Abstract
BackgroundSuccessful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEIx) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked.Methodology/Principal FindingsWith the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEIx derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEIx series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEIx copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI2.7% was as effective as and less cytotoxic than the gold standard 25 kDa bPEI.Conclusions/SignificanceThis work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEIx copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity.
Highlights
With the advent of DNA-based gene therapy as well as the discovery of short interferingRNAs as the key mediators of RNA interference (RNAi), the development of delivery platforms for the systemic application of nucleic acids has gained particular relevance for the establishment of novel therapeutic strategies [1,2]
Kunath and coworkers demonstrated that low molecular weight (MW) (LMW) PEI was less cytotoxic than its high MW (HMW) counterpart [7]
Chitosan was insoluble in water under neutral conditions [23], Chi-g-bPEIx derivatives were completely soluble at physiological pH because of the hydrophilic behavior of branched polyethylenimine (bPEI), as previously reported by others [13]
Summary
With the advent of DNA-based gene therapy as well as the discovery of short interfering (si)RNAs as the key mediators of RNA interference (RNAi), the development of delivery platforms for the systemic application of nucleic acids has gained particular relevance for the establishment of novel therapeutic strategies [1,2]. Cationic polymers display striking advantages as vectors for gene delivery: they can be tailored for the proposed application by selecting an appropriate molecular weight (MW) and/or coupling them to cell or tissue specific targeting moieties [3] Their use in therapy is hampered by their still low transfection efficiency and high toxicity. Chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEIx) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked
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