pH modification of gel mobility shift improves polyplex selection In Vivo.

Area of Science:

• Biomaterials Science
• Gene Therapy
• Polymer Chemistry

Background:

• Cationic polymers form polyplexes with plasmids, protecting DNA and enhancing cellular uptake for gene delivery.
• Gel retardation assays are vital for determining optimal polymer:plasmid ratios, but initial assays showed poor correlation with in vivo efficacy for histidine-lysine (HK) polyplexes.
• The histidine component's pKa suggests pH-dependent binding, necessitating investigation into buffer conditions.

Purpose of the Study:

• To re-evaluate polyplex retardation and stability using electrophoresis assays.
• To investigate the effect of running buffer pH on the gel mobility shift of histidine-lysine (HK) polyplexes.
• To correlate optimized polyplex formation with in vivo gene delivery efficacy and biodistribution.

Main Methods:

• Formation of histidine-lysine (HK) polyplexes at varying peptide:plasmid DNA ratios (1:2 and 1:4 w:w).
• Gel electrophoresis assays were performed using running buffers at standard pH 8.3 and altered pH 7.3.
• In vivo tumor targeting and gene expression analysis were conducted for optimized polyplexes.

Main Results:

• Both HK polyplexes showed significantly greater retardation at pH 7.3 compared to pH 8.3.
• Complete gel retardation was achieved for the 1:2 HK polyplex at pH 7.3.
• Polyplexes formed at the 1:2 ratio demonstrated improved tumor targeting, reduced gene expression variability, and lower lung and liver accumulation in vivo.

Conclusions:

• Electrophoresis buffer pH is a critical parameter for accurately assessing polyplex retardation and optimizing polymer:plasmid ratios.
• A running buffer pH of 7.3 enhances the predictive power of gel mobility shift assays for histidine-lysine polyplexes.
• Optimized polyplex selection using pH 7.3 conditions improves gene delivery efficiency and safety for in vivo applications.