Design, synthesis and evaluation of liposomes modified with dendritic aspartic acid for bone-specific targeting

Article abstract of DOI:10.1016/j.chemphyslip.2019.104832

Bone diseases are notoriously difficult diseases to treat due to the comparatively low blood flows in bone tissue. Therefore, targeting delivery of drugs to bone may not only enhance the treatment efficacy, but also reduce the quantity of drug administered. In order to increase the distribution of paclitaxel (PTX) in bone, in this study, a series of novel dendritic aspartic acid derivatives were designed and synthesized as liposome ligands to deliver PTX to bone effectively. The liposomes were prepared by thin film hydration method and its particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis were also characterized. All the aspartic acid-coated liposomes showed more than 60% binding rates to hydroxyapatite (HAP), especially the PTX-Asp₈-Lip exhibited dramatic binding rates (> 97%) after 24 h. Moreover, the bone-targeting study in vivo indicated that all liposomes could improve the accumulation of PTX in bone, among which, the PTX-Asp₈-Lip showed the best affinity due to the increase of aspartic acid residues exposed on the liposome surface. These results provided a novel and effective entry to the development of bone-targeting drugs.

Full text of DOI:10.1016/j.chemphyslip.2019.104832

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Design, synthesis and evaluation of liposomes modified with dendritic
aspartic acid for bone-specific targeting
Ze Zhao, Changqing Chen, Changwei Xie, Yi Zhao
PII:
S0009-3084(19)30197-5
DOI:
https://doi.org/10.1016/j.chemphyslip.2019.104832
CPL 104832
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Chemistry and Physics of Lipids
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9 August 2019
9 September 2019
21 September 2019
Please cite this article as: Zhao Z, Chen C, Xie C, Zhao Y, Design, synthesis and evaluation of
liposomes modified with dendritic aspartic acid for bone-specific targeting, Chemistry and
Physics of Lipids (2019), doi: https://doi.org/10.1016/j.chemphyslip.2019.104832
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Design, synthesis and evaluation of liposomes modified
with dendritic aspartic acid for bone-specific targeting
Ze Zhaoa,*, Changqing Chena, Changwei Xiea, Yi Zhaob,*
a Department of Orthopedics, the First Affiliated Hospital of Henan Polytechnic University (the Second People's
Hospital of Jiaozuo City), Jiaozuo 454001, China.
b
Department of Translational Medicine Center, the First Affiliated Hospital of Zhengzhou University,
Zhengzhou 450052, China
*Corresponding authors. zezhao0910@163.com (Z. Zhao), zhaoyi0910@163.com (Y. Zhao)
Graphic Abstract
Highlights
A series of novel dendritic aspartic acid derivatives were designed and synthesized as liposome

1

ligands.
The preliminary evaluation in vivo demonstrated that ligand Asp8-coated liposome had an improved
bone-targeting ability.


The quantity of Asp residues will increase the affinity for bone.
Abstract
Bone diseases are notoriously difficult diseases to treat due to the comparatively low blood flows in bone
tissue. Therefore, targeting delivery of drugs to bone may not only enhance the treatment efficacy, but also
reduce the quantity of drug administered. In order to increase the distribution of paclitaxel (PTX) in bone,
in this study, a series of novel dendritic aspartic acid derivatives were designed and synthesized as
liposome ligands to deliver PTX to bone effectively. The liposomes were prepared by thin film hydration
method and its particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis
were also characterized. All the aspartic acid-coated liposomes showed more than 60% binding rates to
hydroxyapatite (HAP), especially the PTX-Asp8-Lip exhibited dramatic binding rates (> 97%) after 24 h.
Moreover, the bone-targeting study in vivo indicated that all liposomes could improve the accumulation of
PTX in bone, among which, the PTX-Asp8-Lip showed the best affinity due to the increase of aspartic acid
residues exposed on the liposome surface. These results provided a novel and effective entry to the
development of bone-targeting drugs.
Keywords: Drug delivery, Bone targeting, Dendritic aspartic acid, Liposome
1. Introduction
Bone diseases, such as bone metastasis, osteomyelitis, osteoporosis, Paget’s disease, are notoriously
difficult diseases to treat due to the comparatively low blood flows in bone tissue [Chastain and Davis,
2019; Zhao et al., 2015; Zhao et al., 2019]. Among them, bone metastasis is a frequent complication in
advanced stage of many cancers, especially breast, prostate, and lung cancers [Sun et al., 2019]. It could
give rise to bone pain, hypercalcemia, pathological fracture and bone malformation because of the
increased activity of osteoclast cells, which leads to the decrease of life quality [Yamashite et al., 2018].
Current treatments for metastatic bone cancers mainly are surgery, radiotherapy and chemotherapy, or the
2

combination of them. Although the chemotherapy has played an important role in increasing the survival
rate and improving the life quality of patients, its low targeting efficiency makes the high and frequent
administration essential, which may lead to severe side effects [Yamashite et al., 2018; Zhao et al., 2019].
Therefore, there is a huge amount of demand of specific delivery systems of chemotherapeutic agents, such
as paclitaxel (PTX), to the site of bone lesions are of great interests for the treatment of metastatic bone
cancer.
To overcome the limitations of chemotherapy for treating bone metastasis, bone-targeting drug delivery
is a promising solution to increase therapeutic efficiency and severe side effects. As we all know, bone is
consist of some unique materials, and the inorganic compound hydroxyapatite (HAP) is the major
ingredient of bone, which is a potential target for selective drug delivery to bone [Zhao et al., 2019]. In our
previous study, we have found that dendritic aspartic acid (Asp) derivatives had an excellent
bone-targeting abilities [Pan et al., 2012]. According to Sarig’s research, the mechanisms for the
bone-affinity of Asp oligopeptides is that the multi-carboxy groups of peptides provide ionic interaction
between negative charges and calcium ions in the mineral component of bone (HAP), and this kind of
affinity only depends on the number of exposed amino acid residues [Sarig 2004]. Simultaneously, we
assumed that the more the quantity of Asp residues exposed on the liposome surface, the stronger the
affinity for bone showed.
With the development of macromolecules, dendrimers, consisting of two dendrons, are widely used in
medical and biomedical fields such as drug delivery [Pan et al., 2012]. Over the years, we has done many
researches on bone targeting or dendrimers specially based on acidic peptides [Pan et al., 2012; Zhao et al.,
2015; and Zhao et al., 2019]. The acidic peptides are biodegradable, biocompatible, and has shown strong
affinity to bone. And we are still exploring how to further improve the carrier's bone targeting ability to
deliver more drugs to bone.
As a drug carrier, liposome has many advantages such as nontoxicity, biocompatibility and
biodegradability, which is widely applied in drug delivery system [Matsuo et al., 2019]. Inspired by the
affinity mechanism and the dendritic effect, we developed a series of novel dendritic aspartic acid
derivatives (Fig. 1) as ligands to increase the quantity of negatively charged groups (carboxy groups of Asp)
exposed on the liposome surface. Using post-insertion technique, the lipophilic steroidal portion was
embedded into the liposome membrane, while the hydrophilic dendritic Asp residues were exposed outside
the membrane. The dendritic Asp-modified liposomes were prepared by the lipid film hydration-ultrasound
3

method. What’s more, to investigate the bone targeting ability, the targeting ability in vitro and in vivo was
evaluated.
Figure 1. The structure of dendritic Asp-modified ligand Asp1-Chol, Asp2-Chol, Asp4-Chol, and Asp8-Chol.
2. Materials and methods
2.1. Materials
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).
Elemental analyses were performed by Atlantic Microlab (Atlanta, GA, 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).
2.2. Methods
2.2.1 Chemistry
2.2.1.1 Synthesis of compound 3-13
The synthesis of compound 3-13 was reported in our previous work [Pan et al., 2012; Zhao et al., 2014;
Zhao et al., 2015; Zhao et al., 2018; and Zhao et al., 2019].
2.2.1.2 Synthesis of compound 14
To a solution of compound 13 (1.50 g, 2.60 mmol) in CH2Cl2 (30 mL) were added isobutyl
chloroformate (IBCF, 0.43 mL, 3.38 mmol) and 4-methylmorpholine (NMM, 0.43 mL, 3.90 mmol), then
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the mixture was stirred at -15 °C for 20 min. Subsequently, Asp(OBn)-OBn (1.14 g, 3.64 mmol) in CH2Cl2
(10 mL) was added to the above reaction mixture slowly and the reaction was continued to stir for 5 h at
room temperature. The reaction was terminated and then filtered. The filtrate was washed with 1 M HCl
(20 mL), 1 M NaHCO3 (20 mL), and brine (20 mL). The organic layer was dried over sodium sulfate,
filtered, concentrated under reduced pressure and purified by silica gel column chromatography to give
compound 14 (1.90 g, 84%) as colorless oil. ESI-MS (m/z): calculated for C53H77NO9Na [M+Na]+
894.5496, found 894.5490. Elemental Analysis: C, 72.99; H, 8.90; N, 1.61, found C, 72.85; H, 9.01; N,
1.73.
2.2.1.3 Synthesis of ligand Asp1-Chol
The mixture of 14 (1.22 g, 1.40 mmol) and 10% Pd/C (0.12 g) in methanol (15 mL) was stirred at the
room temperature under hydrogen atmosphere. After 2 h, the mixture was filtered, and the filtrate was
concentrated to obtain ligand Asp1-Chol (0.90 g, 93%) as colorless oil. ESI-MS (m/z): calculated for
C39H65NO9Na [M+Na]+ 714.4557, found 714.4552. Elemental Analysis: C, 67.70; H, 9.47; N, 2.02, found
C, 67.79; H, 9.38; N, 2.11.
2.2.1.4 Synthesis of compound 15
To a solution of Asp1-Chol (0.45 g, 0.65 mmol) in CH2Cl2 (20 mL) were added IBCF (0.11 mL, 0.85
mmol) and NMM (0.11 mL, 0.98 mmol), then the mixture was stirred at -15 °C for 20 min. Subsequently,
compound 8 (2.92 g, 1.63 mmol) in CH2Cl2 (20 mL) was added to the above reaction mixture slowly and
the reaction was continued to stir for 24 h at room temperature. The reaction was terminated and then
filtered. The filtrate was washed with 1 M HCl (30 mL), 1 M NaHCO3 (30 mL), and brine (30 mL). The
organic layer was dried over Na2SO4, filtered, concentrated under reduced pressure and purified by silica
gel column chromatography to give compound 15 (1.58 g, 57%) as colorless oil. MALDITOF-MS (m/z):
calculated for C231H273N21O57Na [M+Na]+ 4277.907, found 4277.911. Elemental Analysis: C, 65.19; H,
6.47; N, 6.91, found C, 65.05; H, 6.59; N, 6.79.
2.2.1.5 Synthesis of ligand Asp8-Chol
The mixture of 15 (1.00 g, 0.23 mmol) and 10% Pd/C (0.10 g) in methanol (20 mL) was stirred at the
room temperature under hydrogen atmosphere. After 20 h, the mixture was filtered, and the filtrate was
concentrated to obtain ligand Asp8-Chol (0.56 g, 85%) as colorless oil. MALDITOF-MS (m/z): calculated
for C119H177N21O57Na [M+Na]+ 2836.153, found 2836.157. Elemental Analysis: C, 50.80; H, 6.34; N,
10.45, found C, 50.92; H, 6.44; N, 10.32.
5

2.2.1.6 Synthesis of compound 16
Compound 13 (0.92 g, 1.59 mmol) was dissolved in CH2Cl2 (30 mL) and was cooled at -15 °C. Then,
IBCF (0.28 mL, 2.23 mmol) and NMM (0.26 mL, 2.39 mmol) were added, and the mixture was stirred for
15 min. Subsequently, compound 8 (3.43 g, 1.91 mmol) in CH2Cl2 (20 mL) was added dropwise to the
reaction and the mixture was vigorously stirred for another 24 h at room temperature. The reaction was
terminated and then filtered. The filtrate was washed with 1 M HCl (30 mL), 1 M NaHCO3 (30 mL), and
brine (30 mL). The organic layer was dried over Na2SO4, filtered, concentrated under reduced pressure and
purified by silica gel column chromatography to give compound 16 (2.48 g, 66%) as colorless oil.
MALDITOF-MS (m/z): calculated for C131H164N10O30Na [M+Na]+ 2381.155, found 2381.150. Elemental
Analysis: C, 66.71; H, 7.01; N, 5.94, found C, 66.79; H, 7.10; N, 5.86.
2.2.1.7 Synthesis of ligand Asp4-Chol
10% Pd/C (0.20 g) was added into the solution of compound 16 (0.26 g, 0.11 mmol) in methanol (10
mL). The reaction mixture was stirred at the room temperature under hydrogen atmosphere for 20 h, then
filtered. The filtrate was concentrated to obtain ligand Asp4-Chol (0.16 g, 91%) as colorless oil.
MALDITOF-MS (m/z): calculated for C75H116N10O30Na [M+Na]+ 1659.776, found 1659.773. Elemental
Analysis: C, 55.00; H, 7.14; N, 8.55, found C, 55.13; H, 7.25; N, 8.45.
2.2.1.8 Synthesis of compound 17
To a solution of compound 13 (1.44 g, 2.50 mmol) in CH2Cl2 (20 mL) were added IBCF (0.44 mL, 3.50
mmol) and NMM (0.41 mL, 3.74 mmol), then the mixture was stirred at -15 °C for 20 min. Subsequently,
compound 6 (2.43 g, 3.00 mmol) in CH2Cl2 (15 mL) was added to the above reaction mixture slowly and
the reaction was continued to stir for 15 h at room temperature. The reaction was terminated and then
filtered. The filtrate was washed with 1 M HCl (20 mL), 1 M NaHCO3 (20 mL), and brine (20 mL). The
organic layer was dried over sodium sulfate, filtered, concentrated under reduced pressure and purified by
silica gel column chromatography to give compound 17 (2.66 g, 78%) as colorless oil. ESI-MS (m/z):
calculated for C79H106N4O16Na [M+Na]+ 1389.7502, found 1389.7506. Elemental Analysis: C, 69.38; H,
7.81; N, 4.10, found C, 69.46; H, 7.94; N, 4.17.
2.2.1.9 Synthesis of ligand Asp2-Chol
To a solution of compound 17 (1.69 g, 1.24 mmol) in CH3OH (20 mL), Pd/C (0.20 g, 10%) was added.
Then, the mixture was stirred in hydrogen atmosphere at r.t. for 10 h. Pd/C was filtered, and the filtrate was
concentrated to give ligand Asp2-Chol (1.12 g, 90%) as colorless oil. ESI-MS (m/z): calculated for
6

C51H82N4O16Na [M+Na]+ 1029.5624, found 1029.5627. Elemental Analysis: C, 60.82; H, 8.21; N, 5.56,
found C, 60.95; H, 8.07; N, 5.64.
2.2.2 Preparation and characterization of liposomes
PTX-loaded liposomes were prepared through thin film hydration method as our previous reported
[Zhao et al., 2019], including a main lipid composition of SPC, cholesterol, and synthesized ligands. The
component ratio optimized according to our previous report was as follows: (1) uncoated Lip,
SPC/cholesterol/: 62/33; (2) Asp1-Lip, SPC/cholesterol/Asp1-Chol: 62/33/3; (3) Asp2-Lip,
SPC/cholesterol/Asp2-Chol: 62/33/3; (4) Asp4-Lip, SPC/cholesterol/Asp4-Chol: 62/33/3; (5) Asp8-Lip,
SPC/cholesterol/Asp8-Chol: 62/33/3. In brief, the lipid materials were dissolved in the
o
chloroform/methanol (v/v = 2:1), and then dried using a rotary evaporator at 37 C, which was further
dried in vacuum for 24 h. Subsequently, the dried thin film was hydrated with PBS (pH 7.4) at 20 °C for
0.5 h and then sonicated intermittently by a probe sonicator at 80 W for 80 s to form liposomes.
To prepare PTX-loaded liposomes, appropriate amount of paclitaxel was added into the lipid organic
solution prior to the solvent evaporation. Detections about entrapment efficiency (EE%) and drug loading
efficiency (DL%) were conducted using high performance liquid chromatography (HPLC). Measurement
was performed on a LC-10A liquid chromatographic system (Shimadzu). The analytical column was a
reverse-phase HPLC column (ODS-C18 column, 4.6mm × 200 mm, 5 µm, SinoChrom) maintaining at 30
oC. The mobile phase consisted of water-methanol (67:33, v/v) with a low flow rate 1.0 mL/min. The
sample volume injected was 20 µL and the detection wavelength was 227 nm. The EE% and DL% were
calculated using the following equations: EE% = weight of encapsulated PTX / the total weight of PTX,
DL% = weight of encapsulated PTX / the total weight of PTX-loaded liposome. The particle size and zeta
potential of Lip, Asp1-Lip, Asp2-Lip, Asp4-Lip, and Asp8-Lip were characterized by Malvern Zeta sizer
Nano ZS90 (Malvern Instruments Ltd., UK).
2.2.3 In vitro paclitaxel release study of liposomes
The paclitaxel release analysis from PTX-loaded liposomes was performed in PBS (pH 7.4) using
dialysis method. Briefly, each PTX liposomal formulation (0.4 mL) and free paclitaxel were put into a
dialysis bag (MWCO = 8 000 – 14 000 Da) placed in 40 ml release medium (PBS containing 0.1% (v/v)
Tween 80). It was gently oscillated at 37 °C and 0.1 mL sample was taken out and replaced with fresh
medium at predetermined time points (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h). Then, the amount of
PTX was determined using HPLC method as mentioned above.
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2.2.4 Stability of liposomes
To investigate the stability of PTX liposomal formulations, the turbidity variations in the presence of
fetal bovine serum (FBS) and the appearance In brief, 0.1 mL PTX-loaded liposome was incubated in FBS
(0.1 mL) with moderate shaking (45 rpm) at 37 °C. The transmittance of each sample was detected at the
predetermined time points (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h) using a microplate reader (Thermo
Scientific Varioskan Flash, USA) at 750 nm. What’s more, the stability of liposomes stored at 4 °C for
several days was also investigated by monitoring their particle size and polydispersity index (PDI) at the
predetermined time points.
2.2.5 Hemolysis assays
For hemolysis assays, the mouse red blood cells (MRBCs) were obtained according to our previous
report [Zhao et al., 2019]. Briefly, the fresh mouse blood was stabilized and centrifuged at 5 × 103 rpm for
5 min to remove the supernatant. Afterwards, the precipitated MRBCs were washed with PBS, and then
diluted with PBS to a concentration of 2% (w/v). Subsequently, 0.1 mL MRBCs solution was added into
different liposomes with 0.4 mL various concentrations of lipids (10-400 nM). MRBCs incubated with
PBS and Triton X-100 were utilized as negative and positive controls, respectively. All the mixtures were
incubated at 37 °C for 2 h with gentle shaking, and were centrifuged at 1 × 104 rpm for 10 min. Finally, the
absorbance of the supernatant was measured using a microplate reader (Thermo Scientific Varioskan Flash)
at 540 nm. The hemolytic activity percentages of the different samples were calculated using the following
equation: the percent hemolysis (%) = (ASample-ANegative)/(APostive-ANegative) × 100%, where A is the
absorbance of hemoglobin.
2.2.6 Hydroxyapatite binding assay
The hydroxyapatite binding assay of these dendritic aspartic acid-modified liposomal formulations
toward bone matrix was conducted according to our previously reported procedure [Zhao et al., 2015;
Zhao et al., 2019]. In brief, the hydroxyapatite was dissolved in PBS at the concentration of 20 mg/mL.
Then, 0.3 mL PTX-Lip, PTX-Asp1-Lip, PTX-Asp2-Lip, PTX-Asp4-Lip, PTX-Asp8-Lip, and free PTX
(calculated as PTX) were respectively incubated with 0.3 mL HAP suspension and shaken gently at 37 oC.
At the same time, these liposomes were incubated in PBS without HAP used as negative control. At the
predetermined time (0.5 h, 1 h, 2 h, and 24 h), the unbound PTX was separated from the mixture by
centrifugation at 5000 rpm for 3 min. Afterwards, the unbound PTX in the supernatant was determined
quantificationally through HPLC. The binding efficiency was calculated by HAP binding rate (%) =
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(C0-C)/C0.
2.2.7 Cytotoxicity assay
In vitro cytotoxicity of the synthetic ligands (Asp1-Chol, Asp2-Chol, Asp4-Chol, and Asp8-Chol) was
measured on MDA-MB-231 cells using MTT assay. Generally, the cells were seeded into a 96-well plate at
a density of 5 × 103 cells/well, and incubated in a 5% CO2 humidified environment incubator (Thermo
o
Scientific, USA) at 37 C for 24 h. After that, the blank liposomes (PTX-unloaded) contained in fresh
medium were added at different concentrations. After 24 h incubation, 20 μL of MTT solution (5.0 mg/mL)
was added to each well and the cells were cultured for another 4 h at 37 °C. Then, the medium were
removed and the dye was dissolved in DMSO (150 μL). The amount of absorption in each well reflects the
conversion of MTT to formazan by metabolically viable cells, and the cell viability was calculated using
the following equation: ASample / AControl × 100%, where ASample and AControl, where ASample and AControl
represent the absorbance of sample cells and control cells read at 490 nm on an automatic microplate
spectrophotometer, respectively.
2.2.8 Pharmacokinetic study
Pharmacokinetic study was conducted on healthy Sprague Dawley (SD) rats and the concentration of
PTX was determined by HPLC as described previously. Briefly, SD rats (180-220 g) were randomly
divided into six groups with five rats in each group. Free PTX solution (dissolved in
ethanol/polyoxyethylated castor oil: v/v =1/1) and PTX-loaded liposome formulations (PTX-Lip,
PTX-Asp1-Lip, PTX-Asp2-Lip, PTX-Asp4-Lip, and PTX-Asp8-Lip) were intravenous administered to each
group at an equivalent dose of 10 mg/kg PTX, respectively. Blood samples (0.5 mL) from the orbit were
collected at the predetermined time points (5, 15, 30, 60, 120, 240, 480 and 1440 min) and centrifuged at
5000 rpm for 5 min at 4 °C to obtain the plasma samples. Then, the docetaxel internal standard (10 μL, 50
μg/mL) was added into 100 μL plasma samples, respectively. Extraction was performed with 0.2 mL
diethyl ether and the mixture was vortexed for 5 min. After centrifuging at 10 000 rpm for 10 min, the
supernatant was dried at 40 oC under a gentle stream of nitrogen in the pressured gas blowing concentrator.
Then the dried residue was dissolved in methanol (50 µL) and the mixture was further centrifuged at 10
000 rpm for 10 min. Samples were analyzed by HPLC.
2.2.9 Biodistribution and targeting bone in vivo
The evaluation of the bone-targeting ability of these Asp-modified liposomes was performed according
to our previous report [Zhao et al., 2019]. In brief, healthy Balb/c mice (18-22 g) were randomly divided
9

into six groups (n = 5). PTX-Lip, PTX-Asp1-Lip, PTX-Asp2-Lip, PTX-Asp4-Lip, PTX-Asp8-Lip, and the
free paclitaxel solution administered via the tail vein at an equivalent dose of 10 mg/kg PTX. 0.5 h, 1 h, 2 h,
4 h, 8 h, 16 h, 24 h after administration, the mice were killed by perfusion. The heart, liver, spleen, lung,
kidney, and the lower tibia bone was removed, rinsed with saline, blotted dry, and weighed. Then, to the
100 μL tissue homogenate with twice amount of saline was added 10 μL docetaxel (50 μg/mL) as the
internal standard, and the mixture was extracted with 0.2 mL diethyl ether. After vortexing for 5 min, the
o
mixture was centrifuged at 10 000 rpm for 10 min, and the supernatant was dried at 40 C under a gentle
stream of nitrogen in the pressured gas blowing concentrator. Then the dried residue was dissolved in
methanol (100 µL) and the mixture was further centrifuged at 10 000 rpm for 10 min. Samples were
analyzed by HPLC.
2.2.10 Statistical analysis
The concentration efficiency (CE) was used to calculate the bone-targeting capability of liposome. The
value of CE was defined as follows: CE = (Cmax)L / (Cmax)P, where L and P represented the liposomes
loading PTX and free PTX, respectively.
All the data were presented as mean ± standard deviation (SD). The statistical analyses were conducted
using GraphPad Prism 6.0 (San Diego, CA, USA). Statistical comparisons were performed by analysis of
variance (ANOVA) for multiple groups followed by Student’s t-test. The significance was defined as
follows: *P < 0.05, **P < 0.01, ***P < 0.001.
3. Results and discussion
3.1. Chemistry
The synthetic procedure of liposome ligand Asp8-Chol was outlined in Scheme 1. The dendritic
bone-targeting aspartic acid derivative 8 was synthesized according to our previous described work [Pan et
al., 2012]. Briefly, ethyl acrylate 1 and benzylamine 2 underwent a Michael addition reaction at room
temperature to obtain the intermediate 3, which was then hydrogenated, condensated with Boc-Gly-OH,
hydrolyzed to generate the core 4. Subsequently, coupling the core 4 with benzyl protected Asp
(Asp(OBn)-OBn) under the EDCI/HOBt to give the first-generation dendritic Asp 5, which was
deprotected in trifluoroacetic acid (TFA) and coupled with the core 4 to obtain the second-generation
dendron 7. Then, the compound 8 was generated by removing the Boc group in TFA. What’s more, the
acidulated cholesterol derivative 13 was obtained from commercially available cholesterol 9 [Zhao et al.,
2019]. Condensation of compound 13 with Asp(OBn)-OBn was conducted in the presence of IBCF/NMM,
10

followed by the removal of benzyl groups from 14 to give the ligand Asp1-Chol. Subsequently, the
Asp1-Chol was coupled with two equivalents of dendron 8 in the presence of IBCF and NMM to generate
15, which was then debenzylated with 10% Pd/C to give the desired ligand Asp8-Chol.
Scheme 1 Synthesis of ligand Asp8-Chol and Asp1-Chol. Reagents and conditions: (a) C2H5OH, r.t. (B) i) HCOONH4, Pd/C,
CH3OH, r.t.; ii) Boc-Gly-OH, IBCF, NMM, -15 °C - r.t.; iii) NaOH, H2O, r.t. (c) Asp(OBn)-OBn, EDCI, HOBt, CH2Cl2, r.t..
(d) TFA, CH2Cl2, r.t. (e) 4, HBTU, DIPEA, CH2Cl2, r.t. (f) TFA, CH2Cl2, r.t. (g) TsCl, pyridine, 50 °C. (h) Triethylene glycol,
dioxane, reflux. (i) t-butyl bromoacetate, n-Bu4N+HSO4, 50% NaOH, toluene, r.t. (j) TsOH, tolunene, reflux. (k)
11

Asp(OBn)-OBn, IBCF, NMM, CH2Cl2, -15 oC - r.t. (l) H2, Pd/C, CH3OH, r.t. (m) 8, IBCF, NMM, CH2Cl2, -15 oC - r.t. (n) H2,
Pd/C, CH3OH, r.t.
The synthetic route of ligand Asp2-Chol and Asp4-Chol was shown in Scheme 2. The strategy for
synthesis of Asp2-Chol and Asp4-Chol is similar to the preparation of Asp8-Chol. Briefly, the acid 13 was
respectively coupled with first-generation dendron 6 or the second-generation dendron 8 utilizing IBCF
and NMM as the coupling reagents. Afterwards, the benzyl groups of compound 17 and 16 were removed
by catalytic hydrogenolysis to afford Asp2-Chol and Asp4-Chol.
Scheme 2 Synthesis of ligand Asp4-Chol and Asp2-Chol. Reagents and conditions: (a) 8, IBCF, NMM, CH2Cl2, -15 oC - r.t.
(b) H2, Pd/C, CH3OH, r.t. (c) 6, IBCF, NMM, CH2Cl2, -15 oC - r.t. (d) H2, Pd/C, CH3OH, r.t.
3.2. Characterization of liposomes
PTX-loaded liposomes were prepared by the thin-film hydration method. One of the requirments for
liposome to target bone is that they should have proper sizes and uniform distribution [Shi et al., 2015]. As
shown in Table 1, the dynamic light scattering results suggested that all the liposomes had the mean
diameters about 100-120 nm and low polydispersity index (PDI < 0.24). All the Asp-modified liposomes
had the most negative zeta-potential, while the PTX-Lip showed a slightly negative potential. It maybe
attribute to the modification with Asp on the liposome’s surface, which could increase the net negative
charge due to the high density negative carboxyl of Asp and decrease the absorption of the
reticuloendothelial system and immune response [Murugan et al., 2015]. The entrapment efficiency were
greater than 86%, and drug loading efficiency were about 3%, respectively. All of results contributed to
achieve the passive targeting through the enhanced permeability and retention (EPR) effect [Zhang et al.,
2009].
Table 1 The characterization of different PTX-loaded liposomes (n = 3)
Liposomes
Size(nm)
PTX-Lip
PTX-Asp1-Lip
109.2 ± 3.8
PTX-Asp2-Lip
106.5 ± 3.3
PTX-Asp4-Lip
112.4 ± 5.1
PTX-Asp8-Lip
113.6 ± 4.3
102.7 ± 4.9
12

PDI
EE (%)
0.192 ± 0.037
92.54 ± 3.67
3.19 ± 0.17
0.199 ± 0.024
92.11 ± 4.87
3.11 ± 0.25
0.217 ± 0.019
87.46 ± 5.19
2.97 ± 0.22
0.232 ± 0.022
88.90 ± 3.25
3.02 ± 0.27
0.224 ± 0.030
86.82 ± 4.34
2.94 ± 0.30
DL (%)
Zeta potential (mV)
-1.98 ± 0.27
-10.42 ± 1.52
-13.96 ± 2.37
-15.52 ± 3.96
-19.33 ± 3.07
In vitro release studies were used to evaluate entrapment, membrane flexibility and integrity, and
affinity of the drug to the carrier systems. In this study, the PTX release behavior from liposomes was
evaluated using the dialysis method compared with that of the PTX solution (as control). As shown in Fig.
2A, it was revealed that the PTX solution had a fast release characteristic as more 80% of PTX was
released within 10 hours and approximately 100% was released after 24 h. While, the result also showed
that PTX-loaded liposomes achieved sustained release behaviors. The PTX-loaded liposome formulations
released about 50% of the loaded PTX within 8 hours, and the release exhibited a slow release rate in
another 40 hours. What’s more, it was worth noting that there was no significant difference between
PTX-Lip, PTX-Asp1-Lip, PTX-Asp2-Lip, PTX-Asp4-Lip, and PTX-Asp8-Lip on the release property. And
none of the PTX-loaded liposomes displayed burst initial release patterns. All the results indicated that
dendritic aspartic acid modification on the surface of liposome didn't change the release profile of PTX.
Figure 2. In vitro characterization of PTX-loaded liposomes. (A) The PTX release profiles of free PTX, PTX-Lip,
PTX-Asp1-Lip, PTX-Asp2-Lip, PTX-Asp4-Lip, and PTX-Asp8-Lip in PBS containing 0.1% Tween 80. (B) The variations of
transmittance of different modified liposomes in 50% FBS. (C) Size at 4 oC for three days. (D) PDI at 4 oC for three days. (E)
Hemolysis percentage of different liposomes. (n = 3, mean ± SD)
Liposomal particle stability against physiological condition is prerequisite for the further application in
vivo [Taira et al., 2004]. The turbidity variations of different liposomes were measured to investigate the
13

stability of liposomes in serum. As shown in Fig. 2B, the transmittances of all the liposomes were over
90% and these liposomes didn’t show obvious change within 48 h cultured with FBS, which suggested that
no aggregation or sediment was appeared. On the other hand, the results of physical stability was shown in
Fig 2C and 2D. The average size was almost unchanged at 4 °C for three days (Fig. 2C), and PDI increased
slightly (Fig. 2D). All the results implied that the liposomes were sufficient serum stability and physical
stability, which is important for obtaining a longer half-life of blood in vivo.
The hemocompatibility of the Asp-modified liposomes drug delivery system was evaluated using the e
erythrocyte hemolysis test. As expected, hemolysis assay of PTX-loaded liposomes demonstrated that the
five types of liposomes did not show any significant increase in the hemoglobin release with up to 400 nM
of phospholipids (Fig. 2E) and concentration-dependent increase in hemolysis (< 5% hemolysis rate was
regarded as non-toxic) [Li et al., 2018], which suggested that they had a good biosecurity.
3.3. HAP binding assay
As shown in Fig. 3, all the liposomes exhibited some HAP affinity, whereas the free paclitaxel was at
best negligibly bound. Moreover, compared with the uncoated liposome PTX-Lip, the Asp-modified
liposomes showed dramatic binding rates. It was also revealed in Fig. 2 that the HAP binding efficiency
was increased in a time-dependent manner. And the more Asp on the surface of liposomes, the stronger the
HAP binding ability. These results demonstrated that dendritic Asp-modified liposomes had good
bone-targeting ability, and facilitated the next in vivo experiments.
Figure 3. Percentage of PTX from each formulation bound to HAP. *P < 0.05, ***P < 0.001. (n = 3, mean ± SD)
3.4. Cytotoxicity assay
In spite of extensive applicability in biological system, the use of dendron for liposomal drug delivery is
constrained due to its inherent toxicity. Surface engineering, including PEGylation, amino acids
14

conjugation and peptide conjugation, is one of the most effective ways to improve the biocompatibility of
dendron [Pan et al., 2012]. In this study, natural amino acids were chosen to modify liposome for
bone-targeting. Fig. 4 showed the MTT assay results of cytotoxicity of blank liposomes against
MDA-MB-231 cells. As expected, the results suggested that the PTX-unloaded formulations didn’t show
significant cytotoxicity at different concentrations (0.64-2000 ng/mL), and >90% of MDA-MB-231 cells
survived. As a summary, all the unloaded formulations, especially the synthetized aspartic acid dendrons,
were non-toxic or low-toxic in the tested concentrations.
Figure 4. Cytotoxicity of unloaded liposomal formulations (n = 3, mean ± SD)
3.5. Pharmacokinetic studies
For pharmacokinetic study, the HPLC analytical method was established and validated as our previous
report [Zhao et al., 2019]. The pharmacokinetic profiles of paclitaxel from PTX-loaded liposomal
formulations and PTX solution were compared by measuring the concentration of PTX in rat plasma after
administration at 10 mg/kg. The main pharmacokinetic parameters were analyzed by DAS 2.0. As shown
in Fig. 5 and Table 2, the concentration of PTX released from each liposome (PTX-Lip, PTX-Asp1-Lip,
PTX-Asp2-Lip, PTX-Asp4-Lip, and PTX-Asp8-Lip) was much higher than that of naked PTX. But there
were no significant differences between these liposomal formulations. It was also outlined that the
liposomes could increase the area under the concentration-time curve (AUC0–t) of PTX within 24 h
compared with the PTX solution group. What’s more, the Asp-modified liposomes could extend the
elimination half-life (t1/2) of free PTX from 247 min to 395 min (p < 0.01). All the results indicated that the
targeted liposomes might be temporarily restored in the plasma and slowly dissociate from the contents in
plasma, and this consequently lead to the superior circulation time of modified liposomes in vivo.
15

Figure 5. Plasma concentrations of paclitaxel in normal SD rats of different PTX formulations (n = 5, mean ± SD).
Table 2. The pharmacokinetic parameters of different PTX formulations. **P < 0.01. (n = 5, mean ± SD)
AUC(0-t) (μg/mL·min)
Cmax (μg/mL)
t1/2 (min)
PTX
8537.99 ± 1547.26
50.37 ± 5.10
247.52 ± 19.88
PTX-Lip
12589.47 ± 1787.52**
13547.61 ± 2086.11**
13375.65 ± 1439.23**
13586.48 ± 1756.36**
13995.76 ± 1883.61**
56.67 ± 4.64
56.44 ± 2.94
57.67 ± 3.39
59.21 ± 7.79
60.00 ± 1.63
343.29 ± 46.19**
367.11 ± 39.84**
371.58 ± 49.63**
382.47 ± 51.67**
395.87 ± 58.91**
PTX-Asp1-Lip
PTX-Asp2-Lip
PTX-Asp4-Lip
PTX-Asp8-Lip
3.6. Biodistribution and targeting bone in vivo
The biodistribution study was conducted in healthy Balb/c mice to determine whether there was
preferential targeting of the dendritic Asp-modified liposomes to bone in vivo. The paclitaxel was
quantified in each organ and calculated as the percentage of administrated dose per gram of tissue
(%AD·g-1). As shown in Fig. 6, it was observed that a considerable amount of PTX from paclitaxel
solution was accumulated in the heart (Fig. 6A) and kidney (Fig. 6E) at 2 h post-administration. While, the
uptake was reduced by a factor of five (heart) and a factor of seven (kidney) respectively after 8 h. What’s
more, it should be worth noting that there was a relatively lower drug uptake in other organs (Fig. 6B, 6C,
6D), especially in lower tibia bone (Fig. 6F).
For all the liposome formulations, it was shown that both liver and spleen had significant uptake in these
groups (Fig. 6B, 6C). Liver and spleen are considered to be the major macrophage organs, and thus the
intravenously injected liposomes were eliminated rapidly through the two organs. The uptake of
16

Asp-modified liposomes groups in liver was 10% and spleen was about 15% respectively at 2 h
post-administration. But, there was no significant difference between these groups. It was also found that
the uptake of other organs (heart, lung, and kidney) were also more consistent between the dendritic
aspartic acid modified liposomes groups (Fig. 6A, 6D, 6E). Interestingly, the Fig. 6F showed that there was
a significant accumulation in lower tibia bone in the Asp-modified liposomes groups attributing to the bone
targeting ability of aspartic acid. All the PTX-loaded liposomes reached the maximum distribution in the
bone lesion at 2 h after injection. Especially, the concentration efficiencies (CEs) of PTX-Asp8-Lip,
PTX-Asp4-Lip, PTX-Asp2-Lip, PTX-Asp1-Lip, PTX-Lip were enhanced to 4.65 (p < 0.001), 3.56 (p <
0.001), 2.85 (p < 0.01), 2.31 (p < 0.01), 1.97 (p < 0.01) times higher than that of free PTX group. And it is
worth noting that it showed an increasing trend with the increase of the number of aspartic acid residues.
So these data further proved our conjecture that all the ligand-modified liposomes can deliver paclitaxel to
the bone lesions, which had a descending trend as PTX-Asp8-Lip > PTX-Asp4-Lip > PTX-Asp2-Lip >
PTX-Asp1-Lip > PTX-Lip. Moreover, the more quantity of Asp residues exposed to the liposome surface,
the more paclitaxel delivered to bone, which means the stronger affinity to bone. Therefore, the
PTX-Asp8-Lip showed the best affinity, which was consistent with the result of the HAP binding assay.
These results provided a novel and effective entry to the development of bone-targeting drugs.
Figure 6. In vivo distribution of PTX after injection of free PTX, PTX-Lip, PTX-Asp1-Lip, PTX-Asp2-Lip, PTX-Asp4-Lip,
and PTX-Asp8-Lip. (A) heart, (B) liver, (C) spleen, (D) lung, (E) kidney, (F) bone. *P < 0.05, **P < 0.01, ***P < 0.001. (n =
5, mean ± SD)
4. Conclusion
17

In this study, we have designed and successfully synthesized a series of novel dendritic aspartic acid
derivatives with high affinity as ligands for bone targeting liposomes. These liposomes possess good
biosecurity and high HAP binding efficiency in vitro. What’s more, the bone-targeting study in vivo
suggested that all liposomes could increase the accumulation of PTX in bone, among which, the
PTX-Asp8-Lip showed the best affinity due to the more aspartic acid residues exposed on the liposome
surface. Generally, this work presented a way that the synthesis of liposome ligand may be used as
potential means to improve the therapeutic efficiency in bone diseases.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgement
This work was supported by the National Natural Science Foundation of China (No.81903448).
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