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. 2002 Jun;82(6):3105–3117. doi: 10.1016/S0006-3495(02)75652-X

Biophysical and transfection studies of the diC(14)-amidine/DNA complex.

Vadim Cherezov 1, Hong Qiu 1, Veronique Pector 1, Michel Vandenbranden 1, Jean-Marie Ruysschaert 1, Martin Caffrey 1
PMCID: PMC1302099  PMID: 12023234

Abstract

Liposomes of the synthetic cationic lipid, N-t-butyl-N'-tetradecylamino-propionamidine (diC(14)-amidine), efficiently ports DNA into mammalian cells in the absence of other (neutral) lipids. The compositional simplicity of this transfection mix makes it attractive from a formulation perspective. We have used low- and wide-angle x-ray diffraction and polarized light microscopy to characterize the thermotropic phase behavior and microstructure of diC(14)-amidine and of the lipid/DNA (circular plasmid, 5.4 kb) complex with a view to understanding the structure of the complex and its role in transfection. Upon heating, the lipid in buffer undergoes a lamellar crystalline (L(c), d(001) = 41.7 A)-to-lamellar liquid crystal (L(c)(alpha), d(001) depends on hydration and T) transition at approximately 40 degrees C. Sonicated lipid vesicles with a reported transition temperature of approximately 23 degrees C complex with DNA. Complex formation is complete at a DNA/lipid mole ratio (rho) of 0.8. Adding DNA to the lipid causes d(001) of the multilayered complex to drop from 52 to 49 A as rho rises from 0.03 to 1.64. The minimal DNA-DNA duplex separation observed is 26 A, consistent with the close packing of B-DNA. Lipid bilayers in the complex undergo a lamellar gel (L(c)(beta))-to-L(c)(alpha) (superscript c refers to complex) transition at approximately 23 degrees C. Transfection efficiency was maximized at rho = 0.4. The structure and transfection data combined suggest that densely packaged DNA in a net positively charged complex is essential for transfection.

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Selected References

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  1. Allison S. D., Anchordoquy T. J. Mechanisms of protection of cationic lipid-DNA complexes during lyophilization. J Pharm Sci. 2000 May;89(5):682–691. doi: 10.1002/(SICI)1520-6017(200005)89:5<682::AID-JPS14>3.0.CO;2-#. [DOI] [PubMed] [Google Scholar]
  2. Bally MB, Harvie P, Wong FM, Kong S, Wasan EK, Reimer DL. Biological barriers to cellular delivery of lipid-based DNA carriers. Adv Drug Deliv Rev. 1999 Aug 20;38(3):291–315. doi: 10.1016/s0169-409x(99)00034-4. [DOI] [PubMed] [Google Scholar]
  3. Blezinger P., Freimark B. D., Matar M., Wilson E., Singhal A., Min W., Nordstrom J. L., Pericle F. Intratracheal administration of interleukin 12 plasmid-cationic lipid complexes inhibits murine lung metastases. Hum Gene Ther. 1999 Mar 20;10(5):723–731. doi: 10.1089/10430349950018481. [DOI] [PubMed] [Google Scholar]
  4. Boukhnikachvili T., Aguerre-Chariol O., Airiau M., Lesieur S., Ollivon M., Vacus J. Structure of in-serum transfecting DNA-cationic lipid complexes. FEBS Lett. 1997 Jun 9;409(2):188–194. doi: 10.1016/s0014-5793(97)00505-x. [DOI] [PubMed] [Google Scholar]
  5. Byk G., Dubertret C., Escriou V., Frederic M., Jaslin G., Rangara R., Pitard B., Crouzet J., Wils P., Schwartz B. Synthesis, activity, and structure--activity relationship studies of novel cationic lipids for DNA transfer. J Med Chem. 1998 Jan 15;41(2):229–235. doi: 10.1021/jm9704964. [DOI] [PubMed] [Google Scholar]
  6. El Ouahabi A., Pector V., Fuks R., Vandenbranden M., Ruysschaert J. M. Double long-chain amidine liposome-mediated self replicating RNA transfection. FEBS Lett. 1996 Feb 12;380(1-2):108–112. doi: 10.1016/0014-5793(96)00038-5. [DOI] [PubMed] [Google Scholar]
  7. El Ouahabi A., Thiry M., Pector V., Fuks R., Ruysschaert J. M., Vandenbranden M. The role of endosome destabilizing activity in the gene transfer process mediated by cationic lipids. FEBS Lett. 1997 Sep 8;414(2):187–192. doi: 10.1016/s0014-5793(97)00973-3. [DOI] [PubMed] [Google Scholar]
  8. El Ouahabi A., Thiry M., Schiffmann S., Fuks R., Nguyen-Tran H., Ruysschaert J. M., Vandenbranden M. Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes. J Histochem Cytochem. 1999 Sep;47(9):1159–1166. doi: 10.1177/002215549904700908. [DOI] [PubMed] [Google Scholar]
  9. Felgner J. H., Kumar R., Sridhar C. N., Wheeler C. J., Tsai Y. J., Border R., Ramsey P., Martin M., Felgner P. L. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J Biol Chem. 1994 Jan 28;269(4):2550–2561. [PubMed] [Google Scholar]
  10. Felgner P. L. Nonviral strategies for gene therapy. Sci Am. 1997 Jun;276(6):102–106. doi: 10.1038/scientificamerican0697-102. [DOI] [PubMed] [Google Scholar]
  11. Gao X., Huang L. Cationic liposome-mediated gene transfer. Gene Ther. 1995 Dec;2(10):710–722. [PubMed] [Google Scholar]
  12. Harries D., May S., Gelbart W. M., Ben-Shaul A. Structure, stability, and thermodynamics of lamellar DNA-lipid complexes. Biophys J. 1998 Jul;75(1):159–173. doi: 10.1016/S0006-3495(98)77503-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hoffman D. M., Figlin R. A. Intratumoral interleukin 2 for renal-cell carcinoma by direct gene transfer of a plasmid DNA/DMRIE/DOPE lipid complex. World J Urol. 2000 Apr;18(2):152–156. doi: 10.1007/s003450050189. [DOI] [PubMed] [Google Scholar]
  14. Hui S. W., Langner M., Zhao Y. L., Ross P., Hurley E., Chan K. The role of helper lipids in cationic liposome-mediated gene transfer. Biophys J. 1996 Aug;71(2):590–599. doi: 10.1016/S0006-3495(96)79309-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kaganer VM, Ostrovskii BI, de Jeu WH X-ray scattering in smectic liquid crystals: From ideal- to real-structure effects. Phys Rev A. 1991 Dec 15;44(12):8158–8166. doi: 10.1103/physreva.44.8158. [DOI] [PubMed] [Google Scholar]
  16. Koltover I., Salditt T., Rädler J. O., Safinya C. R. An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. Science. 1998 Jul 3;281(5373):78–81. doi: 10.1126/science.281.5373.78. [DOI] [PubMed] [Google Scholar]
  17. Koltover I., Salditt T., Safinya C. R. Phase diagram, stability, and overcharging of lamellar cationic lipid-DNA self-assembled complexes. Biophys J. 1999 Aug;77(2):915–924. doi: 10.1016/S0006-3495(99)76942-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li B., Li S., Tan Y., Stolz D. B., Watkins S. C., Block L. H., Huang L. Lyophilization of cationic lipid-protamine-DNA (LPD) complexes. J Pharm Sci. 2000 Mar;89(3):355–364. doi: 10.1002/(SICI)1520-6017(200003)89:3<355::AID-JPS7>3.0.CO;2-H. [DOI] [PubMed] [Google Scholar]
  19. Lin A. J., Slack N. L., Ahmad A., Koltover I., George C. X., Samuel C. E., Safinya C. R. Structure and structure-function studies of lipid/plasmid DNA complexes. J Drug Target. 2000;8(1):13–27. doi: 10.3109/10611860009009206. [DOI] [PubMed] [Google Scholar]
  20. Liu F., Qi H., Huang L., Liu D. Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration. Gene Ther. 1997 Jun;4(6):517–523. doi: 10.1038/sj.gt.3300424. [DOI] [PubMed] [Google Scholar]
  21. Logan J. J., Bebok Z., Walker L. C., Peng S., Felgner P. L., Siegal G. P., Frizzell R. A., Dong J., Howard M., Matalon Cationic lipids for reporter gene and CFTR transfer to rat pulmonary epithelium. Gene Ther. 1995 Jan;2(1):38–49. [PubMed] [Google Scholar]
  22. MacDonald R. C., Ashley G. W., Shida M. M., Rakhmanova V. A., Tarahovsky Y. S., Pantazatos D. P., Kennedy M. T., Pozharski E. V., Baker K. A., Jones R. D. Physical and biological properties of cationic triesters of phosphatidylcholine. Biophys J. 1999 Nov;77(5):2612–2629. doi: 10.1016/S0006-3495(99)77095-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. May S., Ben-Shaul A. DNA-lipid complexes: stability of honeycomb-like and spaghetti-like structures. Biophys J. 1997 Nov;73(5):2427–2440. doi: 10.1016/S0006-3495(97)78271-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. May S., Harries D., Ben-Shaul A. The phase behavior of cationic lipid-DNA complexes. Biophys J. 2000 Apr;78(4):1681–1697. doi: 10.1016/S0006-3495(00)76720-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pector V., Backmann J., Maes D., Vandenbranden M., Ruysschaert J. M. Biophysical and structural properties of DNA.diC(14)-amidine complexes. Influence of the DNA/lipid ratio. J Biol Chem. 2000 Sep 22;275(38):29533–29538. doi: 10.1074/jbc.M909996199. [DOI] [PubMed] [Google Scholar]
  26. Pector V., Caspers J., Banerjee S., Deriemaeker L., Fuks R., El Ouahabi A., Vandenbranden M., Finsy R., Ruysschaert J. M. Physico-chemical characterization of a double long-chain cationic amphiphile (Vectamidine) by microelectrophoresis. Biochim Biophys Acta. 1998 Jul 17;1372(2):339–346. doi: 10.1016/s0005-2736(98)00074-1. [DOI] [PubMed] [Google Scholar]
  27. Ruysschaert J. M., el Ouahabi A., Willeaume V., Huez G., Fuks R., Vandenbranden M., Di Stefano P. A novel cationic amphiphile for transfection of mammalian cells. Biochem Biophys Res Commun. 1994 Sep 30;203(3):1622–1628. doi: 10.1006/bbrc.1994.2372. [DOI] [PubMed] [Google Scholar]
  28. Rädler J. O., Koltover I., Salditt T., Safinya C. R. Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes. Science. 1997 Feb 7;275(5301):810–814. doi: 10.1126/science.275.5301.810. [DOI] [PubMed] [Google Scholar]
  29. Sasaki S., Fukushima J., Arai H., Kusakabe K. I., Hamajima K., Ishii N., Hirahara F., Okuda K., Kawamoto S., Ruysschaert J. M. Human immunodeficiency virus type-1-specific immune responses induced by DNA vaccination are greatly enhanced by mannan-coated diC14-amidine. Eur J Immunol. 1997 Dec;27(12):3121–3129. doi: 10.1002/eji.1830271207. [DOI] [PubMed] [Google Scholar]
  30. Schmutz M., Durand D., Debin A., Palvadeau Y., Etienne A., Thierry A. R. DNA packing in stable lipid complexes designed for gene transfer imitates DNA compaction in bacteriophage. Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12293–12298. doi: 10.1073/pnas.96.22.12293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tardieu A., Luzzati V., Reman F. C. Structure and polymorphism of the hydrocarbon chains of lipids: a study of lecithin-water phases. J Mol Biol. 1973 Apr 25;75(4):711–733. doi: 10.1016/0022-2836(73)90303-3. [DOI] [PubMed] [Google Scholar]
  32. Zelphati O., Uyechi L. S., Barron L. G., Szoka F. C., Jr Effect of serum components on the physico-chemical properties of cationic lipid/oligonucleotide complexes and on their interactions with cells. Biochim Biophys Acta. 1998 Feb 16;1390(2):119–133. doi: 10.1016/s0005-2760(97)00169-0. [DOI] [PubMed] [Google Scholar]
  33. Zhang R, Suter RM, Nagle JF. Theory of the structure factor of lipid bilayers. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics. 1994 Dec;50(6):5047–5060. doi: 10.1103/physreve.50.5047. [DOI] [PubMed] [Google Scholar]
  34. Zhu T., Caffrey M. Thermodynamic, thermomechanical, and structural properties of a hydrated asymmetric phosphatidylcholine. Biophys J. 1993 Aug;65(2):939–954. doi: 10.1016/S0006-3495(93)81108-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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