Abstract
The aim of this study was to investigate non-viral pDNA carriers based on diblock-copolymers consisting of poly(2-(dimethyl amino)ethyl methacrylate) (pDMAEMA) and poly(2-hydroxyethyl methacrylate) (pHEMA). Specifically the block-lengths and molecular weights were varied to determine the minimal requirements for transfection. Such vectors should allow better transfection at acceptable toxicity levels and the entire diblock-copolymer should be suitable for renal clearance. For this purpose, a library of linear poly(2-(dimethyl amino)ethyl methacrylate-block-poly(2-hydroxyl methacrylate) (pDMAEMA-block-pHEMA) copolymers was synthesized via RAFT (reversible addition-fragmentation chain transfer) polymerization in a molecular weight (Mw) range of 17–35.7 kDa and analyzed using 1H and 13C NMR (nuclear magnetic resonance), ATR (attenuated total reflectance), GPC (gel permeation chromatography) and DSC (differential scanning calorimetry). Copolymers possessing short pDMAEMA-polycation chains were 1.4–9.7 times less toxic in vitro than polyethylenimine (PEI) 25 kDa, and complexed DNA into polyplexes of 100–170 nm, favorable for cellular uptake. The DNA-binding affinity and polyplex stability against competing polyanions was comparable with PEI 25 kDa. The zeta-potential of polyplexes of pDMAEMA-grafted copolymers remained positive (+15–30 mV). In comparison with earlier reported low molecular weight homo pDMAEMA vectors, these diblock-copolymers showed enhanced transfection efficacy under in vitro conditions due to their lower cytotoxicity, efficient cellular uptake and DNA packaging. The homo pDMAEMA115 (18.3 kDa) self-assembled with DNA into small positively charged polyplexes, but was not able to transfect cells. The grafting of 6 and 57 repeating units of pHEMA (0.8 and 7.4 kDa) to pDMAEMA115 increased the transfection efficacy significantly, implying a crucial impact of pHEMA on vector-cell interactions. The intracellular trafficking, in vivo transfection efficacy and kinetics of low molecular weight pDMAEMA-block-pHEMA are subject of ongoing studies.
Highlights
The field of non-viral gene delivery remains a topic of intensive research efforts, and numerous polycations are under investigation [1,2]
Figure 1. 1H nuclear magnetic resonance (NMR) spectra of (A) Cyanopentanoic acid dithiobenzoate (CPT) in CDCl3, (B) pDMAEMA in D2O and (C) pDMAEMA-block-pHEMA in D2O; the numbers designate the atoms within the molecules
It was shown in this study that the in vitro transfection ability of low molecular weight pDMAEMA
Summary
The field of non-viral gene delivery remains a topic of intensive research efforts, and numerous polycations are under investigation [1,2]. One prominent vector in pDNA delivery is poly(ethylene imine) (PEI) 25 kDa, a branched polycation with a high charge density well known for its effective complexation, high transfection, and significant toxicity [3]. Previous reports described pDMAEMA to possess a decreased proton sponge effect compared to PEI 25 kDa [8] implying alternative mechanisms of membrane interaction, such as membrane destabilization [9]. This eventually led to reduced cell death, but the polymer retained the capacity to escape from endosomes [10]
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