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Tabriz University of Medical Sciences
SYNTHESIS, IN VITRO AND CELLULAR
CHARACTERIZATION AND EVALUATION OF
GEMCITABINE-PEPTIDE NANOCONJUGATES
By:
Samad Mussa Farkhani
A Thesis Presented to the
FACULTY OF ADVANCED MEDICAL SCIENCES
TABRIZ UNIVERSITY OF MEDICAL SCIENCES
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE (MEDICAL NANOTECHNOLOGY)
Supervisors:
Dr. Hadi Valizadeh
Dr. Javid Shahbazi Mojarrad
Advisor:
Dr. Parvin Zakeri-Milani
Thesis No: 93/2-11/4 May 2015
I
Tabriz University of Medical Sciences
Faculty of advanced medical sciences
Dissertation submitted for MS.c degree of
Medical Nanotechnology
Synthesis, in vitro and cellular characterization and evaluation of
gemcitabine-peptide Nano conjugates
Author:
Samad Mussa Farkhani
Supervisors:
Dr. Hadi Valizadeh
Dr. Javid Shahbazi Mojarrad
Advisor:
Dr. Parvin Zakeri-Milani
Place of performance:
Faculty of Pharmacy, Research Center for Pharmaceutical Nanotechnology
and Drug Applied Research Center of Tabriz university of Medical Sciences
Dissertation No: 93/2-11/4 May 2015
II
In the name of Allah
Statement of Originality
I certify that the following thesis is based on the results of investigations
performed by me, that this is my own composition, and that it has not previously
been presented for a higher degree.
I hereby confirm above mentioned statement, as a supervisor /
advisor of this thesis.
III
Dedication
This thesis work is dedicated to my parents, who have always loved me
unconditionally and whose good examples have taught me to work hard
for the things that I aspire to achieve.
IV
Acknowledgement
I would like to express my special appreciation and thanks to my
supervisor Professor Hadi Valizadeh, you have been a tremendous mentor
for me. I would like to thank you for encouraging my research and for
allowing me to grow as a research scientist. I must offer my profoundest
gratitude to my second supervisor Dr. Javid Shahbazi Mojarrad who has
been always available for my questions. I would also like to thank my
advisor Dr. Parvin Zakeri-Milani.
I would like to acknowledge financial, academicals and technical support
of Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz
University of medical sciences. I express my warm thanks to Dr. Yadollah
Omidi and Dr. Mohammad Reza Rashidi for their support and guidance
at RCPN.
V
I am also thankful to my good friends Ali Shirani, Samaneh Mohammadi
and Alireza Valizadeh who supported me in all steps of work and also
writing the thesis, and incented me to strive towards my goal. I also thank
Dr. Siavoush Dastmalchi, Dr. Hamed Hamishehkar, Dr. Abolfazl
Akbarzadeh and Dr. Roya Salehi Who accepted to be the reviewer team of
my thesis. At the end a special thanks to my family for all their support.
VI
List of publication:
FARKHANI, S. M., JOHARI-AHAR, M., ZAKERI-MILANI, P., SHAHBAZI
MOJARRAD, J. & VALIZADEH, H. 2015. Enhanced cellular internalization of
CdTe quantum dots mediated by arginine- and tryptophan-rich cell-penetrating
peptides as efficient carriers. Artificial cells, nanomedicine, and biotechnology, 1-5
VII
List of abbreviations
1 Gem: Gemcitabine
2 BOC: Tert-Butyloxycarbonyl
3 CPP: Cell-penetrating peptide
4 DCM: Dichloromethane
5 DIEPA: N, N-Diisopropylethylamine
6 DMF: Dimethylformamide
7 DMSO: Dimethyl sulfoxide
8 EDT: 1, 2-Ethanedithiol
9 EDTA: Ethylenediaminetetraacetic acid
10 FBS: Fetal bovine serum
11 FITC: Fluorescein isothiocyanate
12 FMOC: Fluorenylmethyloxycarbonyl
13 AuNPs: Gold nanoparticles
14 hCNT: Human concentrative nucleoside transporter
15 hENTs: Human equilibrative nucleoside transporters
16 MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
17 SPPS: Solid-phase peptide synthesis
18 AgNPs: Silver nanoparticles
VIII
19 PBS: Phosphate buffered saline
20 QDs: Quantum dots
21 TBTU: N, N, N′, N′-Tetramethyl-O -(benzotriazol-1 - yl)uronium
tetrafluoroborate
22 TFA: trifluoroacetic acid
23 TIS: Triisopropylsilane
24 Arg (R):Arginine
25 Trp (W):Tryptophan
26 Glu (E): Glutamic acid
IX
Table of contents
Abstract ..................................................................................................................... 1
Chapter one: ............................................................................................................. 3
Introduction .............................................................................................................. 3
1. Gemcitabine ...................................................................................................... 4
1.1. Problems in Drug Delivery ........................................................................ 7
Chapter two: ...........................................................................................................10
Literature review ....................................................................................................10
1.1.1. Discovery of CPPs .............................................................................11
1.1.2. Classes of CPPs..................................................................................12
1.1.3. Mechanisms of CPP uptake ...............................................................15
1.1.4. Applications of CPP ...........................................................................16
1.1.4.1. Intracellular delivery .......................................................................16
1.1.4.2. Quantum dots ..................................................................................17
1.1.4.3. Gold nanoparticles ..........................................................................17
1.1.4.4. Nanosilver .......................................................................................18
1.1.4.5. Liposome ........................................................................................18
1.1.4.6. Delivery of biologically active peptides and proteins ....................19
1.1.4.7. Delivery of oligonucleotides, nucleic acids and siRNA ................19
1.1.4.8. CPP as carriers for anticancer drug delivery ..................................20
Chapter three: ........................................................................................................21
Material and methods ............................................................................................21
2.1. Materials and equipment .............................................................................22
2.1.1. Materials ................................................................................................22
2.1.2. Laboratory Equipment ..........................................................................23
2.2. Methods .......................................................................................................24
X
2.2.1. Peptides synthesis by solid-phase peptide synthesis (SPPS) ................24
2.2.1.1. Swelling of the resin .......................................................................24
2.2.1.2. FMOC deprotection ........................................................................25
2.2.1.3. Coupling of amino acid to the resin ................................................26
2.2.1.3.1. Monitoring of Coupling and Capping with ninhydrin test ..........26
2.2.1.4. Cleavage of synthesized peptides from resin .................................28
2.2.1.5. Freeze drying of precipitated peptides ............................................29
2.2.2. Particle Size Measurement ....................................................................30
2.2.3. SEM .......................................................................................................30
2.2.4. FITC labeling of peptides .....................................................................30
2.2.5. Preparation of Gem-peptide conjugates ................................................31
2.2.6. Cell culture ............................................................................................33
2.2.6.1. Thawing A549 cells ........................................................................34
2.2.6.2. Passaging A549 cells ......................................................................34
2.2.6.3. Cell Counting ..................................................................................35
2.2.7. MTT assay .............................................................................................36
2.2.8. Fluorescent Microscopy ........................................................................37
2.2.9. Flow cytometry .....................................................................................38
Chapter four: ..........................................................................................................39
Results .....................................................................................................................39
3.1. Synthesis of CPPs ........................................................................................40
3.2. Cytotoxicity of synthesized CPPs ...............................................................41
3.2.1. Effect of poly-glutamate on toxicity of cationic CPPs .........................42
3.3. Cellular Uptake Studies of CPPs and CPP-E9 conjugates ..........................48
3.4. Cellular Uptake studies of six peptides .......................................................54
3.5. Anti-tumor performance of drug loaded CPPs ............................................58
Chapter five: ...........................................................................................................61
Discussion ................................................................................................................61
XI
4.1. Effect of CPPs on gemcitabine cytotoxicity ...............................................62
4.2. Poly-glutamate interaction with CPPs ........................................................65
4.3. Conclusion ...................................................................................................69
5. References ......................................................................................................71
6. Persian part ...................................................................................................81
XII
List of figures and tables
Figure Page
Figure 1-1. Chemical structure of gemcitabine Page 6
Figure 1-2. Mechanism of gemcitabine action Page 7
Table 1-1. Types and amino acid sequences of some CPPs Page 14
Figure 1-3. Mechanisms of CPP uptake across the cellular membrane Page 16
Figure 2-1. Reaction vessel of peptides synthesis Page 25
Figure 2-2. Schematic representing synthesis of R5W3R4 peptide Page 29
Figure 2-3. Synthesis of FITC-labeled Peptide Page 31
Figure 2-4. Schematic representing preparing of Gem-R5W3R4 conjugate Page 33
Figure 2-5. MTT reduction in live cells by mitochondrial reductase results in the
formation of formazan Page 36
Table 3-1. Sequence and other parameters of the synthesized CPPs Page 40
Figure 3-1. Cytotoxicity of peptides on A549 cells after 72 h incubation Page 42
Figure 3-2. Size distribution of [RW]6/E9, R5W3R4/E9, and R9/E9 nanoparticles
Page 43
XIII
Figure 3-3. SEM images of R9, R9/E9, R5W3R4, R5W3R4/E9, [RW]6 and
[RW]6/E9 Page 45
Figure 3-4. Toxicity of peptides and their nanoconjugates on A549 cells determined
by MTT assay Page 47
Figure 3-5. IC50 of three peptides after and before interaction with E9 Page 48
Figure 3-6. Fluorescent imaging of A549 cells incubated with 10, 25, and 50 µM
FITC-labeled R9 and R9/E9 for 1.5 h at 37 °C Page 51
Figure 3-7. Fluorescent imaging of A549 cells incubated with 10, 25, and 50 µM
FITC-labeled R5W3R4 and R5W3R4/E9 for 1.5 h at 37 °C Page 52
Figure 3-8. Fluorescent imaging of A549 cells incubated with 10, 25, and 50 µM
FITC-labeled [RW]6 and [RW]6/E9 for 1.5 h at 37 °C Page 53
Figure 3-9. Cellular uptake of FITC-labeled peptides and peptides/E9 in live A549
cells after incubation for 1.5 h at 37 °C measured by flow cytometery Page 54
Figure 3-10. Fluorescence microscopy, visualization of FITC-labeled, R5W3R4
(A,B), [RW]6 (C,D), [RW]5 (E,F), [RW]4 (G,H), and [RW]3 (I,J) in A459 cells
line Page 56
Figure 3-11. Cellular uptake of FITC-labeled peptides in live A549 cells after
incubation Page 57
XIV
Table 3-2. Amount of drug loading efficiency Page 58
Figure 3-12. Toxicity of Gem-R5W3R4 (A), Gem-[RW]6 (B), Gem-[RW5] (C),
Gem-[RW]4 (D), Gem-[RW]3 (E) and Gem to A549 cells Page 60
Figure 4-1. Schematic drawing representing the effect of E9 on the uptake and
cytotoxicity of arginine and tryptophan-rich CPPs Page 69
1
Abstract
Background
Gemcitabine is an anticancer drug that displays activity in the treatment of
pancreatic, non-small cell lung, bladder, ovarian and breast cancers. However the
drug has certain drawbacks such as short plasma half-life, its high hydrophilicity and
the induction of resistance related to hENT1 transporters. In recent years it was
showed that the use of cell penetrating peptides (CPPs), a short peptides that improve
the uptake of various covalently and noncovalently attached molecules, could
increase cellular delivery of antitumor drugs.
Aim
The present study was designed to investigate the effect of CPPs conjugation on the
cytotoxicity of gemcitabine in A549 lung carcinoma cell line. Also the effect of poly-
glutamate peptide on cellular uptake and cytotoxicity of the cationic CPPs was
studied.
Methods
The peptides were synthesized manually by solid phase peptide synthesis methods.
Cellular uptake and distribution of peptides was performed by means of flow
2
cytometry and fluorescent microscopy. The gem drug was conjugated to the CPPs
by using succinic linker. Measuring the cytotoxicity of gemcitabine and Gem-CPP
conjugates was performed with MTT assay. Size and morphology of poly-
glutamate/CPP nanoconjugates was carried out by use of DLS.
Results
The data demonstrated that at 15 and 25 µM, Gem-R5W3R4, Gem-[RW]6 and Gem-
[WR]3 conjugates exhibited decreased cell viability compared to Gem alone. Also
E9 interaction could decrease the toxicity of these cationic CPPs at high
concentrations.
Conclusion
The findings in this study support the advantages of using CPPs for improving
intracellular delivery of drugs into tumor as well as their activity. Furthermore, it is
possible to overcome gemcitabine resistance associated with deficiencies in the
expression of hENT1 by using CPP strategy. Additionally, the obtained results
indicate that the cytotoxicity of CPPs could be reduced by poly-glutamic acid (E9)
with slight decrease in uptake efficiency.
Keywords: Cell penetrating peptides, Gemcitabine, Cytotoxicity, Poly-glutamate,
3
Chapter one:
Introduction
4
1. Gemcitabine
Gemcitabine (Gem, dFdC) is a fluorinated nucleoside analogue that displays
anticancer activity in the treatment of pancreatic, non-small cell lung, bladder,
ovarian and breast cancers (Fig. 1-1) (1). Gemcitabine is transported into the cells
by sodium-dependent (concentrative nucleoside transporter hCNTs) and by sodium
independent (equilibrative nucleoside transporter hENTs) mechanisms. It is
recognized that Gemcitabine transportation into cells mediated by five nucleoside
transporters, two equilibrative nucleoside transporters hENT1 and hENT2 and three
concentrative nucleoside transporters (hCNT1, hCNT2, hCNT3) (2). Studies have
shown that most intracellular uptake of gemcitabine is directed by hENT1 and, to a
lesser amount, by hCNT1 and hCNT3 (1). After entering to the cell, the drug
phosphorylated to monophosphate derivative (dFdCMP) by deoxycytidine kinase
(dCK). It then converted to gemcitabine diphosphate (dFdCDP) and gemcitabine
triphosphate (dFdCTP), by nucleoside monophosphate kinase (UMP/CMP) and
diphosphate kinase, respectively (3). The new evidence demonstrates that
incorporation of dFdCDP and dFdCTP into DNA strand is essential for gemcitabine
to inhibit cell replication and induce apoptosis in tumor cells (4). dFdCDP inhibits
ribonucleoside diphosphate reductase liable for catalyzing the reaction that generates
the deoxyribonucleotides required for synthesis and repair of DNA. The triphosphate
form of gemcitabine, dFdCTP, incorporates into DNA as a false nucleoside,
5
inhibiting DNA polymerase and thereby preventing the detection and repair of DNA
repairing enzymes and causing cell death by apoptosis (5-7). Figure 1-2 displayed
the mechanism of gemcitabine action after entering to the cell.
Although the explained mechanism of action contribute to the efficiency of
gemcitabine for cancer treatment, the drug has certain drawbacks that are related to
its unfavorable pharmacokinetic properties. Gemcitabine with a low molecular
weight is rapidly and extensively deaminated in blood and in some tissues by
deoxycytidine deaminase (dCDA) to the inactive and more soluble metabolite 2/, 2/-
difluorodeoxyuridine (dFdU), leading to a short plasma half-life (8, 9). In addition,
it was stated that transport of gemcitabine across the cell membrane requires active
nucleoside transporters. Most of the gemcitabine uptake is mediated by hENT1
transporters and, in fact, hENT1-deficient cells such as human breast and pancreatic
cancer cells are highly resistant to this nucleoside analogue. Deficiency in dCK in
cancer cells is also a frequently described form of intrinsic or acquired resistance to
gemcitabine (10-13). Additionally, the increasing amount of resistance also reduces
the drug cytotoxicity. Because of this, a frequent administration schedule at high
drug doses is required that cause serious side effects such as: myelosuppression, high
levels of hepatoxicity, renal toxicity, gemcitabine-induced arterial thrombosis, and
anemia (14). In recent years various strategies such as liposomes, nanoparticles,
lipidic and nonlipidic derivatives, and polymeric drug conjugates have been
6
investigated to prevent rapid plasma degradation of gemcitabine and enhance
delivery to the tumor tissue (15).
Figure 1-1. Chemical structure of gemcitabine
7
Figure 1-2. Mechanism of gemcitabine action
1.1. Problems in Drug Delivery
Cell-penetrating peptides as drug carriers
Many pharmaceutical molecules need to pass through one or more cell membranes
to reach their site of action. A common characteristic of all plasma membranes is a
phospholipid bilayer, about 10 nm thick, arranged with the hydrophilic heads on the
outside and the lipophilic chains facing inwards. This Feature gives a sandwich
effect, with two hydrophilic layers surrounding the central hydrophobic part (16).
8
The cellular membrane is highly efficient in its role as a selectively permeable
barrier. While this phospholipid bilayer is essential to cell survival and function, it
also presents a major challenge for intracellular delivery of various therapeutic and
diagnostic molecules (17). Although small cancer drugs can cross this membrane via
a natural cellular process or direct diffusion through the lipid bilayer and protein-
based therapeutics can enter cells by membrane mobile transport, in many cases the
effective passage of some bioactive molecules through the plasma membrane
remains a major obstacle for intracellular delivery of cargo (18). Good cellular
uptake often needs the use of high dose of drugs in order to obtain the expected
intracellular biological effect. Therefore, improving the translocation of anticancer
drugs across the cellular membrane will significantly decrease the amount of
administered drug, and the wide side effects on healthy tissues that are currently
observed in most of the cases (19).
Several drug carriers, such as nanospheres, nanocapsules, liposomes, micelles,
lipoproteins, and polymers have been used widely over the past few decades to
deliver a selection of therapeutic and diagnostic agents (20). While several carrier
currently used for drug translocation, cell-penetrating peptides (CPPs) have become
one of the most popular and efficient techniques for intracellular delivery of their
cargo molecules. CPPs are generally defined as short cationic peptides, typically
9
with 5–30 amino acids, which able to penetrate cellular membranes and transfer
covalently or non-covalently attached bioactive molecules into cells (21).
Objectives:
General objective: synthesis of peptide-Gemcitabine nanoconjugates
Specific objectives:
1. Synthesis of cell penetrating peptide containing arginine and tryptophan
amino acids.
2. Coupling of FITC as fluorescent molecule to the CPP for evaluating uptake
of peptides into cells.
3. Evaluating the cytotoxicity of gemcitabine and peptide-gem conjugates on
A549 cancer cells.
Hypothesis:
1- Because of intrinsic ability of these peptides for delivering cargo, our
synthesized peptides efficiently transport gemcitabine into cell.
2- What is the effect of presence of tryptophan and their position within a poly-
arginine influence uptake efficiency as well as cytotoxicity?
10
Chapter two:
Literature review
11
1.1.1. Discovery of CPPs
In 1965, it was found that histones and cationic polyamines such as poly-lysine
stimulate the cellular uptake of albumin by cancer cells in culture. It was revealed
that direct conjugation of albumin and other molecules to poly-lysine made the
transport of the coupled cargo more specific and effective. Subsequent studies
revealed that medium-length polymers of arginine were significantly more effective
at entering cells than the polymers composed of lysine, ornithine or histidine (22).
Initially discovered in 1988, Tat, the HIV transactivator of transcription protein, was
the first sequence found that also passed very efficiently through cell membranes of
cultured mammalian cells (23, 24). Covalently binding the Tat to proteins or
fluorescent probes allowed these molecules to cross the cell membrane (25). In 1991,
the Antennapedia homeodomain (HDAntp) from Drosophila was illustrated to be
internalized by neuronal cells. This helped the discovery in 1994 of the first PTD or
CPP, a 16-merpeptide derived from the third helix of the homeodomain of
Antennapedia named Penetratin. In this year for the first time CPPs used as vectors
when penetratin was employed for the delivery of a small exogenous peptide (26,
27). The research was followed by the identification of the minimal peptide sequence
of TAT (47YGRKKRRQRRR57) required for effective cellular uptake in 1998 (28).
After that, additional polycationic peptides of natural VP22, AntP and synthetic
origin (transportan) have been identified which also facilitate cellular uptake, alone
12
or together with attached cargoes. Since then, the list of available CPPs has grown
intensely and the number continues to increase (table 1-1)
1.1.2. Classes of CPPs
A number of researchers divided CPPs into three categories based on their origin,
which include:
Protein derivatives:
They include natural peptides which exist in organisms and can increase the uptake
of macromolecules into cell.
Synthetic CPPs:
They usually prepared by synthesis from amino acids in the laboratory or industrial
and haven’t exist naturally.
Chimeric:
They are a combination of synthetic peptides and protein derivatives.
In another classification CPPs divided into three group base on Physical–chemical
properties;
Cationic CPPs:
The cationic peptides are short amino acid sequences that are often contains arginine,
lysine and histidine. Arginine as an amino acid has a guanidine head group that can
13
make hydrogen bonds with the negatively charged phosphates and sulfates on the
plasma membrane and might lead to internalization with cell surfaces. Lysine
although has positive charge similar to arginine, but it does not contain the guanidine
head group, and then is less effective at penetrating to the cell membrane when acting
alone. The first CPP discovered was cationic named Tat. Studies on oligoarginine
peptides (from R3 to R12) have shown that the minimal sequence for effective cellular
internalization is octaarginine (R8), and that increasing the number of arginines rises
the level of uptake (29). It was confirmed that the arginine-rich CPP is more efficient
than the other cationic peptides (30). Several studies suggest that at least eight
positive charges are needed for efficient cellular uptake of cationic peptides.
Amphipathic CPPs:
Amphipathic peptides have lipophilic and hydrophilic blocks that are responsible for
mediating the peptide translocation across the plasma membrane. These peptides
mainly are chimeric peptides obtained by covalently attaching a hydrophobic
domain for efficient targeting to cell membranes to a NLS (nuclear localization
sequences).
Hydrophobic CPPs:
14
They contain hydrophobic amino acids and have a low net charge. Compared with
cationic and amphipathic CPPs, only a few hydrophobic CPPs have been discovered
until now.
Table 1-1. Types and amino acid sequences of some CPPs.
Type Name Sequence References
Cationic Polyarginine R8, R9, R10, R12 (31)
TAT 49–57 RKKRRQRRR (32)
Penetratin (pAntp) RQIKIWFQNRRMKWKK (33)
P22N NAKTRRHERRRKLAIER (34)
DPV6 GRPRESGKKRKRKRLKP (35)
DPV3 RKKRRRESRKKRRRES (35)
Amphipathic Transportan GWTLNSAGYLLGKINLKALAALAKKIL (36)
Pep-1 KETWWETWWTEWSQPKKKRKV (37)
MPG GLAFLGFLGAAGSTMGAWSQPKKKRKV (37)
pVEC LLIILRRRIRKQAHAHSK (38)
MAP KLALKLALKALKAALKLA (39)
CADY GLWRALWRLLRSLWRLLWRA (40)
Hydrophobic K-FGF AAVLLPVLLAAP (41)
C105Y CSIPPEVKFNKPFVYLI (42)
15
1.1.3. Mechanisms of CPP uptake
It is obvious that understanding the uptake mechanism and intracellular trafficking
of CPPs is an important step for optimization of these carriers to produce best effect,
and to determine the intracellular behavior and efficiency of the cargo delivery (43).
Although these peptides have been widely used to transport various molecules into
cells, the exact uptake route of CPPs is still challenging lots of questions (44).
Recently, depending on CPP own features, the carried molecule, the cell type and
the membrane lipid composition, the two major intracellular uptake mechanism of
CPP include nonendocytotic and the endocytotic pathways have been proposed (45).
Figure 1-3 has shown different uptake mechanisms proposed to explain the
internalization of free or cargo-conjugated CPPs. It was suggested that CPP with
small cargoes, may enter cells rapidly via direct translocation in addition to the
endocytic way. In contrast, translocation of large molecules coupled to these
peptides tended to be mediated by macropinocytosis in an energy-dependent manner
with slower rates for larger compounds (46).
16
Figure 1-3. Mechanisms of CPP uptake across the cellular membrane (47)
1.1.4. Applications of CPP
1.1.4.1. Intracellular delivery
17
The ability to intracellular delivery of large hydrophilic molecules, such as peptides,
proteins, DNA, siRNA and large particles is a problem because of the bioavailability
restriction imposed by the cell membrane. The discovery of CPPs that can
translocate effectively across cell membrane, hence, opened up fascinating
perspectives for the development of cell delivery. In recent years, these peptides
have been used for efficient cellular delivery of a broad variety of molecules (48).
Delivery of nanoparticles and nanocarriers by CPPs
Recently, CPPs have been employed for cellular delivery of a broad variety of
cargoes including various nanoparticles and pharmaceutical nanocarriers.
1.1.4.2. Quantum dots
In recent years, quantum dots (semiconductor nanocrystals) was used for cancer
targeting and imaging in living animals. They have unique photophysical properties
which make them interesting candidates as fluorescent tags (49). CPPs are used for
improving intracellular delivery of QDs. In one study, it was shown that a CPPs
termed SR9 facilitates the transport of noncovalently associated QDs into A549 cells
(50).
1.1.4.3. Gold nanoparticles
Recently gold nanoparticles (AuNPs) have been employed for cellular applications
including biomedical imaging, clinical diagnostics, and even therapeutics (51).
18
However, to reach their full potential, strong methods must be developed for the
control of gold uptake into cells and for their localization to the cytosol or to specific
organelles. For cellular uptake and targeted delivery, AuNPs have been
functionalized with CPP to improve internalization or with NLS, and TAT peptides
to target the cell nucleus (52).
1.1.4.4. Nanosilver
Silver nanoparticle (AgNP) has been widely used in biomedical, because of its ready
manufacturing process, quality control, and biocompatibility. Recently, AgNP has
been reported for its anticancer effect. A nanopharmaceutical system using TAT
peptide was developed for multidrug-resistant (MDR) cancer treatment, in which
nanosilver with mean size of 8 nm modified with TAT peptide displayed exceptional
antitumor activity in both MDR cells and non-resistant cells (53).
1.1.4.5. Liposome
A liposome is a vesicle usually made of phospholipids and can be filled with drugs,
and used to deliver drugs for cancer and other diseases. To increase control over
drug release, a novel method of constraining a CPP have been developed, which can
be utilize to trigger transport of liposomes into cells upon radiation with UV light.
First, TAT peptide modified on both ends with an alkyl chain and after that was
anchored to the liposomal surface in a forced and deactivated form. When the TAT
19
was engaged in this loop formation no cellular uptake occurred. Disconnection one
of the two alkyl chains that inked to TAT via a UV-cleavable upon irradiation led to
the exposure of the TAT, then induced cellular delivery of the entire liposome
particle into cells (54).
1.1.4.6. Delivery of biologically active peptides and proteins
CPP was used for peptides and protein delivery. Several studies have been used
penetratin to promote the delivery of fragments of protein-inhibiting cyclin-
dependent kinase (Cdk), which have role in regulating the cell cycle. Also, Tat
peptide could stimulate the cellular delivery of Cdk-inhibiting peptide, enabling
arrest of cell proliferation (55, 56).
1.1.4.7. Delivery of oligonucleotides, nucleic acids and siRNA
Recently, CPPs have been used successfully to translocation of oligonucleotides into
cells (57, 58). It has been reported that tow CPP includes transportan and penetratin
were able to transport a peptide nucleic acid (PNA), which was unable to cross the
plasma membrane in its original from, into melanoma cells (59). Cationic CPPs,
interact with the negatively charged phosphate backbone of nucleic acids through
electrostatic interactions (60). Recently, some studies have explored the possibility
to apply CPPs as a gene delivery system. In one study, Liu et al. used arginine-rich
peptides for gene delivery into cells in a noncovalent fashion. They indicated that
20
these peptides are able to transfer plasmid DNA into A549 cancerous cells (61). TAT
peptide also is able to successfully deliver of siRNA to cells for gene silencing used
in the modulation of gene expression (61).
1.1.4.8. CPP as carriers for anticancer drug delivery
Among the various types of cancer treatment, chemotherapy is the most widely used
approaches. Despite the abundant use of chemotherapy, it causing unpleasant side
effects and limited due to its systemic toxicity (61). Since CPPs increase cellular
uptake, the conjugation of anticancer drug with these peptides may also be a
powerful tool for decrease their toxicity in tumor therapy. Several drug such as Taxol
(62), Methotrexate (63), and doxorubicin (64) have shown improved activity when
conjugated to CPP.
In this study, we developed six Gem-CPP conjugates in aim to enhance its
intracellular delivery and therapeutic effects. Six arginine and tryptophan-rich CPPs
were synthesized with solid phase peptide synthesis procedure. The uptake
efficiency of CPPs into cells examined by flow cytometry and fluorescent
microscopy. The synthesized peptides, were chemically conjugated to Gem and
cytotoxicity of conjugates was tested by MTT assay in A594 cell line. Additionally
the effect of electrostatic interaction of poly-glutamate (E9) on cytotoxicity and
cellular uptake of arginine and tryptophan-rich peptides was studied.
21
Chapter three:
Material and methods
22
2.1. Materials and equipment
2.1.1. Materials
Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH and Fmoc-Glu(OtBu)-OH
(AAPPTec, Louisville, USA)
O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate
(TBTU) (Sigma, St. Louis, USA)
N-Ethyldiisopropylamine (Merck, Germany)
Fmoc-Rink-Amide AM resin (AAPPTec, Louisville, USA)
Gemcitabine hydrochloride (Actavis, Italy)
Fluorescein isothiocyanate (FITC) Isomer I (Sigma, St. Louis, USA)
Trifluoroacetic Acid (TFA) (Sigma, St. Louis, USA)
Piperidine (Sigma, St. Louis, USA)
Dimethylformamide(DMF), Dichloromethane (Scharlau, Italy)
Dimethyl sulfoxide (Merck, Germany)
Triisopropylsilane (TIPS) (Sigma, St. Louis, USA)
Phenol (Sigma, St. Louis, USA)
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Roth,
Germany)
RPMI1640 medium (Gibco, USA)
23
Trypsin-EDTA (Invitrogen, Carlsbad, USA)
Penicillin/streptomycin (Applichem, Germany)
Fetal bovine serum (FBS) (Gibco, USA)
Cell culture T-75 flask (Biofil, Canada)
Cell culture 96-well plates, 6-well plates (Biofil, Canada)
A549 lung carcinoma cell line (Pasture, Iran)
2.1.2. Laboratory Equipment
Flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA)
Vacuum pump (Heidolph, Germany)
Rotary evaporator (Heidolph, Germany)
Freeze Dryer (Telstar, Spain)
Centrifuges (Sigma, Germany)
Incubator (Memmert, Germany)
Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK)
Scanning electron microscope (SEM, Mira3 FEG-SEM Tescan, Czech
Republic)
Olympus IX81 fluorescence microscope (Olympus Optical Co., Ltd., Tokyo,
Japan)
24
UV-Vis Spectrophotometers (Shimadzu)
Microplate Reader (Bio-Tek)
Magnetic stirrer
Reaction vessel for peptides synthesis
2.2. Methods
2.2.1. Peptides synthesis by solid-phase
peptide synthesis (SPPS)
All peptides were synthesized manually by solid-phase peptide synthesis method on
a FMOC-Rink-Amide AM resin by FMOC strategy in a fritted glass vessel (fig. 2-
1). The CPPs synthesis steps includes:
2.2.1.1. Swelling of the resin
Resin was swelled in anhydrous DMF for about 1 h under dry nitrogen. For 125 mg
of resin, 2 mL DCM was added. After 30 min resin and DCM was poured into the
reaction vessel and DCM was filtered off by vacuum.
25
Figure 2-1. Reaction vessel of peptides synthesis
2.2.1.2. FMOC deprotection
FMOC deprotection of resin was carried out using piperidine in DMF (20% v/v, 2
mL, 30 min). Ninhydrin test was used to monitor FMOC deprotection. After 30 min
for ensure about completion of deprotection, ninhydrin test carried out. With
completion of deprotection, the reaction solution was filtered off and resin was
washed with DMF (4 × 2 mL) and DCM (4 × 2mL).
26
Calculation the amount of materials required for peptide synthesis
The amount of amino acid required for coupling to resin calculated based on the
loading amount of resin with free amine group. The loading of Rink amid AM resin
was equal to 0.4-0.9 mmol/g and the molar ratio values for synthesis was 1:3:3:3
(free amine group: amino acid: TBTU: DIEPA). Accordingly, for 125 mg resin in
minimum loading the amount of materials calculated was equal to 79, 95, and 63 for
tryptophan, arginine and glutamate respectively. The amount of coupling reagent
including TBTU and DIEPA used for each coupling was equal to 43, and 30 µl
respectively.
2.2.1.3. Coupling of amino acid to the resin
Amino acid was coupled to the resin in the presence of TBTU and DIPEA in DMF
(2 mL) by mixing for 2 h under N2 atmosphere. Then the reaction was monitored
using the ninhydrin test. When the ninhydrin test is negative and coupling was
completed, the reaction solution was filtered off and resin was washed with DMF (4
× 2 mL) and DCM (4 × 2mL). If the ninhydrin test was not negative, the coupling
procedure was repeat. Others amino acids coupled to the amino acid chain by these
deprotection, washing, coupling and washing cycle.
2.2.1.3.1. Monitoring of Coupling and Capping with
ninhydrin test
27
Ninhydrin can be used to monitor deprotection in solid phase peptide synthesis
(Kaiser Test). This test is a qualitative exam for the presence or absence of free
primary amino groups, and it can also be a useful indication about the completeness
of a coupling step. Ninhydrin reacts with the deprotected N-terminal amine group of
the peptide-resin which gives a characteristic dark blue color. The Kaiser test
requires minimal amounts of analyte and is completed within a few minutes.
Ninhydrin Test Solutions:
Reagent A: 1- Dissolve 16.5 mg of KCN in 25 mL of distilled water 2- Dilute 2
mL of above solution with 98 mL of pyridine (freshly distilled from ninhydrin).
Reagent B: dissolve 1.0 g of ninhydrin in 20 mL of n-butanol.
Reagent C: dissolve 20 g of phenol in 10 mL of n-butanol.
Ninhydrin test Method:
First were taken 10-15 beads of resin in a test tube. Then, 2-3 drops of reagents A,
B, and C was added to tube. The tubes were heated at 110°C for 5 minutes.
Interpretation of Results
Colorless or faint blue color (beads): complete coupling, proceed with
synthesis
28
Dark blue solution but beads are colorless: nearly complete coupling, extend
coupling or cap unreacted chains
Solution is light blue but beads are dark blue: coupling incomplete, recouple
Solution is intense blue and all beads are blue: failed coupling, check amino
acid, reagents, then recouple
2.2.1.4. Cleavage of synthesized peptides from
resin
After the coupling of all amino acids, the resin was washed with DMF, DCM and
ethanol respectively (each 2 × 2 mL). The resin was dried under vacuum for 24 h.
Fresh cleavage cocktail, reagent B, TFA/ TIPS / phenol /water (88:2:5:5 v/v/v/v, 3
mL/30mg resin-peptide), was added to the resin for side-chain deprotection and the
final cleavage of the synthesized peptide from the solid support. The mixture was
shaken at room temperature for 2 h. The resin was collected by filtration and washed
with another 2 mL of fresh cleavage cocktail. Combined filtrates were evaporated
by rotary to reduce the volume under dry nitrogen. The crude peptide was
precipitated by adding diethyl ether (10 time of added TFA volume) and centrifuged
at 4000 rpm for 4 min, followed by decantation to obtain the solid precipitates. The
obtained peptide was further washed with ether (2 ×50 mL) for 2 times (fig. 2-2).
29
Figure 2-2. Schematic representing synthesis of R5W3R4 peptide.
2.2.1.5. Freeze drying of precipitated peptides
Freeze-drying or lyophilisation is an effective method of drying various materials
without harming them. This process carried out by sublimation, which involves the
direct transition between the solid state and the gaseous state without passing
through the liquid phase. For long-term preservation, the synthesized CPPs were
lyophilized. The peptides were dissolved in appropriate solution and then freeze-
30
dried at a pressure of 0.5 mbar and with a temperature of -40 °C for 48 h. the freeze-
dried peptides could be stored for about 6 month at -20 °C.
2.2.2. Particle Size Measurement
For the formation of conjugates between peptides and poly glutamate (E9), first three
CPP were dissolved in DMSO and after that poly-glutamate was added to each
peptides in ratios of 1:1, 1:5 and 1:10 (E9:CPPs). Conjugates and free peptides were
diluted in distilled water and particle sizes of each E9/peptides conjugates were
analyzed using a Zetasizer Nano ZS.
2.2.3. SEM
SEM sample was prepared by drop casting a 5 mM aqueous solution (20 µL)
onto the mica surface. Mica surface was lyophilized and analyzed in a Scanning
electron microscopy (SEM, Mira3 FEG-SEM Tescan 5.0 kV). All samples were
imaged after coating with gold, in high vacuum mode.
2.2.4. FITC labeling of peptides
First, N-terminal FMOC deprotection of each prepared peptide was carried out using
piperidine in DMF. A solution of 1.1 equivalent of FITC in pyridine/DMF/DCM
was prepared and added to the peptides-resin then mixed overnight. The completion
31
of reaction was checked using ninhydrin test. The resin was washed and final
cleavage of the CPP-FITC conjugates from the resin was carried out according to
the mentioned protocol (fig. 2-3).
Figure 2-3. Synthesis of FITC-labeled Peptide
2.2.5. Preparation of Gem-peptide conjugates
GEM was coupled to peptides using a succinyl spacer that linked the amine group
of peptides to the hydroxyl (R-OH) group of GEM. The NH2 group of the peptides
32
was changed to a carboxyl moiety by reaction with succinic anhydride as a linker.
After synthesis of peptides on the resin, the final Fmoc removed (20% piperidin, 30
min) and resulted in free amino group. These deprotected peptides were treated with
succinic anhydride (1.5 eq.) and DIEPA (3 eq) in DMF for 2 h. the completion of
reaction controlled with Ninhydrin test. The resin was washed with DMF (4 × 2 mL)
and DCM (4 × 2mL). for conjugation of succinylated peptide to gemcitabine, TEA
(50 µL) were added to a solution of 40 mg Gem in 3 Ml of an 85:15 (v/v) DMA/DMF
mixture and added to succinylated peptides. The reaction was kept under gently
stirring for 48 h. after that the mixture was dried and peptide-GEM conjugates
cleavage from resin with TFA/ TIPS / phenol /water (88:2:5:5 v/v/v/v, 3 mL)
cocktail (fig. 2-4).
33
Figure 2-4. Schematic representing preparing of Gem-R5W3R4 conjugate.
2.2.6. Cell culture
A549 is a human epithelial cell line derived from a lung carcinoma tissue and are
adherent in culture. A549 lung carcinoma cell line was obtained from Pasteur
Institute (Iran). Cells were maintained in RPMI 1640 medium supplemented with
34
10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin and grown at 37
°C in a 5% CO2 humidified atmosphere.
2.2.6.1. Thawing A549 cells
Due to that the frozen cells were in DMSO, which is toxic to the cells, procedures
carried out quickly.
1- First frozen vial of cells placed in 37ºC water bath for ~1 minute until cells
almost completely thawed.
2- The vial contents transferred to a 15 ml conical tube containing 9 ml pre-
warmed media
3- The tube was centrifuged for 5 min in 1,500 rpm to pellet cells. The
supernatant was discarded and cell pellet was resuspended in 10 ml of media
and transferred to flask.
4- Flask placed in incubator (37ºC with 5% CO2).
2.2.6.2. Passaging A549 cells
Cells over growing in flask occupy spaces available for expansion so passage is
necessary. This procedure was performed when cells reached to 80 - 95%
confluency.
35
1- The media was removed using glass pipette vacuum and washed 2 times with
PBS.
2- Appropriate amount of Trypsin/EDTA was added.
3- Flask was incubated from 5 to 30 min (or until cells are visibly detached from
surface) in incubator.
4- Repeatedly pipette up and down was performed with 9 ml pre-warmed media
and transferred cells to 15 ml conical
5- About 1 ml of cells added to a new plate containing 9 ml pre-warmed media
and the cells were returned to incubator.
2.2.6.3. Cell Counting
Many biological applications such as microbiology, cell culture, MTT, fluorescent
microscopy and many others that use cells require that we determine cell
concentration for our experiment. Cell counting is rather straightforward and
requires a counting chamber called a hemocytometer.
First cells were detached by trypsin based on the mentioned protocol. 50 µl of cell
suspension and 50 µl trypan blue were mixed together and then injected into each
side of hemacytometer. The cells counted in 5 out of the 9 boxes of the grid using
10x objective with phase contrast. If a cell is stained blue, then it is dead and should
36
not be counted. Then the average of five boxes was multiplied by 10,000 for amount
of cells per ml.
2.2.7. MTT assay
The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell
viability. This test is based on the ability of viable cells to convert a soluble
tetrazolium salt [3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
(MTT) into an insoluble formazan precipitate. Viable cells with active metabolism
convert MTT into a purple colored formazan product with an absorbance maximum
near 570 nm. The quantity of formazan is measured by recording changes in
absorbance at 570 nm using a plate reading spectrophotometer (fig. 2-5).
Figure 2-5. MTT reduction in live cells by mitochondrial reductase results in the
formation of formazan
37
MTT assay procedure
Cells were trypsinized and centrifuged at 1000 rpm for 5 min. Then cells were
resuspended and counted and diluted to receive 1.5 × 104 cells in 200 µl in each well.
A549 cells were seeded into 96-well plates at a density of 1.5 × 104 cells/ well and
pre-incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO2 in air. The
next day, different concentrations of peptides and Gem-CPP conjugates were added
to the culture medium (all experiments were performed in triplicate). Cells were
incubated at 37 °C for 72 h. Then the medium was removed and the wells were
washed with PBS. The MTT assay was performed by adding 50 μl of 2 mg/ml MTT
to each well for 4 h. MTT solution was removed and resulting formazan crystals
were dissolved by 200 μl DMSO and 25 μl Sorensen buffer then the absorbance of
individual wells was obtained at 570 nm. Untreated cells were defined as 100%
viable. The cell viability was calculated using following formula:
Cell viability = 𝑂𝐷 𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑡𝑒𝑠𝑡/𝑂𝐷 𝑣𝑎𝑙𝑢𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100
2.2.8. Fluorescent Microscopy
The cellular uptake of the FITC-labeled peptide was examined in A549 cell line.
A549 cells were seeded with RPMI 1640 medium on coverslips in 6 well plates and
allowed to adhere overnight. Then the medium was removed and washed with PBS.
The cells were treated with FITC-labeled peptide and their conjugates diluted in
38
serum free RPMI for 1.5 h at 37 °C. After incubation, the media containing the
compound were removed followed by washing with PBS three times. Cells were
fixed on the coverslips by formaldehyde. The most widely used chemical fixative is
formaldehyde, which shows broad specificity for most cellular targets. It reacts with
primary amines on proteins and nucleic acids to form partially-reversible methylene
bridges. To make 10 ml of fixative, 1ml of 37% formaldehyde mixed with 9 ml PBS.
In order to fixing the cells, 2 ml of fixative added to the cells and then the coverslips
were washed three time by PBS. Then coverslips were placed on a microscope slide.
2.2.9. Flow cytometry
A549 cells were seeded with RMPI 1640 medium in 6-well plates (3×105 cells/well)
24 h prior to the experiment. After 24 h, the medium was removed and washed with
PBS. Then FITC-labeled peptide and related nanoconjugates was added to the cells.
The plates were incubated for 1.5 h at 37 °C. After that, the medium containing the
peptide was removed. The cells were washed three times with PBS and detached
with 0.25% trypsin/EDTA (0.53 mM) for 5 min. Then to the each well, 2 mL of the
medium was added and centrifuged for 4 min. Cells were washed twice with PBS
and finally were resuspended in flow cytometry buffer and analyzed by
FACSCalibur.
39
Chapter four:
Results
40
3.1. Synthesis of CPPs
All the peptides were prepared manually by solid phase peptide synthesis on the
Rink-Amide AM resin. Kaiser test was used for controlling the completeness of each
deprotection and coupling step. After synthesis, each resin-bound peptide was dried
overnight, washed, and cleaved by cleavage cocktail to afford CPPs, which were
lyophilized. Table 3-1 display sequence, molecular weight, isoelectric point, and net
charge of the peptides prepared in this work.
Table 3-1. Sequence and other parameters of the synthesized CPPs
CPP Sequence
Molecular
weight (g/mol)
Isoelectric
point
Net charge
at pH 7
Estimated
solubility
R9 RRRRRRRRR 1422.72 pH 14 10
Good water
solubility
R5W3R4 RRRRRWWWRRRR 1981.36 pH 14 10
Good water
solubility
[RW]6 RWRWRWRWRWRW 2071.44 pH 14 7
Good water
solubility
[RW]5 RWRWRWRWRW 1729.04 pH 14 6
Good water
solubility
[RW]4 RWRWRWRW 1386.63 pH 14 5
Good water
solubility
41
[RW]3 RWRWRW 1044.23 pH 14 4
Good water
solubility
E9 EEEEEEEEE 1179.07 pH 3.3 -8
Good water
solubility
R: Arginine, W: Tryptophan, E: Glutamate
3.2. Cytotoxicity of synthesized CPPs
As a functional delivery vector of anti-cancer drugs, blank CPPs must have high
uptake efficiency with low levels of toxicity against cells (65). Therefore, all
peptides were examined for their toxicity in A549 cells before examining the cell
toxicity of Gem-CPP conjugates. The cytotoxicity was determined by the MTT assay
after 72 h of peptide exposure at concentration range between 5 to 50 µM. As shown
in figure 3-1, the peptides exhibited no toxicity up to a concentration of 10 µM.
Among six CPPs, [RW]4 and [RW]3 did not show toxicity even at 50 µM. However
R5W3R4, R9, and [RW]6 exhibited cell toxicity value of 9%, 12% and 16% at
concentration of 25 µM, respectively. At 50 M concentration, cell death caused by
R9, R5W3R4, [RW]6, and [RW]5 were 37%, 28%, 34% and 14% respectively. The
data revealed that R9 has the greatest toxicity on A549 cells at maximum dose.
42
Figure 3-1. Cytotoxicity of peptides on A549 cells after 72 h incubation.
3.2.1. Effect of poly-glutamate on toxicity of
cationic CPPs
One of the aim of this study was to investigate the effect of E9 interaction on the
uptake of synthesized cationic CPPs. For this purpose we selected three of the six
peptides and the effect of E9 was evaluated. Surprisingly, although poly-glutamate
had not significant effect on the uptake of peptides, but dramatically decrease
cytotoxicity of peptides. The following section discuss the effect of E9 on toxicity
and uptake of R9, R5W3R4, and [RW]6 in A549 cells line.
0
20
40
60
80
100
120
R5W3R4 [RW]6 [RW]5 [RW]4 [RW]3 R9
5 µM 10 µM 25 µM 50 µM%
cel
l via
bil
ity
43
Size of nanoconjugates
Zetasizer was used for size determination of CPPs after interaction with poly-
glutamate. Figure 3-2 demonstrates size distribution of three nanoconjugates. The
obtained data showed that after interaction with E9, R9 formed nanosized structures
with diameter of about 300±50 nm. Because of positive charge of R9, it interacts
with E9 electrostatically and forms large nanoparticles. Conjugation of E9 to the
R5W3R4 results in smaller particles than the R9 with diameter of 170±35 nm. In
the case of [RW]6 that contains alternative arginine and tryptophan residues, after
interaction with poly-glutamate smallest nanostructures with diameter of 32±19 nm
were formed.
Figure 3-2. Size distribution of [RW]6/E9, R5W3R4/E9, and R9/E9 nanoparticles.
44
SEM image of CPP-E9
The morphology of nanostructures after interaction of three CPPs with E9 were
characterized using scanning electron microscopy. Figure 3-3 demonstrates the SEM
images of three CPPs. SEM image of R9/E9 exhibited rod-shaped nanosized
structures. Nona-arginine SEM image showed spherical structure with size of about
40 nm. Similar to R9, interaction of poly-glutamate with R5W3R4 resulted in
formation of smaller rod-shape nanostructure. SEM image of [RW]6 revealed that
interaction of E9 has no effect on the shape and size of this peptide. The size of
[RW]6 after and before adding E9 was about 30 nm and its shape was spherical in
both cases. Taken together, the shape of three CPPs before interaction with E9 is
spherical with size below 60 nm. Interaction of E9 resulted in formation of rod-
shaped structure in case of R9 and R5W3R4.
45
Figure 3-3. SEM images of R9, R9/E9, R5W3R4, R5W3R4/E9, [RW]6 and
[RW]6/E9 (10 mM).
Cytotoxicity study of peptides and CPP-E9 conjugates
To investigate the cytotoxicity of peptides and the effect of E9 conjugation on the
cytotoxic effects of peptides, MTT assay was performed in A549 cell line. Peptides
and their conjugates in concentrations up to 50 were exposed to cells for 72 hours.
The results showed that R9 had toxicity at concentrations higher than 25 µM. The
cytotoxicity data demonstrated that the toxicity of R9 was concentration-dependent
(Figure 3-4 A). As shown in figure 3-5, R9 displays IC 50 value of 66 µM against
46
A549 which is increased to 160 µM after conjugation with E9. R5W3R4 (IC50=72
µM) was less toxic against A549 than R9 (Figure 3-4 B, Figure 3-5). Conjugation
with E9 resulted in decreased toxicity of R5W3R4 (IC50=125 µM). [RW]6, that
had no cytotoxicity at 10 µM, showed cell toxicity at 50 µM (Figure 3-4 C),
displaying IC 50 values of 76 µM (Figure 3-5). Conjugation with E9 had no
significant effect on the [RW]6 cytotoxicity (IC50 of 80 vs. 76 µM )
47
Figure 3-4. Toxicity of peptides and their nanoconjugates on A549 cells determined
by MTT assay. The cells were incubated for 3 days in 10% FBS with or without
peptides (10, 25, and 50 µM) and analyzed for proliferation.
48
Figure 3-5. IC50 of three peptides after and before interaction with E9.
3.3. Cellular Uptake Studies of CPPs and CPP-E9
conjugates
To explore the effect of electrostatic interaction of E9 with arginine and tryptophan-
rich CPP on the uptake and intracellular localization of the peptides their
internalization into A549 cells was investigated using fluorescent microscopy.
Figures 3-6, 3-7 and 3-8 show the intracellular uptake of three peptides and their
nanoconjugates following 1.5 h incubation at 37 °C. It is evident that the cellular
0
20
40
60
80
100
120
140
R9 R5W3R4 [RW]6
peptides alone
peptides/E9IC
50
(µ
M)
49
uptake of peptides and their conjugates were enhanced with increasing the
concentration. The interaction of E9 with Nona-arginine decreased the uptake of the
peptide into the cell at 10 and 50 µM but increased it at 25 µM. Fluorescent images
revealed that after interaction of E9 with R9 the uptake of nanoconjugates into the
cell nucleus was reduced compared to the R9 alone (Figure 3-6). The image of FITC-
labeled R5W3R4 showed that the uptake of the peptide was slightly decreased after
conjugation with E9 at 10 µM. However, E9 has no considerable effect on the uptake
of R5W3R4 at higher concentration (Figure 3-7). The peptide with three tryptophan
in the middle of nine arginine showed highest uptake into the cells at 25 and 50 µM.
Similar to R9, translocation of R5W3R4/E9 to the cell nucleus was slightly
decreased compared to the R5W3R4 alone. The image of [RW]6 showed decreased
uptake of peptide at 10 and 25 µM concentration. However interaction of E9 with
[RW]6 at 50 µM did not affect its uptake into the cells (Figure 3-8). In conclusion
the information of the fluorescent microscopy of three peptides and their conjugates
demonstrates that the uptake of R5W3R4 peptide is better than R9 and [RW]6. Poly-
glutamate interaction probably decreases translocation of CPP into the cell nucleus
but have not considerable effect on the intracellular delivery of the peptides.
By means of flow cytometry the relative amounts of internalized peptide in
concentration 10 to 50 μM were obtained after 1.5 h incubation at 37 °C. The mean
cellular fluorescence was increased with increasing the concentration for all tested
50
peptides. Figure 3-9 shows the relative fraction of positive cells (%) after treatment
with peptides and their conjugates. The data of flow cytometry indicated decrease
in percent of positive cells after interaction of R9 with E9 at 10 and 50 and increased
level at 25 µM. The cellular uptake of R5W3R4 was slightly decreased by E9 in all
concentrations. R5W3R4 exhibited best uptakes into the A549 cell at 50 µm. The
flow cytometry data of R9 and R5W3R4 and their conjugates with E9 are consistent
with fluorescent imaging data. E9 exhibited no effect on the cellular uptake of
[RW]6.
51
Figure 3-6. Fluorescent imaging of A549 cells incubated with 10, 25, and 50 µM
FITC-labeled R9 and R9/E9 for 1.5 h at 37 °C. Control cells were incubated in
RPMI medium without the peptides.
52
Figure 3-7. Fluorescent imaging of A549 cells incubated with 10, 25, and 50 µM
FITC-labeled R5W3R4 and R5W3R4/E9 for 1.5 h at 37 °C. Control cells were
incubated in RPMI medium without the peptides.
53
Figure 3-8. Fluorescent imaging of A549 cells incubated with 10, 25, and 50 µM
FITC-labeled [RW]6 and [RW]6/E9 for 1.5 h at 37 °C. Control cells were incubated
in RPMI medium without the peptides.
54
Figure 3-9. Cellular uptake of FITC-labeled peptides and peptides/E9 in live A549
cells after incubation for 1.5 h at 37 °C. The uptake was measured as the relative
fraction of positive cells (%) from flow cytometric analysis of all live cells positive
for the fluorophore.
3.4. Cellular Uptake studies of six peptides
The uptake and intracellular localization of six FITC-labeled peptides were
examined by fluorescent microscopy. Figures 3-10 shows the intracellular
distribution of the prepared peptides following 1.5 h incubation at. The uptake of all
peptides was examined at concentration of 25 µM because of low toxicity of their in
this concentration. [RW]5 exhibited the lowest uptake into the cells among six
55
peptides. After entering the cell, much of [RW]5 accumulates around the nucleus.
R5W3R4, [RW]6 and [RW]4 displayed homogeneous staining throughout the
intercellular space and interestingly also stronger intensity in cellular structures that
are morphologically identified as the cell nucleus and nucleoli. Fluorescent image
revealed that [RW]3 enters mainly into the cell nucleus.
Flow cytometry was used to determine the relative amounts of internalized peptide
after 1.5 h incubation at 37 °C. After the incubation, cells were treated with trypsin
to remove the cell surface-bound peptides. The mean cellular fluorescence was
increased with increasing the concentration of CPPs. Figure 3-11 shows the relative
fraction of positive cells (%) after treatment with peptides. The percentage of cell
fluorescence was increased with increasing concentration of all peptides. This effect
was nearly linear in the tested concentrations. There was an increase in fluorescence
intensity of cells treated with peptides of higher amino acid content. However, the
[RW]5 exhibited lower levels of intracellular fluorescence. R5W3R4 and [RW]3
with three tryptophan showed maximum intracellular fluorescence relative to other
peptides.
56
Figure 3-10. Fluorescence microscopy, visualization of FITC-labeled, R5W3R4
(A,B), [RW]6 (C,D), [RW]5 (E,F), [RW]4 (G,H), and [RW]3 (I,J) in A459 cells.
The top photos show fluorescence microscopy and the bottom bright field of A459
cells. Live cells were treated with 25 µm of peptides for 1.5 h at 37 °C. Control cells
were incubated in RPMI medium without the peptides.
57
Figure 3-11. Cellular uptake of FITC-labeled peptides in live A549 cells after
incubation. The uptake was measured as the relative fraction of positive cells
(%) from flow cytometric analysis of all live cells positive for the fluorophore.
Measurement of drug loading
After completion of coupling reaction between each peptides and GEM, the amount
of drug loading was calculated by using U.V spectroscopy. We determine amount
of drug from the washing (non-conjugated drug) after coupling completion. Then
the loaded drug for each CPPs was calculated which showed in Table 3-2
58
Table 3-2. Amount of drug loading efficiency
CPP-GEM conjugates
Amount of drug in 5 mg of each CPP-GEM conjugates
Wt% drug (mg) in 5 mg of each CPP-GEM conjugates
Drug loading efficiency
R5W3R4-GEM 0.58 11.6 15 %
[RW]6-GEM 0.625 12.5 14 %
[RW]5-GEM 0.46 9.2 13.8 %
[RW]4-GEM 0.525 10.5 16.4 %
[RW]3-GEM 0.7 14 17.6 %
3.5. Anti-tumor performance of drug loaded
CPPs
In this study, the anti-tumor activity of Gem or Gem–CPP were investigated using
the MTT test after 72 h incubation. The activity of drug-peptide conjugates, Gem-
R5W3R4, Gem-[RW]6, Gem-[RW]5, Gem-[RW]4 and Gem-[WR]3, was evaluated
and compared with that of free Gem (Fig. 3-12). Drug loaded CPPs at concentrations
of less than 10 µM Gem did not exhibit increased anti-proliferative activity
compared to the free Gem drug. However at 15 and 25 µM, Gem-R5W3R4, Gem-
[RW]6 and Gem-[WR]3 conjugates exhibited decreased cell viability. Free Gem at
59
concentration of 15 and 25 showed 20% cell viability. The cell viability value was
reduced to 16% and 6% with Gem-R5W3R4 at 15 and 25 µM, respectively. In the
case of Gem-[RW]6, cell viability was decreased to 14% at 15 and 25 µM. Among
the five peptide-drug conjugates, Gem-[RW]3 displayed the highest cytotoxicity at
15 and 25 µM. The cell viability of Gem was decreased to 9% and 5% when it was
coupled to [RW]3 at 15 and 25 µM respectively Gem. In the case of Gem-[RW]5
and Gem-[RW]4 conjugates, cell viability was slightly increased in comparison to
the free Gem drug.
The enhanced cell toxicity of drug-peptide conjugates could be attributed to the high
cellular uptake tendency of the prepared peptides (66) demonstrated by the flow
cytometry and fluorescent microscopy studies.
60
Figure 3-12. Toxicity of Gem-R5W3R4 (A), Gem-[RW]6 (B), Gem-[RW5] (C),
Gem-[RW]4 (D), Gem-[RW]3 (E) and Gem to A549 cells. The cells were incubated
for 3 days in 10% FBS with or without peptides and analyzed for proliferation
by MTT assay.
61
Chapter five:
Discussion
62
4.1. Effect of CPPs on gemcitabine cytotoxicity
Gemcitabine has a therapeutic activity against a variety of solid tumors (67, 68).
However, this anticancer drug suffer from serious limitations. Gemcitabine has very
short plasma circulation time and high hydrophilicity, resulting in limited
intracellular diffusion. In addition, cancer cells acquire resistance over time, which
becomes a major concern for most gemcitabine-related chemotherapies (69). The
resistance of tumor is related to the mechanism of action of this drug. Transport of
gemcitabine into cell requires both the concentrative (hCNT) and equilibrative
(hENT) nucleoside transporters. Considering that most of the gemcitabine uptake
into cells is mediated by hENT1 transporters, hENT1-deficient cells and decreased
expression of hENT1 confers lower gemcitabine toxicity by blocking the cellular
uptake of the drug (70).
Coupling of anticancer drug to CPP may result in numerous advantages, such as
improved solubility, intracellular uptake, bio-distribution and pharmacokinetic
profiles. CPP-based drug delivery system offers great potential for improving
intracellular delivery of therapeutic agents with poor permeability (66, 71). In this
study, in order to protect gemcitabine from rapid metabolic inactivation and to
improve cell absorption, some pro-drugs was designed by coupling gemcitabine
63
drug to CPP. Furthermore this strategy could be used in fighting hENT1-deficient
and resistant tumor cells by increasing transport of the gemcitabine into the cells.
After synthesis of peptides, the uptake efficiency was investigated. Then,
gemcitabine was covalently attached to the peptides by using succinyl hydrolysable
spacer which allow for the drug release after uptake into the cells (72). The cytotoxic
efficacy of Gem and Gem-CPP conjugates were evaluated. The peptides sequences
were chosen to examine how the presence of tryptophans and their position within
poly-arginine influence the cytotoxic of conjugated Gem drug. It was showed that
the addition of tryptophan to oligo-arginine could increase cellular uptake efficiency.
Peptides with tryptophans in the middle, or evenly distributed along the peptide
sequence exhibited higher uptake than that of nona-arginine. This observation was
consistent with earlier reports (73). With increasing the number of amino acids in
the sequences the toxicity was improved so that Gem-R5W3R4 and Gem-[RW]6
conjugates with 12 residues exhibited the best toxicity in cancer cells.
The results showed that three of five peptides improved cytotoxicity of Gem. Gem-
R5W3R4, Gem-[RW]6 and Gem-[RW]3 conjugates displayed increased toxicity
compared to free Gem. The increased toxicity of these drug-CPP conjugates only
seen at the 15 and 25 µm. One of the possible reasons of this effect maybe contribute
to the mechanism of CPP cellular uptake. Recent studies showed endocytic pathways
to be the major route for internalization of CPPs. Although endocytosis pathway
64
may be responsible for the vast majority of cationic peptide internalization,
numerous evidences suggest that direct penetration does occur at threshold
concentrations (74). It was shown that at low concentration, endocytosis of peptides
may occur which result endosomal entrapment peptides (75, 76). The cargo
molecules delivered into cell by CPP that taken up by endocytosis undergo
endosomal entrapment and possible metabolic degradation. But at higher
concentration (above 10 µm), direct translocation into cell is predominant. With the
direct uptake, the drug molecules delivered by CPP would not fall into endosomal
entrapment. Possibly, direct uptake of Gem-CPP conjugates at higher concentration
into cell is one of the reasons that improve toxicity of Gem. In addition that the Gem-
CPP conjugates could be useful for the cytoplasmic delivery, it also will be a
valuable strategy to overcome drug resistance. The main mechanisms recognized for
multidrug resistance, which is due to the presence of P-glycoprotein in plasma
membrane, that is, a ‘‘pump” that can extrude a wide range of anticancer drugs. The
ability of CPP-drug conjugates to evade the P-gp efflux pump was confirmed using
several assays (66, 77). Even though free anticancer drug diffused into the cell more
easily than CPPs–drug, free drug was rapidly pumped from the resistant cell lines.
But, the drug entered into cell mediated by CPP was not pumped from the resistant
cells, leading to higher toxicity in resistant cell lines.
65
4.2. Poly-glutamate interaction with CPPs
According to the obtained results, noncovalent interaction of E9 with arginine and
tryptophan-rich CPPs can reduce the cytotoxicity of the peptides. Previously
arginine-rich CPPs were studied because of their efficient cell internalization
properties (78, 79). Due to the cationic moiety, they can interact with negative part
of cell membrane and efficiently enter to the cell. Therefore at higher concentration,
they can improve the cellular uptake. There is not sufficient number of studies that
could address the toxicity of the present CPPs in cells. In some studies it was
reported that these peptides are non-toxic at low concentration, but in higher dose
they exhibit significant cytotoxicity (80-86). A good CPP to be used as carrier of a
molecule must exhibit no toxicity against cancer cells and healthy cells. Recently, it
was reported that masking a positively charged CPP, with a negatively charged poly-
glutamate that is covalently attached, can reduce toxicity of peptide in vivo (87). We
hypothesized that noncovalent interaction between cationic peptides and poly-
glutamate could reduce the cytotoxicity of the CPPs while maintain their uptake
efficiency in vitro.
Recent studies using mammalian cells showed endocytic pathways particularly
macropinocytosis to be the major route for internalization of CPPs (88, 89). For
66
example transport of short arginine-rich CPPs into the cell was shown to be occurred
via endocytosis-mediated uptake at low concentration (75). According to this
mechanism, cationic peptides are first simply adsorbed to the anionic moieties, such
as heparan sulfate, sialic, or phospholipidic acid of the cell membrane (19, 90).
However, there are evidences indicating that the uptake mechanisms of arginine-
rich peptides could differ according to several factors such as peptide sequence,
peptide concentration, cell type, and culture medium (90-92). Although
endocytosis pathway may be responsible for the vast majority of cationic peptide
internalization, numerous evidences suggest that direct penetration does occur at
threshold concentrations (93). Recently, it was proposed that CPPs enter to the cell
at higher concentration via direct translocation. The direct translocation of peptides
across cellular membranes include the “inverted micelle model”, the models
involving the formation of membrane pores and the “carpet model” (45, 94).
Cationic peptides accumulate on the cell membrane and eventually lead to form a
pore, through which they can enter into the cell. The formation of pore at high dose
of cationic peptides result in destruction of the cell membrane which may lead to
cell death (95).
When these arginine and tryptophan-rich CPPs were used without interaction by E9,
they enter into the cell at doses below 20 µM, and show no toxicity against A549
cell lines. At higher doses, they transport into the cell directly and can disrupt the
67
cell membrane that could result in cell death. For arginine-rich CPP the potential for
direct translocation is thought to be related to the ability of the guanidinium of
arginines to form bidentate hydrogen bonds with membrane lipids and to induce
pores in artificial membranes (96). Regarding the point that E9 has net negative
charge, when it is added to the cationic peptides solution, rod-shaped nanostructures
is formed by electrostatic interaction. The SEM image and zeta size showed that the
shape of peptide before interaction with E9 is spherical with size below 60 nm.
Interaction of R9 and R5W3R4 with E9 resulted in rod-shaped nanostructures with
size range between 130-320 nm. Guterstam et al. have reported that the pathway for
cellular uptake of oligo-arginine is dominated by direct membrane translocation,
whereas the pathway for translocation of negatively charged oligonucleotide
mediated by oligo-arginine is dominated by endocytosis (97). Poly-glutamate also
has negative charge similar to oligonucleotides that could change uptake pathway of
cationic CPPs. It is possible that the uptake of this rod-shaped nanostructure occurs
via endocytosis that reduces toxicity of R9/E9 and R5W3R4/E9 compared to R9 and
R5W3R4 alone (fig. 4-1). The SEM image showed that size and shape of [RW]6 is
not changed after adding E9. The size of [RW]6 after and before adding E9 was
about 32 nm with spherical shape. E9 did not change the IC50 of [RW]6. This might
be due to difference in secondary structure of [RW]6 compare to other tow peptides
which cannot interact with E9 similar to R9 and R5W3R4 (98).
68
Direct translocation is one of the possible reasons for efficient uptake of CPPs by
the cells. Data of FACS analysis showed that in the case of R9 and R5W3R4 uptake
efficiency was slightly decreased after interaction with E9. One of the possible
reasons for decrease uptake of these peptides is the endo-lysosomal entrapment of
the transduced peptides via endocytosis (76). Furthermore, translocation of these
cationic CPPs after interaction with E9 occur via endocytosis that reduce uptake
efficiency of CPPs compared to direct translocation. However in the case of [RW]6,
the uptake efficiency was not changed after conjugation with E9. Likewise the
cytotoxicity of [RW]6 was unchanged after addition of E9.
69
Figure 4-1. Schematic drawing representing the effect of E9 on the uptake and
cytotoxicity of arginine and tryptophan-rich CPPs.
4.3. Conclusion
In conclusion, the results showed coupling of Gem to three of five peptides including
R5W3R4, [RW]6 and [RW]3 caused increase antitumor activity of drug at high
concentration. Collectively, the findings in this study support the advantages of
using CPPs for improving intracellular delivery of drugs into tumor as well as their
70
activity. Furthermore, it is possible to overcome gemcitabine resistance associated
with deficiencies in the expression of hENT1 by using CPP strategy.
Also, the obtained results indicate that the cytotoxicity of CPPs could be reduced by
poly-glutamic acid (E9) with slight decrease in uptake efficiency. This observation
may be attributed to the altered uptake mechanism in the presence of E9 i.e. from
direct translocation (pore formation) to endocytosis (fig4-1). This effect may allow
the use of higher amount of CPPs for more efficient drug and gene delivery with
reduced side effects. However, the usefulness of these nanoconjugates for drug and
gene delivery should be examined using model drugs. Moreover studying the effect
of interaction of E9 with CPP on the secondary structure of peptides could be of
importance to explain the exact mechanism of obtained results.
71
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penetrating peptides: a comparative membrane toxicity study. Analytical biochemistry.
2005;345(1):55-65. Epub 2005/09/03.
81. Stalmans S, Wynendaele E, Bracke N, Gevaert B, D’Hondt M, Peremans K, et al. Chemical-
functional diversity in cell-penetrating peptides. PloS one. 2013;8(8):e71752.
82. Tunnemann G, Ter-Avetisyan G, Martin RM, Stockl M, Herrmann A, Cardoso MC. Live-cell
analysis of cell penetration ability and toxicity of oligo-arginines. Journal of peptide science : an
official publication of the European Peptide Society. 2008;14(4):469-76. Epub 2007/12/12.
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83. Verdurmen WP, Wallbrecher R, Schmidt S, Eilander J, Bovee-Geurts P, Fanghänel S, et al.
Cell surface clustering of heparan sulfate proteoglycans by amphipathic cell-penetrating peptides
does not contribute to uptake. Journal of controlled release. 2013;170(1):83-91.
84. Drin G, Cottin S, Blanc E, Rees AR, Temsamani J. Studies on the internalization mechanism
of cationic cell-penetrating peptides. The Journal of biological chemistry. 2003;278(33):31192-
201. Epub 2003/06/05.
85. Maiolo JR, Ferrer M, Ottinger EA. Effects of cargo molecules on the cellular uptake of
arginine-rich cell-penetrating peptides. Biochimica et biophysica acta. 2005;1712(2):161-72.
Epub 2005/06/07.
86. Lorents A, Kodavali PK, Oskolkov N, Langel U, Hallbrink M, Pooga M. Cell-penetrating
peptides split into two groups based on modulation of intracellular calcium concentration. The
Journal of biological chemistry. 2012;287(20):16880-9. Epub 2012/03/23.
87. Aguilera TA, Olson ES, Timmers MM, Jiang T, Tsien RY. Systemic in vivo distribution of
activatable cell penetrating peptides is superior to that of cell penetrating peptides. Integrative
biology : quantitative biosciences from nano to macro. 2009;1(5-6):371-81. Epub 2009/12/22.
88. Liu BR, Lo S-Y, Liu C-C, Chyan C-L, Huang Y-W, Aronstam RS, et al. Endocytic Trafficking of
Nanoparticles Delivered by Cell-penetrating Peptides Comprised of Nona-arginine and a
Penetration Accelerating Sequence. PloS one. 2013;8(6):e67100.
89. Lee SH, Castagner B, Leroux J-C. Is there a future for cell-penetrating peptides in
oligonucleotide delivery? European Journal of Pharmaceutics and Biopharmaceutics.
2013;85(1):5-11.
90. Kosuge M, Takeuchi T, Nakase I, Jones AT, Futaki S. Cellular internalization and
distribution of arginine-rich peptides as a function of extracellular peptide concentration, serum,
and plasma membrane associated proteoglycans. Bioconjugate chemistry. 2008;19(3):656-64.
Epub 2008/02/14.
91. Hirose H, Takeuchi T, Osakada H, Pujals S, Katayama S, Nakase I, et al. Transient focal
membrane deformation induced by arginine-rich peptides leads to their direct penetration into
cells. Molecular therapy : the journal of the American Society of Gene Therapy. 2012;20(5):984-
93. Epub 2012/02/16.
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93. Palm-Apergi C, Lonn P, Dowdy SF. Do cell-penetrating peptides actually "penetrate"
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2012;20(4):695-7. Epub 2012/04/05.
94. Herce HD, Garcia AE, Litt J, Kane RS, Martin P, Enrique N, et al. Arginine-rich peptides
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cell-penetrating peptides. Biophysical journal. 2009;97(7):1917-25. Epub 2009/10/07.
95. Langel Ü. Cell-Penetrating Peptides: Methods and Protocols: Humana Press; 2010.
96. Verdurmen WP, Thanos M, Ruttekolk IR, Gulbins E, Brock R. Cationic cell-penetrating
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2010/07/14.
97. Guterstam P, Madani F, Hirose H, Takeuchi T, Futaki S, El Andaloussi S, et al. Elucidating
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98. Rydberg HA, Carlsson N, Norden B. Membrane interaction and secondary structure of de
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81
6. Persian part چکیده:
مقدمه:
جمسیتابین یک داروی ضد سرطان بوده که در درمان سرطان های پانکراس، ریه، مثانه و پستان موثر است. با
ناقل جهت عبور از غشا، هیدروفیلیسیته به این وجود این دارو چند عیب از قبیل نیمه عمر پلاسمایی پایین، نیاز
بالا و وجود مقاومت در بعضی سلول های سرطانی دارد. در سال های اخیر نشان داده شده که پپتید های نفوذ گر
سلولی قادر هستند نفوذ پذیری مولکول های اتصال یافته به خود را به درون سلول افزایش دهند.
اهداف:
اسید آمینه آرژنین و تریپتوفان به صورت بلوک و یا 12تا 6پپتید کوچک که حاوی در این مطالعه ابتدا چند
. سپس جمسیتابین به پپتید ها متصل شده و میزان سمیت آن بر روی رده سلولی بود سنتز شدیک در میان
A549 شدپپتید بررسی -در حالت آزاد دارو و کونژوگه دارو.
روش کار:
ا روش سنتز پپتید در فاز جامد ساخته شدند. سپس میزان ورود به سلول و توزیع پپتید ها به صورت دستی ب
ر د به وسیله فلوسیتومتری و میکروسکوپ فلورسنت بررسی شد. FITCدرون سلولی پپتید های لیبل شده با
82
ژوگه ونن سمیت هر پپتید و کمرحله بعد دارو توسط لینکر سوکسینیک آنهیدرید به هر پپتید متصل شده و میزا
مورد بررسی قرار گرفت. MTT دارو با روش-پپتید
:ایجبحث و نت
-GEM-R5W3R4, GEMدارو شامل -پپتید سنتز شده، سه کونژوگه پپتید 5داده ها نشان دادند که از بین
[RW]6, GEM-[RW]3 25 ,15افزایش سمیت را در مقایسه با دارو در غلظت های µM .ایجاد میکند
نفوذ پذیری دارو را به درون سلول افزایش داده و همچنین خروج آزادانه دارو را نیز ار درون سلول این پپتید ها
کاهش میدهند.
نتیجه گیری:
یافته های این مطالعه نشان داد که استفاده از پپتید های نفوذ گر سلولی باعث افزایش اثر بخشی جمسیتابین بر
ز یافته را نی تروش امکان از بین بردن سلول های سرطانی مقاوم روی سلول های سرطانی می شود. همچنین این
افزایش می دهد.
83
چکیده فارسی:
یک آنالوگ مهم دئوکسی سیتیدین بوده که به صورت ساختاری مرتبط (Gemcitabine, dFdC)جمسیتابین
قند دئوکسی ریبوز با فلوئور جایگزین شده ′2به دئوکسی سیتیدین بوده که در آن دو اتم هیدروژن در موقعیت
فعال است. این دارو بر روی سرطان های مختلفی از قبیل ریه، پانکراس، مثانه و پستان فعالیت نشان داده است.
لی د توسط دئوکسی سیتیدین کیناز داخل سلودشدن جمسیتابین در درون سلول نیازمند فسفریلاسیون های متع
(dFdCDP)دی و تری فسفات نوکلئوتید می دهد. در میان آنها جمسیتابین دی فسفات بوده که تشکیل مونو،
میتواند باعث مهار dFdCDPمتابولیت های فعال می باشند. (dFdCTP)و جمسیتابین تری فسفات
یک مهار کننده dFdCTPضروری می باشد. DNAو بازسازی ریبونوکلئوتید ردوکتاز شده که برای سنتز
DNA 1راز بوده و منجر به اتمام زنجیره سازی، شکستن رشته و به دام افتادن کمپلکس برش توپوایزومراز پلیم
که برای آپوپتوزیس القا شده توسط جمسیتابین ضروری است، می شود. با این وجود جمسیتابین چند مشکل در
به طور وسیعی در خون و یکسری درمان سرطان دارد. جمسیتابین با توجه به وزن مولکولی پایین آن به سرعت و
ل شده و بنابراین نیمه عمر پلاسمایی یدتب )difluorodeoxyuridine-/, 2/2(از بافت ها به شکل غیر فعال آن
پایینی دارد. بعلاوه انتقال جمسیتابین از غشا سلولی نیازمند ترانسپورتر های نوکلئوزیدی می باشد. بیشترین
عموما خیلی hENT1انجام میشود و سلول هایی با کمبود hENT1ول توسط انتقال جمسیتابین به درون سل
به این دارو مقاوم هستند. همچنین افزایش مقدار مقاومت باعث کاهش اثر دارو شده و بنابراین دارو در دوزهای
84
استفاده اخیربالا بایستی استفاده گردد که باعث اثرات جانبی مانند سمیت کبدی، آنمی و ... میشود. در سال های
)پپتید های نفوذ گر سلولی( برای افزایش نفوذ پذیری دارو رونق فراوانی یافته است. CPPsاز
CPPs گروهی از پپتیدهای کوچک می باشند که میتوانند از غشا سلولی عبور کرده و در این
راستا قادر به انتقال مولكولهای متصل به خود به داخل سلول هستند. این پپتیدها را با
، توالی گذرنده غشا، پپتید PTDsاسمهای گوناگونی از قبیل دامنه های انتقال پروتئینی
معرفی میکنند. در دو دهه گذشته CPP تروجان یا به طور رایج پپتید نافذ سلولی
عنوان سیستم های دارورسانی جدید راه تازه ای را پیش روی محققین ها بهCPPاستفاده از
گشوده است. تحقیقات گسترده ای در مورد این سیستم های داروسانی، مخصوصا بر روی
داروهای ضدسرطانی انجام شده و در بعضی از حوزه ها به نتایج قابل توجهی دست یافته
کشف (HIV-1) 1از ویروس نقص اکتسابی نوع1982 اند. اولین نمونه این پپتیدها در دهه
پپتید، Tatبعد از کشف نام گذاری کردند Tat وجداسازی شد و آنرا با نام پپتید
مگس سرکه Homeodomain Antennapedia اسید آمینه ای مشتق شده از 16نفوذکننده
کشف و یا ساخته پپتیدی توالیهای خاصهای مختلفی از این و در زمان حاضر گونه آمفی فیلیک جدا سازی شد
.شده است
: شامل که میكنند تقسیم دسته سه به منشا، اساس بر را ها CPP محققین از تعدادی
85
مشتقات پروتئینی : شامل پپتیدهای طبیعی میباشد که در موجودات وجود دارد و باعث افزایش نفوذ -1
ماکرومولكول های به داخل سلول میشوند.
2- : Chimeric شامل ترکیب مشتقات پروتئینی به همراه پپتیدهای سنتزی میباشند.
و میشوند دتولی صنعتی یا آزمایشگاهی شرایط در ها آمینه اسید از پپتید سنتز بوسیله که: سنتتیک -3
.ندارد وجود آنها برای طبیعی نمونه
یا با سلولها را به ورود اجازه ها پروتئین این که است این هست مسلم آنچه CPP مكانیسم مورد در
ووالانیک ایجاد پیوندهای به قادر پپتیدها این .میكنند میسر غشا به جدی آسیب بدون و رسپتورها بدون
کاهش و اثر افزایش به منجر که هستند داروها قبیل از دیگر مواد با داخلی الكترواستاتیكی نیروهای یا
مکانیسم ورود به سلول این پپتید ها میتواند میشود. مطالعات مختلف نشان داده اند که آنها سمیت
توالی به صورت اندوسیتوز و یا ورود مستقیم باشد. ، وتلف از قبیل نوع سلول، بارخبراساس شرایط م
مواد مختلفی از قبیل انواع نانوذرات )مانند نانوذرات طلا، نقره، کوانتوم دات(، نانوحامل های دارو )مانند
و ... توسط این حامل siRNAو DNAوها )مانند دوکسوروبیسین، تاکسول(، لیپوزوم ها، میسل ها(، دار
ها به صورت موثر به درون سلول ها انتقال داده شده اند.
86
اهداف
سلولی تبرداش و تنی برون خصوصیات بررسی و پپتید –جمسیتابین های کونژوگه نانو تهیه :کلی هدف
آنها
طرح اختصاصی اهداف
6 محتوی یتوال با آرژنین تریپتوفان، آمینه اسیدهای برمبنای سلولی نفوذگر پپتید ارزیابی و ساخت -1
.اسید امینو 12 الی
شده نتزس اولیگوپپتیدهای سلولی برداشت میزان بررسی و شده سنتز پپتیدهای نمودن فلورسانس -2
. فلوئورسانس میکروسکپی از استفاده با
صوصیاتخ و بارگیری میزان بررسی و نظر مورد اولیگوپپتیدهای به جمسیتابین شیمیایی بارگیری -3
.دارو رهش
MTT روش به ها نانوکونژوگه سلولی سمیت بررسی -4
روش کار
:سنتز نانوذرات پپتیدی -1
که نقش فاز resin AM Amide Rink میلی گرم 125در ابتدا : متورم سازی رزین
اضافه DCM سی سی 2 قرار داده شد و به آن سی سی 25جامد را برای سنتز داشت در داخل یک بشر
87
دقیقه جهت تورم 30 گردید. بشر مورد نظر در دمای اتاق و تحت شرایط بدون نور به مدت
.رزین نگهداری شد
از رزین بوسیله محلول Fmoc فرایند جدا کردن گروه حفاظت کننده Fmoc:جداکردن
در داخل ظرف واکنش انجام گردید، مدت زمان این (% 20)دی متیل فرمامید و پیپریدین
(DMFبار با 4و DCMبار با 4سپس شستشو انجام میشود ) .فرایند با توجه به تست کایزر میباشد
محاسبه مقادیر مواد لازم برای سنتز پپتید: مقدار اسید آمینه لازم برای اتصال با
.آزاد محاسبه شدبرابر مقدار لودینگ رزین باگروه آمین 3رزین،
پس از وزن TBTU مراحل اتصال اسید آمینه به رزین: اسید آمینه مورد استفاده و
به داخل ظرف واکنش TBTU حل شدند. اسید آمینه به همراه DMF میلی لیتر 2شدن در
ریخته و ظرف روی همزن مغناطیسی قرار داده میشود. این مراحل تحت شرایط اتمسفر خنثی
به ظرف واکنش DIPEA دقیقه ماده 1 گاز آرگون ویا نیتروژن( انجام گرفت و بعد از حدود )
دقیقه بود که برای حصول اطمینان از تست 90 اضافه میگردید. زمان انجام این مرحله حدود
کایزر استفاده شد. چنانچه تست کایزر آزاد بودن گروه های آمین رزین و یا اسید آمینه متصل
رزین را نشان میداد کل این مراحل مجددا تكرار میگردید. بعد از بررسی و مثبت بودن اتصال اسید آمینه به
به گروه های آمین بوسیله تست کایزر مرحله
88
شستشو را انجام و مراحل اتصال اسید آمینه و شستشو به همین طریق
برای اسید آمینه های دیگر تكرار میگردید.
1 /1های متصل به رزین ابتدا معادل به اسید آمینه FITC جهت اتصال :ی پپتیدیبه توالFITC اتصال
.)دی کلرومتان: دی متیل فرمامید: پیریدن( حل میگردید12:7:5در نسبت حجمی FITCبرابرمولی رزین،
FITC هایی که گروه حل شده در محلول فوق الذکر به رزین و اسید آمینه Fmoc ،اضافه آنها جدا شده بود
ساعت بر روی همزن مغناطیسی در شرایط بدون نور قرار داده شد. بعد از زمان 12و ظرف واکنش برای مدت
به گروه آمین انتهای اسید آمینه FITC مورد نظر جهت حصول از اتصال
.از تست کایزر استفاده میگردید
:پپتید-تهیه کونژوگه دارو
رید و پپتید ها متصل شد. بعد از سنتز پپتید، سوکسینیک آنهید جمسیتابین به وسیله لینکر سوکسینیل به
DIEPA وDMF ساعت هم خوردن با انجام 2ساعت افزوده می شوند. بعد از 2رزین برای -به پپتید
پپیتد شستشو میشوند. برای اتصال پپتید سوکسینه شده به -آزمون کایسر در صورت تکمیل واکنش رزین
افزوده شده و به پپتید سوکسینه DMA/DMFبه جمسیتابین حل شده در مخلوطی از TEAجمسیتابین،
ساعت تحت هم زدن شدید قرار داده میشود. سپس مطابق روش های 48شده افزوده میشود. واکنش برای
جمسیتابین از رزین جدا شد.-مرحله بعد پپتید
89
ا نیاز به کوکتل برش میباشد. کوکتل برش ه جدا سازی پپتید از رزین: برای جدا کردن این اسید آمینه
phenol /water TIPS / TFA/ (88:2:5:5 v/v/v/v, 3 mL) پپتید مورد-برای جدا سازی رزین
پپتید شستشو داده شد و تحت شرایط خلا به مدت یک شب خشک و -استفاده قرار گرفت. در ابتدا رزین
میلی لیتر 3پپتید -میلی گرم از رزین 30سپس برای محاسبه دقیق مقدار کوکتل برش وزن و به ازای هر
ل کنش ریخته و کوکتپپتید در داخل ظرف وا-محلول کوکتل برش استفاده شد. جهت فرایند جداسازی رزین
ساعت بود که به صورت 2تحت محیط خنثی به آن اضافه گردید. مدت زمان این فرایند حدود
سپس بالن را جهت کاهش حجم محلول درون آن به روتاری متصل به خلا وصل کردیم تا .شدید همزده شد
مزن مغناطیسی قرار داده حجم اولیه برسد. بعد از کاهش حجم محلول بالن روی ه 4/3حجم آن به حدود
ای دی اتیل اتر اضافه گردید. پپتیده TFAبرابر حجم 10شد و تحت اثر گاز آرگون یا نیتروژن، به آن معادل
دقیقه قرار دادیم. این مرحله مجددا سه بارتكرار 4به مدت RPMI 4000 حاصل را در سانتریفیوژ با دور
میگردید.
MTTبررسی سمیت بوسیله ارزیابی
.انجام گرفت MTT دارو بوسیله ارزیابی -بررسی سمیت نانوذرات پپتیدی، دارو و نانوذرات
خانه انتقال یابند که برای دست یافتن به 96ابتدا لازم بود سلولها به پلیت MTT برای انجام
این امر ابتدا سلول های متصل شده به کف فلاسک باید جدا میگردیدند و بعد از شمارش به
90
یافتند. برای مرحله جداسازی در ابتدا محیط رویی فلاسک بوسیله های پلیت انتقال می خانه
میلی لیتر شستشو داده شدند. جهت 5به مقدار PBS پیپت استریل خارج و دو مرتبه بوسیله
دقیقه 5-3ومدت زمان cm2 25 تریپسین برای هر فلاسک μl 500 جدا کردن سلول ها از
اضافه شد و برای به فلاسک FBSنثی شدن اثرات سمی تریپسین ، محیط کشت حاوی استفاده شد. جهت خ
میلی 15جداسازی تریپسین از محیط کشت، آنها را در داخل فالكن تیوب
.دقیقه قرار داده شد 1و به مدت RPMI 1000 لیتری ریخته و در داخل سانتریفیوژ با سرعت
بعد از سانتریفیوژ، تریپسین همراه با محیط کشت دور ریخته شد و به سلولها، محیط کشت
جهت . جدید اضافه گردید
سلول لحاظ گردید که با توجه به 15000خانه حدود 96کشت سلول در هر خانه از پلیت
.انجام گرفت FBS مقدار سلول شمارش شده و رقیق سازی مقدار آن با محیط کشت حاوی
به هر یک از خانه ها اضافه گردید. بعد از انجام μl 200 محیط کشت وسلولها بوسیله سمپلر
ساعت جهت تیمار 24این مرحله پلیت ها در انكوباتور با شرایط توضیح داده شده برای مدت
دارو از غلظتهای -سازی با نانوذرات و دارو قرارداده شد. جهت تیمارسازی سلولها با نانوذرات پپتیدی
ساعت استفاده شد. 72میكرومولار و در سه پلیت برای زمانهای 25و 15، 10، 5
حیط کشت اضافه شد. پلیت ها به به م MTT ابتدا محیط قبلی خارج گردید و پودر MTT رای انجام ب
ساعت در داخل انكوباتور گذاشته شد تا آنزیم 4مدت
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سوکسینات دهیدروژناز کریستال های زرد را به کریستال های آبی فورمازون تبدیل کند. این
کریستال های آبی رنگ قبل از رنگ سنجی نیاز به محلول سازی دارند که این عمل بوسیله
DMSO 200 μl 25و μl برای هر خانه انجام گرفت. در ارزیابی نبافر سورنسو MTT شدت رنگ
.آبی ایجاد شده با مقدار سلول هایی که دارای فعالیت متابولیكی هستند رابطه مستقیم دارند
نانومتر بوسیله دستگاه 570 خانه های پلیت در طول موج رنگ سنجی
صورت گرفت. Tek Bioاسپكتروفتومتر
میکروسکپ فلورسنت:
ساعت در انکوباتور 24خانه به مدت 6سلول در هر خانه در پلیت 510×3با دانسیته 549Aابتدا سلول ها ی
ساعت بر روی سلول ها تیمار شد. سپس 1.5ساعت پپتید های فلورسنته به مدت 24قرار داده شد. بعد از
سه بار شستشو شده و با فرمالدهید فیکس شدند. تصویر برداری توسط میکروسکپ صورت PBSسلول ها با
گرفت.
فلوسیتومتری:
برای بررسی کمی میزان ورود پپتیدها صورت گرفت.
ساعت در انکوباتور 24خانه به مدت 6در هر خانه در پلیت 510×3را با دانسیته A549ابتدا سلول های
PBSساعت با سلول ها تیمار شدند و سه بار با 1.5د های فلورسنته به مدت قرار داده شد. سپس پپتی
92
بعد از شستشو سلول ها توسط تریپسین از کف پلیت ها جدا شدند و بدنبال آن سانتریفیوژ شستشو شدند.
شدند. سلول ها در دستگاه فلوسیتومتری قرار داده شدند تا بررسی خواص فلورسانس آنها صورت گیرد.
ج و بحث:نتای
پپتید ها با روش فاز جامد سنتز شدند. در هر مرحله از سنتز توسط آزمون کایسر میزان تکمیل کوپلینگ بررسی
شد.
سمیت پپتید های سنتز شده:
و 3[RW]پپتید سنتز شده 6ند. اما از میان دمول سمیتی نشان نداومیکر 10پپتید های سنتز شده در غلظت
[RW]4 اما داشتندنز سمیتی نی 50غلظت های حتی در .R9 ،[RW]6 وR5W3R4 سمیت کمی در غلظت
نشان دادند. 50و سمیت تقریبا قابل توجهی در غلظت 25
اثر توالی پلی گلوتامات بر روی میزان ورود و سمیت پپتید ها:
نتایج نشان داد که اینترکشن پلی گلوتامات با پپتید های کاتیونیک مورد مطالعه در این طرح باعث کاهش سمیت
میکرو مول و بعد از واکنش با گلوتامات به 66برابر بود با R9برای IC50آنها می شوند. به گونه ای که میزان
که بعد از اثر آن به 72ر گلوتامات برابر بود با قبل از اث IC50نیز میزان R5W3R4افزایش یافت. برای 160
میکرولیتر افزایش یافت. 125
93
مطالعه میکروسکپ فلورسنت:
بوده و این پپتید بعد از ورود به درون 5[RW]عکس های فلورسنت نشان داد که کمترین میزان نفوذ توسط
د از ورود رنگ آمیزی همگنی بع 6[RW]و 4[RW]و R5W3R4سلول در اطراف هسته تجمع پیدا میکند.
بیشتر وارد هسته سلول میشود. 3[RW]در سراسر سلول نشان داد. همچنین
فلوسیتومتری:
داده های آن نشان داد که با افزایش تعداد اسید آمینه، شدت فلورسنت محاسبه شده نیز بیشتر می شود. هماهنگ
بیشترین شدت 3[RW]و R5W3R4داشته و کمترین شدت و میزان ورود را 5[RW]با عکس های فلورسنت
فلورسنت را نشان دادند.
دارو:-سمیت سلولی کونژوگه های پپتید
اثر خاصی بر روی سمیت نداشته است. اما 10داده ها نشان داد که دارو متصل شده به پپتید در غلظت های زیر
و Gem-[RW]6و Gem-R5W3R4دارو شامل -کونژوگه پپتید میکرو مول سه 25و 15در غلظت های
Gem-[RW]3 .میزان سمیت بیشتری را نسبت به خود دارو به تنهایی نشان دادند
در مجموع نتایج به ما نشان داد که پپتید ها نفوذ گر سلولی قادر هستند نفوذ پذیری داروهای ضد سرطانی را به
ت پیدا کرده در مقابل مدید بر سلول های مقاودرون سلول افزایش دهند. در کارهای آینده اثرات این کونژوگه ج
94
جمسیتابین بررسی خواهد شد. همچنین تاثیر این پپتید ها بر خصوصیات فارماکوکینیتیک جمسیتابین در محیط
in vivo .بررسی خواهد شد
95
دانشگاه علوم پزشكی تبریز
دانشكده علوم نوین پزشكی
کارشناسی ارشد رشته نانو تكنولوژی پزشكیپایان نامه جهت دریافت درجه
:عنوان
پپتید و بررسی خصوصیات برون تنی و سلولی آنها-تهیه نانوکونژوگه های جمسیتابین
صمد موسی فرخانینگارش:
اساتید راهنما: دکتر هادی ولیزاده ، دکتر جاوید شهبازی مجرد
پروین ذاکری میلانی استاد مشاور: دکتر
دانشكده داروسازی، مرکز ریز فناوری دارویی و مرکزتحقیقات کاربردی داروییمحل اجرا:
دانشگاه علوم پزشكی تبریز
2/93-4/11شماره پایانامه: 1394اردیبهشت
96
دانشگاه علوم پزشكی تبریز
دانشكده علوم نوین پزشكی
ارشد رشته نانو تكنولوژی پزشكیپایان نامه جهت دریافت درجه کارشناسی
:عنوان
پپتید و بررسی خصوصیات برون تنی و سلولی آنها-تهیه نانوکونژوگه های جمسیتابین
نگارش:
صمد موسی فرخانی
اساتید راهنما:
دکتر هادی ولیزاده ، دکتر جاوید شهبازی مجرد
استاد مشاور:
دکتر پروین ذاکری میلانی
2/93-4/11شماره پایانامه: 1394اردیبهشت
