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Cell penetrating peptide-based drug delivery system for targeting mildly acidic pH
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Cell penetrating peptide-based drug delivery system for targeting mildly acidic pH
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Content
CELL PENETRATING PEPTIDE-BASED DRUG DELIVERY SYSTEM
FOR TARGETING MILDLY ACIDIC PH
by
Likun Fei
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
May 2014
Copyright 2014 Likun Fei
ii
DEDICATION
I would like to dedicate this thesis to my grandmothers, Gendi Wu and Yuzhen
Qian, who passed away successively in 2009 when I was in the United States. All the
loving times with them are and will always be the precious memories of mine.
iii
ACKNOWLEDGMENTS
I would like to express my appreciation to my mentor, Dr. Wei-Chiang Shen,
for his guidance, enlightenment and support, during my pursuit of this Ph.D. degree.
My appreciation also goes to Daisy Shen for her advice and assistance during my
research and life. Their kindness and generosity make their laboratory a second home
for me when I am away from my home.
I want to express my gratitude to Dr. Jennica Zaro for the helpful discussions of
experiments and the countless revisions of manuscripts throughout my research. My
gratitude also goes to the other committee members, Dr. Sarah Hamm-Alvarez, Dr.
Curtis Okamoto, Dr. Julio Camarero, and Dr. Kwang-Jin Kim, for their constructive
suggestions during all committee meetings. I am also grateful to other faculty and
staff who have offered their help during my study and research in the School of
Pharmacy.
I would like to thank all my fellow students in the laboratory for their friendship
and support. I want to thank other graduate students who have helped me during
some experiments. In addition, I am also thankful to all the other assistance I have
received during my time at the university.
Finally, I would like to thank my parents, Zhongxue Fei and Meifang Qian, for
their unconditional support. I also want to give special thanks to my wife, Hsin-Fang
Lee, who I met in this very laboratory I did my Ph.D. training. Without her
encouragement and support, I might not finish this thesis in time.
iv
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGMENTS iii
LIST OF FIGURES vi
LIST OF SCHEMES viii
LIST OF TABLES ix
ABBREVIATIONS x
ABSTRACT xi
1. INTRODUCTION 1
1.1. CPP as a carrier for intracellular delivery 1
1.2. Challenges in the clinical application of CPP 2
1.3. Targeting mildly acidic site with CPP 4
1.4. Research project for this thesis: from arginine-rich CPP to HE-MAP 5
2. MATERIALS AND METHODS 8
2.1. Materials 8
2.1.1. Cell lines and cell culture supplies 8
2.1.2. Synthesized DNAs and peptides 8
2.1.3. Other materials 10
2.2. Experimental methods 11
2.2.1. Chemical modification of MAP 11
2.2.2. Construction of expression plasmids 14
2.2.3. Production of fusion protein/peptide 15
2.2.4. Labeling of peptides and proteins 18
2.2.5. In vitro assays 19
2.2.6. Animal studies in mice 24
2.2.7. Statistical analysis 25
3. RESULTS 26
3.1. Study of arginine-rich CPPs 26
3.1.1. Surface binding and cell uptake of peptides 26
3.1.2. Subcellular localization of the internalized peptides 31
3.2. Study of chemically modified MAPs 33
3.2.1. Synthesis and characterization of CA-MAP and CA-MAP-F 33
3.2.2. Cell assays for MAP, CA-MAP, and CA-MAP-F 37
3.3. Study of fusion proteins/peptides containing MAP 42
3.3.1. Construction of plasmids 42
3.3.2. Expression and purification of fusion proteins and peptides 44
3.3.3. Surface binding and cell uptake of GST-HE-MAP at different pH 49
3.3.4. The influence of the length of H/E co-oligopeptide 52
3.3.5. HE-MAP mediated pH-dependent cell association 54
v
3.3.6. Imaging studies in mouse xenograft model of human breast cancer 58
4. DISCUSSIONS 62
4.1. Inspiration from the study of arginine-rich CPPs 62
4.2. Exploration with chemically modified CPPs 68
4.3. Transformation of MAP for targeting mildly acidic pH 74
5. CONCLUSIONS 85
6. FUTURE PERSPECTIVES 87
6.1. Mechanistic study for HE-MAP based delivery system 87
6.2. Pre-clinical study for HE-MAP based delivery system 90
REFERENCES 93
vi
LIST OF FIGURES
Figure 1: Elution profiles of size exclusion chromatography for five peptides
after radiolabeling reactions
27
Figure 2: The influence of net charge on surface binding and cell uptake 29
Figure 3: The influence of arginine sequence distribution on surface binding
and cell uptake
30
Figure 4: The amounts of internalized peptides localized in the vesicular
versus cytosolic fractions
32
Figure 5: Elution profile of reaction mixture on a Sephadex G-15 column
after citraconylation of MAP
34
Figure 6: The amount of free amino groups in CA-MAP after incubation at
various pH conditions
35
Figure 7: The viability of cells after treated with MAP, CA-MAP, and acid
pre-treated CA-MAP at various concentrations
35
Figure 8: Elution profile of reaction mixture on a Sephadex G-15 column
after preparation of CA-MAP-F
36
Figure 9: Surface binding and cell uptake of MAP, CA-MAP, and CA-MAP-
F with or without acid pre-treatment
38
Figure 10: Cell uptake and nuclear internalization of MAP, CA-MAP, and
CA-MAP-F
40
Figure 11: Surface binding and cell uptake of CA-MAP and CA-MAP-F with
or without excess folate competition
41
Figure 12: Plasmid construction for the fusion proteins used in this thesis 43
Figure 13: SDS-PAGE analysis and Coomassie blue staining of purified
proteins
46
Figure 14: Purification of HE-MAP 47
Figure 15: Far-UV CD spectra of 25 μM HE-MAP peptide in phosphate
buffer at pH 6.5 and 7.4
49
Figure 16: Surface binding and cell uptake of GST-HE-MAP at pH 6.0-7.5 50
vii
Figure 17: Confocal analysis of GST-HE-MAP internalization 51
Figure 18: The influence of H/E repeat number on the surface binding and
cell uptake of fusion proteins at pH 6.0-7.5
53
Figure 19: Cell association of GST and GST-HE-MAP at pH 6.0-7.5 54
Figure 20: Total cell associated
125
I-GST-HE,
125
I-GST-MAP, and
125
I-GST-
HE-MAP in HeLa cells at 4 different pH conditions
55
Figure 21: Total cell associated
125
I-labeled fusion proteins in MDA-MB-231
cells at pH 6.5 and 7.4
57
Figure 22: In vivo imaging and organ distribution study for IR800-GST-HE-
MAP in MDA-MB-231 tumor-bearing nude mice
59
Figure 23: In vivo imaging of MDA-MB-231 tumor-bearing nude mice after
intravenous injection of IR800-labeled GST-fusion proteins
61
viii
LIST OF SCHEMES
Scheme 1: Reversible blocking of amino group by citraconic anhydride 69
Scheme 2: Potential applications of HE-MAP based delivery system 89
ix
LIST OF TABLES
Table 1: List of cell lines and culture media used in this thesis 8
Table 2: Sequence of synthesized ssDNAs used in this thesis 9
Table 3: Sequence and molecular weight of synthesized peptides used in this
thesis
10
Table 4: The relative percentage of peptide localized in the vesicular versus
cytosolic compartment
31
Table 5: Cloning works for peptide MAP-G
5
-(HE)
10
versus HE-MAP 42
Table 6: Yields of different fusion proteins in TB media and after purification 45
Table 7: Surface binding and cell uptake of six peptides in CHO cells 63
x
ABBREVIATIONS
CPP: Cell penetrating peptide
MAP: Model amphipathic peptide
CA-MAP: Citraconylated MAP
CA-MAP-F: Folate modified citraconylated MAP
(HE)
10
: HEHEHEHEHEHEHEHEHEHE (one-letter amino acid
sequence)
H/E co-oligopeptide: Histidine-glutamate peptide in the form of (HE)
n
HE-MAP: The fusion peptide of (HE)
10
and MAP with a pentaglycine
linker
GST: Glutathione S-transferase
GST-HE-MAP: The fusion protein of GST and HE-MAP
GST-HE: The fusion protein of GST and (HE)
10
GST-MAP: The fusion protein of GST and MAP
xi
ABSTRACT
Properties of different arginine-rich peptides, including net charge and charge
distribution, were evaluated for their influence on surface binding, internalization,
and intracellular localization. The peptides were radiolabeled and subsequently
tested for surface binding and internalization in cells. Subcellular fractionation
assays were performed to separate the amount of peptides associated with vesicles
from those inside the cytosol. Net neutral charged peptides, YGR
6
E
6
and YG(RE)
6
,
showed large decreases in both surface binding and cell uptake compared to their net
positive charged counterparts, YGR
6
G
6
and YG(RG)
6
. The peptides with clustered
arginine residues, YGR
6
G
6
and YGR
6
E
6
, exhibited significantly higher binding and
uptake than those with alternating arginine and glycine/glutamate residues, YG(RG)
6
and YG(RE)
6
. The intracellular distribution analysis for all of the peptides tested
showed that, regardless of the net uptake, the arginine-rich peptides were
preferentially localized in the cytosolic compartment of the cells. Both net positive
charge and a clustered arginine sequence enhance the surface binding and cell uptake
of peptides, however, the intracellular distribution does not change. These initial
findings inspired the design of the following CPP-based drug delivery systems.
Citraconic anhydride was used to modify different CPPs in order to develop a
pH-sensitive delivery system. After the citraconylation, CA-MAP showed very low
surface binding and cell uptake in cell assays. The binding and internalization could
be recovered when the citracoyl groups in the peptide were removed at acidic pH (3-
4). However, this acid facilitated process may not be very efficient inside endosomes
xii
with a mildly acidic pH (~6.5). The addition of folate to CA-MAP as targeting ligand
could increase its cell uptake and nuclear internalization in folate receptor positive
cells. Although several attempts involving chemical modification didn’t lead to a
feasible pH-sensitive CPP for clinical application, the data from these attempts did
prove the concept that the binding and uptake of CPP could be controlled through the
manipulation of its net charge.
Finally, a pH-sensitive CPP, HE-MAP, was produced as a GST-fusion protein
in bacteria. Both radioactive and fluorescent assays showed pH-dependent cell
uptake for GST-HE-MAP in cells, which could be used for targeting mildly acidic
pH. Cell uptake assays also showed that the pH-dependent binding and uptake
profile of GST-HE-MAP could be shifted when the repeat number of H/E co-
oligopeptide was changed. Compared with two other fusion proteins containing
either (HE)
10
or MAP, only the fused peptide, HE-MAP, could effectively deliver the
cargo GST protein to cells at pH 6.5 or below, while maintaining low delivery to
cells at pH 7.0 and above. Using a xenograft mouse model of human breast cancer,
fluorescent imaging showed that only HE-MAP could effectively target GST to the
tumor site, while reducing non-specific association of MAP in other organs. The in
vitro and in vivo data have demonstrated the diagnostic and/or therapeutic potential
of the fused peptide, HE-MAP, for targeting the acidic tumor microenvironment. The
concise design of this pH-sensitive peptide offers a simple way to overcome CPP’s
lack of selectivity, which could lead to increased application of CPPs and
macromolecular therapeutics.
1
1. INTRODUCTION
With the advancement of research and biotechnology, increasingly more
proteins and peptides have been produced and developed as potential therapeutics.
Amongst the over 130 approved protein/peptide drugs, a majority of their therapeutic
targets, such as hormone receptors or cell surface markers, are outside or on the
surface of cell (Leader et al., 2008). On one hand, it is not surprising considering that
these targets are more readily accessible to protein/peptide drugs. On the other hand,
it emphasizes the challenge for proteins or peptides to cross barriers in the form of
cellular membranes.
1.1. CPP as a carrier for intracellular delivery
CPPs are short peptides, usually no more than 30 amino acids, which are able to
accumulate in different intracellular compartments (e.g., cytosol, endosome, and
nucleus) in mammalian cells. They are usually categorized in two groups: cationic
CPPs and amphipathic CPPs. Cationic CPPs have multiple arginine and/or lysine
residues (e.g., Tat peptide, from trans-activating transcriptional activator of Human
Immunodeficiency Virus 1, HIV-1) while amphipathic CPPs exhibit amphipathic
structures (e.g., MAP) (Patel et al., 2007). The mechanism of internalization has yet
to be established, and has been shown to occur through endocytosis-dependent
mechanism (Lundberg and Johansson, 2001; Nakase et al., 2004; Richard et al.,
2
2005), and endocytosis-independent mechanisms (Cahill, 2009; Rothbard et al., 2004;
Thorén et al., 2003), or a combination of both (Lee et al., 2008b; Wender et al., 2008;
Zaro et al., 2006; Zaro and Shen, 2003, 2005b; Zhang et al., 2009).
Since their discovery, CPPs have been considered as promising carriers for
overcoming the intracellular drug delivery obstacle. Initially found in natural
proteins (Frankel and Pabo, 1988; Green and Loewenstein, 1988; Joliot et al., 1991),
these short peptides are required and sufficient for the membrane transport of their
respective proteins (Derossi et al., 1994; Vivès et al., 1997). Currently, many nature-
derived, synthetic, and chimeric peptides have been identified for their cell
penetrating ability (Futaki et al., 2001; Oehlke et al., 1998; Pooga et al., 1998). Not
only do these peptides accumulate in cells, but they are also able to ferry cargo
molecules into the cells, including small molecule drugs, peptides (Perez et al., 1992;
Zaro and Shen, 2005a), proteins (Fawell et al., 1994), RNAs or DNAs (Mo et al.,
2012), and even large particles, such as liposomes (Torchilin et al., 2001). Many
efforts have been made to develop CPPs as drug carriers for intracellular delivery of
hydrophilic macromolecules (Fischer, 2007; Patel et al., 2009; Patel et al., 2007;
Tung and Weissleder, 2003; Vivès et al., 2008), however, with only very limited
success.
1.2. Challenges in the clinical application of CPP
There are many challenges for the application of CPPs as drug carriers for
intracellular drug delivery. One of such hurdles is the cytotoxicity of these peptides
because of their highly cationic nature. Short cationic CPPs, such as Tat peptide,
3
usually show mild to no cytotoxicity, while amphipathic CPPs (e.g., MAP) exhibit
more significant toxic effects (Saar et al., 2005). Since MAP is a more potent carrier
than Tat peptide (Zaro et al., 2009), enough delivery can be achieved at the
concentration which is well below its toxic level (Scheller et al., 1999). Another
hurdle in the application of CPPs is that the attached cargo molecules may change
the uptake efficiency of the conjugates as compared to free CPPs (El-Andaloussi et
al., 2007; Fischer et al., 2002). The cargos may also alter the original intracellular
localization of CPPs after the conjugation, which may jeopardize the final
effectiveness of the delivery system depending on the intended intracellular target
site (Fischer et al., 2002; Tünnemann et al., 2006).
The most challenging problem in the in vivo application of CPPs is caused by
their universal cell penetrating ability regardless of cell types. Due to their cationic
nature, CPPs bind and get internalized in most cell types tested in vitro, and the
ubiquitous uptake of CPPs by all kinds of cells has also been confirmed in vivo
(Schwarze et al., 1999). This lack of specificity becomes a major hurdle for their
clinical application since non-specific distribution to non-target cells leads to the
waste of therapeutics carried by CPPs and possible side effects. Various attempts in
targeting of the CPP-drug molecules via attachment of antibodies or other ligands
have often failed (Hu et al., 2007; Niesner et al., 2002), because the targeting effect
of these molecules is overridden by the strong interaction between CPPs and
negatively charged cell membranes (Vivès et al., 2008). Therefore, improving
selectivity is crucial for the clinical application of CPPs, especially for use through
systemic delivery.
4
1.3. Targeting mildly acidic site with CPP
Despite of CPP’s lack of specificity, some CPP-based delivery systems have
been developed with great targeting efficiency to specific cells or tissues (reviewed
in Huang et al., 2013; Vivès et al., 2008). In these systems, the CPPs are usually
masked or blocked by other components, which reduces the non-specific binding and
uptake caused by CPPs. At target sites, the components that mask or block the CPPs
are removed, which leads to efficient internalization by target cells. For example, an
activatable CPP has been designed for tumor imaging in which the cationic charge is
reversibly masked by attachment of an anionic sequence through a linkage sensitive
to an enzyme secreted by tumor cells (Jiang et al., 2004), but the application of this
approach may be limited by the presence and cleavage efficiency of the specific
enzyme. Another promising method to activate the masked CPPs is to take advantage
of the relatively acidic pH at target sites, such as inside endosomes or the
microenvironment of tumor cells or sites of inflammation (Gerweck and
Seetharaman, 1996; Grinstein et al., 1991; Menkin, 1960; Sipe and Murphy, 1987).
Take the microenvironment of tumor cells as an example, the pH difference
between tumor and normal tissues offers a great opportunity to achieve targeted
delivery to tumor sites. Since the extracellular pH of tumors is only 0.4-0.8 unit
lower than that of normal tissues (Gerweck and Seetharaman, 1996), a tumor-
targeting delivery system must exhibit a high sensitivity in a relative narrow pH
range of 6.5 to 7.4. Acid-labile chemical bonds have been used to create pH-sensitive
delivery systems (Fei et al., 2009; Kale and Torchilin, 2007), however, the cleavage
rate of these chemical bonds at mildly acidic pH may not be efficient enough for
5
systemic application in vivo. Other types of particulate systems including micelles
and polymers are able to achieve this mild pH sensitivity (Lee et al., 2008a;
Sethuraman and Bae, 2007; Yatvin et al., 1980), but may have fallbacks with
premature release rates and inadequate depth of tumor penetration due to their large
size (Dreher et al., 2006; Helmlinger et al., 1997). All these previous studies offer
possible ways to target CPPs to tumors, but their limitations demand more concise
designs targeting to mildly acidic sites effectively after systemic administration.
1.4. Research project for this thesis: from arginine-rich CPP to HE-MAP
Before this project, the research of CPP in our group focused on the mechanism
of uptake, intracellular localization after internalization, and protein/peptide delivery
via direct conjugation. This project started in 2008 with a specific aim to understand
how the chemical properties of CPPs would affect their specificity of internalization
and function as drug carriers. Conceivably, many physicochemical properties, such
as the net charge, sequence, and conformation of the peptides, are very important for
the development of CPP-based drug delivery systems.
Among the various CPPs, oligoarginines have been shown to be efficiently
shuttled into the cell with or without cargo molecules (Melikov and Chernomordik,
2005; Ter-Avetisyan et al., 2009). The simple sequence of the oligoarginine peptides
provides a good starting point to study the sequence-uptake relationship. In the first
part of this project, four arginine-rich CPPs, i.e., YGRRRRRRGGGGGG (YGR
6
G
6
),
YGRGRGRGRGRGRG (YG(RG)
6
), YGRRRRRREEEEEE (YGR
6
E
6
), and
YGRERERERERERE (YG(RE)
6
), were investigated in CHO cells for surface
6
binding, cell uptake and subcellular localization. The effect of net charge on the
peptides’ uptake was investigated by comparing the glycine-containing versus
glutamate-containing derivatives of YGR
6
. In addition, the peptides are designated as
either a blocked formation, containing a stretch of arginine residues (i.e., YGR
6
G
6
and YGR
6
E
6
), or a mixed formation, with alternating arginine and glycine/glutamate
residues (i.e., YG(RG)
6
and YG(RE)
6
). Two formations were compared to find out
how the distribution of arginine would affect these peptides’ uptake.
Based on the findings from first part (Fei et al., 2011), the aim of project shifted
to developing a pH-sensitive CPP by chemical modification. Citraconylation, an
acid-labile reaction, was used to mask the positively charged lysine residues in CPPs.
Later, folate was added onto the modified CPP as a targeting moiety. The resultant
peptides, CA-MAP and CA-MAP-F, were evaluated in HeLa cells for their surface
binding, cell uptake, and nuclear internalization. Competition assays with excess
folate were performed in order to confirm whether the uptake of CA-MAP-F was
mediated by folate receptor.
After many failed attempts with chemical modifications, a CPP-based carrier
peptide, HE-MAP, was finally produced as a GST-fusion protein. A highly pH-
sensitive co-oligopeptide, (HE)
10
, was fused to MAP with a G
5
linker in order to
mask the positive charges at neutral pH. This idea was based on a paper published by
our group over 20 years ago (Shen, 1990), in which a pH-sensitive complex
formation was reported using polylysine and histamine-modified polyglutamate. The
fusion protein, GST-HE-MAP, was first investigated for surface binding and cell
uptake at various pH conditions. The radioactive assay was then validated by the
7
confocal study of fluorescently labeled fusion protein. Both assays generated
promising results that GST-HE-MAP exhibited highly pH-sensitive binding and
internalization (Zaro et al., 2012), which led to more cell assays in order to confirm
the initial findings. Three GST-fusion proteins containing different length of H/E co-
oligopeptide and MAP were compared for their surface binding and cell uptake in
cell assays. In order to evaluate the contribution from either (HE)
10
or MAP, GST-
HE-MAP was compared with two control proteins, i.e. GST-HE and GST-MAP, in
cell assays and later in a xenograft mouse model of human breast cancer,
demonstrating that HE-MAP can target the cargo protein, GST, to cells at mildly
acidic pH in vitro and to tumor site in vivo.
8
2. MATERIALS AND METHODS
2.1. Materials
2.1.1. Cell lines and cell culture supplies
All cell lines used during this research (Table 1) were purchased from ATCC
(Manassas, VA). All culture media and fetal bovine serum (FBS) were from
Mediatech (Manassas, VA). RPMI 1640 powder, RPMI 1640 folate-free medium, L-
glutamine, penicillin-streptomycin, and trypsin-EDTA were from Invitrogen
(Carlsbad, CA). Culture flasks and plates, serological pipettes were from Greiner
Bio-one (Monroe, NC).
Table 1. List of cell lines and culture media used in this thesis
Cell line (origin) Abbreviation Culture medium
Chinese hamster ovary CHO
Dulbecco’s Modified Eagle
Medium/Ham’s F12
(DMEM/F12)
Human cervical adenocarcinoma HeLa DMEM or RPMI 1640
Human breast adenocarcinoma MCF-7 RPMI 1640
Human breast adenocarcinoma MDA-MB-231 RPMI 1640
2.1.2. Synthesized DNAs and peptides
All single-stranded deoxyribonucleic acids (ssDNAs) used in the cloning were
synthesized by ValueGene (San Diego, CA) (Table 2). YGR
6
, YGR
6
G
6
, YG(RG)
6
,
YGR
6
E
6
, YG(RE)
6
, YGE
6
, YG(RO)
6
, MAP, C(HE)
6
, and (HE)
10
were synthesized
using solid phase synthesis by Genemed (San Antonio, TX) (Table 3).
9
Table 2. Sequence of synthesized ssDNAs used in this thesis
DNA sequence (5’ to 3’) Purpose of the sequence
GATCCTATAAATTAGCATTAAAACTGGCTCT
GAAAGCACTGAAAGCAGCACTGAAACTGGC
AGGTGGTGGTGGAGGTCATGAACACGAACAT
GAGCATGAACATGAACATGAGCACGAACATG
AACACGAGCATGAATAAGC
Sense sequence of DNA encoding
peptide MAP-G
5
-(HE)
10
GGCCGCTTATTCATGCTCGTGTTCATGTTCGT
GCTCATGTTCATGTTCATGCTCATGTTCGTGT
TCATGACCTCCACCACCACCTGCCAGTTTCAG
TGCTGCTTTCAGTGCTTTCAGAGCCAGTTTTA
ATGCTAATTTATAG
Anti-sense sequence of DNA
encoding peptide MAP-G
5
-(HE)
10
GGGCTGGCAAGCCACGTTTGGTG pGEX 5’ sequencing primer
CCGGGAGCTGCATGTGTCAGAGG pGEX 3’ sequencing primer
GCGTGGATCCTATAAATTAGCATTAAAACTG
GCTCTG
5’ PCR primer for DNA encoding
peptide MAP-G
5
-(HE)
10
CGAGGCAGATCGTCAGTCAGTCACG
3’ PCR primer for DNA encoding
peptide MAP-G
5
-(HE)
10
GGGAACGCATCCAGGCACATTGG 3’ sequencing primer
GGATCCCATGAACACGAACATGAGCATGAAC
ATGAACATGAGCACGAACATGAACACGAGC
ATGAAGGTGGTGGTGGAGGTAAATTAG
Partial sense sequence of DNA
encoding peptide HE-MAP
GCGGCCGCTTAATATGCCAGTTTCAGTGCTGC
TTTCAGTGCTTTCAGAGCCAGTTTTAATGCTA
ATTTACCTCCACCACCACCTTCATGCTC
Partial anti-sense sequence of
DNA encoding peptide HE-MAP
GCGTGGATCCCATGAACACGAACATGAG
5’ PCR primer for DNA encoding
HE-MAP
CGATGCGGCCGCTTAATATGCCAGTTTCAG
3’ PCR primer for DNA encoding
HE-MAP
GTGTGGATCCCATGAACACGAACATGAGCAT
GAACATGAACATGAGCACGAACATGAACAC
GAGCATGAATTCTCTC
Sense sequence of DNA encoding
peptide (HE)
10
GAGAGAATTCATGCTCGTGTTCATGTTCGTGC
TCATGTTCATGTTCATGCTCATGTTCGTGTTC
ATGGGATCCACAC
Anti-sense sequence of DNA
encoding peptide (HE)
10
GAGTGAATTCAAATTAGCATTAAAACTGGCT
CTGAAAGCACTGAAAGCAGCACTGAAACTGG
CATATCTCGAGTCAC
Sense sequence of DNA encoding
peptide MAP
GTGACTCGAGATATGCCAGTTTCAGTGCTGCT
TTCAGTGCTTTCAGAGCCAGTTTTAATGCTAA
TTTGAATTCACTC
Anti-sense sequence of DNA
encoding peptide MAP
GATCCCATGAAGGATCCCATGAGCATGAACA
TGAACATGAG
5’ primer for deletion of 12
nucleotides (encoding HEHE)
ATCCTCCAAAATCGGATGGATCCCACGAGCA
TGAGCATGAACACGAACATGAG
5’ primer for addition of 12
nucleotides (encoding HEHE)
10
Table 3. Sequence and molecular weight of synthesized peptides used in this thesis
Peptide Single letter amino acid sequence Molecular weight (Da)
YGR
6
YGRRRRRR 1175.4
YGR
6
G
6
YGRRRRRRGGGGGG 1517.7
YG(RG)
6
YGRGRGRGRGRGRG 1517.7
YGR
6
E
6
YGRRRRRREEEEEE 1950.1
YG(RE)
6
YGRERERERERERE 1950.1
YGE
6
YGEEEEEE 1012.9
YG(RO)
6
* YGRORORORORORO*
1860.3
MAP YKLALKLALKALKAALKLA 2040.7
C(HE)
6
CHEHEHEHEHEHE 1718.7
(HE)
10
HEHEHEHEHEHEHEHEHEHE 2680.5
* O stands for L-ornithine.
2.1.3. Other materials
Sephadex G-10, G-15, G-25, G-50, and LH-20 gels, Sepharcyl S-500 gels,
thrombin, and pGEX-4T-1 vector were from GE Healthcare Life Sciences
(Piscataway, NJ). Pfx DNA polymerase, Taq DNA polymerase, and T4 DNA ligase
were from Invitrogen (Carlsbad, CA). All restriction enzymes were from New
England BioLabs (Ipswich, MA). Ampicillin was from Mediatech (Manassas, VA).
Competent bacteria (E. coli, DH5α and JM109) were from ZYMO Research (Irvine,
CA). PageRuler
TM
prestained protein ladder, Spectra
TM
multicolor low range protein
ladder, glutathione (GSH) agarose, HisPur
TM
nickel nitrilotriacetic acid (Ni-NTA)
resin, and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) were from Thermo
Fisher Scientific (Waltham, MA). Isopropyl β-D-1-thiogalactopyranoside (IPTG),
yeast extract, and sodium lauroyl sarcosine (sarkosyl) were from Amresco (Solon,
OH). Sterile syringe filter with polyethersulfone (PES) membrane (0.22 μm), sterile
Stericup
®
filter unit (0.22 μm, PES, 500 mL), phenylmethylsulfonyl fluoride (PMSF),
11
and acrylamide/bis-acrylamide (19:1) were from EMD Millipore (Billerica, MA). 3-
[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and
acrylamide/bis-acrylamide (29:1) were from JT Baker (Central Valley, PA). Reduced
glutathione was from Alfa Aesar (Ward Hill, MA). Dialysis regenerated cellulose
tubing, 12000-14000 molecular weight cut off (MWCO), was from Spectrum
Laboratories (Rancho Dominguez, CA). Microsep
TM
centrifugal device, MWCO at
10 kDa, was from PALL (Port Washington, NY). Radioactive iodine (
125
I) was from
PerkinElmer (Waltham MA). IRDye 800CW N-hydroxysuccinimide (NHS) ester
and IRDye 800CW carboxylate were from LI-COR Biosciences (Lincoln, NE).
Matrigel
TM
basement membrane matrix and peptone were from BD Biosciences
(Bedford, MA). Competent E. coli BL21-T1
R
, protease inhibitor cocktail, rhodamine
isothiocyanate (RITC), and other chemicals were from Sigma-Aldrich (St. Louis,
MO).
2.2. Experimental methods
2.2.1. Chemical modification of MAP
2.2.1.1. Preparation of CA-MAP and removal of citraconyl groups
MAP was dissolved in dimethylformamide (DMF) or dimethyl sulfoxide
(DMSO) at a concentration of 10 mg/mL in a small clean glass bottle. One hundred-
fold molar excess of citraconic anhydrides (liquid) was added to the MAP solution.
The reaction was stirred overnight at room temperature. Next morning, equal volume
of phosphate buffered saline (PBS, pH 7.4) was added to the bottle. In order to
solubilize the precipitations in the mixture, the pH was adjusted to 7-8 by NaOH.
12
Then, the reaction mixture was loaded onto a Sephadex G-15 column (bed volume =
10 mL, PBS, pH 7.4 as mobile phase). Twenty 1 mL fractions were collected and
measured for absorbance at 280 nm (A280). The fractions containing CA-MAP, first
peak from A280 profile, were stored at 4°C in sealed glass tubes.
The concentration of CA-MAP (C
peptide
) was calculated from A280 using a
tyrosine standard curve. The percentage of amino groups modified during the
reaction was determined using 2, 4, 6-trinitrobenzene sulfonic acid (TNBSA) assay.
Briefly, MAP standards or samples were diluted with 0.1 M NaCO
3
buffer (pH 8).
To 0.5 mL standard/sample solution, 0.25 mL 0.01% (w/v) TNBSA was added, and
the mixture was incubated at 37°C for 2 hours. The reaction was stopped by adding
0.25 mL 10% sodium dodecyl sulfate (SDS) and 0.125 mL 1N HCl before it was
measured for absorbance at 335 nm (A335). The concentration of free amino groups
(C
free
) in CA-MAP sample was calculated from A335 using a MAP standard curve.
Since every MAP molecule has six amino groups, the percentage of citraconylated
amino groups in CA-MAP was calculated according to the following equation:
Citraconylation percentage
6
6
100%
The conditions were tested for the removal of citraconyl groups in CA-MAP.
The peptide solution was adjusted to pH 3-4, 5, 6, or 7, and was incubated at 37°C
for 1, 2, or 3 h. After incubation, the pH of peptide solution was raised to 7-8 to stop
the hydrolysis. TNBSA assay was performed for each sample and A335 was plotted
against incubation time for each pH condition. For all the acid pre-treated peptides
used in cell assays, the peptide solutions were adjusted to pH 3-4 using HCl and
13
incubated for ~18 hours at room temperature prior to experiment (Dixon and Perham,
1968).
2.2.1.2. Preparation of CA-MAP-F
On day 1, 50 mM folic acid was dissolved in DMF in a small brown glass bottle.
While stirring, dicyclohexylcarbodiimide (DCC) and NHS in DMF were added
sequentially to the folate solution. The final concentrations for all three reagents
were 25 mM. The reaction was stirred at dark for 1 hour at room temperature before
an overnight incubation at 4°C. Next morning, the mixture was centrifuged at 5000 g
for 10 min at 4°C. The supernatant containing activated folate (folate-NHS ester)
was stored at 4°C in a sealed tube wrapped with aluminum foil. To link folate onto
MAP, MAP was reacted with 5-fold molar excess of activated folate at dark for 4 h
at room temperature. Without purification, the citraconylation of MAP was carried
out in the same container according to the procedures in section 2.2.1.1. On day 3,
the reaction mixture was diluted with same volume of PBS with 10 mM acetic acid
(pH 7.4). The mixture was then loaded onto a Sephadex G-15 column (bed volume =
10 mL, PBS with 10 mM acetic acid, pH 7.4, as mobile phase) after its pH was
adjusted to 7-8 by NaOH. Thirty-five 1 mL fractions were collected and measured
for absorbance at 280 nm and 370 nm (A370). The fractions containing CA-MAP-F,
first peak from elution profile, were stored at 4°C in sealed glass tubes.
The concentration of CA-MAP-F was calculated from the amount of MAP used
in the reaction and the recovery rate for A280 during size exclusion chromatography.
The concentration of folate in peptide solution was calculated from A370 using a
14
folate standard curve. The percentage of amino groups modified during the reaction
was determined using fluorescamine assay, since folate interferes with TNBSA assay.
Briefly, to 0.75 mL standard/sample solution, 0.25 mL fluorescamine (0.6 mg/mL in
acetone) was added for a reaction time of 15 min at room temperature. All standards
and samples were measured by a fluorescence spectrometer (Hitachi, Tokyo, Japan)
at an excitation wavelength (Ex) of 390 nm and an emission wavelength (Em) of 475
nm. The percentage of modified amino groups in CA-MAP-F was calculated using
the same equation in section 2.2.1.1.
2.2.2. Construction of expression plasmids
For GST-HE-MAP, the full-length double-stranded deoxyribonucleic acid
(dsDNA) encoding HE-MAP peptide was acquired by one single elongation step
after partial annealing of two aforementioned ssDNAs in Table 2. Following
polymerase chain reaction (PCR) amplification, the full sequence was cloned into the
pGEX-4T-1 plasmid through BamHI and NotI sites. The inserted sequence was
confirmed by sequencing (GENEWIZ, San Diego, CA).
For GST-(HE)
8
-MAP or GST-(HE)
12
-MAP, one PCR amplification was used to
generate the full-length dsDNA with a deletion or an addition of 12 nucleotides using
the previous plasmid (encoding GST-HE-MAP) as template. The DNA fragment was
then inserted back to pGEX-4T-1 plasmid between BamHI and NotI sites. The
insertion was confirmed by sequencing.
For GST-HE or GST-MAP, 50 μM of both sense and anti-sense sequences were
mixed in a buffer containing 10 mM tris(hydroxymethyl)aminomethane (Tris) and
15
100 mM NaCl. For annealing, the mixture was kept at 95°C for 5 min before it was
slowly cooled down. At 10°C above the melting temperature (T
m
), the cooling was
slowed down to 0.5°C/min until the temperature was 5°C below T
m
. After cooling
down to room temperature, the mixture containing annealed dsDNA was stored at -
20°C. The DNA fragment encoding peptide (HE)
10
was cloned into pGEX-4T-1
plasmid through BamHI and EcoRI sites, and the DNA fragment encoding MAP was
cloned into pGEX-4T-1 plasmid through EcoRI and XhoI sites. Both inserts were
confirmed after sequencing.
2.2.3. Production of fusion protein/peptide
2.2.3.1. Expression and purification of recombinant proteins
The plasmids with correct insertions were transformed into E. coli expression
strain BL21. A test expression was performed for each protein before large-scale
production using Lysogeny Broth (LB) medium. To express recombinant proteins,
bacteria were incubated in Terrific Broth (TB) media with 75 μg/mL ampicillin at
37ºC with 300 rpm shaking speed until the optical density at 600 nm (OD
600
) of the
media reached 2.5-3.0. IPTG was added into TB media to a final concentration of 0.2
mM. After 3-4 hours of additional incubation, the bacteria were collected, 5000 g for
30 min at 4 ºC, and stored at -80ºC. Expression of GST-fusion proteins was
monitored by SDS polyacrylamide gel electrophoresis (PAGE) followed by
Coomassie blue staining.
To purify GST-HE-MAP by Ni-NTA agarose, bacterial pellets were
resuspended in loading buffer (50 mM NaH
2
PO
4
, 300 mM NaCl, and 10 mM
16
imidazole, pH 8.0), and lysozyme was added to reach a final concentration of 0.25
mg/mL. After ~30 min incubation on ice, PMSF was added to 1 mM and Triton X-
100 was added to a final concentration of 1% (v/v). The bacteria were lysed by
sonication (Misonix Ultrasonic Liquid Processors S-4000, Misonix, Farmingdale,
NY) on ice at amplitude 10 for 4-5 min total working time at a 10 sec on/15 sec off
working cycle. The lysate was centrifuged at 15000 g for 30 min at 4ºC. The
supernatant was loaded on Ni-NTA agarose column pre-equilibrated with loading
buffer. The column was washed with wash buffer (50 mM NaH
2
PO
4
, 300 mM NaCl,
and 20-50 mM imidazole, pH 8.0). Fusion protein was eluted with elution buffer (50
mM NaH
2
PO
4
, 300 mM NaCl, and 250 mM imidazole, pH 8.0), and the excess
imidazole was removed by dialysis against PBS, pH 7.4 (MWCO at 12-14 kDa).
To purify GST-HE-MAP, GST-(HE)
8
-MAP, GST-(HE)
12
-MAP, or GST-HE by
GSH agarose, bacterial pellets were resuspended in PBS, pH 7.4, and were processed,
lysed, and centrifuged according to the aforementioned procedures. The supernatant
was loaded on GSH agarose column pre-equilibrated with PBS, pH 7.4. The column
was washed with 1% Triton X-100 in PBS (pH 7.4) and then PBS (pH 7.4) alone.
Fusion protein was eluted with PBS containing 50 mM GSH and 0.5% CHAPS, pH
7.4. The eluted protein was concentrated and exchanged into PBS, pH 7.4, with
Microsep
TM
centrifugal device, MWCO at 10 kDa. For animal studies, GST-HE-
MAP and GST-HE were further purified by HisPur
TM
Ni-NTA resin to reduce the
endotoxin level according to a similar protocol mentioned above.
To purify GST-MAP by GSH agarose, bacterial pellets were resuspended in
PBS, pH 7.4. After the bacterial suspension was treated with 0.25 mg/mL lysozyme
17
on ice for ~30 min, PMSF was added to 1 mM and sarkosyl was added to a final
concentration of 1.5% (w/v). After sonication and centrifugation, CHAPS and Triton
X-100 were added to the supernatant to final concentrations of 30 mM and 3% (v/v),
respectively (Frangioni and Neel, 1993; Tao et al., 2010). The mixture was loaded on
a GSH agarose column pre-equilibrated with PBS, pH 7.4. As mentioned above, the
column was washed and GST-MAP was eluted, concentrated, and exchanged into
PBS, pH 7.4.
During purification, GST-fusion proteins were monitored by absorbance at a
wavelength of 280 nm, and SDS-PAGE with Coomassie blue staining. The band
densities were measured using Quantity One software (BioRad, Hercules, CA) and
used to estimate the purity of fusion protein. All fusion proteins were sterilized by
passing through 0.22 μm syringe filters and stored at 4ºC in sealed tubes.
2.2.3.2. Purification of fusion peptide HE-MAP
GST-HE-MAP was expressed and purified by GSH agarose according to
section 2.2.3.1 until the washing step. While the fusion protein was bound to the
resin, thrombin diluted in PBS (pH 7.4) was loaded onto the column. The on-column
thrombin cleavage was carried out for ~18 h at room temperature. On the next day,
the cleaved peptide was washed off with PBS, pH 7.4, followed by another
purification using Ni-NTA agarose as described in last section. The eluted HE-MAP
was dialyzed against PBS, pH 7.4 (MWCO at 3500 Da), sterilized by passing
through a 0.22 μm syringe filter, and stored at 4ºC. The molecular weight of purified
peptide was confirmed using liquid chromatography followed by mass spectrometry
18
(LC-MS) with the help from Ken Lo in Dr. Clay Wang’s laboratory. The far
ultraviolet (UV) circular dichroism (CD) spectra of HE-MAP at pH 6.5 and 7.4 were
measured (190 to 260 nm) with the help from Dr. Ralf Langen’s laboratory.
2.2.4. Labeling of peptides and proteins
All peptides and proteins used in radioactive cell assay were labeled with
125
I
using the chloramine T method as previously described (Sonoda and Schlamowitz,
1970) and
125
I-labeled peptides or proteins were purified using size exclusion
chromatography (Sephadex G-50 for all proteins, G-15 or G-10 for all peptides). The
fractions containing
125
I-labeled peptides or proteins were determined using a gamma
counter (Cobra II Auto-Gamma, Packard, Downers Grove, IL).
For confocal study, the fusion protein, GST-HE-MAP, was labeled with RITC
(1.5-fold molar excess) overnight at 4ºC, and rhodamine-labeled GST-HE-MAP was
purified using a Sephadex G-50 column (10 mL bed volume, PBS, pH 7.4, as mobile
phase, avoiding light during purification).
For animal studies, the fusion proteins were labeled with IRDye 800CW NHS
ester (IR800) according to the manufacturer’s protocol. Briefly, to achieve a one-to-
one modification ratio, the reactions were carried out at room temperature for 2 h
with a molar ratio of dye to protein at ~4:1. The IR800-labeled proteins were purified
by either Sephadex G-50 or dialysis (MWCO at 12-14 kDa) and sterilized by passing
through 0.22 μm syringe filters.
After labeling, the concentrations of all labeled proteins were determined by
Micro BCA
TM
protein assay kit (Thermo Fisher Scientific, Waltham, MA)
19
2.2.5. In vitro assays
2.2.5.1. Cell viability assay
CHO cells were grown in 96-well plates in DMEM/F12 medium supplemented
with 10% FBS, 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin.
The cells were incubated at 37ºC at 5% CO
2
, and were replenished with fresh
medium the day before confluence. The cells were treated at 37ºC for 2 h with 0, 2.5,
5, 10, 25, 50, 75, or 100 μg/mL MAP, CA-MAP, or acid pre-treated CA-MAP
(mentioned at the end of section 2.2.1) in serum-free DMEM/F12 medium. Then,
the dosing solution was replaced with 100 μL per well 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) solution (1 mg/mL in serum-free
DMEM/F12) for one-hour incubation at 37ºC. The cells were washed with PBS, pH
7.4, before isopropanol was added to each well. The plate was read for absorbance at
560 nm for each well using Infinite
®
F200 microplate reader (Tecan, Männedorf,
Switzerland).
2.2.5.2. Surface binding and cell uptake assay for peptides
For assays with arginine-rich peptides, CHO cells grown for 4 days in 6-well
plates were first incubated with serum-free medium for 10 min at 37ºC before the
treatment in serum-free medium containing 10μg/mL
125
I-labeled peptide and
protease inhibitor cocktail (final concentration in medium: 4 μM AEBSF, 2 μM
EDTA, 0.3 μM bestatin, 30 nM E-64, 2 μM leupeptin, and 0.6 nM aprotinin). After
one hour incubation at 37ºC, the cell monolayers were washed with 1 mL ice-cold
PBS 3 times and detached by treatment with trypsin-EDTA for 3 min at 37ºC. The
20
cells were then collected and centrifuged (IEC PR-J centrifuge) at 600 g for 3 min.
The trypsin-removable supernatant fractions, designated as the surface bound
oligopeptide, were isolated and the radioactivity was determined using a γ-counter.
The cell pellets were washed with ice-cold PBS containing 0.5 mg/mL heparin
(heparin-PBS) followed by ice-cold PBS (pH 7.4), and dissolved in 1N NaOH. The
total cell uptake was determined by measuring the radioactivity and protein
concentration of the cell pellet using a γ-counter and the BCA assay kit, respectively.
The peptide concentrations used in this thesis were not cytotoxic, as determined by
the MTT assay previously in our laboratory.
For assays involving MAP, CA-MAP, or CA-MAP-F, HeLa cells were grown
for 4 days in 6-well plates in DMEM or RPMI1640 medium supplemented with the
aforementioned amounts of FBS, L-glutamine, penicillin, and streptomycin. The
cells were treated with 5 μg/mL of
125
I-peptide in serum-free RPMI1640. In folate
competition assay, the cells were first incubated with serum-free and folate-free
RPMI1640 for 10 min at 37ºC, and subsequently dosed with 5 μg/mL
125
I-labeled
peptide and 4.4 mg/mL folate (about 4000-fold molar excess of
125
I-peptide) in the
same serum-free medium. After the treatment, the cells were processed with similar
procedures mentioned above except that the heparin-PBS wash was replaced with a
PBS wash. The surface binding and the total cell uptake were calculated from the
radioactivity of samples, and the latter was normalized by the amount of protein in
the cell lysate.
21
2.2.5.3. Subcellular fractionation assay
The amounts of vesicle-associated versus cytosolic arginine-rich peptides were
measured using a subcellular fractionation method that has been previously
described and characterized (Zaro and Shen, 2003, 2005b). Briefly, CHO cells were
grown for 4 days in T-75 flasks and, after confluence, were treated with 10 μg/mL
125
I-peptides at 37ºC for 1 h. Most treatment and wash steps were similar to the cell
uptake assay above except that dosing solutions also contained 0.1 mg/mL
fluorescein isothiocyanate-dextran (70 kDa) (FD). The cell pellets were isolated
following treatment with trypsin to remove the majority of the surface bound CPP
(Mueller et al., 2008), and the trypsin removable supernatant was used to estimate
the amount of tested peptides binding to the cell surface. The cell pellets were
washed by ice-cold heparin-PBS (pH 7.4) and PBS (pH 7.4) to extensively remove
the residual surface bound CPP (Gelman and Blackwell, 1973), and then
homogenized in 1 mL ice-cold homogenization buffer (HB) containing 0.25 M
sucrose, 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),
and 2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4, using a Dounce cell
press (60 passages) (Wheaton, Millville, NJ). The cell homogenates were centrifuged
at 600 g (Eppendorf centrifuge 5415-R) at 4ºC for 10 min, and the supernatants were
fractionated using size exclusion chromatography (Sephacryl S-500, 10 mL bed
volume, HB, pH 7.4, as mobile phase). One milliliter fractions were collected and
measured for radioactivity and fluorescence (Ex 494 nm, Em 518 nm) using a
gamma counter and a fluorescence spectrometer, respectively. The amounts of
22
peptides associated with vesicles or in the cytosol were calculated using the
equations previously described (Zaro and Shen, 2003).
2.2.5.4. Nuclear internalization assay
HeLa cells grown for 4 days in 6-well plates were incubated with 5 μg/mL
125
I-
peptides for 1 h at 37ºC. All treatment and wash steps were similar to the procedures
described in section 2.2.5.2. To isolate the nuclei (Beck et al., 1988), cell pellet from
each well was resuspended in TKM/PIC/PMSF buffer consisted of 200 μL TKM
buffer (50 mM Tris, 25 mM KCl, and 5 mM MgCl
2
, pH 7.5) with 1 mM PMSF and
the same level of protease inhibitor cocktail (PIC) as in dosing solution (section
2.2.5.2). After 15 min incubation on ice, sodium deoxycholate (SD) and Triton X-
100 (TX) were both added to 0.4 %. Following 15 sec vortexing and 10 min
incubation on ice, same volume of 500 mM sucrose in TKM/PIC/PMSF buffer was
added before the sample was centrifuged at 600 g for 10 min at 4ºC. The pellet,
containing nuclei, was washed twice with 200 μL TKM/PIC/PMSF buffer containing
250 mM sucrose, 0.4% SD and TX. The nuclear pellet was further washed with 200
μL 250 mM sucrose in TKM/PIC buffer twice before it was dissolved in 0.5 mL 1N
NaOH. The amount of peptides associated with nuclei was calculated from the
radioactivity of the nuclear pellet, and was normalized with the amount of nuclear
protein estimated by BCA assay.
23
2.2.5.5. pH-dependent cell assays for fusion proteins
For radioactive assays, HeLa or MDA-MB-231 cells grown for 4 days in 6-well
plates were first incubated with serum-free media (self-made from RPMI 1640
powder, without NaHCO
3
, with total 10 mM Na
2
HPO
4
and 10 mM citrate/citric acid,
pH 7.2-7.4) for 10 min at 37ºC. In most cases, the cells were treated with self-made
serum-free RPMI 1640 media, adjusted to various pH values, containing 5 μg/mL or
150 nM
125
I-proteins and protease inhibitor cocktail (same final concentration as
given in section 2.2.5.2). For two experimental groups, the MDA-MB-231 cells were
treated with similar dosing solutions containing 150 nM
125
I-GST-MAP and 150 nM
(HE)
10
peptides. After 1 h incubation at 37ºC, the cell monolayers were washed 3
times with 1 mL ice-cold PBS and detached by treatment with trypsin-EDTA for 3
min at 37ºC. The cells were then collected and centrifuged at 1500 rpm for 3 min.
The trypsin-removable supernatant fractions, designated as the surface bound
proteins, were isolated and counted for radioactivity using a γ-counter. The cell
pellets were washed twice with cold PBS, and dissolved in 1N NaOH. The total cell
uptake was determined by measuring the radioactivity using a γ-counter. The amount
of total cell associated proteins was calculated from the total cell associated
radioactivity (i.e. surface bound and internalized).
For confocal study, HeLa cells grown overnight on coverslips in 12-well plates
were treated with self-made RPMI 1640 media adjusted to pH 6.5 or 7.4 containing
10 μg/mL rhodamine-labeled GST-HE-MAP for 1 hour at 37 ºC. The cells were
washed with ice-cold heparin-PBS (pH 7.4) and PBS (pH 7.4), and fixed in 4%
paraformaldehyde for 15 min. Then, cells were washed with PBS (pH 7.4) and nuclei
24
were stained with 300 nM 4’,6-diamidino-2-phenylindole (DAPI) for 15 min. After
two more PBS (pH 7.4) wash steps, the cover slips were mounted on micro slides
with anti-fade reagent. Images were obtained using confocal laser scanning
microscope (CLSM) with the help from Aaron Hsueh in Dr. Sarah Hamm-Alvarez’s
laboratory (Zeiss LSM 510 Meta).
2.2.6. Animal studies in mice
2.2.6.1. Xenograft mouse model of human breast cancer
All animal studies were performed according to the protocols approved by the
University of Southern California Institutional Animal Care and Use Committee. The
mouse model was generated according to a protocol similar to a previous study
(Chen et al., 2012). Female athymic nude mice (Hsd:Athymic Nude-Foxn1
nu
, 4-6
weeks old) were purchased from Harlan (Livermore, CA). The xenograft model was
generated by subcutaneous injection of ~7 × 10
6
MDA-MB-231 human breast
adenocarcinoma cells suspended in 100 μL Matrigel
TM
into the right flank of mice.
Tumor volume was calculated using the formula (S
2
× L) where S and L represent
the small and large dimensions. Tumors were allowed to grow to 0.5 – 1 cm
3
.
2.2.6.2. In vivo and ex vivo infrared fluorescence imaging
All fluorescence imaging studies were performed using the IVIS SPECTRUM
pre-clinical in vivo imaging system and analyzed using the IVIS Living Imaging 4.2
software (PerkinElmer, Waltham, MA). An infrared filter set (Ex 745 nm, Em 800
nm), was used to acquire the fluorescence of IR800-proteins. All illumination
25
conditions (lamp voltage, filters, f/stop, field of views, binning) were set to the same
levels for all imaging within one experiment. Fluorescence emissions were
normalized and reported as radiant efficiency, unit = (photon/sec/cm
2
/sr)/(μW/cm
2
).
For the first experiment, the control mouse was injected intravenously with 8 nmol
of free dye, IRDye 800CW carboxylate, while another mouse received 5.75 nmol
IR800-labeled GST-HE-MAP (255 nmol/kg). The mice were imaged at 1, 6, 24 h
post-injection before ex vivo imaging of collected tumors and organs (liver, kidneys,
heart, spleen, and pancreas). To compare GST-HE, GST-MAP, and GST-HE-MAP,
each group of mice (n = 3) were injected with 3.65 nmol (160 nmol/kg) of one
IR800-labeled protein intravenously and imaged at various time points post-
injection.
2.2.7. Statistical analysis
For experiments with n ≥ 3, all values are represented as mean ± standard
deviation, and significant differences were evaluated using the Student’s t-test.
Values of p < 0.05 were accepted as statistical significant.
26
3. RESULTS
3.1. Study of arginine-rich CPPs
3.1.1. Surface binding and cell uptake of the peptides
Radioactive iodine (
125
I) was used as a tracer for peptide or protein in most of
the cell assay during this project. Peptides and proteins were labeled with
125
I
according to the procedures described in section 2.2.4. After size exclusion
chromatography, all
125
I-labeled peptides and proteins were stored at -20°C in small
aliquots. A representative figure was shown here for the elution profiles of five
arginine-rich CPPs on 10 mL Sephadex G-15 columns (Figure 1). For each profile
consisting of fifteen 1 mL fractions, there were two peaks. The first peak in each
profile represented
125
I-labeled peptides, which eluted from the column in different
fractions according to peptide’s size and charge. YGR
6
E
6
and YG(RE)
6
both had the
highest molecular weight and zero net charge, which came out in fraction 4 and 5.
YGR
6
G
6
and YG(RG)
6
were smaller than the previous two peptides and had positive
net charge, so they were eluted as later and broader peaks in fraction 5 to 7. For the
smallest sized and positive charged YGR
6
, it was eluted as the latest and broadest
peak in fraction 7 to 10 among all five peptides. The second peak for each peptide
eluted in fraction 11 to 13, which represented the free
125
I left after labeling reactions.
27
Figure 1. Elution profiles of size exclusion chromatography for five peptides after
radiolabeling reactions. YGR
6
, YGR
6
G
6
, YG(RG)
6
, YGR
6
E
6
, YG(RE)
6
were labeled
with
125
I and the reaction mixtures were loaded onto separate Sephadex G-15
columns. One milliliter fractions were collected for each peptides and radioactivity
was measured for a 5 μL sample from each fraction.
The surface binding and cell uptake of six
125
I-labeled peptides (YGR
6
,
YGR
6
G
6
, YG(RG)
6
, YGR
6
E
6
, YG(RE)
6
, YGE
6
), were determined in CHO cells
following a 1 hour incubation with radiolabeled peptides. The effects of charge on
the binding and uptake of the CPPs are presented in Figure 2. The data showed that
there was no significant difference in surface binding or cell uptake between YGR
6
and YGR
6
G
6
. However, significant decreases in surface binding and cell uptake were
observed after the positive charge of the peptide was neutralized in YGR
6
E
6
. The
charge neutralization had a greater effect on surface binding than cell uptake. While
there was a 78% decrease in surface binding from YGR
6
G
6
to YGR
6
E
6
, the neutral
28
peptide showed a 49% decrease in cell uptake compared to its positively charged
counterpart. The binding and uptake of the anionic peptide, YGE
6
, were minimal in
comparison to the arginine-containing CPPs.
Next, the effects of clustering of arginine residues in the CPP sequence on
binding and cellular uptake were evaluated. As seen in Figure 3, the mixed form of
peptides always exhibited lower binding and uptake than those of blocked peptides
(refer to section 1.4 for the definition of blocked or mixed formation). For example,
compared to YGR
6
G
6
, the mixed form YG(RG)
6
showed a 21% decrease in binding
and a 49% decrease in uptake. When the net charge was neutralized, the mixed form,
YG(RE)
6
, showed a 74% decrease in surface binding and a 52% decrease in uptake
as compared to its blocked form counterpart, YGR
6
E
6
. Taken together, these data
indicated that both net positive charge and clustered arginine residues were essential
for the high surface binding and cell uptake of arginine-rich CPPs.
Although the surface binding and cell uptake of the net neutral arginine-
glutamate co-oligopeptides were low in comparison to the net cationic arginine-
glycine co-oligopeptides, there were significant increases compared to YGE
6
. As
shown in Figure 2, the uptake of YGR
6
E
6
and YG(RE)
6
were approximately 18 and
2-fold higher, respectively, than YGE
6
(data not shown for YG(RE)
6
in Figure 2).
Strikingly, the surface binding of YGR
6
E
6
and YG(RE)
6
were 72 and 13-fold higher
than YGE
6
, respectively.
29
Figure 2. The influence of net charge on surface binding and cell uptake. CHO cells
grown for 4 days in 6-well plates were incubated with 10 μg/mL
125
I-peptide for 1 h
at 37ºC. (A) Surface binding was calculated from the radioactivity in the trypsin
removable supernatant; (B) Total cell uptake was determined as ng peptide per mg
cell protein. Data were expressed as means with standard deviations (n = 6).
*indicates a statistically significant difference (p < 0.05) compared to the control
peptide (YGR
6
). (Adapted from Fei et al., 2011, with permission from Informa
Healthcare)
30
Figure 3. The influence of arginine sequence distribution on surface binding and cell
uptake. CHO cells grown for 4 days in 6-well plates were incubated with 10 μg/mL
125
I-peptide in a blocked formation (closed bars) or a mixed formation (open bars)
for 1 h at 37ºC. Both (A) surface binding and (B) cell uptake were determined and
the latter was normalized by cell protein. Data were expressed as means with
standard deviations of triplicates. *indicates a statistically significant difference (p <
0.05). (Adapted from Fei et al., 2011, with permission from Informa Healthcare)
31
3.1.2. Subcellular localization of the internalized peptides
The arginine-rich peptides were also analyzed to determine their subcellular
localization after entering into cells. The subcellular fractionation method was also
performed using polylysine and transferrin by Dr. Jennica Zaro previously in our
laboratory, which are both known to be internalized by endocytosis (Shen et al.,
1992; Widera et al., 2003). The relative percentages localized in the vesicles and in
the cytosol were determined (Table 4). The results showed that all five arginine-rich
peptides were preferentially (>85%) located in the cytosol after internalization
although the exact percentages varied. As expected, both polylysine and transferrin
were both preferentially (>90%) accumulated in the vesicular fraction.
Table 4. The relative percentage of peptide localized in the vesicular versus cytosolic
compartment (Adapted from Fei et al., 2011, with permission from Informa
Healthcare)
Peptide Vesicle associated (%)
*
Cytosolic (%)
*
YGR
6
10.8 ± 6.6 89.2 ± 6.6
YGR
6
G
6
5.5 ± 3.1 94.5 ± 3.1
YG(RG)
6
2.7 ± 1.0 97.3 ± 1.0
YGR
6
E
6
14.6 ± 1.2 85.4 ± 1.2
YG(RE)
6
5.1 ± 3.0 94.9 ± 3.0
Polylysine (13 kDa) 90.5 ± 0.1 9.5 ± 0.1
Transferrin 97 ± 0.02 3 ± 0.02
*
The data are presented as means ± standard deviations of triplicates for polylysine
and transferrin (from previous study of our group), and quadruplicates for all others.
32
Figure 4. The amounts of internalized peptides localized in the vesicular versus
cytosolic fractions. CHO cells grown for 4 days in T-75 flasks were treated with 10
μg/mL
125
I-peptide for 1 h at 37ºC. Cells were fractionated according to the
procedures described in section 2.2.5.3. The amounts of peptides associated with the
vesicles (closed bars) or cytosol (open bars) were calculated and presented as means
with standard deviations of quadruplicates. *indicates a statistically significant
difference (p < 0.05). (Adapted from Fei et al., 2011, with permission from Informa
Healthcare)
Next, the measured amounts of the five different peptides (YGR
6
, YGR
6
G
6
,
YG(RG)
6
, YGR
6
E
6
, YG(RE)
6
) in the cytosol versus the vesicles were compared. The
results from Figure 4 showed that the cytosolic amounts of the peptides in the mixed
configuration or the charge-neutral peptides were significantly lower compared to
YGR
6
, a trend consistent with the total cellular uptake results shown in Figure 2 and
3. These data demonstrated that even though the cytosolic amounts were decreased
33
(Figure 4), charge neutralization and altered distribution of the clustered arginine
residues in an arginine-rich CPP did not significantly alter its subcellular distribution
(i.e. vesicular versus cytosolic accumulation) (Table 4) from the original peptide.
3.2. Study of chemically modified MAPs
3.2.1. Synthesis and characterization of CA-MAP and CA-MAP-F
MAP was modified by an excess amount of citraconic anhydrides, which
formed acid-labile bonds with the amino groups in the peptide. After reaction, the
mixture was purified by a Sephadex G-15 column. The elution profile from one
representative reaction was shown in Figure 5. The first peak of A280 profile
contained CA-MAP, while the second peak consisted of small molecules, such as
citraconic anhydride, citraconic acid, and DMF or DMSO. The modification ratio of
amino groups was determined using TNBSA assay and was always around 90%.
Next, the hydrolysis of citraconyl groups in CA-MAP was evaluated at different
pH conditions. At different incubation time, each sample was tested using TNBSA
assay whose readout, A335, was proportional to the amount of free amino groups in
the sample. As shown in Figure 6, A335 showed no change at pH 6 or 7 and a very
slow increase at pH 5 during the incubation. Only at pH 3-4, the citraconyl groups in
CA-MAP were rapidly removed, which revealed more free amino groups with longer
incubation time. Taken together, in order to remove most of citraconyl groups, CA-
MAP needs to be treated at pH 3-4 for a time period longer than 3 hours at 37°C.
The cytotoxicity of CA-MAP was also evaluated alongside with MAP by MTT
assay (Figure 7). MAP showed a high level of cytotoxicity when the concentration
34
was above 10 μg/mL. The cell viability was already at ~40% after 2 h incubation
with 25 μg/mL MAP. At the same time, compared with control group, no
cytotoxicity was observed for CA-MAP at every concentration tested in this
experiment. After CA-MAP was pre-treated overnight at pH 3-4, the cytotoxicity of
peptide was increased to a similar level as MAP. The data from Figure 6 and 7
demonstrated that the cytotoxicity of MAP was reduced when the amino groups were
blocked by citraconic anhydrides.
Figure 5. Elution profile of reaction mixture on a Sephadex G-15 column after
citraconylation of MAP. MAP was modified by excess citraconic anhydrides
overnight in DMSO or DMF at room temperature. The product, CA-MAP, was
purified by Sephadex G-15. Twenty 1 mL fractions were collected and measured for
absorbance at 280 nm.
35
Figure 6. The amount of free amino groups in CA-MAP after incubation at various
pH conditions. CA-MAP was incubated at pH 3-4, 5, 6, or 7 for 1, 2, or 3 h at 37°C.
After incubation, the amount of free amino groups in each sample was estimated by
TNBSA assay. Higher A335 value indicates more free amino groups in CA-MAP.
Figure 7. The viability of cells after treated with MAP, CA-MAP, or acid pre-treated
CA-MAP at various concentrations. CHO cells were incubated with one of the three
peptides at 2.5-100 μg/mL for 2 h at 37ºC. The cell viability was estimated using
MTT assay. The data were expressed as means ± standard deviations of triplicates.
36
Folate was added to CA-MAP as a targeting moiety, which targets to the cells
overexpressing folate receptors, such as many cancer cells (Leamon and Jackman,
2008). To produce CA-MAP-F, folate was activated and added onto MAP before the
citraconylation of peptide. The product, CA-MAP-F, was purified by size exclusion
chromatography using Sephadex G-15. As shown in Figure 8, 1 mL fractions were
collected and measured for absorbance at 280 and 370 nm. The first peak of A280
profile completely overlapped with the first peak of A370 profile, which
corresponded to CA-MAP-F. Excess folates and other small molecules eluted at
around one bed volume (10 mL), which formed the second peak. As determined by
fluorescamine assay, the modification ratio of amino groups was above 90%. The
conjugation ratio of folate in CA-MAP-F was about one folate per peptide.
Figure 8. Elution profile of reaction mixture on a Sephadex G-15 column after
preparation of CA-MAP-F. The peptide was synthesized and purified according to
the procedures described in section 2.2.1.2. Thirty-five 1 mL fractions were collected
and measured for absorbance at 280 and 370 nm.
37
3.2.2. Cell assays for MAP, CA-MAP, and CA-MAP-F
After iodination, CA-MAP and CA-MAP-F were investigated for their surface
binding and cell uptake in HeLa cells together with the unmodified MAP. All three
peptides, with or without pre-treatment at pH 3-4 overnight at room temperature,
were used to dose the cells for 1 h at 37°C. As shown in Figure 9, MAP showed high
surface binding and cell uptake, and they were not affected by the pre-treatment at
pH 3-4. Compared to MAP, CA-MAP exhibited 64% and 88% decrease in surface
binding and cell uptake, respectively. When the peptides were pre-treated at pH 3-4,
the binding and uptake of CA-MAP recovered to 78% and 54% of MAP’s levels,
respectively, which were about 1-fold and 4-fold increase compared to CA-MAP
without acid pre-treatment. For CA-MAP-F, the levels of surface binding and cell
uptake were comparable to MAP, which were close to 3 and 11 fold of CA-MAP’s
levels, respectively. For acid pre-treated CA-MAP-F, there was a 41% decrease in
surface binding but a 206% increase in cell uptake from untreated CA-MAP-F. In
summary, the citraconylation of MAP significantly reduced both binding and uptake
of the peptide, which could be recovered once the citraconyl groups were removed.
The extra folate molecule on CA-MAP elevated the binding and uptake of the
peptide in HeLa cells.
38
Figure 9. Surface binding and cell uptake of MAP, CA-MAP, and CA-MAP-F with
or without acid pre-treatment. HeLa cells grown for 4 days in 6-well plates were
incubated with 5 μg/mL
125
I-peptide (closed bars) or acid pre-treated
125
I-peptide
(open bars) for 1 h at 37ºC. Both (A) surface binding and (B) total cell uptake were
determined and the latter was normalized by cell protein. Data were expressed as
means with standard deviations of triplicates.
39
Previous research of our group has revealed that MAP has a relative high
nuclear localization compared to arginine-rich CPPs (Zaro et al., 2009). In order to
evaluate the effect of citraconylation and folate on peptide’s nuclear distribution,
CA-MAP and CA-MAP-F were examined for their nuclear internalization together
with MAP in HeLa cells (Figure 10). While MAP showed both high cell uptake and
nuclear internalization, the cell uptake and nuclear internalization of CA-MAP were
only 10% and 7% of MAP’s levels, respectively. CA-MAP-F had a 20% lower level
of cell uptake compared to MAP, but a 42% higher level of nuclear internalization.
The addition of folate to CA-MAP increased not only its cell uptake but also its
subcellular localization to the nucleus.
To confirm whether the increase of surface binding and cell uptake from CA-
MAP to CA-MAP-F was folate-specific, both peptides were tested in HeLa cells
with or without folate competition at 4000-fold molar excess (Figure 11). The results
for peptides without competition were similar to last two experiments (Figure 9 and
10), where both surface binding and cell uptake were very low for CA-MAP but high
for CA-MAP-F. With excess folate competition, CA-MAP showed a 14% decrease
in surface binding and a 3% decrease in cell uptake. Under the same condition, CA-
MAP-F exhibited 27% and 24% reduction in binding and uptake, respectively, which
were still close to 3 and 7 fold of CA-MAP’s levels. Contrastingly, 20-fold molar
excess unlabeled CA-MAP-F could compete off 78% and 75% of the surface binding
and cell uptake of
125
I-labeled CA-MAP-F, respectively (data not shown).
40
Figure 10. Cell uptake and nuclear internalization of MAP, CA-MAP, and CA-MAP-
F. HeLa cells grown for 4 days in 6-well plates were incubated with 5 μg/mL
125
I-
peptide for 1 h at 37ºC. (A) Cell uptake of the peptide was calculated from the
radioactivity in the cell pellet; (B) Total nuclear internalization was determined as ng
peptide inside nucleus per mg nuclear protein. Data were expressed as means with
standard deviations of quadruplicates.
41
Figure 11. Surface binding and cell uptake of CA-MAP and CA-MAP-F with or
without excess folate competition. HeLa cells grown for 4 days in 6-well plates were
incubated with 5μg/mL
125
I-peptide (closed bars) or 5 μg/mL
125
I-peptide with
4.4mg/mL folate (open bars) for 1 h at 37ºC. Both (A) surface binding and (B) total
cell uptake were determined and the latter was normalized by cell protein. Data were
expressed as means with standard deviations of triplicates.
42
3.3. Study of fusion proteins/peptides containing MAP
3.3.1. Construction of plasmids
To produce a fusion peptide containing MAP and (HE)
10
, a DNA sequence
encoding peptide MAP-G
5
-(HE)
10
was first designed, and the codons were optimized
for expression in E. coli. Both sense and anti-sense sequences were synthesized by
company (Genemed), and then annealed to form dsDNA. The DNA fragment was
inserted into pGEX-4T-1 between BamHI and NotI restriction enzyme sites, but no
correct clone was obtained (Table 5). During 4 rounds of cloning, 184 colonies were
screened and 81 plasmids were sent for sequencing. Only 2 plasmids had the correct
insertion, but no fusion protein was expressed after they were transformed into E.
coli BL21 strain. Further sequencing revealed that one plasmid had a deletion in the
coding region for GST protein and the other plasmid had a deletion in the promoter
region of GST gene.
Table 5. Cloning works for peptide MAP-G
5
-(HE)
10
versus HE-MAP
Peptide
Round of
cloning
Colony PCR screening
(positive/total)
DNA sequencing
(correct insertion/total)
MAP-G
5
-(HE)
10
1 34/72 0/25
2 30/40 0/29
3 31/32 0/8
4 20/40 2/19*
HE-MAP 1 36/40 6/10
* 2 clones with correct insertion but no protein expression
43
Figure 12. Plasmid construction for the fusion proteins used in this thesis. DNA
sequences encoding various peptides were inserted into the multiple cloning site of
pGEX-4T-1 using different restriction enzymes. The inserted sequences were
checked by DNA sequencing.
44
Since it seemed impossible to get a correct clone expressing GST-MAP-G
5
-
(HE)
10
fusion protein, a new DNA sequence encoding HE-MAP was designed by
switching two sequence fragments, encoding MAP and (HE)
10
, in the previous
design. The resultant dsDNA fragment was inserted at the same site into pGEX-4T-1
as the previous cloning. This time, correct clones were identified at the first attempt,
which could express fusion proteins with right size (Table 5). Following the first
successful cloning, many other sequences were inserted into the same plasmid,
which resulted in clones expressing different GST-fusion proteins. All the clones
used in this thesis were shown in Figure 12 with the sequences of designed peptides.
The inserted DNAs all contained a stop codon at the end except two sequences
encoding (HE)
10
and MAP. Therefore, the transcription for these two clones stopped
at the in frame stop codon located in the downstream region of the multiple cloning
site. For the clone expressing GST-HE-MAP, thrombin was later used to cleave off
HE-MAP peptide from the fusion protein, which left two amino acids, glycine and
serine, at the N-terminus of the peptide.
3.3.2. Expression and purification of fusion proteins and peptides
All clones were first tested for expression in small scale using LB media. For
large-scale expression, all expression conditions were optimized using TB media in
order to get a higher yield, which required an IPTG induction at high OD
600
(2.5-3.0).
To purify GST-HE-MAP, both Ni-NTA (which binds histidine) and GSH (which
binds GST) agarose resins were tested utilizing a gravity flow method. As shown in
Figure 13A, GST-HE-MAP could be purified using the histidine residues as a
45
recognition site, resulting in ~92% purity as determined by SDS-PAGE (separation
gel with 13% acrylamide/bis-acrylamide, 29:1, for analysis of all fusion proteins)
with Coomassie blue staining. Alternatively, the fusion protein was also purified by
GSH agarose (Figure 13B), resulting in ~94% purity as determined by SDS-PAGE
with Coomassie blue staining. Comparing two resins for GST-HE-MAP purification,
GSH agarose gave a slightly higher purity and a much better yield after purification
(Table 6). For these reasons, all GST-fusion proteins used in the following cell
assays were purified by GSH agarose. As shown in Table 6 and Figure 13, every
purified fusion protein migrated as one major band, which matched its molecular
weight and had over 90% purity as determined by SDS-PAGE with Coomassie blue
staining. For animal studies, GST-HE and GST-HE-MAP were further purified by
Ni-NTA agarose, with extensive wash by 1% Triton X-100, to reduce their
endotoxin levels.
Table 6. Yields of different fusion proteins in TB media and after purification
Fusion protein
Molecular
weight
(kDa)
Yield
(mg/L)
Purified by
Yield after
purification
(mg/L)
GST-HE-MAP 31.3 10-20
Ni-NTA agarose 3-6
GSH agarose 5-15
GST-(HE)
8
-G
5
-MAP 30.7 10-20 GSH agarose 10
GST-(HE)
12
-G
5
-MAP 31.8 10-20 GSH agarose 10
GST-HE 30.3 80-100 GSH agarose 50-80
GST-MAP 29.6 5-10 GSH agarose 1.5-3
46
Figure 13. SDS-PAGE analysis and Coomassie blue staining of purified proteins.
Fusion proteins were purified by Ni-NTA agarose (A) or GSH agarose (B, C, and D).
(A) Lane 1: MW markers. Lane 2: Supernatant. Lane 3: Pellet. Lane 4: Flow through.
Lane 5: Wash. Lane 6: GST-HE-MAP. (B) Lane 1: Bacteria without IPTG induction.
Lane 2: Supernatant. Lane 3: Flow through. Lane 4: GST-HE-MAP. Lane 5: MW
markers. (C) Lane 1: MW markers. Lane 2: GST-(HE)
8
-G
5
-MAP. Lane 3: GST-HE-
MAP. Lane 4: GST-(HE)
12
-G
5
-MAP. (D) Lane 1: MW markers. Lane 2: GST-HE.
Lane 3: GST-MAP. Lane 4: GST-HE-MAP.
47
Figure 14. Purification of HE-MAP. (A) SDS-PAGE of GST-HE-MAP following
GSH agarose column chromatography. Lane 1: MW markers. Lane 2: Bacteria
without IPTG induction. Lane 3: IPTG-induced bacteria. Lane 4: Lysate. Lane 5:
Supernatant. Lane 6: Pellet. Lane 7: Flow through. Lane 8: Wash. Lane 9: GSH
agarose resin. (B) After on-column thrombin cleavage, peptides were eluted with
PBS and analyzed by tricine SDS-PAGE. Lane 1-5: Eluted peptides. Lane 6: MW
markers. (C) The peptides eluted with PBS, pH 7.4, from (B) were further purified
with Ni-NTA agarose column and analyzed by tricine SDS-PAGE. Lane 1: MW
markers. Lane 2: PBS-eluted peptides. Lane 3: Flow through. Lane 4: Wash. Lane 5-
7: Eluted peptides (fractions 1-3). (D) Purified peptides from (C) were analyzed by
LC-MS.
48
To purify HE-MAP peptide, GST-HE-MAP was purified from the bacterial
lysate by GSH agarose (Figure 14A). While binding on the column, the fusion
proteins were treated with thrombin overnight at room temperature. The cleaved
peptides were washed off by PBS, pH 7.4, and purified by Ni-NTA agarose (Figure
14B and 14C). The purity of the final HE-MAP peptide was above 90% for fraction
3 (Lane 7 in Figure 14C) as calculated from the resultant band densities. HE-MAP
moved within tricine gel (separation gel with 16% acrylamide/bis-acrylamide, 19:1,
for all peptide analysis) as a larger-sized band (~10 kDa) than its expected molecular
weight. Therefore, the molecular weight of the peptide was determined by LC-MS,
from which the results confirmed the expected molecular weight of 5132 Da (Figure
14D).
Since the cell uptake of MAP was shown to be dependent on its amphipathic α-
helix structure (Oehlke et al., 1996), it was interesting to see if HE-MAP would still
have similar structure. To study the secondary structure, purified HE-MAP from the
fraction 3 (Figure 14C) was diluted to 25 μM with 10 mM phosphate buffer either at
pH 6.5 or pH 7.4, and was measured using CD spectrometer (Figure 15). After the
background signals from buffer were subtracted, two CD spectra for pH 6.5 and 7.4
were almost identical and both resembled the CD spectrum of α-helix. The data
demonstrated that there was no change in secondary structure for HE-MAP between
pH 6.5 and 7.4.
49
Figure 15. Far-UV CD spectra of 25 μM HE-MAP peptide in phosphate buffer at pH
6.5 and 7.4.
3.3.3. Surface binding and cell uptake of GST-HE-MAP at different pH
In order to evaluate the pH-sensitivity of GST-HE-MAP, cell uptake assays
were carried out at various different pH values. As shown in Figure 16, the
125
I-
labeled GST-HE-MAP fusion protein exhibited a pH-dependent surface binding and
cell uptake profiles. The surface binding of
125
I-GST-HE-MAP increased with
decreasing pH, and leveled off at pH 6.3 and below (Figure 16A). It was increased
by about 2.5-fold when pH was changed from 7.2 to 6.0. Similarly, the cell uptake of
the fusion protein also increased with decreasing pH (Figure 16B). The internalized
125
I-GST-HE-MAP was very low at pH 7.2 and above, but was increased by ~14-fold
at pH 6.6 and ~11-fold at pH 6-6.3.
50
Figure 16. Surface binding and cell uptake of GST-HE-MAP at pH 6.0-7.5. HeLa
cells grown for 4 days in 6-well plates were incubated with 5μg/mL
125
I-GST-HE-
MAP at pH 6.0, 6.3, 6.6, 6.9, 7.2, or 7.5 for 1 h at 37ºC. Both (A) surface binding
and (B) total cell uptake were determined and the latter was normalized by cell
protein. Data were expressed as means ± standard deviations of triplicates. (Adapted
from Zaro et al., 2012, with permission from Elsevier)
51
The results from radioactive assay were verified by the confocal study of cell
uptake of rhodamine-labeled GST-HE-MAP in HeLa cells. As shown in Figure 17,
there was no observable red-fluorescent staining for cells incubated with rhodamine-
labeled fusion protein at pH 7.4. On the other hand, cells incubated with rhodamine-
GST-HE-MAP at pH 6.5 showed punctate vesicular staining throughout the cell.
Combining the results from Figure 16 and 17, GSH-HE-MAP exhibited low surface
binding and cell uptake at neutral pH but significantly higher binding and uptake at
mildly acidic pH.
Figure 17. Confocal analysis of GST-HE-MAP internalization. HeLa cells were
treated with 10μg/mL rhodamine-labeled GST-HE-MAP for 1 h at 37ºC at a pH of
(A) 6.5 or (B) 7.4. After incubation, cells were analyzed by confocal microscopy at
Ex 372/Em 456 (blue DAPI stained nuclei) and Ex 570 nm/Em 595 nm (red punctate
rhodamine-GST-HE-MAP staining). (Adapted from Zaro et al., 2012, with
permission from Elsevier)
52
3.3.4. The influence of the length of H/E co-oligopeptide
GST-(HE)
8
-G
5
-MAP and GST-(HE)
12
-G
5
-MAP were produced and compared
in cell uptake assay, alongside with GST-HE-MAP (same protein as GST-(HE)
10
-G
5
-
MAP), in order to understand how the repeat number of H/E co-oligopeptide in the
fusion protein would affect its surface binding and cell uptake. As shown in Figure
18A, the surface binding of all three
125
I-labeled fusion proteins decreased with
increasing pH. While
125
I-GST-(HE)
8
-G
5
-MAP and
125
I-GST-HE-MAP showed
similar levels of surface binding at pH 6.0 and 6.5, the binding of latter protein had a
steeper drop at pH 7.0 and 7.5.
125
I-GST-(HE)
12
-G
5
-MAP exhibited lower surface
binding at all four pH conditions than the other two proteins and had a big drop from
pH 6.5 to 7.0. The similar trend was observed for the cell uptake profiles of three
fusion proteins, where the differences among three curves were more obvious
(Figure 18B). The cell uptake of
125
I-GST-(HE)
8
-G
5
-MAP was much higher than the
other two proteins at every pH. Although the uptake levels of
125
I-GST-HE-MAP and
125
I-GST-(HE)
12
-G
5
-MAP were close, the former protein still had higher cell uptake
at each pH. These data demonstrated that the fusion protein with a shorter H/E co-
oligopeptide usually showed higher surface binding and cell uptake, and vice versa.
53
Figure 18. The influence of H/E repeat number on the surface binding and cell
uptake of fusion proteins at pH 6.0-7.5. HeLa cells grown for 4 days in 6-well plates
were treated with 5 μg/mL
125
I-GST-(HE)
8
-G
5
-MAP,
125
I-GST-(HE)
10
-G
5
-MAP, or
125
I-GST-(HE)
12
-G
5
-MAP at 37ºC for 1 h at indicated pH. Both (A) surface binding
and (B) total cell uptake were determined and the latter was normalized by cell
protein. Data were expressed as means ± standard deviations of triplicates.
54
3.3.5. HE-MAP mediated pH-dependent cell association
The effect of HE-MAP on the uptake of a cargo protein, GST, was analyzed in
cell association assay. As shown in Figure 19,
125
I-GST-HE-MAP exhibits a pH-
dependent association with HeLa cells while
125
I-GST showed relatively low
association at all pH values. The cell associated
125
I-GST-HE-MAP increased with
decreasing pH, with a sharp increase from pH 6.9 to pH 6.6. The fusion of HE-MAP
peptide to GST increased its cell association by ~1.5-fold at pH 7.2-7.5 and ~4-fold
at pH 6.0, which resulted in a pH-dependent cell association profile.
Figure 19. Cell association of GST and GST-HE-MAP at pH 6.0-7.5. HeLa cells
grown for 4 days in 6-well plates were treated with 5 μg/mL
125
I-GST or
125
I-GST-
HE-MAP for at 37°C 1 h at indicated pH, and the amounts of total cell associated
proteins were determined. Data were expressed as means ± standard deviations of
triplicates.
55
Figure 20. Total cell associated
125
I-GST-HE,
125
I-GST-MAP, and
125
I-GST-HE-
MAP in HeLa cells at 4 different pH conditions. HeLa cells grown for 4 days in 6-
well plates were treated with 150 nM
125
I-labeled fusion proteins at pH 6.0, 6.5, 7.0,
or 7.5 for 1 h at 37°C. The amounts of total cell associated proteins were determined
according to the protocol described in section 2.2.5.5. Data were expressed as means
± standard deviations of triplicates. (Adapted from Fei et al., 2014, with permission
from Elsevier)
Next, GST-HE and GST-MAP were analyzed for cell association at various pH
conditions alongside GST-HE-MAP. Three distinct association curves were observed
for these fusion proteins differed by only ~20 amino acids (Figure 20). For
125
I-GST-
HE, the amount of total cell associated proteins remained low (1-2 pmol) across the
entire pH range. In contrast, the cell association of
125
I-GST-MAP was high across
the entire pH range, where the amounts were 14.5 - 20.6 pmol. The total cell
associated
125
I-GST-HE-MAP showed more significant changes among different pH
56
conditions than the other two fusion proteins. The cell association was low at pH 7.5
to 7.0 (3.3 ± 0.1 and 4.8 ± 0.4 pmol at pH 7.5 and 7.0, respectively), and then
increased 3-6 fold to 16.1 ± 0.3 and 22.4 ± 0.3 pmol at pH 6.5 and 6.0, respectively.
Combined with the results from last assay (Figure 19), the data demonstrated that
both HE and MAP contributed to the high pH-sensitivity of GST-HE-MAP within
the pH range from 6.0 to 7.5.
The
125
I-proteins were also tested for cell association assays in another cell line,
MDA-MB-231, at pH 6.5 or 7.4. As shown in Figure 21A, the trend was similar to
that in HeLa cells, where
125
I-GST-HE had low total cell association at both pH 7.4
and 6.5 (0.7 ± 0.1 and 0.5 ± 0.2 pmol, respectively), and
125
I-GST-MAP had high
cell association at both pH 7.4 and 6.5 (14.7 ± 1.0 and 13.4 ± 0.4 pmol, respectively).
The cell association of
125
I-GST-HE-MAP was pH-sensitive, where the amount was
4.4 ± 0.2 pmol at pH 7.4 which then increased ~3-fold to 14.6 ± 0.5 pmol at a lower
pH of 6.5. Next, the requirement of direct attachment of (HE)
10
to the MAP-fusion
protein was evaluated by mixing
125
I-GST-MAP with (HE)
10
at a 1:1 molar ratio. At
pH 7.4, only
125
I-GST-MAP-HE showed a reduced level of cell associated protein
(4.4 ± 0.1 pmol), while
125
I-GST-MAP and
125
I-GST-MAP mixed with (HE)
10
were
similar (15.2 ± 0.7 and 15.0 ± 1.0 pmol, respectively) (Figure 21B).
125
I-GST-MAP,
125
I-GST-MAP mixed with (HE)
10
, and
125
I-GST-MAP-HE showed similar amounts
of cell association at pH 6.5 (14.1 ± 0.8, 14.4 ± 0.6, and 13.4 ± 1.0 pmol for three
groups, respectively). In summary, pH-dependent cell association of fusion protein,
GST-HE-MAP, was observed only when the (HE)
10
peptide was fused with MAP.
57
Figure 21. Total cell associated
125
I-labeled fusion proteins in MDA-MB-231 cells at
pH 6.5 and 7.4. MDA-MB-231 cells grown for 4 days in 6-well plates were treated
at 37°C for 1 h with (A) 150 nM
125
I-GST-HE,
125
I-GST-MAP, or
125
I-GST-HE-
MAP; (B) 150 nM
125
I-GST-MAP only,
125
I-GST-MAP with (HE)
10
peptide (molar
ratio = 1:1), or
125
I-GST-HE-MAP. The amounts of total cell associated proteins
were determined as described in section 2.2.5.5. Data were expressed as means ±
standard deviations of triplicates. **indicates a statistically significant difference (p
< 0.01) between the two groups. (Adapted from Fei et al., 2014, with permission
from Elsevier)
58
3.3.6. Imaging studies in mouse xenograft model of human breast cancer
In order to determine if similar pH-dependent cell association could be achieved
in vivo, GST-HE, GST-MAP, and GST-HE-MAP were labeled with an infrared
fluorescent dye, IRDye 800CW. After purification, IR800-labeled proteins were
injected intravenously into MDA-MB-231 tumor-bearing nude mice through the tail
vein. With a mildly acidic environment around solid tumors (Gillies et al., 2002), this
model was ideal for the purposes of this study. First, the distribution of free IR800
dye and IR800-GST-HE-MAP in mice was compared at various time points post-
injection. IR800-GST-HE-MAP showed strong fluorescent signals around tumor
sites with a long retention up until 24 h post-injection (Figure 22A), while the free
dye had an extensive distribution in mouse at 1 h post-injection and was quickly
eliminated before 6 h post-injection. Tumor distribution of GST-HE-MAP was
further validated by ex vivo imaging of several organs and tumors at 24 h post-
injection (Figure 22B). The highest level of fluorescence was detected in the tumor
and kidneys. IR800-GST-HE-MAP was also found in the liver, with minimal activity
in the spleen and no detectable activity in the heart or pancreas.
59
Figure 22. In vivo imaging and organ distribution study for IR800-GST-HE-MAP in
MDA-MB-231 tumor-bearing nude mice. Free IR800 dye (control) or IR800-labeled
GST-HE-MAP was injected intravenously. Fluorescent images were acquired (A) 1,
6, 24 h post-injection and (B) ex vivo imaging of tumors and other organs after
euthanizing the mice at 24 h post-injection: 1. Tumor, 2. Heart, 3. Pancreas, 4. Liver,
5. Kidneys, 6. Spleen. Arrows indicate tumor sites. (Adapted from Fei et al., 2014,
with permission from Elsevier)
60
The distribution of GST-HE-MAP in mice was further evaluated alongside
GST-HE and GST-MAP controls (n = 3). Whole-body distribution of the injected
IR800-labeled fusion proteins peaked at 2 h post-injection (Figure 23A), where the
resultant fluorescent images for the three groups showed a pattern similar to what
was observed in cell assays. The fluorescent signals were low with limited whole
body distribution for the IR800-GST-HE group, but high and extensive for the
IR800-GST-MAP group. In contrast, the fluorescent signals in mice injected with
IR800-GST-HE-MAP were relatively low in most organs, but enriched around tumor
sites. The other regions with high fluorescent levels were due to accumulation of the
fusion protein in the kidneys and liver, likely due to the clearance of the protein from
the body. Time-dependent accumulation of IR800-GST-HE-MAP in tumors showed
enrichment in tumor sites as early as 0.5 h post injection, with peak enrichment at
around 1-2 h post injection, and remained in the tumor until 6 h post injection
(Figure 23B). The fluorescent proteins inside tumors had slower clearance than most
normal tissues and organs except the kidneys and liver.
61
Figure 23. In vivo imaging of MDA-MB-231 tumor-bearing nude mice after
intravenous injection of IR800-labeled GST-fusion proteins. Each mouse received
one of the three IR800-labeled fusion proteins at a dose of 3.65 nmol. (A) Images at
2 h post-injection for IR800-GST-HE, IR800-GST-MAP, and IR800-GST-HE-MAP.
Arrows indicate tumor sites. (B) Images for IR800-GST-HE-MAP from 0.5 to 6 h
post-injection. (Adapted from Fei et al., 2014, with permission from Elsevier)
62
4. DISCUSSIONS
4.1. Inspiration from the study of arginine-rich CPPs
The internalization properties of CPPs and the effects of attached cargo are
important, yet controversial, topics in the field of CPP technology (El-Andaloussi et
al., 2007; Fischer et al., 2002; Tünnemann et al., 2006). Previous papers from our
group have shown that cationic CPPs containing the guanidine structure of arginine
can efficiently accumulate in the cytosolic compartment of cells, while cationic CPPs
containing only lysine residues are localized primarily in the vesicular fraction (Zaro
and Shen, 2003, 2005b). At the beginning of this project, the effects of CPP
sequence variation on its internalization properties were characterized to give insight
into the requirements for entry into the cytosol, as well as to the types of cargo
molecules that can be delivered into the cytosol via CPP-carriers. Arginine-rich
peptides with net positive charge, i.e. YGR
6
and YGR
6
G
6
, and net neutral charge, i.e.
YGR
6
E
6
were compared alongside with a negatively charged YGE
6
to determine the
effect of net charge of CPPs on the binding and uptake (Figure 2). Similarly,
peptides in a blocked configuration, i.e. YGR
6
G
6
and YGR
6
E
6
, and a mixed
configuration, i.e. YG(RG)
6
and YG(RE)
6
, were compared to establish the
requirement of the clustering of positive charge in binding and uptake (Figure 3).
Over a dozen cell uptake assays were performed with a part or all of the six
125
I-
labeled peptides (YGR
6
, YGR
6
G
6
, YG(RG)
6
, YGR
6
E
6
, YG(RE)
6
, and YGE
6
). Two
63
representative figures are shown in this thesis (Figure 2 and 3). They were created
using the data from two separate cell uptake assays, in which five or six
125
I-peptides
were compared together in CHO cells. Surface binding and cell uptake data for four
peptides are shown in each figure in order to emphasize the effect of net charge or
charge distribution separately. Among different cell uptake assays, the actual levels
of binding and uptake varied assay to assay, but similar trend was observed in every
assay among all peptides. The peptides used in Figure 2 and 3 were labeled at
different time, therefore, the data for YGR
6
G
6
and YGR
6
E
6
didn’t match very well
between two figures. For the convenience of discussion, the data from two cell
uptake assays are listed in Table 7.
Table 7. Surface binding and cell uptake of six peptides in CHO cells
Peptide
Assay 1 (for Figure 2) Assay 2 (for Figure 3)
Surface binding
(ng)
Cell uptake
(ng/mg)
Surface binding
(ng)
Cell uptake
(ng/mg)
YGR
6
820.0 ± 222.9 226.1 ± 78.9 610.9 ± 46.3 133.2 ± 16.1
YGR
6
G
6
665.0 ± 176.2 249.4 ± 25.7 674.0±41.1 143.5 ± 37.4
YG(RG)
6
609.4 ± 64.8 104.5 ± 34.9 529.1±68.6 73.8 ± 12.5
YGR
6
E
6
146.2 ± 3.3 127.3 ± 56.9 67.8±2.8 47.5 ± 3.2
YG(RE)
6
27.5 ± 1.2 19.9 ± 3.4 17.9±0.4 22.9 ± 0.4
YGE
6
2.0 ± 0.6 6.6 ± 2.1 n.d. n.d.
Data are presented as mean ± standard deviation; n.d. stands for not determined.
Given the fact that the cell surface is negatively charged, it was not surprising
that our results in Figure 2 and 3 showed that all positive charged peptides (YGR
6
,
YGR
6
G
6
, YG(RG)
6
) had high surface binding and cell uptake. Compared to YGR
6
,
the addition of the glycine residues in YGR
6
G
6
did not significantly change the
64
binding or cell uptake, probably because each glycine residue only have a hydrogen
atom as a side chain, which is unlikely to interfere with binding and/or uptake.
However, the insertion of glycine residues between the arginine residues in YG(RG)
6
decreased its binding and uptake to ~80% and ~50% of YGR
6
’s levels, respectively
(Table 7). This result suggests that clustered arginine residues are more favorable to
be internalized by the cells, which has indeed been found in several natural CPPs
(e.g., Tat peptide). On the other hand, the results from the net neutral peptides
(YGR
6
E
6
and YG(RE)
6
) confirm the important role of net positive charge in the
internalization of this type of CPP. Substitution of glycine residues with negatively
charged glutamate residues resulted in at least a 78% loss in surface binding and a 49%
decrease in cell uptake (Table 7). Even between these two net neutral peptides,
YG(RE)
6
showed further decrease in both surface binding and cell uptake, again
indicating the important role of clustered arginine residues in the cell penetrating
ability of these peptides. Additionally, even though these two net neutral peptides
had low cell uptake, it was still several to tens of folds higher than YGE
6
. This result
reiterates the key role of the arginine residues in increasing the cellular uptake of
attached cargo molecules, e.g. G
6
peptide in YGR
6
G
6
can be seen as a cargo
molecule for YGR
6
.
The intracellular localization of these peptides was investigated to determine if
the amount localized in the vesicles versus the cytosolic compartment was altered by
the sequence or net charge of the CPPs. As shown in Figure 4 and Table 4, all of the
peptides derived from YGR
6
were preferentially localized in the cytosol, comparable
to the cytosolic percentage of the original peptide, although the amounts differed.
65
Similar to the results obtained for total cell uptake, the amount of CPP in the cytosol
decreased after neutralizing the net charge, or breaking up the clustered sequence of
arginine residues.
The results of arginine-rich CPPs presented in this dissertation have several
implications. First, charge neutralization substantially decreased both surface binding
and cell uptake. This result is similar to those obtained in a study by Rothbard et al.
(Rothbard et al., 2002), which showed that the internalization of two synthesized
arginine-rich peptides, RDRRDRRDRR and RERRERRERR, was decreased in
comparison to RGRRGRRGRR and R
7
. However, the study determined the
internalization of fluorescently labeled peptides using a suspension cell line, Jurkat.
Under these conditions, the majority of the measured cell-associated peptide was
likely surface bound, since the cells were not treated with trypsin, and only washed
with buffer solution. Therefore, the results presented here clearly demonstrate that
not only surface binding but also cell uptake are affected by the charge
neutralization, however, to a different extent. Second, the sequence of the
oligopeptide in a blocked versus a mixed configuration also affected the binding and
uptake. The binding and uptake of peptides in mixed configuration were lower than
those of peptides in blocked configuration, indicating that the clustering of arginine
residues may initiate cellular uptake more effectively, especially when the overall
charge is neutral in the case of the R/E co-oligopeptides. Possibly, the dispersed
negative carboxyl residues within the peptide interfere with the binding of the
oligopeptide to the biological membrane more effectively, leading to lower uptake
efficiency. The data also show that attachment of a small cargo will decrease the
66
uptake of the CPP conjugate, with a greater effect by a negatively charged cargo than
a neutral one. This finding is consistent with previous reports supporting a direct
penetration mechanism of CPPs into the cytosol through either the plasma membrane
or the endosomes. These reports show that the penetration is dependent on both the
charge or electrostatic binding and the size of the CPP (Cahill, 2009; Rothbard et al.,
2004; Zhang et al., 2009). While the positive charge serves as a driving force to
increase the penetration through the plasma or vesicular membrane, an increase in
size of the cargo or the peptide slows this process. Furthermore, the intracellular
localization of the various arginine-rich peptides tested was similar and still
preferentially localized in the cytosolic compartment (Table 4), although in different
amounts (Figure 4). These data also offer a further insight into the impact of cargo
on the CPP internalization, as the blocked oligoarginine peptides can be considered
as a CPP (R
6
) carrying a small cargo (G
6
, ~360 Da and E
6
, ~790 Da). While the
small neutral cargo in
125
I-YGR
6
G
6
did not statistically significantly affect the
cytosolic amount compared to
125
I-YGR
6
, the larger anionic cargo in
125
I-YGR
6
E
6
resulted in an 80% decrease. CPP-conjugates with larger cargos have also been
tested by our group, including p16 peptide (2.4 kDa) and cytochrome c (~12 kDa),
also showed a reduction in the amount localized to the cytosol compared the carrier
CPP (nona-arginine), further indicating that the size of the cargo is also important in
determining the intracellular fate of the conjugates (Barnes and Shen, 2009; Zaro and
Shen, 2005a). Additionally, these studies illustrate the importance of differentiating
between the amounts internalized in the cytosol versus the vesicles, since the
67
distribution may change depending on the size and charge of the cargo attached to
the CPP.
As mentioned above, our protocol for cell uptake assay includes a trypsin
treatment and a heparin-PBS wash to remove surface bound CPPs, which gives a
more accurate amount for the internalized CPPs. Another advantage of our protocol
is that we use
125
I as the tracer for CPPs, which has a lower detection limit and a
small influence on CPP’s internalization than a conjugated fluorophore. On the other
hand, there are also possible pitfalls while interpreting the data generated by this
protocol. One of such problems is that the presence of radioactivity may not indicate
the presence of
125
I-labeled peptide, since there may be free
125
I in the peptide
solution or
125
I may fall off from the peptide. To avoid this problem, thin layer
chromatography can be performed to check if there is any free
125
I in peptide sample.
Another possible pitfall is how to interpret surface binding data. Since only attached
cells were used in this thesis, the trypsin removable supernatant (refer to section
2.2.5.2 for protocol) contained not only surfaced bound
125
I-peptides but also
125
I-
peptides trapped within the extracellular matrix. This is the major reason that the
data for cell uptake are not proportional to the data for surface binding among
different arginine-rich CPPs (Table 7), because the amount of internalized peptides is
usually proportional to the amount of surface bound peptides but not the amount of
peptides trapped in the extracellular matrix. Last but not least, the extent of adhesion
between different peptides to containers (e.g., 6-well plate and test tube) varies due
to the net charge of peptides. Positively charged peptides bind more tightly to
containers than charge neutral peptides, which may cause more loss during many
68
experimental steps, such as incubation and washing, and lead to underestimated
results for the former peptides.
Based on the knowledge learned from the study of arginine-rich CPPs, I tried to
develop a targeted CPP-based drug delivery system with less non-specific binding.
The rationale behind the idea is the internalization behaviors of arginine-rich CPPs
with net positive charge versus net neutral charge. To achieve a targeted delivery, the
positive charges of CPP in the system should be masked or neutralized at non-
specific sites, and this masking effect should be removed at target sites. In other
words, this CPP-based system should act like YGR
6
G
6
or YG(RG)
6
at target cells
while acting like YGR
6
E
6,
YG(RE)
6
, or YGE
6
at non-target cells. An efficient
targeting requires a high target to background ratio. As shown in Table 7, there are
always larger differences in binding and uptake between YG(RG)
6
and YG(RE)
6
than those between YGR
6
G
6
and YGR
6
E
6
. This result suggests that it may be more
beneficial to use a CPP with dispersed positive charges instead of clustered positive
charges in delivery system, which may lead to a larger target to background ratio.
Taken together, all the findings from this part of research inspire my first design of
CPP-based targeted delivery system.
4.2. Exploration with chemically modified CPPs
Two key challenges must be addressed in order to realize a targeted drug
delivery system based on CPP. One of such challenges is to reduce the non-specific
binding of CPP. The other problem is to provide the targeting ability to delivery
system since CPP doesn’t target to specific cells or tissues. Due to CPP’s strong
69
interaction with all kinds of cell, direct attachment of a targeting moiety to CPP
cannot achieve an intended targeting effect (Vivès et al., 2008). In fact, conjugation
of CPP to an antibody or antibody fragment didn’t improve its target to background
ratio (Hu et al., 2007), or even reduced its specificity in certain case (Niesner et al.,
2002). Therefore, both problems need to be overcome at the same time in order to
achieve a successful targeted CPP-based drug delivery.
Scheme 1. Reversible blocking of amino group by citraconic anhydride
To reduce the non-specific binding of CPP, I tried an acid-labile modification
on CPP using citraconic anhydride (Scheme 1). Citraconic anhydrides form amide
bonds with amino groups in peptides, which leave terminal carboxyl groups (Dixon
and Perham, 1968). Later, all amino groups can be restored after the citracoyl groups
are completely removed at acidic pH in several hours (Palacián et al., 1990). In fact,
similar reaction can be achieved using maleic or dimethylmaleic anhydride. However,
for the purpose of my application, maleyl amino bond is too stable, with a half-life of
11 h at pH 3.5 and 37°C (Butler et al., 1967), while the hydrolysis of dimethylmaleyl
group is too fast, completed within 5 min at pH 3.5 and 20°C (Dixon and Perham,
1968). Arginine-ornithine co-oligopeptide, YG(RO)
6
, was first tested and modified
with an excess amount of citraconic anhydride. Once all the amino groups in peptide
70
were blocked, the citraconylated peptide would have a negative net charge and a
similar charge distribution to YG(RE)
6
, which showed very low surface binding and
cell uptake (Table 7). Therefore, citraconylated YG(RO)
6
should exhibit less non-
specific binding. At acidic pH, the ornithines of peptide would be restored after the
hydrolysis of citraconyl groups, which would lead to high binding and subsequently
high cell uptake. However, the citraconyl groups in the modified YG(RO)
6
were very
stable after overnight treatment at pH 3-4, presumably due to their interaction with
the guanidine groups in adjacent arginine residues.
Although the first attempt with YG(RO)
6
failed, a similar citraconylation
reaction was performed, this time, with MAP. As shown in previous studies of our
group, MAP was preferentially internalized through endocytosis and exhibited
significantly higher cellular uptake and nuclear internalization than arginine-rich
CPP (Zaro et al., 2009). In addition to its high intracellular accumulation, MAP is
also one of the most stable CPPs with a long pharmacokinetic half-life of greater
than 72 h (Sarko et al., 2010). With an amphipathic α-helical structure, the MAP
used in this project (with an extra tyrosine for labeling by
125
I at the N-terminus) has
poor solubility at the pH for citraconylation (pH 8). Therefore, the reaction was
carried out in DMF or DMSO. After CA-MAP was purified (Figure 5), the removal
of citraconyl groups in the peptide was tested at different pH conditions (Figure 6).
Indeed, citraconyl amino bonds were quite stable at pH 7, started to hydrolyze at pH
5, and broke down much faster at pH 3-4 (Palacián et al., 1990). In my hypothesis,
the relative stable citraconyl groups at neutral pH ensure a negative net charge of
CA-MAP, which should result in less non-specific binding. After CA-MAP is
71
internalized through endocytosis, the acidic endosomal environment should facilitate
the hydrolysis of citracoyl groups in CA-MAP, which restores MAP and in turn
facilitates its distribution into nucleus. Another beneficial effect after the
citraconylation of MAP is a reduced cytotoxicity for CA-MAP. As indicated in
Figure 7, compared to MAP, CA-MAP has at least a 10-fold decrease in cytotoxicity
and this reduction results from the presence of citraconyl groups in the peptide,
presumably due to the positive amino groups in MAP are blocked by the negative
citraconyl groups. This charge-dependent reduction of toxicity is consistent to a
previous study, in which the lysine residues in MAP were replaced by glutamate
residues (Scheller et al., 1999).
As mentioned before, a successful targeted CPP-based delivery system requires
low non-specific binding and an efficient targeting ability. Preliminary studies in
cells (results not shown) support that CA-MAP has low binding and uptake at neutral
pH. Therefore, a targeting moiety is needed to facilitate CA-MAP’s internalization at
target cells through endocytosis, and the target moiety used here is folate. Targeted
delivery via folate to folate-receptor overexpressing cells is one of the well-
established methods in active targeting of drug delivery (Leamon and Jackman, 2008;
Sudimack and Lee, 2000). With six folate-targeted drugs in human clinical trials, all
these folate conjugates bind to folate receptors, which are overexpressed on ~40% of
human cancer, and enter into cells through receptor-mediated endocytosis (Low and
Kularatne, 2009). As described in section 2.2.1.2, folate was linked to MAP through
an amide bond between either one of its carboxyl groups and one of the amino
72
groups in MAP. The average modification ratio was around one folate per MAP
molecule (Figure 8).
All three peptides (MAP, CA-MAP, and CA-MAP-F) were compared in HeLa
cells which overexpress folate receptors (Leamon and Low, 1992). As predicted,
CA-MAP showed much lower surface binding and cell uptake than MAP, and the
binding and uptake were restored to comparable levels to MAP when the peptide was
pre-treated overnight at pH 3-4 (Figure 9). Compared with CA-MAP, the conjugated
folate in CA-MAP-F increased the binding and uptake of peptide to same levels as
MAP. The nuclear localization and cell uptake of these three peptides exhibited a
similar pattern (Figure 10), while it seemed that folate may also contributed to the
nuclear localization of CA-MAP-F. It was surprising to find out that CA-MAP-F still
exhibited several-fold higher binding and uptake than CA-MAP in the presence of
4000-fold molar excess free folates (Figure 11). In contrast, there was a close to 80%
reduction in both surface binding and cell uptake for CA-MAP-F, when the cells
were co-treated with 20-fold unlabeled peptides. The results from competition assay
suggest that either a folate-independent mechanism exists for the increased binding
and uptake of CA-MAP-F from CA-MAP, or CA-MAP-F peptides in the solution
have a much higher binding affinity to folate receptors than free folates. Since folates
may self-assemble into aggregates of stacked tetramers (Ciuchi et al., 1994), one
possible explanation for the increased affinity is there are small aggregates in CA-
MAP-F solution which lead to high affinity to folate receptors and increased binding
and uptake.
73
Despite the unexpected competition results for CA-MAP-F, combining CA-
MAP with a targeting moiety seems to be a good targeted delivery system “activated”
inside acidic endosome. However, further studies (data not shown here) and the slow
removal of citraconyl groups at pH 5 indicate that the hydrolysis of CA-MAP into
MAP may not happen efficiently inside endosome. Interestingly, a previous study
found out a very inefficient cleavage of hydrazone bonds, another type of acid-labile
bonds, inside endosome after the folate conjugates were internalized (Yang et al.,
2007). The paper also pointed out that the average pH inside the endosomes, in folate
receptor trafficking pathway, is ~6.5. To avoid this problem, an ideal pH-sensitive
CPP-based delivery system should quickly respond to the mildly acidic pH inside
endosome where the CPP is “activated”, then, help its cargo escape to cytosol or
localize to nucleus. Although citraconylation of MAP has not achieved this goal, it
does offer a real example for managing CPP’s binding and uptake by manipulating
its net charge.
While there are successful stories with acid-labile modifications (Koren et al.,
2012; Leamon et al., 2006), I started developing a pH-responsive MAP triggered by
pH-dependent association and dissociation. Although there were pH-responsive
micelles with a similar concept (Kim et al., 2008; Mohajer et al., 2007; Sethuraman
et al., 2008), the design of peptide was inspired by a previous paper published by Dr.
Shen (Shen, 1990), in which he reported a pH-sensitive complex formation between
polylysine and histamine-modified polyglutamate. With five lysine residues aligned
at one side of its α-helical structure, MAP should associate with negatively charged
peptide C(HE)
6
through charge-charge interaction at neutral pH. At mildly acidic pH,
74
the protonation of histidine residues in C(HE)
6
should destabilize the association
between two peptides, which will release MAP for drug delivery. Since the
interaction between two short peptides is not very strong, they need to be linked
together in order to maintain their interaction at neutral pH. A SPDP linker was used
to attach C(HE)
6
to MAP. However, I didn’t get a good yield for the final product
due to poor solubility of MAP.
4.3. Transformation of MAP for targeting mildly acidic pH
Instead of testing another chemical linkage, I decided to fuse an H/E co-
oligopeptide to MAP through molecular cloning and express the fused peptide as a
GST-fusion protein in E. coli. There were at least three reasons at the time for this
decision. First, without a solid phase synthesizer, molecular cloning offers me an
easy way to change the sequence of peptide. Second, the recombinant peptides are
expressed as uniformed products which have the same amino acid sequence, with a
decent yield. Last but not least, protein expression in E. coli can be easily scaled up
and is more economical.
In order to mask five lysine residues in MAP, a (HE)
10
sequence was first used
because of its matching length to MAP and extra negative charges. The design of
alternating histidine and glutamate residues in the peptide was inspired by the study
of arginine-rich CPPs. The imidazole groups of histidine residues (pK
a
≈ 6) in the
(HE)
10
sequence are neutral at physiological condition, pH 7.4, allowing for the
electrostatic interaction between anionic glutamate residues of (HE)
10
and cationic
lysine residues of MAP. After exposure to mildly acidic pH, the imidazole groups in
75
histidine residues will be protonated, thus partially or fully neutralizing the negative
charges of (HE)
10
and destabilizing the electrostatic interaction between (HE)
10
and
MAP. Therefore, the cell penetrating activity of MAP will be rapidly regenerated at
mildly acidic pH. This regeneration process is similar to the rapid dissociation
between ligands and receptors inside acidic endosomes triggered by the change of
protein conformation (Ciechanover et al., 1983; DiPaola and Maxfield, 1984).
Thanks for my experience of molecular cloning during the undergraduate time,
I picked up the skills for cloning again quickly and began to design the DNA
sequence for the first fusion peptide. In the first design, MAP was at the N-terminus
and linked to (HE)
10
with a short pentaglycine sequence. The codons in DNA were
optimized for the expression in E. coli, and diversified at the repeating region, such
as the sequence encoding (HE)
10
. However, the cloning works for the first peptide,
MAP-G
5
-(HE)
10
, were not successful. During a time period of 6 months, I tested all
possible conditions and did trouble-shooting for each step, including plasmid
contamination, primer’s purity, PCR conditions, bacterial strains, and recipes of
culture media, but I could not get a correct clone (Table 5). For the first and second
rounds of cloning, I tried to insert the DNA fragment into pGEX-4T-1 plasmid. The
majority of sequencing results showed either a deletion or an insertion of one
nucleotide from correct sequence. I picked one clone with a single-nucleotide
deletion near the 5’-terminus of DNA, and tried to insert back the missing nucleotide
in round 3 and 4 of cloning using a mutation primer from two companies. While
round 3 yielded no correct clone, 2 clones showed correct insertion from the
sequencing results of round 4. However, both clones could not express GST-fusion
76
proteins during the test expression. Further sequencing with another primer revealed
deletions in GST gene for both clones. Although it is hard to prove things never
going to happen, the difficulties in the cloning of MAP-G
5
-(HE)
10
strongly argue that
the peptide may be too toxic to be successfully cloned. In contrast, the cloning of
HE-MAP was accomplished in the first attempt. The contrasting results of two
similar peptides, only with the switch between MAP and (HE)
10
, could be explained
in part by the organization of natural antimicrobial peptide precursor structures
(Vassilevski et al., 2008). The antimicrobial peptides typically exhibit amphipathic
structures consisting of clusters of cationic and hydrophobic amino acids (Zasloff,
2002). They are often produced as pro-protein with peptides containing many acidic
amino acids, either at N- or C-terminus, which form complexes with antimicrobial
peptides and prevent their toxic effects before secretion (Vassilevski et al., 2008).
Similar acidic peptides have already been used for the successful productions of
antimicrobial peptides in bacteria. Therefore, the expression of acidic (HE)
10
peptide
before MAP was able to mask the toxic effect of the latter peptide, which ensured the
success of cloning and expression in E. coli. Interestingly, a DNA sequence encoding
MAP was later inserted into pGEX-4T-1 plasmid, and a positive clone expressing
fusion protein GST-MAP was isolated. This cloning was not affected by the toxicity
of MAP because the majority of fusion proteins formed inclusion bodies. All the
findings during cloning suggest that toxic CPPs can be expressed in bacteria when
they are expressed either in inclusion bodies or as fusion peptides with acidic
peptides at proper position.
77
To express and purify various GST-fusion proteins, the conditions were tested
and optimized for higher yields during expression and better recovery and purity
during purification. Due to the antimicrobial activity of MAP (Palm et al., 2006),
many MAP-containing fusion proteins might be toxic to bacteria. In order to achieve
higher yields for these proteins, the leaky expression of fusion proteins should be
avoided and induction with IPTG is more efficient at a higher bacteria density.
Designed to be purified using the GST-tag, many fusion proteins have 10 histidine
residues in their (HE)
10
peptides, which raises the possibility to purify these proteins
using Ni-NTA agarose resin. Indeed, although the histidine residues were mixed with
anionic glutamate residues in (HE)
10
, GST-HE-MAP could be purified by Ni-NTA
resin (Figure 12). Ni-NTA resin-purified GST-HE-MAP tends to have more
precipitations during dialysis than those purified by GSH agarose resin, presumably
due to their different binding sites to different resins. This loss of purified proteins
could be reduced when the eluted proteins are exchanged into PBS, pH 7.4, by a
centrifuge device with MWCO at 10 kDa. Therefore, the incorporation of (HE)
10
to
MAP adds an additional advantage in the purification method following recombinant
production.
It was very exciting to finally get enough purified GST-HE-MAP to test in the
cell assays. To ensure accurate pH values during the treatment, dosing media were
prepared from RPMI1640 powders with additional buffer power at pH 5-7 which
was provided by the extra phosphates, citrates, and citric acids added to the media.
The initial finding with first batch of GST-HE-MAP showed a decreasing amount of
125
I-proteins with an increasing pH value from 6.0 to 7.5 both in surface binding and
78
cell uptake (Figure 16). There was a drop in cell uptake at pH levels below 6.3,
which was most likely due to inhibition of endocytosis in this pH range (Davoust et
al., 1987). In fact, the surface binding correlates well with the protonation level of
histidine residues in the fusion protein from pH 6.0 to 7.5. For the cell uptake of
GST-HE-MAP, the low uptake at neutral pH and the high uptake at mildly acidic pH
fit the hypothetical working model of HE-MAP peptide very well. Interestingly, the
pH profile of GST-HE-MAP showed a 10-fold difference in cell uptake between pH
6.5-7.0, which was much larger than I had expected. A possible explanation is that
the increased protonation of histidine residues from pH 7.0 to 6.5 suddenly
destabilizes the association between (HE)
10
and MAP, which results in a dramatic
increase in cell uptake. Off course, there are other possibilities which require further
study.
Following the radioactive assay, GST-HE-MAP was labeled with fluorescent
tag for confocal study. As shown in Figure 17, the fluorescent assay validated the
pH-dependent cell uptake of the fusion protein. Only cells incubated with the fusion
protein at pH 6.5, but not pH 7.4, showed punctate vesicular staining throughout the
cell, which is consistent with the localization of MAP and its protein conjugates
(Kenien et al., 2012; Zaro et al., 2009). Both radioactive and fluorescent assays
strongly indicate that GST-HE-MAP exhibits pH-sensitive binding and cell uptake at
mildly acidic pH expected in tumor microenvironment or early endosome (Cairns et
al., 2006; Coffey et al., 2006; Gerweck and Seetharaman, 1996; Sipe and Murphy,
1987).
79
Before a systematic study of different GST-fusion proteins, fusion peptide, HE-
MAP was purified from GST-HE-MAP for several simple characterizations (Figure
14). HE-MAP migrated on tricine gel as a higher molecular weight band (~10 kDa)
than expected (~5 kDa) due to the high number of polar residues in the sequence,
which is known to cause anomalous migration. After thrombin cleavage, several
bands appeared as lower molecular weight bands than HE-MAP on the gel, probably
due to the cleavage action of thrombin beyond the designed recognition site. This
problem may be avoided by adjusting the treatment conditions for thrombin or
optimization of peptide sequence. HE-MAP solution with a higher purity was
subjected to LC-MS analysis. The calculated mass from multiple-charge ions is the
same as the theoretical value predicted for HE-MAP, which strongly suggests the
expressed peptide should match the designed sequence. For a direct proof, amino
acid sequencing is required in future. The purified HE-MAP was also tested for its
far-UV CD spectra at pH 6.5 and 7.4 (Figure 15), in order to find out whether there
is a change in secondary structure between these two pH conditions. The near
identical spectra indicate that α-helical structures within HE-MAP at pH 6.5 and 7.4
are about the same.
It was fortunate that a correct length for histidine-glutamate co-oligopeptide
was chosen at the beginning of peptide design. To further study the effect of H/E co-
oligopeptide’s length, GST-fusion proteins with a shorter and longer H/E sequence,
(HE)
8
and (HE)
12
, were produced and compared with GST-HE-MAP. With different
repeat number (i.e., 8, 10, and 12) for H/E co-oligopeptide, three proteins showed a
similar trend at pH 6.0-7.5 in surface binding and cell uptake with significantly
80
different values (Figure 18). Compared with GST-HE-MAP, the deletion or insertion
of four amino acids, HEHE, increases or decreases the isoelectric point (pI) of the
respective fusion protein, GST-(HE)
8
-G
5
-MAP or GST-(HE)
12
-G
5
-MAP, which
shifts the pH profile of surface binding in the basic or acidic direction along the pH
axis in Figure 18A, respectively. The difference in pI among three fusion proteins
also contributes to the divergence of their cell uptake at various pH conditions.
However, the several-fold increase of cell uptake from GST-HE-MAP to GST-
(HE)
8
-G
5
-MAP at each pH value is unlikely caused only by the small pI change
between two fusion proteins. This suggests that the removal of HEHE from HE-
MAP significantly weakens the interaction between H/E co-oligopeptide and MAP.
The loss of masking for MAP in turn triggers a big increase in uptake. In summary,
the study of fusion proteins with different repeat number for H/E co-oligopeptide
reveals that the binding and internalization of GST-HE-MAP could be increased or
decreased by decreasing or increasing the length of H/E co-oligopeptide.
Since the first cell assay for GST-HE-MAP, pH-dependent binding and cell
uptake have been found for all different GST-fusion proteins containing the fusion
peptide of MAP and H/E co-oligopeptide. However, these results are only indirect
evidence for the HE-MAP mediated pH-dependent binding and uptake of cargo
protein, GST. At the same time, the cell assays for HE-MAP also reveal a similar
pH-dependent cell uptake for the peptide (data not shown). In order to get direct
evidence for the HE-MAP mediated pH-sensitive delivery of cargo protein, GST-
HE-MAP was compared with GST, GST-HE, and GST-MAP in cell assay at
different pH values. Total cell association is used for the comparison among different
81
fusion proteins, because previous studies from our group have shown that there may
be over-estimation or under-estimation in cell uptake or surface binding data due to
the different abilities of proteins or peptides to interact with cell membranes or to
withstand the trypsin treatment. In addition, the cell association data from cell assays
are more relevant to the fluorescent imaging data from animal studies.
GST and GST-HE-MAP were first compared for cell association at 6 different
pH conditions in HeLa cells (Figure 19). The consistent low cell association of GST
across pH 6.0-7.5 clearly indicates that the pH-dependent cell association of GST-
HE-MAP is mediated by HE-MAP peptide in the fusion protein. Next, three fusion
proteins, GST-HE, GST-MAP, and GST-HE-MAP, were purified together for the
following cell assay. Even before the cell study, they already showed distinct
differences in the expression level (Table 6) and during the purification (Figure 13D).
While GST-HE and GST-HE-MAP could be purified easily with GSH agarose, a
special protocol was used to solubilize and purify the GST-MAP, which mainly
formed inclusion bodies in E. coli. The large reduction in the expression level of
GST-MAP or GST-HE-MAP compared to GST-HE is presumably due to MAP,
which is known to have a certain level of antimicrobial activity (Palm et al., 2006).
The attachment of (HE)
10
to MAP in GST-HE-MAP increased its yield compared to
GST-MAP, which is indirect evidence that MAP is masked by (HE)
10
, thus reducing
its cytotoxicity. While MAP also showed toxicity in mammalian cells at a peptide
concentration over 2 μM (Scheller et al., 1999), the concentrations used in this thesis
were below this level and showed no cytotoxicity by MTT assay (data not shown).
82
To understand how (HE)
10
and/or MAP affected the cell association of GST,
125
I-labeled GST-HE, GST-MAP, and GST-HE-MAP were compared at 4 different
pH conditions in HeLa cells (Figure 20). With ten negative charges from (HE)
10
peptide, it was not surprising that GST-HE showed low cell association at pH 6.5-7.5
in HeLa cells. The cell associated GST-HE slightly increased at pH 6.0, presumably
because the protonation of histidine residues neutralized the negative charges in the
protein. The addition of (HE)
10
peptide to GST does not change its cell association
within this pH range (Fei et al., 2012). In comparison to GST-HE, GST-MAP
exhibited about 8-18 fold higher cell association at each pH tested. These results
support MAP as a potent carrier for proteins beyond neutral pH conditions (Kenien
et al., 2012; Oehlke et al., 1998). GST-MAP was slightly pH-sensitive, with a ~40%
increase at pH 6 compared to pH 7.5. GST-HE-MAP on the other hand had low cell
association, close to GST-HE, at neutral pH but high cell association, comparable to
GST-MAP, at mildly acidic pH. The combined contribution of (HE)
10
and MAP
indeed resulted in the pH-sensitive delivery of the GST-fusion protein, which
preferentially bound and entered cells at mildly acidic environment. This pH
sensitivity of GST-HE-MAP was also confirmed in other cell lines, i.e. MDA-MB-
231 (Figure 21A) and MCF-7 (data not shown). According to the hypothesis
published in 2012 (Zaro et al., 2012), the charge interaction between (HE)
10
and
MAP masks MAP and its association with cells at neutral pH. In order to determine
if this interaction requires a chemical linkage between the two peptides, equimolar
amounts of GST-MAP and (HE)
10
were mixed prior to dosing cells, where it was
shown that only the directly linked treatment group (GST-MAP-HE) exhibited pH
83
sensitivity (Figure 21B). This result strongly suggests that fusion of (HE)
10
and MAP
is required for their interaction at neutral pH.
To evaluate the pH-sensitive cell association in vivo, a mouse xenograft human
breast cancer model was generated using MDA-MB-231 cells with the help from Dr.
Li-Peng Yap from Dr. Peter Conti’s laboratory in the Molecular Imaging Center at
USC. The tumors were allowed to grow to a certain size so that they would provide a
mildly acidic microenvironment as established in previous publications (Gerweck
and Seetharaman, 1996; Raghunand et al., 1999). As shown in Figure 22A, the
kinetics of the free dye was completely different from that of IR800-labeled GST-
HE-MAP. This result suggests that the conjugation of dye is stable enough to
represent the distribution of fusion protein in mouse during this study, and the
increased fluorescence signal in tumors is unlikely due to the property of the dye
itself. Additionally, both in vivo imaging and ex vivo organ distribution (Figure 22, A
and B) indicate the tumor retention of GST-HE-MAP can be detected for more than
24 hours. To further investigate and confirm the results in vitro, equimolar doses of
three IR800-labeled fusion proteins were injected intravenously into mice (n = 3) and
imaged serially. At 2 h post-injection, the three groups injected with different IR800
fusion proteins showed distinct patterns of infrared fluorescent signals (Figure 23A),
which correlate well with the in vitro results (Figure 21A). Consistent with the in
vitro results, GST-HE showed a low fluorescent signal throughout the body except
for the kidneys, liver, and bladder where the protein was presumably processed
and/or excreted. For GST-MAP, the fluorescent signal was widespread throughout
the body and above the maximum of the scale. This extensive in vivo distribution of
84
CPPs is consistent with a previous finding (Schwarze et al., 1999), and serves as
additional evidence for CPP’s lack of specificity. GST-HE-MAP exhibited an
elevated tumor distribution compared to GST-HE, and had reduced distributions to
other normal organs and tissues when compared to GST-MAP (Figure 23A), which
was consistent with ex vivo imaging (Figure 22B). The comparison among three
fusion proteins in vivo clearly supports the notion that (HE)
10
can mask MAP at
normal pH, which leads to a selective distribution of cargo protein to the mildly
acidic tumor site (Zaro et al., 2012). In addition, the time course data indicate that
GST-HE-MAP can target efficiently (within 30 min post injection) to the tumor site
in vivo and has a long retention there (up to 24 hours) (Figure 22A and 23B).
The data presented in the last part of this dissertation establish GST-HE-MAP
as a first CPP-based recombinant protein, which targets the mildly acidic tumor
environment in vivo. Although the fusion protein is heavily distributed to kidneys,
presumably due to its small size (Maack et al., 1979), this issue could conceivably be
significantly improved by increasing the protein size above the renal filtration cutoff
of ~60 kDa. After optimization of in vivo distribution, this HE-CPP based system
could be used for diagnostic and/or therapeutic purposes for treatment of cancer.
Conceivably, the H/E co-oligopeptide can be applied to any CPP sequence that
depends on lysine and/or arginine residues for efficient internalization. The length of
the H/E repeats can be easily customizable to accommodate the number of cationic
charges in the CPP sequence. Therefore, this HE-MAP based system serves as a
model to study and develop other CPP-based delivery systems depending on pH-
sensitive association/dissociation.
85
5. CONCLUSIONS
In the first part of this dissertation, arginine-rich peptides were tested in CHO
cells for their surface binding, cell uptake, and intracellular localization. The data
provided here demonstrated that both positive charge and distribution of arginine
residues within a CPP sequence play important roles in the high cell uptake of the
arginine-rich CPPs. Although different arginine-containing peptides showed
different uptake efficiencies, all of the peptides tested in this part were able to reach
the cytosol inside the cells. The results give insights to understanding the mechanism
of the uptake, and also lay the foundation for the rational design of targeted CPP-
based drug delivery systems.
During the second period of this dissertation, several different chemically
modified CPPs were tested for their potential as pH-sensitive drug delivery systems.
Although all the attempts were not very successful, the data from the study of CA-
MAP provide an example for the management of surface binding and cell uptake of
MAP through altering the net charge. The possible inefficient conversion of acid-
labile chemical bonds inside mildly acidic endosome led to the pursuit of a targeted
delivery system based on the pH-sensitive association and dissociation between
MAP and histidine-glutamate co-oligopeptide.
In the last part of this dissertation, a pH-sensitive targeted CPP, HE-MAP, was
designed and produced as a GST-fusion protein. The pH-sensitivity of this fusion
86
protein could be changed by altering the length of H/E co-oligopeptide. The in vitro
data unambiguously support that HE-MAP can deliver a cargo protein, GST, to cells
within a mildly acidic environment (pH ~6.5). The in vivo data support that only HE-
MAP can target GST to tumor site with reduced distributions to other normal tissues
and organs except the kidneys and liver. This pH-sensitive peptide can be developed
as a diagnostic and/or therapeutic tool for acidic tumors. Moreover, the design of
HE-MAP offers a simple and effective way to reduce the non-specific binding and
uptake of CPPs at normal physiological pH. This design not only brings forward a
concise delivery platform for targeting of peptide/protein drugs, but also pushes
forward the application of CPPs and potential macromolecular therapeutics.
87
6. FUTURE PERSPECTIVES
At the end of this dissertation, a pH-sensitive CPP, HE-MAP, has been
developed, which can deliver a cargo protein to cells in mildly acidic pH (~6.5) in
vitro and tumors in vivo, presumably also in a mildly acidic environment. The
hypothetical model and potential applications of this pH-sensitive HE-MAP peptide
are summarized in Scheme 2 (Zaro et al., 2012). The data presented in this thesis
have provided important supporting evidence for the hypothetical model and the
potential application of this peptide in tumor microenvironment (Scheme 2A). At the
same time, many interesting findings and challenging problems encountered during
the research demand further studies and better solutions. In addition, the proposed
model and other potential applications of HE-MAP bring forward more questions
and possibilities which need to be answered and realized.
6.1. Mechanistic study for HE-MAP based delivery system
The results from cell assays strongly support the pH-dependent dissociation
between (HE)
10
and MAP from neutral to mildly acidic pH, as depicted in the
hypothetical model (Scheme 2). At pH 7.4, the huge difference between the cell
association under two treatment conditions (Figure 21B), GST-HE-MAP and GST-
MAP mixed with equimolar (HE)
10
peptide, emphasizes that the fusion between two
parts are required for the pH-sensitivity of HE-MAP. However, there are still many
88
details need to be filled in for this proposed model. For example, after its
internalization through endocytosis by cells at mildly acidic site, where does this HE-
MAP based delivery system go? The intracellular localization after internalization is
important because it dictates which type of delivery system is more suitable for a
specific drug in term of the intracellular localization of drug’s therapeutic target.
Since MAP has been showed to distribute to the nucleus (Zaro et al., 2009), this HE-
MAP based delivery system is a good carrier for cargos with a nuclear target. In
another study from our group, the results provide evidence that the distribution of
MAP-protein conjugates can be altered by different linkers between MAP and cargo
protein (Kenien et al., 2012). Based on this study, a disulfide linker can be used to
increase the cytosolic delivery of cargos by HE-MAP based system.
Besides the intracellular localization, it is also interesting to see whether HE-
MAP can be “activated” inside the endosome, once it is internalized through
receptor-mediated endocytosis (RME) after a targeting moiety is put onto the
delivery system. Basically, this is a proof of concept for the hypothetical model and
the potential application described in Scheme 2B. A pH insensitive MAP-based
system may be needed as control for this assay, with or without endosomotropic
agent which can abolish the acidic pH inside endosome. In addition to assays that
characterize the delivery system, different cargos need to be attached to the system
and tested for their activity after the delivery. At the same time, the effect of different
cargos on the internalization properties and intracellular localization of the HE-MAP
based delivery system need to be investigated during the study of cargo delivery.
89
Scheme 2. Potential applications of HE-MAP based delivery system. (A) At the
surface of normal (non-tumorigenic) cells (pH 7.4), the delivery system will be in its
inactive form, and not bind to the surface or be internalized. At the surface of tumor
cells, where the pH is mildly acidic (pH 6.5-7), the delivery system will be activated,
exposing the membrane-permeable CPP for subsequent binding and internalization.
(B) At the surface of non-target cells that do not express targeted receptors for RME,
the delivery system will be in its inactive form and not bind to the surface or be
internalized. Ligand attachment to un-masked CPPs has shown to be ineffective in
biological targeting methods via RME, resulting in high internalization in non-target
cells due to the high-cationic charge of CPPs. At the surface of target cells, the
delivery system will be internalized via RME where exposure to the acidic
endosomal environment will lead to activation of the membrane-permeability of the
CPP. (Adapted from Zaro et al., 2012, with permission from Elsevier)
90
Compared to its surface binding (Figure 16A), GST-HE-MAP has a much
higher pH-sensitivity in cell uptake (Figure 16B). A similar phenomenon is seen
when the CPP in the system is changed to Tat peptide or arginine-rich CPP, however,
to a much lower extent (results from Chunmeng Sun in our group). Therefore, this
extra pH sensitivity in cell uptake must link to certain property of MAP. Although no
difference in secondary structure is found for HE-MAP between pH 6.5 and 7.4
(Figure 15), further study is needed to understand how the structure of this peptide
would affect its binding and uptake at different pH conditions. It is possible that HE-
MAP have different levels of membrane permeability at these two pH values, which
cannot be revealed without the presence of membrane lipids. Changing the repeat
number for H/E co-oligopeptide may also provide some information about how the
interaction between MAP and H/E co-oligopeptide will affect the internalization of
fusion peptide as a whole.
6.2. Pre-clinical study for HE-MAP based delivery system
Although the imaging study in a mouse tumor model has shown that HE-MAP
can target a cargo protein to the tumor site in vivo, there are still many questions
need to be answered. The first one of such questions is to confirm the HE-MAP
mediated distribution in vivo in pH-dependent. In order to establish a direct
relationship between the protein distribution and pH at various tissues and organs in
vivo, both parameters need to be monitored simultaneously in the same study.
Another relevant question is how HE-MAP based delivery system is distributed
within the tumor mass and at the cellular level. To answer this question, other animal
91
imaging technologies may be required. Moreover, the stability of this delivery
system also needs a full evaluation.
While the imaging study in mice (Figure 22 and 23) has clearly shown the
potential of GST-HE-MAP as a diagnostic (imaging) tool of tumors inside body,
more experiments will be needed for pre-clinical study. First, IR800-GST-HE-MAP
has a very high distribution to the kidneys, which may cause the interference of
signals during the imaging for tumor near the kidneys. Therefore, other fusion
partners for HE-MAP or further modifications for GST-HE-MAP need to be tested
in order to reduce the distribution to the kidneys. Another required experiment will
be the in vivo toxicity study (e.g. using mice). The optimized system should exhibit
minimum to no toxicity for all major organs, such as the kidneys and liver. At the
same time, a complete study is needed for the pharmacokinetics (PK) of candidate
HE-MAP based system.
To further develop HE-MAP into a potential therapeutic for cancer, a proper
drug needs to be selected and linked with HE-MAP delivery system. While a protein
or peptide drug is easier to produce with the system, a small-molecule drug may also
be used. For the latter drug class, the drug molecules need to be incorporated into the
delivery system, which should only be released at target site. Then, the drug loaded
system needs to be tested in cell assay in order to demonstrate the following two
properties: (1) it should be highly effective against various types of cancer cell at
mildly acidic condition; (2) it should exhibit little to no toxicity to normal cells at
physiological pH. After in vitro assays, animal experiments are required for its
toxicity, PK, and pharmacodynamics (PD). Since the pH at inflammation sites is also
92
mildly acidic (Menkin, 1960), the HE-MAP delivery system may also be applied to
inflammation diseases after similar in vitro and in vivo tests.
When HE-MAP is used as a pH-sensitive delivery system that gets activated
inside endosome, the in vivo targeting of cargos is mainly dependent on the property
of the targeting moiety used in the system. Similarly, all the aforementioned studies
are required for its pre-clinical evaluation. The real interesting point is the potential
that HE-MAP can bring to the targeting moiety. Without being overwhelmed by the
strong non-specific interaction between CPP and cell membrane, the HE-MAP based
delivery system maintains the original efficiency of targeting moiety, and is activated
inside endosome and ensures delivery of cargos, as long as the system is internalized
by any kind of endocytosis. For those well-established targeting ligands which
trigger receptor-mediated endocytosis, such as folate, the HE-MAP based delivery
system can compensate the inability of folate to deliver macromolecular therapeutics
outside endosome (Low and Kularatne, 2009). For the emerging cell-targeting
peptides (Vivès et al., 2008), on top of the advantage mentioned above, the HE-MAP
base delivery system can fuse with anyone of these peptide and offers a simple
production method.
In summary, further study of HE-MAP will increase the understanding of
rational design of pH-sensitive CPPs, and pave the road for the potential clinical
applications of CPPs.
93
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Abstract (if available)
Abstract
Properties of different arginine‐rich peptides, including net charge and charge distribution, were evaluated for their influence on surface binding, internalization, and intracellular localization. The peptides were radiolabeled and subsequently tested for surface binding and internalization in cells. Subcellular fractionation assays were performed to separate the amount of peptides associated with vesicles from those inside the cytosol. Net neutral charged peptides, YGR₆E₆ and YG(RE)₆, showed large decreases in both surface binding and cell uptake compared to their net positive charged counterparts, YGR₆G₆ and YG(RG)₆. The peptides with clustered arginine residues, YGR₆G₆ and YGR₆E₆, exhibited significantly higher binding and uptake than those with alternating arginine and glycine/glutamate residues, YG(RG)₆ and YG(RE)₆. The intracellular distribution analysis for all of the peptides tested showed that, regardless of the net uptake, the arginine‐rich peptides were preferentially localized in the cytosolic compartment of the cells. Both net positive charge and a clustered arginine sequence enhance the surface binding and cell uptake of peptides, however, the intracellular distribution does not change. These initial findings inspired the design of the following CPP‐based drug delivery systems. ❧ Citraconic anhydride was used to modify different CPPs in order to develop a pH‐sensitive delivery system. After the citraconylation, CA-MAP showed very low surface binding and cell uptake in cell assays. The binding and internalization could be recovered when the citracoyl groups in the peptide were removed at acidic pH (3-4). However, this acid facilitated process may not be very efficient inside endosomes with a mildly acidic pH (~6.5). The addition of folate to CA-MAP as targeting ligand could increase its cell uptake and nuclear internalization in folate receptor positive cells. Although several attempts involving chemical modification didn't lead to a feasible pH‐sensitive CPP for clinical application, the data from these attempts did prove the concept that the binding and uptake of CPP could be controlled through the manipulation of its net charge. ❧ Finally, a pH‐sensitive CPP, HE-MAP, was produced as a GST‐fusion protein in bacteria. Both radioactive and fluorescent assays showed pH‐dependent cell uptake for GST-HE-MAP in cells, which could be used for targeting mildly acidic pH. Cell uptake assays also showed that the pH‐dependent binding and uptake profile of GST-HE-MAP could be shifted when the repeat number of H/E co‐oligopeptide was changed. Compared with two other fusion proteins containing either (HE)₁₀ or MAP, only the fused peptide, HE-MAP, could effectively deliver the cargo GST protein to cells at pH 6.5 or below, while maintaining low delivery to cells at pH 7.0 and above. Using a xenograft mouse model of human breast cancer, fluorescent imaging showed that only HE-MAP could effectively target GST to the tumor site, while reducing non‐specific association of MAP in other organs. The in vitro and in vivo data have demonstrated the diagnostic and/or therapeutic potential of the fused peptide, HE-MAP, for targeting the acidic tumor microenvironment. The concise design of this pH‐sensitive peptide offers a simple way to overcome CPP's lack of selectivity, which could lead to increased application of CPPs and macromolecular therapeutics.
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Fei, Likun
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Cell penetrating peptide-based drug delivery system for targeting mildly acidic pH
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