Lipid-derived nanoparticles for immunostimulatory RNA adjuvant delivery

Purpose: TLR7/8 agonists are promising vaccine adjuvants and antivirals, but RNA agonists are unstable and small-molecule agonists cause systemic toxicity. A delivery system specifically designed to deliver immunostimulatory RNA (isRNA) to TLR-expressing cells could enable safer and more localized innate immune activation.

Hypothesis: Lipid-like materials (“lipidoids”) can be designed to deliver isRNA to TLR7/8 within endosomes, inducing type I interferon and adaptive immune responses. Different lipidoid structures may target distinct immune cell types, enabling tailored innate and adaptive immune activation.

Aims: Synthesize and screen a lipidoid library for isRNA delivery in human PBMCs. - Optimize lipidoid-RNA nanoparticle formulation and characterize in vivo cytokine responses. - Evaluate antiviral prophylaxis against influenza and vaccine adjuvant activity with ovalbumin. - Determine TLR/MyD88 dependence and cell-type specificity of immune activation.

Delivery system: Platform: Lipidoid-RNA nanoparticles (LRNPs), also called lipid-derived nanoparticles. - Chemistry: Combinatorial lipidoids made by reacting amine cores with alkyl-acrylate or alkyl-acrylamide tails. Second-generation lead lipidoids: I-4 and II-4 based on the 100-core diamine with mixed tails. - Formulation: Lipidoid, cholesterol, and C16 mPEG2000 ceramide; extruded through 80 nm membranes; typical lipid:RNA mass ratio 15:1. - Payload: Immunostimulatory single-stranded RNA R-006 (TLR7/8 agonist); control R-1263; no RNA controls. - Targeting ligand: None; cell-specific delivery inferred from lipidoid structure. - Route: Subcutaneous or intravenous injection in mice.

Approach: In vitro screening: ~96 lipidoids complexed with R-006 or R-1263 at multiple ratios; human PBMCs; type I IFN readout. - In vivo innate immune response: BALB/c and 129sv mice; s.c. or i.v. injection; serum IFN-α, IP-10, IL-6 measured 6–24 h. - Antiviral study: 129sv mice; s.c. LRNP-II with R-006, R-1263, or blank; 9 h later intranasal influenza A/PR8; lung viral titer at 24 h. - Vaccine adjuvant study: C57BL/6 and MyD88−/− mice; Ova protein mixed with LRNP-I or LRNP-II, DOTAP-RNA, or CpG ODN 1826; i.m. days 0, 14, 21; serum IgG and splenic CD8+ T cells day 28. - Mechanism: HEK293 cells expressing human TLRs; MyD88−/−, TLR7−/−, TLR4−/−, TLR9−/− mice.

Key methods: Combinatorial lipidoid synthesis. - High-throughput type I IFN cell-based assay. - Cytokine ELISA for IFN-α, IP-10, IL-6. - Influenza lung viral titer assay. - Ova-specific IgG, IgG1, IgG2c ELISA. - Chromium-release CTL assay and tetramer flow cytometry for antigen-specific CD8+ T cells. - TLR-overexpressing HEK293 activation assays.

Key results: 14 lipidoid formulations matched or exceeded Lipofectamine 2000 for isRNA-induced type I IFN in PBMCs; 100-core lipidoids were enriched. - Second-generation LRNP-I and LRNP-II were superior to DOTAP and ND(5)-98-1 for isRNA delivery. - LRNP-II induced 10-fold greater IFN-α and IP-10 than LRNP-I in vivo; LRNP-I potently induced IL-6. - Influenza prophylaxis: R-006 LRNP reduced lung viral titer 10-fold vs untreated; control R-1263 or empty LRNP did not. - Ova vaccination: LRNPs increased Ova-specific IgG by 3–4 log orders over protein alone; LRNP-I and LRNP-II enhanced antigen-specific CD8+ T cells more than CpG ODN 1826 or DOTAP-RNA. - Mechanism: IFN-α and IP-10 were MyD88- and TLR7-dependent; LRNP-II IL-6 was fully TLR7-dependent; LRNP-I IL-6 was partially TLR7-dependent and possibly involved TLR8. TLR4 and TLR9 were not required.

Interpretation: Lipidoid-RNA nanoparticles are effective isRNA delivery vehicles for TLR7/8 activation, can suppress viral replication, and act as potent vaccine adjuvants. Lipidoid structure influences cell-specific delivery, with LRNP-II favoring PDC/TLR7 responses and LRNP-I favoring monocyte/myeloid cell activation. This provides a platform for manipulating innate and adaptive immunity in vaccines and antiviral therapy.

Limitations: No large-animal or human in vivo validation. - Long-term safety, repeated-dose toxicity, and dose–response effects were not fully evaluated. - TLR8 role in mouse is indirect; no TLR8−/− mice were available. - Some adjuvant activity was MyD88-independent, and the mechanism remains unclear. - Cell-specific targeting is inferred from cytokine profiles and in vitro experiments, not direct in vivo cell-type delivery quantification. - Formulations were heterogeneous and not fully characterized for size/stability in all conditions.