Effect of molecular weight of chitosan on the physicochemical, morphological, and biological properties of polyplex nanoparticles intended for gene delivery

Aranda-Barradas, ME; Trejo-Lopez, SE; Del Real, A; Alvarez-Almazan, S; Mendez-Albores, A; Garcia-Tovar, CG; Gonzalez-Diaz, FR; Miranda-Castro, SP

Abstract

The use of viral vectors in gene therapy has many disadvantages that have been widely described. The development of new nonviral vectors using safe polymers such as chitosan provides a viable alternative. Chitosan molecular weight and nitrogen/phosphorus (N/P) ratio are key factors that determine the shape, size, and surface charge of polyplex nanoparticles (NPs) assembled by complex coacervation for gene therapy; and at the same time, their transfection efficiency is affected by these characteristics, besides the cell line used for transfection. However, there are some inconsistencies in previous reports about the effect of chitosan molecular weight on all these parameters. Enhanced Green Fluorescent Protein plasmid (pEGFP-N1) and two molecular weight (MW) chitosan (20.6 and 57.5 kDa) with a high degree of deacetylation (>= 85%) were used to design and assembly nanoparticles by complex coacervation with N/P ratios of 43 and 8. NPs were characterized determining morphology by Scanning Electron Microscopy (SEM), size by Dynamic Light Scattering (DLS) and zeta-potential by Laser-Doppler velocimetry (LDV). Also, biological functionality was assessed by in vitro transfection assays. NPs assembled using the lowest MW chitosan (20.6NPs) and N/P ratio = 8 had spherical shape, smaller mean size and polydispersity index (119 +/- 20 nm; 0.257, respectively), and lower zeta-potential (17.65 +/- 1.21 mV). Moreover, no significant difference was observed in size, polydispersity index and zeta-potential after 30 days of storage, in contrast with NPs assembled using the 57.5 kDa chitosan (57.5NPs). In vitro transfection assays using two cervical cancer cell lines (HeLa and SiHa) showed that 20.6 NPs leaded to a higher expression of Green Fluorescent Protein (GFP) in contrast to 57.5NPs for both cell lines, and expression in HeLa cells was higher than in SiHa cell line. It was confirmed that transfection efficiency depends on all these physical parameters given by MW of chitosan, cell physiology, and the addition of fetal bovine serum in the transfection formulation. These results indicate that low MW chitosan and a low N/P ratio are more suitable to design chitosan-based nonviral vectors for gene therapy, given that physicochemical and biological properties, so as the stability of these NPs are better that those formulated with chitosan of higher MW.

Keywords: Chitosan; Nanoparticles; Gene therapy; Polyplex; Coacervation; In vitro transfection

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Chitosans

Chitosan is a natural biopolymer, usually extracted from shells and insect shells. Its main components include glucosamine and acetylglucosamine. In the pharmaceutical field, chitosan is widely used in many applications due to its numerous beneficial properties, including biocompatibility, biodegradability, low toxicity, and adsorption properties.

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