Gene-Delivery Ability of New Hydrogenated and Partially Fluorinated Gemini bispyridinium Surfactants with Six Methylene Spacers

Abstract

The pandemic emergency determined by the spreading worldwide of the SARS-CoV-2 virus has focused the scientific and economic efforts of the pharmaceutical industry and governments on the possibility to fight the virus by genetic immunization. The genetic material must be delivered inside the cells by means of vectors. Due to the risk of adverse or immunogenic reaction or replication connected with the more efficient viral vectors, non-viral vectors are in many cases considered as a preferred strategy for gene delivery into eukaryotic cells. This paper is devoted to the evaluation of the gene delivery ability of new synthesized gemini bis-pyridinium surfactants with six methylene spacers, both hydrogenated and fluorinated, in comparison with compounds with spacers of different lengths, previously studied. Results from MTT proliferation assay, electrophoresis mobility shift assay (EMSA), transient transfection assay tests and atomic force microscopy (AFM) imaging confirm that pyridinium gemini surfactants could be a valuable tool for gene delivery purposes, but their performance is highly dependent on the spacer length and strictly related to their structure in solution. All the fluorinated compounds are unable to transfect RD-4 cells, if used alone, but they are all able to deliver a plasmid carrying an enhanced green fluorescent protein (EGFP) expression cassette, when co-formulated with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 1:2 ratio. The fluorinated compounds with spacers formed by six (FGP6) and eight carbon atoms (FGP8) give rise to a very interesting gene delivery activity, greater to that of the commercial reagent, when formulated with DOPE. The hydrogenated compound GP16_6 is unable to sufficiently compact the DNA, as shown by AFM images.

Keywords: DNA-surfactant interaction; atomic force microscopy on DNA; gene delivery; gene therapy; heterocyclic gemini cationic surfactants; nonviral vectors; partially fluorinated gemini surfactants.

Figure 1

Compounds under study, n is the number of carbon atoms in the spacer.

Figure 2

MTT test on RD4 (left) and A549 cells (right) for GP16_6: (a) untreated cells; (b) 40 µM; (c) 20 µM; (d) 10 µM; (e) 5 µM; (f) 2.5 µM; (g) 1.25 µM; (h) only DOPE. In gray, GP16_6 alone; in black, GP16_6: DOPE = 1:2. Values are the mean ± S.D. of three independent experiments (n = 8 per treatment, p < 0.05).

Figure 3

Effects of FGP6 (left) and FGP8 (right) on RD4 (top), LA4 (middle) and A549 (bottom) cell lines proliferation measured by MTT test after 48 h of incubation: (a) untreated cells; (b) 40 µM; (c) 20 µM; (d) 10 µM; (e) 5 µM; (f) 2.5 µM; (g) 1.25 µM; (h) only DOPE. In gray, FGPn alone; in black, FGPn: DOPE = 1:2. Values are the mean ± S.D. of three independent experiments (n = 8 per treatment, p < 0.05).

Figure 4

EMSA experiments showing complexation of GP16_6, FGP6 and FGP8 with circular plasmid pEGFP-C1. Shifting is observable as a function of concentration (μM). As a negative control, only the plasmid was used, which is completely unshifted. 100 μM cationic lipid concentration corresponds to N/P ≈ 1.

Figure 5

(a–d) are AFM images showing the effect induced on DNA plasmid by incubation with the compounds under study: (a) plasmid alone; (b) plasmid + GP16_6; (c) plasmid + FGP6; (d) plasmid + FGP8. All images were obtained with supercoiled 0.5 nM pEGFP-C1 plasmid deposited onto mica and with the microscope operating in tapping mode in air at the same N/P ratio as transfection. For image d, see Ref. [21]. (e) DLS volume distribution of plamid + FPG6, the same sample as (c).

Figure 6

Transfection of RD4 (left) and A549 cells (right) by GP16_6, FB6 and FB8 5 μM. For each kind of cells, on the left, phase contrast and, on the right, fluorescence microscope observation of the transfected cells (as shown by green cells expressing EGFP) is shown. The experiments were done with the surfactant alone and with surfactant:DOPE = 1:2. Cells are not transfected with DOPE alone (see [14,18,21]). Transfection rate of FB8 5 μM + DOPE is not indicated because it is <1%. The scale bars = 20 μm are common for all of the micrographs.

Figure 7

Gene delivery ability on RD4 cells of GP16_n hydrogenated gemini surfactants and partially fluorinated gemini surfactants FGPn as a function of the spacer length, n: (a) experiments done only with the surfactants and (b) with surfactant:DOPE ratio = 1:2. Positive control by a commercial reagent is in green. Spacer 12 (not shown) is unable to deliver DNA. See [18,21].