Ligand-tethered lipid nanoparticles for targeted RNA delivery to treat liver fibrosis

Purpose: Liver fibrosis has no approved antifibrotic therapies. Activated hepatic stellate cells (HSCs) drive fibrosis by producing collagen, and HSP47 is a key collagen chaperone in these cells. Targeted delivery of siRNA to activated HSCs remains challenging, so a ligand-tethered LNP platform was developed to deliver RNA selectively to activated fibroblasts/HSCs.

Hypothesis: Incorporating an anisamide ligand into the lipidoid itself will enable targeted siRNA-LNP delivery to activated HSCs via sigma receptor binding. The lead anisamide-tethered lipidoid LNP will silence HSP47, reduce collagen deposition, and alleviate liver fibrosis better than the benchmark MC3 LNP.

Aims: Develop a one-pot, two-step modular synthesis of anisamide-tethered lipidoids (AA-lipidoids). - Screen a combinatorial library for potency and sigma receptor-dependent selectivity in activated fibroblasts. - Characterize the lead AA-T3A-C12 LNP and its cellular uptake, transfection, and gene silencing. - Evaluate in vivo HSP47 silencing, antifibrotic efficacy, biodistribution, and safety in a CCl₄-induced mouse liver fibrosis model.

Delivery system: Platform: Ligand-tethered ionizable lipid nanoparticles (LNPs). - Lead lipidoid: AA-T3A-C12 (anisamide-tethered, T3A polyamine core, C12 epoxide tails). - Excipients: DSPC, cholesterol, C14-PEG2000. - Payloads: HSP47 siRNA, GFP siRNA, Cy5-siRNA, luciferase mRNA. - Formulation: Microfluidic mixing; ionizable lipid:RNA weight ratio 10:1. - Physicochemical properties: ~65.6 nm, PDI 0.018, siRNA encapsulation efficiency 87.4%, pKa 5.72, neutral surface charge. - Targeting ligand: Anisamide, binds sigma receptor overexpressed on activated fibroblasts/HSCs. - Route: Intravenous injection in mice.

Approach: In vitro: Activated 3T3-GFP fibroblasts and primary murine HSCs; two-round screening: first GFP knockdown, then haloperidol (sigma antagonist) blocking to assess selectivity. Co-culture with H2.35 hepatocytes to model competitive uptake. - In vivo: CCl₄-induced liver fibrosis in male Balb/c mice. Groups: G1 healthy, G2 PBS, G3 AA-T3A-C12/siGFP, G4 AA-T3A-C12/siHSP47, G5 MC3/siHSP47. LNPs given IV twice weekly for 2 weeks at 5 µg siRNA/mouse; n = 5/group. - Controls: MC3 LNP, non-targeted T3A-C12 LNP, PBS, healthy mice.

Key methods: DLS, TEM, RiboGreen, TNS pKa for LNP characterization. - Flow cytometry for cellular uptake and GFP expression. - Immunofluorescence for HSP47 and α-SMA. - Western blot for HSP47 and GAPDH. - IVIS/ex vivo fluorescence for biodistribution. - H&E and Picrosirius red staining for liver histology/collagen. - Serum ALT, AST, TBIL, IL-6, TNF-α for safety.

Key results: Lead AA-T3A-C12 LNP achieved >80% GFP knockdown at 50 nM in activated 3T3-GFP fibroblasts; knockdown significantly reduced by haloperidol (p = 0.009), confirming sigma receptor dependence. - In fibroblast/hepatocyte co-culture, AA-T3A-C12 LNP delivered 1.6-fold more Cy5-siRNA to fibroblasts than MC3 LNP; fibroblast selectivity ratio was 0.56 vs 0.34 for MC3. - HSP47 knockdown: ~60% in activated 3T3 fibroblasts and 65% in primary HSCs with AA-T3A-C12/siHSP47, vs ~35% and 34% with MC3/siHSP47. - In vivo: AA-T3A-C12/siHSP47 achieved ~65% HSP47 knockdown in fibrotic liver vs 31% for MC3, with reduced collagen deposition and improved liver histology. - Safety: no significant worsening of ALT, AST, TBIL, or inflammatory cytokines; no histological damage to heart, spleen, lung, or kidney.

Interpretation: AA-T3A-C12 LNP enables targeted RNA delivery to activated fibroblasts/HSCs via anisamide–sigma receptor interaction. It produces superior HSP47 silencing and antifibrotic efficacy compared with MC3 LNP, supporting ligand-tethered LNPs as a promising strategy for liver fibrosis and other hard-to-target cell types.

Limitations: Screening used 3T3 fibroblasts rather than primary HSCs; TGF-β-stimulated 3T3 cells mimic but do not fully replace activated HSCs. - Anisamide conjugation changes lipidoid structure/hydrophobicity, complicating strict comparisons with non-targeted analogs. - Non-targeted T3A-C12 LNP had poor siRNA encapsulation and liver accumulation, limiting in vivo comparison. - Only a CCl₄-induced mouse fibrosis model; no large-animal or human validation. - No long-term survival, chronic dosing, or fibrosis reversal durability data. - Potential immunogenicity and repeated-dose safety of the ligand-tethered LNP were not extensively evaluated.