Abstract
Cationic polymers remain attractive tools for non-viral gene transfer. The effectiveness of these vectors rely on the ability to deliver plasmid DNA (pDNA) into the nucleus of cells. While we have previously demonstrated the potential of Lignin-PGEA-PEGMA as a non-viral gene delivery vector, alterations of cellular phenotype and cytotoxicity were observed post transfection. The present study aims to explore transfection conditions for high efficiency and low toxicity of the Lignin-PGEA-PEGMA based gene delivery system. Cellular toxicity was significantly reduced by using the centrifugation protocol, which enables rapid deposition of DNA complexes. Replacement of media post centrifugation resulted in minimal exposure of cells to excess polymers, which were toxic to cells. At an optimized DNA amount (500–750 ng) and molar ratios of nitrogen (N) in polymer to phosphate (P) in pDNA (N/P = 30–40), with the use of a novel transfection enhancer that facilitates endosomal escape and nuclear trafficking, the efficiency of gene delivery was increased significantly 24 h post transfection. The present study demonstrated an appropriately optimized protocol that enabled the utility of a novel cationic polymer blend with a mixture of fusogenic lipids and a histone deacetylate inhibitor in non-viral transfection, thereby providing an attractive alternative to costly commercial gene carriers.
Highlights
Despite the significant disadvantages associated with clinical applications of viral gene carriers, they remain as the mainstream use in most gene-based applications [1,2,3]
We hypothesized that bio-based polymers with functional groups that reduce reactive oxygen species (ROS) post transfection would be useful in gene delivery
Instead of size-exclusion chromatography (SEC), we explored the feasibility of mild centrifugation in depositing LG100 polyplexes onto the cell surface and subsequently removing excess polymer
Summary
Despite the significant disadvantages associated with clinical applications of viral gene carriers, they remain as the mainstream use in most gene-based applications [1,2,3]. HEK293T cells were transfected with a plasmid encoding GFP reporter gene in 24-well tissue culture vessels to monitor the efficiencies of various parameters. Despite the high transfection efficiency, alteration of cell morphology and reduction in cell number were observed with increasing DNA amount (Figure 1).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
