Improving transfection efficiency of ultrapure oligochitosan/DNA polyplexes by medium acidification

Abstract

Context: Ultrapure oligochitosans (UOCs) have recently been reported as efficient nonviral vectors for corneal and retinal gene delivery. However, the influence of some physicochemical factors on the transfection efficiency, such as the pH, remains unclear. Deeper in vitro research of these factors could provide valuable information for future clinical applications. Objective: The aim of this study is to determine the influence of the pH decrease on the transfection efficiency of UOC/pDNA polyplexes in HEK293 and ARPE19 cells. Materials and methods: We elaborated self-assembled UOC/pCMS-EGFP polyplexes. The influence of the most important factors on the particle size and the zeta potential was studied by an orthogonal experimental design. We evaluated, in vitro, the cellular uptake and the transfection efficiency by flow cytometry, and the cytotoxicity of the vectors by CCK-8 assay.Results and discussion: The pH of the medium strongly influences the physicochemical properties of the polyplexes, and by its modulation we are able to control their superficial charge. A significant increase on the cellular uptake and transfection efficiency of UOCs was obtained when the pH was acidified. Neither of our UOC/pCMS-EGFP polyplexes caused cytotoxicity; however, cells treated with Lipofectamine 2000ä showed decreased cell viability. Conclusion: This kind of UOC vectors could be useful to transfect cells that are in an acidic environment, such as tumor cells. However, additional in vivo studies may be required in order to obtain an effective and safe medicine for nonviral gene therapy purpose.

Evaluation of cystamine-modified hyaluronic acid/chitosan polyplex as retinal gene vector

“…Also, cellular uptake is one of the major obstacles for a successful gene delivery strategy and polyplexes are thought to enter the cell via endocytosis in a process that may be dependent on polyplex charge [26]. A higher surface charge can facilitate electrostatic interactions with the negatively charged proteoglycans in cellular membranes and hence promote polyplex internalization [27]. Our results show a positive correlation between polyplex surface charge and transfection efficiency that is in agreement with the literature.…”

Low Molecular Weight Chitosan (LMWC)-based Polyplexes for pDNA Delivery: From Bench to Bedside

“…Koping-Hoggard et al observed a size increase from 68 to 174 nm when chitosan Mw increased from 2 to 7 kDa for polyplexes prepared at N/P ratio of 60 [22]. Our research group has recently described that particle size of HMWC is around 4-hold higher than the size of polyplexes based on LMWC (5.3 kDa; 7kDa), prepared at N/P of 20 [30]. However, other authors have reported a decrease in size of polyplexes with increased Ch M w [19].…”

“…According to Lavertu and colleagues the best mixing technique was found to be adding Ch over pDNA, pippeting up and down and tapping the tube gently [26]. Other authors, however, have reported that for the correct formation of LMWC-pDNA polyplexes a more vigorous vortex agitation is preferred [27][28][29][30]. The incubation time required for the correct formation of the polyplexes is another factor to consider.…”

“…LMWC-pDNA polyplexes tend to aggregate after several hours of incubation due to the physical instability of aqueous suspensions over time [31,32]. Therefore, an incubation time between 15 and 30 min has been chosen by several authors as the optimal for the correct formation of the polyplexes avoiding the formation of aggregates [27,30,33,34]. Nevertheless, according to Lavertu et al there were no observed differences in the gene expression with incubation times in the range of 30 and 120 min [26].…”

“…Typically, the size of Ch based polyplexes is measured by dynamic light scattering (DLS). It is described in the literature that Ch-pDNA polyplex size can range from a few ten nm [18] to a few hundred nm [30] depending on several factors such as the mixing concentration, the N/P ratio, the pH of the medium and properties of the chosen Ch and pDNA. Electron microscopy has also been employed to measure particle size, but smaller results in particle size are obtained compared to DLS.…”