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.

Keywords: 1,4-dihydropyridines; DNA transfection; cationic amphiphiles; cytotoxicity; gene delivery; lipoplexes.

Scheme 1

Strategies for the synthesis of cationic 1,4-DHP derivatives D19 and C12-Man-Q. Reaction conditions: (a) NBS, MeOH, r.t., 20 h; (b) pyridine, dry acetone, r.t. 48 h [26]; (c) paraformaldehyde, dimethylamine hydrochloride, EtOH, conc.HCl, reflux, 24 h; and (d) 1-bromooctane, nitromethane, DMF, reflux, 48 h.

Figure 1

pDNA (pEGFP-C1) and cationic amphiphiles affinity test obtained by gel electrophoresis assay at various N/P (0.25–3) ratios. Supercoiled (sc) and linear (l) forms of plasmid DNA indicated by arrows. (A) pDNA retardation assay: (a) for C12-Man-Q/pDNA complexes without NaCl; (b) for C12-Man-Q/pDNA complexes with 0.15 M NaCl; (c) for D19/pDNA complexes with NaCl; and (B) quantification of the pDNA binding shift by densitometry (the percentage of the bonded supercoiled DNA was assessed by the density of bands on agarose gel using the open-access image processing program ImageJ) [41].

Figure 2

Transmission electron microscopy (TEM) images of lipoplexes formed by cationic lipid and pDNA (pEGFP-C1) at optimised N/P ratio. (A) lipoplexes formed by amphiphile C12-Man-Q (N/P ratio of 0.9); and (B) lipoplexes formed by amphiphile D19 (N/P ratio of 2). Scale bars represent 100 nm.

Figure 3

Viability of BHK-21 cells (%) 24 h post transfection determined by MTT assay. (A) C12-Man-Q/DNA complexes; (B) D19/DNA complexes. DNA-pEGFP-C1. For each value of the N/P ratio three independent repeats were performed.

Figure 4

Cos-7 cell morphology evaluated by fluorescent microscopy 24 h post transfection with 1,4-DHP/DNA (pEGFP-C1) lipoplexes. (A) C12-Man-Q/DNA lipoplexes; and (B) D19/DNA lipoplexes. Scale bars represent 100 μm.

Figure 5

Transfection efficiency achieved using 1,4-DHP amphiphiles determined by luciferase enzyme activity 24 h post transfection. (A) Compound C12-Man-Q (1-without NaCl; 2-with NaCl additive); and (B) compound D19. For each value of the N/P ratio three independent repeats were performed.

Figure 6

BHK-21 morphology changes (24 h after transfection) under the influence of lipoplexes of C12-Man-Q (A) and D19 (B) analysed by confocal fluorescent microscopy. Blue (DAPI)—nucleus of the cell, green EGFP expression in transfected cells transfection complex. Scale bars represent 100 μm.

Figure 7

Influence of the ionic strength of the lipoplex formation medium on BHK-21 cell morphology changes 24 h after transfection by C12-Man-Q/pEGFP-C1 lipoplexes. (A) lipoplexes prepared in medium containing 0.15 M NaCl; and (B) lipoplexes prepared in medium without the addition of NaCl. Green cells represent transfected cells, which express pEGFP. Scale bars represent 100 μm.

Figure 8

Optimisation of the C12-Man-Q/pDNA (pRL-CMV) lipoplexes/cell contact time on BHK-21 cells; N/P ratio of 0.9; sample prepared without additional NaCl. (A) The impact of the lipoplexes/cell contact time on the cell viability, determined 24 h post transfection by MTT test; and (B) the impact of lipoplexes/cell contact time on transfection efficiency determined by luciferase enzyme activity.

Figure 9

Influence of transfection parameters (lipoplex formation medium and lipoplex/cell contact time) on transfection efficiency of C12-Man-Q in BHK-21 cells in comparison to the reference compound D19. (I): evaluation of EGFP production in cells by fluorescent microscopy (scale bars represent 100 μm): (A) transfection by C12-Man-Q lipoplexes performed under standard conditions; (B) transfection by C12-Man-Q after optimisation of the key parameters of the transfection protocol; (C) transfection by the reference compound D19; (II): quantification of the influence of transfection parameters was assessed by amount of fluorescent cells using the open-access image processing program ImageJ.

Figure 10

Assessment of transfection efficiency of the C12-Man-Q lipoplexes in cancer cell lines by fluorescent microscopy: B16 and H2-35 (both of mouse origin), and MCF-7 and HepG2 (human origin). Scale bars reflect 100 μm.

Figure 11

The comparison of the transfection efficiency of the studied amphiphilic 1,4-DHP derivatives C12-Man-Q and D19 in B16, H2-35, Cos-7, MCF-7, HeLa, Huh-7, and HepG2 cell lines. Image analysing program ImageJ was used to count fluorescent cells in the field of view.