[en] We used a 32P-labeled pCMV-CAT plasmid DNA to estimate the DNA uptake efficiency and unlabeled pCMV-CAT plasmid DNA to quantify the CAT activity after transfection of COS cells using each of the three following cationic compounds: [1] vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine, and previously described as diC14-amidine [1]), [2] lipofectin (a 1:1 mixture of N-(1-2,3-dioleyloxypropyl)-N,N,N-triethylammonium (DOTMA) and dioleylphosphatidylethanolamine (DOPE)), and [3] DMRIE-C (a 1:1 mixture of N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammonium bromide (DMRIE) and cholesterol). Surprisingly, a high CAT activity was observed with vectamidine although the DNA uptake efficiency was lower as compared to lipofectin and DMRIE-C. Transmission electron microscopy (TEM) revealed endocytosis as the major pathway of DNA-cationic lipid complex entry into COS cells for the three cationic lipids. However, the endosomal membrane in contact with complexes containing vectamidine or DMRIE-C often exhibited a disrupted morphology. This disruption of endosomes was much less frequently observed with the DNA-lipofectin complexes. This comparison of the three compounds demonstrate that efficient transfection mediated by cationic lipids is not only correlated to their percentage of uptake but also to their ability to destabilize and escape from endosomes.
[1] Ruysschaert, J.M., El Ouahabi, A., Willeaume, V., Huez, G., Fuks, R., Vandenbranden, M. and Di Stefano, P. (1994) Biochem. Biophys. Res. Commun. 203, 1622-1628.
[2] Felgner, J.H., Kumar, R., Sridhar, C.N., Wheeler, C.J., Tsai, Y.J., Border, R., Ramsey, P., Martin, M. and Felgner, P.L. (1994) J. Biol. Chem. 269, 2550-2561.
[3] Gao, X. and Huang, L. (1995) Gene Ther. 2, 710-722.
[4] Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M. and Danielsen, M. (1987) Proc. Natl. Acad. Sci. USA 84, 7413-7417.
[5] Leventis, R. and Silvius, J.R. (1990) Biochim. Biophys. Acta 1023, 124-132.
[6] Behr, J.P., Demeneix, B., Loeffler, J.P. and Perez-Mutul, J. (1989) Proc. Natl. Acad. Sci. USA 86, 6982-6986.
[7] Malone, R.W., Felgner, P.L. and Verma, I.M. (1989) Proc. Natl. Acad. Sci. USA 86, 6077-6081.
[8] El Ouahabi, A., Pector, V., Fuks, R., Vandenbranden, M. and Ruysschaert, J.M. (1996) FEBS Lett. 380, 108-112.
[9] Capaccioli, S., Di Pasquale, G., Mini, E., Mazzei, T. and Quattrone, A. (1993) Biochem. Biophys. Res. Commun. 197, 818-825.
[10] Debs, R.J., Freedman, L.P., Edmunds, S., Gaensler, K.L., Duzgunes, N. and Yamamoto, K.R. (1990) J. Biol. Chem. 265, 10189-10192.
[11] Walker, C. (1992) Proc. Natl. Acad. Sci. USA 89, 7915-7919.
[12] Zhu, N., Liggitt, D. and Debs, R. (1993) Science 261, 209-211.
[13] Hyde, S.C. et al. (1993) Nature 362, 250-255.
[14] Solodin, I., Brown, C.S., Bruno, M.S., Chow, C.Y., Jang, E.H., Debs, R.J. and Heath, T.D. (1995) Biochemistry 34, 13537-13544.
[15] Nabel, G.L. et al. (1993) Proc. Natl. Acad. Sci. USA 90, 11307-11311.
[16] Caplen, N.J. et al. (1995) Nature Med. 1, 39-46.
[17] Nabel, G.J., Chang, A.E., Nabel, E.G., Plautz, G.E., William, E., Fox, B.A., Felgner, P., Shu, S. and Cho, K. (1994) Hum. Gene Ther. 5, 57-77.
[18] Gershon, H., Ghirlando, R., Guttman, S.B. and Minsky, A. (1993) Biochemistry 32, 7143-7151.
[19] Sternberg, B., Sorgi, F.L. and Huang, L. (1994) FEBS Lett. 356, 361-366.
[20] Gustafsson, J., Arvidson, G., Karlsson, G. and Almgren, M. (1995) Biochim. Biophys. Acta 1235, 305-312.
[21] Mahato, R.I., Kawabata, K., Nomura, T., Takakura, Y. and Hashida, M.J. (1995) Pharmacol. Sci. 84, 1267-1271.
[22] Friend, D.S., Papahadjopoulos, D. and Debs, R.J. (1996) Biochim. Biophys. Acta 1278, 41-50.
[23] Zhou, X. and Huang, L. (1994) Biochim. Biophys. Acta 1189, 195-203.
[24] Worbel, I. and Collms, D. (1995) Biochim. Biophys. Acta 1235, 296-304.
[25] Hui, S.W., Langner, M., Zhao, Y.L., Ross, P., Hurley, E. and Chan, K. (1996) Biophys. J. 71, 590-599.
[26] Zabner, J., Fasbender, A.J., Moninger, T., Poellinger, K.A. and Welsh, M.J. (1995) J. Biol. Chem. 270, 18997-19007.