Lipopeptide with a RGDK tetrapeptide sequence can selectively target genes to proangiogenic α5β1 integrin receptor and mouse tumor vasculature

Article abstract of DOI:10.1021/jm800915y

Integrins, the major class of αβ heterodimeric transmembrane glycoprotein receptors, play crucial roles in mediating tumor angiogenesis. Genetic ablation experiments combined with use of antibodies/peptide ligands for blocking either α₅ or β

Full text of DOI:10.1021/jm800915y

7298
J. Med. Chem. 2008, 51, 7298–7302
Brief Articles
Lipopeptide with a RGDK Tetrapeptide Sequence Can Selectively Target Genes to
Proangiogenic r5ꢀ1 Integrin Receptor and Mouse Tumor Vasculature
,‡
Dipankar Pramanik,†,‡ Bharat K. Majeti,†, Goutam Mondal,‡ Priya P. Karmali,#,‡ Ramakrishna Sistla,§ Obula G. Ramprasad,|
Gunda Srinivas,| Gopal Pande,| and Arabinda Chaudhuri*,‡
DiVision of Lipid Science and Technology, Pharmacology DiVision, Indian Institute of Chemical Technology, Hyderabad 500607, India,
Centre for Cellular and Molecular Biology, Hyderabad 500607, India
ReceiVed July 23, 2008
Integrins, the major class of Rꢀ heterodimeric transmembrane glycoprotein receptors, play crucial roles in
mediating tumor angiogenesis. Genetic ablation experiments combined with use of antibodies/peptide ligands
for blocking either R5 or ꢀ1 integrins have convincingly demonstrated R5ꢀ1 integrin to be unquestionably
proangiogenic among the 24 known integrin receptors. Herein, we report on a novel RGDK-lipopeptide 1
that targets selectively R5ꢀ1 integrin and is capable of targeting genes to mouse tumor vasculatures.
Scheme 1. Structure of the R5ꢀ1 integrin receptor and tumor
vasculature targeting RGDK-lipopeptide 1
Introduction
Angiogenesis, the sprouting of new blood vessels from pre-
existing vessels, is a remarkable feature of tumor growth.1
Prevention of new blood vessel formation around tumor tissues
is a promising antiangiogenic therapeutic approach to combat
cancers. In such antiangiogenic cancer therapy, selective target-
ing of anticancer drugs/genes to tumor vasculatures is ac-
complished by exploiting unique molecular markers overex-
pressed in the tumor endothelial cells. Integrins, the major class
of Rꢀ heterodimeric transmembrane glycoprotein receptors,
belong to one such unique class of molecular markers. Among
the total 24 integrins, Rvꢀ3, Rvꢀ5, and R5ꢀ1 integrins have
received extensive exploitations as antiangiogenic drug targets.
Integrins mediate cellular adhesion to ECM or to adjacent cells
and can regulate cell survival and proliferation by modulating
intracellular signaling processes.2-6 Because many integrins and
their specific extracellular matrix ligands play crucial roles in
mediating tumor angiogenesis, they are gaining increasing
importance as drug targets in antiangiogenic cancer therapy.7
Brooks et al. and others reported that the monoclonal antibody
(LM609) as well as the various RGD-tripeptide based low-
molecular-weight reagents are recognized by the integrins Rvꢀ3
and Rvꢀ5 and these reagents are capable of blocking tumor and
retinal angiogenesis in response to growth factors.8-10 However,
the findings in a series of gene ablation experiments involving
the use of Rv or ꢀ3 and/or ꢀ5 deficient mice have seriously
questioned the proangiogenic nature of these two integrins. Mice
lacking Rv or ꢀ3 and/or ꢀ5 deficient genes have been shown to
be viable and fertile.11,12 Perhaps the proangiogenic nature of
the Rvꢀ3 and Rvꢀ5 integrins has been most seriously challenged
by the findings that mice lacking Rv integrins exhibit extensive
tumor growth and angiogenesis.13 Importantly, no such dis-
crepancy between the genetic results and those obtained using
antibodies or low-molecular weight reagents exist for the integrin
R5ꢀ1 and its ligands. Use of antibodies/peptide ligands for
blocking either R5 or ꢀ1 integrins inhibited angiogenesis.14
Genetic ablation of either R5 or ꢀ1 integrins in mice led to
embryonic lethality with major vascular defects.15,16 Thus, gene
ablation experiments combined with the use of antibodies/
peptide ligands have convincingly demonstrated R5ꢀ1 integrin
as an unquestionably proangiogenic integrin receptor. Ever since
these reports on the distinct proangiogenic roles of the R5ꢀ1
integrins were disclosed, efforts toward developing efficient
R5ꢀ1 integrin receptor ligands and antagonists targeting systems
are gaining increasing importance. Reports on the developments
of efficient and selective nonpeptidic ligands and antagonists
of R5ꢀ1 integrin receptor have been published.17-20 Zimmer-
mann et al. reported their in vitro studies on the development
of two cyclic penta- and hexapeptide ligands containing RGD
sequence and a ꢀ-amino acid with considerable affinity for R5ꢀ1
integrin receptor.21 Herein, we show that lipopeptide containing
a lysine functionality immediately after the RGD sequence
(RGDK-lipopeptide 1, Scheme 1) in its polar headgroup region
can selectively target genes to R5ꢀ1 integrin receptors. When
the lysine residue of the RGDK tetrapeptide sequence was
replaced with a leucine residue, the resulting RGDL-lipopeptide
3 did not show any R5ꢀ1 integrin receptors selective transfection
* To whom correspondence should be addressed. Phone: 91 40 27193201.
Fax: 91 40 27193370. E-mail: arabinda@iict.res.in.
†
Equally contributing authors.
Division of Lipid Science and Technology, Indian Institute of Chemical
‡
Technology.
§
Pharmacology Division, Indian Institute of Chemical Technology.
Centre for Cellular and Molecular Biology.
|
Present address: Laboratory of David A. Cheresh, Department of
Pathology, University of California, San Diego, School of Medicine &
Moores UCSD Cancer Center, 3855 Health Sciences Drive 0803, La Jolla,
CA 92093-0803.
#
Present address: Laboratory of Erkki Ruoslahti, Cancer Research Center,
The Burnham Institute, La Jolla, CA 92037.
10.1021/jm800915y CCC: $40.75
2008 American Chemical Society
Published on Web 10/29/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7299
Figure 1. RGDK-lipopeptide 1 targets genes selectively to R5ꢀ1 integrin receptors. (A) Evaluation of the R5ꢀ1, Rvꢀ3, and Rvꢀ5 integrins in
A549 cells by FACS. In each of the three experiments, about one million cells were fixed and labeled with monoclonal antibodies against R5ꢀ1,
Rvꢀ3, and Rvꢀ5 integrins and then examined by flow cytometry. The blue shaded areas on the left side in each FACS profile represents the signal
of the control isotype antibody, and the traces on the right represent the signal with anti-R5ꢀ1 (green), antiRvꢀ3 (red), and antiRvꢀ5 (indigo)
integrin antibodies. (B) Relative efficacies of the RGDK-lipopeptide 1 and the control RGEK-lipopeptide 2 in transfecting A549 cells (*P <
0.0005). (C) Gene transfer efficiencies of the RGDK-lipopeptide 1 in A549 cells (measured using the lipofection method described in ref 32) in the
presence of monoclonal antibodies against R5ꢀ1, Rvꢀ3, and Rvꢀ5 integrins (*P < 0.005 when compared to the control transfection experiments
in absence of any added antibody).
properties. This demonstrates that the lysine functionality in
RGDK-lipopeptide 1 plays a crucial role behind its R5ꢀ1
integrin receptor specificity. Immunohistochemical findings
demonstrate that the RGDK-lipopeptide 1 is capable of deliver-
ing genes to the mouse tumor vasculatures under systemic
settings. Importantly, remarkable inhibition of tumor growth was
observed when the electrostatic complex of the RGDK-
lipopeptide 1 and the anticancer p53 gene was intravenously
administered in C57BL/6 mice bearing an aggressive B16F1
tumor.
confirmed by reversed phase analytical HPLC (Figures S1-S3,
Supporting Information). Details of syntheses, purifications, 1H
NMR, and high-resolution mass spectral characterizations for
the RGDK-lipopeptide 1 and the control lipopeptides, namely
RGEK-lipopeptide 2 and RGDL-lipeptide 3, are provided in
the Supporting Information.
To begin with, we tested the efficiencies of the RGDK-
lipopeptide 1 in delivering genes into A549 cells (human lung
carcinoma cells) following previously described liposomal gene
delivery protocol.32 Briefly, electrostatic complex of a plasmid
DNA (pCMV-SPORT-ꢀ-Gal reporter gene encoding the enzyme
ꢀ-galactosidase) and the cationic liposomes of the lipopeptides
(containing 2:1 mole ratio of RGDK-lipopeptide 1 and Chola)
were incubated with the cells. After 48 h (which ensured
transgene expression in the transfected cells), cells were lysed
and the activities of the expressed ꢀ-galactosidase were assayed
by adding a spectrophotometric substrate of ꢀ-galactosidase
(ortho-nitrophenyl-ꢀ-galactoside). Fluorescence-assisted cell
sorting (FACS) analysis revealed the presence of all the three
Rvꢀ3, Rvꢀ5, and R5ꢀ1 integrin receptors expressed on its
surface (Figure 1A). As depicted in Figure 1B, the RGDK-
lipopeptide 1 was found to be efficient in transfecting A549
cells. RGD and not RGE is the ligand for integrin receptors.33
With a view to confirm the involvement of integrin receptor in
the cellular uptake of RGDK-lipopeptide 1:p-CMV-SPORT-ꢀ-
Gal gene complex, we synthesized the control RGEK-lipopep-
tide 2 and evaluated its gene transfer property in A549 cells.
The gene transfer efficacy of the control RGEK-lipopeptide 2
was found to be remarkably compromised compared to that of
Results and Discussion
The amino acid sequence arginine-glycine-aspartic acid
(RGD) is the most evolutionary conserved feature of many
natural integrin-binding extracellular matrix proteins, and in-
tegrin receptors potentiate cellular internalization processes for
many viruses.22-24 On the basis of this rationale, efficient
integrin receptor targeting systems consisting of RGD-modified
liposomes,25-27 cRGD-functionalized polymeric micelles,28 and
lipid based nanoparticles containing a covalently grafted integrin
ligands29,30 have been reported. However, most of these prior
reported lipopeptide systems containing the RGD sequence are
Rvꢀ3 integrin receptor specific and none of these RGD-
lipopeptide systems are capable of selectively targeting the
unquestionably proangiogenic R5ꢀ1 integrin receptors. Herein,
we demonstrate for the first time that a cationic lipopeptide
containing a RGDK tetrapeptide sequence in its polar headgroup
region can selectively target genes to the proangiogenic R5ꢀ1
integrin receptors. The RGDK-lipopeptide 1 was synthesized
by covalently grafting the RGD-tripeptide sequence to the lysine
residue of a previously described monolysinylated cationic
amphiphile.31 The purities of all the final lipopeptides were
a Abbreviations: Chol: cholesterol; DCM: dichloromethane; DMEM:
Dulbecco’s modified Eagles medium; FBS: fetal bovine serum; ONPG:
o-nitrophenyl-ꢀ-D-galactopyranoside; PBS: phosphate buffered saline.

7300 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Brief Articles
Figure 2. Enhanced transfection efficiencies of the RGDK-lipopeptide 1 in L27 cells with overexpressed R5ꢀ1 integrins. (A) FACS profiles of the
R5ꢀ1 integrins in S180 and L27 (S180 cells transformed with human R5 cDNA) cells. (B) Enhanced gene transfer efficiencies of the RGDK-
lipopeptide 1 in L27 cells compared to that in S180 cells (*P < 0.001). (C) Gene transfer efficiencies of the RGDK-lipopeptide 1 in L27 cells
pretreated with monoclonal antibodies against R5ꢀ1, Rvꢀ3, and Rvꢀ5 integrins (*P < 0.001 compared to transfection efficacies in untreated cells).
the RGDK-lipopeptide 1 (Figure 1B). Such significantly reduced
gene delivery efficiency of the control RGEK-lipopeptide 2
(compared to that of RGDK-lipopeptide 1) supports the notion
that the cellular uptake of the RGDK-lipopeptide 1:DNA
complexes is mediated via integrin receptors
Next, with a view to gaining insights into whether the RGDK-
lipopeptide 1 is capable of delivering genes into cells via any
specific integrin receptor among the Rvꢀ3, Rvꢀ5, and R5ꢀ1
integrin receptors, we measured the gene transfer efficiencies
of the RGDK-lipopeptide 1 in A549 cells preincubated with
the monoclonal antibodies against these three integrins. The
transfection efficiencies of the RGDK-lipopeptide 1 were found
to be minimally affected when A549 cells were preincubated
with monoclonal antibodies against Rvꢀ3 and Rvꢀ5 integrins
(Figure 1C). Thus, the cellular uptake of the lipopeptide:DNA
complexes is unlikely to be mediated via either a Rvꢀ3 or a
Rvꢀ5 integrin receptor. Contrastingly, the RGDK-lipopeptide
1 was found to be significantly less efficient in delivering genes
(by ∼60%) when A549 cells were preincubated with the
monoclonal anti-R5ꢀ1 integrin antibodies (Figure 1C). Thus,
the findings summarized in Figure 1C provided convincing
evidence for the R5ꢀ1 integrin receptor specific transfection
properties of the RGDK-lipopeptide 1. The R5ꢀ1 integrin
selectivity of the RGDK-lipopeptide 1 was further confirmed
by conducting the transfection experiments in S180 (murine
sarcoma cells) and L27 cells (S180 cells stably transfected with
human R5).34 That the degree of R5ꢀ1 integrin receptors
expression in L27 cell surface is significantly higher than that
in S180 cells was confirmed by the findings in the conventional
Figure 3. Tumor growth inhibition and tumor vasculature targeting
properties of the RGDK-lipopeptide 1. (A) Six-eight week old female
FACS analysis (Figure 2A). Consistent with the R5ꢀ1 integrin
receptor selectivity of the RGDK-lipopeptide 1 and the higher
amount of R5ꢀ1 integrin receptor expression in L27 cell surface
(compared to that in S180 cells), the RGDK-lipopeptide 1 was
found to be significantly more efficacious in transfecting L27
cells than in transfecting S180 cells (Figure 2B). Importantly,
the transfection efficiencies of the RGDK-lipopeptide 1 were
significantly reduced when L27 cells were preincubated with
monoclonal anti-R5ꢀ1 integrin antibodies and not with mono-
clonal antibodies raised against the integrins Rvꢀ3 and Rvꢀ5
(Figure 2C).
C57BL/6 mice (each weighing 20-22 g) with aggressive B16F1 tumors
(produced by subcutaneous injections of 1 × 105 B16F1 cells in 100
µL of Hank’s buffer salt solution (HBSS) into the left flanks on day 0)
were randomly sorted into four groups and each group (n ) 5) was
administered intravenously with: RGDK-lipopeptide 1:p53 lipoplex in
5% aqueous glucose (filled diamonds), RGEK-lipopeptide 2:p53
lipoplex in 5% aqueous glucose (filled squares), liposomes of RGDK-
lipopeptide 1 alone (filled triangles) and 5% aqueous glucose alone
(filled circles) on days 5, 7, 9, 11, and 14. Tumor volumes (V ) 1/2 ·ab2,
where a ) maximum length of the tumor and b ) minimum length of
the tumor measured perpendicular to each other) were measured with
a slide calipers for up to 22 days. Results represent the means ( SD
(n ) 5, *P < 0.005 compared to RGEK/p53). (B) Representative
samples of B16F1 tumors excised on day 22. (I) Tumor treated with
vehicle only. (II) Tumors treated with RGDK-liposomes only. (III)
Tumors treated with RGEK-liposomes/p53 complexes. (IV) Tumors
treated with RGDK-liposomes/p53 complexes.
We envisaged that the lysine group might be playing an
important role behind the above-mentioned R5ꢀ1 integrin
receptor specific gene delivery efficiencies of the RGDK-
lipopeptide 1. To address this issue, we synthesized a second

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7301
Figure 4. (A,C). Immunohistochemical staining of tumor endothelial cells. On day 22 after tumor inoculation, mice were intravenously injected
with the complex of RGDK-lipopeptide 1 and R5-GFP plasmid DNA. After 24 h, tumors were excised, sectioned, and the representative tumor
sections were immunostained for tumor endothelial cells with anti-vWF antibodies. (B,D) RGDK-lipopeptide 1 targets genes to tumor neovasculature.
The stained tumor slides shown in parts (A) and (C) when observed in the same positions under fluorescent microscope (at 400 magnification)
using a green filter revealed GFP expression in the tumor vasculatures. Bar ) 1 µm.
control RGDL-lipopeptide 3 following the same peptide cou-
pling strategy as adopted for synthesis of the RGDK-lipopeptide
1 in which the lysine residue was substituted with leucine.
Unlike in the case of the RGDK-lipopeptide 1, the transfection
efficiencies of the control RGDL-lipopeptide 3 in A549 cells
remained essentially unaffected when the cells were preincu-
bated with monoclonal antibodies against all the three integrins
namely, R5ꢀ1, Rvꢀ3, and Rvꢀ5 integrins (Figure S4, Supporting
Information). Clearly, the control RGDL-lipopeptide 3 does not
show any specificity toward Rvꢀ3, Rvꢀ5, and R5ꢀ1 integrin
receptors. This finding strongly supports the notion that the
lysine (K) residue plays a crucial role in imparting selectively
R5ꢀ1 integrin receptor targeting efficacy to the RGDK-
lipopeptide 1. Future binding studies using purified R5ꢀ1
integrin receptors will provide more quantitative information
on the relative binding affinities of RGDK- lipopeptide 1 and
RGDL-lipopeptide 3 for R5ꢀ1 integrin receptor.
Next, with a view to address the systemic potential of the
R5ꢀ1 integrin receptor specific RGDK-lipopeptide 1, anticancer
p-CMV-p53 gene in complexation with RGDK-lipopeptide 1
was intravenously administered in C57BL/6 mice bearing the
aggressive B16F1 tumors (the sizes of the RGDK-lipopeptide
1:p53 lipoplexes varied within 300-400 nm as measured by
dynamic laser light scattering instrument, Malvern, UK). As
depicted in Figure 3 (parts A and B), remarkable tumor growth
inhibition was observed. The degree of the tumor growth
inhibition was low when p-CMV-p53 gene was administered
in complexation with the control RGEK-lipopeptide 2. Because
RGD and not RGE is a integrin receptor ligand, this finding is
consistent with the involvement of integrin receptor mediated
cellular uptake of the RGDK-lipopeptide:p53 complex in the
systemic setting too. No significant inhibition of tumor growth
was observed when naked p53 plasmid alone (i.e., not in
complexation with the RGDK-lipopeptide 1) were intravenously
administered (data not shown), presumably due to its suscep-
tibility to serum nucleases. Mice intravenously administered with
vehicle alone (5% aqueous glucose solution) developed large
tumors on day 22 (Figure 3, parts A and B) and were sacrificed
at that point. Importantly, extent of tumor growth inhibition was
much less pronounced for mice intravenously injected with the
control RGDL-lipopeptide 3:p53 complexes (Figure S5, Sup-
porting Information).
lipopeptide 1:p-R5GFP (plasmid DNA encoding green fluores-
cence protein) via tail-vein injection.
Fixed tumor sections upon immunohistochemical staining
with blood vessel marker (anti-vWF antibodies) revealed the
presence of the tumor vasculatures when observed in bright field
(Figure 4A,C) immunohistochemically stained tumor sections
when observed under a fluorescent microscope revealed GFP
expression (green fluorescence) in the same tumor vasculatures
(Figure 4B,D). Thus, the findings summarized in Figure 4 (parts
A-D) convincingly demonstrated the ability of the RGDK-
lipopeptide 1 in delivering genes to tumor vasculatures.
Conclusions
Clinical success of antiangiogenic cancer therapy critically
depends upon selective delivery of cytotoxic genes/drugs to the
endothelial cells of the sprouting new blood vessels around the
tumors. Potential targets include mediators of angiogenesis and
protein molecules overexpressed in the endothelial cells of tumor
neovasculatures. Integrins, the Rꢀ heterodimeric transmembrane
glycoprotein receptors, belong to such a class of molecules that
play crucial roles in mediating tumor angiogenesis. Gene
ablation experiments combined with use of antibodies/peptide
ligands have convincingly demonstrated R5ꢀ1 integrin to be the
most proangiogenic among these three integrin receptors. Herein,
we demonstrate for the first time that lipopeptide containing a
lysine functionality after the RGD sequence in its polar
headgroup region (RGDK-lipopeptide 1) can selectively target
genes to the proangiogenic R5ꢀ1 integrin receptors. When the
lysine residue of the RGDK tetrapeptide sequence was replaced
with a leucine residue, the resulting RGDL-lipopeptide 3 did
not show any R5ꢀ1 integrin receptor selective transfection
properties. This demonstrates that the lysine functionality in
RGDK-lipopeptide 1 plays a crucial role behinds its R5ꢀ1
integrin receptor specificity. Immunohistochemical staining of
mice tumor sections revealed efficacy of RGDK-lipopeptide 1
in delivering genes to tumor vasculatures. Importantly, remark-
able inhibition of tumor growth was observed when the
electrostatic complex of the RGDK-lipopeptide 1 and the
anticancer p53 gene was intravenously administered in C57BL/6
mice bearing aggressive B16F1 tumor. Availability of the
presently described RGDK-lipopeptide 1 is expected to find
future clinical exploitation in antiangiogenic cancer therapy
including noninvasive imaging of tumor vasculatures.
Finally, toward probing whether the presently described
RGDK-lipopeptide 1 is capable of targeting tumor vasculatures
under systemic settings, aggressive B16F1 tumors (murine
melanoma tumor) were produced in 6-8 weeks old female
C57BL/6 mice (each weighing 20-22 g) by subcutaneous
injections of 1 × 105 B16F1 cells in 100 µL of Hank’s buffer
salt solution (HBSS) into their left flanks. When large tumors
were grown (on day 22 after tumor cell inoculation), mice were
administered with the electrostatic complexes of RGDK-
Acknowledgment. This work was supported by the Depart-
ment of Biotechnology, Government of India, New Delhi, and
the Council of Scientific and Industrial Research, Government
of India, New Delhi (NWP 0036). We thank Erkki Ruoslahti
for suggesting to us in vivo experiments on C57BL/6 mice
bearing aggressive B16F1 tumors, Robert. J. Debs and Nalam.
M. Rao for providing the p-CMV-p53 and p-CMV-SPORT-ꢀ-

7302 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Brief Articles
development in mouse embryos lacking fibronectin. DeVelopment
1993119, 1079–1091.
(16) Yang, J. T.; Rayburn, H.; Hynes, R. O. Embryonic mesodermal defects
in alpha 5 integrin-deficient mice. DeVelopment 1993, 119, 1093–
1105.
(17) Heckmann, D.; Meyer, A.; Marinelli, L.; Zahn, G.; Stragies, R.;
Kessler, H. Probing integrin selectivity: rational design of highly active
and selective ligands for the a5ꢀ1 and avꢀ3 intrgrin receptor. Angew.
Chem., Int. Ed. 2007, 46, 3571–3574.
(18) Stragies, R.; Osterkamp, F.; Zischinsky, G.; Vossmeyer, D.; Kalkhof,
H.; Reimer, U.; Zahn, G. Design and synthesis of a new class of
selective integrin alpha5beta1 antagonists. J. Med. Chem. 2007, 50,
3786–3794.
(19) Meyer, A.; Auernheimer, J.; Modlinger, A.; Kessler, H. Targeting RGD
recognizing integrins: drug development, biomaterial research, tumor
imaging and targeting. Curr. Pharm. Des. 2006, 12, 2723–2747.
(20) Smallheer, J. M.; Weigelt, C. A.; Woerner, F. J.; Wells, J. S.; Daneker,
W. F.; Mousa, S. A.; Wexler, R. R.; Jadhav, P. K. Synthesis and
biological evaluation of nonpeptide integrin antagonists containing
spirocyclic scaffolds. Bioorg. Med. Chem. Lett. 2004, 14, 383–387.
(21) Zimmermann, D.; Gutho¨hrlein, E. W.; Malesevic´, M.; Sewald, K.;
Wobbe, L.; Heggemann, C.; Sewald, N. Integrin R5ꢀ1 ligands:
biological evaluation and conformational analysis. ChemBioChem.
2005, 6, 272–276.
(22) Ruoslahti, E. RGD and other recognition sequences for integrins. Annu.
ReV. Cell DeV. Biol. 1996, 12, 697–715.
(23) Triantafilou, K.; Takada, Y.; Triantafilou, M. Mechanisms of integrin-
mediated virus attachment and internalization process. Crit. ReV.
Immunol. 2001, 21, 311–322.
(24) Garrigues, H. J.; Rubinchikova, Y. E.; DiPersio, C. M.; Rose, T. M.
Integrin alphaVbeta3 Binds to the RGD motif of glycoprotein B of
Kaposi’s sarcoma-associated herpesvirus and functions as an RGD-
dependent entry receptor. J. Virol. 2008, 82, 1570–1580.
(25) Xiong, X.; Huang, Y.; Lu, W. L.; Zhang, X.; Zhang, H.; Nagai, T.;
Zhang, Q. Intracellular delivery of doxorubicin with RGD-modified
sterically stabilized liposomes for an improved antitumor efficacy: in
vitro and in vivo. J. Pharm. Sci. 2005, 94, 1782–1793, and the
references 16-20 cited therein.
(26) Koppitz, M.; Huenges, M.; Gratias, R.; Kessler, H. Synthesis of
Unnatural Lipohilic N-(9H-Fluoren-9-ylmethoxy)carbonyl-Substituted
Amino Acids and Their Incorporation into Cyclic RGD-Peptides: A
Structure-Activity Study. HelV. Chim. Acta 1997, 80, 1280–1300.
(27) Hu, B.; Finsinger, D.; Peter, K.; Guttenberg, Z.; Ba¨rmann, M.; Kessler,
H.; Escherich, A.; Moroder, L.; Bo¨hm, J.; Baumeister, W.; Sui, S. F.;
Sackmann, E. Intervesicle cross-linking with integrin alpha IIb beta 3
and cyclic-RGD-lipopeptide. A model of cell-adhesion processes.
Biochemistry 2000, 39, 12284–12294.
(28) Nasongkla, N.; Shuai, X.; Ai, H.; Weinberg, B. D.; Pink, J.; Boothman,
D. A.; Gao, J. cRGD-functionalized polymer micelles for targeted
doxorubicin delivery. Angew. Chem., Int. Ed. 2004, 43, 6323–6327.
(29) Hood, J. D.; Bednarski, M.; Frausto, R.; Guccione, S.; Reisfeld, R. A.;
Xiang, R.; Cheresh, D. A. Tumor regression by targeted gene delivery
to the neovasculature. Science 2002, 296, 2404–2407.
(30) Murphy, E. A.; Majeti, B. K.; Barnes, L. A.; Makale, M.; Weis, S. M.;
Lutu-Fuga, K.; Wrasidlo, W.; Cheresh, D. A. Nanoparticle-mediated
drug delivery to tumor vasculature suppresses metastasis. Proc. Natl.
Acad. Sci. U.S.A. 2008, 105, 9343–9348.
(31) Karmali, P. P.; Valluripalli, V. K.; Chaudhuri, A. Design, syntheses
and in vitro gene delivery efficacies of novel mono-, di-, and
trilysinated cationic lipids: a structure-activity investigation. J. Med.
Chem. 2004, 47, 2123–2132.
(32) Rajesh, M.; Sen, J.; Srujan, M.; Mukherjee, K.; Sreedhar, B.;
Chaudhuri, A. Dramatic influence of the orientation of linker between
hydrophilic and hydrophobic lipid moiety in liposomal gene delivery.
J. Am. Chem. Soc. 2007, 129, 11408–11420.
(33) Cherny, R. C.; Honan, M. A.; Thiagarajan, P. Site-directed mutagenesis
of the arginine-glycine-aspartic acid in vitronectin abolishes cell
adhesion. J. Biol. Chem. 1993, 268, 9725–9729.
(34) Beauvais, A.; Erickson, C. A.; Goins, T.; Craig, S. E.; Humphries,
M. J.; Thiery, J. P.; Dufour, S. Changes in the fibronectin-specific
integrin expression pattern modify the migratory behavior of sarcoma
S180 cells in vitro and in the embryonic environment. J. Cell. Biol.
1995, 128, 699–713.
Gal plasmids, respectively, and Dr. Rajkumar Banerjee for his
assistance in tumor growth inhibition experiments. D.P., B.K.M.,
and G.M. thank the Council of Scientific and Industrial
Research, Government of India, New Delhi, and P.P.K. thanks
the University Grant Commission, Government of India, New
Delhi, for providing doctoral research fellowships.
Supporting Information Available: Details of syntheses,
1
purifications, H NMR, and high-resolution mass spectral charac-
terizations for the RGDK-lipopeptide 1 and the control lipopeptides,
namely RGEK-lipopeptide 2 and RGDL-lipopeptide 3, reverse
phase HPLC chromatograms for RGDK-lipopeptide 1, RGEK-
lipopeptide 2, RGDK-lipopeptide 3 in two mobile phases, and the
1
details of the HPLC conditions; H NMR and mass spectral data
for RGE-lipopeptide 2 and RGDK-lipopeptide 3, methods for
preparation of liposomes, details of transfection and FACS protocol,
General methods and materials, results in the antibody saturation
experiments and in vivo tumor regression experiments with RGDL-
lipopeptide 3, and details for antibody saturation experiments. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Carmeliet, P. Angiogenesis in life, disease and medicine. Nature 2005,
438, 932–936.
(2) Hynes, R. O. Integrins: bidirectional, allosteric signaling machines.
Cell 2002, 110, 673–687.
(3) Giancotti, F. G.; Ruoslahti, E. Integrin Signaling. Science 1999, 285,
1028–1032.
(4) van der, F. A.; Sonnenberg, A. Function and interactions of integrins.
Cell. Tissue Res. 2001, 305, 285–298.
(5) Varner, J. A.; Emerson, D. A.; Juliano, R. L. Integrin alpha 5 beta 1
expression negatively regulates cell growth: reversal by attachment
to fibronectin. Mol. Biol. Cell 1995, 6, 725–740.
(6) Friedlander, M.; Brooks, P. C.; Shaffer, R. W.; Kincaid, C. M.; Varner,
J. A.; Cheresh, D. A. Definition of two angiogenic pathways by distinct
alpha v integrins. Science 1995, 270, 1500–1502.
(7) Stupack, D. J.; Cheresh, D. A. Integrins and angiogenesis. Curr. Top.
DeV. Biol. 2004, 64, 207–238.
(8) Brooks, P. C.; Montgomery, A. M.; Rosenfeld, M.; Reisfeld, R. A.;
Hu, T.; Klier, G.; Cheresh, D. A. Integrin alpha v beta 3 antagonists
promote tumor regression by inducing apoptosis of angiogenic blood
vessels. Cell 1994, 79, 1157–1164.
(9) Brooks, P. C.; Stro¨mblad, S.; Klemke, R.; Visscher, D.; Sarkar, F. H.;
Cheresh, D. A. Antiintegrin alpha v beta 3 blocks human breast cancer
growth and angiogenesis in human skin. J. Clin. InVest. 1995, 96,
1815–1822.
(10) Friedlander, M.; Theesfeld, C. L.; Sugita, M.; Fruttiger, M.; Thomas,
M. A.; Chang, S.; Cheresh, D. A. Involvement of integrins alpha v
beta 3 and alpha v beta 5 in ocular neovascular diseases. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 9764–9769.
(11) Bader, B. L.; Rayburn, H.; Crowley, D.; Hynes, R. O. Extensive
Vasculogenesis, Angiogenesis, and Organogenesis Precede Lethality
in Mice Lacking All Rv Integrins. Cell 1998, 95, 507–519.
(12) Hodivala-Dilke, K. M.; McHugh, K. P.; Tsakiris, D. A.; Rayburn,
H.; Crowley, D.; Ullman-Cullere´, M.; Ross, F. P.; Coller, B. S.;
Teitelbaum, S.; Hynes, R. O. Beta3-integrin-deficient mice are a model
for Glanzmann thrombasthenia showing placental defects and reduced
survival. J. Clin. InVest. 1999, 103, 229–238.
(13) Reynolds, L. E.; Wyder, L.; Lively, J. C.; Taverna, D.; Robinson,
S. D.; Huang, X.; Sheppard, D.; Hynes, R. O.; Hodivala-Dilke, K. M.
Enhanced pathological angiogenesis in mice lacking beta3 integrin or
beta3 and beta5 integrins. Nat. Med. 2002, 8, 27–34.
(14) Kim, S.; Bell, K.; Mousa, S. A.; Varner, J. A. Regulation of
angiogenesis in vivo by ligation of integrin alpha5beta1 with the central
cell-binding domain of fibronectin. Am. J. Pathol. 2000, 156, 1345–
1362.
(15) George, E. L.; Georges-Labouesse, E. N.; Patel-King, R. S.; Rayburn,
H.; Hynes, R. O. Defects in mesoderm, neural tube and vascular
JM800915Y