Dual-active targeting liposomes drug delivery system for bone metastatic breast cancer: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.chemphyslip.2019.104785

Bone is the most common organ affected by metastatic breast cancer. Targeting cancers within the bone remains a great challenge due to the inefficient delivery of therapeutic to bone. In order to increase the distribution of paclitaxel (PTX) in bone metastases, in this study, a novel bone-targeted glutamic oligopeptides-RGD peptide (Glu₆-RGD) derivative was designed and synthesized as liposome ligand for preparing liposome to effectively deliver PTX to bone metastases. The liposome was prepared and its particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis and cytotoxicity were also characterized. What's more, the Glu₆-RGD-Lip showed superior targeting ability in vitro and in vivo evaluation as compared to naked PTX, non-coated, singly-modified and co-modified by physical blending liposomes. All the results suggested that Glu₆-RGD-modified liposome showed excellent targeting activity to metastatic bone cancer. This study may be conducive to the field of bone-targeting drugs delivery.

Full text of DOI:10.1016/j.chemphyslip.2019.104785

Accepted Manuscript
Title: Dual-active targeting liposomes drug delivery system
for bone metastatic breast cancer: synthesis and biological
evaluation
Authors: Ze Zhao, Yi Zhao, Changwei Xie, Changqing Chen,
Dong Lin, Sheng Wang, Dong Lin, Xinhua Cui, Zhongshuai
Guo, Junfeng Zhou
PII:
S0009-3084(19)30095-7
DOI:
Article Number:
https://doi.org/10.1016/j.chemphyslip.2019.104785
104785
Reference:
CPL 104785
To appear in:
Chemistry and Physics of Lipids
Received date:
Revised date:
Accepted date:
10 April 2019
15 May 2019
9 June 2019
Please cite this article as: Zhao Z, Zhao Y, Xie C, Chen C, Lin D, Wang S, Lin D,
Cui X, Guo Z, Zhou J, Dual-active targeting liposomes drug delivery system for bone
metastatic breast cancer: synthesis and biological evaluation, Chemistry and Physics of
Lipids (2019), https://doi.org/10.1016/j.chemphyslip.2019.104785
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Dual-active targeting liposomes drug delivery system for
bone metastatic breast cancer: synthesis and biological
evaluation
Ze Zhao1*, Yi Zhao2*, Changwei Xie1, Changqing Chen1, Dong Lin1, Sheng Wang1, Dong Lin1,
Xinhua Cui1, Zhongshuai Guo1, Junfeng Zhou1
1Department of Orthopedics, the First Affiliated Hospital of Henan Polytechnic University (the Second People's
Hospital of Jiaozuo City), No.17 Minzhu South Road, Jiaozuo 454001, China.
2the First Affiliated Hospital of Zhengzhou University, No.1 Jianshe East Road, Zhengzhou 450052, China
*Corresponding authors. zezhao0910@163.com (Z. Zhao), zhaoyi0910@163.com (Y. Zhao).
Graphical Abstract
Highlights
A novel bone-targeted glutamic oligopeptides-RGD peptide (Glu6-RGD) derivative was designed and
synthesized as liposome ligand.



The preliminary evaluation in vivo demonstrated that ligand Glu6-RGD-coated liposome had an
improved targeting ability.
Glu6-RGD could act as a vector to enhance the delivery of drugs bone metastasis.
1

Abstract
Bone is the most common organ affected by metastatic breast cancer. Targeting cancers within the bone
remains a great challenge due to the inefficient delivery of therapeutic to bone. In order to increase the
distribution of paclitaxel (PTX) in bone metastases, in this study, a novel bone-targeted glutamic
oligopeptides-RGD peptide (Glu6-RGD) derivative was designed and synthesized as liposome ligand for
preparing liposome to effectively deliver PTX to bone metastases. The liposome was prepared and its
particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis and cytotoxicity
were also characterized. What's more, the Glu6-RGD-Lip showed superior targeting ability in vitro and in
vivo evaluation as compared to naked PTX, non-coated, singly-modified and co-modified by physical
blending liposomes. All the results suggested that Glu6-RGD-modified liposome showed excellent
targeting activity to metastatic bone cancer. This study may be conducive to the field of bone-targeting
drugs delivery.
Keywords: Bone metastases, Bone-targeting, Glutamic oligopeptides, RGD peptide, Drug delivery
1. Introduction
Bone metastasis is a frequent complication in advanced stage of many cancers, especially breast cancer
and prostate cancer [Zhao et al. 2017]. This is because the physiological microenvironment of bone is very
suitable for tumor cells to support tumor inoculation and progress [Wang et al. 2014]. The patients with
bone metastases suffer from bone pain, hypercalcemia and pathologic fractures, which lead to the decrease
of life quality [Anada et al. 2009; Liao and Xu 2017]. At present, metastatic bone cancer is mainly treated
by surgery, radiotherapy and chemotherapy. Therefore, strategies that can effectively deliver drugs, such as
paclitaxel (PTX), to the site of metastasis are of great interests for the treatment of metastatic bone cancer
due to lack of target specificity of anticancer drugs [Liu et al. 2017].
Desired accumulation at tumor sites is the principal purpose of anticancer drug delivery systems. The
inorganic compound hydroxyapatite (HAP), which is the main component of bone, is a promising target
for selective drug delivery to bone [Duan et al. 2009]. In our previous study, we have found that glutamic
acid oligopeptides has an excellent bone-targeted ability [Zhao et al. 2015]. The mechanisms of the
targeting ability to bone was showed by acidic oligopeptides: the ionic interaction with Ca2+ in the mineral
component of bone was built by the peptides bearing negative charges [Sarig 2004]. Moreover, the ability
of glutamic acid oligopeptides targeting to bone was depended on the number of exposed carboxyl residues
2

[Sarig 2004].
What’s more, it is well known that RGD peptide is one of the most frequently-used neoplastic targeting
ligands in drug delivery system [Wang et al. 2014]. The RGD sequence, an arginine-glycine-aspartic
(Arg-Gly-Asp) tripeptide, has been identified as a high affinity αvβ3 selective ligand [Fu et al. 2019]. The
αvβ3, one of the intergrin receptor family, is proved to be over-expressed in the most malignant tumors. As
a results, a lot of interesting researches had been done on the nanocarriers modified by RGD to target drug
to the tumors [Fu et al. 2019; Xiao et al. 2018]. In addition, the αvβ3 is also highly expressed in bone
metastatic cells and osteoclasts, so that the αvβ3 integrin is also an attractive target for bone [Jiang et al.
2012]. There the RGD peptide may also have the property of bone targeting.
Over the years, we has done a lot of researches on bone targeting and cancer targeting moieties specially
based on RGD or peptides by active process [Fu et al. 2019; Zhao et al. 2015]. The advantageous property
of peptides such as biocompatibility, non-cytotoxicity, and enzymatic degradation can make the drug more
biocompatible and no unexpected long-term effects. And we are still exploring how to further improve the
carrier's bone targeting ability. As parts of our continuing efforts, we focused on utilizing glutamic
oligopeptide and RGD peptide to selectively deliver drug to bone metastases. Herein, we developed a
PTX-loaded liposome co-modified with glutamic oligopeptide and RGD peptide. Such delivery system had
a potential bone-targeted affinity through the Glu6 and RGD exposed on the surface of the liposome, and
facilitated bone metastases cellular uptake by the modification of RGD. To investigate the further bone
targeting, the in vitro bone targeting was evaluated through hydroxyapatite (HAP) binding assay. What’s
more, the Glu6-RGD modified (Figure 1), non-coated, singly-modified and co-modified by physical
blending liposomes (Glu6+RGD) were prepared by the lipid film hydration-ultrasound method, and
targeting ability in vivo were also investigated.
2. Materials and methods
2.1. Materials
All liquid reagents were distilled before use. All unspecified reagents were from commercial resources.
TLC was performed using precoated silica gel GF254 (0.2 mm), while column chromatography was
performed using silica gel (100-200 mesh). The melting point was measured on a YRT-3 melting point
apparatus (Shantou Keyi instrument & Equipment Co. Ltd, Shantou, China). Elemental analyses were
performed by Atlantic Microlab (Atlanta, GA, USA). NMR spectra were taken on a Varian INOVA 400
3

MHz (Varian, PaloAlto, CA, USA). Mass spectra were recorded on an Agilent 1946B ESI-MS instrument
(Agilent, PaloAlto, CA, USA). Reversed-phase chromatography performed on C18 chromatographic
analysis was carried out using the HPLC system (Alltech, IL, USA) consisted of a RF-530 fluorescence
detector (Shimadzu, Japan) and Allchorom plus data operator, respectively. A Diamonsil column (200 ×
4.6 mm, 5 μm) was used. Soybean phospholipids (SPC) were purchased from Shanghai Taiwei Chemical
Company (Shanghai, China). Paclitaxel (PTX) was purchased from AP Pharmaceutical Co. Ltd.
(Chongqing, China). 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased
from Beyotime Institute Biotechnology (Haimen, China). Kunming mice were purchased from Experiment
Animal Center of Henan Polytechnic University (P.R. China). All animal procedures for this study were
approved by the Experiment Animal Administrative Committee of Henan Polytechnic University (P.R.
China).
2.2. Methods
2.2.1 Chemistry
2.2.1.1 Synthesis of compound 3-17
The synthesis of compound 3-17 was reported in our previous work [Fu et al. 2019; Xiao et al. 2019;
Zhao et al. 2014; Zhao et al. 2015; Zhao et al. 2018].
2.2.1.2 Synthesis of compound 18
To a solution of compound 11 (0.10 g, 0.15 mmol) in CH2Cl2 (5 mL) were added
dicyclohexylcarbodiimide (DCC, 37 mg, 0.18 mmol) and cat. dimethylaminopyridine (DMAP), then the
mixture was stirred at -5 °C for 30 min. Compound 17 (0.11 g, 0.17 mmol) in CH2Cl2 (5 mL) was added to
the above reaction mixture slowly. After stirring for 10 h at room temperature, the reaction was terminated
and then filtered. The filtrate was concentrated, and the residue was purified by flash chromatography to
give 18 (128 mg, 65%) as white solid. HRMS: (ESI+) calculated for C69H104N8O17Na [M+Na]+ 1339.7417,
found 1339.7414. Elemental Analysis: C, 62.90; H, 7.96; N, 8.50, found C, 62.76; H, 8.11; N, 8.39.
2.2.1.3 Synthesis of compound 19
To a solution of compound 5 (0.27 g, 0.18 mmol) in CH2Cl2 (10 mL) were added DCC (55 mg, 0.27
mmol) and cat. DMAP, then the mixture was stirred at -5 °C for 30 min. Compound 18 (0.24 g, 0.18 mmol)
in CH2Cl2 (5 mL) was added to the above reaction mixture slowly. After stirring for 20 h at room
temperature, the reaction was terminated and then filtered. The filtrate was concentrated, and the residue
was purified by flash chromatography to give 19 (0.18 g, 36%) as white waxy solid. HRMS: (ESI+)
4

calculated for C152H192N14O38Na [M+Na]+ 2845.3453, found 2845.3450. Elemental Analysis: C, 64.67; H,
6.86; N, 6.95, found C, 64.79; H, 6.96; N, 6.77.
2.2.1.4 Synthesis of ligand Glu6-RGD-Chol
To a solution of compound 19 (50 mg, 0.018 mmol) in CH3OH (5 mL), Pd/C (20 mg, 10%) was added.
Then, the mixture was stirred in hydrogen atmosphere at 50 °C for 40 h. Pd/C was filtered, and the filtrate
was concentrated to give ligand Glu6-RGD-Chol (18 mg, 52%) as white waxy solid. HRMS: (ESI+)
calculated for C89H139N13O36Na [M+Na]+ 1988.9343, found 1988.9347. Elemental Analysis: C, 54.34; H,
7.12; N, 9.26, found C, 54.22; H, 7.29; N, 9.37.
2.2.1.5 Synthesis of compound 20
To a solution of compound 16 (1.00 g, 1.73 mmol) in CH2Cl2 (25 mL) was added N-methylmorpholine
(NMM, 0.29 mL, 2.60 mmol) and isobutyl chlorocarbonate (IBCF, 0.26 mL, 2.08 mmol), and the reaction
o
was stirred at -10 C for 15 min. Then compound 4 (2.22 g, 1.56 mmol) in CH2Cl2 (10 mL) was added
slowly. After stirring for another 10 h at r.t., the mixture was washed with 1 mol/L HCl, saturated NaHCO3
and saturated NaCl. The organic layer was dried over anhydrous Na2SO4, and concentrated in vacuo. The
residue was purified by flash column chromatography to afford 20 (1.75 g, 56%) as white waxy solid.
HRMS: (ESI+) calculated for C114H144N6O25Na [M+Na]+ 2021.0112, found 2021.0115. Elemental Analysis:
C, 68.52; H, 7.26; N, 4.21, found C, 68.37; H, 7.39; N, 4.28.
2.2.1.6 Synthesis of ligand Glu6-Chol
To a solution of compound 20 (0.10 g, 0.050 mmol) in CH3OH (5 mL), Pd/C (20 mg, 10%) was added.
Then, the mixture was stirred in hydrogen atmosphere at r.t. for 20 h. Pd/C was filtered, and the filtrate was
concentrated to give ligand Glu6-Chol (46 mg, 68%) as white waxy solid. HRMS: (ESI+) calculated for
C65H102N6O25Na [M+Na]+ 1389.6792, found 1389.6790. Elemental Analysis: C, 57.09; H, 7.52; N, 6.15,
found C, 57.22; H, 7.35 N, 6.01.
2.2.1.7 Synthesis of ligand RGD-Chol
The synthesis of RGD-Chol was reported in our previous work [Fu et al. 2019].
2.2.2 Preparation and characterization of liposomes
Liposomes were prepared by thin film hydration method as our previous reported [Fu et al. 2019; Peng
et al. 2018; Xiao et al. 2019]. Lipid compositions of the prepared liposomes were as follows: (1)
conventional liposomes (Lip), SPC/cholesterol/ (molar ratio = 62: 33); (2) Ligand Glu6-Chol modified
liposomes (Glu6-Lip), SPC/cholesterol/ligand Glu6-Chol (molar ratio = 62: 33: 3); (3) Ligand RGD-Chol
5

modified liposomes (RGD-Lip), SPC/cholesterol/Ligand RGD-Chol (molar ratio = 62: 33: 3); (4) Ligand
Glu6-Chol and RGD-Chol co-modified liposomes (Glu6+RGD-Lip), SPC/cholesterol/Ligand
Glu6-Chol/RGD-Chol (molar ratio = 62: 33: 3: 3); (5) Ligand Glu6-RGD-Chol modified liposomes
(Glu6-RGD-Lip), SPC/cholesterol/Ligand Glu6-RGD-Chol (molar ratio = 62: 33: 3). All lipid materials
were dissolved in the chloroform/methanol (v/v = 2:1), and then the organic solvent was removed by rotary
evaporation to get a lipid film, which was further dried in vacuum overnight. Next, the film was hydrated
in PBS (pH 7.4) for 0.5 h at 20 °C. Finally, it was further sonicated intermittently by a probe sonicator at
80 W for 80 s to form liposomes. PTX-loaded liposomes were prepared with paclitaxel added to the lipid
organic solution prior to the solvent evaporation. The entrapment efficiency of paclitaxel was determined
by HPLC. The mean size and zeta potential of Lip, Glu6-Lip, RGD-Lip, Glu6+RGD-Lip and
Glu6-RGD-Lip were detected by Malvern Zeta sizer Nano ZS90 (Malvern Instruments Ltd., UK).
2.2.3 Release of free PTX in vitro
In vitro PTX release study was performed using dialysis method. Each PTX-loaded liposomes (0.4 mL)
or free PTX were placed into dialysis tube (MWCO = 8 000 – 14 000 Da) and tightly sealed. Then the
dialysis tubes were placed into 40 mL PBS containing 0.1% (v/v) Tween 80 and incubated under 37 °C for
48 h with gentle oscillating at 50 rpm. At predetermined time points (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and
48 h), 0.1 mL release medium was taken out and replaced with equal volume of fresh release medium.
Then the samples were diluted with acetonitrile and the concentrations of PTX were determined at the
wavelength of 225 nm by HPLC.
2.2.4 In vitro stability of liposomes in serum
Turbidity variations were measured to demonstrate the serum stability of liposomes in the presence of
fetal bovine serum (FBS). Briefly, liposomes were mixed with equal volume of FBS under 37 °C with
moderate shaking at 45 rpm. The transmittance of the mixture was measured at predetermined time points
(0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h) at 750 nm by a microplate reader (Thermo Scientific Varioskan
Flash, USA).
2.2.5 Hemolysis assays
To evaluate the safety of ligand-modified liposomes during body circulation, hemolysis assay was
performed. Fresh mouse blood was collected in heparin sodium-containing tubes. The red blood cells
(RBCs) were separated and collected by centrifugation at 5 × 103 rpm for 5 min and washed several times
with PBS until the supernatant became colorless. After the last wash, the RBCs were diluted with PBS to a
6

concentration of 2% (v/v). Various concentrations of liposomes were incubated with equal volume of 2%
RBCs solutions for 1 h at 37 °C with gentle shaking, followed by centrifugation at 1 × 104 rpm for 10 min.
Absorbance of hemoglobin was measured using a microplate reader (Thermo Scientific Varioskan Flash) at
540 nm. The values for 0% and 100% hemolysis were determined by incubating erythrocytes with PBS or
1% (v/v) Triton X-100. The hemolysis percentage was calculated using the following equation:
A
Sample
A
A
Negative
The percent hemolysis =
× 100%
APostive
Negative
where A is the ultraviolet absorbance of hemoglobin.
2.2.6 Cytotoxicity assay
MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented
o
with 10% fetal bovine serum (FBS), 100 U/mL streptomycin and 100 U/mL penicillin at 37 C in a
humidified incubator containing 5% CO2 (Thermo Scientific, USA). The cytotoxicity of PTX-loaded
liposomes was measured with MTT assay. Generally, the cells were seeded in a 96-well tissue culture plate
at a density of 5 × 103 cells/well and cultured for 24 h at 37 oC. PTX-loaded liposomes and free PTX were
diluted to predetermined concentrations with PBS and added into each well. The final concentrations of
PTX were in the range of 0.1-20 μg/mL. After 24 h incubation, 20 μL MTT solution (5.0 mg/mL) was
added to each well and incubated for 4 h at 37 °C. After removal of the culture medium, the reduced MTT
dye was solubi1ized by DMSO (150 μL) and the absorbance was read at 490 nm wavelength on an
automatic microplate spectrophotometer. Cell viability was calculated using the following equation: Atest
/
Acontrol × 100%, where Atest and Acontrol represent the absorbance of treated cells and control cells,
respectively.
2.2.7 HAP binding assay
An in vitro HAP binding assay was set up using the method described by our previous report to evaluate
the binding of Glu6-RGD modified liposomes to HAP [Zhao et al. 2015]. Briefly, 0.6 mL PTX or
liposomes (calculated as PTX) were mixed with 0.3 mL HAP suspension (20 mg/mL) or 0.3 mL PBS as a
o
control, followed by gentle shaking for 2 h at 37 C. After centrifugation at 5000 rpm for 3 min, the HAP
precipitate was separated from the unbound liposome in the supernatant. Unbound liposome were
quantified through HPLC to measure PTX encapsulated in the liposome. The degree of HAP binding was
calculated according to the following formula:
M
C
M
M
E
HAP binding rate (%) =
× 100%
C
7

where MC is the amount of PTX in the control group and ME is the amount of PTX in the experimental
groups.
2.2.8 Targeting metastatic bone in vivo
Six-week-old female Balb/c mice bearing MDA-MB-231 tumors in the tibia were divided into six
groups with five mice in each group. PTX-Lip, PTX-Glu6-Lip, PTX-RGD-Lip, PTX-Glu6+RGD-Lip,
PTX-Glu6-RGD-Lip and paclitaxel were given to the mice via the tail vein and each was equivalent to the
administration dose of 10 mg/kg PTX. At 0.5 h, 1 h, 2 h, 4 h and 8 h after injection, the mice were killed
by cervical dislocation. The bone metastatic lesions and normal bone lesions were excised, rinsed with
0.9% NaCl, dried over filter paper and weighed. The bone tissues were homogenized with twice amount of
saline. An aliquot of 10 μL of internal standard (docetaxel, 100 μg/mL) was added into 100 μL bone
homogenate, and extracted with 200 μL ether. The mixture was vortexed for 5 min, and centrifuged at 10
000 rpm for 10 min. The supernatant was transferred to another centrifuge tube, and dried under air stream
at room temperature. The dry residue was reconstituted with 100 μL of methanol. The solution was
centrifuged at 10 000 rpm for 10 min, and then 20 μL of the supernatant was injected into the HPLC
system for analysis.
3. Results and discussion
3.1. Chemistry
The synthetic pathway of liposome ligand Glu6-RGD-Chol was outlined in Scheme 1. Briefly,
bone-targeting peptide 5 with carboxyl groups protected by benzyl groups was synthesized by a
conventional liquid-phase peptide synthetic method which was described in our previous work [Zhao et al.
2015]. The synthesis of RGD intermediate 11 started from the available material Boc-Gly,
Asp(OBzl)-OBzl and Boc-Arg(NO2)-OH by a series of segment condensation which were described in our
previous work [Fu et al. 2019]. Cholesterol 12 underwent five steps to generate compound 17 [Fu et al.
2019]. Subsequently, condensation of compound 17 and RGD derivative 11 in the presence of dicyclohexyl
carbodiimide (DCC) and 4-dimethylamiopyridine (DMAP) gave 18, which was then coupled with Glu6
peptide intermediate 5 to generate 19. Finally, debenzylation of the benzyl groups with 10% Pd/C reached
the desired ligand Glu6-RGD-Chol.
What’s more, compound 16 was conjugated with peptide 4 in the presence of IBCF and NMM to give
8

compound 20, which was underwent a deprotection reaction to get the target ligand Glu6-Chol (Scheme 2).
The ligand RGD-Chol was synthesized according to our previous report [Fu et al. 2019].
3.2. Preparation and characterization of liposomes
One of the requirment for liposome to target bone is that they should have proper sizes and uniform
distribution. The particle sizes and zeta potentials of different liposomes in this study were listed in Table 1.
The encapsulation efficiencies (EE) of PTX were greater than 88%. The average particle sizes of all
liposomes were close to or less than 120 nm, and the values of PDI were close to 0.2. To our knowledge,
the particle size and zeta potential of liposomes were crucial to in vitro and in vivo study [Shi et al. 2015].
What's more, modification of the liposome with Glu6 significantly increased the net negative charge due to
the high density negative charge of glutamic oligopeptides [Zhang et al. 2017].
3.3. Release of free PTX in vitro
PTX release properties were evaluated in PBS containing 0.1% Tween 80. As shown in Fig. 2, free PTX
exhibited a rapid release, with over 80% of the drug released into the media within 12 h incubation. On the
other hand, PTX-loaded liposomes achieved sustained release behaviors that the cumulative PTX release
of drug loaded liposomes was about 60% after 48 h incubation in PBS. No significant difference on release
properties was observed between PTX-Lip, PTX-Glu6-Lip, PTX-RGD-Lip, PTX-Glu6+RGD-Lip and
PTX-Glu6-RGD-Lip, and none of the PTX-loaded liposomes displayed burst initial release patterns.
3.4. In vitro stability of liposomes in serum
It is important for liposome to have superior stability in biological conditions, which is closely related to
governing the activity of the associated therapeutic agent. Transmittance of different liposomes were
monitored in the presence of 50% FBS. As shown in Fig. 3, the transmittance of the liposomes were above
90% and hardly changed after 48 h incubation with 50% FBS. This indicated that the liposomes were
stable enough to prevent the interaction between liposomes and serum protein, which is important for a
long blood half-life in vivo.
3.5. Hemolysis assays
9

Hemocompatibility is a key point for in vivo applications of liposomes. As shown in Fig. 4, hemolysis
assay of PTX-loaded liposomes demonstrated that the two types of liposomes did not show any significant
increase in the hemoglobin release with up to 600 nmoles of phospholipids. PTX-Glu6-RGD-Lip did not
display concentration-dependent increase in hemolysis as well and less than 10% hemolysis was always
regarded as non-toxic, suggesting that it had a good biosecurity.
3.6. Cytotoxicity
The cytotoxicity of different liposomes on MDA-MB-231 cells was evaluated using MTT assay. As
shown in Fig. 5, free PTX showed higher inhibition rate than PTX-loaded liposomes, because free drugs
could be transported into the cells directly by passive diffusion, without a drug release process.
Furthermore, the cytotoxicity of blank liposomes was also measured, and it was shown that all the kinds of
blank liposomes had no significant cytotoxicity. Hence, our liposomal drug delivery system was safe and
non-toxic to be further used in vivo.
3.7. HAP binding assay
HAP is a main component in bone tissue, investigation of the HAP affinity has been widely used to
evaluate the potential bone targeting of nano-sized drug delivery system. In this experiment, we compared
the HAP binding efficiency between PTX, PTX-Lip, PTX-Glu6-Lip, PTX-RGD-Lip, PTX-Glu6+RGD-Lip
and PTX-Glu6-RGD-Lip. As shown in Figure 6, compared with PTX, PTX-Lip and PTX-RGD-Lip,
PTX-Glu6-Lip, PTX-Glu6+RGD-Lip and PTX-Glu6-RGD-Lip exhibited a significantly higher HAP
binding efficiency with hydroxyapatite power. These results demonstrated that Glu6-modified liposome
had good bone-targeting ability, and facilitated the next in vivo experiments.
3.8. Targeting metastatic bone in vivo
To determine whether the liposomes PTX-Glu6-RGD-Lip could promote the accumulation of paclitaxel
in the bone metastatic lesions, a bone metastasis model was established by injecting MDA-MB-231 cancer
cells into the tibia of Balb/c mice. The PTX concentration in bone metastatic lesions and normal bone was
detected at 0.5 h, 1 h, 2 h, 4 h and 8 h after intravenous administration of different formulations. It was
obviously found that all PTX-loaded liposomes had a higher concentration than that of PTX injection. As
shown in Fig. 7A-B, the PTX concentration both in the metastatic bone and normal bone lesions of
10

Glu6-modified liposomes groups was obviously increased compared with PTX-Lip and PTX-RGD-Lip
attributing to the bone targeting ability of glutamic oligopeptides, which was consistent with the result of
the HAP binding assay. What’s more, the results showed that it had an obviously increased accumulation in
bone metastatic lesions than in normal bone. Furthermore, PTX-Glu6-RGD-Lip had an increased
concentration compared to PTX-Glu6-Lip and PTX-Glu6+RGD-Lip groups due to the synergistic effect of
the dual-mediated endocytosis. As shown in Fig. 7B, the PTX in metastatic bones following the injection
of PTX-Glu6-RGD-Lip was 5-8 times higher than that following PTX injected, 3-5 times higher than that
following PTX-Lip injected, and 1-3 times higher than that following PTX-Glu6-Lip, PTX-RGD-Lip and
PTX-Glu6+RGD-Lip. These results indicated the targeting effect of PTX-Glu6-RGD-Lip for metastatic
bones, which could lead to the improved efficacy in bone metastasis and reduced toxicity to other tissues.
4. Conclusion
In this study, we have developed Glu6-RGD-modified liposomal drug delivery system. The liposome
formulation was capable of bone affinity through glutamic oligopeptides, and specific tumor recognition
by RGD modification on the surface of the particles. PTX-Glu6-RGD-Lip possess good stability, high
HAP binding efficiency, as well as improved cytotoxicity. More importantly, the in vivo targeting
metastatic bone study suggested that PTX-Glu6-RGD-Lip favored accumulation of loaded paclitaxel in the
metastatic bones. In general, this work has a promising application in bone metastasis targeting delivery
systems and more work is in progress.
Conflict of Interest
The authors declare no competing financial interest.
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Figure 1. The structure of the ligand Glu6-RGD-Chol
Figure 2. The PTX release profiles of free PTX, PTX-Lip, PTX-Glu6-Lip, PTX-RGD-Lip, PTX-Glu6+RGD-Lip and
PTX-Glu6-RGD-Lip in PBS (pH 7.4) containing 0.1% Tween 80 over 48 h (n = 3, mean ± SD).
Figure 3. The variations of transmittance of different modified liposomes in 50% FBS (n=3, mean ± SD).
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Figure 4. Hemolysis percentage of different liposomes (n = 3, mean ± SD).
Figure 5. (A) The cytotoxicity study of PTX-loaded liposomes and free PTX against MDA-MB-231 cells. (B) The
cytotoxicity study of blank liposomes against MDA-MB-231 cells (n = 3, mean ± SD).
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Figure 6. HAP binding ratio of different types of PTX-loaded liposomes and PTX after incubation with HAP for 2 h (n = 3,
mean ± SD).
Figure 7. PTX concentration 0.5 h, 1 h, 2 h, 4 h and 8 h after injection of free PTX, PTX-Lip, PTX-Glu6-Lip, PTX-RGD-Lip,
PTX-Glu6+RGD-Lip and PTX-Glu6-RGD-Lip in the normal bone (A) and bone metastatic lesions (B). (Five mice in each
group, mean ± SD)
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Scheme 1 Synthesis of ligand Glu6-RGD-Chol. Reagents and conditions: (a) i) IBCF, NMM, THF, -15 °C - r.t., 20 h; ii) TFA,
CH2Cl2, r.t., 1 h. (b) Repeat steps as Step (a) for four times. (c) succinic anhydride, CH2Cl2, 50 oC, 2 h. (d) Asp(OBzl)-OBzl,
IBCF, NMM, CH2Cl2, -10 oC - r.t., 2 h. (e) HCl, THF, r.t., 2 h. (f) Boc-Arg(NO2)-OH, IBCF, NMM, DMF, -10 oC - r.t., 2 h.
(g) TFA, CH2Cl2, r.t., 4 h. (h) succinic anhydride, dioxane, 80 oC, 0.5 h. (i) TsCl, pyridine, 50°C, 5 h. (j) Triethylene glycol,
dioxane, reflux, 6 h. (k) t-butyl bromoacetate, n-Bu4N+HSO4, 50% NaOH, toluene, r.t., 16 h. (l) TsOH, tolunene, reflux, 8 h.
o
o
(m) diethanol amine, IBCF, NMM, CH2Cl2, -10 C - r.t., 5 h. (n) 11, DCC, DMAP, CH2Cl2, -5 C - r.t., 10 h. (o) 5, DCC,
DMAP, CH2Cl2, -5 oC - r.t., 20 h. (p) H2, Pd/C, CH3OH, 50 oC, 40 h.
Scheme 2 Synthesis of ligand Glu6-Chol. Reagents and conditions: (a) 4, IBCF, NMM, THF, -15 °C -r.t., 10 h. (b) H2, Pd/C,
CH3OH, r.t., 20 h.
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Table 1 The characterization of different PTX-loaded liposomes (n = 3)
Liposomes
Size(nm)
PTX-Lip
103.5 ± 4.2
0.185 ± 0.019
91.85 ± 2.78
-2.39 ± 0.22
PTX- Glu6-Lip
119.3 ± 3.9
PTX-RGD-Lip
108.2 ± 2.8
PTX-Glu6+RGD -Lip
115.3 ± 3.4
PTX-Glu6-RGD -Lip
121.9 ± 4.7
PDI
2.012 ± 0.009
89.21 ± 5.04
-15.69 ± 2.34
0.190 ± 0.022
90.77 ± 4.31
-4.52 ± 0.57
0.194 ± 0.038
89.57 ± 3.54
0.197 ± 0.034
88.62 ± 6.26
EE (%)
Zeta potential (mV)
-15.11 ± 2.68
-14.37 ± 4.85
18