Cationic Poly(organo)phosphazenes for Gene Delivery

Gene delivery represents a promising therapeutic strategy for the treatment of inherited, acquired as well as infectious disorders, e.g., cystic fibrosis, Parkinson’s disease and cancer. Effective delivery systems are essential to protect nucleic acids from degradation, ensure cellular uptake, and enable controlled release. While viral vectors exhibit high efficiency, their clinical translation is limited by safety concerns such as immunogenicity and toxicity. Consequently, non-viral carriers, including synthetic polymers, have attracted significant interest. Among these, poly(organo)phosphazenes are particularly promising due to their tunable biodegradability to non-toxic degradation products, multifunctionality and high structural flexibility, which enables good binding to biomolecules. In this work, ten differently substituted cationic poly(organo)phosphazenes were synthesized as potential carriers for the delivery of monomeric G-quadruplex-based cytosine-phosphate-guanine oligodeoxynucleotides. The polymeric precursor, poly(dichloro)phosphazene, was prepared via living cationic polymerization of trichloro(trimethylsilyl)phosphoranimine, resulting in narrow dispersities and controlled molecular weights. Side chains, including glycine-ethylester, Nϵ-Boc-L-lysine-methylester, and 2-[2-(Boc-amino)ethoxy]ethanol, were introduced via nucleophilic macromolecular substitution, and successful synthesis was confirmed by 1H- and 31P-NMR spectroscopy and GPC analysis. Additionally, a thymine-functionalized poly(organo)phosphazene was synthesized to enable base-pairing with adenine moieties. All polymers were further evaluated regarding their degradation behavior and DNA-binding capabilities. Both the pure polymers and their corresponding polyplexes were investigated regarding their cytotoxicity, whereas immunostimulatory activity was assessed only for the polyplexes. L-lysine-methylester-based (Lys) polyphosphazenes exhibited higher cytotoxicity than 2-(2-aminoethoxy)ethanol-based polyphosphazenes, while polyplex formation significantly increased cell viability for all polymers. Immunostimulation assays indicated Lys25 and Lys40 as the most promising candidates. Preliminary DNA-binding experiments suggested stronger interactions for L-lysine-methylester-containing polyphosphazenes. Overall, this study provides an initial screening of poly(organo)phosphazenes as gene delivery vectors for monomeric G-quadruplex-based cytosine-phosphate-guanine oligodeoxynucleotides.