Synthesis and Comparative Evaluation of Novel Cationic Amphiphile C12-Man-Q as an Efficient DNA Delivery Agent In Vitro

Abstract

New amphiphilic 1,4-DHP derivative C12-Man-Q with remoted cationic moieties at positions 2 and 6 was synthesised to study DNA delivery activity. The results were compared with data obtained for cationic 1,4-DHP derivative D19, which is known to be the most efficient one among the previously tested 1,4-DHP amphiphiles. We analysed the effects of C12-Man-Q concentration, complexation media, and complex/cell contact time on the gene delivery effectiveness and cell viability. Transmission electron microscopy data confirms that lipoplexes formed by the compound C12-Man-Q were quite uniform, vesicular-like structures with sizes of about 50 nm, and lipoplexes produced by compound D19 were of irregular shapes, varied in size in the range of 25–80 nm. Additionally, confocal microscopy results revealed that both amphiphiles effectively delivered green fluorescent protein expression plasmid into BHK-21 cells and produced a fluorescent signal with satisfactory efficiency, although compound C12-Man-Q was more cytotoxic to the BHK-21 cells with an increase of concentration. It can be concluded that optimal conditions for C12-Man-Q lipoplexes delivery in BHK-21 cells were the serum free media without 0.15 M NaCl, at an N/P ratio of 0.9. Compound D19 showed higher transfection efficiency to transfect BHK-21 and Cos-7 cell lines, when transfecting active proliferating cells. Although D19 was not able to transfect all studied cell lines we propose that it could be cell type specific. The compound C12-Man-Q showed modest delivery activity in all used cell lines, and higher activity was obtained in the case of H2-35 and B16 cells. The transfection efficiency in cell lines MCF-7, HeLa, and Huh-7 appears to be comparable to the reference compound D19 and minimal in the HepG2 cell line.

Pleiotropic Properties of Amphiphilic Dihydropyridines, Dihydropyridones, and Aminovinylcarbonyl Compounds

“…Previously, 3,5-bis(dodecyloxycarbonyl)-1,4-DHPs containing cationic moieties were elaborated as synthetic lipid-like compounds having promising gene delivery properties for DNA transfection; the basic structure-activity relationships have been verified for the cationic 1,4-DHP derivatives as gene delivery systems, and it has been shown that the molecular structure affected their self-assembling properties, pDNA-binding ability, and properties of the formed 1,4-DHP amphiphile-pDNA complexes [ 13 , 15 , 28 ]. The synthetic procedure for the amphiphilic compounds 1 – 7 , 9 – 22 , and 26 is displayed in Scheme 1 via a multistep sequence.…”

“…Additionally, for studies of targeted changes in the structure of corresponding dicationic compound 10 , which was found to be more active for DNA delivery among the tested 1,4-DHP amphiphiles [ 13 , 15 , 28 ], 1,1′,1 ″ ,1‴-((1,4-phenylenebis(3,5-bis((dodecyloxy)carbonyl)-1,4-dihydropyridine-4,2,6-triyl))tetrakis(methylene))tetrakis(pyridin-1-ium) tetrabromide ( 26 ) was synthesized as a double or a “dimeric” molecule of compound 10 and 1,1′-((3,5-bis(dodecyloxycarbonyl)-4-phenylpyridine-2,6-diyl)bis(methylene))-bis(pyridin-1-ium)dibromide ( 27 ) was prepared in accordance with what was reported in [ 14 ] as the oxidized form of compound 10 . For evaluation of the anion influence on cytotoxicity, 1,1′-((3,5-bis((dodecyloxy)carbonyl)-4-phenyl-1,4-dihydropyridine-2,6-diyl)bis(methylene))bis(pyridin-1-ium) ditetrafluoroborate ( 11 ) was synthesized from compound 10 after treating with NH 4 BF 4 .…”

“…Our research group has developed liposome-forming cationic 1,4-dihydropyridine (1,4-DHP) amphiphiles capable of transfecting pDNA into different cell lines in vitro . To assess the influence of different molecular architectures on gene delivery properties, numerous 1,4-DHP amphiphiles were studied [ 13 – 15 ]. Besides, some of these amphiphilic compounds revealed anticancer activity in HT-1080 and MH-22A cells and antiradical activities (27–40% in DPPH tests) [ 13 , 16 ].…”

Synthesis and Characterisation of 1,1′-{[3,5-Bis(dodecyloxy-carbonyl)-4-(thiophen-3-yl)-1,4-dihydropyridine-2,6-diyl]bis-(methylene)}bis(pyridin-1-ium) Dibromide

“…Some representatives of this class show pDNA transfection activity; however, it is lower than for the corresponding 1,4-DHP amphiphiles [8]. Minor modifications, such as elongation by one methylene group of the distance between cationic moieties and the linker, also led to notable changes in the delivery activity of cationic 1,4-DHP [9]. It is possible to fill liposomes formed by amphiphilic 1,4-DHP derivatives with magnetic particles, which show targeted accumulation under the guidance of external magnetic fields [10].…”

Recent Approaches to Chiral 1,4-Dihydropyridines and their Fused Analogues

“…Consequently, during the last decades, the development of new pyridinium moieties containing compounds based on a 1,4-DHP core has become an interesting area for medicinal chemistry research. Cationic 1,4-DHP amphiphiles having one or two cationic moieties and various length ester appendages, were found to be capable of transfecting pDNA into different cell lines in vitro [31,32], due to their self-assembling properties [33,34].…”