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Role of polymer microstructure in shaping the biological performance of lipophilic polycations in intracellular plasmid delivery, The
Wright, Aryelle R. E.
Wright, Aryelle R. E.
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2024
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2026-04-04
Abstract
In the quest for safer and more efficient non-viral gene therapy vectors, cationic polymers have gained significant attention due to their versatility and multifunctionality. Customizing polymeric vectors by altering their microstructures, or monomer dispersity, can enhance delivery efficiency. While statistical and block polymer microstructures have been investigated, gradient remain underexplored. This study examines the impact of linear copolymer microstructures via delivery efficiency of plasmid DNA (pDNA), cell viability, hemolysis, and complement activation. Copolymers composed of lipophilic 2-(diisopropylamino)ethyl methacrylate (DIP) and hydrophilic 2-(hydroxyethyl) methacrylate (HEMA) were synthesized via reversible addition-fragmentation chain transfer (RAFT). Five copolymers (statistical—S, block—B, and gradient—G1, G2, G3) were created with varying spatial arrangements of cationic DIP repeat units. These copolymers were complexed with pDNA to form polyplexes, which were then characterized for size, charge, and binding affinity. Characterization in water showed positively charged nanoparticles (+27
mV±4) with an average hydrodynamic radius of 51±2 nm. B and higher N/P ratios demonstrated higher binding affinities (5% ±2 fluorescence intensity). Delivery efficiency and cytotoxicity were assessed through transfection experiments in human embryonic kidney (HEK293T) cells and the CCK8 assay. S demonstrated the highest delivery efficiency (69±1%) but the lowest cell viability (26±1%). B did not transfect effectively (0±0%) but had the highest cell viability (93±3%). In contrast, G1–G3 showed intermediate delivery efficiencies (63±1%, 54±1%, and 49±2%, respectively) and viabilities (29±1%, 25±1%, and 24±3%). Hemocompatibility experiments mirrored our cell viability results: S was the most hemolytic (50±1% RBC lysis), while G1–G3 had lower RBC lysis (12±1%, 10±1%, and 8±1%, respectively), and B had the lowest (0±0%). Interestingly, complement activation was comparable across all polyplex microstructures (46 ng/mL±4 in SC5b-9 and 213 ng/mL±9 in C4d), indicating our plasmid initiated complement activation, not our polymer component. Overall, G1–G3 demonstrated high transfection efficiency comparable to S, while also providing moderate cell viability and hemocompatibility similar to B. Our findings show how the spatial distribution of lipophilic cations affects their biological performance, suggesting that optimizing polycation microstructures can enhance gene delivery efficacy.
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