Chitosan nanoparticles for siRNA delivery Optimizing formulation to increase stability and efficiency

Purpose: Naked siRNA is unstable and inefficient, and chitosan-based carriers are limited by poor solubility at physiological pH and weak endosomal disruption. The study aimed to optimize chitosan nanoparticle formulations for intravenous siRNA delivery that achieve high gene silencing without cytotoxicity and remain stable in biological fluids including blood.

Hypothesis: If chitosan nanoparticles are formulated with PEGylated chitosan, an endosomal-disrupting agent such as PEI, and a negatively charged polymer such as hyaluronic acid, then they will show improved plasma stability, efficient cellular uptake, high gene silencing, and low cytotoxicity suitable for intravenous siRNA delivery.

Aims: Assess the influence of chitosan/TPP ratio, chitosan molecular weight/origin, PEGylation, addition of an endosomal disrupting agent, and addition of a negatively charged polymer on nanoparticle properties. - Evaluate gene silencing activity and cytotoxicity in B16F10 melanoma cells expressing luciferase. - Monitor siRNA nanoparticle integrity and size behavior in human plasma. - Develop chitosan-based formulations with high in vitro silencing, low cytotoxicity, and high plasma stability for potential intravenous siRNA delivery.

Delivery system: Platform: Chitosan-based nanoparticles prepared by ionic gelation with sodium tripolyphosphate (TPP). - Key components: Chitosan of various molecular weights/origins; PEGylated chitosan (C_PEG; ~30 mol% PEG grafting, PEG 1000 Da); branched PEI (25 kDa) as endosomal-disrupting agent; hyaluronic acid (HA, 234.4 kDa) as negatively charged polymer; siRNA payload. - Formulations tested: C, C/C_PEG, C/C_PEG/PEI, C_PEG/PEI (low/high TPP), C_PEG/PEI/HA. - Payload: siRNA fixed at 4% of chitosan weight. - Physicochemical properties: Size 120–290 nm; PDI 0.18–0.29; encapsulation efficiency generally >90%; zeta potential ranged from positive to near-neutral to negative depending on PEG/HA content.

Approach: In vitro only: B16F10 murine melanoma cells stably expressing firefly luciferase (B16F10 luc) for silencing/cytotoxicity; SiHa human cervical tumor cells expressing GFP for secondary silencing confirmation. - Transfection: 4 h incubation with nanoparticles, 48 h readout; siRNA doses 100 or 200 nM/well for main silencing; dose range 1–400 nM for optimization. - Controls: INTERFERin positive control; PEI/siRNA complexes; control/scramble siRNA; untreated cells; Triton 1% for LDH. - Stability: Incubation in 80% human plasma at 37°C; no in vivo animal model.

Key methods: Dynamic light scattering (DLS) for size and zeta potential. - Oligreen assay and fluorescence fluctuation spectroscopy (FFS) for encapsulation/complexation efficiency. - Atomic force microscopy (AFM) for morphology. - ¹H and ³¹P NMR for surface/core structure. - Luciferase inhibition assay for gene silencing. - GFP silencing in SiHa cells by fluorescence microscopy. - Confocal microscopy for intracellular siRNA/endosome localization. - Flow cytometry for cellular uptake. - MTT and LDH assays for cytotoxicity. - Single particle tracking (SPT) for size distribution in buffer and plasma.

Key results: Basic chitosan/TPP nanoparticles showed poor transfection regardless of chitosan type. - PEI-containing PEGylated formulations achieved high silencing: C_PEG/PEI l = 71%, C_PEG/PEI h = 62%, C3/C_PEG/PEI = 66% luciferase inhibition at 200 nM, compared with INTERFERin = 78%. - Higher chitosan molecular weight improved silencing: ~63% inhibition with 120 kDa vs ~38% with 36 kDa. - Plasma stability: C_PEG-free C3 nanoparticles released ~70% of siRNA within 30 min; PEGylated nanoparticles maintained ~100% complexation after 2 h in 80% plasma. C_PEG/PEI/HA showed no size change in plasma, while C_PEG/PEI aggregated. - Cytotoxicity: nanoparticles caused 10–20% cell mortality by LDH; PEI/siRNA complexes caused >40%. MTT viability: C_PEG/PEI = 82%, C_PEG/PEI/HA = 72%, C/C_PEG/PEI = 60%. - Uptake: C2/C_PEG/PEI was taken up by only 58% of cells; other optimized formulations showed ~100% uptake.

Interpretation: The authors conclude that PEGylation and PEI inclusion are essential for high gene silencing, and that plasma stability is a crucial parameter for intravenous siRNA delivery. Chitosan/TPP-only systems are not suitable for intravenous use, whereas high-molecular-weight chitosan, sufficient PEG, and hyaluronic acid produce stable, efficient, low-toxicity nanoparticles promising for future in vivo systemic gene silencing.

Limitations: No in vivo validation; all efficacy and stability data are in vitro or in human plasma, not in animals. - No biodistribution, tumor accumulation, or therapeutic efficacy in a disease model. - No targeting ligand was included. - The authors note that further in vivo studies are needed before clinical translation.