Development of a nontoxic and efficient gene delivery vector based on histidine grafted chitosan

Purpose: siRNA therapeutics are limited by poor cellular uptake, serum nuclease degradation, and inefficient endosomal/lysosomal escape. Chitosan is biocompatible and biodegradable but has low transfection efficiency, largely due to poor buffering capacity and weak endosomal escape. Histidine grafting was explored to add imidazole groups (pKa ~6) that enhance proton-sponge buffering and siRNA delivery.

Hypothesis: If histidine is grafted onto chitosan at an optimal feed ratio, then the resulting histidine-grafted chitosan (HGCS) polymers will show improved water solubility, stronger endosomal buffering capacity, efficient siRNA binding, low cytotoxicity, and enhanced cellular uptake compared with unmodified chitosan.

Aims: Synthesize a series of HGCS polymers by EDC/NHS coupling with varying histidine/chitosan feed ratios (1, 2, 5, 10). - Characterize polymer structure, water solubility, and endosomal buffering capacity. - Form HGCS/siRNA complexes at different polymer/siRNA (P/R) weight ratios and evaluate particle size, zeta potential, morphology, and siRNA-binding stability. - Assess cytotoxicity in RSC96 cells and cellular uptake in B16F1 and HeLa cells, with PEI and a commercial cationic liposome as comparisons.

Delivery system: Platform: Histidine-grafted chitosan (HGCS) cationic polymers for siRNA delivery. - Polymer chemistry: Chitosan (deacetylation ≥95%, viscosity 100–200 mPa·s) grafted with L-histidine via EDC/NHS-mediated acylation. Feed weight ratios histidine/chitosan = 1, 2, 5, 10, labeled HGCS1, HGCS2, HGCS5, HGCS10. - Payload: siRNA (sense: 5′-UUGUUUUGGAGCGAAAAdTdT-3′; antisense: 5′-UUUGCGUCCCAAAACAAdTdT-3′); CY3-labeled siRNA used for uptake imaging. - Complex formation: HGCS/siRNA complexes self-assembled by electrostatic interaction at P/R weight ratios of 0.2, 1, 2, 5, and 10. - Targeting ligand: None.

Approach: In vitro only. - Cell lines: RSC96 rat Schwann cells for cytotoxicity; B16F1 mouse melanoma and HeLa human cervical carcinoma cells for cellular uptake. - Controls/comparators: Naked siRNA; linear PEI (25 kDa) for cytotoxicity; GP-siRNA-Mate plus commercial cationic liposome for uptake. - Complex doses: Final siRNA concentration 2.6 µg/mL for characterization; uptake studies used 19 µmol siRNA per well. - Replicates: Most measurements n = 3.

Key methods: FT-IR and XRD for polymer structure and crystallinity. - Acid-base titration for endosomal buffering capacity (pH 3–11). - Laser particle size analyzer and zeta potential for complex size/surface charge. - TEM for nanoparticle morphology. - Agarose gel retardation assay for siRNA binding. - MTT assay for cytotoxicity. - Inverted fluorescence microscopy with DAPI nuclear staining and CY3-siRNA; ImageJ for mean fluorescence intensity.

Key results: Synthesis: FT-IR showed amide C=O at 1640 cm⁻¹ and N–H bending shift from 1590 to 1522 cm⁻¹; histidine O–H at 3016 cm⁻¹ disappeared. XRD showed loss of histidine crystal peaks at 18.8° and 24.3°, and HGCS polymers were amorphous. - Solubility/buffering: Unmodified chitosan dissolved only at pH < 4; HGCS dissolved over a broader pH range. HGCS polymers had stronger buffering than CS, with HGCS2 showing the best buffering capacity. - Particle size/zeta: Most HGCS/siRNA complexes were 100–200 nm. HGCS2/siRNA at P/R = 5 was smallest, ~95 nm. At P/R = 0.2, zeta was negative; at P/R ≥ 1, zeta became positive, mostly > 25 mV. TEM showed regular, smooth nanoparticles without adhesion. - siRNA binding: At P/R = 0.2, partial dissociation occurred; at P/R ≥ 2, no dissociated siRNA was detected. HGCS2 had the best siRNA-binding capacity. - Cytotoxicity: HGCS polymers at up to 400 µg/mL maintained > 80% RSC96 viability, considered nontoxic by ISO 10993-5. PEI caused a dramatic concentration-dependent decrease in viability. - Cellular uptake: Naked siRNA uptake was low. HGCS2 and HGCS5 achieved uptake close to commercial liposome in B16F1 cells; in HeLa cells, HGCS2/siRNA fluorescence was even stronger than the commercial liposome. Uptake increased with P/R ratio up to 5; HGCS10 did not improve delivery. siRNA signal appeared in the cytoplasm.

Interpretation: HGCS polymers are promising non-viral gene delivery vectors. They exhibited good solubility, high buffering capacity, efficient siRNA binding, and low cytotoxicity. The amount of histidine affected particle size, siRNA binding, and cellular uptake, with histidine/chitosan ratios of 2–5 giving the best performance. Histidine-grafted chitosan has excellent potential for gene therapy.

Limitations: In vitro only; no in vivo biodistribution, safety, or therapeutic efficacy. - No functional gene silencing/knockdown assay; the study reports cellular uptake rather than target-gene inhibition. - No disease-targeting siRNA or targeting ligand. - Limited cell panel (RSC96, B16F1, HeLa) and short experimental time points. - No serum stability or long-term storage stability data.