Rational Design of a New Class of Cyclodextrin-Containing Polymers for Gene Delivery

The transfer of gene therapy from an academic exercise to a clinical setting demands the development of an efficient, biocompatible gene delivery vector. Current non-viral systems suffer from toxicity, low transfection efficiency, and in vivo instability. In this work, a new class of polymers was designed to address these issues. Linear, polyamidine, β-cyclodextrin (βCD)-containing polymers (βCDPs) are synthesized by polymerizing difunctionalized cyclodextrins with other difunctionalized comonomers. The inclusion of βCD in the backbone of the polyamidine polymers decreases the IC₅₀s by three orders of magnitude, resulting in a polymer with very low in vitro and in vivo toxicity. The cationic βCDPs are able to self-assemble with and condense DNA into small particles (100-150 nm in diameter). When formulated at a positive charge, the complexes are readily internalized by nearly all exposed cultured cells. The transgene expression from the delivered complexes was increased by fine-tuning the βCDP structure for optimum reporter gene activity and by modifying the polymer to enhance endosomal release. The function of the βCDPs was found to be highly dependent on the polymer structure; changes in position of the amidine charge centers by a few angstroms resulted in transfection and toxicity differences of one order of magnitude. The highest transfection is achieved with the βCDP6 polymer, that contains a 2 methylene spacer between the cyclodextrin and amidine group and a 6 methylene spacer between adjacent amidine functionalities. The conjugation of a pH-sensitive moiety, histidine, to βCDP6 endgroups also increases transgene expression by 20-fold without a change in polymer toxicity. Flow cytometry and confocal microscopy experiments with fluorescently-Iabeled DNA suggest that histidylation of βCDP6 enhances transfection by buffering the endosomal pH, thereby delaying lysosomal degradation and allowing for more endosome release. The βCDP-based particles (βCD-polyplexes) were modified for in vivo stability by using the ability of cyclodextrins to form inclusion complexes with hydrophobic guest molecules. Various compounds were conjugated to adamantane, a molecule that has a high cyclodextrin association constant. The adamantane conjugates, when added to preformed βCD-polyplexes, are able to self-assemble with the βCD-polyplexes without disrupting the polymer/DNA binding interactions. Using this method, βCD-polyplexes were modified with adamantane-polyethylene glycol (PEG) conjugates. The PEGylated particles were salt stabilized in a PEG length-dependent manner. In a second example, modification of βCD-polyplexes with anionic peptide-adamantane conjugates prevented non-specific cellular uptake in cultured cells. The assembly of the three components, DNA, βCDP, and adamantane-based modifer, results in a particle with the potential for achieving systemic, in vivo gene delivery. Finally, a small molecule, fluorescein, was conjugated to adamantane and co-delivered with βCD-polyplexes to cultured cells, thus demonstrating the possibility for therapeutic pouches of small molecule and gene-drugs.