A new helper phospholipid for gene delivery

Article abstract of DOI:10.1039/b716247b

Enhanced gene transfection activity is observed when using a new helper lipid with DOTAP, compared to DOPE. The Royal Society of Chemistry.

Full text of DOI:10.1039/b716247b

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A new helper phospholipid for gene deliveryw
Carla A. H. Prata,a Yougen Li,b Dan Luo,b Thomas J. McIntosh,c
Philippe Barthelemyd and Mark W. Grinstaff*a
Received (in Austin, TX, USA) 22nd October 2007, Accepted 27th December 2007
First published as an Advance Article on the web 29th January 2008
DOI: 10.1039/b716247b
Enhanced gene transfection activity is observed when using a
new helper lipid with DOTAP, compared to DOPE.
for 1 day with trimethylamine in acetonitrile to give compound
4.
Given the structural similarity between 4 and lipids 2 and 3,
it is likely that 4 will form bilayers. A differential scanning
calorimeter (DSC) trace of hydrated amphiphile 4 shows a
phase-transition temperature at B44 1C. Next, we prepared
vesicles of 4, 4/DOTAP, and DOPC/DOTAP. The milky
aqueous suspension of 4, 4/DOTAP, or DOPC/DOTAP was
extruded through a 100 nm polycarbonate membrane using a
mini-extruder and after 20 extrusions, a homogeneous lipo-
some solution was observed (see ESI for detailsw). The average
diameter, determined by dynamic light scattering, of the
liposomes prepared from 4, 4/DOTAP, and DOPC/DOTAP
was 106, 127, and 139 nm, respectively. Transmission electron
micrographs of 4 and 4/DOTAP showed vesicular organiza-
tions in both samples of several hundred nanometres. The
structure of the bilayers formed by 4 was investigated at 25 1C
by X-ray diffraction. The diffraction patterns of oriented
multilayers of 4 show a lamellar structure with a d spacing
of 6.1 nm. We were unable to obtain patterns from pellets of
4/DOTAP. In the presence of DNA, the 4/DOTAP/DNA
assembly d spacing increases to 7.6 nm. This 1.5 nm increase
in repeat period is similar to that observed for other bilayers
containing cationic lipids when DNA is incorporated between
adjacent bilayers.16
The delivery of nucleic acids to a cell offers the potential to
correct a defective gene or introduce a new gene for a specific
biological activity.1–7 As such, in vitro gene delivery is widely
used in research laboratories, and in vivo gene therapy holds
promise for the cure of genetic diseases including cancer. The
current delivery approaches include viral vectors, synthetic
cationic vectors, calcium phosphate particles, surface-
mediated vectors, and electroporation.1,2,8,9 Of these, synthetic
cationic vectors offer the advantages of minimal toxicity, non-
immunogenicity, ease of synthesis, and large nucleic acid
payloads; but suffer from low transfection activities.1–4,10–20
This low activity reflects inefficiencies in the overall transfec-
tion pathway.21 Today, many synthetic cationic vectors such
as 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP;
1) are used in conjunction with ‘‘helper’’ phospholipids which
allow fusion of the bilayer with the membrane of the endo-
some, to increase the transfection efficacy.22,23 These helper
lipids are typically zwitterionic lipids such as dioleoyl-
phosphatidylethanolamine (DOPE; 2) or dioleoylphosphati-
dylcholine (DOPC; 3). Herein, we describe the synthesis and
characterization of a new helper lipid that displays enhanced
gene transfection activity when used with DOTAP.
The propensity of the lipids to bind DNA was measured via
an ethidium bromide displacement fluorescence assay. This
assay entailed measuring the reduction of the fluorescence
The zwitterionic phospholipid 4 is shown in Fig. 1. The
complete synthetic procedure to lipid 4 can be found in the
ESIw. Briefly stated, benzyl formate was added to an octane
solution of dodecanoic diacid in the presence of Dowex 50W-
X2, and the resulting monobenzylated fatty acid was coupled
to 3-(tert-butyldiphenylsilyl)glycerol in the presence of DCC
and DMAP in CH2Cl2. The silyl group was removed with
n-tetrabutylammonium fluoride in tetrahydrofuran. The
resulting compound was reacted with chloro-oxo-dioxa-
phospholane in THF at 0 1C for 18 h, and then the reaction
mixture was transferred to a pressure tube and heated to 60 1C
a Departments of Biomedical Engineering and Chemistry, Boston
University, Boston, MA 02215, USA. E-mail: mgrin@bu.edu;
Tel: +1 617-358-3429
b Department of Biological and Environmental Engineering, Cornell
University, Ithaca, NY 14853, USA
c Department of Cell Biology, Duke University Medical Center,
Durham, NC 27710, USA
d
´
INSERM U869, Bordeaux, Universite Victor Segalen, F-33076
Bordeaux, France
w Electronic supplementary information (ESI) available: Complete
experimental details, characterization data, and cytotoxicity experi-
ments. See DOI: 10.1039/b716247b
Fig. 1 Cationic and zwitterionic amphiphiles under investigation.
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intensity of the DNA-intercalated ethidium bromide, as this
fluorescent probe is displaced by the cationic amphiphile. The
fluorescence intensity decreases upon addition of DOTAP,
4/DOTAP, DOTAP/DOPE, and DOPC/DOTAP. The results
obtained with DOTAP, DOTAP/DOPE, and DOPC/DOTAP
are consistent with previous reports. A 1 : 1 assembly is formed
between the lipids and DNA. The zwitterionic lipids 4, DOPC,
and DOPE do not displace EtBr, consistent with the unfavor-
able electrostatic interactions with the anionic DNA (Fig. 2).
Transfection experiments with 4, 4/DOTAP, DOPE/
DOTAP and DOTAP using the reporter gene, b-galactosidase
(b-gal, pVax-LacZ1, Invitrogen), were performed with Chi-
nese hamster ovarian (CHO) cells and L6 human muscle cells.
The DOPE/DOTAP system was used instead of DOPC/
DOTAP since the former is reported to be more active.22
The reporter gene was first mixed with the lipids in potassium
phosphate buffer (PBS) at room temperature. We tested two
different lipid/DNA ratios (5 : 1 and 20 : 1) while keeping the
zwitterionic/DOTAP ratio constant at 1 : 1. Next, the lipid/
DNA complexes were added to the cells. The amount of DNA
used was the same as used in the naked DNA control (no
lipid), and the negative control was compound 4 without
DNA. After incubation at 37 1C and 5% CO2 for 2 h, the
medium was removed and fresh growth medium was added.
Transfection efficiencies were assessed after 48 h using the
b-galactosidase enzyme assay in conjunction with a standard
curve. The efficiency of each transfection was calculated as
b-gal activity normalized to total protein. The zwitterionic
lipid 4 by itself does not transfect DNA. As expected upon
addition of DOPE to DOTAP, the transfection efficacy
increased.22 In the presence of 4/DOTAP at a ratio of 1 : 1
(with amphiphile/DNA ratio of 20 : 1), the transfection
increased B400% when compared to DOPE/DOTAP under
the same conditions (Fig. 3). Increasing the 4/DOTAP ratio
from 1 : 1 to 2 : 1 afforded higher activity, but further increases
in the ratio yielded less transfection. With these encouraging
results, we evaluated the 4/DOTAP vector system for gene
transfection in L6 cells. As shown in Fig. 3, the use of the
zwitterionic lipid 4 and DOTAP increased the transfection
level by about four-fold compared to DOPE/DOTAP. Cyto-
toxicity experiments were also performed with CHO cells, and
Fig. 3 (Top) gene transfection results after 48 h in CHO cells (cation/
zwitterion ratio in parentheses) and (bottom) gene transfection results
after 48 h in L6 cells (cation/zwitterion ratio in parentheses). Amphi-
phile/DNA ratio 1 : 20; N = 3.
none of the amphiphiles showed significant cytotoxicity, with
results similar to the non-treated cells (see ESIw).
In summary, we have synthesized and characterized a new
helper zwitterionic phospholipid for use in the delivery of
nucleic acids into cells. When combined with DOTAP, this
phospholipid affords an increase in gene delivery as measured
by new protein expression in two different cell lines. Mechan-
istic studies including cell trafficking experiments are ongoing
to elucidate the pathway and the role this new helper lipid
plays in gene transfection. The investigation of new synthetic
vector systems provides opportunities to prepare specific
amphiphilic structures, study their self-assembly properties,
characterize their interactions with nucleic acids, and evaluate
new strategies for increasing transfection efficiency.
This work was supported by BU and grants from the
National Institutes of Health (MWG and TJM).
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Fig. 2 Ethidium bromide displacement assay for the amphiphiles
under investigation. N = 3. Error bars per point range from 5 to 10%.
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