Chemico-Physical Properties of Some 1,1'-Bis-alkyl-2,2'-hexane-1,6-diyl-bispyridinium Chlorides Hydrogenated and Partially Fluorinated for Gene Delivery.

The development of very efficient and safe non-viral vectors, constituted mainly by cationic lipids bearing multiple charges, is a landmark for in vivo gene-based medicine. To understand the effect of the hydrophobic chain's length, we here report the synthesis, and the chemico-physical and biological characterization, of a new term of the homologous series of hydrogenated gemini bispyridinium surfactants, the 1,1'-bis-dodecyl-2,2'-hexane-1,6-diyl-bispyridinium chloride (GP12_6). Moreover, we have collected and compared the thermodynamic micellization parameters (cmc, changes in enthalpy, free energy, and entropy of micellization) obtained by isothermal titration calorimetry (ITC) experiments for hydrogenated surfactants GP12_6 and GP16_6, and for the partially fluorinated ones, FGPn (where n is the spacer length). The data obtained for GP12_6 by EMSA, MTT, transient transfection assays, and AFM imaging show that in this class of compounds, the gene delivery ability strictly depends on the spacer length but barely on the hydrophobic tail length. CD spectra have been shown to be a useful tool to verify the formation of lipoplexes due to the presence of a "tail" in the 288-320 nm region attributed to a chiroptical feature named ψ-phase. Ellipsometric measurements suggest that FGP6 and FGP8 (showing a very interesting gene delivery activity, when formulated with DOPE) act in a very similar way, and dissimilar from FGP4, exactly as in the case of transfection, and confirm the hypothesis suggested by previously obtained thermodynamic data about the requirement of a proper length of the spacer to allow the molecule to form a sort of molecular tong able to intercalate DNA.

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

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.