Assessment of Cholesterol-Derived Ionic Copolymers as Potential Vectors for Gene Delivery

Abstract

A library of cholesterol-derived ionic copolymers were previously synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization as 'smart' gene delivery vehicles that hold diverse surface charges. Polyplex systems formed with anionic poly(methacrylic acid-co-cholesteryl methacrylate) (P(MAA-co-CMA)) and cationic poly(dimethylamino ethyl methacrylate-cocholesteryl methacrylate) (Q-P(DMAEMA-co-CMA)) copolymer series were evaluated for their therapeutic efficiency. Cell viability assays, conducted on SHEP, HepG2, H460, and MRC5 cell lines, revealed that alterations in the copolymer composition (CMA mol %) affected the cytotoxicity profile. Increasing the number of cholesterol moieties in Q-P(DMAEMA-co-CMA) copolymers reduced the overall toxicity (in H460 and HepG2 cells) while P(MAA-co-CMA) series displayed no significant toxicity regardless of the CMA content. Agarose gel electrophoresis was employed to investigate the formation of stable polyplexes and determine their complete conjugation ratios. P(MAA-co-CMA) copolymer series were conjugated to DNA through a cationic linker, oligolysine, while Q-P(DMAEMA-co-CMA)-siRNA complexes were readily formed via electrostatic interactions at conjugation ratios beginning from 6:1:1 (oligolysine-P(MAA-co-CMA)-DNA) and 20:1 (Q-P(DMAEMA-co-CMA)-siRNA), respectively. The hydrodynamic diameter, ζ potential and complex stability of the polyplexes were evaluated in accordance to complexation ratios and copolymer composition by dynamic light scattering (DLS). The therapeutic efficiency of the conjugates was assessed in SHEP cells via transfection and imaging assays using RTqPCR, Western blotting, flow cytometry, and confocal microscopy. DNA transfection studies revealed P(MAA-co-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhere to the cell surface as opposed to internalizing and partaking in endosomal disrupting activity. The transfection efficiency of Q-P(DMAEMA-co-CMA)-GFP siRNA complexes were found to be polymer composition and N/P ratio dependent, with Q-2% CMA-GFP siRNA polyplexes at N/P ratio 20:1 showing the highest gene suppression in GFP expressing SHEP cells. Cellular internalization studies suggested that Q-P(DMAEMA-co-CMA)-siRNA conjugates efficiently escaped the endolysosomal pathway and released siRNA into the cytoplasm. The gene delivery profile, reported herein, illuminates the positive and negative attributes of each therapeutic design and strongly suggests Q-P(DMAEMA-co-CMA)-siRNA particles are extremely promising candidates for in vivo applications of siRNA therapy.

Cholesterol Modified Self-Assemblies and Their Application to Nanomedicine

“…More recently, the same authors examined the library of P(DMAEMA-co-CMA) copolymers as delivery vectors for siRNA. 319 Initial complexing studies showed that increasing CMA content drives siRNA complexation ratios to higher N/P values due to the decreasing amounts of amine groups in the polymer composition that can be protonated. The gene suppression level of the compacted complexes was evaluated in vitro in GFP-SHEP cells utilizing anti-GFP siRNA.…”

The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers

“…Previous cell toxicity studies conducted with P(MAA-co-CMA) copolymer series demonstrated high levels of viability, between concentrations of 0.05 μM and 50 μM, in the designated cell lines. 53 The half-maximal inhibitory concentration (IC 50 ) doses of the polymers were not calculated due to their exceptionally low toxicity values. The variance of CMA ratio in the copolymer compositions, did not exhibit any major differences in toxicity amongst each other, as polymers or as nanocomplexes (Fig.…”

pH-Responsive Amphiphilic Carboxylate Polymers: Design and Potential for Endosomal Escape

“…One of the biggest achievements is the development of controlled radical polymerization, especially reversible addition-fragmentation chain transfer (RAFT) polymerization (Fairbanks et al, 2015;Perrier, 2017). Because RAFT polymerization is compatible with carboxylate monomers and suitable at various conditions (such as in aqueous solutions or at ambient temperature), it has been widely adopted in the amphiphilic carboxylate polymers synthesis, including PPAA derivatives and copolymers of MAA or AA mentioned in the previous section (Convertine et al, 2009;Tai et al, 2012;Sevimli et al, 2013;Wannasarit et al, 2019;Dailing et al, 2020;Wang et al, 2020).…”

“…Regarding the applications of these MAA or AA-containing amphiphilic copolymers, a common approach is to decorate these polymers on the surface of liposomes and boost the delivery efficiency by enhancing the endosomal escape (Yessine et al, 2006;Yamazaki et al, 2017). Due to the anionic nature of these polymers, it is difficult to condensate DNA or RNA directly, but adding a cationic polymer in the formulation such as polylysine solves the problem by forming tertiary polyplexes via electrostatic interactions (Sevimli et al, 2013). A recent study indicated that these polymers can modify cell membranes by hydrophobic interactions and facilitate the delivery of cationic peptides (Dailing et al, 2020).…”