Novel tetrapeptide, RGDF, mediated tumor specific liposomal doxorubicin (DOX) preparations

Article abstract of DOI:10.1021/mp200039s

Arginine-glycine-aspartate (RGD) has been shown to possess a strong affinity for the integrins overexpressed in tumor cells, especially during tumor invasion, angiogenesis and metasis. Based on work from others, a novel tetrapeptide, arginine-glycine-aspa

Full text of DOI:10.1021/mp200039s

ARTICLE
pubs.acs.org/molecularpharmaceutics
Novel Tetrapeptide, RGDF, Mediated Tumor Specific Liposomal
Doxorubicin (DOX) Preparations
Huirui Du,†,‡ Chunying Cui,†,‡ Lili Wang,§ Hu Liu,*,§ and Guohui Cui*,†
†School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing, China. 100069
§School of Pharmacy, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3V6
S Supporting Information
b
ABSTRACT: Arginine-glycine-aspartate (RGD) has been shown to
possess a strong affinity for the integrins overexpressed in tumor cells,
especially during tumor invasion, angiogenesis and metasis. Based on
work from others, a novel tetrapeptide, arginine-glycine-aspartate-
phenylanaline (RGDF), has been designed and studied as a homing
device to direct liposomal doxorubicin (DOX) to tumor cells in this work.
In order to incorporate RGDF into liposomal DOX preparations, RGDF
was conjugated with three different fatty alcohols to achieve RGDFꢀfatty
alcohol conjugates. Glycine-glycine-aspartate-phenylanaline (GGDF)-
lauryl alcohol conjugate was synthesized as a negative control.
RGDFꢀfatty alcohol conjugates (RGDFO(CH2)nCH3) and GGDFꢀ
lauryl alcohol conjugate (L-GGDFC12-DOX) incorporated liposomal
preparations were obtained by first preparing liposomes using the film
dispersion method followed by loading DOX using a transmembrane pH gradient method. Because of their amphipathic nature,
RGDFꢀ or GGDFꢀfatty alcohol conjugates are expected to be readily incorporated into liposomes with their fatty alkanyl chains
being intercalated between fatty acyl chains of liposomal bilayers and the hydrophilic peptide moiety (RGDF or GGDF) being
anchored on the surface of liposomes. The particle size and zeta potential of liposomal DOX preparations containing RGDFꢀfatty
alcohol conjugate (L-RGDF-DOXs) or L-GGDFC12-DOX were measured, and their morphology was studied using transmission
electron microscopy. In vitro DOX release profile from RGDF incorporated liposomal DOX was measured. The antitumor activities
of RGDF incorporated liposomal DOX preparations were evaluated in ICR mice inoculated with sarcoma S180, which is known to
express Rvβ3 integrin. Both conventional liposomal DOX preparation (L-DOX) without RGDFO(CH2)nCH3 and L-GGDFC12-
DOX were used as negative controls. Our results showed improved tumor growth inhibition with L-RGDF-DOXs over doxorubicin
hydrochloride solution, L-DOX and L-GGDFC12-DOX. Pathological examination of tumor biopsy demonstrated that L-RGDF-
DOXs induced enhanced tumor cell death in comparison to negative controls. Pharmacokinetic studies showed that the
concentrations of DOX found in tumor sites were increased by 1.7ꢀ4.5-fold when liposomal DOX preparation containing
RGDFꢀlauryl alcohol conjugate (L-RGDFC12-DOX) was administered in comparison to when L-GGDFC12-DOX or
doxorubicin hydrochloride solution was administered. The concentrations of DOX found in the heart, which is the main site of
toxic effects of DOX, were significantly reduced when L-RGDFC12-DOX was administered in comparison to when L-GGDFC12-
DOX or doxorubicin hydrochloride solution was administered.
KEYWORDS: RGD, tumor specific liposomes, doxorubicin, antitumor, selective delivery of liposomes, drug targeting
of DOX include nausea, vomiting and heart arrhythmias. It also
inhibits bone marrow functions causing neutropenia. Accumula-
tive doses of DOX dramatically increase the risk of cardiac side
effects, including congestive heart failure, dilated cardiomyopathy
and death.4ꢀ6
Various targeted drug delivery strategies have been explored to
improve the therapeutic index of anticancer agents. Liposomal
systems of anticancer drugs have been marketed and shown to
1. INTRODUCTION
Doxorubicin (DOX) is a commonly used anticancer agent and
belongs to anthracycline antibiotics. Like other anthracyclines,
DOX is known to interact with DNA by intercalation and
inhibition of DNA biosynthesis.1 DOX inhibits the progression
of the DNA topoisomerase II, which unwinds DNA for tran-
scription. In addition, DOX stabilizes the topoisomerase II
complex after the DNA chain has been broken for replication,
preventing the DNA double helix from being resealed and
thereby stopping the process of replication.2 DOX is commonly
used to treat certain types of leukemia, Hodgkin’s lymphoma, and
cancers of the bladder, breast, stomach, lung, ovaries, thyroid, soft
tissue sarcoma, multiple myeloma, and others.3 Major side effects
Received: January 25, 2011
Accepted: June 1, 2011
Revised:
April 26, 2011
Published: June 01, 2011
r
2011 American Chemical Society
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have reduced toxicity.7,8 However, better strategies are needed to
improve the selectivity of existing systems. Immunoliposomes
have been studied for active delivery of anticancer agents to
tumors, in which tumor-specific antibodies are anchored on the
surface of liposomal vesicles. After administration, they are
expected to be actively associated with tumor specific antigens
present on the surface of tumor cells. Therefore, anticancer
agents encapsulated in such liposomes are expected to be actively
delivered to tumor cells, resulting in reduced toxicity in normal
cells.9,10
2. MATERIALS AND METHODS
2.1. Materials. Doxorubicin hydrochloride (pharmaceutical
grade) was obtained from Beijing Huafeng United Technologies
Ltd. (Beijing, China). Capryl alcohol (1-octanol, CH3(CH2)7ꢀ
OH), lauryl alcohol (1-decocanol, CH3(CH2)11ꢀOH) and cetyl
alcohol (1-hexadecanol, palmityl alcohol, CH3(CH2)15ꢀOH)
were obtained from Beijing Chaoyang Xudong Chemicals Inc.
(Beijing, China). All amino acids and their protected forms were
of L-configuration and were from Sichuan Sangao Biochemical
Co. Ltd. (Chengdu, Sichuan, China). Dicyclohexylcarbodiimide
(DCC), 1-hydroxybenzotriazole (HOBt), N-methylmorpholine
(NMM), BocArg(NO2)OH and BocAsp(OBzl)OH were
from GL Biochem Ltd. (Shanghai, China). Egg lecithin (MW =
750.00) was purchased from ACROS ORGANICS (Geel,
Belgium). Cholesterol was purchased from Beijing Aoboxing
Biotech Co. Ltd. (Beijing, China). All reagents were of chemical
grade unless otherwise specified.
Integrins, a group of transmembrane proteins, are receptors
that mediate an attachment between a cell and the tissues
surrounding it, which may be other cells or the extracellular
matrix. They are heterodimers containing two distinct chains,
named R- and β-subunits,11,12 and play an important role in
tumor invasion and metastasis, a complex multistage process
involving tumorꢀhost interaction which includes adhesion,
angiogenesis and proteolysis. Increased expression of integrins
facilitates adhesion of tumor cells to the endothelial linings of
blood vessels to colonize host organs.13 It is known that the
tripeptide arginine-glycine-asparate (Arg-Gly-Asp, RGD) has a
strong affinity for integrins and demonstrated an inhibitory effect
on the adhesion and angiogenesis of tumor cells.14,15 RGD,
however, also promotes the detachment of invasive tumor cells
from the primary tumor site leading to the colonization of other
organs.16,17 Such dual and opposite effects on the progression
of tumor limited the therapeutic potential of RGD in cancer
treatment. Nevertheless, the elevated expression of integrins
found in many tumors is well documented.18,19 RGD has been
conjugated to phospholipids and polyethylene glycols to
achieve site specific liposomes.20,21 However, studies so far
involved complicated chemistry and unpredictable characteris-
tics for formulations. In this work, a new tetrapeptide, arginine-
glycine-aspartate-phenylalanine (Arg-Gly-Asp-Phe, RGDF), was
designed to achieve improved affinity for integrins in tumor cells
and to be used as a homing device on liposomes for targeting
tumor cells. Phenylalanine, an aromatic amino acid, was added to
RGD based on the findings from others that hydrophobic amino
acids flanking the RGD motif increased binding affinity of RGD
to integrins.22,23 Liposomal DOX preparation was used to study
the targeting potential of RGDF to tumor cells. In order to
incorporate RGDF into liposomal DOX preparation to direct
its delivery to tumor cells, RGDFꢀfatty alcohol conjugates
(RGDFO(CH2)nCH3) were designed as amphiphilic com-
pounds (with both hydrophobic and hydrophilic characteristics)
so that they would intercalate into the bilayers of liposomal DOX.
Fatty alcohols of different chain lengths (C8, C12 and C16) were
used in this work. Liposomal DOX preparations containing
RGDFO(CH2)nCH3 were prepared and evaluated. Glycine-
glycine-aspartate-phenylanaline (GGDF) was synthesized as a
negative control, in which arginine, a positively charged and
bulky amino acid, found in RGDF was replaced by glycine, a
neutral and much smaller amino acid. Mice inoculated with S180
sarcoma, which is known to express Rvβ3 integrin, were used to
examine the anticancer activity and targeting potential of the
RGDFꢀfatty alcohol conjugate incorporated liposomal DOX
preparations (L-RGDF-DOXs). Tissue distribution and phar-
macokinetic profile of L-RGDF-DOXs in mice were also studied.
The results were compared with those of conventional liposomal
DOX preparation (L-DOX, containing no homing device) and
GGDFꢀlauryl alcohol conjugate (L-GGDFC12-DOX) incorpo-
rated liposomal DOX preparation.
Animal experiments were carried out according to a protocol
approved by the Experimental Animal Care Committee of Capital
Medical University. Male Kunming and ICR mice purchased from
the Animal Services, Capital Medical University were supplied
with food and water ad libitum.
Laborota 4000 rotary evaporator was from Heidolph Instru-
ments (Schwabach, Germany). An IKA.MS1 minishaker was
purchased from Guangzhou, China. VCX500 ultrasonicator was
a product of Sonics & Materials, Inc. (Newtown, CT, USA). Waters
2695 HPLC was used for quantitative analysis. An OLYMPUS IX71
inverted microscope equipped with a digital camera (OLYMPUS
CAMEDIA C-7070) was purchased from Olympus Co. (Tokyo,
Japan). A transmission electron microscope (TEM), JEM-1230,
JEOL, was purchased from Japan. A particle size analyzer, Zetasizer
Nano ZS-90, was purchased from Malvern Inc. (United Kingdom).
A steam sterilizer was a product of VARIOKLAV (Germany).
2.2. Synthesis of RGDFO(CH2)nCH3 and GGDFO(CH2)11CH3.
RGDFO(CH2)nCH3 and GGDFO(CH2)11CH3 were synthe-
sized according to the schemes shown in Figures 1 and 2,
respectively. The respective fatty alcohols of different carbon chain
lengths (C8, C12 or C16) were first connected to the N-terminus
protected phenylalanine (Boc-Phe-OH) in the presence of DCC,
HOBt and NMM to yield N-terminus protected (Boc) phenyla-
lanyl esters [Boc-FO(CH2)nCH3, n = 7, 11 or 15]. After deprotec-
tion, H2N- FO(CH2)nCH3 (n = 7, 11 or 15) reacted with N-tert-
butoxycarbonyl-L-aspartic acid 4-benzyl ester (Boc-Asp-OBzl) in
the presence of DCC, HOBt and NMM to yield N-terminus
protected 4-benzyl aspartylphenyalanyl esters, [Boc-D(OBzl)FO-
(CH2)nCH3, n = 7, 11 or 15]. Protected dipeptides, arginine-
glycine (RG) or glycine-glycine (GG), were formed by dissolving
N-R-tert-butyloxycarbonyl-N-γ-nitro-L-arginine [Boc-Arg(NO2)-
OH] or N-tert-butoxycarbonyl-glycine (Boc-Gly-OH) and glycine
benzyl ester tosylate (H-Gly-OBzl) in dimethylformamide
(DMF). Condensation was completed in the presence of DCC
and HOBt which resulted in Boc-Arg(NO2)-Gly-OBzl [Boc-
R(NO2)G-OBzl] or Boc-Gly-Gly-OBzl [Boc-GG-OBzl]. Boc-R-
(NO2)G-OBzl or Boc-GG-OBzl was then subjected to selective
deprotection in the presence of 4 N NaOH (0 °C) to remove
benzyl esters, which provided Boc-R(NO2)G-OH or Boc-GG-
OH. Previously synthesized N-terminus protected 4-benzyl aspar-
tylphenyalanyl esters [Boc-D(OBzl)FO(CH2)nCH3, n = 7, 11 or
15] were subjected to treatment with 4 N HClꢀEtAc to remove
the N-terminus protection to yield 4-benzyl aspartylphenyalanyl
esters [H2N-D(OBzl)FO(CH2)nCH3, n = 7, 11 or 15], which
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Figure 1. Synthesis of RGDFO(CH2)nCH3 (n = 7, 11, 15): (I) DCC, HOBt, NMM and CH3(CH2)nOH, n = 7, 11, 15; (II) DCC, HOBt, NMM and
Tos Gly-OBzl; (III) DCC, HOBt, NMM and BocAsp(OBzl)OH; (IV) 2 N NaOH; (V) 4 N HClꢀEtOAc; (VI) 5% H2 (0.02 M Ba) Pd/C.
3
Figure 2. Synthesis of GGDFO(CH2)11CH3: (I) DCC, HOBt, NMM and CH3(CH2)11OH; (II) DCC, HOBt, NMM and Tos Gly-OBzl; (III) DCC,
3
HOBt, NMM and BocAsp(OBzl)OH; (IV) 2 N NaOH; (V) 4 N HClꢀEtOAc; (VI) 5% H2 (0.02 M Ba) Pd/C.
were then subjected to condensation with Boc-R(NO2)G-OH or
Boc-GG-OH in the presence of DCC and HOBt to yield N-R-tert-
butyloxycarbonyl-N-γ-nitro-arginyl-glycyl-4-benzylaspartyl-phe-
nylalanyl fatty alcohol esters [Boc-R(NO2)GD(OBzl)FO-
(CH2)nCH3, n = 7, 11 or 15] (Figure 1) or [Boc-GGD(OBzl)
FO(CH2)11CH3] (Figure 2). Both nitro and benzyl protection
groups were removed via catalytic hydrogenation in the presence
of Pd/C to yield [Boc-RGDFO(CH2)nCH3, n = 7, 11 or 15]
(Figure 1) or [Boc-GGDFO(CH2)11CH3] (Figure 2). Finally
the N-tert-butyloxycarbonyl protection group was removed in
4 N HClꢀEtOAc to yield [RGDFO(CH2)nCH3, n = 7, 11 or 15]
(Figure 1) or [GGDFO(CH2)11CH3] (Figure 2).
2.3. Preparation of Liposomal Formulations. L-RGDF-
DOXs were prepared according to a method reported by
others24,25 with modifications. In brief, phospholipids, cholester-
ol and RGDFO(CH2)nCH3 (n = 7, 11, 15) (molar ratio:
14.25:4.75:1) were dissolved in chloroform in a flask. Chloro-
form was evaporated under reduced pressure to form a lipid film,
which was subsequently mixed with 120 mM ammonium sulfate.
The mixture was vortexed. The fully hydrated mixture was
sonicated for 10 min to obtain an opaque liposomal preparation,
which was sealed in a dialysis bag (molecular weight cutoff:
8,000ꢀ14,000 Da) and dialyzed against saline for 8 h. After five
times of repeated dialysis, the resultant liposomal preparation
was placed in a flask. In a separate flask, doxorubicin hydro-
chloride (DOX:phospholipids 1:20 w/w) was dissolved in a
small quantity of distilled water. The above liposomal prepara-
tion and DOX were mixed and shaken at 37 °C in a water bath
for 30 min to yield L-RGDF-DOXs. Conventional L-DOX or
liposomal DOX preparation without RGDFꢀfatty alcohol con-
jugate incorporated was obtained using the same protocol except
that RGDFO(CH2)nCH3 was not included in the procedure.
To study the effect of the amount of RGDF on the anticancer
activity and targeting potential in liposomal DOX, a second
RGDFꢀlauryl conjugate incorporated liposomal DOX prepara-
tion (L-RGDFC12-DOX) was also obtained using phospholi-
pids, cholesterol and RGDFO(CH2)11CH3 at a molar ratio of
14.25:4.75:0.5. Using the same protocol, GGDFꢀlauryl alcohol
conjugate incorporated liposomal DOX preparation (L-GGDF-
DOX) was also prepared. DOX encapsulation efficiency was
determined according to a previously reported method.26 In
brief, 0.5 mL of the respective liposomal preparation was loaded
onto a Sephadex G50 column, which was eluted with normal
saline solution at a flow rate of 1.5 mL/min to separate free DOX
from liposomes. The liposomal fraction or liposomes loaded with
DOX were collected and transferred to a 25 mL flask into which
0.1 mL of 10% Triton X-100 was added, and the final volume
was adjusted to 25 mL using normal saline. The mixture was
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thoroughly mixed to allow DOX that was incorporated into the
liposomes to be released, and the amount of DOX released
(which is referred to as DOX encapsulated) was determined at
496 nm using a spectrophotometer. In a separate test, 0.5 mL of
the respective liposomal preparation was placed in a 25 mL flask,
into which 0.1 mL of 10% TritonX-100 was added, and the final
volume was adjusted to 25 mL using normal saline. The mixture
was mixed thoroughly to allow any DOX that was incorporated
into the liposomes to be released. The amount of DOX was
determined and considered as total DOX (free DOX and DOX
encapsulated). The encapsulation efficiency was calculated as the
amount of DOX encapsulated/total DOX. The experiment was
repeated three times. The morphology of liposomes was exam-
ined by TEM. Particle sizes and zeta-potentials of liposomes were
analyzed by a particle size analyzer, Zetasizer Nano ZS-90.
2.4. In Vitro Release of DOX from Its L-RGDF-DOXs. The
release of DOX from its L-RGDF-DOXs was determined using
L-RGDFC12-DOX according to the method reported by Kim
et al.27 Briefly, 2 mL of L-RGDFC12-DOX was placed and sealed
in a dialysis bag, which was placed in a beaker containing 50 mL
of phosphate buffered saline (PBS, pH 7.4) and dialyzed in a
shaker at 37.5 °C. At different time intervals (1, 2, 4, 6, 12, 24, 48,
and 72 h), 50 mL of the PBS in the beaker was removed and
replaced with 50 mL of fresh PBS to maintain a sink condition.
Concentrations of DOX in the PBS withdrawn were analyzed at
496 nm using a spectrophotometer. Total DOX released over the
72 h monitored was calculated, and results were plotted.
2.5. In Vivo Anticancer Activities of L-RGDF-DOXs in Mice
Inoculated with S180 Sarcoma. The anticancer activity was
determined in ICR mice (Swiss) inoculated with S180 sarcoma.
Mice were maintained in accordance with institutional guide-
lines. S180 tumor cells passaged in Kunming mice abdomen
were harvested on the eighth day and suspended in saline at 2.0 ꢁ
107/mL, which was injected to ICR mice (0.2 mL/per mouse).
The mice inoculated with S180 tumor cells were randomly divided
into 12 different groups with 10 in each group. Twenty-four hours
after inoculation (day 1), mice were injected (0.2 mL/animal)
with 2 mg/kg of DOX in doxorubicin hydrochloride solution
(S-DOX), L-DOX, L-RGDFC12-DOX, RGDFꢀcapryl alcohol
conjugate incorporated liposomal DOX (L-RGDFC8-DOX) or
RGDFꢀcetyl alcohol conjugate incorporated liposomal DOX
(L-RGDFC16-DOX) via the tail vein, and injection was repeated
on days 4 and 7. Two days after the final injection, mice were
sacrificed and tumors were weighed. Blank liposomal preparation
with no RGDFO(CH2)nCH3 or DOX (L) and saline (NS) were
used as negative controls. Furthermore, L-GGDFC12-DOX
(2 mg/kg of DOX) was also used as a control. To study the
effect of the concentration of RGDF in the liposomal pre-
parations on anticancer activity, 2 mg/kg of DOX in L-RGDFC12-
DOX prepared using different amounts of RGDFC12 as de-
scribed in section 2.3 was injected to mice inoculated with S180
tumor cells. The anticancer activities of L-RGDF-DOXs given
with different dosages of DOX (1 mg/kg, 2 mg/kg and 3 mg/kg)
were also studied. Data are expressed as X ( SD, and statistical
significance was determined using unpaired two-sided Student’s
t-test. Removed tumor tissues were washed with saline, wrapped
with tinfoil and stored at ꢀ80 °C for subsequent tissue dissection
and hematoxylinꢀeosin staining. Sliced tissues were examined
and photographed under a microscope.
reported by others.9 In brief, S180 tumor cells passaged in the
abdomens of the Kunming mice were harvested on the eighth
day and suspended at 2.0 ꢁ 107/mL. The tumor cell preparation
(∼2 ꢁ 106 tumor cells) was injected subcutaneously under the
right armpit of each of the 120 ICR mice (22ꢀ24 g). Seven days
later, mice were randomly divided into three different groups
with 40 in each group which were injected via the tail vein with
7.5 mg/kg of DOX in S-DOX, L-GGDFC12-DOX or
L-RGDFC12-DOX (40 mice for each preparation). At each time
interval (5 min, 15 min, 30 min, 1 h, 4 h, 8 h, 24 h and 48 h) after
injection of the respective formulations, blood samples from five
mice were collected, and mice were then sacrificed. Both the
heart and tumor were removed, washed with distilled water, dried
with paper towel, weighed and frozen at ꢀ20 °C. Heart and
tumor tissues were homogenized with PBS (1 g of tissue:3 mL of
PBS), and homogenized tissue samples were transferred to
centrifuge tubes. To each centrifuge tube, 50 μL of methanol
and 50 μL of daunorubicin (1 μg/mL) as an internal standard
were added and vortexed. The homogenates were subjected to
extraction using chloroform and methanol (4:1) (3 mL for tumor
and 1.5 mL for heart). The tubes were vortexed for 3 min and
centrifuged at 1000g for 10 min. An aliquot of the organic layer
was removed and dried. The residue was dissolved in 180 μL of
HPLC grade methanol, and the solution was analyzed by HPLC
(21) using a C-18 column and acetonitrile:water (30:70)28,29 as
mobile phase at 0.8 mL/min with UV detection at λmax = 254 nm.
DOX concentrations versus time were plotted.
According to the method proposed by Hunt et al.,30 the
following calculations were carried out to obtain therapeutic
availability (TA) and drug targeting index (DTI):
TA ¼ AUCtargetDC=AUCtargetD
DTI ¼ ðAUCtargetDC=AUCtoxDCÞ=ðAUCtargetD=AUCtoxD
Þ
where “target” represents the targeted site, which is the tumor in
this study, “tox” represents the major toxic organ/tissue, which is
the heart in this study,4ꢀ6 “DC” represents the site specific
formulations being studied, which are L-RGDF-DOX pre-
parations, and “D” represents the same amount of free drug or
negative controls used, which are conventional L-DOX and
L-GGDFC12-DOX. When TA is >1, the site specific formula-
tions studied are considered having targeting capability.
3. RESULTS AND DISCUSSION
To synthesize RGDFO(CH2)nCH3 (n = 7, 11 and 15) and
GGDFO(CH2)11CH3, the N-termini of the respective peptides
were protected with (Boc)2O and C-termini were protected with
benzyl esters. In most of the condensation procedures where
peptide amide bonds were formed, excess amounts of DCC were
added. The esterification of the respective fatty alcohols to
phenylalanine was found to be slow and incomplete, and excess
amounts of fatty alcohols were used. Because of the difficulty in
removing unreacted fatty alcohols, the ratio of fatty alcohol to
phenylalanine was kept at not more than 1.5. The products,
RGDFO(CH2)nCH3 (n = 7, 11 or 15) and GGDFO-
(CH2)11CH3, were purified using silica gel columns eluted with
gradient chloroform and methanol (120:1, 100:1, 90:1 and 80:1).
The purities of compounds were found to be more than 90% as
determined by HPLC. Purified compounds were characterized
using 1H and 13C nuclear magnetic resonance (NMR) and mass
spectrometry (MS). Melting points and specific optical rotation
2.6. Biodistribution of S-DOX, L-GGDFC12-DOX and
L-RGDFC12-DOX in Tumor Bearing Mice. Tissue distribu-
tion of L-RGDF-DOXs was determined according to a method
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Figure 4. In vitro release of DOX from L-RGDFC12-DOX (N = 3).
Figure 3. TEM photograph of L-RGDFC12-DOX showing spherical
shaped particles.
F) amino acids. Given the molar ratio of phospholipids to RGDF
in RGDF incorporated liposomal preparations being 14.25:1 (or
14.25:0.5), the zeta potential of RGDF incorporated liposomal
preparations reflected the charge of phospholipids found in the
liposomes. Although GGDF possesses a negative charge, the zeta
potential of GGDF incorporated liposomal preparation is pri-
marily determined by the charge of the phospholipids since the
amount of GGDF in the GGDF incorporated liposomal pre-
paration was very small in comparison to that of phospholipids
(1:14.25). The encapsulation efficiency of DOX into liposomes
was about 94%.
The release of DOX from L-RGDF-DOX preparations over
time was studied using L-RGDFC12-DOX, and it was found that
DOX was slowly released as shown in Figure 4. About 60% of
encapsulated DOX was released within the first 12 h. At 72 h,
almost 90% of encapsulated DOX was released.
The antitumor activities of L-RGDF-DOXs were evaluated in
ICR mice inoculated with S180 sarcoma. Tumor weights were
determined and compared among mice administered with
various preparations. As shown in Figure 5, all liposomal DOX
preparations were found to have improved effect in reducing tumor
growth in comparison with S-DOX. There was little difference
between L-DOX and L-GGDFC12-DOX, suggesting that GGDF
had little effect on the anticancer activity of liposomal DOX prepara-
tion. However, L-RGDF-DOXs were foundtobemoreeffective than
L-DOX and L-GGDFC12-DOX. The results suggest that RGDF
might have delivered more DOX to tumor cells due to its affinity for
the integrins overexpressed in tumor cells. There was no significant
difference found among the various RGDFꢀfatty alcohol conjugates
studied.
To further examine the targeting potential of RGDF, three
different doses of DOX (1, 2, and 3 mg/kg) in L-RDGFC12-
DOX were administered to tumor-bearing mice, and results are
shown in Figure 6. It was found that there was little difference
between 2 and 3 mg/kg of DOX although the effect of 1 mg/kg of
DOX was found to be significantly lower. By comparing the
results in Figures 5 and 6, it was found that the tumor size
following 1 mg/kg of DOX in L-RGDFC12-DOX formulation
was similar to that of 2 mg/kg of DOX in L-DOX, suggesting that
RGDF incorporated liposomal DOX was able to achieve the
same effect as higher L-DOX perhaps by directing more DOX to
tumor sites.
Table 1. Particle Sizes and Zeta Potentials of Various DOX
Liposomes
formulation
particle size (nm)
zeta potential (mV)
L-RGDFC8
233.0 ( 1.1
191.0 ( 2.4
227.6 ( 7.4
143.9 ( 0.6
244.0 ( 2.9
176.5 ( 1.4
216.0 ( 7.8
248.5 ( 1.2
250.2 ( 7.2
223.9 ( 9.9
ꢀ44.80 ( 0.66
ꢀ46.33 ( 2.51
ꢀ41.78 ( 1.32
ꢀ52.30 ( 1.76
ꢀ48.76 ( 3.72
ꢀ43.15 ( 2.04
ꢀ59.79 ( 1.33
ꢀ44.73 ( 2.43
ꢀ43.63 ( 2.49
ꢀ49.70 ( 1.64
L
L-DOX
L-RGDFC8-DOX
L-RGDFC12-DOX
L-RGDFC16- DOX
L-GGDFC12-DOX
2.5% L-RGDFC8-DOX
L-RGDFC8-DOX 1 mg
L-RGDFC8-DOX 3 mg
of the purified final products were also determined. Detailed
chemistry data of the four peptide fatty alcohol conjugates are
provided in the Supporting Information.
Because of the amphipathic nature of RGDFꢀ or
GGDFꢀfatty alcohol conjugates, they are expected to be readily
incorporated into liposomes. The fatty alkanyl chains are likely to
be intercalated between fatty acyl chains of liposomal bilayers
while the hydrophilic peptide moiety (RGDF or GGDF) is likely
to be anchored on the surface of liposomes and projected to an
aqueous environment.
DOX was loaded into liposomes including RGDFꢀfatty
alcohol conjugate incorporated, GGDFꢀlauryl alcohol conju-
gate incorporated and conventional liposome using a transmem-
brane pH gradient method as described above. The morphology
of the RGDFꢀfatty alcohol conjugate incorporated liposomal
DOX preparations was studied, and results of L-RGDFC12-
DOX are shown in Figure 3 as an example. Under the TEM, the
liposomal DOX preparations were found to exist as spherical
particles with an average size of ∼200 nm. The particle size and
zeta-potential of the liposomal formulations prepared were
examined, and the results are summarized in Table 1. It was
found that the particle sizes of most formulations were between
190 and 250 nm, and the zeta-potentials were found to be about
ꢀ45 mV. It should be noted that RGDF is electronically neutral
as it contains one acidic (D), one basic (R) and 2 neutral (G and
The effect of the amount of RGDFꢀlauryl alcohol conjugate
in liposomal DOX on tumor size was also examined with
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Figure 5. Tumor weights in mice inoculated with S180 following tail vein injection of 2 mg/kg of L-RGDF-DOXs on days 1, 4, and 7 (N = 10).
*Significant compared to S-DOX (P > 0.05). #Significant compared to liposomal DOX preparation or L-DOX (P > 0.01). &Significant compared to
L-GGDFC12-DOX (P > 0.01).
Figure 8. Tumor biopsy (H&E staining, 10 ꢁ 10 magnification) after
tail vein injection of saline, 2 mg/kg of DOX in L-DOX and
L-RGDFC12-DOX in S180 inoculated mice.
described above. As shown in Figure 7, there was little difference
Figure 6. Tumor weights in tumor-bearing mice administered with
between the two L-RGDFC12-DOX preparations studied, which
L-RGDFC12-DOX preparations containing 1, 2, and 3 mg/kg of DOX
suggested that the number of receptors on the surface of tumor
(N = 10). *Significant compared to 1 mg/kg of DOX (P > 0.01).
#Insignificant compared to 2 mg/kg of DOX (P > 0.05).
cells is limited and the cellular uptake is saturable.
In addition to tumor size, pathological examination is also
important in assessing the antitumor potential of cytotoxic
agents. Figure 8 shows the H&E stained images of tumor tissues
dissected from ICR mice inoculated with S180 after treatment
with different DOX formulations. The left panel in Figure 8 is
tumor cells treated with saline in which nuclei of tumor cells were
clearly stained indicating viable tumor cells. Following the
treatment of L-DOX (middle panel), localized tumor cell death
and cytolysis were observed. However, there were still a sig-
nificant number of unlysed tumor cells with intact nuclei shown.
Treatment with L-GGDFC12-DOX resulted in an image similar
to that with L-DOX (image not shown). Following the treatment
of L-RGDFC12-DOX (right panel), more dead tumor cells were
observed and the structure of tumor tissues was significantly
damaged.
The results of pathological examinations were consistent with
those of tumor size. Compared to L-DOX, L-RGDFC12-DOX
was shown to be more effective, suggesting that elevated concen-
trations of DOX may have been delivered to the tumor site via
RGDF moiety.
The pharmacokinetic profiles and tissue distribution of S-
DOX, L-GGDFC12-DOX and L-RGDFC12-DOX were studied.
Concentrations of DOX in plasma, tumor and heart at differ-
ent time intervals after tail vein injection of DOX formulations
were determined using HPLC. Figure 9 shows plasma concen-
trations of DOX following administration of S-DOX, L-GGDFC12-
DOX and L-RGDFC12-DOX. A pharmacokinetic software,
3P87, was used to calculate pharmacokinetic parameters as listed
in Table 2.
Figure 7. Tumor weights in mice inoculated with S180 sarcoma follow-
ing tail vein injection of two L-RGDFC12-DOX preparations, A and B
(N = 10). *Insignificant compared to 5.0% (P > 0.05). A: prepared with
phospholipids, cholesterol and RGDFO(CH2)11CH3 at a molar ratio of
14.25:4.75:0.5. B: prepared with phospholipids, cholesterol and
RGDFO(CH2)11CH3 at a molar ratio of 14.25:4.75:1.
L-RGDFC12-DOX containing two different amounts of RGDF,
one prepared with phospholipids, cholesterol and RGDFO-
(CH2)11CH3 at a molar ratio of 14.25:4.75:1 and the other
with phospholipids, cholesterol and RGDFO(CH2)11CH3 at
14.25:4.75:0.5. Both preparations were administered to mice
inoculated with S180 sarcoma, and tumor size was determined as
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Figure 9. DOX concentrations in plasma in mice inoculated with S180
sarcoma following tail vein injection of 7.5 mg/kg of DOX in S-DOX,
L-GGDFC12-DOX and L-RGDFC12-DOX (N = 5).
Figure 10. DOX concentrations in the tumor following tail vein injection
of S-DOX, L-GGDFC12-DOX and L-RGDFC12-DOX (N = 5).
Table 2. Pharmacokinetic Parameters of DOX in Plasma
Following Tail Vein Administration of 7.5 mg/kg of DOX
in S-DOX, L-GGDFC12- DOX and L-RGDFC12- DOX
Table 3. Pharmacokinetic Parameters in the Tumor
Following Tail Vein Injection of DOX, L-GGDFC12-DOX
and L-RGDFC12- DOX
L-GGDFC12- L-RGDFC12-
S-
L-GGDFC12- L-RGDFC12-
parameter
unit
S-DOX
2.7131
DOX
DOX
parameter
unit
DOX
DOX
DOX
A
μg/mL
0.3812
14.368
207.537
0.03614
37.5397
0.1989
A
μg/mL
3.0002
1.6657
4.4412
3.6514
2.0540
0.04121
3.1166
7.5412
0.9945
V/F(c) (mg/kg)/(μg/mL) 2.6046
V/F(c)
AUC
CL
(mg/kg)/(μg/mL) 5.2873
(μg/mL)h 1.6435
mg/kg/h/(μg/mL) 4.5635
AUC
CL
(μg/mL)h
38.5630
86.8256
0.08638
mg/kg/h/(μg/mL) 0.1944
after the administration of RGDF incorporated liposomes
(C(max)L-RGDFC12-DOX) was 2.4- and 1.3-fold of that of doxor-
ubicin hydrochloride solution (C(max)S-DOX) and nontargeted
peptide incorporated liposomes (C(max)L-GGDFC12-DOX), respec-
As shown in Table 2, the area under the curve (AUC) fol-
lowing L-RGDFC12-DOX was 2.25-fold of that following S-
DOX while the clearance rate (CL) of L-RGDFC12-DOX was
44% of that of S-DOX. The results indicated that L-RGDFC12-
DOX is associated with a reduced plasma clearance or increased
circulation time of DOX in comparison to S-DOX. This observa-
tion is consistent with the results observed in the in vitro release
study (Figure 4), which showed a gradual release of DOX from
L-RGDFC12-DOX.
To evaluate the targeting capability of RGDF peptide, a
tetrapeptide with a sequence of GGRF was designed as a non-
targeting moiety or control. By comparison, it was observed that
AUCL-RGDFC12-DOX was about 42% of AUCL-GGDFC12-DOX while
tively. The clearance rate of L-RGDFC12-DOX (CLL-RGDFC12-DOX
)
in tumor tissues was 22% and 48% of that of doxorubicin hydro-
chloride solution (CLS-DOX) and nontargeted peptide incorpo-
rated liposomes (CLL-GGDFC12-DOX). The results indicated that
the RGDF incorporated liposomal DOX (L-RGDFC12-DOX)
resulted in elevated concentrations of DOX in the tumor
and reduced clearance of DOX from the tumor in comparison
to both doxorubicin hydrochloride solution (S-DOX) and non-
targeted peptide incorporated liposomes (L-GGDFC12-DOX).
The higher concentration and longer residence time in the tumor
are likely responsible for the improved antitumor activities
observed as shown in Figures 5 and 8.
A very important mechanism of doxorubicin cardiotoxicity
involves the formation of cytotoxic free radicals such as super-
oxide radical anion and hydroxyl radical, both of which are
formed via reduction of the anthracyclinone quinine to hydro-
quinone by NADPH/CYP450 reductase. The production of
hydroxyl radicals inside tumor cell might augment the antineo-
plastic effect of the anthracyclines, but such formation is un-
common at standard antineoplastic doses.4 Cytotoxic radicals
generated in the heart are believed to be responsible for acute and
often severe cardiotoxicity. Cardiac tissue is vulnerable to the
attack of free radicals because it lacks the catalase enzyme,4,6
which converts hydrogen peroxide to harmless water and oxygen.
The hydrogen peroxide generated in the myocardium will
eventually become highly reactive hydroxyl radicals. In addition,
the active secondary C13- alcohol metabolites of anthracycline
the plasma clearance of L-RGDFC12-DOX (CLL-RGDFC12-DOX
)
was 2.39-fold of that of L-GGDFC12-DOX (CLL-GGDFC12-DOX),
suggesting that nontargeted peptide incorporated liposomes
have a longer circulation time in plasma than RGDF incorporated
liposomes (L-RGDFC12-DOX). The RGDFꢀfatty alcohol con-
jugate anchored on the surface of liposomes is likely responsible
for the reduced circulation time as it may have rapidly directed
liposomal DOX to tumor sites and consequently its residence
time in plasma was reduced.
Figure 10 shows the concentrations of DOX found in the
tumor after tail vein injection of three formulations. The calcu-
lated pharmacokinetic parameters of three formulations in tumor
tissues are shown in Table 3, which demonstrated that the AUC
following RGDF incorporated liposomes (AUCL-RGDFC12-DOX
)
was 4.59- and 2.25-fold of that of nontargeted peptide
(GGDF) incorporated liposomes (AUCL-GGDFC12-DOX) and
doxorubicin hydrochloride solution (AUCS-DOX), respectively.
The maximum concentration of DOX in the tumor observed
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ARTICLE
and L-GGDFC12-DOX. The results indicated that liposomal
formulations of DOX significantly reduced DOX accumulation
in the heart in comparison to free DOX solution. Compared to
GGDF incorporated liposomes, RGDF incorporated liposomes
(L-RDGFC12-DOX) diverted more DOX to tumor tissues and
reduced accumulation of DOX in the heart.
The targeting potential of L-RGDFC12-DOX in comparison
to S-DOX and L-GGDFC12-DOX was determined according to
the method proposed by Hunt and his research group,30 and
targeting parameters including TA and DTI were calculated. The
results are summarized in Table 5
The TA values of L-RGDFC12-DOX in comparison to
S-DOX and L-GGDFC12-DOX are 4.59 and 2.07, respectively,
indicating the targeting capacity of RGDF for tumor site. The
DTI values of L-RGDFC12-DOX in comparison to S-DOX and
L-GGDFC12-DOX are 2.43 and 1.71, respectively, again indi-
cating the targeting capacity of RGDF.
Figure 11. DOX concentrations in the heart following tail vein injection
of S-DOX, L-GGDFC12-DOX and L-RGDFC12-DOX (N = 5).
Liposomal DOX formulations have been studied by several
research groups.31,32 In most studies, the dosage of DOX needed
to exhibit significant inhibition of tumor growth was 5 mg/kg. In
this study, a dose of 2 mg/kg of DOX in RGDF incorporated
liposomal preparations was shown to have a significant effect on
tumor growth, suggesting that RGDF improved the efficacy of
liposomal DOX, which is likely a result of more DOX molecules
delivered to tumor sites when RGDF was incorporated in the
liposomal preparation. The results supported the hypothesis that
RGDF, as a tumor specific peptide, can direct the delivery of
liposomal DOX to tumors.
Table 4. Pharmacokinetic Parameters of DOX in the Heart
Following Tail Vein Administration of 7.5 mg/kg of DOX
in S-DOX, L-GGDFC12- DOX and L-RGDFC12- DOX
L-GGDFC12- L-RGDFC12-
parameter
unit
S-DOX
4.993
DOX
DOX
A
μg/mL
2.0137
3.7244
60.7558
0.1488
2.3632
3.1737
50.4140
0.1234
V/F(c) (mg/kg)/(μg/mL) 1.5020
AUC
CL
(μg/mL)h
32.1275
In conclusion, RGDF modified DOX liposomal preparation
achieved much improved delivery to tumors while levels of DOX
to the heart were significantly reduced in comparison to con-
ventional liposomal preparations.
mg/kg/h/(μg/mL) 0.2334
Table 5. Targeting Parameters of L-RGDFC12-DOX in Mice
Inoculated with S180 Sarcoma
TA
DTI
’ ASSOCIATED CONTENT
compared with S-DOX
4.59
2.07
2.43
1.71
S
Supporting Information. Detailed chemistry data of the
b
compared with L-GGDFC12-DOX
four peptide fatty alcohol conjugates. This material is available
free of charge via the Internet at http://pubs.acs.org.
anticancer agents can also lead to chronic cardiomyopathy. For
example, C13-hydroxyl doxorubicin (doxirubicinol) formed in-
duces a prolonged inhibition of calcium loading, opens selective
ion channels leading to increased cytosolic levels of Ca2þ in the
sarcoplasmic reticulum, and inhibits Naþ/Kþ-ATPase action in
the sarcolemma. These important cellular events can induce a
chronic cardiomyopathy that presents a severe congestive heart
failure.5 In the current study RGDF was designed to direct
more DOX in liposomes to tumor cells and reduce the DOX
accumulation in the heart. Figure 11 shows the concentrations of
DOX found in the heart after tail vein injection of RGDF
incorporated liposomal DOX in comparison to DOX solu-
tion and GGDF incorporated liposomal DOX. The calculated
pharmacokinetic parameters of the three formulations in the
heart are shown in Table 4, and the concentrations of DOX in
the heart are represented in Figure 11. DOX from S-DOX
reached its peak concentration within minutes, and the peak
concentration was much higher than that after liposomal for-
mulations (L-GGDFC12-DOX and L-RGDFC12-DOX). The
liposomal preparations reached their peak concentrations in
about 4 h after tail vein administration. It was also found that
the cardiac concentration of DOX following the administration
of L-RGDFC12-DOX was the lowest in comparison to S-DOX
’ AUTHOR INFORMATION
Corresponding Author
*H.L.: Memorial University of Newfoundland, School of Phar-
macy, 300 Prince Philip Drive, St. John's, Newfoundland, Canada
A1B 3V6; tel, þ1 709 777 6382; fax, þ1 709 777 7044; e-mail,
hliu@mun.ca. G.C.: tel, þ86 010 83911538; fax, þ86 10 8391
1533; e-mail, cuiguohui@bjmu.edu.cn or cgh@ccmu.edu.cn.
G.C.: School of Chemical Biology and Pharmaceutical Sciences,
Capital Medical University, Beijing, China. 100069.
Author Contributions
‡Authors who contributed equally to the study.
’ ACKNOWLEDGMENT
This research was supported by the National Natural Science
Foundation of China (30873202). The authors are also grateful
to the Key Laboratory on Peptides and Small Drug Molecules
and the Modern Drug Analysis Laboratory at the Capital Medical
University for their generous help.
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ARTICLE
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