Novel galactosylated poly(ethylene glycol)-cholesterol for liposomes as a drug carrier for hepatocyte-targeting

Article abstract of DOI:10.1166/jnn.2015.9707

In this study, three types of galactosylated cholesterol (i.e., gal-PEG194-chol, gal-PEG1000-chol and gal-PEG2000-chol) were synthesized with one terminal of polyethylene glycol of various chain lengths conjugated to the galactoside moiety, and the other terminal conjugated to the cholesterol. The galactose-modified liposomes were prepared by thin film-hydration method and doxorubicin (DOX) was loaded to the liposomes by using a ammonium sulfate gradient procedure. The liposomal formulations with galactosylated cholesterol were characterized. Flow cytometry and laser confocal scanning microscopy analyses showed that the galactose-modified liposomes facilitated the intracellular uptake of liposomes into HepG2 via asialoglycoprotein receptor (ASGP-R) mediated endocytosis. Cytotoxicity assay showed that the cell proliferation inhibition effect of galactosemodified liposomes was higher than that of the unmodified liposomes. Additionally, the study on frozen section of liver showed that the galactose-modified liposomes enhanced the intracellular uptake of liposomes into hepatocytes. Taken together, these results suggested that liposomes containing such galactosylated cholesterol (i.e., gal-PEG-chol), had a great potential as drug delivery carriers for hepatocyte-selective targeting.

Full text of DOI:10.1166/jnn.2015.9707

Article
Journal of
Nanoscience and Nanotechnology
Vol. 15, 4058–4069, 2015
Copyright © 2015 American Scientific Publishers
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Printed in the United States of America
Novel Galactosylated Poly(ethylene glycol)-Cholesterol
for Liposomes as a Drug Carrier for Hepatocyte-Targeting
Huafang Zhang1ꢀ†, Yan Xiao1ꢀ†, Shengmiao Cui2, Yuefang Zhou1, Ke Zeng1,
Mina Yan1, and Chunshun Zhao1ꢀ∗
1Guangzhou Higher Education Mega Center, School of Pharmaceutical Sciences,
Sun Yat-Sen University, Guang Zhou 510006, P. R. China
2Guangzhou Higher Education Mega Center, School of Traditional Chinese Medicine,
Guangdong Pharmaceutical University, Guang Zhou 510006, P. R. China
In this study, three types of galactosylated cholesterol (i.e., gal-PEG194-chol, gal-PEG1000-chol
and gal-PEG2000-chol) were synthesized with one terminal of polyethylene glycol of various chain
lengths conjugated to the galactoside moiety, and the other terminal conjugated to the cholesterol.
The galactose-modified liposomes were prepared by thin film-hydration method and doxorubicin
(DOX) was loaded to the liposomes by using a ammonium sulfate gradient procedure. The lipo-
somal formulations with galactosylated cholesterol were characterized. Flow cytometry and laser
confocal scanning microscopy analyses showed that the galactose-modified liposomes facilitated
the intracellular uptake of liposomes into HepG2 via asialoglycoprotein receptor (ASGP-R) medi-
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ated endocytosis. Cytotoxicity assay showed that the cell proliferation inhibition effect of galactose-
IP: 45.52.164.210 On: Thu, 05 Nov 2015 17:05:09
modified liposomes was higher than that of the unmodified liposomes. Additionally, the study on
Copyright: American Scientific Publishers
frozen section of liver showed that the galactose-modified liposomes enhanced the intracellular
uptake of liposomes into hepatocytes. Taken together, these results suggested that liposomes con-
taining such galactosylated cholesterol (i.e., gal-PEG-chol), had a great potential as drug delivery
carriers for hepatocyte-selective targeting.
Keywords: Hepatocyte-Targeting, Galactose, Doxorubicin, Liposomes, Asialoglycoprotein
Receptor.
1. INTRODUCTION
decreasing Kd values of ∼10−3, ∼10−6, ∼5 × 10−9, and
∼10−9 M, respectively.5ꢀ10
Human hepatocellular carcinoma (HCC) is one of the most
deadly and most rapidly increasing cancer in the world, it
is frequently a terminal complication of chronic inflamma-
tory and fibrotic liver diseases.1ꢀ2
The asialoglycoprotein receptor (ASGP-R) is expressed
by liver parenchymal cells, which contain 1–5×105 bind-
ing sites per cell.3–5 It can bind desialylated proteins from
the serum and internalize them in the cell interior.6ꢀ7
The ASGP-R can recognize a wide variety of desialy-
lated glycoproteins and neoglycoproteins that contain ter-
minal ꢁ-D-galactose or N-acetylgalactosamine residues.8ꢀ9
The affinity of the ASGP-R increases for mono-, di-, tri-,
and tetra-antennary oligosaccharides with correspondingly
Liposomes can alter the distribution and bioavailability
of encapsulated drugs, which represents a good approach
for treatment of several diseases.14–16 Liposomes are com-
monly cleared by the reticular endothelial system (RES)
when administered intravenously.13ꢀ17 Kupffer cells in the
liver are part of the RES. Relatively high accumulation
of administered liposomes is usually observed in the liver,
mostly in non-parenchymal cells.13ꢀ18
Polyethylene glycol (PEG)-coated liposomes can remain
in the circulating blood for long periods and have pas-
sive targeting effect to tumor sites, which improve the
therapeutic effects of encapsulated drugs. In addition to
passive targeting, active targeting of liposomes is a promis-
ing approach to improving the pharmacological effect of
drugs and reducing their side-effects during treatment.19ꢀ20
Receptor-mediated drug targeting is a promising approach
∗Author to whom correspondence should be addressed.
†These two authors contributed equally to this work.
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doi:10.1166/jnn.2015.9707

Zhang et al.
Novel Galactosylated Poly(ethylene glycol)-Cholesterol for Liposomes as a Drug Carrier for Hepatocyte-Targeting
to obtain selective drug delivery to diseased tissues in vivo,
leading to a reduction in drug toxicity and improved thera-
peutic outcomes. Uptake of ligand-incorporated liposomes
is significantly higher than that of liposomes without lig-
ands. Delivery of drugs using liposomes bound to ASGP-R
in a specific manner would provide significant therapeu-
tic benefits in hepatic diseases. Galactose-terminated com-
pounds, such as lactosylceramide,21 asialofetuin (AF)22 or
synthetic glycolipids23 have been used to modify lipo-
somes for selective accumulation in the liver, i.e., targeting
hepatocytes.13 Since the recognition of galactosylated lipo-
somes by the receptors is highly efficient, the liposomes
can be rapidly delivered to the target cells.
Chemical Co. (St. Louis, MO, USA). PEG194 and
PEG1000 were purchased from Aladdin Chemistry Co.,
Ltd.; Galactose was purchased from Aladdin Chemistry
Co., Ltd. and Shanghai Baoman Biological technology
Co., Ltd.; DSPC was purchased from Genzyme Phar-
maceuticals. DSPE-PEG2000 was purchased from Avanti
Polar Lipids, Inc.
HepG2 cells and HeLa cells were purchased from
the laboratory animal center of Sun Yat-Sen University.
Cells were cultured in DMEM medium supplemented
with 10% fetal bovine serum and antibiotꢀics (streptomycin
100 lg/mL, penicillin 100 U/mL) at 37 C in humidified
air with 5% CO2.
In recent decades, different types of glycolipids with
lipophilic anchor moieties have been synthesized for incor-
poration into liposomes.24ꢀ25 Cholesterol, one of the lipid
components used to form liposomes, is usually selected
as the lipophilic anchor moiety for stably introducing the
galactosyl moiety into liposomes.9ꢀ20ꢀ24ꢀ26
2.2. Experimental Animals
Male KM mice aged 6–8 weeks (18–20 g) were pur-
chased from the laboratory animal center of Sun Yat-Sen
University.
In this study, we synthesized a novel multi-functional
galactosylated cholesterol derivative (Gal-PEG2000-chol)
containing
(i) a lipophilic anchor moiety (cholesterol) for stable
incorporation into liposomes,
(ii) a polyethylene glycol for steric stabilization and long-
circulating effect, and
(iii) a galactose moiety for the cell surface receptors in
2.3. Synthesis of Galactose-PEG-Cholesterol
Conjugates
The route of synthesis of galactose-PEG-cholesterol con-
jugates is shown in Figure 1.
(2S,3R,4S,5S,6R)-6-(acetoxymethyl)tetrahydro-2 H-py-
ran-2,3,4,5-tetrayl tetraacetate (1). Anhydrous sodium
acetate (2 g) was slowly dissolved in 20 mL of acetic
anhydride under reflux. D-galactose (2 g) was then added
to the solution and the mixture was left to react under
reflux for 1.5 h. The reaction mixture was poured into ice,
and stirred to attain a yellow powder, recrystallizated with
ethanol to obtain compound 1 (85% yield).
(2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-hydroxytetrahydro-
2 H-pyran-3,4,5-triyl triacetate (2). To the solution of
ethylenediamine (195 ꢂl) in anhydrous DCM (30 mL),
glacial acetic acid (185 ꢂl) was added dropwise. Then,
compound 1 (908.7 mg, 2.33 mmol) was added. After the
completion of the reaction monitored by TLC, the reaction
mixture was diluted with water (50 ml) and extracted
with DCM (3 × 50 ml). The combined organic layer was
washed with 1 N HCl (2 × 50 ml) and sat. NaHCO3
(2 × 50 ml), and dried over anhydrous Na2SO4. Then,
organic layer was filtered and condensed under reduced
pressure without further purification to provide desired
product 2 as a white oil (462 mg, 57%).
(2R,3S,4S,5R,6S)-2-(acetoxymethyl)-6-(2,2,2-trichloro-
1-iminoethoxy)tetrahydro-2 H-pyran-3,4,5-triyl triacetate
(3). To a stirred solution of compound 2 (462 mg,
1.33 mmol) in dry CH2Cl2 (3 mL), powdered K2CO3
(309.2 mg, 2.24 mmol) and trichloroacetonitrile (98%,
0.39 mL, 3.8 mmol) were added at room temperature. The
reaction mixture was stirred for 22 h and filtered and the
filtrate was evaporated in vacuo to dryness. The residue
was purified by column chromatography (petroleum
ether–EtOAc, 3:1). Compound 3 was obtained as a white
powder (320 mg, 49%). 1 H NMR (400 MHz, CDCl3),
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hepatocytes.27
IP: 45.52.164.210 On: Thu, 05 Nov 2015 17:05:09
It is well known that DOX can be efficiently encapsu-
lated in liposomes via transmembrane sulfate ammonium
gradients and form a stable drug-sulfate gel in the lipo-
somes interior, which results in the greater stability of
DOX liposomes in plasma and during storage.9ꢀ28 There-
fore, DOX was chosen as the model compound for the
demonstration of contents delivery because of its appeal
as a cancer chemotherapeutic agent and its fluorescence
allows it to be identified within tissues and cells.
Copyright: American Scientific Publishers
The goal of this study is to develop a galactose modified
liposomal formulation for doxorubicin (DOX) delivery and
evaluate its hepatocyte targeting effect in vitro and in vivo.
Gal-modified liposomes are composed of 1,2-distearoyl-
sn-glycero-3-phosphocholine (DSPC), Cholesterol (Chol)
and Gal-PEG2000-chol. In vitro cellular uptake of Gal-
modified liposomes was investigated in human hepatoma
HepG2 cells which are known to express asialoglycopro-
tein receptors. Flow cytometry and laser scanning confo-
cal microscope were used to evaluate the cellular uptake
of DOX liposomes in HepG2 cells. In vivo liver-targeting
effects of a variety of DOX liposomal formulations were
studied in mice.
2. MATERIALS AND METHODS
2.1. Materials
Cholesterol was purchased from Guangzhou Pharmaceu-
ticals Corporation. PEG2000 was purchased from Sigma
J. Nanosci. Nanotechnol. 15, 4058–4069, 2015
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Novel Galactosylated Poly(ethylene glycol)-Cholesterol for Liposomes as a Drug Carrier for Hepatocyte-Targeting
Zhang et al.
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Copyright: American Scientific Publishers
Figure 1. Synthesis of galactose-PEG-cholesterol conjugates.
ꢃ/ppm: 8.67 (s, 1 H), 6.61 (d, 1 H), 5.57 (d, 1 H), 5.42
(m, 2 H), 4.46 (m, 1 H), 4.20 (m, 1 H), 4.08 (m, 1 H),
2.17 (s, 3 H), 2.02 (d, 9 H).
reflux for 24 h in an inert atmosphere. The solution was
cooled and the solvent removed in vacuo. The residue
was partitioned between CHCl3 (100 mL) and water
(100 mL), washed sequentially with saturated NaHCO3
(2×50 mL), water (50 mL), and saturated brine (50 mL),
and dried over anhydrous Na2SO4, and the solvent was
removed in vacuo. The residue was purified by column
chromatography on silica gel (200–300 mesh) by using
petroleum ether/ethyl acetate (10:1) → CH2Cl2:MeOH
(35:1) to obtain compound 5 (54.2% yield). 1 H NMR
(400 MHz, CDCl3), ꢃ/ppm: 5.34 (d, 1 H), 3.66 (m, 16 H),
3.17 (m, 1 H), 0.65 (s, 3 H).
Ms-PEG194-chol (6). To a solution of 5 (1.65 g,
2.93 mmol) and triethylamine (2.0 ml, 14.6 mmol) in
anhydrous THF (146 ml) in ice bath, methanesulfonyl
chloride (340 ꢂl, 4.4 mmol) was added. After 10 min,
the solution was allowed to attain room temperature to
stirred overnight. The reaction mixture was concentrated.
The resulting precipitation was partitioned between EtOAc
and H2O (1:1, v/v). The aqueous layer was then extracted
with EtOAc. The combined organic layer was dried on
Cholest-5-en-3ꢁ-ol-p-toluenesulfonate (4). A solution
of cholest-5-en-3b-ol (10 g, 25.86 mmol) and p-toluene-
sulfonylchloride (10 g, 52.47 mmol) in dry pyridine
(100 ml) was stirred for 16 h at room temperature.
After adding water (25 ml) to the reaction, the mix-
ture was extracted with chloroform (50 ml, three times).
The organic phase was dried over anhydrous sodium sul-
fate and concentrated in vacuo. The residue was puri-
fied by flash SiO2 column chromatography with elution
of petroleum ether/ethyl acetate (20:1). Compound 4 was
obtained as a white powder (80% yield). 1 H NMR
(400 MHz, CDCl3), ꢃ/ppm: 7.81 (d, 2 H), 7.34 (d, 2 H),
5.31 (d, 1 H), 4.32 (m, 1 H), 0.96 (s, 3 H), 0.91 (s, 3 H),
0.89 (d, 6 H), 0.65 (s, 3 H).
11-(cholest-5-en-3b-yloxy)-3,6,9-trioxaundecan-1-ol (5)
.
Tetraethyleneglycol (6 mL) was added to a solution of
cholest-5-ene-3ꢁ-tosylate (3 g, 5.4 mmol) in anhydrous
1,4-dioxane (54 mL), and the mixture was stirred under
4060
J. Nanosci. Nanotechnol. 15, 4058–4069, 2015

Zhang et al.
Novel Galactosylated Poly(ethylene glycol)-Cholesterol for Liposomes as a Drug Carrier for Hepatocyte-Targeting
anhydrous Na2SO4 and concentrated. The residue was
purified by flash SiO2 column chromatography with elu-
tion of petroleum ether/ethyl acetate (5:6) to obtain com-
pound 6 (37.9% yield). 1 H NMR (400 MHz, CDCl3),
ꢃ/ppm: 5.34 (d, 1 H), 3.76 (m, 16 H), 3.08 (s, 3 H), 0.68
(s, 3 H).
30 min in an N2 atmosphere and then cooled to 0 ꢀC.
The promoter BF3 ·OEt2 (100 ꢂL) in dry CH2Cl2 (2.6 mL)
was added dropwise, and the mixture was stirred for 3.5 h.
Additional BF3 · OEt2 (200 ꢂL) in CH2Cl2 (2.6 mL) was
then added, and the mixture was allowed to be warmed to
room temperature and stirred for 6 h. The reaction mix-
ture was filtered and washed with CH2Cl2 (30 mL) and the
filtrate was evaporated in vacuo to dryness. The residue
was purified by column chromatography (CH2Cl2/MeOH
15:1) to give a mixture (this step was not to get a purity
of compound 10).
To a solution of the mixture (433 mg) in 1,4-dioxide
(15 ml), sodium methoxide (0.1 M in methanol, 5.5 ml)
was added, and the mixture was stirred for 4 h at room
temperature in an N2 atmosphere. After neutralization
with HCl, the mixture was concentrated in vacuo and
purified by silica gel chromatography (CH2Cl2/MeOH
8:1) to obtain compound 11 (16.5% yield). 1 H NMR
(400 MHz, DMSO-D6), ꢃ/ppm: 5.31 (d, 1 H), 4.78 (d,
1 H), 4.64 (d, 1 H), 4.54 (m, 1 H), 4.30 (d, 1 H), 4.10
(d, 1 H), 3.55 (m, 115 H), 0.65 (s, 3 H). 13C NMR
11-(Cholest-5-en-3b-yloxy)-3,6,9-trioxaundecanyl-
2,3,4,6-tetra-O-acetyl-b-D-galactopyranoside (7). A mix-
ture of compound 5 (704 mg, 1.25 mmol), compound 3
(741.6 mg, 1.51 mmol) and freshly activated molecular
sieves (4 Å, 3.6 g) in dry CH2Cl2 (36 ml) was stirred at
room temperaturꢀe for 30 min in an N2 atmosphere and
then cooled to 0 C. The promoter BF3 ·OEt2 (100 ꢂL) in
dry CH2Cl2 (8 mL) was added dropwise, and the mixture
was stirred for 3.5 h. Additional BF3 · OEt2 (500 ꢂL) in
CH2Cl2 (8 mL) was then added, and the mixture was
allowed to be warmed to room temperature and stirred
for 6 h. The reaction mixture was filtered and washed
with CH2Cl2 (30 mL) and the filtrate was evaporated in
vacuo to dryness. The residue was purified by column
chromatography (petroleum ether–EtOAc, 1:1) to obtain
compound 7 (227 mg, 20.3%).
(400 MHz, DMSO-D6), ꢃ/ppm: 140.50 (–O
C
C–),
O–, gala). IR
1069.
11-(cholest-5-en-3b-yloxy)-3,6,9-trioxaundecanyl-b-D
-
120.99 (–C C–, chol), 103.55 (–O
C
galactopyranoside(gal-PEG194-chol) (8). To a solution of
7 (227 mg, 0.254 mmol) in 1,4-dioxide (15 ml), sodium
methoxide (0.1 M in methanol, 6 ml) was added and the
(cm−1): ꢄO
3436, ꢄC 2944, ꢄC
H
H
O
PEG2000-chol (12). A mixture of PEG2000 (8.9 g,
4.45 mmol) and NaH (60%, 182.1 mg, 4.55 mmol) in
mixture was stirred for 4 h at room temperature in an N
dry THF (20 ml) and DMSO (20 ml) was stirred at room
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2
atmosphere. After neutralizatioIPn:w4i5th.5H2.C1l6, 4th.2e1m0iOxtnur:eThu,te0m5peNraotvur2e0f1or53107:m0i5n:0in9 an N2 atmosphere. Then the
Copyright: American Scientific Publishers
was concentrated in vacuo and purified by silica gel chro-
matography (CH2Cl2/MeOH 10:1) to obtain compound 8
(80% yield). 1 H NMR (400 MHz, CDCl3), ꢃ/ppm: 5.34
(d, 1 H), 4.32 (d, 1 H), 4.02 (m, 2 H), 3.85 (m, 3 H), 3.65
(m, 18 H), 0.68 (s, 3 H). 13C NMR (400 MHz, DMSO-
solution of compound 6 (711 mg, 1.11 mmol) in THF
ꢀ
(10 ml) was added, and the mixture was stirred at 60 C
for 8 h. The reaction mixture was cooled to room tempera-
ture and poured into 50 ml ice water. The mixture was then
extracted with CH2Cl2 (3×100 ml). The combined organic
layer was dried on anhydrous Na2SO4 and concentrated.
The residue was purified by SiO2 column chromatography
with elution of CH2Cl2/MeOH 25:1 to give compound 12
(45% yield). 1 H NMR (400 MHz, CDCl3), ꢃ/ppm: 5.34
(d, 1 H), 3.64 (m, 182 H), 0.68 (s, 3 H).
D6), ꢃ/ppm: 163.12 (–O
C
C–), 125.95 (–C C–,
, gala). MS: [M+Na] 747.5.
3432, ꢄC 2930, ꢄC 1100.
chol), 103.38 (
O
C
O
IR (cm−1): ꢄO
H
H
O
PEG1000-chol (9). A mixture of PEG1000 (10 g,
10 mmol) and NaH (60%, 430 mg, 10.75 mmol) in dry
THF (50 ml) and DMSO (50 ml), was stirred at room
temperature for 30 min in an N2 atmosphere. Then the
solution of compound 6 (1.708 g, 2.66 mmol) in THF
gal-PEG2000-chol (14). A mixture of compound 12
(756.5 mg, 0.3 mmol), compound
3 (239.2 mg,
0.49 mmol) and freshly activated molecular sieves (4 Å,
1.3 g) in dry CH2Cl2 (9 ml) was stirred at room temper-
atureꢀ for 30 min in an N2 atmosphere and then cooled
to 0 C. The promoter BF3 ·OEt2 (40 ꢂL) in dry CH2Cl2
(1.8 mL) was added dropwise, and the mixture was stirred
for 3.5 h. Additional BF3 · OEt2 (115 ꢂL) in CH2Cl2
(1.8 mL) was then added, and the mixture was allowed
to be warmed to room temperature and stirred for 6 h.
The reaction mixture was filtered and washed with CH2Cl2
(30 mL) and the filtrate was evaporated in vacuo to dry-
ness. The residue was purified by column chromatography
(CH2Cl2/MeOH 10:1) to give a mixture (this step was not
to get a purity of compound 13).
ꢀ
(20 ml) was added, and the mixture was stirred at 60 C
for 8 h. The reaction mixture was cooled to room temper-
ature and poured into 50 ml ice water. The mixture was
then extracted with CH2Cl2, (3 × 100 ml). The combined
organic layer were dried on anhydrous Na2SO4 and con-
centrated. The residue was purified by SiO2 column chro-
matography with elution of CH2Cl2/MeOH 35:1 to obtain
compound 9 (48% yield). 1 H NMR (400 MHz, CDCl3),
ꢃ/ppm: 5.34 (d, 1 H), 3.64 (m, 90 H), 3.17 (m, 1 H), 0.68
(s, 3 H).
gal-PEG1000-chol (11). A mixture of compound 9
(689 mg, 0.44 mmol), compound 3 (282.7 mg, 0.57 mmol)
and freshly activated molecular sieves (4 Å, 1.2 g) in
dry CH2Cl2 (13 ml) was stirred at room temperature for
To a solution of the mixture (710 mg) in 1,4-dioxide
(13 ml), sodium methoxide (0.1 M in methanol, 5.25 ml)
J. Nanosci. Nanotechnol. 15, 4058–4069, 2015
4061

Novel Galactosylated Poly(ethylene glycol)-Cholesterol for Liposomes as a Drug Carrier for Hepatocyte-Targeting
Zhang et al.
was added, and the mixture was stirred for 4 h at room
temperature in an N2 atmosphere. After neutralization with
HCl, the mixture was concentrated in vacuo and puri-
fied by silica gel chromatography (CH2Cl2/MeOH 5:1) to
obtain compound 14 (15.8% yield). 1 H NMR (400 MHz,
DMSO-D6), ꢃ/ppm: 5.31 (d, 1 H), 4.75 (d, 1 H), 4.61
(d, 1 H), 4.50 (m, 1 H), 4.28 (d, 1 H), 4.10 (d, 1 H),
3.83 (d, 1 H), 3.51 (m, 192 H), 0.65 (s, 3 H). 13C NMR
2.5. Characterization of DOX Liposomes
The particle size and zeta-potential of the DOX-liposomes
were analyzed by using a Malvern Zetasizer Nano ZS90
(Malvern Instruments Ltd., UK). To determine the encap-
sulation efficacy, unencapsulated DOX was separated from
liposomes by size exclusion chromatography using a
Sephadex G-50 column. PBS (pH 7.4) was used as the elu-
ent. The eluted liposomes were collected and lysed with
Triton X-100 (1% v/v). The DOX concentration was deter-
mined by UV spectrophotometry (480 nm). The envelop-
ment efficiency (EE%) of DOX was calculated based on
the ratio of liposomal drug to total drug.
(400 MHz, DMSO-D6), ꢃ/ppm: 140.51 (–O
C
C–),
121.00 (–C C–, chol), 103.56 (–O
C
O–, gala). IR
1100. HNMR,
1
(cm−1): ꢄO
3443, ꢄC 2913, ꢄC
H
H
O
13C NMR spectrum and IR spectra were seen in supple-
mentary information.
2.6. Intracellular Distribution of Liposomes by
Laser Confocal Microscopy Analysis
2.4. Preparation of Liposomes
Two different cell lines were used in this study. HepG2
cells expressing ASGP-R were derived from a human hep-
atocellular carcinoma. Hela cells without ASGP-R were
used as a control. Cells were seeded on cover glass in a
24-well culture plate at a density of 7×104 cells/well. The
cells were incubated for 24 h to 50% confluence and then
treated with free-DOX and a variety of liposomal DOX
formulations for 2 h. The cells were then washed three
times with cold PBS, fixed by 4% paraformaldehyde at
room temperature, and permeabilized with 0.5% Triton X-
100 in PBS. The cells were stained with DAPI (1 ꢂg/ml) to
visualize the nuclei. Zeiss LSM710 laser confocal micro-
scope was used to investigate the intracellular uptake and
subcellular distribution of DOX (excitation/emission wave-
length: 488 nm/560 nm).
The galactose-modified liposomes were prepared by thin-
film hydration, followed by remote loading of DOX with
ammonium sulfate gradient. Lipids were dissolved in
CHCl3 (gal-PEG-chol was dissolved in CHCl3, MeOH
(1:2)) and dried under a N2 stream. A trace amount
of chloroform was removed by keeping the lipid film
under a vacuum. The lipid film was hydrated with
250 mM (NH4ꢅ2SO4 to obtain a blank liposomes sus-
pension. The liposomes suspension was then sequentially
extruded through polycarbonate membranes with pore size
of 200 nm and 100 nm. The resulting liposomes were
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dialyzed (molecular weight cutoff size 14,000) against
IP: 45.52.164.210 On: Thu, 05 Nov 2015 17:05:09
PBS (pH 7.4) at 37 C For drug loading, DOX was dis-
Copyright: American Scientific Publishers
ꢀ
solved in a small volume of deionized water and added
to the liposomes to achieve a drug/lipid ratio of 1/ꢀ10
(mol/mol). The loading process was carried out at 65 C
for 30 min and DOX liposomes was obtained. The lipids
composition of the DOX liposomes was shown in Table I.
Gal-194-liposomes (2%), Gal-194-liposomes (4%), Gal-
1000-liposomes (2%), Gal-1000-liposomes (4%), Gal-
2000-liposomes (2%) and Gal-2000-liposomes (4%) were
prepared to investigate the effect of the liver target-
ing of galactosylated cholesterol with various PEG chain
lengths (i.e., gal-PEG194-chol, gal-PEG1000-chol and gal-
PEG2000-chol).
2.7. Cellular Uptake of Liposomes by
Flow Cytometry Analysis
Cells suspension (8×105 cells/well) were seeded in a 24-
ꢀ
well culture plate and incubated for 24 h at 37 C in 5%
CO2 until 80% confluence. The cells were then treated
with free DOX and a variety of liposomal DOX formula-
tions for 2 h. The cells were harvested and washed three
times with cold PBS. The cellular uptake of DOX was
measured by using a flow cytometer EPICS XL (Beckman
Coulter). The intracellular DOX was excited with an argon
Table I. The lipids composition of liposomes.
Mol
DSPC
Cholesterol
DSPE-PEG2000
5
Gal-PEG 194-chol
Gal-PEG 1000-chol
Gal-PEG 2000-chol
5
Common-liposomes
PEG-liposomes
55
50
55
55
55
52
54
52
54
52
54
45
45
40
40
40
43
41
43
41
43
41
Gal-2000-liposomes (5%)
Gal-194-liposomes (5%)
Gal-1000-liposomes (5%)
Gal-194-liposomes (2%)
Gal-194-liposomes (4%)
Gal-1000-liposomes (2%)
Gal-1000-liposomes (4%)
Gal-2000-liposomes (2%)
Gal-2000-liposomes (4%)
5
5
3
1
3
1
3
1
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Zhang et al.
Novel Galactosylated Poly(ethylene glycol)-Cholesterol for Liposomes as a Drug Carrier for Hepatocyte-Targeting
laser at the wavelength of 488 nm and the fluorescence
was detected at 575 nm. Files were collected for 10,000
gated events and analyzed with the FlowJo software.
Three types of galactosylated cholesterol (i.e., gal-
PEG194-chol, gal-PEG1000-chol and gal-PEG2000-chol)
were successfully synthesized. The 1 HNMR, 13CHMR
and IR identification results showed that the structures of
the three types of galactosylated cholesterol were correct.
1 HNMR, 13CNMR spectrum and IR spectra were seen
in supplementary information.
2.8. Competitive Inhibition Experiments of Galactose
Galactose was used as a competitive inhibitor, to study
whether the cellular uptake of the Gal-2000-liposomes was
via ASGP-R, HepG2 cells were seeded in 24-well plates
and incubated for 24 h, to which 100 mM galactose solu-
tion was added and then Gal-2000-liposomes was added
to incubate for 2 h. The treatment of the confocal and flow
cytometry samples was the same as that in 2.6 and 2.7.
3.2. Characteristics of the Liposomes
Liposomes were prepared by thin film hydration followed
by the extrusion as described above. The liposomes had
a mean diameter of approximately 150 nm and relatively
narrow distribution. The Zeta potential of the liposomes
ranged from −36 to −18 mV. The negative zeta potentials
might be due to the presence of the negatively charged
lipid mPEG-DSPE in the formulation. Entrapment effi-
ciency of DOX into liposomes was >93% at the drug/lipid
ratio of 1:10. The results were summarized in Table II.
After loading the DOX, the characteristics of the
liposomes did not change. DOX was encapsulated into
liposomes by transmembrane sulfate ammonium gradients
with high encapsulation efficiency. DOX and the sulfate
formed a stable drug-sulfate gel in the liposomes interior,
which resulted in greater stability of DOX liposomes dur-
ing storage. The drug loading efficiencies were similar for
liposomes with and without galactose modification.
2.9. In Vitro Cytotoxicity Assay
The cytotoxicities of various liposomes were tested on
HepG2 cells and Hela cells, respectively. Briefly, each well
of 96-well plates was seeded with 4000 cells and incubated
for 24 h. The cells were then exposed to serial concentra-
tions of free DOX or a variety of liposomal DOX formula-
tions. After for a 48 h period of incubation, 10 ꢂl of MTT
solution (5 mg/mL) was added to each well, followed by
incubation for another 4 h. Then the medium was removed
and 150 ꢂl of DMSO was added to each well for to dis-
solve the blue formazan crystals. The absorbance was read
on a microplate reader at a wavelength of 570 nm. The
experiment was carried out in triplicate. The data reported
represented the mean of triplicate measurement.
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3.3. Intracellular Uptake of the Liposomes
2.10. Study on Frozen Section of Liver
Copyright: American Scientific Publishers
Intracellular uptake of the various liposomal formulations
All in vivo protocols were reviewed and approved by
Animal Ethical and Welfare Committee of Sun Yat-
sen University. Free-DOX and a variety of liposomal
DOX formulations were administrated via tail vein at
a dose of 5 mg/ml into mice. Mice were sacrificed at
1 h after injection. The liver was excised and frozen
rapidly in dry ice, allowing the generation of ten-ꢂm-
thick cryosections. The tissue sections were fixed in
cold acetone, washed with PBS, stained with FITC-
Phalloidin (sigma), and mounted with the DAPI con-
taining medium (VECTASHIELD® with DAPI). Images
were captured by using Zeiss LSM710 laser confocal
microscope.
was evaluated by using confocal microscopy and flow
cytometry analysis. Figure 2, showed the intracellular
uptake of liposomes containing DOX (excitation/emission:
488/560 nm). The distribution pattern of fluorescent sig-
nals in HepG2 cells were significantly different among
non-modified-liposomes (Fig. 2(A1)), PEG-liposomes
(Fig. 2(B1)) and Gal-2000-liposomes (Fig. 2(C1)). Strong
DOX fluorescence intensity was observed in the nuclei of
HepG2 cells treated with galactose-modified liposomes.
To confirm the intracellular uptake of DOX-loaded lipo-
somes, the fluorescence intensity of DOX against HepG2
cells was evaluated by using flow cytometry. As shown in
Figure 3, the fluorescence intensity of DOX delivered by
the Gal-liposomes (including Gal-2000-liposomes (5%),
Gal-194-liposomes (5%), and Gal-1000-liposomes (5%))
in HepG2 cells was higher than that of other liposomes,
indicating that galactose on liposomes surface could bind
with ASGP-R, which is frequently expressed on the sur-
face of hepatocytes.
3. RESULTS AND DISCUSSION
3.1. Synthesis of Galactose-PEG-Cholesterol
Conjugates
Cholesterol is an important component of the liposomes
membrane and can improve the stability of the liposomes.
Cholesterol is more stable than phospholipids, which can
resist harsh reaction conditions. Thus we used galactose,
polyethylene glycol (PEG) and cholesterol as the starting
materials to synthesize galactosylated cholesterol of gal-
PEG-chol. The conjugates can be inserted into the lipid
bilayer membranes (lipophilic groups), and functionalize
the liposomes with a liver targeting moiety (i.e., galactose).
Gal-2000-liposomes (5%) was more efficiently taken
up by the cells compared to non-modified liposomes.
The uptake could be blocked by 100 mM free galac-
tose (Fig. 2(D)). Similarly, flow cytometry results showed
that the cellular uptake of galactose modified liposomes
was higher than that of non-modified liposomes and could
be blocked by free galactose (Fig. 3). Taken together,
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Novel Galactosylated Poly(ethylene glycol)-Cholesterol for Liposomes as a Drug Carrier for Hepatocyte-Targeting
Zhang et al.
Table II. The characteristic of the DOX liposomes (n = 3).
DOX-liposomes
Mean diameter (nm)
PDI
Z-potential (mV)
EE (%)
Common-liposomes
PEG-liposomes
189.3 4.3
173.6 5.6
162.2 3.2
164.6 5.1
161.9 5.4
180.1 1.3
184.5 1.4
158.5 0.6
156.8 0.7
146.8 0.9
164.0 1.9
0.080 0.020
0.030 0.027
0.099 0.028
0.177 0.008
0.145 0.030
0.093 0.035
0.039 0.013
0.086 0.036
0.050 0.034
0.107 0.003
0.080 0.020
−24.2 1.2
−28.9 1.5
−18.2 2.6
−23.2 2.3
−17.4 2.0
−36.7 0.3
−35.0 1.2
−34.6 1.0
−31.0 1.8
−26.6 1.2
−24.2 1.2
96.55
96.52
94.53
93.17
95.19
94.39
95.70
97.32
98.52
96.36
93.22
Gal-2000-liposomes (5%)
Gal-194-liposomes (5%)
Gal-1000-liposomes (5%)
Gal-194-liposomes (2%)
Gal-194-liposomes (4%)
Gal-1000-liposomes (2%)
Gal-1000-liposomes (4%)
Gal-2000-liposomes (2%)
Gal-2000-liposomes (4%)
these results indicated that liposomes which contained Gal-
PEG2000-chol could effectively target the HepG2 cells via
the ASGP-R.
The enhanced cellular uptake of DOX by Gal-modified
liposomes is due to receptor-mediated endocytosis. The
nuclei of the cells treated with galactose-modified lipo-
somes (Fig. 2(C1)) showed stronger DOX fluorescence
than that with PEG-modified liposomes (Fig. 2(B1)) within
2 h of incubation, indicating that process of cellular uptake
was rapid and that DOX was quickly released from the
endosomes and accumulated in the nuclei. In agreement
with confocal microscopy observation, cellular uptake effi-
ciency measured by flow cytometry revealed a remark-
able difference in DOX level in cancer cells treated with
PEG-liposomes and Gal-modified liposomes. As shown in
Figure 2, the red fluorescence intensity of Gal-modified
The mechanism of drug cellular uptake for drug-loaded
liposomes was explained by diffusion, endocytosis or
membrane fusion, depending on the characteristics of
liposomes and cells. HepG2 cells treated with PEG-
modified liposomes did not show any DOX fluorescence
in cytoplasm (Fig. 2(B1)) suggesting that non-specific
endocytosis was not involved in the DOX uptake of
liposomal formulations. Therefore, the DOX uptake for
PEG-modified liposomes might be mainly through the
mechanism of diffusion.
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Figure 2. Confocal microscopy images of HepG2 cells and Hela cells incubated with common-liposomes (A1), (A2), PEG-liposomes (B1), (B2) and
Gal-2000-liposomes (5%) (C1), (C2), 100 mM galactose+Gal-2000-liposomes (5%) (D) for 2 h at 37 ꢀC. Cells were fixed and then treated with DAPI
(blue) for nuclei staining. Red: Fluorescence of Dox. Blue: fluorescence of DAPI. Pink: The merger fluorescence of blue and red.
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Zhang et al.
Novel Galactosylated Poly(ethylene glycol)-Cholesterol for Liposomes as a Drug Carrier for Hepatocyte-Targeting
with Gal-194-liposomes (2%), Gal-194-liposomes (4%),
Gal-1000-liposomes (2%) and Gal-1000-liposomes (4%),
which showed weaker DOX fluorescence than Gal-2000-
liposomes (2%) and Gal-2000-liposomes (4%). Greater
DOX fluorescence intensity was observed in HepG2 cells
treated with Gal-2000-liposomes (4%) compared to that
treated with Gal-2000-liposomes (2%). The flow cytom-
etry data shown in Figure 4 agreed with the confocal
microscopy imaging results.
Although Gal-194 (2%), Gal-194 (4%), Gal-1000
(2%) and Gal-1000 (4%) liposomes contained galacto-
sylated cholesterol (gal-PEG194-chol and gal-PEG1000-
chol), DSPE-PEG2000 with a longer PEG chain was also
added to the liposomes. It is likely that the galactose lig-
and could not be exposed on the surface of the liposomes,
and therefore the galactose ligand could not be interacted
with the ASGP-R which was expressed on HepG2 cells.
There was no significant difference between the fluores-
cence intensity of DOX in HepG2 cells treated with Gal-
194 (2%), Gal-194 (4%), Gal-1000 (2%) and Gal-1000
(4%) liposomes and that of PEG-liposomes.
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Figure 3. Flow cytometry analysis of HepG2 cells after incubated
with PEG-liposomes and Gal-modified liposomes for 2 h with 10%FBS
medium (n = 3).
liposomes in HepG2 cells was much higher than that of
PEG-liposomes, suggesting that receptor-mediated endo-
cytosis was responsible for the improved cellular uptake
of DOX for Gal-modified liposomes but not for PEG-
liposomes because terminal galactoside residue was able
to bind with surface ASGP-R of HepG2 cells.
Galactosylated cholesterol was conjugated to PEG
with various chain lengths and their effects on cellular
uptake were investigated. To prepare Gal-194-liposomes
(2%), Gal-194-liposomes (4%), Gal-1000-liposomes (2%),
Gal-1000-liposomes (4%), Gal-2000-liposomes (2%)
and Gal-2000-liposomes (4%), galactosylated cholesterol
conjugated with various PEG chain lengths was mixed
with DSPE-PEG2000 at different concentrations. We spec-
ulated that due to the various length of the PEG chain
of the galactosylated cholesterol, the galactose ligand
on the liposomes exposure to varying degrees when
DSPE-PEG2000 was incorporated into the liposomes, thus
influenced the cellular uptake of the galactose-modified
liposomes.
The confocal images of cellular uptake in HepG2 cells
were shown in supplementary information. As expected,
there was no significant difference among cells treated
Figure 4. Flow cytometry analysis of HepG2 cells after incubated with
different PEG chain length of galactose modified DOX liposomes for 2 h
with 10%FBS medium (n = 3).
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Novel Galactosylated Poly(ethylene glycol)-Cholesterol for Liposomes as a Drug Carrier for Hepatocyte-Targeting
Zhang et al.
Gal-2000 (2%) and Gal-2000-liposomes (4%) contained
a certain amount of gal-PEG2000-chol. Although a certain
amount of DSPE-PEG200 was added, gal-PEG2000-chol
had longer PEG chains, therefore galactose ligand could
be exposed on the liposomes surface and could be inter-
acted with ASGP-R on the HepG2 cells. Hence, the flu-
orescence intensity of DOX in HepG2 cells treated with
Gal-2000 (2%) and Gal-2000-liposomes (4%) was higher
than that of the liposomes which contained galactosylated
cholesterol with a shorter PEG chains.
In this study, we choosed Hela cell lines which
lacked ASGP-R to further confirm that cellular uptake
of the galactose-modified liposomes was via the ASGP-
R
interaction. We could speculate that there was
no significant difference among the DOX fluorescence
intensity was observed in Hela cells treated with
galactose-modified liposomes, common liposomes and
PEG-modified liposomes.
Figure 5. Cell proliferation inhibition of free DOX and DOX liposomes
in HepG2 cells (n = 3).
As expected, Gal-modified liposomes (Gal-2000-
liposomes (5%)) (Fig. 2(C2)) showed a minor tendency to
be internalized by Hela cells, and there was no significant
difference between PEG-liposomes (Fig. 2(B2)) and Gal-
modified liposomes (Fig. 2(C2)). The fluorescence inten-
sity of Gal-modified liposomes in Hela cells was weaker
than that in HepG2 cells. And the flow cytometry results
(shown in supplementary information) agreed with the
result of confocal microgragh images.
shown in supplementary information. Free DOX showed
the highest inhibition effect. No significantly difference
in the inhibition of Hela cells was shown among non-
modified liposomes and gal-modified liposomes (i.e., gal-
PEG194-chol, gal-PEG1000-chol or gal-PEG2000-chol).
Hela cells do not express the ASGP-R, therefore the galac-
tose modified liposomes did not have the effect of increas-
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Hela cells did not express the ASGP-R, thus Gal-
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ing the inhibition of Hela cells.
modifed liposomes did not have thCe oepffyecritgohft:inAcmreeasriincgan Scientific Publishers
cellular uptake via receptor-mediated endocytosis. On the
other hand, HepG2 cells expressed the ASGP-R, and the
galactose ligand on the surface of Gal-modified liposomes
could be interacted with the ASGP-R and increase the
HepG2 cells uptake.
3.5. Study on Frozen Section of Liver
Confocal microscopy was used to evaluate the hepatocyte
uptake of free-DOX and DOX-liposomes in vivo. Figure 6
showed the images of liver frozen sections of KM mice
harvested 1 h after intravenous injection of free-DOX and
DOX-liposomes. The nuclei of the cells were stained with
DAPI, the F-actin outlining the cell membrane was stained
green with FITC Phalloidin, and the DOX was shown
in red. Figure 6(A) showed the image of blank control.
And the other images demonstrated that virtually all cell
nuclei (as detected by DAPI staining) were also labeled by
doxorubicin.
Examination of the images in Figure 6 revealed that
virtually all the cell nuclei, irrespective of shape, which
were stained by DAPI were also labeled by doxorubicin.
Some labeled nuclei were large and round (presumed hep-
atocyte) and brightly stained, while other nuclei were
oblong, oval (presumed non-parenchymal), or in some
cases indented.29ꢀ30 Thus, the non-parenchymal cells and
hepatocytes can be distinguished by their distinct mor-
phologies as indicated by the arrow.
3.4. Cell Cytotoxicity Assay
Figure 5 showed the cell proliferation inhibition of HepG2
cells with increasing concentration of DOX solution and
DOX-liposomes. Inhibition of cells proliferation after
exposure to Gal-modifed liposomes was much more sig-
nificant than that of non-modifed liposomes. The cytotoxi-
city of Gal-2000-liposomes (5%) and Gal-2000-liposomes
(4%) was greater than that of other liposomes. These
results indicated that Gal-2000-liposomes (5%) and Gal-
2000-liposomes (4%) could enter HepG2 cells efficiently.
These results were consistent with the observation that
liposomes which contained gal-PEG2000-chol exhibited
higher intracellular uptake in HepG2 cells than that of non-
modified liposomes. Free DOX showed the highest inhi-
bition. Doxorubicin is a small molecule drugs with highly
nucleophilic nature. It could rapidly enter the cells by dif-
fusion and inhibit the synthesis of RNA and DNA, leading
to significantly higher inhibition.
Distribution of relatively strong DOX fluorescence to
nuclei could be observed in the hepatocytes treated with
galactose-modified liposomes, indicating that the lipo-
somes incorporated with the Gal-PEG2000-chol showed
The cell proliferation inhibition of free DOX and DOX
liposomes in Hela cells after incubation of 48 h were
a
remarkably specific targeting effect to the liver
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Zhang et al.
Novel Galactosylated Poly(ethylene glycol)-Cholesterol for Liposomes as a Drug Carrier for Hepatocyte-Targeting
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Copyright: American Scientific Publishers
Figure 6. Confocal microscopy images of liver sections of free DOX and DOX liposomes, Blank (A), free DOX (B), common-liposomes (C),
PEG-liposomes (D), Gal-2000-liposomes (5%) (E), Gal-194-liposomes (2%) (F), Gal-194-liposomes (4%) (G), Gal-1000-liposomes (2%) (H) , Gal-
1000-liposomes (4%) (I), Gal-2000-liposomes (2%) (J), Gal-2000-liposomes (4%) (K). Nuclei were stained blue with DAPI, FITC was shown as green
fluorescence, doxorubicin was shown with red fluorescence, and the merger image was on the bottom right.
tissue. The DOX fluorescence was observed in the fol-
lowing order of intensity: Gal-2000-liposomes (5%) >
common-liposomes > PEG-liposomes. There was no sig-
nificant difference in the detected fluorescent intensity
between Gal-2000-liposomes (4%) (Fig. 6(K)) and Gal-
2000-liposomes (5%) (Fig. 6(E)), but they were rela-
tively stronger than that of Gal-2000-liposomes (2%).
Weak fluorescence intensity was observed in the hep-
atocytes treated with PEG-liposomes (Fig. 6(D)), Gal-
194-liposomes (2%) (Fig. 6(F)), Gal-194-liposomes (4%)
(Fig. 6(G)), Gal-1000-liposomes (2%) (Fig. 6(H)) and Gal-
1000-liposomes (4%) (Fig. 6(I)).
The kinetics of DOX entering the cell nuclei was
also investigated by detecting the fluorescence at various
time points after intravenous injection. Figure 7 presented
the images showing doxorubicin labeling in sections of
liver tissue from animals sacrificed at different time point
following intravenous injection of Gal-2000-liposomes
(5%) containing doxorubicin. Figure 7(A) demonstrated
that after 10 min following intravenous injection, liver
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Novel Galactosylated Poly(ethylene glycol)-Cholesterol for Liposomes as a Drug Carrier for Hepatocyte-Targeting
Zhang et al.
Figure 7. Confocal microscopy images of liver sections of Gal-2000-liposomes (5%), 10 min (A), 30 min (B), 1 h (C), 4 h (D), 6 h (E). Nuclei were
stained blue with DAPI, FITC was shown as green fluorescence, doxorubicin was shown as red fluorescence, and the merger image is on the bottom
right.
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cell nuclei were already clearly labeled. As shown in
Figure 7(C), cell nuclei were clearly and intensely labeled
Copyright: American Scientific Publishers
1 h after administration. Intensity of doxorubicin label-
ing appeared to decline between 4 h (Fig. 7(D)) and 6 h
(Fig. 7(E)).
4. CONCLUSION
In this study, three types of novel galactosylated
IP: 45.52.164.210 On: Thu, 05 Nov 2015 17:05:09
cholesterol with a mono-galactoside moiety have been
synthesized, including gal-PEG194-chol, gal-PEG1000-
chol and gal-PEG2000-chol. The incorporation of gal-
PEG2000-chol (4% and 5% mol/mol) into liposomes
enhanced the liver target ability of liposomal DOX. More-
over, the results of intracellular uptake study, cell cytotox-
icity assay in vitro and liver frozen sectioned study in vivo
provided evidence that the recognition of galactosylated
residues of gal-PEG2000-chol by the ASGP-R on the sur-
face of parenchymal cells accounted for the liver accumu-
lation of Gal-2000-liposomes. These results suggested that
liposomes containing gal-PEG2000-chol could be a useful
drug carrier system for hepatocyte selective targeting.
Within the liver, histologic analysis indicated doxoru-
bicin was present in the nuclei of all identifiable cell types
(Figs. 6 and 7). The release of doxorubicin from the Gal-
2000-liposomes (5%) was apparently quite rapid, as nuclei
were labeled with doxorubicin fluorescence within 10 min
after systemic administration (Fig. 7(A)).
The release of doxorubicin and its appearance within
cell nuclei was remarkably rapid (within 10 min), and did
not require an externally applied stimulus such as ultra-
sound. At present, the mechanism of this apparent trig-
gered release is not known. One hypothesis, which remains
to be tested, is that the interaction of the liposomes with
the ASGP-R is sufficiently strong so that the liposomes
bilayer is destabilized, resulting in release of entrapped
contents.30
Doxorubicin proved to be an excellent compound for
the demonstration of targeted liposomes content delivery.
It could be conveniently loaded into liposomes at high con-
centration. The purpose of this investigation was to test
whether content delivery of doxorubicin could be targeted
to normal liver. The next step of this study will be to
explore the targeted delivery characteristics of this formu-
lation in liver tumor animal models.
Acknowledgments: We gratefully thank Yang Liu
Ph.D. from School of Medicine University of north Car-
olina at Chapel Hill, for her kindness in carefully revis-
ing the language of this article. We acknowledge financial
support from the National Natural Science Foundation of
China (Grant No. 81173003/H3008).
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Received: 6 May 2013. Accepted: 3 November 2013.
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