Development of glycyrrhetinic acid-modified stealth cationic liposomes for gene delivery

Article abstract of DOI:10.1016/j.ijpharm.2010.06.029

The glycyrrhetinic acid-modified stealth cationic liposomes (GA-PEG-CLs) loaded with pDNA (GA-PEG-CLPs) were developed and found to transfect human hepatocellular carcinoma cell line HepG2 with high efficiency. GA-PEG-CLs were comprised of DOTAP, cholesterol (Chol) and glycyrrhetinic acid-polyethyleneglycol-cholesterol conjugate (GA-PEG-Chol). Agarose gel electrophoresis revealed that 5% GA-PEG-CLs constituted by DOTAP/Chol/GA-PEG-Chol at molar ratio of 50:45:5 could completely entrap pDNA at a lower liposomes/pDNA weight ratios of 4:1 (N/P ratio: 1.14). Compared to ordinary cationic liposomes (CLs), steric cationic liposomes (PEG-CLs) and 1% GA-PEG-CLs made from DOTAP/Chol/MPEG2000-Chol/GA-PEG-Chol at molar ratio of 50:45:4:1, 5% GA-PEG-CLs were found to possess the highest transfection efficiency as gene vectors in serum-free or serum-containing medium in PKCα over-expressed HepG2 cells but no significance difference in human embryonic kidney cell line HEK 293. Additionally, 5% GA-PEG-CLs have the lowest cytotoxicity on human normal hepatocyte cell line L02. The competitive inhibition experiments mediated by GA were carried out in HepG2 cells, which demonstrated that GA-PEG-CLs could deliver selectively pDNA to hepatoma cells by the targeting moiety GA. In conclusion, GA-PEG-CLs containing 5% GA-PEG-Chol might be one of the most potential gene vectors as hepatoma targeting therapy.

Full text of DOI:10.1016/j.ijpharm.2010.06.029

International Journal of Pharmaceutics 397 (2010) 147–154
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical Nanotechnology
Development of glycyrrhetinic acid-modified stealth cationic liposomes for gene
delivery
Zhi Yao Heb,1, Xi Zheng , Xiao Hua Wu , Xiang Rong Song , Gu He , Wen Fang Wu , Shui Yu ,
b,1
a
a,∗
a,∗
c
a
b
a
Sheng Jun Mao , Yu Quan Wei
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, PR China
West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, PR China
CSPC Pharmaceutical Group Limited, Shijiazhuang, Hebei 050051, PR China
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The glycyrrhetinic acid-modified stealth cationic liposomes (GA–PEG–CLs) loaded with pDNA
Received 4 March 2010
Received in revised form 11 June 2010
Accepted 18 June 2010
(GA–PEG–CLPs) were developed and found to transfect human hepatocellular carcinoma cell line HepG2
with high efficiency. GA–PEG–CLs were comprised of DOTAP, cholesterol (Chol) and glycyrrhetinic
acid–polyethyleneglycol–cholesterol conjugate (GA–PEG–Chol). Agarose gel electrophoresis revealed
that 5% GA–PEG–CLs constituted by DOTAP/Chol/GA–PEG–Chol at molar ratio of 50:45:5 could com-
pletely entrap pDNA at a lower liposomes/pDNA weight ratios of 4:1 (N/P ratio: 1.14). Compared to
ordinary cationic liposomes (CLs), steric cationic liposomes (PEG–CLs) and 1% GA–PEG–CLs made from
DOTAP/Chol/MPEG2000-Chol/GA–PEG–Chol at molar ratio of 50:45:4:1, 5% GA–PEG–CLs were found to
possess the highest transfection efficiency as gene vectors in serum-free or serum-containing medium
in PKC␣ over-expressed HepG2 cells but no significance difference in human embryonic kidney cell line
HEK 293. Additionally, 5% GA–PEG–CLs have the lowest cytotoxicity on human normal hepatocyte cell
line L02. The competitive inhibition experiments mediated by GA were carried out in HepG2 cells, which
demonstrated that GA–PEG–CLs could deliver selectively pDNA to hepatoma cells by the targeting moi-
ety GA. In conclusion, GA–PEG–CLs containing 5% GA–PEG–Chol might be one of the most potential gene
vectors as hepatoma targeting therapy.
Available online 27 July 2010
Keywords:
Glycyrrhetinic acid
Cationic liposomes
Gene transfection
PEG
Hepatocellular carcinoma
©
2010 Elsevier B.V. All rights reserved.
1
. Introduction
trostatic interactions between the lipoplexes and cells or tissues
leaded to non-specific cell or tissue uptake, non-specific immune
response (a toxic response), a shorter half-life in vivo (Lv et al.,
2006; Karmali and Chaudhuri, 2007; Morille et al., 2008; Ditto et
al., 2009). Therefore, many researchers focused on the development
of novel, effective, long-circulating and targeted-delivery cationic
liposomes.
According to literatures, cationic liposomes tagged with appro-
priate targeting ligands could function as an efficient targeted gene
delivery system (Sudimack and Lee, 2000; Karmali and Chaudhuri,
2007; Morille et al., 2008; Yan and Qi, 2008; Pathak et al., 2009). The
published ligands used to modify cationic liposomes included folate
(Yan and Qi, 2008), galactosyl (Fumoto et al., 2003), integrins (Hood
et al., 2002), sigma ligands (Mukherjee et al., 2005), estradiol (Reddy
and Banerjee, 2005), transferrin (Penacho et al., 2008), TAT (MacKay
et al., 2008), and so on. In this study, glycyrrhetinic acid (GA) was
selected as the specific ligand of cationic liposomes. GA is one of
the main compounds extracted from the root of Glycyrrhiza glabra
L. (licorice) (Mao et al., 2007), which could inhibit liver carcinogen-
esis and cell proliferation of the human hepatocellular carcinoma
Cationic liposomes, one of the promising and the most represen-
tative non-viral gene delivery vectors, have many advantages over
viral ones such as less-immunogenicity, non-oncogenicity, large
DNA capacity, non-restriction on the size of the DNA molecule,
ability of evading random integration of vector DNA into host
chromosome (avoiding any insertional mutagenesis) and large-
scale production (Templeton, 2003; Lv et al., 2006; Karmali
and Chaudhuri, 2007). Cationic liposomes could interact with
negatively charged plasmids via electrostatic attractions at the
physiological pH to form positively charged lipid–DNA complexes
(
lipoplexes) and get endocytosed by the negatively charged cell
membrane (Karmali and Chaudhuri, 2007). Lipoplexes represent
one kind of attractive alternative to viral vectors for cell transfec-
tion in vitro and in vivo but still suffer from relatively low efficiency
(
Duan et al., 2009) and certain toxicity. The non-specific elec-
∗ Corresponding authors. Tel.: +86 28 85164063; fax: +86 28 85164060.
(HCC) cell line HepG2 (Satomi et al., 2005). It has been proved that
E-mail addresses: xiangrong 11@126.com (X.R. Song), hegu@scu.edu.cn (G. He).
These authors contributed equally.
1
protein kinase C (PKC) ␣, the target binding sites of GA, expressed
0
378-5173/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2010.06.029

1
48
Z.Y. He et al. / International Journal of Pharmaceutics 397 (2010) 147–154
more highly in HCC cells than that in the adjacent non-tumor liver
cells (O’Brian et al., 1990; Yang et al., 2003; Ying et al., 2008). There-
fore, we hypothesized that GA-modified cationic liposomes could
target selectively to HCC cells by the specific interaction of GA with
PKC␣ over-expressed in HCC cells.
To increase the HCC cells-targeted efficiency in vivo prob-
ably by decreasing the clearance of reticuloendothelial system,
poly(ethyleneglycol) (PEG) was selected as the linker between
GA and cationic liposomes according to literatures (Green et al.,
Chol-suc. PEG–Chol was purified on a silica-gel column, eluting
with a mixed solvent system of DCM and methanol. GA–PEG–Chol
(compound 7) was prepared through the chemical reaction of mGA-
suc and PEG–Chol. The purification process of GA–PEG–Chol was
similar to that of PEG–Chol.
1H NMR spectra of GA–PEG–Chol, mGA-suc and PEG–Chol were
recorded on a JEOL JNM-a 400 instrument (Tokyo, Japan) at room
temperature. All the samples were dissolved in CDCl3.
2
007; Morille et al., 2009). PEG could dissimulate the positive
charges by shielding cationic liposomes surface with hydrophilic
chain, thereby weakening the toxic response caused by non-specific
electrostatic interactions of lipoplexes with normal cells. Addition-
ally, the introduction of PEG to targeted cationic liposomes would
have many other advantages such as extending the half-life of
lipoplexes by reducing opsonization and enhancing serum stabi-
lization (Bombelli et al., 2007), avoiding the tendency of lipoplexes
to form large aggregates (de Lima et al., 2001), improving their bio-
compatibility with biological fluids (Sudimack and Lee, 2000) and
achieving the higher targeting efficiency (Green et al., 2007; Morille
et al., 2009).
To our knowledge, no gene vectors modified with GA have
been published so far. Thus, we aimed to develop GA-modified
stealth cationic liposomes (GA–PEG–CLs) for gene delivery. In this
paper, cholesterol (Chol) and PEG2000 were covalently linked to
form PEG–Chol, and then GA was covalently conjugated to the
distal end of PEG–Chol to produce GA–PEG–Chol. GA–PEG–CLs
were made from GA–PEG–Chol, cholesterol and 1,2-dioleoyl-3-
trimethylammonium-propane (chloride salt) (DOTAP). The toxicity
of GA–PEG–CLs as gene vector was investigated. Additionally,
in vitro cell transfection efficiency of gene loaded GA–PEG–CLs
2.3. Preparation of blank cationic liposomes
GA–PEG–CLs were prepared by a film dispersion method
(Templeton et al., 1997). Briefly, DOTAP, Chol and GA–PEG–Chol
at different molar ratios were dissolved in chloroform. The lipids
solution was evaporated on a Büchi rotary evaporator to remove
the solvent and then the formed thin film was further dried under
high vacuum for 6 h. The dry lipid film was immediately hydrated
in 5% (w/v) glucose solution. The suspension of lipids was sonicated
by probe until a translucent lipid suspension was obtained. There-
after, GA–PEG–CLs were passed through a 0.22 ␮m microporous
◦
membrane and stored at 4 C until use.
The lipid compositions of ordinary cationic liposomes (CLs) and
steric cationic liposomes (PEG–CLs) were DOTAP/Chol at molar
ratio of 50:50 and DOTAP/Chol/MPEG2000-Chol at molar ratio of
50:45:5, respectively. CLs and PEG–CLs were prepared by the same
method as GA–PEG–CLs. Experiments were performed in triplicate.
2.4. Preparation of lipoplexes
(
GA–PEG–CLPs) was assessed on HCC HepG2 cell line and human
The 5% (w/v) glucose solution of pDNA was added into the stock
suspensions of different blank cationic liposomes (CLs, PEG–CLs
and GA–PEG–CLs) respectively, and then incubated at room tem-
perature for 30 min to form different lipoplexes (CLPs, PEG–CLPs
and GA–PEG–CLPs). The weight ratios of pDNA to blank cationic
liposomes were optimized by electrophoresis experiments. Elec-
trophoresis was conducted on 1% (w/v) agarose gel in TAE buffer
embryonic kidney cell line HEK 293.
2
. Materials and methods
2
.1. Materials
(
40 mM Tris/HCl, 1% acetic acid, 1 mM EDTA, pH 7.4) containing
1
,2-Dioleoyl-3-trimethylammonium-propane (chloride salt)
DOTAP) was purchased from Avanti Polar Lipids Inc. (Alabaster,
AL, USA). Cholesterol (Chol) was obtained from Shanghai Bio
Life Science Technology Co. Ltd. (Shanghai, China). 1-(3-
ethidium bromide as nucleic acid stain. 10 ␮L of lipoplexes mixed
with 3 ␮l of loading buffer were applied to agarose at a constant
voltage of 120 V for 20 min. The electrophoresis gel was visualized
and digitally photographed by a gel documentation system (Gel
Doc 1000, Bio-Rad Laboratories, Hercules, CA, USA).
(
&
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
and dimethylaminopyridine (DMAP) were purchased from Sigma.
Green fluorescent protein plasmid DNA (pDNA) was kindly donated
by Dr. Hong Xin Deng (Sichuan University). All reagents and sol-
vents were of analytical grade and were used without further
purification except chloroform.
2
.5. Characterization of blank cationic liposomes and lipoplexes
The mean particle size and zeta potential were measured using a
Zetasizer Nano ZS90 (Malvern Instruments, Ltd., Malvern, Worces-
tershire, UK). The samples were suitably diluted with 5% (w/v)
isotonic glucose solution (pH 7). The mean particle size was mea-
2
.2. Synthesis of GA–PEG–Chol conjugates
◦
The synthesis process of GA–PEG–Chol conjugates was dis-
sured by dynamic light scattering at a fixed angle of 90 . The zeta
played in Scheme 1. Synthesis of 18␤-glycyrrhetinic acid succinic
anhydride ester (mGA-suc, compound 3) was carried out according
to the literature (Tian et al., 2009). PEG–Chol (compound 6) was
prepared as follows: Firstly, the mixture containing cholesterol,
DMAP and succinic anhydride in DCM solution was stirred for 48 h,
thereafter DMAP was removed by washing with 1 mol/l HCl solu-
tion and saturated NaHCO3 solution. The combined organic layer
potential was automatically calculated from the electrophoretic
mobility. Experiments were performed in triplicate.
2.6. Cell culture
Human hepatoma cell line HepG2, human normal hepatocyte
cell line L02 and human embryonic kidney cell line HEK 293 were
obtained from State Key Laboratory of Biotherapy, Sichuan Univer-
sity. The cells were cultured as a monolayer in Dulbecco’s Modified
Eagles’s Medium (DMEM) supplemented with penicillin, strepto-
mycin, and 10% fetal bovine serum and in a humidified atmosphere
was dehydrated with anhydrous Na SO4 and then concentrated to
2
yield cholesterol succinic anhydride ester (Chol-suc, compound 5).
Secondly, Chol-suc, DMAP, EDCI and PEG2000 dissolved in chloro-
form were refluxed for 24 h. The mixture was concentrated under
vacuum, and then re-dissolved with DCM. The DMAP and other
by-products were removed as described in preparation process of
◦
containing 5% CO2 at 37 C.

Z.Y. He et al. / International Journal of Pharmaceutics 397 (2010) 147–154
149
Scheme 1. Synthetic scheme for GA–PEG–Chol.
2
.7. Cytotoxicity experiments
a Bio-Tek ␮Quant Universal Microplate Spectrophotometer (Bio-
Tek Instruments Inc., Winooski, VT, USA). The relative cell viability
compared to control was calculated as (Asample/Acontrol) × 100%. All
the experiments were repeated in triplicates.
Cytotoxicity of various blank cationic liposomes was deter-
mined by MTT assay on L02 and HepG2 cell lines as described before
(
Song et al., 2009). Cells were seeded on 96-well plates (Corning
3
Inc., Corning, NY, USA) at a density of 3 × 10 cells per well. Fol-
lowing attachment for 24 h, cells were treated by various blank
cationic liposomes with different concentrations. These treatment
agents were incubated with cells for an additional 48 h. Then 20 ␮l
of MTT stock solution (5 mg/ml in saline) was added to each well
2.8. Transfection experiments
HepG2 cells and HEK 293 cells were seeded on a Costar 6-
5
well plate (Corning Incorporated, Corning, NY, USA) at 1.5 × 10
cells/well in 2 ml of culture medium. After 24 h of growth, the
culture medium was replaced by 800 ␮l DMEM in each well.
CLPs, PEG–CLPs and GA–PEG–CLPs in a final volume of 200 ␮l
containing 1 ␮g pDNA were subsequently added to the wells.
After 6 h incubation (in typical culture conditions), cell culture
◦
and the cells were further incubated at 37 C for 4 h. Finally, the
culture medium was removed by aspiration and then 150 ␮l of
dimethyl sulfoxide (DMSO) was added to each well to dissolve for-
mazan crystals. The absorbance of each well was read at 490 nm on

1
50
Z.Y. He et al. / International Journal of Pharmaceutics 397 (2010) 147–154
Fig. 1. Comparison of the 1H NMR spectra of Chol-b-PEG, GA–PEG–Chol and mGA-suc in CDCl3.
Fig. 2. The average diameters, PDI and zeta potentials of four kinds of blank cationic liposomes.
medium was changed to complete culture medium again and
the cells were incubated for an additional 48 h. The expressed
green fluorescent protein (GFP) was observed under an Olympus
IX 70 inverted fluorescence microscope (Olympus Corp., Shinjuku,
Tokyo, Japan). After that, the cell suspensions were collected after
trypsinized with 0.25% trypsin-EDTA and centrifuged. And then
the cell sediments were washed three times with PBS. The resus-
pended cell suspensions in PBS were analyzed by flow cytometry
on a Coulter EPICS Elite ESP Flow Cytometer (Beckman coulter,
Miami, FL, USA) to determine the transfection efficacy of the
lipoplexes.
To assess the transfection ability of CLPs, PEG–CLPs and
GA–PEG–CLPs in medium containing 10% serum, the transfection
experiment of HepG2 cells was carried out as described above
except that 800 ␮l DMEM was changed to 800 ␮l complete culture
medium.
The competitive inhibition experiments mediated by GA were
carried out in HepG2 cells. HepG2 cells were incubated with various
Fig. 3. The average diameters, PDI and zeta potentials of four kinds of lipoplexes.

Z.Y. He et al. / International Journal of Pharmaceutics 397 (2010) 147–154
151
Fig. 4. Agarose gel electrophoresis of four different lipoplexes (CLs/pDNA, PEG–CLs/pDNA, 1% GA–PEG–CLs/pDNA and 5% GA–PEG–CLs/pDNA) at weight ratios ranging from
:1 to 7:1.
2
amounts of GA (2.58 ␮M to 10.32 ␮M) for 30 min in serum-free
medium, and then 5% GA–PEG–CLPs were added. The other process
was accordant with the above-mentioned method.
3.3. Preparation and characterization of lipoplexes
Agarose gel electrophoresis was used to study and compare
abilities of the four different cationic liposomes (CLs, PEG–CLs, 1%
GA–PEG–CLs and 5% GA–PEG–CLs) to condense pDNA (Fig. 4). It
turned out that CLs, PEG–CLs, 1% GA–PEG–CLs and 5% GA–PEG–CLs
completely retarded DNA migration at liposomes/pDNA weight
ratios of 4:1 (N/P ratio which is the ratio between the nitrogen
in the cationic lipid and the phosphate in pDNA: 1.14), 7:1 (N/P
ratio: 1.99), 7:1 (N/P ratio: 1.99) and 4:1 (N/P ratio: 1.14) respec-
tively. This result might be due to the different surface potential
of the four kinds of cationic liposomes. To elucidate the above
phenomenon, the four kinds of lipoplexes were further character-
ized by average diameter, PDI and zeta potential. The dispersion
medium of four cationic liposomes was 5% (w/v) isotonic glucose
solution (pH 7), which simulated the physiological condition. The
results were displayed in Fig. 3. The average diameter of all the
four lipoplexes increased, while PDI had no significant change.
Compared to corresponding blank cationic liposomes, the zeta
potentials of CLPs and PEG–CLPs did not change dramatically, while
that of 1% GA–PEG–CLPs increased significantly but that of 5%
GA–PEG–CLPs decreased significantly. According to literatures, the
interaction of cationic liposomes with pDNA was dominated by
the electrostatic attachment of the negatively charged DNA and
positively charged liposomes (Zuidam and Barenholz, 1997) and
hydrophobic interaction. The targeting moiety GA was hydropho-
bic. 1% GA–PEG–CLPs and 5% GA–PEG–CLPs contained different
amounts of GA, therefore the hydrophobic interaction of the two
liposomes with pDNA might be different, which resulted in differ-
ent zeta potential change.
2.9. Data analysis
Data analysis was performed using Student’s t-test on Statistical
Product and Service Solutions (SPSS V13.0, SPSS Inc., Chicago, USA).
Differences were considered significant at p < 0.05.
3
. Results and discussion
3.1. Synthesis of GA–PEG–Chol conjugates
Fig. 1 shows the 1H NMR spectra of GA–PEG–Chol, mGA-suc
and PEG–Chol in CDCl . The single peaks at ı 5.69 and 5.60 (a)
3
were attributed to the protons of olefinic bond (–(C O)–CH C–)
in GA. The single peak at ı 5.30 (b) was attributed to the pro-
tons of olefinic bond (–CH –CH C–) in cholesterol. The peaks
2
at ı 2.58–2.66 (c) came from the protons of succinate linkage
(
–(C O)–CH –CH –(C O)–). The peaks at ı 3.52–3.76 (d) were
2 2
attributed to the protons from the glycol unit (–O-CH –CH –O–) in
2
2
PEG chain. Thus GA–PEG–Chol conjugates have been successfully
synthesized.
3.2. Preparation and characterization of blank cationic liposomes
Two kinds of GA–PEG–CLs containing different ligand den-
sity were prepared. One was 1% GA–PEG–CLs which were made
from DOTAP/Chol/MPEG2000-Chol/GA–PEG–Chol at molar ratio
of 50:45:4:1, the other was 5% GA–PEG–CLs constituted by
DOTAP/Chol/GA–PEG–Chol at molar ratio of 50:45:5. As seen in
Fig. 2, the size of blank cationic liposomes slightly increased
with introduction of PEG, while the zeta potential decreased in
contrast. This phenomenon, accordant with that reported in the
literature (Morille et al., 2009), might result from the PEG steric
shield around the surface of the cationic liposomes which could
increase the diameter of liposomes and partly shield the positive
charge.
3.4. In vitro cell transfection efficiency
To assess the gene transfection efficiency of GA–PEG–CLPs con-
structed above and optimize better density of GA, the in vitro cell
transfection experiment was carried out using HCC HepG2 cell
line as model. The samples investigated in the experiment were
CLPs, PEG–CLPs, 1% GA–PEG–CLPs and 5% GA–PEG–CLPs at the
optimal liposomes/pDNA weight ratios in which pDNA was com-
pletely entrapped. After treatment with four kinds of lipoplexes,
the fluorescent photos were presented in Fig. 5 and the transfection

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Z.Y. He et al. / International Journal of Pharmaceutics 397 (2010) 147–154
Fig. 7. GFP gene expression efficiency of HEK293 after treatment with four kinds of
lipoplexes.
Fig. 5. The GFP expression fluorescent photos of HepG2 after treatment with four
kinds of lipoplexes.
In order to demonstrate the mechanism of GA–PEG–CLPs
HCC targeting, the transfection efficiency in HEK 293 cell line
and the competitive inhibition experiments mediated by GA in
HepG2 cell line were performed. HEK 293 cell line does not
express the GA receptor according to the HEK 293 cell database
record. The GFP gene expression efficiency of HEK293 after treat-
ment with four kinds of lipoplexes (Fig. 7) showed that two
GA–PEG–CLPs had lower transfection efficiency than CLPs and
PEG–CLPs. GA–PEG–CLPs could achieve higher gene transfection
efficiency in HCC other than normal tissues, which indicated that
GA–PEG–CLPs might be lower toxic in normal tissues than CLPs
and PEG–CLPs. The competitive inhibition experimental result
mediated by GA in HepG2 cell line was presented in Fig. 8.
Before transfected by 5% GA–PEG–CLPs, HepG2 cells was firstly
treated with four different concentrations of GA (2.58 ␮M, 5.16 ␮M,
efficiency was displayed in Fig. 6. It was easy to see that the transfec-
tion efficiency increased when GA ligand density increased from 0%
to 5%. This phenomenon indicated that GA could improve the gene
transfection efficiency on HepG2 cell line with a density-dependent
feature. The zeta potentials of PEG–CLPs, 1% GA–PEG–CLPs and 5%
GA–PEG–CLPs had no significant difference (p > 0.05) (Fig. 4), while
the average diameters of the above three lipoplexes increased with
increase in GA–PEG–Chol content (Fig. 3). Theoretically, the endo-
cytosis of 5% GA–PEG–CLPs might decrease due to the biggest size.
However, 5% GA–PEG–CLPs achieved the highest transfection effi-
ciency. It might be deduced that the uptake of 5% GA–PEG–CLPs
was enhanced by the over-expressing GA receptors PKC␣ on the
membrane of hepatocellular carcinoma cells.
The transfection efficiency of PEG–CLPs was one time lower than
CLPs, probably resulting from the steric shielding of the vector’s
cationic charge from the highly anionic cell surface proteoglycans
7
.74 ␮M and 10.32 ␮M) for 30 min in order to saturate the GA bind-
ing site of PKC␣. The GFP gene expression efficiency in HepG2 cells
pretreated by GA dramatically decreased (p < 0.05). As seen in Fig. 8,
(
as seen in Fig. 3, the zeta potential of CLPs was higher than that
2
.58 ␮M GA which was two times GA that 5% GA–PEG–CLPs con-
of PEG–CLPs p < 0.05) (Mounkes et al., 1998). After introduction of
GA–PEG–Chol, the transfection efficiency increased. When the con-
tent of GA–PEG–Chol was up to 5%, the transfection efficiency of
stealth lipoplexes was not dramatically different from that of CLPs.
This suggested that the PEG-dependent shielding of charge inter-
actions may have been compensated by the high affinity binding of
GA to PKC␣ on HepG2 cells (Reddy et al., 2002).
tained, was enough to inhibit the PKC␣ activity. Further increase
in GA concentration, no further decrease in GFP gene expression
efficiency was observed. Thus, it might be concluded that HepG2
cells over-expressed PKC␣ and GA–PEG–CLPs HCC targeting was
mediated by the specific interaction of PKC␣ with GA targeting
moiety.
Fig. 6. GFP gene expression efficiency of HepG2 after treatment with four kinds of
lipoplexes.
Fig. 8. GFP gene expression efficiency of HepG2 after treatment with different con-
centrations of GA and 5% GA–PEG–CLPs.

Z.Y. He et al. / International Journal of Pharmaceutics 397 (2010) 147–154
153
cell line L02 by comparing with CLs, PEG–CLs and 1% GA–PEG–CLs.
The results were shown in Figs. 10 and 11. On both cell lines, the
cytotoxicity of four blank cationic liposomes were dependent on
the concentration. The cytotoxicity of 5% GA–PEG–CLs was similar
as PEG–CLs and 1% GA–PEG–CLs on HepG2 cell line, but lower than
CLs, PEG–CLs and 1% GA–PEG–CLs on L02 cell line. This indicated
that 5% GA–PEG–CLs was a low-toxicity gene carrier suitable for
hepatoma targeting therapy.
4. Conclusion
As a novel hepatoma targeting gene vector, GA–PEG–CLs could
entrap pDNA with high efficiency and enhance the uptake of
pDNA in hepatocellular carcinoma cells and then achieve high
transfection efficiency. The gene entrapment and transfection effi-
ciencies of GA–PEG–CLs were dependent on the amount of GA
ligand and had a positive relationship. 5% GA–PEG–CLs, comprising
DOTAP/Chol/GA–PEG–Chol at molar ratio of 50:45:5, had higher
gene transfection efficiency and lower cytotoxicity of normal hep-
atocyte cells, which might be one of the most potential gene vectors
as hepatoma targeting therapy.
Fig. 9. GFP gene expression efficiency of HepG2 after treatment with four kinds of
lipoplexes in the medium containing 10% serum.
Acknowledgements
This research has received financial support from the National
Natural Science Foundation of China (Nos. 30901868 and
3
0772668), National 863 Project (No. 2007AA021810) and the Doc-
toral Program of Higher Education of China (No. 20090181120114).
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