Preparation and biological evaluation of tumor-specific Ara-C liposomal preparations containing RGDV motif

Article abstract of DOI:10.1002/jps.23326

Arginine-glycine-aspartate (RGD) has been shown to be essential for the recognition of integrins overexpressed in tumor cells, especially during tumor invasion, angiogenesis, and metasis. In this study, a novel tetrapeptide, RGD-valine (RGDV), was designe

Full text of DOI:10.1002/jps.23326

Preparation and Biological Evaluation of Tumor-Specific Ara-C
Liposomal Preparations Containing RGDV Motif
FEI WANG,1 CHUNYING CUI,1 ZHAO REN,1 LILI WANG,2 HU LIU,2 GUOHUI CUI1
1School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing 100069, China
2School of Pharmacy, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador A1B 3V6, Canada
Received 24 May 2012; revised 26 August 2012; accepted 5 September 2012
Published online 28 September 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23326
ABSTRACT: Arginine–glycine–aspartate (RGD) has been shown to be essential for the recog-
nition of integrins overexpressed in tumor cells, especially during tumor invasion, angiogene-
sis, and metasis. In this study, a novel tetrapeptide, RGD–valine (RGDV), was designed and
attached to the N position of 1-$-D-arabinofuranosylcytosine (Ara-C) at the valine end, as
a homing device for the delivery of Ara-C to tumor cells. Furthermore, fatty acids of vari-
ous chain lengths (CnH2n+1COOH, n = 7, 9, 11, 13, and 15) were attached to the arginine
end of RGDV to form a series of CnH2n+1CO–RGDV–Ara-C compounds. The structures of
CnH2n+1CO–RGDV–Ara-C compounds were confirmed using mass spectrometry and nuclear
magnetic resonance. The liposomal preparations of the synthesized CnH2n+1CO–RGDV–Ara-C
compounds were obtained using the film dispersion method in the presence of phospholipids. The
particle size, zeta potential, and dispersity index of the liposomes formed were found to be ap-
proximately 215 nm (diameter), approximately −30 mV, and <0.3, respectively. The antitumor
activity of the liposomal preparations containing the respective CnH2n+1CO–RGDV–Ara-C com-
pounds was evaluated in mice inoculated with sarcoma S180. Liposomal Ara-C preparation, lipo-
somal C11H23CO–V–Ara-C preparation, Ara-C, and C11H23CO–V–Ara-C sodium carboxymethyl
cellulose (CMC-Na) suspensions were used as controls. CnH2n+1CO–RGDV–Ara-C containing li-
posomal preparations were shown with an enhanced antitumor activity, likely because of the tar-
geting effect of RGDV. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
J Pharm Sci 101:4559–4568, 2012
Keywords: drug delivery systems; drug targeting; liposomes; prodrugs; 1-$-D-
arabinofuranosylcytosine (Ara-C); RGD; antitumor
Drugs with a balanced solubility are, therefore, pre-
ferred.
INTRODUCTION
Although drugs may cross cell membranes by vari-
ous mechanisms such as passive diffusion, carrier-
mediated transport, and phagocytosis, most drugs
rely on passive diffusion. Consequently, drug’s solubil-
ity characteristics are essential for drug absorption.1
Drugs of high hydrophilicity are usually associated
with a poor membrane-penetrating ability, whereas
drugs of high lipophilicity generally have a poor affin-
ity for biological fluids resulting in poor distribution.
Cytarabine (1-$-D-Arabinofuranosylcytosine, Ara-
C, Fig. 1), a pyrimidine analogue, is an antimetabolic
antitumor agent. It has been used for the treatment
of leukemia for over 40 years. It is also used for acute
nonlymphocytic leukemia. However, Ara-C suffers
from a low bioavailability due to its poor lipophilic-
ity. Following absorption, Ara-C undergoes deamina-
tion resulting in 1-$-D-arabinofuranosyluracil, which
is inactive. It is also known that solid tumors are not
sensitive to Ara-C.2
Various strategies have been explored to improve
the effectiveness of anticancer agents including Ara-
C. There have been many reports wherein chemical
modifications of Ara-C have been attempted and var-
ious moieties have been attached to the NH2 and/
or 3ꢀ, 5ꢀ-OH groups3–9 among which are Enocitabine
and N-palmitoyl-Ara-C. It has been reported that the
Additional Supporting Information may be found in the online
version of this article. Supporting Information
Fei Wang and Chunying Cui contributed equally to this study.
Correspondence to: Hu Liu (Telephone: +709-777-6382;
Fax: +709-777-7044; E-mail: hliu@mun.ca); Guohui Cui
(Telephone: +86-1083911538; Fax: +86-1083911533; E-mail:
cgh@ccmu.edu.cn)
Journal of Pharmaceutical Sciences, Vol. 101, 4559–4568 (2012)
© 2012 Wiley Periodicals, Inc. and the American Pharmacists Association
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Figure 1. The structure of Cytarabine (1-$-D-
Arabinofuranosylcytosine or Ara-C). Systematic (IU-
PAC) name: {4-amino-1-[(2R,3S,4R,5R)-3,4-dihydroxy-5-
(hydroxymethyl)oxolan-2-yl] pyrimidin-2-one}.
Figure 2. General structure of CnH2n+1CO–RGDV–Ara-C
compounds synthesized.
signed as a tumor-specific homing device and at-
tached to the N4 position of Ara-C via valine, which
was added to the RGD motif. It has been demon-
strated that nonpolar amino acids flanking the RGD
moiety increased the binding affinity of RGD to
integrins27,28 and valine is a hydrophobic amino
acid. In addition, fatty acids of various chain lengths
(CnH2n+1COOH) were attached to the arginine end of
RGDV to form a series of CnH2n+1CO–RGDV–Ara-C
compounds (Fig. 2). As such, the amine group on Ara-
C is protected from deamination, whereas RGDV is
used for tumor recognition. Liposomal systems of an-
ticancer drugs have been marketed and shown with
reduced toxicity.29,30 The fatty acids in the design
were used to improve the overall lipophilicity of the
compounds synthesized so that they can be easily in-
corporated into liposomes.
introduction of fatty acids into Ara-C resulted in gen-
erally improved antitumor activities.10
It is known that the growth of many types of can-
cer is closely related to the integrity of integrins,11,12
a group of transmembrane proteins that mediates the
attachment between a cell and the tissues surround-
ing it including other cells or the extracellular ma-
trix. Integrins are heterodimers containing two dis-
tinct chains named "- and $-subunits,13,14 and play an
important role in tumor invasion and metastasis, a
complex multistage process involving tumor–host in-
teraction, which includes adhesion, angiogenesis, and
proteolysis. Increased expression of integrins facili-
tates the adhesion of tumor cells to the endothelial
linings of blood vessels to colonize host organs.15 It
is known that tripeptide, arginine–glycine–asparate
(Arg–Gly–Asp, RGD), is essential for the recognition
of integrins. Synthetic inhibitors that contain an RGD
motif demonstrated an inhibitory effect on the ad-
hesion and angiogenesis of tumor cells.15–20 RGD,
however, is also known to promote the detachment
of invasive tumor cells from the primary tumor site
leading to the colonization of other organs.21,22 Such
dual and opposite effects on the progression of tu-
mor limit the therapeutic potential of RGD in cancer
treatment. Nevertheless, elevated expression of inte-
grins in many types of tumor is well documented.23,24
RGD’s affinity for integrins has led researchers to
investigate the possibility of using RGD to achieve
site-specific delivery of liposomes by conjugating RGD
to phospholipids and polyethylene glycols in the
liposomes.20,25 However, studies so far are noncon-
clusive.
MATERIALS AND METHODS
Materials
All chemicals were of chemical grade unless other-
wise specified. Ara-C was obtained from Shanghai
Hanhong Chemicals and Technology Company, Ltd.
(Shanghai, China). Caprylic acid [CH3(CH2)6COOH],
decanoic acid [CH3(CH2)8-COOH], lauric acid
[CH3(CH2)10-COOH], myristic acid [CH3(CH2)12-
COOH], and palmitic acid [CH3(CH2)14-COOH] were
purchased from Beijing Chaoyang District Xudong
Chemical Plant (Beijing, China). All amino acids were
of L-configuration and were obtained from Sichuan
Sangao Biochemical Company Ltd. (Chengdu, Sich-
uan, China). Dicyclohexylcarbodiimide (DCC), 1-ethyle-
3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC·HCl), 1-hydroxybenzotriazole (HOBt), N-alpha-
tert-butyloxycarbonyl-N-gamma-nitro-L-arginine [Boc–
Arg(NO2)OH], H–Gly–OH, H–Asp(OBzl)2, and Boc–
Val–OH were from GL Biochem Ltd. (Shanghai,
Studies previously reported by this group revealed
that fatty acid–amino acid–Ara-C compounds were
more effective than Ara-C in solid tumors.26 This
study is a natural progression of the previous work.
A novel tetrapeptide, RGD–valine (RGDV), was de-
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rotection, N-gamma-nitro–L-argininyl–glycyl–benzyl
ester [H–Arg(NO2)–Gly–OBzyl] was obtained.
CnH2n+1COOH and Arg(NO2)–CO–Gly–COOBzl were
condensed in the presence of DCC, HOBt, and N-
methylmorpholine (NMM) to yield CnH2n+1CO–
Arg(NO2)–CO–Gly–COOBzl, which was subsequently
subject to partial deprotection to remove the benzyl
group to obtain CnH2n+1CO–Arg(NO2)–Gly–COOH.
CnH2n+1CO–Arg(NO2)–Gly–COOH was then conden-
sed with H–Asp(COOBzl)–COOBzl in the presence of
DCC, HOBt, and NMM to yield CnH2n+1CO–Arg–
Gly–Asp(COOBzl)–COOBzl. The protective benzyl
ester groups in CnH2n+1CO–Arg–Gly–Asp(COOBzl)–
COOBzl were removed via hydrogenation (Pd/C) to
yield CnH2n+1CO–Arg–Gly–Asp(COOH)–COOH (or
CnH2n+1CORGD).
China). Acetonitrile (chromatography grade) was
from the Fisher Scientific China (Beijing, China).
Water was triple distilled. Sodium chloride solu-
tion for injection and sodium carboxymethyl cel-
lulose (CMC-Da) were from Qidu Pharmaceuticals
Company, Ltd. (Zibo, Shandong, China). Egg lecithin
(molecular weight = 750 Dalton) was purchased from
Acros Organics (Geel, Belgium). Cholesterol was pur-
chased from Beijing Aoboxing Biotech Company Ltd.
(Beijing, China).
Laborota 4003 rotary evaporator was from Hei-
dolph Instruments (Schwabach, Germany). Water cir-
culating vacuum pump SHB-III was from Zhenzhou
Great Wall Scientific Industrial and Trade Com-
pany Ltd. (Henan, China). Transmission electron
microscope (TEM), JEM-1230, was purchased from
JEOL (Tokyo, Japan). Zetasizer Nano ZS-90 with
DTS v.4.00 software was purchased from Malvern
Inc. (Worcestershire, United Kingdom). Waters 2695
HPLC (Milford, Massachusetts) with C18 (4.6 ×
250 mm2, 5 :m; Capcell Pak (Shiseido, Tokyo, Japan)
was used for quantitative analysis and Waters 600
Multisolvent Delivery System with C18 (20 × 250
mm2, 5 :m; Capcell Pak (Shiseido, Tokyo, Japan)
was used for preparation purposes. Magnetic stir-
rer S21-2 was from Shanghai Sile Instrument Com-
pany, Ltd. (Shanghai, China). Nuclear magnetic res-
onance (NMR) was recorded using an Advance II
In a separate experiment, the N-terminus pro-
tected valine (Boc–Val–COOH) was conjugated with
Ara-C in the presence of EDC, HOBt, and NMM in
anhydrous DMF as solvent to yield Boc–Val–CO–Ara-
C).26 The protective Boc group was then removed to
yield H–Val–CO–Ara-C.
Finally, CnH2n+1CO–Arg–Gly–Asp(COOH)–COOH
was conjugated with H–Val–CO–Ara-C to yield
CnH2n+1CO–RGDV–Ara-C. Because there are two
COOH groups on aspartate (D), two series of isomers
for each of CnH2n+1CO–RGDV–Ara-C compounds as
illustrated in Figure 4 were obtained.
The two corresponding isomers for each of the
CnH2n+1CO–RGDV–Ara-C compounds synthesized
¨
500 NMR instrument (Bruker, Fallanden, Switzer-
land) and mass spectrometry (MS) data were ob-
tained using a Waters Micromass Quattro Micro 2000
API LC–MS–MS system (Milford, Massachusetts).
Melting point was determined using a microscopic
melting point apparatus (XT5) from Beijing Keyi
Electro-optic Instrument Plant (Beijing, China). Opti-
cal rotation was measured using a P-1020 Polarimeter
from Jasco (Tokyo, Japan). Disposable 1.0 mL sterile
syringes were purchased from Shanghai Kangshou
Medical Devices Company, Ltd (Shanghai, China).
Animal experiments were carried out according
to a protocol approved by the Experimental Animal
Care Committee of Capital Medical University, Bei-
jing, China. Male Kunming mice (18–22 g) from the
Animal Services of Capital Medical University were
supplied with food and water ad libitum.
(A and
B series) were separated using high-
performance liquid chromatography (HPLC). As an
example, Figure 5 shows the HPLC chromatogram
of C9H19CO–RGDV–Ara-C isomers (peak 1: A series
and peak 2: B series) detected at 251 nm and using
acetonitrile–water (7:13) as mobile phase with a flow
rate of 0.4 mL/min. The purity of the purified isomers
was determined to be at least 95%.
The CnH2n+1CO–RGDV–Ara-C compounds synthe-
sized were characterized by their melting points, opti-
cal rotation characteristics, and MS and NMR results.
Detailed chemistry can be found in the Supplemen-
tary Information.
Preparation and Characterization of Liposomal
Preparations Containing CnH2n+1CO–RGDV–Ara-C
Compounds Synthesized
Synthesis and Characterization of
CnH2n+1CO–RGDV–Ara-C
Liposomal preparations containing CnH2n+1CO–
RGDV–Ara-C compounds synthesized were prepared
according to a previously reported method32–34 with
minor modifications. In brief, egg lecithin and
CnH2n+1CO–RGDV–Ara-C (n = 7, 9, 11, 13, and 15)
(molar ratio: 1:1) were dissolved in chloroform in
a flask. Chloroform was evaporated under reduced
pressure at 35◦C to form a film, which was sub-
sequently mixed with phosphate buffer (pH 7.4).
The mixture was vortexed, and the fully hydrated
According to a previously reported method,26,31 CH3
(CH2)nCO–RGD and H–V–Ara-C were first synthes-
ized, and the two parts were then condensed to pro-
duce CH3(CH2)nCO–RGDV–Ara-C as illustrated
in Figure 3. Using dimethylformamide (DMF) as solv-
ent, Boc–Arg(NO2)–OH reacted with glycyl–benzyl
ester (H–Gly–OBzl) to yield Boc–Arg(NO2)–Gly–OBzl
in the presence of DCC and HOBt. After selective dep-
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Figure 3. Synthesis of CnH2n+1CO–RGDV–Ara-C and C11H25CO–V–Ara-C (I) DCC, HOBt,
NMM/anhydrous DMF, and THF; (II) 4N HCl-EtOAc; (III) 2N NaOH/ice bath; (IV) 5% H2
(0.02 Mba) Pd/C/ethanol; and (V) EDC, HOBt, and NMM/anhydrous DMF. Abbreviations:
DCC, dicyclohexylcarbodiimide; EDC, 1-ethyle-3-(3-dimethylaminopropyl)carbodiimide; HOBt,
1-hydroxybenzotriazole; NMM, N-methylmorpholine.
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Figure 4. The structures of CnH2n+1CO–RGDV–Ara-C isomers.
mixture was sonicated for 20 min to obtain an opaque
liposomal preparation.
Biological Evaluation of CnH2n+1CO–RGDV–Ara-C
Compounds
Liposomal preparations containing CnH2n+1CO–
RGDV–Ara-C compounds at 3.58 mmol/mL were
characterized by their particle size and zeta poten-
tial at 25◦C. Polydispersity index (PI) was also deter-
mined as a measure of the breadth of the particle size
distribution.35,36 Results were averages from three
measurements.
The morphology of the liposomal preparations was
examined. About 5 :L of the preparation was placed
on a filter paper supported by a copper grid to allow
the sample to dry before being viewed under TEM
(80 kV). Images were obtained.
Animal model: Biological evaluation was determined
using male Kunming mice (18–22 g) inoculated with
S180 sarcoma. Mice were maintained in accordance
with institutional guidelines. S180 tumor cells pas-
saged in Kunming mice abdomen were harvested on
the eighth day and suspended in about 3 mL of ster-
ilized saline in a 15 mL centrifuge tube. The final
volume was adjusted to 10 mL using sterilized saline
and a homogenous mixture was obtained, which was
then centrifuged at 1000 rpm (rotor diameter 15 cm,
83xg) for 5 min. Supernatant was removed and pre-
cipitate was mixed with saline, and mixture was
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Figure 5. HPLC chromatogram of C9H19CO–RGDV–Ara-C isomers (peak 1: series A, peak 2:
series B). Mobile phase: acetonitrile–water, 7:13; flow rate: 0.4 mL/min; UV detection: 251 nm.
recentrifuged. Following centrifugation, precipitate
was suspended in about 9 mL of sterilized saline from
which 0.1 mL of cell suspension was collected and
mixed with 9.9 mL of sterilized saline. Cell suspen-
sion thus obtained was stored in ice until use. Cell
count was determined by transferring 0.10 mL of
the cell suspension obtained above into an Eppendorf
tube into which 100 :L of Trypan Blue was added
before cell number was counted. Tumor cells har-
vested were suspended in sterilized saline at 2.0 ×
107/mL and used for inoculation by injecting 0.2 mL
of the cell suspension into the abdomen of each mouse
under aseptic conditions. Mice inoculated with S180
tumor cells were randomly divided into 25 groups
with 10 mice in each group. Mice were given (via
the tail vein) 0.2 mL of 3.58 mmol/L of the respec-
tive CnH2n+1CO–RGDV–Ara-C containing liposomal
preparations for which liposomal Ara-C preparation
of equal molar concentration was used as positive
control and blank liposome as negative control, or
CnH2n+1CO–RGDV–Ara-C suspensions prepared in
0.5% of CMC-Na for which Ara-C suspension in CMC-
Na was used as positive control and CMC-Na solution
(0.5%) as negative control. First administration was
given 24 h after inoculation, and administration was
repeated every other day for a total of four treatments.
On day 8, mice were sacrificed, and tumors were re-
moved and weighed.
Antitumor activity was defined by tumor growth
inhibition (TGI%), which was calculated as [(tumor
weight of negative control − tumor weight of treat-
ment group)/tumor weight of negative control] ×
100%. Results are expressed as X SD, and statistical
significance was determined using Student’s t-test.
RESULTS AND DISCUSSION
Particle Size and Zeta Potential
As shown in Table 1, the particle size of liposomes con-
taining various CnH2n+1CO–RGDV–Ara-C was found
to be approximately 215 nm, which is similar to that
of Ara-C liposomes. The zeta potential of the prepa-
rations was approximately −30 mV, which is slightly
higher than that of Ara-C. The PI values of the prepa-
rations are also listed in Table 1. Generally speaking,
PI values less than 0.7 indicate a rather homogenous
system. The relatively low PI values associated with
the liposomal preparations prepared in this study
(<0.3) suggested more uniform particles.35,36 There
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Table 1. Particle size, Zeta Potential, and Polydispersity Index (PI) of CnH2n+1CO–RGDV–Ara-C Liposomes
Liposomal Preparation
Particle Size (nm, X SD)
Zeta Potential (mV, X SD)
PI (X SD)
C7H15CO–RGDV–Ara-C(A)
C7H15CO–RGDV–Ara-C(B)
C9H19CO–RGDV–Ara-C(A)
C9H19CO–RGDV–Ara-C(B)
235.0 25
236.6 29
200.1 22
176.2 19
198.0 15
210.5 19
238.2 27
221.0 26
216.3 18
195.6 22
220.2 21
234.5 25
−30.56 4.36
−30.99 4.82
−31.06 5.21
−26.92 3.58
−28.79 3.65
−29.81 3.91
−31.15 5.50
−27.50 4.42
−25.16 4.53
−27.78 3.89
−25.56 4.51
−25.18 3.26
0.283 0.028
0.276 0.023
0.216 0.019
0.255 0.028
0.238 0.022
0.236 0.19
0.219 0.039
0.233 0.036
0.266 0.028
0.286 0.021
0.246 0.056
0.334 0.052
C
C
11H23CO–RGDV–Ara-C(A)
11H23CO–RGDV–Ara-C(B)
C13H25CO–RGDV–Ara-C(A)
C13H25CO–RGDV–Ara-C(B)
C
C
15H29CO–RGDV–Ara-C(A)
15H29CO–RGDV–Ara-C(B)
C11H23CO–V–Ara-C
Ara-C
n = 3.
does not appear to be a relationship between parti-
cle sizes/zeta potential and types of fatty acids. The
liposomal preparations were stable in a refrigerator
for at least 3 weeks. Particle size and zeta potential
of preparations administered to animals were moni-
tored to ensure quality.
Evaluation of Antitumor Activity
Inhibition of Tumor Growth
The tumor inhibition results of liposomal prepara-
tions and suspension containing CnH2n+1CO–
RGDV–Ara-C are provided in Tables 2 and 3.
Table 2. Tumor Growth Inhibition (TGI) of CnH2n+1CO–RGDV–Ara-C
Liposomes in S180-Bearing Mice
Liposomal Preparation
Tumor Weight (g, X SD)
TGI (%)
C7H15CO–RGDV–Ara-C(A)
C7H15CO–RGDV–Ara-C(B)
C9H19CO–RGDV–Ara-C(A)
C9H19CO–RGDV–Ara-C(B)
0.4651 0.2428∗∗
56.8
53.8
65.3
64.4
64.0
61.5
66.1
59.5
61.9
54.5
32.9
24.1
0
0.4978 0.1406∗,∗∗,∗∗∗
0.3741 0.2049∗,∗∗,∗∗∗
0.3836 0.1037∗,∗∗,∗∗∗
0.3875 0.2040∗,∗∗,∗∗∗
0.4145 0.2154
C11H23CO–RGDV–Ara-C(A)
C11H23CO–RGDV–Ara-C(B)
C13H25CO–RGDV–Ara-C(A)
C13H25CO–RGDV–Ara-C(B)
C15H29CO–RGDV–Ara-C(A)
C15H29CO–RGDV–Ara-C(B)
C11H23CO–V–Ara-C
0.3645 0.1285∗,∗∗,∗∗∗
0.4356 0.2641∗,∗∗
0.4103 0.2030∗,∗∗,∗∗∗
0.4904 0.1187∗,∗∗
0.7224 0.3085
Ara-C
Blank liposome
0.8173 0.2593
1.077 0.2858
n = 8.
∗Significant compared with blank liposome, p < 0.01.
∗∗Significant compared with Ara-C liposome, p < 0.05.
∗∗∗Significant compared with C12–V–Ara-C liposome, p < 0.05.
Table 3. TGI of CnH2n+1CO–RGDV–Ara-C Suspensions in S180-Bearing Mice
Suspension
Tumor Weight (g, X SD)
TGI (100%)
C7H15CO–RGDV–Ara-C(A)
C7H15CO–RGDV–Ara-C(B)
C9H19CO–RGDV–Ara-C(A)
C9H19CO–RGDV–Ara-C(B)
0.5981 0.1756
0.6065 0.3328
0.5325 0.1753
0.5590 0.2220
0.5768 0.2605
0.6246 0.1777
0.5968 0.2282
0.5964 0.2573
0.5531 0.1854
0.5839 0.1508
0.6039 0.1679
0.6041 0.1592
0.7139 0.2724
16.2
15.0
25.4
21.7
19.2
12.5
16.4
16.5
22.5
18.2
15.4
15.4
0
C11H23CO–RGDV–Ara-C(A)
C11H23CO–RGDV–Ara-C(B)
C13H25CO–RGDV–Ara-C(A)
C13H25CO–RGDV–Ara-C(B)
C15H29CO–RGDV–Ara-C(A)
C15H29CO–RGDV–Ara-C(B)
C11H23CO–V–Ara-C suspension
Ara-C suspension
Blank CMC-Na solution
n = 8.
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Figure 6. Biopsy results of tumor tissue of S180 inoculated mice following tail vein adminis-
tration of blank liposomes (a), C11H23CO–RGDV–Ara-C(A) liposomes (b), Ara-C liposomes (c),
and C11H23CO–Val–Ara-C liposomes (d) (H&E staining, 10 × 10 magnification).
As shown in Table 2, all CnH2n+1CO–RGDV–Ara-
the image of tumor cells treated with blank liposome
in which the structure of tumor tissue was not affected
and the nuclei of tumor cells were clearly stained
indicating viable tumor cells. Following the treat-
ment of C11H23CO–RGDV–Ara (Fig. 6b), the struc-
ture of the tumor tissue was significantly damaged
and cell death was clearly observed, whereas Ara-C
and C11H23CO–V–Ara-C (A) resulted in localized tu-
mor cell death and cytolysis (Figs. 6c and 6d). How-
ever, there were still a significant number of unlysed
tumor cells with intact nuclei shown.
C liposomal preparations were found to be more
effective than Ara-C and C11H23CO–V–Ara-C in
the inhibition of tumor growth in mice, whereas
there was little difference between Ara-C and
C11H23CO–V–Ara-C liposomes. The data in Table 3
showed that there was little difference between
CnH2n+1CO–RGDV–Ara-C suspensions and Ara-C
suspension or C11H23CO–V–Ara-C suspension. The
results clearly demonstrated that RGDV was able to
improve the antitumor activity of Ara-C in its lipo-
somal formulations, whereas it made little difference
in the antitumor activity of Ara-C in its suspensions.
It was also found that the length of the fatty acid in
CnH2n+1CO–RGDV–Ara-C had little impact on their
activity against tumor growth. In addition, there was
little difference observed between the two isomers for
each of the CnH2n+1CO–RGDV–Ara-C.
CONCLUSIONS
This study focused on the potential of RGD to
enhance the antitumor activity of Ara-C by at-
taching RGD to Ara-C via another amino acid,
valine. In addition, fatty acids of various chain
lengths were introduced into the structure. A se-
ries of CnH2n+1CO–RGDV–Ara-C were synthesized
and characterized. They were subsequently pre-
pared into liposomal preparations and suspensions,
respectively. The fatty acid moiety in the struc-
ture was used to improve the overall lipophilicity
of CnH2n+1CO–RGDV–Ara-C so that they can be
more readily incorporated in the vesicles of liposome,
Biopsy of Tumor Tissue
In addition to tumor size, pathological examinations
are also important in assessing the antitumor po-
tential of cytotoxic agents or formulations. Figure 6
shows the H&E stained images of tumor tissues dis-
sected from mice inoculated with S180 sarcoma after
treatment with different formulations. Figure 6a is
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DOI 10.1002/jps

A TETRA PEPTIDE MEDIATED TUMOR TARGETING OF ARA-C PRODRUG LIPOSOMES
4567
whereas RGD was used for its unique specificity for
the integrins overexpressed in cancer cells to achieve
more selective delivery. CnH2n+1CO–RGDV–Ara-C
would undergo hydrolysis to release Ara-C, which is
cytotoxic.
As noted earlier, there are two isomers for each
of the CnH2n+1CO–RGDV–Ara-C compounds, which
were successfully separated using HPLC. The parti-
cle size, zeta potential, and PI of the liposomal prepa-
rations of CnH2n+1CO–RGDV–Ara-C were found to
be approximately 215 nm (diameter), approximately
−30 mV, and <0.3, respectively.
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The antitumor activities of CnH2n+1CO–
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sarcoma. It was found that CnH2n+1CO–RGDV–Ara-
C liposomes were more effective (TGI and biopsy
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and Ara-C suspension. The improved antitumor
activity of CnH2n+1CO–RGDV–Ara-C liposomes over
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the fact that liposomes possess a better penetrating
capability through tumor cell membranes.26 The cur-
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ACKNOWLEDGMENTS
This research was supported by the National Nat-
ural Science Foundation of China (30873202). The
authors are also grateful to the Key Laboratory on
Peptides and Small Drug Molecules, and the Mod-
ern Drug Analysis Laboratory at the Capital Medical
University for their generous help.
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