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

Cationic polymers that bind with the plasmids to form polyplexes protect the DNA from enzymatic degradation and improve cellular and tissue uptake. Complete or near complete gel retardation of the polyplex is an important assay to determine the optimal polymer: plasmid ratio for in vitro and in vivo studies. Nevertheless, despite minimal to moderate gel retardation of histidine-lysine (HK) polyplexes formed with low peptide: plasmid DNA ratios (1:2 and 1:4; w:w), the polyplexes effectively targeted the tumor in vivo. To understand the lack of predictability of the initial gel mobility shift assays, we revisited the retardation and stability of polyplexes with these electrophoresis assays. Because the histidine component with a pKa of about 6.0 will have a greater positive charge and may bind plasmids with a higher affinity at lower pHs, we compared the retardation of the two HK polyplexes when the pH of the running buffer of the gel mobility shift assay was altered. Both HK polyplexes were retarded significantly more when the running buffer had a pH of 7.3 instead of the standard pH of 8.3. Indeed, the HK polyplexes formed at the 1:2 ratio showed complete retardation at pH 7.3. Consequently, while both HK polyplexes formed at these low ratios targeted the tumor, the polyplex formed with the 1:2 ratio had reduced tumor gene expression variability and lower lung and liver values. Thus, the selection of the optimal ratios for the linear HK and plasmid for transfection studies in vivo was improved with a running buffer pH of 7.3.
Competing Interests: Declaration of competing interest The authors of this manuscript declare that there are no competing interests.
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