Manipulating the delivery and immunogenicity of DNA vaccines through the addition of CB[8] to cationic polymers

Challenges with vaccine reactogenicity, stability, and access have highlighted the need to develop alternative strategies for formulation and delivery. We explored the incorporation of cucurbit[n]urils (CBs), as supramolecular "hosts," into nucleic acid-polymer polyplexes. CBs are small, non-toxic, barrel-shaped molecules that transiently crosslink polymers containing supramolecular "guests," thereby increasing molecular weight (MW) of the complex, a correlate of transfection efficiency. We tested whether the supramolecular interactions of CB[8] impact polyplex function. We generated a library of different CB[8] polyplexes using plasmid DNA (pDNA), varying N/P (the ratio of polymer to plasmid), the length, and guest (phenylalanine [Phe]) group frequency of the polyethylenimine (PEI) polymer backbone. We found that N/P 32 and the 20Phe1 (20kDa PEI with 1 mol% Phe) gave optimal gene expression and that incorporating CB[8] in polyplex formulations improved gene expression, both <i>in vitro</i> and <i>in vivo</i>. Despite increases in gene expression, inclusion of CB[8] in formulations with higher guest-binding capacity led to decreased immunogenicity, possibly as a result of dampened innate immune responses. Our data show that CB[8] polyplexes increase gene delivery and expression but alter inflammatory responses. These findings highlight that rational design of the CB[8] polymer system can enable nucleic acid delivery for both vaccine and therapeutic applications.