elastins like polypeptides are similar in structure and have hydrophobic and cross
linking regions [20-22]. The hydrophobic domains have repeats of nonpolar amino acids
and have β turns whereas the cross linking regions are mostly hydrophilic and have α
turn. The hydrophobic domains determine the structure of elastin.
ELPs are genetically engineered pentapeptide motif of Valine –Proline –Glycine –Xaa-
Glycine (VPGXG) where X can be replaced by any amino acid except Proline. They are
12
soluble at lower temperature but when the temperature reaches a critical temperature-
inverse transition temperature, they aggregate. This is a reversible process. They can be
synthesized such that they respond to a variety of stimuli such as pH, ionic strength,
temperature, light. ELPs can be used for targeted drug delivery and protein purification.
They can be designed such that they have appropriate half life and route of clearance.
They are cost effective in production and biocompatible [23, 24].
Figure 3: Phase Transition of ELPs. Tt is the transition temperature. At a temperature below Tt,
it exists in soluble state. Above Tt, it aggregates and shows turbidity. [This image was taken from
Dr. Mackay’s lab]
The ELP-fusion protein can be purified by simple and economical process called Inverse
Transition Cycling. It is subjected to alternate rounds of hot spin and cold spin. The phase
13
transition is accelerated by addition of NaCl salt and by raising the temperature. This is
then centrifuged to separate the pellet containing ELP from the supernatant with the
E.coli biomolecules. This is then centrifuged at a temperature lower than the transition
temperature to discard the contaminants in the ELP pellet. The hot cycle and the cold
cycles are repeated till ELP is pure. The di-block ELP can form a spherical micelle with a
transition temperature between the hydrophilic and hydrophobic di blocks [23, 24].
Since they are biocompatible and they can be genetically engineered to have a specific
molecular weight, their degradation can be controlled. Thermally responsive ELP
generally have a transition temperature between 37°C and 42°C. ELPs have been used for
tumor targeting after hypothermia[23]. ELPs can be efficiently used in bulk implants and
injectibles and encapsulations and release of proteins, DNA and Drugs. Aggregated ELP
which has a longer half life can be used as an injectable depot. ELPs can also be used in
synthesis and retention of matrix and provide an environment for tissue regeneration
[24].ELPs can be used for targeting specific cells via the affinity ligand through receptor
mediated endocytosis [25].
ELPs are biodegradable. It was found that the S48I48 had a terminal half-life of 8.7 h,
with a degradation rate of 2.49%/day in vitro and 2.46%/day in vivo[26].
2.6 Specific Aim:
The objective of my present study was to test the hypothesis that ELP trafficking occurs
through the pIgR receptor when ELP fusion proteins containing pIgR binding domains
were generated. The ELP fusion protein containing the affinity ligand which binds to
14
pIgR was selected. The ELP fusion proteins Hex-S48I48 and dmIgA-S48I48 were
characterized and its cellular uptake was observed in Calu-3 cells.
15
CHAPTER 3
MATERIALS AND METHODS
3.1 Materials:
The oligonucleotides for Hex-S48I48 and dmIgA-S48I48 were ordered from Integrated
DNA Technology (IDT). The pET25b+ vector and BLR cells were acquired from
Novagen (Madison, WI). Top 10 cells were obtained from Invitrogen (Carlsbad, CA).The
ELPs dmIgA-S48I48 and Hex-S48I48 were designed by Dr. Guoyong Sun. NHS-
Rhodamine was acquired from Thermos Scientific (Rockford, IL). The Calu-3 cell line
was obtained from ATCC (American Type Culture Collection). Dulbecco’s Modified
Eagle Medium (DMEM) and Fetal Bovine Serum (FBS) were obtained from Cellgro
(Manassas, VA). All the reagents were acquired from New England Biolabs unless
specified.
3.2 Design of the Plasmid for Hex-S48I48 and dmIgA-S48I48:
They are designed using recursive directional ligation. In this method, short gene
fragments are combined using recombinant DNA techniques. This is continued until a
desired gene fragment of a particular length is obtained [17, 18 and 28].
Hex-S48I48: The sequence of Hex-S48I48 is YRNYPT G (VPGSG)48(VPGIG)48 Y.
Here YRNYPT is the Hex and (VPGSG)48(VPGIG)48 is S48I48. The molecular weight
is 40.22kD
Oligonucleotide for Hex
The forward motif
CTCCTCGG and the r
GTAGTTACGG TAACCca
were NdeI and Bam HI. Their site of
NdeI Bam HI
tatgGGTTACCGTAACTACCCGACCGGTTACTGATCTCCTCGG
acCCAATGGCATTGATGGGCTGGCCAATGACTAGAGGAGCCctag
NdeI
Oligonucleotide for Hex-S48I48:
motif tatgGGTTACCGTAACTACCCGACCGGTTACTGAT
and the reverse motif gatcCCGAGGAGATCAGTAACCGGTCGG
TAACCca were obtained from IDT. The restriction enzymes used
were NdeI and Bam HI. Their site of action is as represented below.
NdeI Bam HI
tatgGGTTACCGTAACTACCCGACCGGTTACTGATCTCCTCGG
acCCAATGGCATTGATGGGCTGGCCAATGACTAGAGGAGCCctag
BamHI
16
tatgGGTTACCGTAACTACCCGACCGGTTACTGAT-
gatcCCGAGGAGATCAGTAACCGGTCGG-
were obtained from IDT. The restriction enzymes used
acCCAATGGCATTGATGGGCTGGCCAATGACTAGAGGAGCCctag
BamHI
17
Plasmid Map:
Figure 4: The plasmid map with its restriction sites
18
The oligonucleotide for Hex-S48I48 was annealed after heating the forward and reverse
primers and T4 DNA ligase buffer to 90°C and then cooled to form double stranded
DNA. The plasmid vector was cut with the enzymes NdeI and BamHI to create the sticky
ends. This sequence was inserted into the Pet 25 b+ plasmid between the cutting sites of
NdeI and BamHI through ligation by using T4 DNA ligase . The plasmid vector with the
sequence of Hexamer was then transformed to E. coli Top 10 cells and plated on agar
which is supplemented by Ampicillin. After overnight incubation, few colonies were
selected. They were purified using Qiagen Plasmid Purification kit. The plasmid was sent
for sequencing and the sequence was confirmed.
The ligated combination was then digested with NdeI and BamH I at 37 °C for 3 hours
and loaded onto agarose gel QIAquick Gel Extraction Kit Was used to extract the DNA
from the gel. S48I48 was ligated into a vector and then transformed to E. coli Top 10
cells and plated on agar containing Ampicillin. After overnight incubation, few colonies
were selected. They were purified using Qiagen Plasmid Purification kit. The sequence
was confirmed.
dmIgA-S48I48
Amino acid sequence of dmIgA-S48I48 is G (TWASRQEPSQGTTTFAVTSGPGGG-
GGGPG)2(VPGSG)48(VPGIG)48Y. Here TWASRQEPSQGTTTFAVTS corresponds
to mIgA and GPGGGGGGPG is Linker. The molecular weight according to the formula
is 45.02kD. The forward and reverse sequence of oligonucleotides of mIgA and Linker
was ordered from IDT.
Oligonucleotide for mIgA
The forward motif is
ACCACCACCTTCGCTGTTACCTC
gatcctgaagatcattatcagtaAGAGGTAACAGCGAAGGTGGTGGTACCCTGAGACGGTT
CCTGACGAGAAGCCCAGGT
Oligonucleotide for Linker:
The forward motif is
tactgataatgatcttcag
gatcctgaagatcattatcagtaACCCGGACCACCACCACCACCACCCGGACC
The oligonucleotide for mIgA and Linker
forward and reverse primers
enzymes BseRI and BamHI
The recognition sites are:
BseRI
The annealed oligonucleotides
cutting sites of BseRI and BamHI
buffer. The plasmid vector now has the
mIgA:
motif is TACCTGGGCTTCTCGTCAGGAACCGTCTCAGGGT
ACCACCACCTTCGCTGTTACCTCTtactgataatgatcttcag and the reverse
AGAGGTAACAGCGAAGGTGGTGGTACCCTGAGACGGTT
CCTGACGAGAAGCCCAGGTACC.The underlined part represents the mIgA sequence.
Linker:
The forward motif is TGGTCCGGGTGGTGGTGGTGGTGGTCCGGG
and the reverse motif is
ACCCGGACCACCACCACCACCACCCGGACC
for mIgA and Linker was annealed separately after heating
forward and reverse primers to 90 °C and then cooled to form double stranded
BseRI and BamHI were used to cut the plasmid vector to create the sticky ends.
:
AcuI BamHI
annealed oligonucleotides were inserted into the pet 25 b+ plasmid
BseRI and BamHI through ligation by using T4 DNA ligase and ligase
buffer. The plasmid vector now has the sequence of mIgA- and Linker.
19
ACCTGGGCTTCTCGTCAGGAACCGTCTCAGGGT-
everse motif is
AGAGGTAACAGCGAAGGTGGTGGTACCCTGAGACGGTT
The underlined part represents the mIgA sequence.
GGTCCGGGTGGTGGTGGTGGTGGTCCGGGT-
and the reverse motif is
ACCCGGACCACCACCACCACCACCCGGACCACC
after heating the
then cooled to form double stranded DNA. The
to create the sticky ends.
BamHI
plasmid between the
by using T4 DNA ligase and ligase
and Linker. It was then
20
transformed to E.coli Top 10 cells and plated on agar containing Ampicillin. After
overnight incubation, few colonies were selected. They were purified using Qiagen
Plasmid Purification kit. The plasmid was sent for sequencing and the sequence was
confirmed.
The ligated combination was then digested with AcuI, BserI and BssHII at 37 °C for 3
hours and loaded to agarose gel. The gel was extracted using QIAGEN gel extraction kit.
The extracted DNA was ligated into a vector using T4 DNA ligase (Invitrogen) and then
transformed to E.coli Top 10 cells and plated on agar containing Ampicillin. After
overnight incubation, few colonies were selected. They were purified using Qiagen
Plasmid Purification kit. It was then ligated with S48I48.
3.3 Expression of the ELP Fusion protein:
The purified plasmid was transformed into freshly thawed BLR cells and plated on agar
coated with Ampicillin and incubated overnight. It was then inoculated into an
Erlenmayer flask containing 50ml of L.B medium. It was incubated overnight in shaking
incubator at 37°C. This medium is taken and added to 1L flask containing T.B dry
medium. This was also incubated at 37°C overnight in the shaking incubator.
3.4 ELP Purification using Inverse Transition Cycling (ITC):
ITC is a simple and cost effective process to purify proteins. It makes use of the special
property of the ELP which displays phase transition behavior. When the temperature is
below the critical transition temperature, they are soluble in aqueous solution. Above this
21
critical temperature, ELPs undergo aggregation. The transition temperature of ELP is
dependent on a number of factors like the molecular weight, the amino acid X and the
salt concentration of the solution[27].
This suspension was subjected to discontinuous ultrasonication for cell lysis. The lysed
product was subjected to a cold spin at 12000 rpm at 4°C for 15 min. The pellet which
contains the cellular debris is discarded and the supernatant which contains the ELP is
retained. Cold polyethyleneimine (PEI) was added at a concentration of 0.5%. The
solution changes to white color due to the co precipitation of the PEI and the DNA. It is
again subjected to cold spin. The pellet obtained would have the cellular debris and the
precipitated DNA.
The supernatant was placed warmed to 37°C in the water bath for 10 min and 3M NaCl
was added. Turbidity was observed. It was then subjected to hot spin at 4000 rpm at
37°C for 10 min. The supernatant with E.coli biomolecules was disposed off and the
pellet which contains the aggregated ELP was resuspended in 40 ml of cold PBS. It was
suspended in cold PBS. The hot and cold spins were repeatedly performed to attain a high
level of purity. Thus, repeated aggregation, centrifugation and resolubilization help in the
purification of ELP [27].
22
Figure 5: Purification of ELP: ELP containing impurities is subjected to hot spin. The pellet
contains the ELP and the supernatant is discarded. It is then subjected to cold spin. These cycles
are repeated to obtain pure ELP.
3.5 SDS PAGE:
10% SDS Gels was prepared manually. The sample buffer-sodium dodecyl sulphate (4X)
which contains glycerol and bromophenol blue was added to the ELPs and heated at
95°C for 5 min to denature them. Around 100 µg of ELP was loaded into the wells. They
were stained with Copper Chloride and Commassie Blue. The molecular weight marker
used was Kaleidoscope protein ladder (from Bio-rad laboratories). The Versa-Doc
instrument was used to image these gels.
23
3.6 ELP Characterization using UV-Vis Spectrophotometer:
Different concentrations of the ELP were prepared (5µM, 10µM, 25µM, 50µM,
100µM).The blank used was PBS buffer. Temperature was raised at the rate of 1°C /min
starting from 15°C till it reached 80°C. The optical density was measured at these
temperatures.
3.7 ELP Characterization using Dynamic Light Scattering (DLS):
The ELPs Hex-S48I48 and dmIgA-S48I48 were diluted by PBS to obtain a concentration
of 25µM. It was then filtered through a 20 nm membrane at a temperature of 4°C and
transferred to a 384 well microplate. It was centrifuged at 4°C to remove air bubbles and
then mineral oil (ice cold) was added. It was then centrifuged again. The particle size was
measured in Dyna Pro Plate reader (Wyatt Inc., Santa Barbara, CA).
3.8 ELP Characterization using MALDI-TOF:
The matrix used here was sinapinic acid. BSA was used as control and 2 µl of the
samples Hex-S48I48, dmIgA-S48I48, Hex-S48I48-BSA and dmIgA-S48I48 in matrix
was added to the MALDI plates and analyzed.
3.9 Cell Culture:
Calu-3 cells are human bronchiole epithelial cells. They were obtained from a Caucasian
white male. They polarize into apical and basolateral layers. They form monolayers
which show high electric resistance and indicate the formation of tight epithelial barriers.
This cell model has been widely used for drug transport studies [42, 43].
24
Calu-3 cells were cultured using (minimum essential medium (MEM) supplemented with
10% FBS, 1% Penicillin, 1% Glutamine, 1% Non Essential Amino Acids.
3.10 Rhodamine Labelling of the ELP:
The ELP was mixed with 100 µM borate buffer to maintain pH at 8.5. It was mixed with
Rhodamine (Thermo Fisher Scientific Inc, Rockford, IL) dissolved in DMSO and kept on
ice for 3 hours. It was then passed through a PD 10 desalting column (GE Healthcare,
Piscataway, NJ) to separate the free Rhodamine. The concentration is measured on
nanodrop spetrophotomter at the wavelengths of 280 and 555 nm. The concentration of
Rh-dmIgA-S48I48 and Rh-Hex-S48I48 was calculated.
3.11 Scanning with Confocal Fluorescence Microscopy:
The Calu-3 cells were allowed to grow on dishes of 35mm diameter in DMEM medium.
It takes 4 days to form a small cluster. The cells were washed with warm medium .The
Rh-Hex-S48I48 and Rh-dmIgA-S48I48 were thawed on ice. The medium was also
cooled on ice at the same temperature. The Rh-ELP of 25µM concentration was added to
the dishes and incubated at 37°C for 1 hour. After that, the cells were washed with
medium three times to remove the free Rh-ELP and Lysotracker green, an indicator of
lysosomes and early endosomes, is added. The cells were imaged using Zeiss LSM 510
Confocal Microscope. The argon and Neon Laser was used at the excitation wavelength
of 488 and 534 nm. The objective lens used was 63X Oil Immersion.
4.1 Confirmation of the purity and molecular
ELPs were constructed using Recursive and Directional Ligation. Five rounds of Inverse
Transition Cycling were performed to obtain pure Hex
purity was confirmed using SDS PAGE. Around 100 µg of ELP was loaded o
The gel showed single thick band indicating that dmIgA
were pure.
Figure 6: SDS PAGE of S48I48Hex
CHAPTER 4
RESULTS
4.1 Confirmation of the purity and molecular weight of ELPs:
ELPs were constructed using Recursive and Directional Ligation. Five rounds of Inverse
Transition Cycling were performed to obtain pure Hex-S48I48 and dmIgA
purity was confirmed using SDS PAGE. Around 100 µg of ELP was loaded o
The gel showed single thick band indicating that dmIgA-S48I48, Hex-S48I48
SDS PAGE of S48I48Hex-S48I48, dmIgA-S48I48
25
ELPs were constructed using Recursive and Directional Ligation. Five rounds of Inverse
and dmIgA-S48I48. The
purity was confirmed using SDS PAGE. Around 100 µg of ELP was loaded onto the gel.
S48I48 and S48I48
26
4.2 Characterization of Hex-S48I48 and dmIgA using UV-Vis spectrophotometer:
Hex-S48I48 and dmIgA-S48I48 were characterized using different concentrations (5 µM,
10 µM, 25µM, 50 µM, 100 µM in PBS buffer). ELPs are soluble below their transition
temperature and they are aggregate when heated above the transition temperature and
turbidity can be observed. Optical Density at wavelength of 350 nm was used to
characterize their transition temperatures. Both Hex-S48I48 and dmIgA-S48I48 have two
transition temperatures. This is because of the presence of diblock that that consists of
Isoleucine in the guest residue of the first repeating sequences of the ELP and Serine in
the guest residue of the second repeating sequence. The first increase in absorbance is
due to the Isoleucine which shows lower transition temperature and the second increase
in absorbance is due to Serine which forms bigger aggregates and shows higher transition
temperature.
27
Figure 7: Turbidity plot for Hex-S48I48: As the temperature increases, the transition
temperature decreases .The two transition temperatures observed were Tt1= 31
o
C and
Tt2=67
o
C
Figure 8: Turbidity plot for dmIgA-S48I48: As the temperature increases, the transition
temperature decreases. The two transition temperatures observed were Tt1= 23
o
C and
Tt2=50
o
C
28
Figure 9: Log plot of turbidity of Hex S48I48 [35].From the graph, it is evident that the transition
temperature decreases when the concentration of the ELPs increase.
Figure 10: Log plot of turbidity of dmIgA S48I48 [35].From the graph, it is evident that the
transition temperature decreases when the concentration of the ELPs increase.
29
Figure 11: Log plot of turbidity of S48I48 [35].From the graph, it is evident that the transition
temperature decreases when the concentration of the ELPs increase.
4.3 Characterization of ELP using Dyamic Light Scattering:
Hex S48-I48:
Figure 12: There is an aggregation of ELP but it does not form a nanosized micelle.
30
dmIgA-S48I48:
Figure 13: dmIgA-S48 I48 forms a nanosized micelle.
S48I48 :
Figure 14: Formation of nano sized micelle for S48I48 with increase of temperature [35].
31
4.4 Characterization of ELP using MALDI-TOF:
Figure 15: The observed molecular mass of dmIgAS48I48 is 44kDa.
Figure 16: The observed molecular mass of Hex-S48I48 is 39.09 kDa
32
4.5 Cellular Uptake of Hex-S48I48 and dmIgA:
Figure 17: Uptake of ELPs- Hex-S48I48, dmIgA-S48I48 and S48I48: Rh labeled ELPs were
incubated for 1 hour and then their uptake was observed. Both Hex-S48I48 and S48I48 were
internalized. Hex-S48I48 appeared to have more internalization.
Lysotracker is used to track lysosomes and early endosomes. Rhodamine was used to
label the Elastin like polypeptides. DIC is the Differential Interface Contrast. Overlay
Overlay DIC Rhodamine Lysotracker
Hex-SI
Control
SI dmIgA-SI
33
was the superimposition of both Lysotracker and Rhodamine. Hex-S48I48 was observed
to have more uptake than dmIgA-S48I48 which in turn appeared to have better uptake
than S48I48.
34
CHAPTER 5
DISCUSSIONS
We wanted to show that targeted ELPs could be effectively delivered to cells expressing
the targeting receptor, and be used as drug delivery vehicle. We chose the pIgR to
transcytose the ELPs across the mucosal epithelial cells. dmIgA is an immunologlobulin
which binds to pIgR on domain 1 and Hex-S48I48 binds to domain 2. We chose them to
be the affinity ligands to effectively target the pIgR.
I obtained the sequence for dmIgA-S48I48 and Hex-S48I48 from Dr.Guoyong Sun which
was constructed using recursive directional ligation. Five round of ITC were performed
to obtain pure ELP. I then characterized the ELP and ELP-fusion protein. I confirmed
their purity and estimated their molecular weight through SDS PAGE. A single clear
band was obtained indicating high levels of purity.
ELPs are soluble below a certain temperature “transition temperature” and above this
temperature; they aggregate and appear to be in a turbid state. UV-Vis spectrophotometer
was used to characterize the transition temperature. Since S48I48 is a di-block polymer, it
shows two transition temperatures, due to the presence of Isoleucine and Serine. The
transition temperatures of Hex-S48I48 were 31
o
C and 67
o
C and the transition
temperatures of dmIgA were: 23
o
C and 50
o
C. Log concentration Vs temperature graphs
35
were plotted and they clearly showed that the transition temperature decrease with an
increase in temperature.
Dynamic Light Scattering was used to find the hydrodynamic radius of the ELPs. dmIgA-
S48I48 formed nano sized micelles when the temperature was increased from 10
o
C to
37
o
C. Hex-S48I48 formed particles but it was not of nanoscale.
MALDI-TOF was used to confirm the molecular masses of the ELPs. From cellular
uptake data observed through confocal microscope, it is clear that Hex-S48I48 shows
better cellular uptake than S48I48 and dmIgA-S48I48.
36
CHAPTER 6
CONCLUSIONS
Hex S48I48 was observed to have better uptake even though it did not form nano-
micelles. This may be because probably only few nanoparticles are formed which were
responsible for its uptake. Another possibility is that the uptake was probably by
macropinocytosis and not by pIgR receptor. Another explanation is that the transcytosis
that we actually observed might be apical to basolateral. More experiments need to be
conducted to confirm this.
37
CHAPTER 7
FUTURE DIRECTIONS
Cellular uptake experiments have confirmed that the ELP fusion proteins Hex-S48I48
and dmIgA-S48I48 enter the Calu-3 cell and colocalizes in the lysosome. However,
further experiments are needed to confirm that ELP fusion protein trafficking occurs
through pIgR. This can be verified by inhibition of anti human pIgR antibody.
The cellular uptake experiments were conducted in Calu-3 cells. They can be performed
in other cell lines which express pIgR. Transcytosis assays can be conducted to verify
that the ELP fusion proteins are actually being transcytosed.
38
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Abstract (if available)
Abstract
The polymeric Immunoglobulin Receptor (pIgR) plays an important role in the mucosal defense system. It transports immunoglobulins across many epithelial cells into their secretions. It has domains which facilitate the binding and help in transport from one side of the cell to another via transcytosis. ❧ Elastin Like Polypeptides are peptapeptide repeats of (VPGXG) and are biocompatible. Above their transition temperature, particular sequences of these peptides may undergo phase transition and form micelles. They can be combined genetically with affinity ligands to achieve targeted delivery. For my thesis, I focused on the ligands dmIgA and Hexamer which bind to pIgR. ❧ I constructed the diblock ELP-fusion proteins, Hex-S48I48 and dmIgA-S48I48, through recursive directional ligation. I purified them using Inverse Transition Cycling which has alternate rounds of hot spin and cold spin and their purity was confirmed through SDS-PAGE. ❧ They were characterized using UV-Vis Spectrophotometry to measure their transition temperatures. Dynamic Light Scattering was used to measure their hydrodynamic radius and their ability to form a nano-sized micelle. Mass spectrometry was used to measure their molecular weight. These properties of these fusion proteins were then compared to S48I48, the backbone without targeting ligands. ❧ To explore their trafficking in cells expressing pIgR, diblock ELP fusion proteins were labeled with Rhodamine dye. From confocal fluorescence microscopy, it was observed that Hex-S48I48, although it does not form nanoparticles, shows good cellular uptake. Though dmIgA formed nanosized micelles, their cellular uptake was not optimum.
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Firdos, Juhi
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Polymeric immunoglobulin receptor mediated drug carrier based on the genetically engineered temperature sensitive polypeptides
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School of Pharmacy
Degree
Master of Science
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Pharmaceutical Sciences
Publication Date
05/09/2014
Defense Date
03/23/2012
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