Chitosan as a non-viral co-transfection system in a cystic fibrosis cell line

Abstract

Successful gene therapy requires the development of suitable vehicles for the selective and efficient delivery of genes to specific target cells at the expense of minimal toxicity. In this work, we investigated a non-viral gene delivery system based on chitosan (CS) to specifically address cystic fibrosis (CF). Thus, electrostatic self-assembled CS-pEGFP and CS-pEGFPsiRNA complexes were prepared from high-pure fully characterized CS (Mw ~20 kDa and degree of acetylation ~30%). The average diameter of positively-charged complexes (i.e.  ~ +25 mV) was ~200 nm. The complexes were found relatively stable over 14 h in OptiMEM. Cell viability study did not show any significant cytotoxic effect of the CS-based complexes in a human bronchial cystic fibrosis cell line (s). We evaluated the transfection efficiency of this cell line with both CS-pEGFP and co-transfected with CS-pEGFP-siRNA complexes at (N/P) charge ratio of 12. We reported an increase in the fluorescence intensity of CS-pEGFP and a reduction in the cells co-transfected with CS-pEGFP-siRNA. This study shows proof-of-principle that co-transfection with chitosan might be an effective delivery system in a human CF cell line. It also offers a potential alternative to further develop therapeutic strategies for inherited disease treatments, such as CF.

Chitosan Nanocomplexes for the Delivery of ENaC Antisense Oligonucleotides to Airway Epithelial Cells

“…Results revealed that the nucleic acid was retained in the gel pocket by CS with advanced efficiency in higher P/N charge ratios (Figure 4). The findings of the strong binding efficiency of nucleic acids and CS correspond to previous reports [23,33]. Deng et al even demonstrated protection of plasmid DNA from DNases by complexation with CS and hyaluronic acid [34].…”

“…The increase in zeta potential with increasing P/N charge ratio indicates that nanocomplexes were successfully formed, as the positive charge arises from the protonated amino groups that were not neutralized by the negatively charged phosphate groups of the ASO. With increasing P/N charge ratio, the amount of CS is increased and therefore an increasing excess of positively charged amino groups leads to an increasing zeta potential as shown in previous studies [23,30]. Furthermore, it was observed that nanocomplexes prepared with NaCl had a slightly lower zeta potential.…”

“…This increase can be explained by interaction of the nanocomplexes with electrolytes and proteins in the medium also observed before [47]. The supplements in the medium likely accumulate in proximity to the hydrophilic surface of the nanocomplexes, producing short-range repulsive hydration forces and thereby stabilizing the nanocomplexes [23,47]. Based on the results of the stability measurements, transfection experiments should be performed with the supplemented medium.…”

“…However, to the best of our knowledge, we provide the first attempt at delivering ASOs using CS to address the imbalanced ion transport in CF. Previously, we showed that transfection of nucleic acids using CS is an effective delivery system in a human CF cell line [23,24]. Now, we are interested in gleaning understanding of whether this approach is adaptable to an air-liquid interface (ALI) culture system, which is characterized by an extensive mucociliary differentiation process resulting in an in vitro model that is representative of the in vivo airway physiological context.…”

Dual Tumor-Targeting Nanocarrier System for siRNA Delivery Based on pRNA and Modified Chitosan

“…RNAi, a potent and highly specific gene-silencing phenomenon triggered by double-stranded RNA helix, 1 has emerged as a promising strategy for treatment of a wide range of diseases 2 . Small interfering RNA (siRNA) with 21–23 base pairs has been proven to be more efficient in gene silencing than other RNAi molecules and opens wide perspectives in therapeutics for the treatment of many diseases linked to elevated expression of identified genes, including cancer and infectious, inflammatory, and neurodegenerative diseases 3, 4.…”