Bioconjugate Chemistry
ARTICLE
present study gradually diminished after 24 h post injection; data
not shown), the presently described coconut amphiphiles 1 may
find future use in nonviral gene therapy of lung diseases.
Abbreviations:
Chol,cholesterol;
DCM,dichloromethane;
DMEM,Dulbecco’s Modified Eagles Medium;
MEM,Minimum Essential Medium;
DMF,N,N-dimethyl formamide;
FBS,fetal bovine serum;
’ CONCLUSIONS
We have demonstrated for the first time that cationic amphi-
philes designed with the natural fatty acyl chain asymmetry of
food-grade coconut oil are less cytotoxic and deliver genes
selectively to mouse lung. Despite lauroyl chains being the major
fatty acyl chains of coconut oil, both the in vitro and In Vivo gene
transfer efficiencies of such cationic amphiphiles were found to
be remarkably superior (>4-fold) to those of their pure dilauroyl
analogue. Our findings demonstrate for the first time that the
transfection enhancing influence of asymmetric hydrocarbon
chains previously observed in pure, synthetic cationic amphi-
philes containing two defined aliphatic hydrocarbon chains of
different lengths also works for cationic amphiphiles designed
with the natural fatty acyl chain asymmetry of a food-grade oil.
Mechanistic studies involving the technique of fluorescence
resonance energy transfer (FRET) revealed higher biomembrane
fusibility of the cationic liposomes of the coconut amphiphiles
than that of the symmetric dilauroyl analogue. AFM study revea-
led pronounced fusogenic nonlamellar structures of the lipo-
somes of coconut amphiphiles. Findings in the FRET and cellular
uptake study, taken together, support the notion that the higher
cellular uptake resulting from the more fusogenic nature of the
liposomes of coconut amphiphiles 1 is likely to play a dominant
role in making the coconut amphiphiles transfection competent
under in vitro conditions. However, further In Vivo experiments
using additional control amphiphiles with hydrophobic chain
asymmetry need to be carried out in the future to confirm the
transfection-enhancing role of hydrophobic chain asymmetry
under systemic settings.
ONPG,o-nitrophenyl-β-D-galactopyranoside;
PBS,phosphate buffered saline;
GFP,green fluorescent protein.
’ REFERENCES
(1) Check, E. (2003) Harmful potential of viral vectors fuels doubts
over gene therapy. Nature 423, 573–574.
(2) Bell, P. C., Bergsma, I. P., Bras, W., Stuart, M. C. A., Rowan,
M. C., Feiters, M. C., and Engberts, J. B. F. N. (2003) Transfection
mediated by gemini surfactants, engineered escape from the endosomal
compartment. J. Am. Chem. Soc. 125, 1551–1558.
(3) Martin, B., Sainlos, M., Aissaoui, A., Oudrhiri, N., Hauchecorne,
M., Vigneron, J, P., Lehn, J. M., and Lehn, P. (2005) The design of
cationic lipids for gene delivery. Curr. Pharm. Des. 11, 375–94.
(4) Kirby, A. J. (2003) et al. Gemini surfactants: new synthetic
vectors for gene transfection. Angew. Chem., Int. Ed. Engl. 42, 1448–1457.
(5) Prata, C. A. H., Zhao, Y., Barthelemy, P., Li, Y., Luo, D.,
McIntosh, T. J., Lee, S. J., and Grinstaff, M. W. (2004) Charge-reversal
amphiphiles for gene delivery. J. Am. Chem. Soc. 126, 12196–12197.
(6) Desigaux, L., Sainlos, M., Lambert, O., Chevre, R., Letrou-
Bonneval, E., Vigneron, J. P., Lehn, P., Lehn, J. M., and Pitard, B.
(2007) Self-assembled lamellar complexes of siRNA with lipidic ami-
noglycoside derivatives promote efficient siRNA delivery and interfer-
ence. Proc. Natl. Acad. Sci. U.S.A. 104, 16534–16539.
(7) Unciti-Broceta, A., Holder, E., Jones, L. J., Stevenson, B., Turner,
A. R., Porteous, D. J., Boyd, A. C., and Bradley, M. (2008) Tripod-like
cationic lipids as novel gene carriers. J. Med. Chem. 51, 4076–4084.
(8) Lynn, D. M., Anderson, D. G., Putman, D., and Langer, R.
(2001) Accelerated discovery of synthetic transfectionvectors: Parallel
synthesis and screening of a degradable polymer library. J. Am. Chem.
Soc. 123, 8155–8156.
(9) Choi, J. S., Joo, D. K., Kim, C. H., Kim, K., and Park, J. S. (2000)
Synthesis of a barbell-like triblock copolymer, poly(L-lysine) dendrimer-
block-poly(ethylene glycol)-block-poly(L-lysine) dendrimer, and its
self-assembly with plasmid DNA. J. Am. Chem. Soc. 122, 474–480.
(10) Luo, D., Haverstick, K., Belcheva, N., Han, E., and Saltzman,
W. M. (2002) Poly (ethylene glycol)-conjugated PAMAM dendrimer
for biocompatible, high-efficiency DNA delivery. Macromolecules
35, 3456–3462.
(11) Srinivas, R., Samanta, S., and Chaudhuri, A. (2009) Cationic
amphiphiles: promising carriers of genetic materials in gene therapy.
Chem. Soc. Rev. 38, 3326–3338.
(12) Bhattacharya, S., and Bajaj, A. (2009) Advances in gene delivery
through molecular design of cationic lipids. Chem. Commun 31, 4632–
4656.
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR and ESI-MS mass
S
b
spectral characterizations for coconut amphiphiles 1 and Control
dilauroyl amphiphile 2 as well as for their tertiary amine precur-
sors (Figures S1-S8), reverse-phase HPLC chromatograms and
HPLC conditions for control dilauroyl amphiphile 2 in two
mobile phases (Figure S9), DNA binding assay and DNase I
sensitivity assay (Figure S10), size and surface potential data
(Tables S1 and S2) (total 17 pages). This material is available free
’ AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed. E-mail: arabin-
da@iict.res.in, Tel: 91-40-27193201, Fax: 91-40-27160757.
(13) Singh, R. S., Mukherjee, K., Banerjee, R., Chaudhuri, A., Hait,
S. K., Moulik, S., Ramadas, Y., Vijayalakshmi, A., and Rao, N. M. (2002)
Anchor dependency for non-glycerol based cationic lipofectins: mixed
bag of regular and anomalous transfection profiles. Chem.—Eur. J.
8, 900–909.
’ ACKNOWLEDGMENT
(14) Kukowska-Latallo, J. F., Bielinska, A. U., Johnson, J., Spinder,
R., Tomalia, D. A., and Baker, J. R. (1996) Efficient transfer of genetic
material into mammalian cells using Star burst polyamidoamine den-
drimers. Proc. Natl. Acad. Sci. U.S.A. 93, 4897–4902.
(15) Byk, G., Dubertret, C., Escriou, V., Frederic, M., Jaslin, G.,
Rangara, R., Pitard, B., Crouzet, J., Wils, P., Schwartz, B., and Scherman,
D. (1998) Synthesis, activity, and structure-activity relationship studies
of novel cationic lipids for DNA transfer. J. Med. Chem. 41, 224–235.
This work was supported by the Council of Scientific and
Industrial Research, Government of India, New Delhi (NWP-
0036). We thank N. M. Rao, Centre for Cellular and Molecular
Biology, Hyderabad, India for providing us p-CMV-SPORT-β-
Gal; VC thanks University Grants Commission, MS and KR
thank CSIR, Government of India, New Delhi, for providing
doctoral research fellowships.
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dx.doi.org/10.1021/bc100537r |Bioconjugate Chem. 2011, 22, 497–509