Cationic Bottlebrush Polymers Outperform Linear Polycation Analogues for pDNA Delivery and Gene Expression

Abstract

Cationic polymer vehicles have emerged as promising platforms for nucleic acid delivery because of their scalability, biocompatibility, and chemical versatility. Advancements in synthetic polymer chemistry allow us to precisely tune chemical functionality with various macromolecular architectures to increase the efficacy of nonviral-based gene delivery. Herein, we demonstrate the first cationic bottlebrush polymer-mediated pDNA delivery by comparing unimolecular, synthetically defined bottlebrush polymers to their linear building blocks. We successfully synthesized poly­(2-(dimethyl­amino)­ethyl methacrylate) (pDMAEMA) bottlebrushes through ring-opening metathesis polymerization to afford four bottlebrush polymers with systematic increases in backbone degree of polymerization (N bb = 13, 20, 26, and 37), while keeping the side-chain degree of polymerization constant (N sc = 57). Physical and chemical properties were characterized, and subsequently, the toxicity and delivery efficiency of pDNA into HEK293 cells were evaluated. The bottlebrush-pDNA complex (bottleplex) with the highest N bb, BB_37, displayed up to a 60-fold increase in %EGFP+ cells in comparison to linear macromonomer. Additionally, we observed a trend of increasing EGFP expression with increasing polymer molecular weight. Bottleplexes and polyplexes both displayed high pDNA internalization as measured via payload enumeration per cell; however, quantitative confocal analysis revealed that bottlebrushes were able to shuttle pDNA into and around the nucleus more successfully than pDNA delivered via linear analogues. Overall, a canonical cationic monomer, such as DMAEMA, synthesized in the form of cationic bottlebrush polymers proved to be far more efficient in functional pDNA delivery and expression than linear pDMAEMA. This work underscores the importance of architectural modifications and the potential of bottlebrushes to serve as effective biomacromolecule delivery vehicles.

Polycation-Mediated Transfection: Mechanisms of Internalization and Intracellular Trafficking

“…Smaller (17 kDa) PDMAEMA is essentially inactive as a transfectant . Modifications used to make this class of polymers into useful transfectants include altering the architecture to stars or combs (which do not condense DNA as well), − acid labile copolymers, which break down in the endosome to release the DNA, or increasing the steric hindrance (and hydrophobicity)/decreasing the concentration of the amine. ,,− In all these cases the objective is to weaken the binding between the polycation and payload to allow for release. Grafting PEI to PDMAEMA makes it quite efficient, with it being more efficient the more PEI is incorporated .…”

Protection of Double-Stranded RNA via Complexation with Double Hydrophilic Block Copolymers: Influence of Neutral Block Length in Biologically Relevant Environments

“…However, cationic homopolymers can exhibit high levels of cytotoxicity, and the polyplexes they form with DNA/RNA can be unstable, with the likeliness of electroneutralization upon complexation with DNA/RNA leading to increased aggregation. 6 , 20 − 22 , 24 − 26 Thus, tailored polymer architectures including branched, 16 , 27 , 28 dendritic, 29 or block copolymers 30 − 33 have recently been evaluated for improving the stabilization of polyplexes for the protection and targeted release of genetic material.…”

Polymer-based non-viral vectors for gene therapy in the skin

“…A wide range of structurally diverse polymers has been used in NVV development. Commonly employed cationic polymers used as NVVs include polyethylenimine (PEI) and its derivatives, poly-(β-amino ester) (PBAEs), cationic poly amino acids (PAAs), 73,74 polyacrylates, 75,76 and chitosan. 77 While poly( propylene imine) (PPI), 78 polyacrylamides 79 and polyamidoamines (PAMAMs), 80 also represent commonly employed polymers with great promise, their application to skin-based approaches remains relatively undescribed.…”