Functionalized chitosan derivatives as nonviral vectors: physicochemical properties of acylated N,N,N-trimethyl chitosan/oligonucleotide nanopolyplexes (original) (raw)

Cationic polymers have recently attracted attention due to their proven potential for nonviral gene delivery. In this study, we report novel biocompatible nanocomplexes produced using chemically functionalized N,N,N-trimethyl chitosan (TMC) with different N-acyl chain lengths (C5-C18) associated with single-stranded oligonucleotides. The TMC derivatives were synthesized by covalent coupling reactions of quaternized chitosan with n-pentanoic (C5), n-decanoic (C10), and noctadecanoic (C18) fatty acids, which were extensively characterized by Fourier transform-infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance (1 H NMR). These N-acylated TMC derivatives (TMCn) were used as cationic polymeric matrices for encapsulating anionic 18-base singlestranded thiophosphorylated oligonucleotides (ssONs), leading to the formation of polyplexes further characterized by zeta potential (ZP), dynamic light scattering (DLS), binding affinity, transfection efficiency and in vitro cytotoxicity assays. The results demonstrated that the length of Version: Postprint (identical content as published paper) This is a self-archived document from i3S-Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 the grafted hydrophobic N-acyl chain and the relative amino:phosphate groups ratio (N/P ratio) between the TMC derivatives and ssON played crucial roles in determining the physicochemical properties of the obtained nanocomplexes. While none of the tested derivatives showed appreciable cytotoxicity, the type of acyl chain had a remarkable influence on the cell transfection capacity of TMC-ssON nanocomplexes with the derivatives based on stearic acid showing the best performance based on the results of in vitro assays using a model cell line expressing luciferase (HeLa/Luc705). Version: Postprint (identical content as published paper) This is a self-archived document from i3S-Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 fluorescence. Cells treated with TMC-C18-based nanoparticles showed the highest fluorescence intensity for all N/P ratios in comparison to all other TMC nanoparticles and control free ON, but below L2k. The higher hydrophobicity imparted by the C18 chains seems to play an important role in the initial interaction of the nanocomplexes with the cells. Still, in general, an increase in hydrophobicity improves cellular interactions of the TMC/ssON nanocomplexes as verified for the TMC-C5s and TMC-C18. The nanocomplexes formed with TMC-C10 did not follow this rule, which, however, goes in line with the overall instability of such nanocomplexes demonstrated in the previous experiments. 3.8. Cell toxicity (metabolic activity) Cell viability was evaluated by measuring metabolic activity after incubation with the TMC/ssON nanocomplexes at different N/P ratios (Fig. 9). Independently of the nanocomplexes tested no significant alterations of the metabolic activity of the cells have been observed indicating absence of cytotoxicity by the TMC/ssON nanocomplexes.