Synthesis, Biophysical Characterization and in Vitro Transfection Activity of Novel Bivalent Amine Cationic Lipids in the Absence of Dioleoylphosphatidylethanolamine (DOPE)
Affiliation(s)
1 Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, New York, USA. 2 Faculty of Applied Sciences, Department of Applied Chemistry and Biochemistry, University for Development Studies, Navrongo Campus, Upper East Region, Ghana.
1 Division of Pharmaceutical Sciences, Arnold & Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, New York, USA. 2 Faculty of Applied Sciences, Department of Applied Chemistry and Biochemistry, University for Development Studies, Navrongo Campus, Upper East Region, Ghana.
ABSTRACT
In this paper, a novel series of bis [(aminoethyl)]-amine cationic lipid derivatives have been synthesized and identified to purity by NMR and Elemental analysis. B16-F0 cells were transfected with cationic lipid/pEGFP-N1 and cationic lipid/ß-gal lipoplexes complexed at +/- charge ratios of 1:1, 2:1, and 4:1. Dimyristoyl derivative showed highest activity at charge ratio 2:1 and both dimyristoyl and dioleoyl derivatives showed similar ß-gal activity at charge ratios 4:1. In 40 mM tris buffer pH 7.2 the dioleoyl derivative was able to fully complex with and retard pDNA at charge ratios above 2:1. None of the other lipid derivatives, dilauroyl, dimyristoyl, dipalmitoyl and distearoyl were able to fully neutralize the plasmid DNA at charge ratios similar to those used in the transfection experiment. The gel-to-liquid phase transition temperatures for dimyristoyl, dipalmitoyl and distearoyl were determined by a fluorescence anisotropy method to be 27.5°C, 32.5°C and 39°C, respectively. A gel-to-liquid crystalline phase transition temperature below 37°C, appears to be the crucial property that cationic lipids have to possess in order to mediate high levels of in vitro transfection activity in the absence of other helper lipids.
In this paper, a novel series of bis [(aminoethyl)]-amine cationic lipid derivatives have been synthesized and identified to purity by NMR and Elemental analysis. B16-F0 cells were transfected with cationic lipid/pEGFP-N1 and cationic lipid/ß-gal lipoplexes complexed at +/- charge ratios of 1:1, 2:1, and 4:1. Dimyristoyl derivative showed highest activity at charge ratio 2:1 and both dimyristoyl and dioleoyl derivatives showed similar ß-gal activity at charge ratios 4:1. In 40 mM tris buffer pH 7.2 the dioleoyl derivative was able to fully complex with and retard pDNA at charge ratios above 2:1. None of the other lipid derivatives, dilauroyl, dimyristoyl, dipalmitoyl and distearoyl were able to fully neutralize the plasmid DNA at charge ratios similar to those used in the transfection experiment. The gel-to-liquid phase transition temperatures for dimyristoyl, dipalmitoyl and distearoyl were determined by a fluorescence anisotropy method to be 27.5°C, 32.5°C and 39°C, respectively. A gel-to-liquid crystalline phase transition temperature below 37°C, appears to be the crucial property that cationic lipids have to possess in order to mediate high levels of in vitro transfection activity in the absence of other helper lipids.
KEYWORDS
Gene Delivery, Cationic Lipid, Acyl Chain Fluidity, Elasticity, Lipoplex, Transfection, Non-Viral Vector, DOPE, Endosomal Release, TNS
Gene Delivery, Cationic Lipid, Acyl Chain Fluidity, Elasticity, Lipoplex, Transfection, Non-Viral Vector, DOPE, Endosomal Release, TNS
Cite this paper
Donkor, A. , Spelios, M. and Savva, M. (2014) Synthesis, Biophysical Characterization and in Vitro Transfection Activity of Novel Bivalent Amine Cationic Lipids in the Absence of Dioleoylphosphatidylethanolamine (DOPE). Pharmacology & Pharmacy, 5, 1015-1028. doi: 10.4236/pp.2014.511113.
Donkor, A. , Spelios, M. and Savva, M. (2014) Synthesis, Biophysical Characterization and in Vitro Transfection Activity of Novel Bivalent Amine Cationic Lipids in the Absence of Dioleoylphosphatidylethanolamine (DOPE). Pharmacology & Pharmacy, 5, 1015-1028. doi: 10.4236/pp.2014.511113.
References
[1] Patil, P.M., Chaudhari, P.D., Megha, S. and Duragkar, N.J. (2012) Review Article on Gene Therapy. International Journal of Genetics, 4, 74-79. [2] Mahato, R.I., Rolland, A. and Tomlinson, E. (1997) Cationic Lipid-Based Gene Delivery Systems: Pharmaceutical Perspectives. Pharmaceutical Research, 14, 853-859.
http://dx.doi.org/10.1023/A:1012187414126 [3] Lee, E.R., Marshall, J., Siegel, C.S., Jiang, C., Yew, N.S., Nichols, M.R., Nietupski, J.B., Ziegler, R.J., Lane, M.B., Wang, K.X., Wan, N.C., Scheule, R.K., Harris, D.J., Smith, A.E. and Cheng, S.H. (1996) Detailed Analysis of Structures and Formulations of Cationic Lipids for Efficient Gene Transfer to the Lung. Human Gene Therapy, 7, 1701-1717.
http://dx.doi.org/10.1089/hum.1996.7.14-1701 [4] 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) Enhanced Gene Delivery and Mechanism Studies with a Novel Series of Cationic Lipid Formulations. Journal of Biological Chemistry, 269, 2550-2561. [5] Ajit. S.N., Thoma, L., Miller, D.D. and Mahato, R.I. (2005) Cationic Lipids with Increased DNA Binding Affinity for Nonviral Gene Transfer in Dividing and Nondividing Cells. Bioconjugate Chemistry, 16, 156-168.
http://dx.doi.org/10.1021/bc049818q [6] 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) Lipofection: A Highly Efficient, Lipidmediated DNA-Transfection Procedure. Proceedings of the National Academy of Sciences of the United States of America, 84, 7413-7417.
http://dx.doi.org/10.1073/pnas.84.21.7413 [7] Gao, X. and Huang, L. (1991) A Novel Cationic Liposome Reagent for Efficient Transfection of Mammalian Cells. Biochemical and Biophysical Research Communications, 179, 280-285.
http://dx.doi.org/10.1016/0006-291X(91)91366-K [8] Behr, J.P., Demeneix, B., Loeffler, J.P. and Perez-Mutul, J. (1989) Efficient Gene Transfer into Mammalian Primary Endocrine Cells with Lipopolyamine-Coated DNA. Proceedings of the National Academy of Sciences of the United States of America, 86, 6982-6986.
http://dx.doi.org/10.1073/pnas.86.18.6982 [9] Edelstein, M.L., Abedi, M.R., Wixon, J. and Edelstein, R.M. (2004) Gene Therapy Clinical Trials Worldwide 1989-2004—An Overview. Journal of Gene Medicine, 6, 597-602.
http://dx.doi.org/10.1002/jgm.619 [10] Sheikh, M., Feig, J., Gee, B. and Savva, M. (2003) In Vitro Lipofection with Novel Series of Symmetric 1,3-Diakylamidopropane-Based Cationic Surfactants Containing Single Primary and Tertiary Amine Polar Head Groups. Chemistry and Physics of Lipids, 124, 49-61.
http://dx.doi.org/10.1016/S0009-3084(03)00033-1 [11] Savva, M., Aljaberi, A., Feig, J. and Stolz, D.B. (2005) Correlation of the Physicochemical Properties of Symmetric 1,3-dialkoylamidopropane-Based Cationic Lipids Containing Single Primary and Tertiary Amine Polar Head Groups with in Vitro Transfection Activity. Colloids and Surfaces B: Biointerfaces, 43, 43-56.
http://dx.doi.org/10.1016/j.colsurfb.2005.03.008 [12] Sakaitani, M. and Ohfune, Y. (1990) Syntheses and Reactions of Silyl Carbamates. 1. Chemoselective Transformation of Amino Protecting Groups via Tert-Butyldimethylsilyl Carbamates. The Journal of Organic Chemistry, 55, 870-876.
http://dx.doi.org/10.1021/jo00290a015 [13] Kearns, M., Donkor, A.-M. and Savva, M. (2007) Structure-Transfection Activity Studies of Novel Cholesterol Based Amphiphiles. Molecular Pharmaceutics, 5, 128-139.
http://dx.doi.org/10.1021/mp700131c [14] Spelios, M. and Savva, M. (2007) Novel N,N’-diacyl-1,3-diaminopropyl-2-carbamoyl Bivalent Cationic Lipids for Gene Delivery—Synthesis, in Vitro Transfection Activity and Physicochemical Characterization. FEBS Journal, 275, 148-162.
http://dx.doi.org/10.1111/j.1742-4658.2007.06185.x [15] Savva, M., Chen, P., Aljaberi, A., Selvi, B. and Spelios, M. (2005) In Vitro Lipofection with Novel Asymmetric Series of 1,2-dialkoylamidopropane-Based Cytofectins Containing Single Symmetric Bis-(2-dimethylaminoethane) Polar Head Groups. Bioconjugate Chemistry, 16, 1411-1422.
http://dx.doi.org/10.1021/bc050138c [16] Aljaberi, A. and Savva, M. (2005) Synthesis, in Vitro Transfection Activity and Physicochemical Characterization of Novel N,N’-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) Amphiphilic Derivatives. Chemistry and Physics of Lipids, 133, 135-149.
http://dx.doi.org/10.1016/j.chemphyslip.2004.09.017 [17] Aljaberi, A., Spelios, M., Kearns, M., Selvi, B. and Savva, M. (2007) Physicochemical Properties Affecting Lipofection Potency of a New Series of 1,2-dialkoylamidopropane-Based Cationic Lipids. Colloids and Surfaces B: Biointerfaces, 57, 108-117.
http://dx.doi.org/10.1016/j.colsurfb.2007.01.012
[1] Patil, P.M., Chaudhari, P.D., Megha, S. and Duragkar, N.J. (2012) Review Article on Gene Therapy. International Journal of Genetics, 4, 74-79. [2] Mahato, R.I., Rolland, A. and Tomlinson, E. (1997) Cationic Lipid-Based Gene Delivery Systems: Pharmaceutical Perspectives. Pharmaceutical Research, 14, 853-859.
http://dx.doi.org/10.1023/A:1012187414126 [3] Lee, E.R., Marshall, J., Siegel, C.S., Jiang, C., Yew, N.S., Nichols, M.R., Nietupski, J.B., Ziegler, R.J., Lane, M.B., Wang, K.X., Wan, N.C., Scheule, R.K., Harris, D.J., Smith, A.E. and Cheng, S.H. (1996) Detailed Analysis of Structures and Formulations of Cationic Lipids for Efficient Gene Transfer to the Lung. Human Gene Therapy, 7, 1701-1717.
http://dx.doi.org/10.1089/hum.1996.7.14-1701 [4] 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) Enhanced Gene Delivery and Mechanism Studies with a Novel Series of Cationic Lipid Formulations. Journal of Biological Chemistry, 269, 2550-2561. [5] Ajit. S.N., Thoma, L., Miller, D.D. and Mahato, R.I. (2005) Cationic Lipids with Increased DNA Binding Affinity for Nonviral Gene Transfer in Dividing and Nondividing Cells. Bioconjugate Chemistry, 16, 156-168.
http://dx.doi.org/10.1021/bc049818q [6] 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) Lipofection: A Highly Efficient, Lipidmediated DNA-Transfection Procedure. Proceedings of the National Academy of Sciences of the United States of America, 84, 7413-7417.
http://dx.doi.org/10.1073/pnas.84.21.7413 [7] Gao, X. and Huang, L. (1991) A Novel Cationic Liposome Reagent for Efficient Transfection of Mammalian Cells. Biochemical and Biophysical Research Communications, 179, 280-285.
http://dx.doi.org/10.1016/0006-291X(91)91366-K [8] Behr, J.P., Demeneix, B., Loeffler, J.P. and Perez-Mutul, J. (1989) Efficient Gene Transfer into Mammalian Primary Endocrine Cells with Lipopolyamine-Coated DNA. Proceedings of the National Academy of Sciences of the United States of America, 86, 6982-6986.
http://dx.doi.org/10.1073/pnas.86.18.6982 [9] Edelstein, M.L., Abedi, M.R., Wixon, J. and Edelstein, R.M. (2004) Gene Therapy Clinical Trials Worldwide 1989-2004—An Overview. Journal of Gene Medicine, 6, 597-602.
http://dx.doi.org/10.1002/jgm.619 [10] Sheikh, M., Feig, J., Gee, B. and Savva, M. (2003) In Vitro Lipofection with Novel Series of Symmetric 1,3-Diakylamidopropane-Based Cationic Surfactants Containing Single Primary and Tertiary Amine Polar Head Groups. Chemistry and Physics of Lipids, 124, 49-61.
http://dx.doi.org/10.1016/S0009-3084(03)00033-1 [11] Savva, M., Aljaberi, A., Feig, J. and Stolz, D.B. (2005) Correlation of the Physicochemical Properties of Symmetric 1,3-dialkoylamidopropane-Based Cationic Lipids Containing Single Primary and Tertiary Amine Polar Head Groups with in Vitro Transfection Activity. Colloids and Surfaces B: Biointerfaces, 43, 43-56.
http://dx.doi.org/10.1016/j.colsurfb.2005.03.008 [12] Sakaitani, M. and Ohfune, Y. (1990) Syntheses and Reactions of Silyl Carbamates. 1. Chemoselective Transformation of Amino Protecting Groups via Tert-Butyldimethylsilyl Carbamates. The Journal of Organic Chemistry, 55, 870-876.
http://dx.doi.org/10.1021/jo00290a015 [13] Kearns, M., Donkor, A.-M. and Savva, M. (2007) Structure-Transfection Activity Studies of Novel Cholesterol Based Amphiphiles. Molecular Pharmaceutics, 5, 128-139.
http://dx.doi.org/10.1021/mp700131c [14] Spelios, M. and Savva, M. (2007) Novel N,N’-diacyl-1,3-diaminopropyl-2-carbamoyl Bivalent Cationic Lipids for Gene Delivery—Synthesis, in Vitro Transfection Activity and Physicochemical Characterization. FEBS Journal, 275, 148-162.
http://dx.doi.org/10.1111/j.1742-4658.2007.06185.x [15] Savva, M., Chen, P., Aljaberi, A., Selvi, B. and Spelios, M. (2005) In Vitro Lipofection with Novel Asymmetric Series of 1,2-dialkoylamidopropane-Based Cytofectins Containing Single Symmetric Bis-(2-dimethylaminoethane) Polar Head Groups. Bioconjugate Chemistry, 16, 1411-1422.
http://dx.doi.org/10.1021/bc050138c [16] Aljaberi, A. and Savva, M. (2005) Synthesis, in Vitro Transfection Activity and Physicochemical Characterization of Novel N,N’-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) Amphiphilic Derivatives. Chemistry and Physics of Lipids, 133, 135-149.
http://dx.doi.org/10.1016/j.chemphyslip.2004.09.017 [17] Aljaberi, A., Spelios, M., Kearns, M., Selvi, B. and Savva, M. (2007) Physicochemical Properties Affecting Lipofection Potency of a New Series of 1,2-dialkoylamidopropane-Based Cationic Lipids. Colloids and Surfaces B: Biointerfaces, 57, 108-117.
http://dx.doi.org/10.1016/j.colsurfb.2007.01.012