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.
Highlights
Gene therapy is based on the transfer of genetic material into cells to treat an inherited or acquired disease, or at least to improve the clinical status of a patient, or to prevent a disease
In length having in mind the optimization of the delivery ability of both hydrogenated and the following, we examine the effect of the spacer length having in mind the optimization partially fluorinated dipyridinium gemini surfactants with chlorides as counterions (GP16_n of the delivery ability of both hydrogenated and partially fluorinated dipyridinium gemini and FGPn, respectively, where n is the number of methylene groups in the spacer)
We were amazed by the tight relationship we have found between solution thermodynamics of dipyridinium gemini surfactants and their DNA compacting ability, making “old” macroscopic thermodynamic methods essential to rationalize their biological behavior at a molecular level [14,16–18,21]
Summary
Gene therapy is based on the transfer of genetic material into cells to treat an inherited or acquired disease, or at least to improve the clinical status of a patient, or to prevent a disease. With the rapid conclusion of the Human Genome Sequencing Project, the idea has matured to treat diseases caused by a known genetic defect by delivering to the diseased cells or body organs a correct copy of the defective gene. This is the rationale of gene therapy, a clinical reality, after the approval by FDA of the in vivo treatment of ADA-SCID and Leber congenital amaurosis, or ex vivo CAR-T therapy for treating. The expression of the protein can take place after the delivery of the genetic material inside mammalian cells. Whereas in RNA vaccines the genetic material is packaged in lipid nanoparticles, the DNA ones use viral vectors because they need more efficient delivery devices to overcome the cell membrane, and to reach the nucleus for expression
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