Development and characterisation of chitosan nanoparticles for siRNA delivery

Purpose: siRNA has therapeutic potential but is limited by poor cellular uptake, rapid nuclease degradation, and poor blood stability. Chitosan was explored as a non-viral siRNA vector because of its low toxicity, biodegradability, biocompatibility, and cationic nature, but its use for siRNA delivery had not been systematically characterized.

Hypothesis: If siRNA is associated with chitosan by ionic gelation/TPP entrapment rather than by simple complexation or surface adsorption, then it will bind more strongly, be better protected from serum nucleases, and produce higher gene silencing. Chitosan type and molecular weight were also expected to influence particle size and biological activity.

Aims: Prepare chitosan-siRNA nanoparticles by simple complexation, ionic gelation with TPP (entrapped siRNA), and adsorption onto preformed chitosan-TPP nanoparticles. - Characterize particle size, zeta potential, pH effects, siRNA binding, and loading efficiency. - Assess siRNA stability in serum. - Evaluate gene silencing and cytotoxicity in CHO K1 and HEK 293 cells in vitro.

Delivery system: Platform: Chitosan-based nanoparticles for siRNA delivery. - Chitosan types: Medical-grade chitosan (~86% deacetylation): chitosan hydrochloride 270 kDa (Cl213) and 110 kDa (Cl113); chitosan glutamate 470 kDa (G213) and 160 kDa (G113). - Crosslinker: Sodium tripolyphosphate (TPP) for ionic gelation. - Payload: siRNA targeting pGL3 luciferase; control/non-silencing siRNA. - Association methods: (1) simple complexation, (2) siRNA entrapment during ionic gelation, (3) siRNA adsorption onto preformed chitosan-TPP nanoparticles. - No targeting ligand.

Approach: In vitro only. CHO K1 (Chinese hamster ovary) cells for main silencing studies; HEK 293 (human kidney) cells for comparison. - Cells co-transfected with pGL3-control (firefly luciferase) and pRL-TK (renilla luciferase) using Lipofectamine 2000, then treated with chitosan-siRNA formulations. - siRNA dose: 4 pmol/well; silencing measured at 24 and 48 h. - Controls: naked siRNA, Lipofectamine 2000–siRNA, mismatch siRNA, untreated cells. - Serum stability: incubation in 5% and 50% FBS at 37°C up to 72 h. - Cytotoxicity: MTT assay.

Key methods: Particle size and zeta potential by Malvern particle analyzer/Zetasizer. - Agarose gel retardation assay for siRNA binding. - UV absorbance at 260 nm for siRNA loading efficiency. - Polyacrylamide-urea gel electrophoresis for serum stability. - Dual-Glo luciferase assay for gene silencing (firefly normalized to renilla). - MTT assay for cell viability.

Key results: Particle size: Chitosan-TPP nanoparticles were 510 ± 22.9 nm (G213), 276 ± 17.9 nm (G113), 709 ± 50.3 nm (Cl213), and 415 ± 44.6 nm (Cl113) at chitosan:TPP 6:1. siRNA entrapment did not significantly change size. - Zeta potential: Chitosan-TPP nanoparticles ranged from ~+40 to +60 mV; adding siRNA reduced charge to ~+30 mV. Lowering pH from 6 to 4 increased surface charge ~twofold to +40 mV. - Binding/loading: Entrapped siRNA showed complete binding; loading efficiency was 100% for all entrapped chitosan-TPP nanoparticles. Adsorbed siRNA required ~100:1 chitosan:siRNA for complete binding and gave 83% (G213), 90% (G113), 72% (Cl213), and 59% (Cl113) loading. - Serum stability: Naked siRNA was intact only up to 30 min in 5% FBS and fully degraded by 48 h. Chitosan-TPP-siRNA began degrading after 24 h and was fully degraded by 72 h in 5% FBS. In 50% FBS, naked siRNA degraded at time 0, whereas chitosan-TPP protected siRNA up to 7 h, with full degradation after 48 h. - Gene silencing: Chitosan glutamate G213 (470 kDa) was most effective. At 24 h in CHO K1 cells: simple complexation = 51% knockdown; ionic gelation entrapment = 82%; adsorption = 63%. Entrapped G213 was slightly more efficient than Lipofectamine 2000. Naked siRNA and mismatch siRNA showed negligible silencing. HEK 293 cells showed lower silencing. - Cytotoxicity: >90% viability for chitosan-siRNA complexes and naked siRNA; 18–40% viability loss for siRNA associated with chitosan-TPP nanoparticles, although TPP/chitosan alone caused no loss. Some formulations showed cell recovery at 48 h.

Interpretation: Chitosan can serve as a delivery system for siRNA. Chitosan glutamate with higher molecular weight (G213) was the best candidate vector. Transfection efficiency depended on the method of siRNA association, and entrapping siRNA by ionic gelation produced better biological effects than simple complexation or surface adsorption. Chitosan-TPP nanoparticles are presented as potentially safer and more cost-effective siRNA delivery vectors.

Limitations: In vitro only; no in vivo validation, biodistribution, or therapeutic efficacy. - Silencing was against a reporter gene (luciferase), not a disease target. - Chitosan-TPP nanoparticle formulations caused some loss of cell viability (18–40%), despite low toxicity of chitosan/siRNA complexes. - No long-term stability, targeting, or systemic administration data. - Chitosan source, salt form, and molecular weight strongly influenced performance, which may limit reproducibility.