RGD-fatty alcohol-modified docetaxel liposomes improve tumor selectivity in vivo

Article abstract of DOI:10.1016/j.ijpharm.2014.04.001

The tripeptide arginine-glycine-aspartate (RGD) was conjugated with various fatty alcohols to obtain RGDOC_nH_{2n + 1} (n = 8, 10, 12, 14, 16, 18), which were incorporated into the bilayer of docetaxel liposomes to improve their tumor sp

Full text of DOI:10.1016/j.ijpharm.2014.04.001

International Journal of Pharmaceutics 468 (2014) 133–141
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
RGD-fatty alcohol-modified docetaxel liposomes improve tumor
selectivity in vivo
1
1
*
Yinghuan Li , Xuelian Zheng , Yi Sun, Zhao Ren, Xuemei Li, Guohui Cui
School of Chemical Biology and Pharmaceutical Science, Capital Medical University, Beijing 100069, China
A R T I C L E I N F O
A B S T R A C T
Article history:
The tripeptide arginine-glycine-aspartate (RGD) was conjugated with various fatty alcohols to obtain
RGDOCnH2 (n = 8, 10, 12, 14, 16, 18), which were incorporated into the bilayer of docetaxel liposomes
Received 26 December 2013
Received in revised form 11 March 2014
Accepted 2 April 2014
n + 1
to improve their tumor specificity. The fatty alcohols were accepted as linking groups to insert the
tetrapeptide RGDX (X = amino acid) into the bilayer of liposomes. RGDX was previously shown to be a
potent ligand to target tumor cell-surface integrin receptors, whereas RGD was not shown to have this
ability. We hypothesized that RGD-fatty alcohol conjugates lacking the fourth amine X can guide
liposomes to tumors without reducing their binding affinity to integrins. Antitumor activity,
pharmacokinetics and biodistribution studies were evaluated in mice inoculated with S180 sarcoma.
Compared with unmodified liposomes, RGD-fatty alcohol-modified liposomes successfully delivered
significantly more docetaxel to tumors, which led to significant tumor weight loss and increased tumor
docetaxel concentrations accompanied by reduced liver accumulation. Improved affinity of RGD-fatty
alcohols to integrins was also confirmed on A375 cell model. Further comparisons among the tumor-
targeting capacities of liposomes containing RGD-fatty alcohols, RGDF-fatty alcohols and RGDV-fatty
acids demonstrated that RGD-fatty alcohols were as effective as the other two tetrapeptide derivatives.
Therefore, a simplified tumor-targeting delivery system using RGD-fatty alcohols was developed.
ã 2014 Elsevier B.V. All rights reserved.
Available online 04 April 2014
Keywords:
RGD
Fatty alcohol
Docetaxel liposomes
Tumor targeting
Pharmacokinetics
In vivo
1. Introduction
a specific conformation for binding to different sub-types of
integrins (Ruoslahti and Pierschbacher, 1987). The hydrophobicity
The tripeptide arginine-glycine-aspartate (Arg-Gly-Asp, RGD)
has been widely studied as an adhesive peptide. RGD is highly
hydrophilic, easily degraded by enzymes and has a short half-life in
vivo. RGD has been conjugated to various amino acids such as
cysteine, glutamine, serine, phenylalanine, valine, tyrosine, leucine
and tryptophan to form RGDX (Aguzzi et al., 2010; Du et al., 2011;
Kim et al., 2011; Park et al., 2002; Pozzetto et al., 2005; Wang et al.,
2012). The RGDX tetrapeptides are usually considered to be potent
ligands for adhering to cell-surface integrin receptors, which are
over-expressed on various tumor cell surfaces (Barczyk et al., 2010;
Bellis, 2011). Integrin receptors recognize RGD as a primary
binding sequence, and the fourth amino acid X modulates the
binding capacity for different sub-types of integrins through two
mechanisms. The fourth amino acid X is thought to force RGD into
of X was found to affect the affinity of RGDX to integrins: increased
hydrophobicity resulted in increased affinity (Spurio et al., 2004).
Both mechanisms suggest that the fourth amino acid in RGDX is
important for binding to integrins. A study on the adhesion of RGD,
RGD-serine (RGDS), RGD-valine (RGDV) and RGD-threonine
(RGDT) to Chinese hamster ovary cells demonstrated that RGD
had the lowest adhesion (Ventura et al., 2012), which suggested
that the addition of a hydrophobic amino acid to the RGD sequence
increased the binding affinity of RGD for integrins, likely by
affecting the conformation or directly changing binding capacity.
Fatty alcohols/acids/amines may be further conjugated to RGDX
(RGDX-fatty alcohols/acids/amines) to form a bridge between
RGDX and the bilayer of lipids. Thus, the resultant conjugates,
RGDX-fatty alcohols/acids/amines, with both hydrophilic and
hydrophobic characteristics, can be easily incorporated into
formulations such as liposomes. Given the affinity of RGDX for
integrins overexpressed on the surface of tumor cells, these
peptides can improve the tumor selectivity of formulations.
RGD was thought to have limited binding affinity to integrins
(Chen et al., 2012; Park et al., 2002). This report examined whether
* Corresponding author at: Capital Medical University, School of Chemical
Biology and Pharmaceutical Science, 10 Xitoutiao, You Anmen, Fengtai District,
Beijing 100069, China. Tel.: +86 10 83911538; fax: +86 10 83911533.
E-mail address: cgh@ccmu.edu.cn (G. Cui).
1
Authors who contributed equally to the study.
http://dx.doi.org/10.1016/j.ijpharm.2014.04.001
0378-5173/ã 2014 Elsevier B.V. All rights reserved.

134
Y. Li et al. / International Journal of Pharmaceutics 468 (2014) 133–141
the lipophilic fatty alcohol linked to RGD (RGD-fatty alcohol)
would function in the same manner as RGDX-fatty alcohols to
effectively target liposomes to tumors. RGD-fatty alcohols were
designed and incorporated into the bilayer of liposomes, which
were examined for their tumor-targeting capacity. A series of
mice, weighing 18–22 g, were purchased from Animal Services,
Capital Medical University.
2.2. Synthesis of RGD-fatty alcohols (RGDOCnH2
n + 1
)
CnH2
OH with different chain lengths (n = 8–18) were investi-
RGDOCnH2
(n = 8, 10, 12, 14, 16, 18) were synthesized as
described in Fig.1, which was simplified from the reported methods
n + 1
n + 1
gated in this work. Glycine-glycine-aspartate (GGD) and fatty
alcohol conjugates were also synthesized by changing R to G in
RGD-fatty alcohols to parallel examine the influence of amino acid
sequence on the tumor-targeting capacity. Docetaxel, a clinically
well-established anti-mitotic chemotherapy drug, was used as a
model drug (Lefrancois et al., 2011). The in vivo anticancer activity,
pharmacokinetics, and biodistribution of docetaxel liposomes
containing various RGD-fatty alcohols were determined.
for the synthesis of tetrapeptide RGDXOCnH2
Lin et al., 2002; Wang et al., 2012). Briefly, N-
(Du et al., 2011;
n + 1
a-tert-butyloxycar-
bonyl-N- -nitro-L-arginine [Boc-Arg(NO2)-OH] and tosylated gly-
g
cine benzyl ester (Tos-Gly-OBzl) were first dissolved in DMF and
reacted in the presence of DCC, HOBt and NMM to yield the double-
protected dipeptide, i.e., Boc-Arg(NO2)-Gly-OBzl. After the selective
removal of the protectivegroupat the C-terminus in the presence of
2N NaOH in an ice bath, Boc-Arg(NO2)-Gly-OH was obtained. A
2. Materials and methods
series of fatty alcohols, CnH2 OH (n = 8, 10, 12, 14, 16, 18), were
n + 1
connected to the N-terminally protected benzyl aspartate [Boc-Asp
(OBzl)-OH] in THF in the presence of DCC, HOBt and NMM to yield
2.1. Materials
Boc-Asp(OBzl)-OCnH2
H-Asp(OBzl)-OCnH2
. After deprotecting the N-terminus,
were obtained and reacted with previous-
n + 1
Docetaxel and paclitaxel were purchased from Beijing Xinze
n + 1
Technology Co., Ltd. (Beijing, China). Fatty alcohols CnH2
OH
ly synthesized Boc-Arg(NO2)-Gly-OH to yield N-
ycarbonyl-N- -nitro-arginyl-glycyl-benzylaspartyl fatty alcohol
esters, i.e., Boc-Arg(NO2)-Gly-Asp(OBzl)-OCnH2 . Nitro, benzyl
and N-tert-butyloxycarbonyl (Boc) protecting groups were then
removed via a two-step process to yield the final products,
a-tert-butylox-
n + 1
(n = 8, 10, 12, 14, 16, 18; i.e., 1-octanol, 1-decanol, 1-dodecanol, 1-
tetradecanol, 1-hexadecanol and 1-octadecanol) were purchased
from Chaoyang Xudong Chemicals Inc. (Beijing, China). All amino
acids and their protected forms were L isomers and were obtained
from GL Biochem Ltd. (Shanghai, China). Dicyclohexylcarbodiimide
(DCC), 1-hydroxybenzotriazole (HOBt), and N-methylmorpholine
(NMM) were alsofromGL BiochemLtd. Dimethylformamide (DMF),
tetrahydrofuran (THF), NaOH, ethyl acetate (EA), chloroform and all
the ingredients needed for phosphate buffered saline (PBS) were
purchased from Beijing Chemical Works (Beijing, China). Acetoni-
trile was from Fisher Chemicals. Egg lecithin (MW = 750 Daltons)
was purchased from Acros Organics (Geel, Belgium). Cholesterol
was from Bjkebio Ltd. (Beijing, China). 1,2-Dioleoyl-sn-glycero-3-
phospho-ethanolamine-N-(lissamine rhodamine B sulfonyl) am-
monium salt (Rhod-DOPE) and 1,2-dioleoyl-3-trimethylammo-
niumpropane (DOTAP) were from Avanti Polar Lipids, Inc. 40,6-
diamidino-2-phenylindole, dihydrochloride (DAPI) was from
Roche. Tert-butyl methyl ether was from Tianjing Fucheng
Chemicals (Tianjing, China). CMC-Na, Tween 80 and Pd/C were
obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing,
China). All reagents and solvents were of analytical or HPLC grade.
All materials were used as received.
g
n + 1
RGDOCnH2
. Using the same method, GGDOC14H29 was synthe-
n+ 1
sized as a control and is depicted in Fig. 1.
To examine the amphipathy of RGD-fatty alcohols, the morphol-
ogy of the self-assembled particles synthesized in deionized water
was observed under a transmission electron microscope (TEM, JEM-
1230, JEOL, Japan).
2.3. Preparation of docetaxel liposomes containing RGD-fatty alcohols
[Lip-RGD(Cn)]
Docetaxel liposomes containing the RGD-fatty alcohols synthe-
sized above were prepared using the standard thin-film hydration
method (Li et al., 2012). In brief, docetaxel, lecithin and
RGDOCnH2
(n = 8, 10, 12, 14, 16, 18) (1:18:1, molar ratio) were
n + 1
dissolved in chloroform and transferred into a suitable round
bottom flask. The solvent was removed by rotary evaporation, and
samples were dried overnight under vacuum. The thin film was
rehydratedwithPBS(pH7.4)andthensonicatedfor20 mintoobtain
an opaque liposomal preparation (1 mg/mL docetaxel) of Lip-RGD
(Cn) (n = 8, 10, 12, 14, 16, 18) (Table 1). Conventional docetaxel
liposomes without the RGD motif (Lip-Con) were also prepared. In
Animal experiments were conducted in accordance with the
protocol approved by the Experimental Animal Care Committee of
Capital Medical University (Beijing, China). Male Kunming and ICR
Fig. 1. Synthesis of RGDOCnH2
(n = 8, 10, 12, 14, 16, 18) and GGDOC14H29. (I) DCC, HOBt and NMM; (II) 2N NaOH (ice bath); (III) 4N HCl-EA; (IV) DCC and HOBt; (V) 5% Pd/C
n + 1
and H2 (0.02 MBa).

Y. Li et al. / International Journal of Pharmaceutics 468 (2014) 133–141
135
Table 1
sarcoma growing in the abdomen (Vital River Laboratory Animal
Technology Co., Ltd. Beijing, China) were sacrificed on the eighth
day after tumor implantation, and ascites containing tumor cells
were collected. After centrifugation, tumor cells were resuspended
in saline at 2.0 ꢀ107/mL, and 0.2 mL was injected subcutaneously
(s.c.) into the right armpit of each ICR mouse (day 0).
Properties of RGD-linked liposomes and various control liposomes.
Formulations Composition Molar ratio Size
Zeta potential
(mV, X ꢃ SD)
of targeting
motif
of targeting (nm, X ꢃ SD)
motif (%)
Lip-RGD(C8)
Lip-RGD(C10)
Lip-RGD(C12)
Lip-RGD(C14)
Lip-RGD(C16)
Lip-RGD(C18)
Lip-GGD(C14)
Lip-(half)RGD
(C14)
RGDOC8H17
RGDOC10H21
RGDOC12H25
RGDOC14H29
RGDOC16H33
RGDOC18H37
GGDOC14H29
5
5
5
5
5
5
5
227.3 ꢃ 3.1
262.7 ꢃ2.5
273.7 ꢃ3.1
241.0 ꢃ1.0
284.2 ꢃ1.9
328.7 ꢃ 8.5
242.0 ꢃ 5.0
263.3 ꢃ 3.1
ꢂ54.8 ꢃ 2.6
ꢂ54.8 ꢃ 1.9
ꢂ57.2 ꢃ 2.6
ꢂ60.5 ꢃ 2.7
ꢂ48.4 ꢃ7.3
ꢂ52.4 ꢃ 3.5
ꢂ55.8 ꢃ 5.0
ꢂ44.1 ꢃ3.7
2.6. In vivo anti-tumor activity of Lip-RGD(Cn)
On day 1 (24 h after inoculation), mice were randomly divided
into 14 groups (n = 10). Mice were injected intravenously (i.v.) via
the tail vein with various preparations as described in Table 2 on
days 1, 3, 5 and 7. Mice administered with saline (group A) and E-
lip-RGD(C8) (group B) were used as negative controls. Mice
administered with a docetaxel suspension (group C, docetaxel
suspended in 0.5% CMC-Na solution with the aid of Tween 80 as the
wetting agent), Lip-Con (group D) and Lip-GGD(C14) (group K)
were used as positive controls. In addition, the anti-tumor activity
of Lip-RGD(C14) containing three different doses of docetaxel was
determined. On day 9, mice were sacrificed, and tumors were
dissected and weighed. The anti-tumor activity was determined
with the tumor growth inhibition (TGI%), which was calculated
with the formula: (1 ꢂ tumor weight of treated group/tumor
weight of negative control group B) ꢀ 100% (Wang et al., 2012).
RGDOC14H29 2.5
Lip-Con
E-lip-RGD(C8) RGDOC8H17
–
0
5
252.8 ꢃ 2.6
239.0 ꢃ1.0
ꢂ66.6 ꢃ 7.4
ꢂ49.8 ꢃ 1.5
addition, docetaxel liposomes containing GGDOC14H29 [Lip-GGD
(C14)] were prepared using docetaxel, lecithin and GGDOC14H29
(1:18:1, molar ratio) and the same protocol. Lip-(half)RGD(C14) was
a liposomal preparation containing 1:18.5:0.5 docetaxel, lecithin
and RGDOC14H29
RGDOCnH2 (n = 8, 10, 12, 14, 16, 18) (18:1, molar ratio) were
calledasE-lip-RGD(Cn)(n = 8,10,12,14,16,18)andusedasanegative
control. Empty liposomes composed of 100% lecithin were called as
E-lip. Liposomal preparations were purified by size exclusion
chromatography on a Sephadex G50 column equilibrated in PBS to
removefreedrugs. The concentration of docetaxel in liposomes was
analyzed by reverse phase HPLC (Agilent Technologies 1200 Series)
with UV detectionat 226 nm, a flowrate of 1 mL/min on a symmetry
. Empty liposomes containing lecithin and
n + 1
2.7. Pharmacokinetic and biodistribution studies of Lip-RGD(Cn)
On day 8 following inoculation with S180 tumor cells as
described in Section 2.5, mice received i.v. tail vein injections of
docetaxel (10 mg/kg body weight) in suspension, Lip-RGD(C14)
and Lip-GGD(C14) (Li et al., 2012; van Tellingen et al., 1999). Blood
samples were collected in heparin-treated tubes at various time
intervals (0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24 h), and five mice were
used for each time interval. The plasma was isolated by
centrifugation (1000 ꢀ g, 10 min) and stored at ꢂ20 ꢁC. Mice were
sacrificed, and tumors and livers were collected, washed and
weighed.
C18 column (4.6 mm ꢀ 150 mm, 5
mm particle size), and an isocratic
program in an acetonitrile:water (42:58, v/v) solvent system.
The particle size and zeta potential were determined on a
ZetaPALS instrument (Brookhaven Instruments Corp., Worcester-
shire, NY). The insertion of RGD-fatty alcohols in liposomes was
assessed by differential scanning calorimetry (DSC, Netzsch,
Germany).
2.4. The binding of RGD-fatty alcohols to integrins on A375 tumor cell
To measure the docetaxel concentration in the plasma, 10
mL of
paclitaxel (10 g/mL in acetonitrile) was added to 0.2 mL of plasma
m
Empty liposomes containing RGDOC12H25, DOTAP, lecithin,
Rhod-DOPE and cholesterol (1:40:30:1:28, molar ratio) were
prepared using the same method in Section 2.3 and named as E’-
lip-RGD(C12). Empty liposomes containing DOTAP, lecithin, Rhod-
DOPE and cholesterol (40:30:1:29, molar ratio) were also prepared
for comparison and named as E’-lip. Rhod-DOPE (red fluorescence)
was added to enable detection. DOTAP as a cationic lipid was added
to magnify fluorescence intensity signals for observation, based on
the knowledge that positive surface charge enhances liposomes
uptake into cells. We compared the binding and uptake difference
of E’-lip-RGD(C12) and E’-lip on human melanoma A375 cells.
A375 cells are known as over-expressing avb3 integrin receptors.
A375 cells were seeded on glass bottom micro-well dishes (Mat
Tek Corporation) overnight to allow the formation of a monolayer
and then incubated with different formulations at a final medium
as an internal standard (Zhang et al., 2012). The mixture was
vortexed for 30 s after the addition of 2 mL of tert-butyl methyl
ether and then centrifuged at 1000 ꢀ g for 10 min. A volume of
1.8 mL of supernatant was transferred to a centrifuge tube and
dried under vacuum. The residue was dissolved in 0.2 mL of mobile
phase and then analyzed by HPLC on a symmetry C18 column
Table 2
Experimental design of anti-tumor activity in S180 mice.
Group no.
Formulations (i.v.)
Dose of docetaxel
(mg/kg body weight)
A
B
C
D
E
Saline
0a
0b
5
5
5
5
5
5
3.5
2
E-lip-RGD(C8)
Docetaxel Suspension
Lip-Con
concentration of 15 m
g/mL lipids at 37 ꢁC and 4 ꢁC for 0.5, 2 and 4 h,
washed three times with ice-cold PBS, and fixed in 10% formalin for
20 min. After staining the cell nuclei with DAPI (100 ng/mL in PBS)
for 10 min, cells were covered with glycerol (90%) and examined
under a confocal microscope (LEICA TCS SP5, Germany). The
excitation/emission wavelengths were 405/458 and 561/633 nm
for DAPI (blue) and rhodamine (red), respectively.
Lip-RGD(C8)
Lip-RGD(C10)
Lip-RGD(C12)
Lip-RGD(C14)
Lip-RGD(C14)
Lip-RGD(C14)
Lip-(half)RGD(C14)
Lip-RGD(C16)
Lip-RGD(C18)
Lip-GGD(C14)
F
G
H1
H2
H3
H4
I
5
5
5
5
J
K
2.5. Animals and tumors models
a
Male ICR mice (18–22 g) were inoculated with S180 sarcoma
according to institutional guidelines. Briefly, mice with S180
The dose of saline is 0.2 mL per mouse.
The dose of lipids is equivalent to that of group E.
b

136
Y. Li et al. / International Journal of Pharmaceutics 468 (2014) 133–141
(150 mm ꢀ 4.6 mm, 5
m
m). The mobile phase was acetonitrile:
Targeting index (TI) is defined as:
water (42:58, v/v) with a flow rate of 1 mL/min, and the injection
i
ðAUC0ꢂ1ÞTDDS
volume was 100 mL (Immordino et al., 2003; Zhao et al., 2009). The
detection wavelength was 226 nm.
Tumors and livers collected above were homogenized with
internal standard paclitaxel (10 mL, 10 mg/mL in acetonitrile) and
saline-acetonitrile solution (50:50, v/v,1 mL per 0.6 g of tissue) and
then centrifuged at 1000 ꢀ g for 10 min. The supernatant was
removed, and the residue was redissolved and re-pelleted. The
combined supernatants were dried under vacuum. The residue was
dissolved in 0.5 mL of mobile phase and 2 mL tert-butyl methyl
ether. After centrifugation, two layers were formed, and the
organic phase (1.8 mL) was collected, dried and re-dissolved in
0.2 mL of mobile phase for analysis in an HPLC system as described
above.
TI ¼
i
ðAUC0ꢂ1ÞCDDS
Relative targeting index (RTI) is defined as:
ðAUC0ꢂ1ÞtTuDmDoSr=ðAUC0ꢂ1ÞTDDS
liver
RTI ¼
liver
ðAUC0ꢂ1ÞtCuDmDoSr=ðAUC0ꢂ1ÞCDDS
where i represents the specific tissue, e.g., blood, liver or tumor.
TDDS indicates the targeted drug delivery system, i.e., Lip-RGD
(C14), and CDDS indicates the conventional drug delivery system,
i.e., docetaxel suspension or Lip-GGD(C14). The higher the values
for these parameters (except TI for blood and liver), the better the
tumor targeting ability of the formulation.
The ratio of the measured peak areas of docetaxel to paclitaxel
(As/Ai) was converted to the amount of docetaxel in the plasma (or
tissue) using standard curves constructed with known amounts of
docetaxel and an equal amount of paclitaxel added in the blank
plasma (or tissue). Docetaxel concentrations were plotted vs. time.
2.10. Statistical analysis
Statistical analysis was performed with Student’s t-test, and
statistical significance was defined as p < 0.05 or p < 0.01. Data
reported are the means ꢃ standard deviation (X ꢃ SD).
2.8. Fitting of pharmacokinetic parameters
3. Results
The fitting of a concentration–time (c–t) curve was performed
using a nonlinear least square (NLS) model program developed by
Dr. Jia-bi Zhu and based on the Gauss–Newton method (Li and Zhu,
2004). The minimum deviation sum of square (SS) and the
minimum Akaike’s Information Criterion (AIC) were used for the
program’s model fitting. For docetaxel c–t curves in the plasma
after i.v. injections, the equations c ¼ c0 ꢀ eꢂkt (one-compartment
3.1. Synthesis and characterization of RGD-fatty alcohols
RGDOCnH2
(n = 8, 10, 12, 14, 16, 18), and GGDOC14H29 were
n + 1
synthesized and purified via silica gel columns. The products were
characterized by 1H and 13C nuclear magnetic resonance (NMR),
mass spectrometry (MS), melting point (mp) analysis and optical
a
t
b
model) and c ¼ Aeꢂ þ Beꢂ t(two-compartment model) were
compared in the NLS model. For the amount of docetaxel in
tissues, formula zero and formula first were selected in the model
as follows:
rotation,
a. Two key signal peaks in MS demonstrated the stable
ester linkage of RGD-fatty alcohols, for example, 459.5 [M + H]+ and
917.6 [2M + H]+ of RGDOC8H17. The peaks of RGD (379[M + H]+, 757
[2M + H]+) and C8H17OH (131[M + H]+, 261[2M + H]+) were not
found in MS. The 1HNMR data showed the newly generated peptide
bond of RGDOC8H17 deducted one hydrogen atom (H) from two
carboxyl hydroxyls (ꢂꢂCOOH) with one single peak at 8.95 (s, 1H).
Furthermore, the 1HNMR signals of two methylenes (ꢂꢂCH2ꢂꢂ)
following the hydroxyl (ꢂꢂOH) in C8H17OH were moved from 3.53
to 4.08 and from 1.48 to 1.55 since the influence of newly formed
peptide bond was stronger than that of hydroxyl group. The
Formula zero:
1t
c ¼ Að1 ꢂ eꢂk Þ . . . t ꢄ T
ꢀ
ꢁ
1T
c ¼ A 1 ꢂ eꢂk
ꢀ eꢂk ðtꢂTÞ . . . t > T
2
Formula first:
ꢀ
ꢁ
13CNMR data clearly showed four carbonyl (
172.04, 171.86, 171.14, 170.88, 170.34) and one amide (
without “H” in RGDOC8H17, and the multiplet strong peaks (
d
= 172.63, 172.39,
= 157.59)
= 14–
1t
2t
c ¼ A eꢂk ꢂ eꢂk
d
where T is the duration for zero-order absorption, and A is a
coefficient. In formula zero, k1 is the absorption rate constant in the
ascending part, and k2 is the elimination rate constant in the
declining region after T of the c–t curve. In formula first, k1 is the
elimination rate constant, and k2 is the absorption rate constant.
The area under the curve (AUC) is included in the program
based on the calculation of the logarithmic trapezoidal method, i.e.,
d
65) of carbon atom (C) in “ꢂꢂC8H17” group. The purity of
compounds was greater than 90% as determined by HPLC. Detailed
data for the 7 conjugates can be found in the Supplementary data.
RGDOC14H29 in deionized water (0.5 mg/mL) was able to self-
assemble into particles of 270.5 nm in diameter and with a ZP of
ꢂ73.9 mV. The morphology of the particles observed by TEM is
shown in Fig. 2, demonstrating that RGDOC14H29 peptide
amphiphiles (PAs) assembled in water into helical structures that
appear as coiled fiber bundles. This structural arrangement may be
due to hydrogen-bonding based on the fundamental principles of
supramolecular chemistry (Kang and Jhon, 2000; Lehn, 1993). This
result is similar to a report that PAs assemble in water into
cylindrical or quadruple helical fibers with a braided morphology
(Palmer and Stupp, 2008).
n
X
ðCi ꢂ Ciꢂ1Þðti ꢂ tiꢂ1
lnCi ꢂ lnCiꢂ1
Þ
Cn
ktrm
AUC0ꢂ1
¼
þ
,
i¼1
where n is the group number of experimental data, and ktrm is the
disposition rate constant of the terminal linear phase.
2.9. Tumor-targeting capacity evaluation
The targeting ability of the various formulations was defined by
the following parameters (Du et al., 2011).
Two targeting efficiency (TE) parameters, TE1 and TE2, are
defined as follows:
3.2. Characterization of docetaxel liposomes with RGD-fatty alcohols
incorporated
The efficiency of docetaxel incorporation into RGD-fatty
alcohol-containing liposomes was determined by HPLC analysis
on a Sephadex G50 column to be greater than 95%. Table 1 shows
ðAUC0ꢂ1Þ
ðAUC0ꢂ1Þtumor
ðAUC0ꢂ1Þliver
TE1 ¼
tumor ; TE2 ¼
ðAUC0ꢂ1Þplasma

Y. Li et al. / International Journal of Pharmaceutics 468 (2014) 133–141
137
Fig. 2. TEM of RGDOC14H29 self-assembly morphology in deionized water (0.5 mg/mL).
the particle size and zeta potential of various docetaxel liposomes
with RGD-fatty alcohols incorporated. All liposomes showed a
narrow size distribution with mean particle sizes of 220–330 nm
and zeta potentials less than ꢂ40 mV, which were contributed
from the RGD/GGD-motif and phospholipids in the liposomes
(Warr, 2011).
ꢅ100 ꢁC (
D
H = 88 J/g) and ꢅ205 ꢁC (
D
H = 57 J/g) (curve 4). The
physical mixture of RGDOC12H25 and empty liposomes were
prepared by mixing RGDOC12H25 and lyophilized E-lip at 1:18
(molar ratio). The corresponding endothermic peaks of the
physical mixture were ꢅ105 ꢁC
(
D
H = 29 J/g) and ꢅ230 ꢁC
(DH = 15 J/g) (curve 2). The physical mixture produced a more flat
DSC thermograms are broadly used to clarify the interaction of
molecules (Budai et al., 2003). In Fig. 3, the pre-transition and main
phase transition temperature of empty liposomes (E-lip) were
DSC curve with decreased altitudes and enthalpy of endothermic
peaks than E-lip or RGDOC12H25. E-lip-RGD(C12) only showed one
DH = 67 J/g) (curve 3), which
represented the main phase transition of liposomes with increased
enthalpy. The disappearance of the RGDOC12H25 endothermic peak
endothermic peak at ꢅ225 ꢁC (
ꢅ95 ꢁC (
D
H = 30 J/g) and ꢅ235 ꢁC (
D
H = 25 J/g), respectively (curve
1), whereas RGDOC12H25 showed two endothermic peaks at
Fig. 3. DSC thermograms of blank liposomes containing RGDOC12H25. (1) Denotes blank liposomes; (2) represents the physical mixture of RGDOC12H25 and blank liposomes;
(3) denotes RGDOC12H25-linked blank liposomes; (4) represents RGDOC12H25. (#) Points to the endothermic peaks.

138
Y. Li et al. / International Journal of Pharmaceutics 468 (2014) 133–141
suggested that RGD-fatty alcohols are effectively inserted into the
bilayer of liposomes.
3.3. Improved affinity of RGD-fatty alcohols to integrins on A375 cells
The confocal microscopy images of E’-lip-RGD(C12) and E’-lip
demonstrate the red fluorescence signals (rhodamine-labeled
liposomes) were observed both on the cell membrane and inside
the cell at 37 ꢁC and only on the cell membrane at 4 ꢁC (Fig. 4). The
published method from literature has confirmed that the red
fluorescence intensities (FI) in cells at 37 ꢁC represented the total
cell-associated liposomes (TC) and the red FI in cells at 4 ꢁC
represented cell membrane-bound liposomes (B) (Li et al., 2011).
The red FI in quantitative cells were measured by NIH ImageJ
software (Macintosh, USA) and calculated as FI/cell. The results of
TC and B time course studies showed RGD-fatty alcohol facilitated
more liposomes binding to cell membrane and uptake into the cell,
compared to unmodified liposomes.
Fig. 5. Tumor weights (X ꢃ SD) and TGI% in groups A–K (Table 2) after the
administration of different formulations to mice bearing S180 sarcoma tumors
(n = 10). * p < 0.05, # p < 0.01: vs. group D (i.e., Lip-Con).
liposomes containing RGD-fatty alcohols were found to be more
effective in reducing tumor size (groups E, F, G, H1, I and J; p < 0.01
vs. group D), likely due to their improved affinity for the integrins
over-expressed on tumor cells (Bellis, 2011). There was no
significant difference observed among docetaxel liposomes con-
taining different RGD-fatty alcohols (groups E, F, G, H1, I and J;
p > 0.05).
3.4. Anti-tumor activity in vivo
Fig. 5 shows the results for tumor weights and TGI% following
the administration of various formulations as described in Table 2.
Groups A (saline) and B (empty liposomes containing RGDOC8H17
had the same effect on tumor weights, suggesting lipids and RGD-
fatty alcohols did not inhibit tumor growth under experimental
)
Lip-RGD(C14) was selected to further examine the effect of
RGD-fatty alcohols on docetaxel liposomes of varied concentra-
tions (2, 3.5 and 5 mg/kg) in tumor-bearing mice (groups H3, H2
and H1). Although docetaxel at 5 mg/kg showed improved
antitumor activity, there was little difference found between
docetaxel at 2 and 3.5 mg/kg. Both groups significantly reduced
tumor weights compared to group D (5 mg/kg of docetaxel in Lip-
Con, p < 0.05). This result demonstrates that RGD-fatty alcohol-
modified liposomes can inhibit tumor growth with less docetaxel
than conventional liposomes.
conditions. There was little difference between groups
D
(docetaxel liposome) and (docetaxel liposome containing
K
GGDOC14H29) with respect to tumor weights, suggesting a minimal
effect of GGDOC14H29 on the anticancer activity of docetaxel
liposomes. Compared to group D (docetaxel liposome), docetaxel
The effect of the quantity of RGD-fatty alcohols incorporated in
docetaxel liposomes was investigated by comparing groups H1 and
H4. The amount of RGDOC14H29 incorporated in group H4 was one
half that in group H1. There was little difference observed between
the two groups for tumor size. An increase in the RGD-fatty alcohol
concentration did not improve antitumor activity, suggesting that
the specific binding between RGD-fatty alcohols and integrins on
tumor cell membranes was near saturation under experimental
concentrations.
TGI% values corresponding to various treatments are also
shown in Fig. 5. The TGI%s for groups E, F, H1, H4, I and J were
greater than 60%.
3.5. Pharmacokinetic and biodistribution properties
Fig. 6 shows the concentrations of docetaxel at different time
intervals in the blood, liver and tumors after the administration of
different docetaxel formulations. Lip-RGD(C14) resulted in the
greatest docetaxel concentrations, including Cmax in the tumor and
Cmin in the liver, and Lip-GGD(C14) also showed higher concen-
trations in the tumor and lower concentrations in the liver than
docetaxel suspension alone. These results suggested that lip-
osomes delivered more docetaxel to the tumor site, and RGD-fatty
alcohols were able to further increase the docetaxel concentrations
in the tumor, likely due to the active targeting of RGD-fatty alcohol
to the integrins expressed at the tumor site. Different fitting curves
are also shown. With the criteria of minimal SS and AIC, the two-
compartment model, formula first and formula zero were selected
as the best-fit equations for docetaxel concentrations in the blood,
liver and tumors, respectively. Interestingly, the best fitting curves
Fig. 4. Cellular uptake of E’-lip-RGD(C12) and E’-lip at 37 ꢁC and 4 ꢁC (1890 ꢀ mag).
Rhodamine-labeled liposomes appear in red fluorescence, nuclei appear in blue
fluorescence. The red fluorescence intensities (FI) in A375 cells were measured by
ImageJ. The value of red FI in cells at 37 ꢁC represented the total cell-associated
liposomes (TC) and that at 4 ꢁC represented cell membrane-bound liposomes (B).

Y. Li et al. / International Journal of Pharmaceutics 468 (2014) 133–141
139
Fig. 6. Docetaxel concentration (C) time (t) course studies in blood, liver and tumors. Diamond dots represent experimental data. Mean ꢃ SD, n = 5. Some SDs are smaller than
symbols. Dotted lines denote fitting curves by the one-compartment model (blood) and by formula first (liver and tumor), and solid lines denote fitting curves by the two-
compartment model (blood) and by formula zero (liver and tumor).
for different formulations in one organ are achieved with the same
model, suggesting that liposomes altered the amount of docetaxel
distributed in various tissues without changing the drug release
mode.
Tables 3–5 listed the fitting parameters of docetaxel concen-
trations in the blood, liver and tumors following the administration
of docetaxel suspension, Lip-RGD(C14) and Lip-GGD(C14). Lip-RGD
(C14) had the highest values of fast and slow disposition rate
3.6. Tumor selectivity evaluation of Lip-RGD(Cn)
Statistical analysis shows Lip-RGD(C14) has a higher blood
concentration at 5 min than docetaxel suspension (p < 0.01), while
no difference between Lip-RGD(C14) and Lip-GGD(C14). Further-
more, Lip-RGD(C14) showed a higher tumor concentration at 5 min
than docetaxel suspension or Lip-GGD(C14) (p < 0.01). These
results demonstrate hydrophilic RGD-motif modified liposomes
may partly avoid the effect of macrophages, similar as the
hydration effect of pegylation, and thus increase the blood
concentration of Lip-RGD(C14). On the other hand, RGD-fatty
alcohols linked liposomes increase tumor-targeting capacities and
quickly aggregate in the tumor, which decrease the liposomes
amount in systemic circulation and also decreased phagocytosis of
macrophage.
constants (a and b) for docetaxel c–t fitting curves in blood,
indicating RGD-fatty alcohol accelerated docetaxel-loaded lipo-
some distribution into tissues. Correspondingly, Lip-RGD(C14) also
showed the maximal AUC0–1 in tumors and the minimal AUC0–1
in the liver compared with the other two formulations. This result
further illustrated the active targeting of RGD-fatty alcohols to
tumors.
Table 3
Fitting parameters of best-fit c–t equations for docetaxel-loaded formulations in
Table 4
blood.
Fitting parameters of best-fit c–t equations for docetaxel-loaded formulations in
liver.
Parameters
Suspension
Lip-RGD (C14)
Lip-GGD (C14)
Parameters
Suspension
Lip-RGD (C14)
Lip-GGD (C14)
A (
a
m
g/mL)
)
1.289
1.795
0.170
0.206
0.010
4.156
4.875
0.421
0.328
0.019
2.744
2.163
0.125
0.022
0.348
1.666
2.418
(hꢂ1
A (
m
g/g)
)
)
20.813
0.100
0.888
5.429
0.127
1.037
2.181
11.457
39.542
9.359
0.080
0.521
9.470
4.988
B (
b
m
g/mL)
K1 (hꢂ1
K2 (hꢂ1
SS
(hꢂ1
)
SS
28.732
29.506
175.307
AIC
AUC0–1 [(
ꢂ19.763
ꢂ15.746
AIC
m
g/mL) h]
1.574
2.209
AUC0–1 [(
m
g/g) h]
115.303
ꢂ
ꢃ
Best-fit equation: c ¼ Aeꢂ þ Beꢂ (two-compartment model)
Best-fit equation: c ¼ A eꢂk ꢂ eꢂk (Formula first)
a
t
b
t
1t
2t

140
Y. Li et al. / International Journal of Pharmaceutics 468 (2014) 133–141
Table 5
close TGI% achieved with conventional liposomes and GGD-motif-
linked (non-targeted) liposomes indicated that the TGI% could be
used to compare the efficacy of liposomes containing doxorubicin,
cytarabine and docetaxel. Fig. 7 appears to suggest that the TGI% of
various liposomes containing RGDF-fatty alcohols, RGDV-fatty
acids and RGD-fatty alcohols are similar. In addition, the
calculations of the TI values for tumors from pharmacokinetic
data are similar between RGDF-fatty alcohol and RGD-fatty alcohol
groups, e.g., 2.07 vs. 2.36 compared to GGD-motif-linked lip-
osomes. These results reveal that RGD is the necessary sequence to
ensure an affinity for integrins, not the tetrapeptide RGDX. The
fourth amino acid can be replaced by a fatty alcohol, and the
alcohol’s lipophilicity may facilitate the insertion of the RGD-motif
into the membrane of liposomes with a preferred conformation.
Compared to the method reported for the synthesis of
Fitting parameters of best-fit c–t equations for docetaxel-loaded formulations in
tumor.
Parameters
Suspension
Lip-RGD (C14)
Lip-GGD (C14)
A (
m
g/g)
1.645
7.857
5.299
11.608
3.282
0.500
0.571
5.199
1.923
31.544
3.558
0.500
0.010
K1 (hꢂ1
K2 (hꢂ1
T (h)
SS
)
)
3.605
0.500
0.053
ꢂ9.625
1.119
AIC
AUC0–1 [(
ꢂ19.876
m
g/g) h]
3.912
1.659
ꢂ
ꢃ
ꢂ
ꢃ
Best-fit equation: c ¼ A 1 ꢂ eꢂk . . . t ꢄ T; c ¼ A 1 ꢂ eꢂk
ꢀ eꢂk ðtꢂTÞ . . . t > T
1t
1T
2
(Formula zero)
Table 6 demonstrates the calculated parameters TE, TI and RTI.
TE evaluated the tumor selectivity of one formulation compared to
the blood and liver. Lip-RGD(C14) showed a better TE value than
other formulations, with TE1 greater than 1 and TE2 nearly 0.1.
These results revealed that the bulk of the docetaxel in liposomes
distributed to the liver, but RGD-fatty alcohol increased its tumor
selectivity. TI and RTI further compared the tumor-targeting
capacity of various formulations. The TI values for tumors were the
highest at greater than 2, whereas those for blood were
approximately 1, and those for the liver were the lowest, below
0.4. This result showed that RGD-fatty alcohol linked liposomes
had good tumor-targeting capacity accompanied by decreased
liver accumulation compared with docetaxel suspension or
conventional liposomes. The inspiring results of RTI extended this
concept. Obviously, Lip-RGD(C14) is superior to docetaxel suspen-
sion or unmodified liposomes [Lip-GGD(C14)] for tumor selectivi-
ty.
tetrapeptide-fatty alcohols, RGDFOCnH2
(Du et al., 2011), the
n + 1
synthesis of RGDOCnH2
(n = 8–18) analogues in the present
n + 1
work was simpler. To synthesize RGDFOCnH2
,
Boc-Phe-
n + 1
OCnH2
were synthesized to protect the N-terminus of
n + 1
phenylalanine (Phe, F) and then connected with Boc-Asp(OBzl)-
OH to form Boc-Asp(OBzl)-Phe-OCnH2 In this paper,
OH were used directly without protection through a
.
n + 1
CnH2
n + 1
carbonyl condensation reaction.
It is known that the increased expression of avb3 integrins on
various tumor cell surfaces activates tumor invasion, including cell
adhesion, proliferation, migration, metastatic dissemination,
survival and angiogenesis (Renner et al., 2004; Xu et al., 2012).
RGD motif-containing peptides compete for integrin-binding with
the endogenous RGD sequence present in ECM substituents, such
as fibronectin, vitronectin, fibrinogen, and osteopontin, and this
inhibits integrin-mediated tumor cell adhesion and induces
adhesion-dependent apoptosis (Hynes, 2002). Therefore, the
RGD motif may be partially endowed with anti-tumor activity.
However, 5% RGDOC8H17 without docetaxel in liposomes did not
have an anti-tumor effect under the experimental conditions
(group B, Fig. 5).
4. Discussion
It was believed that a fourth amino acid appended to the RGD
tripeptide was necessary for specific binding to integrins overex-
pressed on the surface of tumor cells. The results of this study
demonstrated that docetaxel liposomes modified with tripeptide-
Severalpapershavechosenaone-compartmentmodeltoanalyze
tissue distribution kinetics following i.v. injection because two-
compartment and other models are complicated. In this study,
formula zero and formula first were selected to model docetaxel
fatty alcohol conjugates, RGDOCnH2
(n = 8–18), were specifi-
n + 1
cally targeted to tumors, which suggested that fatty alcohols could
provide the binding affinity for integrins. Furthermore, an effort
was made to compare the tumor-targeting capacities of RGDF-fatty
alcohols, RGDV-fatty acids and RGD-fatty alcohols using the TGI%,
despite that these values were obtained with different anti-cancer
agents. The TGI%s of RGDV-fatty acids were obtained from prior
studies by our group on C CO-RGDV-cytarabine (n = 8,
H2
n ꢂ 1 n ꢂ 1
10, 12, 14, 16)-conjugated liposomes, whereas those for RGDF-fatty
alcohols were determined with RGDFOCnH2 (n = 8, 12, 16)-
n + 1
modified doxorubicin liposomes (Du et al., 2011; Wang et al., 2012).
Fig. 7 shows the TGI% histogram of different liposomes containing
various RGD motifs. The calculation of TGI% can compensate for the
different animal tumor models (See Section 2.6). Moreover, the
Table 6
Comparison of targeting parameters between docetaxel-loaded formulations.
Parameters
Suspension
Lip-RGD(C14)
Lip-GGD(C14)
TE1
0.711
0.006
1
1.771
0.099
15.501
6.875
0.686
0.014
–
TE2
RTIa
RTIb
Fig. 7. TGI% comparison of liposomes containing various RGD motifs. RGDF-fatty
alcohols are RGDFOCnH2
(n = 8–16)-modified doxorubicin liposomes. RGDV-
CO-RGDV-cytarabine (n = 8–16)-conjugated liposomes.
n + 1
–
1
fatty acids are C
H2
RGD-fatty alcohols are RGDOCnH2
n ꢂ 1 n ꢂ 1
(n = 8–18)-modified docetaxel liposomes. C8
n + 1
Parameters
Blood
Liver
Tumor
indicates an alkyl chain length of n = 8. Con are conventional liposomes without RGD
motifs. GGD are GGDFOC12H25-modified doxorubicin liposomes for the RGDF-fatty
alcohols group, C11H23CO-V-cytarabine-conjugated liposomes for the RGDV-fatty
acids group and GGDOC14H29-modified docetaxel liposomes for the RGD-fatty
alcohols group. The data for RGDF-fatty alcohol and RGDV-fatty acid groups are
deduced from Du et al., 2011 and Wang et al., 2012.
TIa
TIb
1.404
0.913
0.226
0.343
3.497
2.358
a
CDDS is suspension in the equation.
CDDS is Lip-GGD(C14) in the equation.
b

Y. Li et al. / International Journal of Pharmaceutics 468 (2014) 133–141
141
Chen, Y.L., Tang, J., Kesler, M.J., Sham, Y.Y., Vince, R., Geraghty, R.J., Wang, Z., 2012.
The design, synthesis and biological evaluations of C-6 or C-7 substituted 2-
hydroxyisoquinoline-1,3-diones as inhibitors of hepatitis C virus. Bioorganic
and Medicinal Chemistry 20, 467–479.
Du, H., Cui, C., Wang, L., Liu, H., Cui, G., 2011. Novel tetrapeptide, RGDF, mediated
tumor specific liposomal doxorubicin (DOX) preparations. Molecular Pharma-
ceutics 8, 1224–1232.
concentration time courses in tissues. Formula first has a format
similar to the one-compartment model’s first-order absorption
equation and is good at fitting docetaxel c–t profiles of tested
formulations in the liver. Formula zero uses T as a parameter to
describe the duration of zero-order absorption when the com-
poundsslowlyenterthetissue,e.g.,tumorsinthispaper,followedby
first-order elimination. Formula zero and formula first have
demonstrated powerful fitting capacities in various time courses
(Li and Zhu, 2004). The nonlinear fitting results of formula zero and
formula first in this study were much better than those of classic
compartment models (data not shown). The accurate fitting curves
in Fig. 6 ensure the calculation of various targeting parameters.
Hynes, R.O., 2002. Integrins: bidirectional, allosteric signaling machines. Cell 110,
673–687.
Immordino, M.L., Brusa, P., Arpicco, S., Stella, B., Dosio, F., Cattel, L., 2003.
Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes
containing docetaxel. Journal of Controlled Release 91, 417–429.
Kang, Y.K., Jhon, J.S., 2000. Preferred conformations of a linear RGD tripeptide.
Journal of Peptide Research 56, 360–372.
Kim, J., Nam, H.Y., Kim, T.I., Kim, P.H., Ryu, J., Yun, C.O., Kim, S.W., 2011. Active
targeting of RGD-conjugated bioreducible polymer for delivery of oncolytic
adenovirus expressing shRNA against IL-8 mRNA. Biomaterials 32, 5158–
5166.
Lefrancois, L.H., Pujol, C., Bodier, C.C., Teixeira-Gomez, A.P., Drobecq, H., Rosso, M.L.,
Raze, D., Dias, A.A., Hugot, J.P., Chacon, O., Barletta, R.G., Locht, C., Vidal
Pessolani, M.C., Biet, F., 2011. Characterization of the Mycobacterium avium
subsp. paratuberculosis laminin-binding/histone-like protein (Lbp/Hlp) which
reacts with sera from patients with Crohn’s disease. Microbes and Infection 13,
585–594.
Lehn, J.M., 1993. Supramolecular chemistry. Science 260, 1762–1763.
Li, Y.H., Zhu, J.B., 2004. Modulation of combined-release behaviors from a novel
“tablets-in-capsule system”. Journal of Controlled Release 95, 381–389.
Li, Y., Wang, J., Gao, Y., Zhu, J., Wientjes, M.G., Au, J.L., 2011. Relationships between
liposome properties, cell membrane binding, intracellular processing, and
intracellular bioavailability. The AAPS Journal 13, 585–597.
Li, J., Xu, H., Ke, X., Tian, J., 2012. The anti-tumor performance of docetaxel liposomes
surface-modified with glycyrrhetinic acid. Journal of Drug Targeting 20, 467–
473.
Lin, N., Zhao, M., Wang, C., Peng, S., 2002. Synthesis and antithrombotic activity of
carbolinecarboxyl RGD sequence. Bioorganic and Medicinal Chemistry Letters
12, 585–587.
Palmer, L.C., Stupp, S.I., 2008. Molecular self-assembly into one-dimensional
nanostructures. Accounts of Chemical Research 41, 1674–1684.
Park, H.S., Kim, C., Kang, Y.K., 2002. Preferred conformations of RGDX
tetrapeptides to inhibit the binding of fibrinogen to platelets. Biopolymers
63, 298–313.
Pozzetto, U., Aguzzi, M.S., Maggiano, N., Scala, E., Capelli, A., Castagneto, M.,
Capogrossi, M.C., Citterio, F., Serino, F., Facchiano, A., 2005. RGDS peptide
inhibits activation of lymphocytes and adhesion of activated lymphocytes to
human umbilical vein endothelial cells in vitro. Immunology and Cell Biology
83, 25–32.
Renner, C., Sacca, B., Moroder, L., 2004. Synthetic heterotrimeric collagen peptides
as mimics of cell adhesion sites of the basement membrane. Biopolymers 76,
34–47.
5. Conclusions
A simplified RGD motif-modified liposome was developed for
improved tumor selectivity in vivo. RGD-fatty alcohols incorporat-
ed into docetaxel liposomes were effective at improving the
antitumor activity of docetaxel liposomes in mice bearing S180
sarcoma. RGD-fatty alcohols were as effective as RGDF-fatty
alcohols and RGDV-fatty acids at targeting to tumors. The length of
the fatty alcohol (C8–C18) linked to RGD had little impact on the
antitumor activity of docetaxel liposomes. An increase in the
concentration of RGD-fatty alcohols in docetaxel liposomes from
2.5 to 5% (molar ratio) did not improve the antitumor activity.
A two-compartment model, formula first and formula zero
were selected as the best-fit mathematic models for the docetaxel
c–t profiles of tested formulations in the blood, liver and tumors,
respectively. These models expanded the frequently used equa-
tions for pharmacokinetic data analysis and showed good fitting
results for various processes in vivo. The calculations of AUC, TE, TI
and RTI based on the fitting parameters further demonstrated that
RGD-fatty alcohol-modified liposomes strongly improved tumor
selectivity and significantly decreased liver metabolism compared
to conventional liposomes.
Acknowledgments
Ruoslahti, E., Pierschbacher, M.D., 1987. New perspectives in cell adhesion: RGD and
integrins. Science 238, 491–497.
Spurio, M., Palou, J., Angerri, O., Parada, R., Salvador, J., Villavicencio, H., 2004.
This research was supported by the National Natural Science
Foundation of China Grant 81202486 and Beijing Natural Science
Foundation Grant 7133228.
Mucosal fold as
a mechanical cause of obstruction in orthotopic ileal
neobladder. Archivio Italiano di Urologia, Andrologia 76, 34–36.
Ventura, M., Turroni, F., Motherway, M.O., MacSharry, J., van Sinderen, D., 2012.
Host-microbe interactions that facilitate gut colonization by commensal
bifidobacteria. Trends in Microbiology 20, 467–476.
Appendix A. Supplementary data
Wang, F., Cui, C., Ren, Z., Wang, L., Liu, H., Cui, G., 2012. Preparation and biological
evaluation of tumor-specific Ara-C liposomal preparations containing RGDV
motif. Journal of Pharmaceutical Sciences 101, 4559–4568.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ijpharm.2014.
04.001.
Warr, G.W., 2011. Introduction to a special issue in memory of Paul S. Gross. Mar
Biotechnol (NY) 13, 585–586.
Xu, Q., Liu, Y., Su, S., Li, W., Chen, C., Wu, Y., 2012. Anti-tumor activity of paclitaxel
through dual-targeting carrier of cyclic RGD and transferrin conjugated
hyperbranched copolymer nanoparticles. Biomaterials 33, 1627–1639.
Zhang, H., Li, R.Y., Lu, X., Mou, Z.Z., Lin, G.M., 2012. Docetaxel-loaded liposomes:
preparation, pH sensitivity, pharmacokinetics, and tissue distribution. Journal
of Zhejiang University Science B 13, 981–989.
Zhao, L., Wei, Y.M., Zhong, X.D., Liang, Y., Zhang, X.M., Li, W., Li, B.B., Wang, Y., Yu, Y.,
2009. PK and tissue distribution of docetaxel in rabbits after i. v. administration
of liposomal and injectable formulations. Journal of Pharmaceutical and
Biomedical Analysis 49, 989–996.
References
Aguzzi, M.S., Fortugno, P., Giampietri, C., Ragone, G., Capogrossi, M.C., Facchiano, A.,
2010. Intracellular targets of RGDS peptide in melanoma cells. Molecular Cancer
9, 84.
Barczyk, M., Carracedo, S., Gullberg, D., 2010. Integrins. Cell and Tissue Research 339,
269–280.
Bellis, S.L., 2011. Advantages of RGD peptides for directing cell association with
biomaterials. Biomaterials 32, 4205–4210.
Budai, M., Szabo, Z., Szogyi, M., Grof, P., 2003. Molecular interactions between DPPC
and morphine derivatives: a DSC and EPR study. International Journal of
Pharmaceutics 250, 239–250.
van Tellingen, O., Beijnen, J.H., Verweij, J., Scherrenburg, E.J., Nooijen, W.J.,
Sparreboom, A., 1999. Rapid esterase-sensitive breakdown of polysorbate 80
and its impact on the plasma pharmacokinetics of docetaxel and metabolites in
mice. Clinical Cancer Research 5, 2918–2924.