Efficient Delivery of Plasmid DNA Using Incorporated Nucleotides for Precise Conjugation of Targeted Nanoparticles

Many obstacles restrict development of DNA plasmid-based therapeutic delivery, involving but not limited to poor cellular uptake, premature material dissociation, and inefficient response. Additionally, lack of precision loading of the plasmids on the carrier nanoparticle may affect the overall nonspecificity in terms of loading as well as the site of loading. Here we report a strategy using the incorporation of a biotin-modified nucleotide into a 4.7 kb plasmid sequence for the site-specific nanoparticle conjugation as an improvement on targeted DNA plasmid delivery. Initially, a designed 80-nucleotide sequence was elongated by incorporating biotin-16-aminoallyl-2'-dCTP that facilitated streptavidin binding as determined via polyacrylamide gel electrophoresis (PAGE). This modified sequence was ligated into a specific location of the EGFP plasmid to avoid possible interference with important functional elements and gene expression off of the plasmid. In parallel, a gold nanoparticle complex comprising of either a CD44 or mutant DNA conjugated aptamer, a PEGylated streptavidin, and a derivatized hyaluronic acid stabilizing polymer was synthesized. To delineate the ability of this nanoparticle-plasmid complex to exhibit an improved cellular delivery, MDA-MB-231 cells were treated with a set of plasmid and plasmid-nanoparticle complexes. Successful expression of EGFP was only observed in cells treated with the biotin-modified EGFP plasmid and a streptavidin-CD44 aptamer-nanoparticle. This demonstrated the need for the specific biotin-streptavidin binding to avoid nanoparticle-plasmid dissociation for improved efficacy. This proof-of-principle concept creates a flexible scaffold that can be assimilated into any plasmid and can produce small RNAs or encoding a therapeutic gene via an installation of a design that uses incorporated modified nucleotides as tethering points for nanoparticles which can play host to stabilizing ligands, additional therapeutic molecules and antibody conjugates among other possibilities. In our system, the nanoparticles are vehicles for the addition of targeting ligands that were essential for cell specificity and enhanced cellular uptake.