Design and Synthesis of Galactose-Biotin Lipid Materials for Liposomes to Promote the Hepatoma Cell–Targeting Effect

Article abstract of DOI:10.1016/j.xphs.2019.04.007

A series of novel low-toxic hepatoma cell–targeting lipid materials were designed and synthesized, in which monogalactose, digalactose, and galactose-biotin were used as targeting moieties and hydrophilic heads while stearate was used as hydrophobic tail (Mono-Gal-ST, Di-Gal-ST, and Gal-Biotin-ST). The corresponding galactose-biotin-modified liposomes (Mono-Gal-LPs, Di-Gal-LPs, and Gal-Biotin-LPs) and conventional liposomes (LPs) were prepared. These galactose-biotin-modified liposomes can distinguish hepatoma cells from other tissue cells owing to the recognition of asialoglycoprotein receptor by galactose group. Moreover, the ability of liposomes to distinguish hepatoma cells from normal hepatocytes follows a trend of LPs < Mono-Gal-LPs < Di-Gal-LPs < Gal-Biotin-LPs, which is attributed to the cluster glycoside effect and the synergistic effect of galactose and biotin. In addition, the endocytosis of these galactose-biotin-modified liposomes were competitively inhibited by galactose, further confirming these liposomes entered hepatoma cells via asialoglycoprotein receptor–mediated pathway.

Full text of DOI:10.1016/j.xphs.2019.04.007

Journal of Pharmaceutical Sciences xxx (2019) 1-8
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences
journal homepage: www.jpharmsci.org
Pharmaceutical Nanotechnology
Design and Synthesis of Galactose-Biotin Lipid Materials for
Liposomes to Promote the Hepatoma CelleTargeting Effect
1
1
2
,
3
,
*
4
4
Ruihua Ding , Zhenjie Li , Jianyi Wang
Mian Wang
1
, Xueyan Zhu , Zhuang Zhao ,
1, *
College of Life Science and Technology, Guangxi University, Nanning 530004, China
Medical College, Guangxi University, Nanning 530004, China
School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China
Guangxi Institute for Food and Drug Control, Nanning 530021, China
2
3
4
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel low-toxic hepatoma celletargeting lipid materials were designed and synthesized, in
which monogalactose, digalactose, and galactose-biotin were used as targeting moieties and hydrophilic
heads while stearate was used as hydrophobic tail (Mono-Gal-ST, Di-Gal-ST, and Gal-Biotin-ST). The
corresponding galactose-biotin-modified liposomes (Mono-Gal-LPs, Di-Gal-LPs, and Gal-Biotin-LPs) and
conventional liposomes (LPs) were prepared. These galactose-biotin-modified liposomes can distinguish
hepatoma cells from other tissue cells owing to the recognition of asialoglycoprotein receptor by
galactose group. Moreover, the ability of liposomes to distinguish hepatoma cells from normal hepato-
cytes follows a trend of LPs < Mono-Gal-LPs < Di-Gal-LPs < Gal-Biotin-LPs, which is attributed to the
cluster glycoside effect and the synergistic effect of galactose and biotin. In addition, the endocytosis of
these galactose-biotin-modified liposomes were competitively inhibited by galactose, further confirming
Received 8 October 2018
Revised 17 March 2019
Accepted 4 April 2019
Keywords:
hepatoma celletargeting
galactose
biotin
lipid material
these liposomes entered hepatoma cells via asialoglycoprotein receptoremediated pathway.
®
©
2019 American Pharmacists Association . Published by Elsevier Inc. All rights reserved.
Introduction
the systemic toxicity of doxorubicin,16 but the materials of most
nanoparticles and polymeric micelles are short of enough safety for
17,18
Hepatocellular carcinoma (HCC) annually results in more than 1
million deaths in the world, being the third place of malignant
tumor mortality.1 10-Hydroxycamptothecin (HCPT) shows high
cytotoxicity against hepatoma cells through inhibiting topoisom-
erase I and inducing cell apoptosis. However, the widespread
clinical application of HCPT as liver cancer chemotherapeutics is
limited by its poor solubility, low bioavailability, poor selectivity,
their clinical application.
By contrast, liposomes are mainly
composed of natural phospholipids which are biologically safe.
Therefore, liposomes were the first nanocarriers used in clinical
applications. To date, some liposomes encapsulated with antitumor
drugs (doxorubicin, paclitaxel, mitoxantrone, etc) have been in
,2
3
,4
18,19
clinical trials or approved into markets.
However, these con-
ventional liposomes are still unsatisfactory in the targeting effect to
tumor cells, particularly to hepatoma cells.
5
,6
and significant toxic side effects.
As we known, nanoparticular, micellar, and liposomal formu-
lations can help to improve the bioavailability of chemotherapeu-
tics and reduce the systemic toxicity of chemotherapeutics to some
To improve the hepatoma-cell selectivity and reduce systemic
toxicity of anticancer drugs, some liver-targeting drug delivery
systems have been developed through the modification with
extent.7
-14
For example, the pectin nanoparticles can improved
15
galactose, glycyrrhetinic acid, cholic acid, and so on.
20-25
Among
the water solubility of HCPT and the chitosan micelles can reduce
these liver-targeting moieties, galactose can specially recognize the
asialoglycoprotein receptor (ASGPR) overexpressed on the surfaces
of hepatocytes, being one of the efficient liver-targeting group. For
example, arabinogalactan-modified doxorubicin liposomes
This article contains supplementary material available from the authors by request
or via the Internet at https://doi.org/10.1016/j.xphs.2019.04.007.
exhibited higher uptakes in the mice liver than the conventional
*
Correspondence to: Mian Wang (Telephone: þ86-771-3270736) and Jianyi Wang
26
liposomes.
The galactosyl modification promoted polymer
(
(
Telephone: þ86-771-3270736).
27
microbubbles to accumulate in the hepatoma cells. Doxorubicin
E-mail addresses: jianyiwang@gxu.edu.cn (J. Wang), mianwang@gxu.edu.cn
M. Wang).
nanoparticle modified with galactosamine showed higher
https://doi.org/10.1016/j.xphs.2019.04.007
022-3549/© 2019 American Pharmacists Association . Published by Elsevier Inc. All rights reserved.
®
0

2
R. Ding et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-8
Figure 1. (a) Chemical structures of targeting lipid materials. (b) Schematic illustration for the liposomes modified with targeting lipid materials.
therapeutic effect in xenograft-bearing nude mice than non-
targeted nanoparticles and free doxorubicin.
lipid materials on the hepatoma celletargeting potential of the
corresponding liposomes are still in the shadow.
2
8
The modification of drug delivery systems with tumor-targeting
group is another strategy to improve the hepatoma celletargeting
effect. For instance, a novel prodrug modified by biotin, which
can specifically recognized the biotin receptors overexpressed on
To address these issues, several novel hepatoma celletargeting
lipid materials (Mono-Gal-ST, Di-Gal-ST, and Gal-Biotin-ST) are
designed (Fig. 1) and synthesized (Scheme 1). Then, the HCPT-
loaded liposomes assembled by these targeting lipid materials
were constructed and characterized.
2
9
the surface of hepatoma cells, was developed.
The biotin-
modified doxorubicin liposomes can obviously enhance tumor
accumulation and decrease the undesired distribution in other
Experimental
3
0
tissues.
Biotin-modified
rhodamine-triphenylphosphonium
31
probes showed an excellent tumor-targeting effect.
Clearly,
Materials and Instruments
galactose- or biotin-modified materials can improve the hepatoma
celletargeting capability of drug delivery systems. However, the
influences of glycocluster-modified or galactose-biotin comodified
The chemicals and solvents were commercially available and
used without further purification. 10-Hydroxycamptothecine was
Scheme 1. Synthetic Routes of targeting lipid materials. Reagents and conditions: a: NaH, 3-bromo-1-propyne; b: CH
CuSO $5H O, sodium ascorbate; e: biotin, EDCI, DMAP; f: (BOC) O, CH OH g: KOH, 3-bromo-1-propyne; h: dry CH Cl
CH OH, CH ONa.
3
OH, ion-exchange resin; c: stearic acid, EDCI, DMAP; d: Gal-N
3
,
4
2
2
3
2
2
, CF COOH; i: acetyl-Gal-N , CuSO $5H O, sodium ascorbate; j:
3
3
4
2
3
3

R. Ding et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-8
3
Table 1
Lipid Formulation, Particle Size, Zeta Potential, Encapsulation Efficiency, and Drug Loading of Liposomes
Liposomes
SPC/CHOL/TLM/HCPT (molar Ratio)
Particle Size (nm)
Zeta (mV)
EE (%)
DL (%)
LPs
10:1:1:1
10:1:1:1
10:1:1:1
10:1:1:1
213.5 ± 1.4
230.3 ± 1.8
218.7 ± 3.4
213.6 ± 2.9
ꢂ18.2 ± 1.6
ꢂ36.9 ± 2.2
ꢂ17.0 ± 2.8
ꢂ38.7 ± 2.3
67.2 ± 3.5
65.4 ± 3.7
71.7 ± 3.6
68.2 ± 4.2
7.1 ± 1.1
6.7 ± 1.3
7.9 ± 1.3
7.2 ± 1.7
Mono-Gal-LPs
Di-Gal-LPs
Gal-Biotin-LPs
TLM, targeting lipid materials; EE%, encapsulation efficiency; DL%, drug loading; SPC, soy phosphatidylcholine; CHOL, cholesterol; TLM, targeting lipid materials.
purchased from Chengdu Yuancheng Bio-Tech co., Ltd. (Sichuan,
China). Galactose was obtained from Xilong chemical co., Ltd.
esterification reaction or an acylation reaction to give the alkynyl
chain compound. Finally, the galactose or biotin was introduced by
a click reaction or an esterification reaction to give the aimed ma-
(
Guangdong, China). Biotin was purchased from Bide Pharmatech
1
Ltd. (Shanghai, China). Stearic acid was purchased from Guang-
terials. The structures of these compounds were confirmed by H
1
13
dong Guanghua Sci-Tech Co., Ltd. (Guangdong, China). H NMR
NMR, C NMR, and MS (Supporting Information).
13
and C NMR spectra were recorded on a Bruker 600 MHz
ꢀ
(
Switzerland) at 25 C, with Tetramethylsilane (Shanghai, China)
Preparation of Liposomes
as the internal standard in CDCl
3
, and the solvent peak as the
internal reference in CD OD. MS analysis was performed with a
3
All liposomes were prepared by thin-film evaporation.32,33 For
the preparation of the conventional liposomes (LPs), the mixture of
soy phosphatidylcholine, cholesterol, and HCPT were dissolved in
the mixed solvent of chloroform and methanol (5:1, v/v) with a
Thermo Fisher Scientific LTQ FT Ultra. The cytotoxicity assays were
conducted using ELꢁ800 absorbance microplate reader. Cell im-
age assays were performed by multiphoton laser confocal scan-
ning microscopy (Leica-TCS-SP8MP, Germany).
molar ratio of about 10:1:1. The solvent was then evaporated at
ꢀ
4
5 C for 1 h to form a dry lipid film. Next, the film was hydrated for
Synthesis of Targeting Lipid Materials
2 h with PBS solution (pH 6.86) and ultrasonicated in an iceewater
bath for 3 times (10 min each time) to decrease the particle size.
The final lipid concentration was 10 mg/mL. The unentrapped HCPT
was removed by dialysis. Mono-Gal-LPs, Di-Gal-LPs, and Gal-
Biotin-LPs were prepared using the similar protocol besides add-
ing different targeting lipid materials, and the molar ratio of soy
As shown in Scheme 1, DL-1,2-isopropylidene-glycerol or 2-
amino-1,3-propanediol scaffold, which was used as starting mate-
rial, reacted with propargyl bromide to give the alkynyl com-
pounds. The alkynyl compounds and stearic acid then undergo an
Figure 2. Size distributions of liposomes in aqueous solution. (a) LPs. (b) Mono-Gal-LPs. (c) Di-Gal-LPs. (d) Gal-Biotin-LPs.

4
R. Ding et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-8
In Vitro Drug Release
In vitro release of HCPT was investigated via dialysis. First, 2 mL
of LPs, Mono-Gal-LPs, Di-Gal-LPs, Gal-Biotin-LPs, and free HCPT
solution were placed in dialysis bags (molecular weight:
8000~14,000). Then, these dialysis bags were put into 100 mL of
PBS buffer solution (pH 6.86) containing 30% methanol in a shaker
ꢀ
(
37 C, 120 rpm) for 48 h. Samples (2 mL) were withdrawn at
different time and replaced with the same volume of fresh medium.
These samples were analyzed by high-performance liquid
chromatography.
Targeting Effect and Galactose or Biotin Competition Assays
LO2, HepG2, A549, Hela, and SGC-7901 cells were seeded in 24-
well plates and cultured for 24 h. Then, the culture medium was
replaced with fresh medium containing LPs, Mono-Gal-LPs, Di-Gal-
LPs, or Gal-Biotin-LPs with equivalent HCPT concentrations (20
m
g/
ꢀ
mL). After that, the cells were incubated for another 3 h at 37 C.
Subsequently, culture medium was removed, and cells were
washed with PBS for 3 times. Finally, the cells were fixed with 4%
paraformaldehyde for 15 min at room temperature and rinsed with
PBS for 3 times.
Figure 3. Scanning electron microscopic micrograph of liposomes. (a) LPs; (b) Mono-
Gal-LPs; (c) Di-Gal-LPs; (d) Gal-Biotin-LPs.
HepG2 cells were seeded in a 24-well plate and cultured for
2
4 h. After that, the cells were divided into 2 groups: control
phosphatidylcholine/cholesterol/targeting lipid materials/HCPT
was 10:1:1:1 (Table 1).
The encapsulation efficiency and drug loading of HCPT were
calculated according to the following equations.
group and galactose competition group. Galactose competition
group was preincubated with galactose (a competitive inhibitor
of ASGPR, 40 mM) for 2 h at 37 C. Then, both control group and
ꢀ
galactose competition group were incubated with Mono-Gal-LPs,
Di-Gal-LPs, and Gal-Biotin-LPs (containing 50 mg/mL of HCPT) for
ꢀ
C
C
e
2 h at 37 C. After washing with PBS for 3 times, the cells were
fixed with 4% paraformaldehyde and then rinsed with PBS for 3
times.
EEð%Þ ¼ ꢁ 100%
t
where C
t
stands for the total drug amount added and C
the drug amount encapsulated in the liposomes.
e
stands for
For biotin competition assays, HepG2 cells were preincubated
with 0, 50, 100, and 200
biotin receptor) for 2 h at 37 C. After that, HepG2 cells were incu-
m
g/mL of biotin (a competitive inhibitor of
ꢀ
bated with Mono-Gal-LPs, Di-Gal-LPs, and Gal-Biotin-LPs (con-
weight of encapsulated HCPT
ꢀ
DLð%Þ ¼
ꢁ 100%
taining 50
mg/mL of HCPT) for 2 h at 37 C. After washing with PBS
weight of liposomes
for 3 times, the cells were fixed with 4% paraformaldehyde and
then rinsed with PBS for 3 times.
All of the cell images were captured by multiphoton laser
confocal scanning microscopy. The fluorescence quantitative anal-
ysis was performed by using ImageJ software to calculate the
average fluorescence intensity of cells in each picture.
Particle size and size distribution were measured by 3D DLS
Spectrometer (Switzerland) and zeta potential was measured by
zeta-phase analysis light scattering. Samples were diluted
using water and measured in triplicate. The morphology of
freeze-drying liposomes was observed by scanning electron
microscopy.
Cytotoxicity Assays of Targeting Lipid Materials and Liposomes
MTT assays34 were carried out to evaluate the cytotoxicity of
targeting lipid materials. Briefly, HepG2 cells were seeded into 96-
well plates at a density of 6000 cells per well and incubated for
24 h. Subsequently, 150
mL culture medium containing different
concentrations of targeting lipid materials (0-40
mM) were added in
9
6-well plates to incubate for 48 h. Then, 10
m
L of MTT solution (5
ꢀ
mg/mL) was added. After 4 h coincubation at 37 C, the medium was
replaced with 150
mL of dimethyl sulfoxide followed by gentle
shaking for 10 min. The absorbance was measured via an ELꢁ800
absorbance microplate reader at 490 nm.
The MTT assays were also performed to investigate the anti-
tumor efficacy of liposomes and free HCPT. The HepG2 cells were
treated with Mono-Gal-LPs, Di-Gal-LPs, Gal-Biotin-LPs, and free
HCPT for 24 h, and then 10 mL of MTT solution (5 mg/mL) was added
to coincubate for 4 h. The formed formazan was dissolved by
dimethyl sulfoxide. The absorbance was measured at 490 nm.
Figure 4. In vitro HCPT release profiles of different formulations.

R. Ding et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-8
5
Figure 5. (a) Confocal fluorescence microscopy images of LO2, HepG2, A549, Hela, and SGC-7901 cells incubated with LPs, Mono-Gal-LPs, Di-Gal-LPs, and Gal-Biotin-LPs for 3 h at
ꢀ
3
7 C. (b) The average fluorescence intensity of each image was calculated using Image J software. Scale bar: 50
mm. For the fluorescence intensities of Mono-Gal-LPs, Di-Gal-LPs, and
Gal-Biotin-LPs in HepG2 versus those in A549, Hela, or SGC-7901, p < 0.01.
Results and Discussion
In vitro release profiles of free HCPT and HCPT-loaded liposomes
are shown in Figure 4. In the first hour, the free HCPT solution
rapidly released about 35% HCPT, whereas HCPT-loaded liposomes
released about 5% HCPT, indicating that HCPT-loaded liposomes
had no burst-release phenomenon. The accumulative drug release
of the free HCPT solution reached the peak after 5 h, whereas those
of HCPT-loaded liposomes reached the peak after 24 h, indicating
that the liposomes possess sustained-release properties. Similar
release patterns were observed among LPs, Mono-Gal-LPs, Di-Gal-
LPs, and Gal-Biotin-LPs, implying that the addition of targeting lipid
materials did not apparently affect the drug release behaviors of
liposomes. The final accumulative drug release of the free HCPT
solution and HCPT-load liposomes were less than 50%, which is
because that the concentrations on both sides of the dialysis bag
reached balance.
Characterization of Liposomes
As shown in Table 1, the average particle sizes of LPs, Mono-Gal-
LPs, Di-Gal-LPs, and Gal-Biotin-LPs were 213, 230, 218, and 213 nm,
respectively. The liposomes showed relatively narrow particle size
distributions, with the order of distribution width being LPs z Di-
Gal-LPs < Mono-Gal-LPs < Gal-Biotin-LPs (Fig. 2). Four types of li-
posomes were negatively charged (ꢂ17.0 ~ ꢂ38.7 mV), suggesting
that the galactose or biotin modifications did not reverse the
electrical potentials of liposomes. Although the HCPT encapsulation
efficiency of Mono-Gal-LPs (65.4%) is a little lower than that of LPs
(
67.2%), the encapsulation efficiencies of Di-Gal-LPs (71.7%) and
Gal-Biotin-LPs (68.2%) are higher than LPs, implying that dual-
targeting groupemodified lipid material may slightly promote the
encapsulation efficiencies of liposome. In addition, the drug-
loading efficiencies of these liposomes were similar with the
values ranging from 6.7% to 7.9%. Furthermore, all 4 types of lipo-
somes had spherical morphology revealed by scanning electron
microscopy images (Fig. 3).
Targeting Effects of Liposomes
To evaluate the hepatoma celletargeting effects of galactose-
biotin-modified liposomes, we investigated their cellular uptakes in
HepG2 (hepatoma cells), LO2 (normal hepatocytes), A549 (lung
ꢀ
Figure 6. (a) Confocal fluorescence microscopy images of HepG2 cells pretreated with 40 mM galactose (Gal) or 0 mM Gal for 2 h at 37 C and then incubated with LPs, Mono-Gal-
ꢀ
LPs, Di-Gal-LPs, and Gal-Biotin-LPs for 2 h at 37 C. (b) The average fluorescence intensity of each image was calculated using ImageJ software. Scale bar: 50
mm. **p < 0.01.

6
R. Ding et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-8
ꢀ
Figure 7. (a) Confocal fluorescence microscopy images of HepG2 cells pretreated with 0, 50, 100, and 200
m
g/mL biotin for 2 h at 37 C and then incubated with Gal-Biotin-LPs for 2 h
ꢀ
at 37 C. (b) The average fluorescence intensity of each image was calculated using Image J software. Data are presented as mean ± SD, **p < 0.01.
cancer cells), Hela (cervical carcinoma cells), and SGC-7901 (gastric
cancer cells). As shown in fluorescence images and fluorescence
quantification (Fig. 5), the HepG2 cellular uptake of Mono-Gal-LPs
was higher than that of LPs (fluorescence intensity ratio 3.9:1),
indicating that galactose modification could promote liposomes to
recognize hepatoma cells. The possible reason is that the galactosyl
group of Mono-Gal-LPs can specifically recognize ASGPR overex-
receptor on hepatoma cells were higher than those on normal
hepatocytes. Obviously, Gal-Biotin-LPs are more promising for
precise treatment of HCC.
To further illuminate the contribution of galactose group on the
hepatoma celletargeting capacity of these galactose-biotin-
modified liposomes, we also performed galactose competition ex-
periments (galactose is a competitive inhibitor of ASGPR). As shown
in Figure 6a, after preincubation with galactose, the HepG2 cellular
uptakes of Mono-Gal-LPs, Di-Gal-LPs, and Gal-Biotin-LPs were
significantly reduced, indicating that liposomes modified with
galactose group entered hepatoma cells via ASGPR-mediated
endocytosis. The fluorescence quantification also gave a similar
conclusion. Interestingly, with the preincubation of 40 mM galac-
tose, the HepG2 cellular uptakes decreased 80% for Mono-Gal-LPs,
72% for Di-Gal-LPs, and 66% for Gal-Biotin-LPs (Fig. 6b). These data
imply that Mono-Gal-LPs enter HepG2 cells mainly mediated by
ASGPR, Di-Gal-LPs possess higher HepG2-endocytosis capacity
than Mono-Gal-LPs owing to the galactosyl cluster effect, and Gal-
Biotin-LPs enter HepG2 cells not only mediated by ASGPR but also
mediated by biotin receptor.
3
5
pressed on the surface of hepatoma cells. The fluorescence in-
tensity of Di-Gal-LPs in HepG2 was 1.4 times that of Mono-Gal-LPs,
indicating the hepatoma celletargeting ability of liposomes was
improved with the increasing of galactosyl groups. This is because
that multivalent galactose group has stronger affinity to ASGPR of
3
6,37
hepatoma cells (cluster glycoside effect).
In addition, the fluo-
rescence intensity of Gal-Biotin-LPs in HepG2 was 1.3 times that of
Mono-Gal-LPs as well as 5.0 times that of LPs. These data implied that
the targeting lipid materials with biotin group could be favorable for
liposomes to recognize hepatoma cells, and liposomes comodified
with galactose and biotin groups had synergistic targeting effects.
As shown in Figure 5, the uptakes of LPs in several cell lines were
less and did not show obvious differences among them. However,
because of the HepG2 cellular uptakes of Mono-Gal-LPs, Di-Gal-LPs,
and Gal-Biotin-LPs were more than several other tissue cells
To elucidate the contribution of biotin group on the hepatoma
celletargeting capacity of the galactose-biotin-modified liposomes,
we also performed biotin-blocking experiments (biotin is a
competitive inhibitor of biotin receptor). As shown in Figure 7, after
(Fig. 5), further demonstrating that galactose modification or
galactose-biotin comodification could promote liposomes to accu-
rately recognize hepatoma cells via ASGPR-mediated pathway
preincubation with 0, 50, 100, and 200 mg/mL biotin, the HepG2
(
HepG2 has high expression of ASGPR, whereas A549, Hela, and
cellular uptakes of Gal-Biotin-LPs were progressively decreased,
indicating that liposomes modified with biotin group can enter
HepG2 cells via biotin-receptorsemediated endocytosis.
37-39
SGC-7901 have low expression of ASGPR
). Interestingly, the
cellular uptakes of LPs, Mono-Gal-LPs, Di-Gal-LPs, and Gal-Biotin-
LPs in HepG2 were 0.9, 1.9, 2.1, and 2.2 times those in LO2,
respectively (Fig. 5b), indicating that the capacity of liposomes to
distinguish hepatoma cells from normal hepatocytes follows a
trend of LPs < Mono-Gal-LPs < Di-Gal-LPs < Gal-Biotin-LPs. The
possible reason was that the expressions of ASGPR and biotin
Cytotoxicity of Targeting Lipid Materials and Liposomes
As shown in Figure 8, when the concentrations of galactose or
biotin lipid materials reached 40 mM, the cell viabilities were still
Figure 8. In vitro cells viability of HepG2 cells treated (for 48 h) with different con-
Figure 9. In vitro cells viability of HepG2 cells treated with different concentrations of
centrations of Mono-Gal-ST, Di-Gal-ST, and Gal-Biotin-ST.
Mono-Gal-LPs, Di-Gal-LPs, and Gal-Biotin-LPs for 24 h.

R. Ding et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-8
7
above 90%, indicating that these materials have relatively high safety
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A comparison of these liposomes (Mono-Gal-LPs, Di-Gal-LPs,
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summarized in Table S1. First, the targeting lipid materials syn-
thesized in this work were small molecular compounds which can
be precisely determined molecular weight and molecular structure.
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(
galactose and biotin) against 2 cellular targets (ASGPR and biotin
receptor), showing a synergistic hepatoma celletargeting effect.
Furthermore, Mono-Gal-LPs, Di-Gal-LPs, and Gal-Biotin-LPs
exhibited excellent hepatoma celletargeting effects. More impor-
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1
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1
Conclusions
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In summary, hepatoma celletargeting lipid materials (Mono-
Gal-ST, Di-Gal-ST, and Gal-Biotin-ST) were designed and synthe-
sized, and the corresponding galactose-biotin-modified liposomes
were prepared. In vitro cellular uptake assays showed these
galactose-biotin-modified liposomes can distinguish hepatoma
cells from other tissue cells because of the introduction of galactose
group. The capacity of liposomes to distinguish hepatoma cells
from normal hepatocytes follows a trend of LPs < Mono-Gal-LPs
1
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(
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<
Di-Gal-LPs (cluster glycoside effect)
<
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ꢀ ꢀ
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The authors acknowledge the supported for this work by the
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