Novel integrin-targeted binding-triggered drug delivery system for methotrexate

Article abstract of DOI:10.1007/s11095-011-0495-5

Purpose: To design a binding-induced conformation change drug delivery system for integrin-targeted delivery of methotrexate and prove the feasibility of using hairpin peptide structure for binding triggered drug delivery. Methods: Methotrexate prodrugs were synthesized using solid phase peptide synthesis techniques by conjugating methotrexate to Arg-Gly-Asp (RGD) or a hairpin peptide, RWQYV^DPGKFTVQRGD (hairpin-RGD). Levels of integrin α_Vβ₃ in HUVEC were up-regulated using adenoviral system and knocked down using siRNA. Stability of prodrugs and methotrexate release from prodrugs were evaluated in plasma, in presence or absence of integrin α_Vβ₃-expressing cells. Molecular modeling was performed to support experimental results using MOE. Results: Prodrugs recognized and bound to integrin α_Vβ ₃-expressing cells in integrin α_Vβ₃ expression level-dependent manner. Prodrug with hairpin peptide could resist Streptomyces griseus-derived glutamic acid-specific endopeptidase (SGPE) and plasma enzyme hydrolysis. Drug release was triggered in presence of HUVEC cells and SGPE. Analysis of conformation energy supported that conformational change in MTX-hairpin-RGD led to exposure of labile link upon binding to integrin α_Vβ₃-expressing cells. Conclusions: Binding-induced conformation change of hairpin peptide can be used to design integrin-targeted drug delivery system.

Full text of DOI:10.1007/s11095-011-0495-5

Pharm Res (2011) 28:3208–3219
DOI 10.1007/s11095-011-0495-5
RESEARCH PAPER
Novel Integrin-Targeted Binding-Triggered Drug Delivery
System for Methotrexate
Phanidhara Kotamraj & Wade A. Russu & Bhaskara Jasti & Jay Wu & Xiaoling Li
Received: 8 March 2011 /Accepted: 27 May 2011 /Published online: 22 June 2011
#
Springer Science+Business Media, LLC 2011
ABSTRACT
griseus-derived glutamic acid-specific endopeptidase (SGPE) and
plasma enzyme hydrolysis. Drug release was triggered in
presence of HUVEC cells and SGPE. Analysis of conformation
energy supported that conformational change in MTX-hairpin-
RGD led to exposure of labile link upon binding to integrin
αVβ3-expressing cells.
Conclusions Binding-induced conformation change of hairpin
peptide can be used to design integrin-targeted drug delivery
system.
Purpose To design a binding-induced conformation change
drug delivery system for integrin-targeted delivery of metho-
trexate and prove the feasibility of using hairpin peptide
structure for binding triggered drug delivery.
Methods Methotrexate prodrugs were synthesized using solid
phase peptide synthesis techniques by conjugating methotrex-
ate to Arg-Gly-Asp (RGD) or a hairpin peptide,
RWQYVDPGKFTVQRGD (hairpin-RGD). Levels of integrin
αVβ3 in HUVEC were up-regulated using adenoviral system
and knocked down using siRNA. Stability of prodrugs and
methotrexate release from prodrugs were evaluated in plasma,
in presence or absence of integrin αVβ3-expressing cells.
Molecular modeling was performed to support experimental
results using MOE.
.
.
.
KEY WORDS hairpin peptide integrin αVβ3 methotrexate
.
targeted delivery triggered release
INTRODUCTION
Results Prodrugs recognized and bound to integrin αVβ3-
expressing cells in integrin αVβ3 expression level-dependent
manner. Prodrug with hairpin peptide could resist Streptomyces
In cancer therapy, achieving high tumor to non-tumor
tissues drug concentration ratio is required to reduce side
effects. Targeted drug delivery of anticancer agents can
be designed using prodrug strategy to release the active
drug in the vicinity of tumor via tumor-targeted
enzymatic activation (1). Several ligands and monoclonal
antibodies specific to receptors and antigens associated
with tumors have been reported to bring anticancer agents
specifically to tumors and then release the anticancer
agent by enzymatic activation (1–3). Though these
strategies have been shown to be promising in achieving
high tumor to blood drug ratios, they suffer from serious
limitations, such as immunogenicity posed by antibodies
and premature prodrug activation by residual enzyme
present in the blood (1–3). Due to these drawbacks,
refining the reported methods and search for new
approaches based on the ligand-specific interaction and
enzymatic activation continues.
Electronic Supplementary Material The online version of this article
(doi:10.1007/s11095-011-0495-5) contains Supplementary Material,
which is available to authorized users.
:
:
:
P. Kotamraj W. A. Russu B. Jasti X. Li (*)
Department of Pharmaceutics & Medicinal Chemistry, T. J. Long
School of Pharmacy & Health Sciences, University of the Pacific
Stockton, California 95211, USA
e-mail: xli@pacific.edu
J. Wu
VM Discovery
Fremont, California, USA
Present Address:
P. Kotamraj
Abon Pharmaceuticals
Northvale, New Jersey, USA

Binding Triggered Drug Release for Integrin Targeted Delivery
3209
Most improvements for tumor-targeted enzyme-based
prodrug activation delivery methods have been focused on
optimizing specificity (Kcat/KM) and reducing the half-life in
order to reduce prodrug activation away from the tumor
environment (4, 5). In addition, introducing clearing agents
to expedite the removal of residual enzyme antibody
conjugate (E-Ab) from blood, restricting the enzyme expres-
sion within the tumor cell, and using humanized antibodies
have alleviated these problems to some extent (1, 3, 6, 7).
Methotrexate (MTX)-amino acid prodrugs have been
designed to be specifically cleaved by carboxypeptidase
(CPA) enzyme for tumor targeting. These prodrugs were
made by allowing glutamic acid residue, α-COOH of MTX,
to react with an additional amino acid. When phenylalanine
(MTX-α-Phe) is used as the additional amino acid, it was
shown to be a better substrate for CPA among all other single
amino acid conjugated prodrugs (4). The bulkier structure of
the labile link in MTX-α-Phe prodrug made it insensitive to
the endogenous carboxypeptidase enzyme and hence meta-
bolically more stable in the plasma.
enzyme, which eliminates the need for clearing agent. This
design provides significant advantages over antibody-mediated
enzyme targeting. Absence of antibodies circumvents the
immunogenicity of antibody-mediated enzyme targeting. The
objectives of this study are to design and synthesize the RGD-
targeted MTX prodrug with β-hairpin peptide structure and
demonstrate binding-induced targeted release of MTX in vitro.
MATERIALS AND METHODS
Materials
All 9-fluorenylmethyloxycarbonyl (Fmoc) protected amino
acids and Rink amide resin were obtained from Novabio-
chem (Darmstadt, Germany). Dimethylformamide (DMF),
diisopropylcarbodiimide (DICI), piperidine, hydroxybenzo-
triazole (HOBT), dimethylchloride (DMC), trifluoroacetic
acid (TFA), diisopropylethylamine (DIPEA), 4-amino-4-
deoxy-10-methyl pteroic acid HCl (MAPA), 2,4-dinitro-
phenol, ethylcarbodiimide hydrochloride (EDC), bovine
serum albumin, methotrexate, azo-casein, cell lysis RIPA
buffer and alkaline phosphatase secondary antibodies were
purchased from Sigma-Aldrich (St. Louis, MO, Milwaukee,
WI). Thioanisole and 1, 2 ethanedithiole were obtained
from TCI (Portland, OR) and Alfa Aesar (Ward Hill, MA),
respectively. Streptomyces griseus-derived glutamic acid endo-
peptidase (SGPE) was purchased from Fluka. GRGDS was
purchased from polypeptide laboratories. Human Umbili-
cal Vein Endothelial Cells (HUVEC, pooled), EGM
complete medium, and bovine brain extract (BBE) were
obtained from Lonza (Walkersville, MD). VnRC3 cells were
kindly donated by Dr. Ginsberg (Scripps Research Institute,
La Jolla, CA). All other cells were obtained from ATCC.
AdEasy™ adenoviral vector system kit was obtained from
Stratagene (Stratagene, La Jolla, CA). The lipofectamine and
primers were purchased from Invitrogen. The integrin β3
siRNA (human) was purchased from Santa Cruz biotechnol-
ogy (Santa Cruz, CA). The αVβ3 Integrin Investigator Kit,
rabbit polyclonal antibody against integrin β5 and Immobilon
P (polyvinylidene difluoride, PVDF) for electroblotting were
purchased from Millipore. Fetal bovine serum (FBS) was
purchased from Innovative Research (Novi, MI); Fibrinogen
was purchased from Calbiochem (Gibbstown, NJ).
Several tumors, such as gliomas and ovarian cancers,
have been shown to over-express integrin αVβ3 receptor (8),
a membrane-bound hetero-dimer associated with angio-
genesis and tumor metastasis (9, 10). Arg-Gly-Asp (RGD), a
tripeptide sequence commonly conserved in endogenous
αVβ3 binding ligands such as firbronectin and vitronectin
(11), has been used for radioimaging (12), viral transfection
(13) and drug delivery (14).
In this study, a two-pronged approach for drug
accumulation at tumor site was investigated which could
potentially eliminate the immunogenicity problems associ-
ated with tumor-targeted enzyme-based prodrug activation.
First, prodrugs are designed to target a specific receptor
using a small ligand that can negate the necessity for enzyme-
antibody (E-Ab) conjugation to eliminate immunogenicity-
related problems. Second, a beta-hairpin peptide structure is
introduced to the delivery system to reduce the premature
activation of prodrug in the blood circulation. A prodrug built
on the β-hairpin peptide structure is designed to achieve the
above-mentioned characteristics. The prodrug consisted of
MTX as a model anticancer agent, RGD as targeting moiety,
and β-hairpin peptide to protect the enzymatic labile linkage
to MTX. It is hypothesized that prodrug would resist
enzymatic hydrolysis prior to reaching the target and reduce
the premature activation by residual enzyme in the blood
circulation. Upon binding to the target, overexpressed
integrin αVβ3 on tumor cells, the β-hairpin structure would
be perturbed to expose the labile linkage of MTX for
enzymatic cleavage. A twelve amino acid peptide,
RWQYVDPGKFTVQ, previously demonstrated to fold into
an anti-parallel β-hairpin in aqueous environment (15), is
used to mask enzymatic hydrolysis. The tumor binding
would trigger the prodrug activation by specific foreign
Synthesis of MTX Prodrugs
Two prodrugs were synthesized by conjugating MTX with
either RGD (referred as MTX-RGD) or a twelve amino acid
peptide-RGD, RWQYVDPGKFTVQ-RGD (referred to as
MTX-hairpin-RGD). Peptides were built from C to N
terminus on Rink amide resin using standard N-9-
fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide syn-

3210
Kotamraj et al.
thesis (SPPS) protocols at room temperature. Briefly, coupling
of amino acid was carried out in Chemglass® synthesizer for
one hour with intermittent shaking by adding 2.5 mol
equivalents of amino acid, diisopropylcarbodiimide (DICI)
and 1-hydroxy-benzotriazole (HOBt) in dimethylformamide
(DMF) to one mol of resin. Before coupling of a new amino
acid, amine groups were deprotected by treating with 20%
piperidine in DMF. After the coupling, the unreacted groups
were blocked by acylation (335 μL acetic anhydride and
530 μL diisopropylethylamine mixture in DMF for each mol.
of resin). After each step, the resin was successively washed
with DMF and dichloromethane (CH2Cl2) three times each
to remove residual reactants. Completion of reaction was
confirmed by ninhydrin test. Typical coupling cycle involved
repetition of coupling step two times with 2.5 equivalents of
each amino acid followed by capping. The resin-bound
peptide was used for further coupling of dinitrophenyl ester of
4-amino-4-deoxy-10-methyl pteroic acid (MAPA) and 2, 4
dinitrophenol ester (DNPE).
Activation of COOH of MAPA was achieved by forming
an active ester of 2, 4 dinitrophenol (DNPE). The DNPE was
synthesized by carefully heating reaction mixture consisting of
MAPA and 2, 4-diniotrophenol (DNP) in 2:3 mol ratio and 3-
(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride
(EDC, 1.5 parts in mol) in DMF at 48 2°C for 72 h. For every
24 h, additional amounts of EDC and DNP were added. After
72 h, solvent was evaporated under vacuum using rotary
evaporator, and residue was triturated in isopropanol. Com-
pletion of reaction was monitored by TLC. The molecular
weight of final product was confirmed by Matrix-Assisted
Laser Desorption Ionization—Time of Flight (MALDI-TOF)
mass spectrometry. The DNPE (1 part) was condensed with
free amine group of N-terminal glutamic acid (E) on the
resin (1 part) in presence of diisopropylethylamine
(DIPEA, 1.5 parts) in DMF at room temperature with
stirring for 72 h. Schematic is shown in Fig. 1.
high performance liquid chromatography (RP-HPLC,
Thermo separation products, P4000, AS3000, UV2000,
ChromQest v2.51, Agilent C18, 5 μ, 4.6×250 mm,
detection at 305 and 214 nm). Separation was achieved
by using a mobile phase gradient method. Peaks were
characterized by either MALDI-TOF (Shimadzu-Kratos
PC Axima CFR V2.2.1) or Electro Spray Ionization Mass
Spectrometry (ESI-MS, Varian 500-MS Ion Trap Mass
Spectrometer equipped with an ESI source, MS Worksta-
tion Version 6.9.1) to confirm the molecular weight.
Elutions containing the prodrugs were pooled, evaporated
and finally lyophilized to obtain prodrug with a purity of
95% or higher. They were stored at −20°C till use.
Cell Culture
All cancerous cell lines (VnRC3, A2058, MCF7, A375 and
HTB 129) were grown in 75 cm2 cell culture flasks obtained
from TPP in Dulbecco’s Modified Eagles Complete
Growth Medium (DMEM) supplemented with 10% FBS,
glutamine (1%v/v, 200 mM) and antibiotics (1%v/v,
5,000 I.U/mL of penicillin and 5,000 μg/mL of strepto-
mycin) in 5% CO2 incubator at 37°C. HUVEC cells were
grown under similar conditions using the EGM complete
growth medium supplemented with BBE.
Characterization of Integrin Expression
Western blot analysis was carried out on the cell lysates of
VnRC3, HUVEC, A2058, MCF7, A375 and HTB129 to
determine the expression pattern of αV, β3, and β5. Cell lysate
was prepared using RIPA buffer from the centrifuged cell
pellet of confluent cells by subjecting the pellet to four freeze-
thaw cycles (5 min in −80°C and 1 min at 37°C). The protein
content was determined by using BCA assay kit. Proteins were
analyzed using 8% SDS-PAGE gel at 200 mV for 30 min.
PVDF membranes were used to transfer the proteins, and
they were blocked with 15 mL of SEA BLOCK blocking
buffer in TBST (SB-TBST) for 1 h. The PVDF membranes
were then incubated with primary antibodies (αV, β3 or β5
diluted in SB-TBST, 1:1000 times) overnight at 4°C, followed
by incubation for 1 hr with alkaline phosphatase-coupled
rabbit anti-goat antibody. Antibody-protein complexes were
then detected using the Lumiphos-based chemiluminescence
technique to determine integrin protein levels.
The prodrug was cleaved from the resin after coupling
with DNPE using a cleavage cocktail containing, 2.5%
triisopropylsilane (TIS), and 2.5% water containing suffi-
cient quantity of thioanisol and ethanedithiol in trifluoro-
acetic acid (TFA). Resin was filtered, and filtrate was
evaporated carefully using rotary evaporator under low
pressure below 30°C to obtain crude concentrated prodrug.
Purification and Characterization
The concentrated prodrug was precipitated by adding it
into chilled isopropylether. This procedure was repeated
three times to remove ether-soluble impurities. The water-
soluble peptide was lyophilized to obtain a dry solid.
Prodrug was stored at −20°C until further processing.
Possible deletion products and unconjugated peptide
without MAPA portion were separated by reverse phase
Modulation of β3 Expression
The cDNA for β3 was cloned into adenovirus using
AdEasy™ adenoviral vector system according to the
manufacturer’s recommendations. Briefly, 2.5 μL of 105
times diluted pc-DNA3 stock containing β3 cDNA (a gift
from Dr. David Wilcox, Medical College of Wisconsin

Binding Triggered Drug Release for Integrin Targeted Delivery
3211
Fig. 1 Synthetic scheme for
MTX-hairpin-RGD.
O
HO
NH2
N
N
N
N
N
NH2
MAPA
2,4-DNP
EDC
DMF
O2N
O
O
NH2
NO2
N
N
N
N
N
NH2
Solid phase peptide synthesis
1. H2N-Glu-Peptide-NH-Resin
DIPEA
2. Cleavage from resin
RGD
RGly
RLys
RThr
RGln
O
O
O
O
O
H
H
N
H
N
H
N
N
N
H
N
H
N
H
N
H
NH2
O
RPhe
RTyr
O
RVal
O
RArg
RGlu
O
RAsp
HN
O
O
O
RTrp
O
O
H
N
H
N
H
N
N
N
H
N
H
N
H
NH2
N
RVal
O
RGln
O
RArg
O
N
N
N
N
NH2
Labile link
MTX
MTX-hairpin-RGD
Cancer Center) was electroporated (Eppendorf, Electro-
porator 2510) into 40 μL of electrocompetent JM109
bacteria to produce the plasmid for further use. The β3
cDNA was amplified from pc-DNA3 by the forward and
reverse primers (I-F and I-R, Table I) with EcoRV and
XhoI sites at 5′ and 3′ ends, respectively. The cDNA
collected from pGEM-T-β3 was then ligated between
EcoRV and XhoI sites with T4 DNA ligase at the multiple
cloning site of pCMV-Tag 2A with the FLAG tag sequence
at the 5′ end of the insert. The resultant pCMV-Tag 2A-β3
construct was used to amplify the FLAG-β3 cDNA with
BglII and XhoI sites on forward and reverse primers (II-F
and 1-R, Table I). The FLAG-β3 cDNA was harvested
from pGEMT-FLAG-β3 by restriction digestion with XhoI
and BglII. The cDNA was then ligated to the linearized
pShuttle between BglII and XhoI sites in the multiple
cloning sites. The product (pShuttle-FLAG-β3) was con-
firmed by EcoRV digestion (positions 1007 and 3404)
after selecting the transformants from the electroporated
JM109 cells grown on 0.03% Kanamycin plates.
BJ5183-AD1 cells containing pAdEasy-1 vector were
electroporated with linearized pShuttle-CMV-flag-β3.
The pAdEasy-FLAG-β3 constructs were purified using
large prep, and AD-293 cells were transfected with the
purified constructs using Lipofectamine PLUS. After four
days of incubation, the AD-293 cells were harvested and
lysed. To overexpress β3, 50 μL of the lysate was added to
T-25 flask, and the expression of β3 was confirmed by
western blot after 48 h.
Downregulation of β3 was achieved by treating the cells
in T-25 flask with 16 μL of 10 μM siRNA specific for β3
and lipofectamine® in Optimem medium (2 mL) for 5 h.

3212
Kotamraj et al.
Table I Primers for PCR
Primer
Sequence
Tm (°C)
I – F
I – R
II – F
CG ATA TCA ATG CGA GCG CGG CCG CGG CC
96
106
100
A Adenine; C Cytosine; G Gua-
nine; T Thiamine; F Forward
primer; R Reverse primer; Tm
Melting temperature (approx.)
CG CTC GAG TTA AGT GCC CCG GTA CGT GAT ATT GG
CG AGA TCT GCC ACC ATG GAT TAC AAG GAT GAC G
Then, the medium was replaced with complete growth
medium, and the expression was examined by western blot
after 48 hrs. The suppression of β3 expression was
determined by western blot analysis.
points and compared with those obtained in the growth
medium without cells.
Plasma Stability Studies
Image J (Version 1.44p, Wayne Rasband, NIH, USA)
was used to quantify western blots. Image densities were
corrected for background, and the relative density com-
pared to the siRNA-treated sample was reported.
Blood was collected into heparinized vacutainer (Becton
Dickinson) from anesthetized rabbits, and plasma was
collected by centrifugation. Prodrugs were incubated in
plasma at 37°C for 24 h at 100 μM final concentrations.
Amounts of released MTX at different time points were
estimated by HPLC following a reported sample prepara-
tion method (16). Briefly, at predetermined time points,
25 μL of sample and 50 μL of chilled acetonitrile (ACN)
were collected. The samples were centrifuged, and super-
natant was added to chloroform (four times volume of
supernatant). Aqueous supernatant was analyzed by HPLC
for MTX. Stability was expressed as the amount of MTX
released at different time points. Free MTX was also
incubated as a control to observe the degradation of MTX
in the plasma. The proteolytic enzyme activity of the
plasma was confirmed using standard azo-casein method.
MTX recovery for the extraction procedure was deter-
mined to be not less than 97%.
Binding Specificity of Prodrugs
Binding specificity of the prodrugs to αVβ3 integrins was
demonstrated by endothelial adhesion assay as described
previously (17) with modifications. Untreated 96-well plate
wells were coated with 50 μL each of fibrinogen (FG,
1 mg/mL), bovine serum albumin (BSA, 3% w/v), MTX-
RGD (10 μmolar) and MTX-hairpin-RGD (10 μmolar)
dissolved in Hank’s balanced buffer saline (HBSS) and
incubated at 4°C overnight. Before the experiment, wells
were briefly blocked by 0.5% w/v of BSA. Each well was
incubated with 20,000 trypsinized cells expressing different
levels of αVβ3 (overexpressed, wild or knockdown) in
complete growth medium for 90 min with or without a
pre-incubation step (1 h at 4°C) with RGD peptide. Non-
adherent cells were washed with HBSS. Adhered cells were
fixed in methanol for 30 min at 4°C and were stained with
crystal violet for 15 min after HBSS washings. Absorbance
was read at 565 nm after dissolving in 200 μL of 1%
sodium dodecyl sulfate (SDS).
Molecular Modeling
Molecular Operating Environment (MOE-2006.08,
Chemical Computing Group, Inc.) software was used
for building molecules, energy minimization, peptide-
integrin docking and dynamics. A twelve amino acid
peptide with sequence of RWQYVDPGKFTVQ was
built. The anti-parallel β-hairpin conformation was in-
duced by applying restrains for distances between alpha
carbon atoms on opposite strands based on the NMR
NOEs reported for this sequence in the literature (15).
Arg-Gly-Asp (RGD-NH2) and MTX were attached at the
C and N terminus, respectively. Energy was minimized
using AMBER99 forcefield without restrains for distances
in aqueous environment to observe the propensity to form
hairpin. Local energy minimum was determined by
repeated in silico heating and cooling with concomitant
search for low energy conformations followed by energy
minimization of all molecules in the database. Binding
simulation studies were carried out with integrin αVβ3 X-ray
crystal structure (Protein Data Base file, 1L5G) using cyclic-
RGD as template for bound RGD conformation. β-hairpin
SGPE Enzyme Hydrolysis Studies
One mL of 100 μM MTX-hairpin-RGD was incubated
with 8 mU of SGPE in presence or absence of αVβ3-
expressing HUVEC in a 12-well culture plate. At prede-
termined time points, 25 μL of samples were drawn and
immediately added to 100 μL of cold acetonitrile to
inactivate enzyme. These samples were frozen until the
analysis. MTX-RGD was used as control to estimate the
hydrolysis rate. Samples were thawed, appropriately diluted
and analyzed for MTX by HPLC (Thermo Separation
Products, P4000 pump, AS3000 autosampler and UV2000
detector; SN4000 Spectra System; ChromQuest v.2.51)
using Agilent (C-18, 4.6×250 mm) column at 305 nm.
Initial hydrolysis rates of prodrugs were estimated by
measuring the amount of MTX released at different time

Binding Triggered Drug Release for Integrin Targeted Delivery
3213
Table II Synthesis of Methotrexate Prodrugs
peptide with MTX was coupled to bound RGD
conformation for the simulation. Energy minimization
was carried on selected atoms with the entire receptor
and RGDs kept fixed. The preferred conformation upon
ligand binding was determined by comparing the
potential energy values in open and folded conformations in
the bound state.
Molecule Sequence
Yield (%)
[M+H]+a
Purity (%)
MTX-RGD
MTX-RWQYV-DP-GKFTVQ-
RGD
47
31
782.6
96
93
2273.3
D Asp; E Glu; F Phe; G Gly; K Lys; D P d-Pro; Q Gln; R Arg; T Thr; V Val;
W Trp; Y Ty r
a MALDI-TOF molecular ion peak
RESULTS AND DISCUSSION
Synthesis of Prodrugs
cells with altered level of α subunit also exhibited similar
altered β expression. The 22C4 antibody, specific to
uncomplexed β chain failed to bind to any of the cells
when α subunit was knocked down. This suggested that β
subunit cannot be expressed on the membrane alone
without its complementary α subunit (22). These studies
suggest that by altering one of the subunits of heterodimeric
receptor, it is possible to control the membrane expression
of a specific integrin dimer. Similarly, in our case, increase
in β subunit expression is expected to translate into the
overexpression of the whole integrin. While siRNA could
almost completely knock down this subunit expression, the
overexpression of β3 was increased with increasing Adβ3
viral volume to transfect the cells. The results of knockdown
and overexpression of β3 are shown in Fig. 2. The integrin
β3 expression was modulated to create three levels of
integrin αVβ3 expression (knockdown, wild-type, and over-
expressed) for testing the responses of RGD-specific binding
on the amount of expressed integrin αVβ3. As shown in
Fig. 3, at any given expression level, it is evident that
significantly higher (t-test P<0.05) numbers of cells express-
ing αVβ3 bound to RGD-containing peptides, such as
fibrinogen, MTX-RGD, and MTX-hairpin-RGD com-
pared to a non-specific substrate, BSA. Within each
substrate group (coating), the number of bound cells
decreased significantly (ANOVA P<0.05) with decreased
expression of αVβ3. Within each level of expression, there
was no binding difference between prodrugs. Apart from
RGD, other portions and higher order of conformations of
fibrinogen are attributed to higher cell binding compared
to RGD prodrugs. The specificity of RGD binding
dependence was demonstrated by preincubating wild-type
HUVEC with RGD peptide prior to binding to prodrugs.
The addition of amino acid residues to the MTX-α-
carboxylate has been shown to generate prodrugs that
can be easily activated by selective enzymatic hydrolysis
(4). Many synthetic schemes have been reported to
selectively obtain MTX-α-amino acid derivatives in high
yield (17, 18). Using the method reported by Coughlin et
al. (19), α-amino acid-conjugated prodrugs were synthe-
sized (Fig. 1). The MTX was synthesized in situ by
conjugating Glu and DNPE. The Fmoc-based peptide
synthesis method involved synthesis from C to N terminus.
Accordingly, the synthesis was started with coupling α-
COOH of Asp of the RGD onto the Rink amide resin. In
every step of coupling, the free alpha-COOH was linked
to deprotected NH2 of the previously coupled amino acid.
All other reactive groups, such as γ-COOH in the case of
Glu, do not interfere in the synthesis since they are
protected orthogonally. Once the coupling of RGD and
12 amino acids of the hairpin peptide completed, Glu was
added where only the α-COOH is reacted with NH2 of
Arg of the hairpin peptide. Thus, when the pteroic acid
portion was reacted with NH2 of Glu, it formed MTX-α-
hairpin-RGD. The MTX-RGD, without bearing a hair-
pin structure, was used as control for MTX-hairpin-RGD
peptide. Characterization data of these synthesized MTX
prodrugs are summarized in Table II.
Specific Binding of RGD to Integrin αVβ3
After screening the expression pattern for integrin αV, β3
and β5 in several cell lines, HUVEC was found to be the
most suitable for binding experiments, as the expression of
integrins is prone to fewer fluctuations in normal cells. Also,
HUVEC cell line has been widely used to test the RGD-
specific recognition and binding (14, 20). Wild-type
HUVEC expresses both αVβ3 and αVβ5. Cheresh et al.
carried out experiments to modify the integrin αVβ3
expression by altering the αV subunit. LM609, an antibody
specific to integrin αVβ3, bound to M21 cells proportional
to the receptor expression level (21). From the study using
AP3 antibody directed to β subunit, it was found that the
Fig. 2 Modulation of integrin β3 in HUVEC. 1, Molecular weight
standards; 2, HUVEC wild; 3, SiRNA treated; 4, 20 μL Adβ3; 5,40 μL
Adβ3; 6, 80 μL Adβ3.

3214
Kotamraj et al.
0.8
the bond between Arg (R) and Glu (E) (23) was used. It is
evident from the structure of prodrugs that both (MTX-
RGD and MTX-hairpin-RGD) of them have MTX (with
a Glu) attached to an Arg moiety and hence are cleavable
by SGPE. The protection of labile linkage between MTX
and β-hairpin was demonstrated by measuring the initial
MTX hydrolysis rates in presence of SGPE. In the
absence of integrin binding (absence of HUVEC cells),
the rate of MTX release was significantly (P<0.05) slower
from MTX-hairpin-RGD (0.7 0.1 ng/hr) than from MTX-
RGD (1.0 0.1 ng/hr) (Fig. 4). The bare hairpin structure
was shown not to self-associate at 1.6 mM concentration (15).
Therefore, self-association can be ruled out as a possible
reason for slower hydrolysis rate in the present study, since
all experiments were conducted at 15 times diluted concen-
trations compared to the concentration-reported result in
association. The prodrug can also protect the labile linkage
from non-specific enzymes usually present in plasma. The
appearance rate of free MTX from different prodrugs in
plasma was used as an indicator for the stability of prodrug
in plasma. The rate of MTX appearance was significantly
(P<0.05 at each time point and for the slope) lower for
MTX-hairpin-RGD than that of MTX-RGD (Fig. 5). The
higher stability or resistance to non-specific hydrolysis of
MTX-hairpin-RGD can be attributed to several reasons.
The masking effect of hairpin conformation plays an
important role in preventing the labile linkage from
endopeptidase hydrolysis. Though it is challenging to avoid
endopeptidase-mediated hydrolysis, literature evidence
showed that creating a steric hindrance to mask the labile
linkage can improve the stability. Rozek and co-workers
designed an anti-microbial cyclic peptide by joining two ends
with a disulfide bond to stabilize lipid-bound structure as
well as improve the stability of peptides in presence of
protease. The stability of the cyclic peptide was improved 4
to 5 times in presence of trypsin. From structural analysis of
cyclic peptide it was revealed that the lysine and tryptophan
HUVEC_Ad
HUVEC_wild
HUVEC_siRNA
HUVEC_wild_PI
3
0.6
0.4
0.2
0.0
FG
MR
MDR
BSA
Coat
Fig. 3 Cell adhesion assay with HUVEC. Y-axis represents the
absorbance of crystal violet proportional to the number of cells bound.
The bars represent means (n=6), and error bars represent standard
deviation. Ab = Absorbance; HUVEC_Adβ3 = HUVEC transfected with
Adβ3 virus; HUVEC_siRNA = HUVEC treated with siRNA; HUVEC_-
wild = HUVEC wild type; HUVEC_wild_PI = HUVEC wild type
preincubated with RGD containing peptides.
The cell binding to prodrug-coated surface abolished com-
pletely for pre-incubated cells. Therefore, it can be concluded
that the prodrugs can specifically recognize and bind to αVβ3-
expressing cells in an RGD-dependent manner.
The prodrug was designed to exist in blood circulation
with labile linkage between MTX and β-hairpin (Bond
between MTX’s Glu and hairpin’s Arg) protected by steric
hindrance, and the labile linkage will be exposed after
conformation change upon binding to integrin αVβ3. These
two conformations of prodrug were studied experimentally
and supported by the molecular modeling results.
1.4
The β-Hairpin-Based Steric Hindrance to Labile Link
No cells
With Cells
#
#
1.2
1.0
0.8
0.6
* #
The MTX-α amino acid prodrugs reported in literature
utilized CPA as the specific enzyme for activation, since
it can cleave the terminal amino acid with a free α-
carboxylic acid group (COOH) (4). Prodrugs of the
current study differ from MTX- α amino acid prodrugs
in two ways and hence cannot use CPA enzyme to activate
the prodrug. First, MTX is attached to a β-hairpin-RGD
peptide consisting of 15 amino acids instead of a single
amino acid. Second, the bond between MTX and Arg of
hairpin peptide does not have a free α-carboxylic group.
Therefore, Streptomyces griseus-derived glutamic acid-specif-
ic endopeptidase (SGPE) capable of preferentially cleaving
*
MTX-RGD MTX-hairpin-RGD
Prodrug
Fig. 4 SGPE mediated specific hydrolysis of prodrugs. The MTX (in ng)
initial release rate was determined and compared in presence and absence
of cells. Statistics: *: t-test P<0.05; #: ANOVA, No significant difference.

Binding Triggered Drug Release for Integrin Targeted Delivery
3215
10
binding-induced conformational change. The increase in
enzymatic hydrolysis and MTX release in such scenario
was demonstrated by incubation of MTX-hairpin-RGD
peptide and MTX-RGD with SGPE in presence of αVβ3-
expressing HUVEC as shown in Fig. 4. Initial MTX release
rates suggested that the hairpin peptide did not pose any
steric hindrance when bound to αVβ3, and the release rate
(1.0 0.1 ng/hr) was not significantly different from the
MTX-RGD (1.1 0.1 ng/hr) that does not exhibit any
hairpin-based steric hindrance (Fig. 4). Compared to the
MTX release rate from MTX-hairpin-RGD in the absence
of cells (0.7 0.1 ng/hr), there was 40% increase in the
release rate in presence of cells. This result is comparable to
previously reported model system (28). These results
support exposure of labile link due to unfolding upon
binding of RGD to integrin αVβ3. Previously, it has been
shown that equilibrium is established between the hairpin
(folded) and straight chain (unfolded) conformations of
hairpin structure peptide (15). In presence of αVβ3 integrin,
straight chain conformation preferentially binds with
integrin αVβ3, which triggers the equilibrium shift towards
the unfolded species leading to more folded forms to
unfold. A mechanism for inducing drug release from
MTX-hairpin-RGD via this route is proposed in Fig. 6
(Mechanism A). It is also possible that binding induces
unfolding of MTX-hairpin-RGD to expose the labile
linkage and resulted in enzymatic hydrolysis; therefore, an
alternative mechanism was also proposed (Fig. 6, Mecha-
nism B). Binding of folded form brings the RGD end of
MTX-hairpin-RGD very close to the receptor, which may
result in an unfavorable conformation that leads to
unfolding of the hairpin. It may also be possible that these
two mechanisms operate simultaneously. The preferred
mechanism is not clear, and further studies will be needed
to understand the exact mechanism. Nevertheless, the
experiment results support the design concept of binding-
triggered drug release for integrin-targeted delivery.
Since the drug release occurs upon binding to integrin
αVβ3-overexpressing cells, drug will be released in the
vicinity of the target cells. MTX is a folate mimic and is
known to be taken up by cells via active (folate receptor)
and passive transport mechanisms. Preferential binding of
MTX prodrugs followed by a specific enzyme mediate
hydrolysis results in accumulation of MTX in the cells
overexpressing the integrin αVβ3. Apart from achieving
targeted delivery of anti-cancer drug, due to small size,
hairpin peptide is unlikely to elicit immunological reactions.
Furthermore, unlike in ADEPT (antibody directed enzyme
prodrug therapy), the current strategy does not utilize any
antibody or anti-antibodies (clearing agents) to either target
the enzyme to tumor or clear the residual enzyme in the
blood. This may lead to significant reduction of immuno-
logical complications associated with ADEPT.
MTX-RGD
8
MTX-hairpin-RGD
6
4
2
0
0
2
4
6
8
10
Time (h)
Fig. 5 MTX release in plasma.
sidechains are compactly packed and might contribute to the
higher protease resistance (24). A simple way to avoid
hydrolysis by exopeptidases such as carboxypeptidases and
aminopeptidases is to block the C and N termini. Making the
C-terminus an amide is one of such methods to block the
C-terminus (25). After MTX-hairpin-RGD was cleaved
from Rink amide resin, MTX-hairpin-RGD was left with
an amide at its C-terminus. On the N-terminus, Glu and
pteroic acid were attached to form MTX. These structures
in MTX-hairpin-RGD contribute to the exopeptidase
hydrolysis masking. Furthermore, the introduction of an
un-natural amino acid isomer, D-Pro, in the sequence was
also playing a role in stabilizing the prodrug. It has been
shown that substitution of L-amino acids with their D-isomers
improved the plasma stability of Cetrorelix, a decapeptide
(25). Radiolabelled compounds conjugated to RGD peptides
were shown to accumulate near the tumor within 2 h of IV
administration (12, 26, 27). Hence, it is desirable for RGD-
conjugated prodrugs to be stable for at least 2 h so that they
can reach the target tumor. Both MTX-hairpin-RGD and
MTX-RGD retained at least 80% prodrug form in plasma
for 2 h; however, MTX-hairpin-RGD had significantly
higher stability (Fig. 5). This suggested that the introduction
of hairpin peptide-based stearic hindrance in the prodrug
was beneficial in improving the plasma stability Non-specific
plasma enzyme hydrolysis of unfolded (straight chain)
conformer that exists in equilibrium with hairpin conformer
when prodrug is not bound to cells may cause the release of
MTX from MTX-hairpinm-RGD. Compared to similar
targeting strategies, this approach can curb the non-specific
hydrolysis to a greater extent, leading to reduced availability
of drug non-specifically.
Binding-Induced Conformation Change
In addition to imparting masking capability (stability in
plasma) to the prodrug, the hairpin structure was also
introduced with an intent to expose the labile link (bond
between MTX’s Glu and hairpin’s Arg) by an integrin

3216
Kotamraj et al.
Fig. 6 Proposed mechanism of
binding induced unfolding and
exposure of labile link. (a) Equi-
librium shift towards the unfolded
species is triggered by preferential
binding of unfolded structure. (b)
Hairpin unfolds due to high free
energy of bound hairpin confor-
mation. RGD = Arg-Gly-Asp;
SGPE
SGPE
Cell
αVβ3
a
Binding-induced equilibrium shift
SGPE = Streptomyces griseus-
derived Glu specific endopeptidase.
SGPE
SGPE
αVβ3
αVβ3
Binding-induced conformation change
b
RGD
Drug
Molecular Modeling Studies
core formation comprising side chains away from the
external aqueous environment. The NOEs observed in
previous proton NMR studies of the core peptide in
aqueous environment support the distance restraints between
these amino acids (15). In aqueous environment, the energy
of MTX-hairpin-RGD in hairpin conformation was
31 kcal/mol less than its straight chain counterpart
(Table III and Fig. 7). Due to the strong beta turn
propensity of DPro-Gly sequence, the straight chain
conformer also showed a U-shaped structure. However,
larger distance between two strands, absence of both H-
bonds and alternatively arranged 10 and 14 member rings
suggest that it is not a β-hairpin conformation. Therefore,
this conformation was referred to as “straight chain”
conformer in the subsequent paragraphs. In contrast, the
hairpin conformer (Fig. 7b) showed H-bonds forming
typical 10-and 14-member rings. The predicted lower
energy of hairpin structure in aqueous environment com-
pared to its straight chain conformation suggested that this
peptide preferentially adopts a hairpin structure in aqueous
environment.
The molecular modeling was used as secondary supporting
evidence to show the propensity of hairpin formation and
the binding-induced unfolding of MTX-hairpin-RGD. The
12-amino acid peptide was built using Arg-Trp-Gln-Tyr-
Val-DPro-Gly-Lys-Phe-Thr-Val-Gln (referred as “peptide”
from now on) sequence using MOE to analyze the energetic
state for preferentially forming a hairpin conformation in
aqueous medium after conjugating MTX and RGD. A 6Å
distance restraint was applied between the alpha C atoms of
Trp-Val and Tyr-Phe to bring the two strands sufficiently
close to form a hairpin conformation. The energy was
minimized using MOE after placing the molecule in water
using “WATER SOAK” function without distance
restraints to verify its propensity to remain in a hairpin
conformation. Previous studies suggested that the hairpin
conformation is facilitated by hydrophobic side chain
interactions and hydrogen bonding between the strands
(15). These studies suggested that the entropic penalty of
more compact structure is compensated by hydrophobic
Table III Potential Energy Values
of Different Conformers
Molecule
Energy (kcal/mol)a
MTX-hairpin-RGD straight chain conformer in water
MTX-hairpin-RGD hairpin conformer in water
−4606
−4637
20872
5×106
21104
Integrin αVβ3 bound RGD (from Protein data bank file, IL5G)
Integrin αVβ3 bound MTX-hairpin-RGD
MTX Methotrexate; RGD Arg-
Gly-Asp
a Local minimum
Integrin αVβ3 bound MTX-straight chain-RGD

Binding Triggered Drug Release for Integrin Targeted Delivery
3217
determined by MOE without any further modification and
was used as reference for the minimum possible energy.
The MTX-peptide was coupled to the bound N terminus of
RGD either in its hairpin or straight chain conformation.
The energy of these two structures was minimized while
Fig. 7 Minimum energy conformations of MTX-peptide-RGD in aqueous
environment. (a) No restraints were applied for distances before energy
minimization, and it represents straight chain conformer (non-hairpin
structure). (b) Restraints were applied for distances between two strands
to bring them close, and energy was minimized without any restrains. This
represents anti-parallel beta hairpin conformation (Note: Rings formed by
H-bonds are characteristic for an anti-parallel beta hairpin.)
Data for integrin αVβ3 receptor crystal structure bound
with cyclic RGD are available from protein databank.
Cyclic RGD chemical structure was modified to obtain
RGD by deleting the linking molecule between R and D
amino acids without disturbing the conformation. This
resulted in RGD tripeptide in its bound conformation to
αVβ3 receptor. The α-COOH of Asp is not essential for
integrin binding. In cyclic RGD the alpha COOH of Asp is
involved in cyclization and thus not free. Additionally,
guanidine group of Arg and β-COOH of Asp, essential for
prominent interaction, are intact in RGD. Therefore,
amide modification may not adversely affect the binding
specificity of RGD ligand, and it can be safely assumed that
both cyclic RGD and RGD-NH2 would bind to integrin
receptor in a similar fashion though with different affinities.
The free energy of RGD bound to αVβ3 receptor was
Fig. 8 Molecular modeling of binding induced unfolding. Top: Integrin
αVβ3 bound RGD; Middle: Integrin αVβ3 bound hairpin conformer
(folded); Bottom: Integrin αVβ3 bound straight chain conformer.

3218
Kotamraj et al.
specific activation by L-49-sFv-[beta]-Lactamase fusion protein.
Bioorg Med Chem Lett. 2003;13:539–42.
keeping the RGD and αVβ3 receptor fixed. Keeping the
RGD and αVβ3 receptor unchanged (fixed) allowed the
distal end (MTX end) of the peptide to adopt minimum
energy conformation in bound state. The straight chain
conformation could re-arrange itself and reach an energy
value close to the reference, whereas hairpin structure
coupled to bound RGD had higher energy compared to
straight chain conformer after minimization. Probably,
hairpin conformation could not rearrange itself to straight
chain conformation, as it was unable to overcome the energy
barrier of its local minimum under the MOE experimental
conditions (Table III, Fig. 8). In reality, the same energy
barrier may prevent it from assuming hairpin conformation
in bound state. These results suggest that the hairpin peptide
prefers to be in straight chain conformation (unfolded) when
it is bound to integrin αVβ3 receptor, since staying close to
the receptor in its hairpin form was not energetically
favorable. Therefore, hairpin structure adopts straight chain
conformation, which leads to disruption of hairpin confor-
mation. The molecular modeling results support the exper-
imental observations and are consistent with design concept
to create a binding-triggered targeted delivery of anticancer
agent to integrin αVβ3 overexpressed cells.
In summary, methotrexate prodrugs conjugated to
bare RGD or RGD with a hairpin peptide were
synthesized, and these prodrugs recognize and bind to
integrin αVβ3-expressing cells. The MTX-hairpin-RGD
prodrug can resist non-specific enzymatic hydrolysis in the
plasma as well as specific hydrolysis mediated by SGPE
when compared with MTX-RGD prodrug. Enzymatic
hydrolysis studies and molecular modeling supported that
the MTX-hairpin-RGD prodrug prefers to assume a
straight chain conformation when bound to integrin,
leading to the SGPE hydrolysis-mediated drug release.
These results suggested that the design of an integrin
binding-triggered conformation change is feasible and
may pave the way to a new group of stimuli-sensitive
drug-release-based carrier systems.
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ACKNOWLEDGMENTS & DISCLOSURES
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The authors would like to thank Dr. William Chan and
Dr. Shiladitya Bhattacharya for their contributions and
supports for the development of AdEasy viral vector.
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