Evaluation of Chitosan-Tripolyphosphate Nanoparticles as a p-shRNA Delivery Vector: Formulation, Optimization and Cellular Uptake Study

Abstract

Polysaccharides (especially chitosan) have recently attracted much attention as gene therapy delivery vehicles for their unique properties such as biocompatibility, biodegradability, low toxicity, and controlled release. Nanoparticles have strong potential as a carrier of plasmid short hairpin RNA (p-shRNA). This study aimed to find the optimum conditions for obtaining Chitosan/triphosphate (TPP)/p-shRNA nanoparticles by the ionic gelation method, and investigating the cellular uptake of the optimized nanoparticles. After applying the central composite design of response surface methodology (RSM), the optimum conditions for preparation of nanoparticles with small size and high loading efficiency were: chitosan/TPP ratio = 10, pH = 5.5 and N/P ratio = 11. The resulting nanoparticles had an average size of 172.8 ± 7 nm and loading efficiency of 71.5 ± 5%. SEM images showed spherical and smooth nanoparticles. The nanoparticles complexed with p-shRNA and may protect it against nuclease digestion. Cytotoxicity studies with HeLa and PC3 human cancer cells demonstrated that chitosan/TPP nanoparticles had low toxicity. Cellular uptake studies using HeLa cells showed that the nanoparticles entered the cells (cellular uptake) and delivered DNA, probably due to their favorable Zeta potential (approximately +28 mV) and small size.

Keywords: Cellular Uptake; Chitosan; Gene Delivery; Nanoparticle; Response Surface Methodology (RSM); p-shRNA.

Figure 1

Schematic view of the constructed p-shRNA-EGFR plasmid.

Figure 2

Schematic diagram of chitosan/TPP/p-shRNA nanoparticles. The SEM image is shown in the lower-left part of the figure.

Figure 3

Electrophoretic mobility of chitosan/TPP/p-shRNA nanoparticles with different N/P ratios. 1: ladder, 2: nanoparticles with N/P ratio = 0.5, 3: nanoparticles with N/P ratio 5, 4: nanoparticles with N/P ratio = 12.5: nanoparticles with N/P ration = 19.

Figure 4

Response surface analysis. The plots (A, B, C, D, E and F) show the effect of chitosan/TPP ratio, pH and N/P ratio on the nanoparticles size and loading efficiency.

Figure 5

FTIR spectra. (A) Purified chitosan and (B) chitosan/TPP nanoparticles.

Figure 6

Cytotoxicity of nanoparticles. MTT assay for different concentrations of the optimized nanoparticles with Hela and PC3 cell lines.

Figure 7

Cellular uptake of chitosan/TPP nanoparticles. HeLa cells treated with nanoparticles for 4 h and the uptake was assessed by flow cytometry.

Figure 8

Cellular uptake study. FITC-labeled chitosan/TPP nanoparticles after 4 h of incubation. (a) Nucleus of cells have been identified through Hoechst staining. (b) Fluorescent image of HeLa cells. (c) a and b merged.