Development of siRNA-loaded chitosan nanoparticles targeting Galectin-1 for the treatment of glioblastoma multiforme via intranasal administration

Purpose: Galectin-1 (Gal-1) is overexpressed in glioblastoma multiforme (GBM), promotes tumor progression, angiogenesis, chemoresistance, and immune suppression. A non-invasive delivery strategy is needed to reach CNS tumors with siRNA while limiting systemic effects. The intranasal route was explored as an underexplored pathway for delivering siRNA-loaded chitosan nanoparticles to GBM.

Hypothesis: If anti-Gal-1 siRNA is loaded into concentrated chitosan nanoparticles and administered intranasally, then the nanoparticles will protect siRNA from RNase degradation, transiently open nasal epithelial tight junctions, transport siRNA into the CNS tumor microenvironment within hours, and reduce Gal-1 expression via sequence-specific RNA interference.

Aims: Develop chitosan nanoparticles that complex and protect Gal-1-specific siRNA. - Evaluate transfection potential in murine and human GBM cell lines. - Investigate permeation-enhancing properties on an epithelial layer. - Assess in vivo distribution of fluorophore-tagged siRNA formulation in the CNS tumor after intranasal instillation. - Verify whether anti-Gal-1 siRNA produces intratumoral Gal-1 reduction via sequence-specific mRNA degradation.

Delivery system: Platform: Chitosan nanoparticles prepared by ionic gelation with sodium tripolyphosphate (TPP). - Chitosan: 50 kDa, viscosity 10 mPa·s for 1% w/v in 1% acetic acid, degree of acetylation reported as 85.2%. - Formulation: Chitosan 0.7 mg/mL, TPP 1 mg/mL, chitosan:TPP weight ratio 2.625:1; siRNA 24 µg/mL (1.4% theoretical payload); stirred at 1300 RPM, 25°C. - Payload: Anti-Gal-1 siRNA (human and mouse sequences) or scrambled siRNA; fluorescein- or dye 547-labeled siRNA for imaging. - Post-processing: Ultracentrifugation, redispersion, freeze-drying with sucrose as lyoprotectant (nanoparticle/lyoprotectant 1/8), reconstitution before use. - Targeting ligand: None; reliance on chitosan mucoadhesion/permeation enhancement and intranasal route.

Approach: In vitro: Murine GL261 glioma cells; human primary glioblastoma cultures; Calu-3 bronchial epithelial cells for barrier integrity; primary nasal epithelium. - In vivo: C57BL/6J mice; orthotopic intracranial GL261-WT or GL261-BFP tumors (0.5 × 10^6 cells injected stereotactically). - Intranasal dosing: 8 × 3 µL drops at 3-min intervals. For distribution: healthy mice sacrificed 4 h after last administration. For efficacy: tumor-bearing mice treated at days 5, 8, 12, and 15 after tumor inoculation and sacrificed at day 20. - Controls: Untreated, naked siRNA, scrambled siRNA, ± siRNA; n = 3 for characterization, n = 12 for migration assay, n = 10/group for Western quantification, n = 5/group for immunofluorescence.

Key methods: Dynamic light scattering and laser Doppler electrophoresis for size, PDI, zeta potential. - TEM for morphology. - SYBR green assay for siRNA encapsulation efficiency. - Gel retardation assay for RNase protection. - Confocal microscopy for cellular uptake and CNS/tumor distribution. - RT-qPCR and Western blot for Gal-1 mRNA/protein. - Scratch migration assay. - TEER and FITC-dextran 4 kDa permeability for epithelial barrier integrity; ZO-1 immunofluorescence. - GeneRACE-PCR and sequencing for sequence-specific Gal-1 mRNA cleavage.

Key results: Nanoparticle characteristics: Optimal formulation had Z-average 141 ± 5 nm, PDI 0.3, zeta potential +32 mV, siRNA payload 24 µg/mL, and encapsulation efficiency 81 ± 3%. Freeze-dried particles stored at 4°C remained stable for at least 8 weeks. - RNase protection: Free siRNA was rapidly degraded and not observed after 30 min; siRNA loaded in chitosan nanoparticles showed no degradation for at least 3 h. - In vitro activity: Anti-Gal-1 siRNA-loaded nanoparticles reduced Gal-1 mRNA and protein in murine GL261 and human primary GBM cells. Gal-1 knockdown significantly reduced GL261 migration at 48 h: surface area ~40% for +siRNA vs ~20% for −siRNA/buffer (p < 0.05). - Epithelial modulation: Chitosan nanoparticles (0.06% w/v) significantly reduced TEER at 2 h and increased FD4 flux; TEER recovered by 24 h. ZO-1 staining was lost, suggesting transient tight-junction opening. - In vivo distribution: After intranasal administration, siRNA was detected in nasal mucosa, olfactory bulb, and hindbrain within 4 h; in tumor-bearing mice, siRNA reached the tumor microenvironment by 4 h and 8 h and co-localized with GL261-BFP tumor cells and tumor-associated vessels. - In vivo Gal-1 reduction: Four intranasal doses of anti-Gal-1 siRNA nanoparticles significantly decreased Gal-1 protein in tumor-bearing brains by Western blot (p < 0.001; n = 10/group) and immunofluorescence. GeneRACE-PCR showed sequence-specific cleavage of Gal-1 mRNA near the siRNA binding site, though total mRNA reduction was not significant.

Interpretation: The authors conclude that concentrated siRNA-loaded chitosan nanoparticles can deliver anti-Gal-1 RNA interference to the CNS via the intranasal route, reduce Gal-1 in the GBM tumor microenvironment, and decrease tumor cell migration. This supports the intranasal pathway as a feasible non-invasive strategy for delivering biologically active agents to CNS neoplasms such as glioblastoma.

Limitations: No survival benefit or tumor growth inhibition data are reported; follow-up ends at day 20. - In vivo Gal-1 mRNA reduction was not statistically significant, despite evidence of sequence-specific cleavage. - It remains unclear whether intact nanoparticles or released siRNA reach the tumor. - No long-term toxicity, safety, or biodistribution/clearance data are provided. - Only one orthotopic murine glioma model was used; human translation remains uncertain. - No targeting ligand or active tumor-selective delivery mechanism. - A patent application is declared by the authors.