Engineering, 2012, 5, 172-175
doi:10.4236/eng.2012.410B045 Published Online October 2012 (
Copyright © 2012 SciRes. ENG
The Mechanism of Lipofectamine 2000 Mediated
Transmembran e Gene Delivery
Shaohui Cui, Shubiao Zhang, Hui ying Chen, Bing Wa ng, Yinan Zhao, Def u Zhi
Key Laboratory of Bio-chemistry Engineering - The State Ethnic Affairs Commission-Ministry of Education,
Dalian Nationalities University, Dal ian, C hina
Received 2012
In this paper, the relatived mechanism between lipofectamine 2000 mediated transmembrane gene delivery and endocytic pathway
were invest ig ated . Cl ath rin and caveolae-mediated endocytic pathway contributions to transfection efficiency were studied. The inhi-
bitors of endocytosis were used to treat HEp-2 cells before lipofectamine 2000/pGFP-N2 transfection. Transfection efficiency was
evaluated with green flu ores cence p rotein (GFP) expression assays. Cell viability and cytotoxicity were evaluated with MTT method.
The results indicated that inhibitors of clathrin (chlorpromazine or wortmannin) and caveolin (genistein) could reduce the cell trans-
fection efficiency o bservabl y. Both clathrin and caveolae-mediated endocytic pathways play important roles in transmembrane gene
Keywords: Gene Delivery; Cationic Liposomes; Transfection Efficienc y; Endocytic Pathwa y; Inhibitor
1. Introduction
Non-viral DNA delivery systems have been developed to faci-
litate gene entry into mammalian cells[1]. It has shown that
non-viral vectors have the advantages over viral ones as they
are non-immunogenic, easy to produce and not oncogenic.
However, they have the major limitation of inefficien t tran sfec-
tion[2]. Indeed, non-viral vectors showing much lower toxicity
to cell proliferati on were commerciall y available for li poplexes
mediated transfection (lipofection) of cultured cells. However,
despite several studies investigating the mechanisms of uptake
and intracellular trafficking of non-viral vectors to improve
delivery, the current understanding of these processes was still
limited. Because cell membrane was the first barrier for lipop-
lexes entry into the cell, the mechanism of cationic liposomes
mediated transmembrane gene delivery was important as the
study could contribute to improving transfection efficiency.
There is convincing evidence that endocytosis represents the
major pathway of lipoplexes entry into cells that leads to pro-
ductive gene expressio n, rather than that genes are tr anslocated
into the cytoso l at the level o f the pl asma membrane [3-4]. How-
ever, ther e are vario u s endo cytic p ath ways op erated in eukaryo-
tic cells, such as clathrin-dependent and clathrin-independent
pathways, the latter includes phagocytosis, macropinocytosis
and caveolae-mediated internalization[5]. The relative contribu-
tion of each pathway in lipoplexes internalization has been
poorly defined to date, although involvement of the clath-
rin-mediated pathway has been firmly established, while evi-
dence was emerging that entry may also occur via macropino-
cytosis[4-6]. Endocytotic inhibitors such as chloroquine, chlor-
promazine are very helpful for studying drug intracellular re-
lease or elucidating a specific endocytosis route[7]. There are
still many problems that have not been elucidated on the bio-
logical barriers and properties of gene delivery through non-
viral vector s, alth ough much effort has been exp ended on them.
Understanding the mechanism of transmembrane might play a
significant role in overcoming the hurdles in gene delivery.
This study was performed with cationic liposome, lipofecta-
mine 2000 which showed higher transfection efficiency. The
inhibitors of clathrin and caveolin, such as chlorpromazine,
wortmannin and genistein were used to treat with cells. Via
evaluated inhibitory effects of lipoplexes transfection to explore
the underlying transmembrane mech anism in HEp-2 cells.
2. Experimental
2.1. Materials and Equipment
Human throat epidermis (HEp-2) cancer cell line was pur-
chased from Cell Bank of Chinese Academy of Sciences.
RPMI-1640, DMEM, Fetal bovine serum (FBS) were pur-
chased from HyClone. Lipofectamine 2000 transfection reagent
were purchased from Invitrogen Corporation. Plasmid
pGFP-N2 (containing green fluorescent protein gene) was pur-
chased from Clontech Company. MTT, Wortmannin, Chlor-
promazin e, Genistein were purchased from sig maAldrich.
Inverted fluorescence microscope (Olympus IX 71) was
purchased from Japan. Microplate reader (Sunrise Tecan) was
purchased from Australia. Carbon dioxide incubator (NAPCO
7100) was purchased from France.
2.2. Cell Culture
HEp-2 cells were grown in 100 ml culture flask in RPMI-1640
supplemented with 10% fetal bovine and antibiotics (100 U
penicillin/ml and 100 mg streptomycin/ml) at 37 in 5% CO2
2.3. Lipoplexes P reparat ion
Copyright © 2012 SciRes. E NG
Preparation of lipoplexes (50μl/well amount of calculation):
The plasmid DNA (pGFP-N2) 0.2μg and transfection reagents
Lipofectamine 2000 were diluted in Opti-MEM 25μl, and then
the diluted transfection reagent dropping 25μl dilutions of
plasmid DNA to 25μl, the side with the vortex oscillator plus
side, at room temperature and incubated for 20 min. The weight
ratio of lipofectamine 2000 to pDNA was kept constant at 3:1.
2.4. Transfection Procedure
HEp-2 cells ( 1×105 cells /ml) were s eeded int o 96-well plates in
100μl growth medium (RPMI-1640) until the required cell
number was obtained (80% confluence) at the time of transfec-
tion. Cells were washed once with PBS and 50μl transfection
lipoplexes (prepared as described above) were added to each
well. It was mixed gently by rocking the plate back and forth.
The cells were incubated in DMEM (not containing FBS) under
5% CO2 at 37 in a humidified incubator for 4-6 h, then cell s
were washed by PBS once and medium was replaced by com-
plete medium and cells were further cultured for 48h, prior to
analysis. Fi nal ly, assay for t ran sgene exp ressio n or selecti on for
stable transfection was do ne according to report gene.
2.5. Green Fluorescence Protein (GFP) Assay
The expression of green fluorescence protein was imaged by
inverted fluorescence microscope and transfection efficiency
was given as relative efficiency. The transfection efficiency
was determined based on cell percent with GFP-express ing.
The number of GFP-expressing cells versus the total cell quan-
tity in th e micros cop e was defin ed as th e tran sfectio n efficien cy.
Cell counting was performed randomly in microscopic observa-
tion scope under 10×16 magnifications with three repeats. Data
were presented as the mean ± sta ndard deviatio n ( SD).
2.6. Cytotoxicity Assay
The cytotoxicity was evaluated by MTT assay. Hep-2 cells
(1×105 cells /ml) were seeded into 96-well plates at 200μl /well
and incubated 24 h after. 50μl MTT (5mg/ml in PBS) was add-
ed and incubated for an additional 4 h at 37. MTT-containing
medium was aspirated off and 150μl DMSO was added to dis-
solve the formazan and the absorbance was meas ured at 570 nm
to determine cell viability as percentage of control. Non-treated
cells as contro l.
3. Results
3.1. Inhibitors of Cl athrin In fluenced Tra nsfect i on
Efficiency of Lipoplexes
Endocytosis is the process by which cells take up macro- mo-
lecules from the surrounding medium. The best character- ized
process is the so-called clathrin-dependent endocytosis. To
assess whether clathrin-mediated endocytosis influenced trans-
fection efficiency two inhibitors of clathrin (chlorprom-azine
and wortmannin) were used to treated HEp-2 cells 1 hour be-
fore transfection. The transfection efficiency of lipofecta-
mine2000/pGFP-N2 complexes was evaluated by GFP expres-
sion which showed significantly higher inhibition than lipofec-
tamine2000 control. Figure 1 showed the represent-ative fluo-
rescence profiling of positive GFP-expressing cells and whole
cell contrast. Chlorpromazine (10-100μM) could observably
lower transfection efficiency 1-12-fold (Figure 1 a, b) and
wortmannin (10-100nM) had 0.8-15-fold (Fig. 1 c, d) com-
pared with control. These results suggested that both chlorpro-
mazine and wortmannin could lower transfection efficiency
through inhibiting clathrin pathway. Endocytosis is also cur-
rently known about clathrin-independent endocytic processes
such as those involving caveolae and lipid rafts. An under-
standing of endocytosis and the cellular trafficking that occurs
thereafter has a great deal of relevance to current molecular
Fig ure 1 . Effects of treatment with diff erent concentration s of chlorpromazine (10100μM ) and wortmannin (10100 nM) o n HE p-2 cells
1 hour after transfected lipofectamine 2000/pGFP-N2 complexes experimental image ( inverted fluorescent microscope photos:20×10) con-
trast livin g cell.
Copyright © 2012 SciRes. ENG
In addition, the cytotoxicity was tested with chlorproma-zine
and wortmannin by MTT assay which could influenced cell
viability and the valuation of cell transfection efficiency. Cell
viability were 85%, 80%, 74%, 70%, 65%, 60% respectively
when treated cell with chlorpromazine10, 20, 40, 60, 80,
100µM(Figure not showed). The cytotoxicity of chlorproma-
zine was lower. The cytotoxicity of wortmannin was tested
from 10-100nM. When the concentrations of wortmannin ex-
ceeded 60nM, cell survival rate was under 50% (Figur e 2).
Therefore, the cytot oxicit y could influ ence to eval uate tran sfec-
tion efficiency seriously. But, between 10nM and 60nM, wort-
mannin could inhibite the transfection efficiency.
3.2. The Effects of Inhibitors of Caveolin on
Lipoplexes Cell Transfe c tion
In order to explore caveolae-dependent internalization pathway
effects on transfection processes. Genistein, the inhibitor of
caveolin, was used to treat HEp-2 cells 1 hour before cell
transfection. Caveolins are essential for the formation and sta-
bili ty of caveolae. Figure 4 showed (part of pictures) the trans-
fection efficiency was reduced 80% , 73% , 65% , 45% with
genistein (10, 50, 100, 200μM), respetivley, in terms of GFP
expression than control. These results demonstrated the me-
chanisms of transmembrane transport of lipoplexes by caveo-
lae-dependent pathway into the cell membrane channels.
Through inhibiting the activities of caveolin Genistein could
reduce caveolae-mediated endocytosis and transfection effi-
ciency compared with Lipofecamine 2000 control. The cyto-
toxicity of genistein10-100µMwas very small with the cell
viability of above 80% , but genistein with the concentrations
over 100µM could cause cell viability to lower much (Figure
control1020 40 60 80100
Wortmannin conc.(nmol.L-1)
Cell viability(%)
control10 20 40 60 80100
Chloromazin conc.(umol.L
Cell viability(%)
Figure 2. HEp-2 cells treated with different concentrations of inhi bitor : wortmannin and chlorpromazine1hr, then was determined with the
tetrazolium-based colorimetric cell viability. Non-treated cells as control. Error bars show standard deviation (n = 4).
control10 30 50 80100150200
Cell viability(%)
Figure 3. HEp-2 cells treated with different concentrations of genistein 1hr, then was determined with the tetrazolium-based col orimetric cell
viability. Non-treated cells as control. Error bars show standard deviation (n = 4).
Figure 4. Effects of different concentrations of genistein on HEp-2 cells transfected Lipofectamine 2000/pGFP-N2 complexes experimental
image ( inverted fluorescent microscope photos:20×10).
Copyright © 2012 SciRes. E NG
4. Discussion
Knowledge about the transmembrane uptake mechanism is
important for the development of more efficient carriers. Cell
membrane was the first barrier for internalization of lipoplexes.
Some resul ts indicat ed that endo cytosis was the majo r mechan-
ism of entry. Lipoplexes via which endocytic pathways entry
into cell is of great importance for the design of further im-
proved non-viral gene delivery systems. There are various en-
docytic pathways in eukaryotic cells[8-9]. Therefore, the aim of
this study was to investigate the endocytic pathways were in-
volved in cationic liposome mediated transmembrane gene
delivery. A correlation between clathrin-mediated or Caveo-
lae-dependent endocytosis and lipoplexes transfection was
supported convincingly by several pieces of evidence, including
the use of inhibitors of endocytosis, co-localization with path-
way-specific markers and defective in clathrin-mediated endo-
cytosis. They failed to internalize lipoplexes and concomitantly
showed a proportional decrease in transfection efficiency. A
combined approach was essential, since the use of inhibitors
alone might give rise to unambiguous outcome because o f to xic
side effects. Our research focused on plasma membrane endo-
cytic pathways to determine the endocytic pathways contri-
buted to lipoplexes gene delivery. The results indicated that
transfection efficiency was decreased after incubation with
inhibitors, both through the clathrin and caveolae-mediated
pathways inhibition. However, inhibition of caveolin signifi-
cantly reduced the uptake of lipoplexes. It was suggested that
the mechanism of cationic liposomes mediated transmembrane
gene delivery mainly dependent caveolae-mediated pathway.
Taken together, these results demonstrated that uptake of li-
poplexes in HEp-2 cells was mediated via both the clath-
rin-mediated and caveolae-mediated pathway. It is not definite
which is the main. These results are of i mportan ce for success-
ful gene transport via clathrin-mediated and caveolae-mediated
uptake and rational design of cationic lipid gene delivery sys-
5. Acknowledgements
The study was supported by the National Natural Science
Foundation of China (21046008 and 21176046) and the Fun-
damental Research Funds for the Central Universities
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