Advances in Materials Physics and Chemistry, 2012, 2, 229-232
doi:10.4236/ampc.2012.24B058 Published Online December 2012 (http://www.SciRP.org/journal/ampc)
In Vitro Study of Carbamate-Linked Cationic Lipid for Gene
Delivery Against Cervical Cancer Cells
Defu Zhi1, Shuibao Zhang1, Yinan Zhao1, Shaohui Cui1, Bing Wang1, Huiying Chen1, Defu Zhi2, Defeng Zhao2
1SEAC-ME Key Laboratory of Biochemistry Engineering, Dalian Nationalities University, Dalian, China
2State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, China
Received 2012
ABSTRACT
Design and synthesis of a carbamate-linked cationic lipid DDCTMA (N-[1-(2,3-didodecylcarbamoyloxy)propyl]-N,N,N-trimethyl-
ammonium iodide) as gene delivery carriers was described in this work. The transfection efficiency of cationic liposome increased
dramatically with the increase in the content of DOPE. In addition, the transfection efficiency of some of cationic lipoplexes was
superior or parallel to that of two commercial transfection agents, Lipofectamine2000 and DOTAP. The carbamate-linked cationic
lipid DDCTMA/DOPE may be a promising gene carrier that has high transfection efficiency as well as low cytotoxicity.
Keywords: Cationic Lipids; DNA Condensation; Gene Delivery; Transfection Efficiency
1. Introduction
As an important non-viral gene vector, cationic lipids have
attracted increasing attention because they have many potential
advantages compared with other non-viral vectors, including
significant simplicity and ease of production, good repeatability,
potential commercial value, and wide range of clinical applica-
tion and safety [1]. Felgner et al. [2] have demonstrated that
cationic lipid—DOTMA (N-[1-(2, 3-dioleoyloxy)propyl]-N,N,
N-trimethylammonium chloride) was an effective gene vector
in the study of complex transfection, since then numerous cati-
onic lipids with different structures have been synthesized and
used for the delivery of nucleic acids into cells during the last
25 years [3].
Cationic lipids have three basic chemical functional domains:
a hydrophilic headgroup, a hydrophobic domain, and a linker
bond that tethers the cationic headgroup and hydrophobic tail
domain [4]. Linker bonds of cationic lipids have large influence
on the transfection efficiency, biodegradability and the stability
of cationic lipids. Most of the linker bonds in some synthesized
lipids are ether, ester, or amide bonds, which are either too
stable to be biodegraded and thus cause cytotoxicity (e.g., ether
linkers) or prone to decompose during systemic circulation (e.g.,
ester linkers). In contrast with these strategies, in a previous
paper [5] we developed two carbamate-linked lipids (DDC-
TMA and DDCEDMA) bearing quaternary ammonium head-
group and identical length of hydrocarbon chains for the pur-
pose of taking advantage of the pH sensitivity of the carbamate
bond (Scheme 1), which has high transfection efficiency in
Hela and Hep-2 cells as well as low cytotoxicity. To advance
the study of this lipid for gene delivery we investigated the
influence of the N/P ratios on the characteristics of the
DDCTMA/DOPE/DNA complex compared with two commer-
cial transfection agents, Lipofectamine2000 and DOTAP.
2. Materials and Methods
2.1. Instruments and Reagents
Most chemicals were obtained from Sinopharm Holding Co.
Ltd. (Shanghai, China). 3-Chloro-1,2-propanediol was pur-
chased from Johnson Matthey (Hong Kong, China). N,N-car-
bonyldiimidazole (CDI) was purchased from Medpep Co. Ltd.
(Shanghai, China). Cell culture media and fetal bovine sera
(FBS) were purchased from Invitrogen Corporation (Carlsbad,
CA, USA). Dulbecco’s modified Eagle’s medium (DMEM)
was purchased from Sigma Co. Ltd. (USA).
Lipofectamine 2000 reagent was purchased from Invitrogen
Corporation (Shanghai). DOTAP reagent was purchased from
Roche Diagnostics GmbH.
Scheme 1. Synthetic routes of cationic lipid. Reagents and condi-
tions: (a) 2.5 equiv. 33% dimethylamine in aqueous solution, 1.0
equiv. sodium hydroxide, 4 h at 50 ºC, (70%); (b) 2.1 equiv. CDI in
toluene solution, 3 h at 40 ºC with N2; (c) 2.1 equiv. alkylamine in
toluene solution, 3 h at 40 ºC with N2, (50-60%); (d) 40 equiv. halo-
genated hydrocarbons, 24 h, 80 ºC (95%).

D. F. ZHI ET AL.
230
2.2. Characterization
FTIR spectral studies were carried out with a Thermo Nicolet
370 DTGS (USA) spectrometer in the range between 4000 and
500 cm-1. All powder samples were compressed into KBr pel-
lets for the FTIR measurements. ESI-MS was detected by
SHIMADZU LCMS-2010EV (Japan) in methanol (MeOH)
depending on sample solubility at room temperature. Electros-
pray ionization was achieved by application of a potential of
3.5 kV to a stainless needle. A Harvard apparatus syringe pump
system was set at 3.0 mL/min. Nitrogen as a nebulizer gas was
delivered to the spectrometer by a nitrogen line. HPLC-ELSD
was detected by SHIMADZU LCMS-2010EV (Japan) and
SFD-ZAM3000 (Schambeck SFD GmbH, Germany). Nuclear
magnetic resonance (NMR) spectrum was recorded on a Varian
Mercury plus 400-MHz NMR (USA) spectrometer in chloro-
form (CDCl3) depending on sample solubility at room tem-
perature.
2.3. Synthesis and Characterization of Cationic Lipid
(DDCTMA)
Synthesis and characterization of cationic lipid DDCTMA
were as reported earlier [5]. The synthesis of cationic lipid
DDCTMA is shown in Scheme 1.
2.4. Preparation of Liposomes and Plasmid DNA
A solution of cationic lipid (1mg) in chloroform (1mL) was
evaporated under a stream of nitrogen, and the residual solvent
was removed under vacuum overnight. Liposomes were pre-
pared by resuspending the lipid in distilled water (1mL) at 55°C
and sonicating them to clarity at this temperature for 2 h in a
closed vial.
Plasmid pGL3 coding for luciferase gene was purchased
from Clontech (USA). Plasmid DNA was isolated using a BBI
DNA purification kit. The DNA concentration was determined
by measuring UV absorbance at 260nm and 280nm, and the
purity was confirmed by agarose gel electrophoresis and
OD260/280 measurement.
2.5. DNA Binding Assay
DNA-lipid complexes were formed by mixing 2 μg of plasmid
DNA (0.1 μg/μL in 10mM Hepes buffer, pH 7.4) with varying
amounts of cationic lipid so that the final lipid/DNA charge
ratios were maintained at 0.5/1 to 8/1 in a total volume of 50 μL.
Complexes were incubated for 30 min at room temperature
after which 20 μL of each lipoplex was loaded on a 1.2% aga-
rose gel and subjected to electrophoresis. The samples were
electrophoresed at 90 V for 1h, and the bands were visualized
with ethidium bromide staining.
2.6. Transfection Assay
Human cervical cancer cells (7721) were obtained from ATCC
(American Type Culture Collection Shanghai Representative
Office) and seeded in 100μl of growth medium (RPMI1640)
without antibiotics. The cells were incubated at 37 ºC in a fully
humidified atmosphere containing 5% CO2, up to 80% conflu-
ence prior to use.
To measure transfection efficiency, liposomes and 0.5μg
plasmid DNA were diluted in 25μl DMEM without serum,
respectively, and mixed gently. Five minutes after dilution, the
diluted liposomes were added to the diluted DNA and mixed
together with vortex. The mixture was held for 20 min at room
temperature to enable the lipoplex formation. The original cell
culture media were replaced with the lipoplex solution contain-
ing the transfection lipoplexes (prepared as described above)
and phosphate buffered saline (PBS) for each well. They were
incubated at 37ºC in a humidified incubator with 5% CO2 for
4-6 h, then cells were washed by PBS or DMEM once and the
medium was exchanged with fresh and complete DMEM cul-
ture media and cells were further cultured for 48 h, prior to
analysis. Finally, the transfection efficiency of some of the
cationic liposomes, as % relative light units (RLU) was meas-
ured by luciferase assay.
3. Results and Discussion
We have described the synthesis of lipid DDCTMA and re-
ported some of its physicochemical characteristics and transfec-
tion efficiencies in vitro and cytotoxicity using Hela and Hep-2
cell lines [5]. Herein, we not only investigated its DNA-binding
ability and transfection efficiencies using 7721 cell lines but
also researched on the influence of the N/P ratios on the char-
acteristics of the DDCTMA/DOPE/DNA complex compared
with two commercial transfection agents, Lipofectamine2000
and DOTAP.
3.1. Properties of cationic liposomes
In order to determine the effect of DOPE and cationic lipo-
somes to pGFP-N2 plasmid charge ratios (N/P ratios), pGL3
plasmid complexes were prepared by adjusting the stoichiome-
try of cationic liposomes and plasmid (N/P, 0.5/1, 1/1, 1.5/1,
2/1, 3/1, 4/1, 6/1and 8/1), using liposomes prepared from cati-
onic lipid (DDCTMA) and a co-lipid (DOPE) at different molar
ratios (N/D, 2/1, 1/1 and 1/2).
As shown in Figure 1, the DNA-binding ability of cationic
liposome increased with an increase in the N/P ratio, indicating
that these liposomes have an ability to form a lipoplex with
plasmid DNA. When the N/P ratio of DDCTMA/DOPE (2/1)
liposome/DNA was greater than 2, liposome and DNA bound
tightly and completely. When N/P was equal to 4, the band of
DDCTMA/DOPE (1/1) liposome/DNA was fainter than the
N/P ratios of 6/1 and above. However, DDCTMA/DOPE (1/2)
liposome was found to be the weakest in DNA binding, as the
band of plasmid DNA disappeared at the N/P ratio of 8/1. Ob-
viously, the DNA-binding ability of cationic lipid decreased
with an increase in the content of DOPE. It is postulated that
the increase in the content of DOPE induces significant changes
in morphologies and structural parameters of the lipoplexes
(such as, particle size and ζ-potential as showed in Table 1) and
hence influences their DNA-binding ability [6].
3.2. In Vitro Transfection
In constructing gene complex vectors, the molar ratio of cati-
onic lipid to co-lipid may play an important role in influencing
the phase transition temperature of cationic liposomes and the

D. F. ZHI ET AL. 231
structure of lipoplexes. To determine the most appropriate for-
mulations, we prepared cationic liposomes of DDCTMA and
DOPE with different molar ratios (N/D ratios, 2/1, 1/1 and 1/2),
as these ratios are the most commonly used proportions in
cationic liposomes preparation [7], and transfection efficiency
was reported as % relative light unit (RLU) per mg of total
protein content as shown in Figure 2. Transfection efficiency
of cationic liposome increased dramatically with the increase in
the content of DOPE. It may be because the DNA-binding abil-
ity of cationic liposomes was too strong to release DNA from
complex lower DOPE ratios, leading to relative lower transfec-
tion efficiency and the agarose gel electrophoresis experiments
also supplement this fact.
N/D=2/1 N/D=1/1 N/D=1/2
Figure 1. Gel electrophoresis of cationic liposomes (DDCTMA
DOPE=2/1, DDCTMA/DOPE=1/1, and DDCTMA/DOPE=1/2)
/pGFP- N2 complexes at various weight ratios. Lane 1: marker (λ
DNA/EcoR I + Hind III Markers from SABC), lane 2: naked plas-
mid DNA (2 μg) and lanes 3→10: lipoplexes of plasmid DNA (2 μg)
with progressively increasing proportions (N/P, 0.5/1, 1/1, 1.5/1, 2/1,
3/1, 4/1, 6/1and 8/1) of cationic liposomes.
Table 1. Particle Size and Zeta Potential of Liposomes.
ζ-potential
(mV)
Particle size
(nm) PDIa
Lipofectamine 2000 46.4 144 0.230
DOTAP 48.6 170 0.477
DDCTMA/DOPE=2/1 40.3 259 0.572
DDCTMA/DOPE=1/2 43 233 0.551
0
10000
20000
30000
40000
50000
60000
0.5/11/1 2/1 3/14/1 6/1 8/1
Liposomes
RLU/m g
DDCTMA/DOPE=2/1 DDCT MA/DOPE=1/1
DDCTMA/DOPE=1/2 Lipofectamine 2000
DOTAP
Figure 2. Effect of DOPE composition in DDCTMA lipoplexes
(DDCTMA/DOPE=2/1, DDCTMA/DOPE=1/1 and DDCTMA/
DOPE=1/2) on transfection efficiencies in 7721 cells and compared
with transfection efficiencies of Lipofectamine2000 and DOTAP.
The N/P ratio is another important factor to affect the trans-
fection efficiency. With respect to the influence of weight ratios,
these cationic liposomes showed a maximum transfection level
at the N/P ratio of 2/1. As the N/P ratio influences the property
of lipoplexes in constructing gene complex vectors, the N/P
ratio has an impact on transfection efficacy.
Under certain conditions, the transfection efficiency of car-
bamate-linked cationic lipid was superior or similar to that of
the two commercial transfection agents. For example, DDC-
TMA/DOPE=1/2-liposome demonstrated higher transfection
efficiency at the N/P ratios of 2/1 compared with Lipofec-
tamine2000 and DOTAP. Therefore, the synthetic carbamate-
linked cationic lipid has a great potential for DNA complexa-
tion and may be useful as non-viral vectors for clinical therapy
applications.
4. Conclusions
We developed an efficient method of transfection by combining
cationic liposome and DOPE and showed that the N/P ratio of
cationic liposome/DNA may markedly influence the character-
istics of the complex vector. The combination of cationic lipo-
some and DOPE resulted in the high gene transfection effi-
ciency in vitro gene delivery. The results demonstrate that the
DNA-binding ability of cationic liposomes was too strong to
release DNA from complex in the transfection, leading to rela-
tive lower transfection efficiency. Additionally, the transfection
efficiency of some cationic liposomes was superior or similar to
that of the two commercial transfection agents, which also
suggested that the complex vector might be a promising gene
carrier and can be considered for use in gene transfer in vivo.
5. Acknowledgements
The study was supported by the National Natural Science
Foundation of China (20876027 and 21176046), Roche and the
Fundamental Research Funds for the Central Universities
(DC12010104).
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