Bioinspired Lipid Nanocarriers for RNA Delivery

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TL;DR

This review discusses bioinspired strategies to improve lipid-based RNA delivery, including substituting LNP components with natural lipids, mimicking endogenous molecules, viruses, and exosomes. Key findings show that replacing cholesterol with β-sitosterol can increase transfection efficiency up to 211-fold, and incorporating phosphatidylserine enhances cellular uptake and targets secondary lymphoid organs. These approaches aim to overcome current limitations such as poor endosomal escape (1–2%) and liver-dominant biodistribution, potentially enabling safer and more effective RNA therapeutics.

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**Background:** RNA therapeutics hold great promise for treating a wide range of diseases, but their clinical translation is hindered by delivery challenges. Naked RNA is large, negatively charged, and susceptible to degradation, requiring effective delivery vehicles. Current state-of-the-art carriers, such as ionizable lipid nanoparticles (LNPs), have enabled FDA-approved drugs like Onpattro and COVID-19 vaccines, yet they face significant limitations: frequent localization to clearance-associated organs (primarily the liver), and limited endosomal escape (only 1–2% of internalized RNA reaches the cytosol). To address these issues, researchers are exploring bioinspired design principles that incorporate biological elements into lipid nanocarriers to improve tissue targeting, cellular uptake, and endosomal escape.

**Methods:** This narrative review summarizes recent literature on bioinspired lipid-based nanocarriers for RNA delivery. The authors categorize strategies into four main approaches: (1) substituting LNP components with natural lipids (e.g., replacing cholesterol with β-sitosterol, using polysarcosine instead of PEG, or incorporating natural helper lipids like DGTS); (2) mimicking endogenous molecules (e.g., high-density lipoprotein (HDL)-mimicking particles, ginger-derived nanoparticle (GDNP)-mimicking LNPs); (3) mimicking viruses (e.g., virosomes containing viral fusion proteins, LNPs incorporating phosphatidylserine (PS) to mimic viral membranes); and (4) mimicking exosomes (e.g., liposomes with exosome-like lipid compositions or transmembrane proteins like Connexin 43). The review evaluates each strategy based on key delivery criteria: encapsulation efficiency, biocompatibility, cellular uptake, endosomal escape, organ targeting, and scalability.

**Key Results:** - **Natural lipid substitution:** Replacing cholesterol with β-sitosterol (a phytosterol differing by one ethyl group) in LNPs (enhanced LNPs, eLNPs) resulted in an 11- to 211-fold improvement in transfection in vitro, depending on the alkyl derivative and mRNA dosage. Substituting PEG-lipids with polysarcosine (pSar) lipids maintained comparable performance while reducing proinflammatory cytokine secretion and complement activation. Using the natural helper lipid DGTS instead of DSPC showed worse in vitro transfection but better in vivo performance after nebulization. Incorporating the ionizable phospholipid PL1 enabled effective mRNA delivery to T-cells within tumors. - **Mimicking endogenous molecules:** HDL-mimicking particles (TLPs) successfully targeted SR-B1 receptors and delivered siRNA to prostate cancer cells, reducing tumor volume in a xenograft model after 13 doses of 0.7 mg siRNA/kg over 26 days with no off-target toxicity. Another HDL-mimic (siRNA-CaP-rHDLs) exploiting the Ras pathway delivered ATF5 siRNA to glioblastoma cells, extending survival time in mice at a 0.36 mg/kg dose. GDNP-mimicking LNPs (nLNPs) containing PA, MGDG, and DGDG (5:2:3 ratio) delivered IL-22 mRNA to the colon, accelerating healing in a colitis model. - **Mimicking viruses:** Virosomes incorporating influenza hemagglutinin (HA) achieved siRNA delivery comparable to lipofectamine in vitro, with cell viability between 80–100% but low encapsulation efficiency (~37%). Cationic virosomes with Sendai virus proteins (viroplexes) showed significant tumor growth inhibition in mice when combined with doxorubicin. LNPs containing 2.5% PS increased EGFP signal 3-fold in vitro and showed a 13.5% increase in cellular uptake. In vivo, PS-LNPs achieved a 3.7-fold increase in transfection potency in zebrafish embryos and, importantly, redirected delivery from the liver to secondary lymphoid organs (spleen and lymph nodes), with 45-fold stronger spleen signal compared to PA-LNPs. - **Mimicking exosomes:** Exosome-mimetic liposomes (DOPC/SM/Chol/DOPS/DOPE at 21/17.5/30/14/17.5 molar ratio) showed >3-fold higher gene silencing than conventional PC/Chol liposomes but lower than cationic DOTAP liposomes. Encapsulation efficiency was only 31% for conventional preparation but improved to >50% using a microfluidic method. Incorporating Connexin 43 (Cx43) transmembrane protein via cell-free synthesis enabled 58% dye-positive cells (vs. 5% without Cx43) and ~30% VEGF knockdown in U87 MG cells.

**Clinical Implications:** Bioinspired lipid nanocarriers offer promising avenues to overcome key barriers in RNA delivery. Natural lipid substitution, particularly with β-sitosterol and PS, provides relatively simple modifications that can significantly enhance transfection efficiency and enable extra-hepatic targeting (e.g., spleen, lymph nodes). These strategies maintain the scalability and synthesis simplicity of conventional LNPs, making them attractive for clinical translation. Mimicking endogenous molecules like HDL may unlock delivery to hard-to-reach tissues such as the brain and tumors, while virus- and exosome-mimicking approaches could improve cellular uptake and endosomal escape. However, many of these strategies are still in early preclinical stages and face challenges in scalability, encapsulation efficiency, and in vivo performance compared to established LNPs. Further research should focus on optimizing formulations, developing predictive in vitro models, and elucidating cellular uptake and endosomal escape pathways to enable rational design of next-generation RNA carriers.