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Phytosterol Substitution in Lipid Nanoparticles Enhances mRNA Delivery, Lyophilization Stability, and Ocular Gene Therapy

  • 12 hours ago
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Technical Review | Materials Today Bio, Vol. 38, 2026 | Wang, Li, Chen et al. | Featured Tool: PreciGenome NanoGenerator®


Background

Lipid nanoparticles (LNPs) are the established delivery platform for mRNA therapeutics. Each LNP is composed of four core lipid components: an ionizable lipid, a phospholipid, a PEG-lipid, and a structural sterol — typically cholesterol. Cholesterol stabilizes the bilayer, modulates membrane fluidity, and facilitates endosomal escape, the rate-limiting step in which encapsulated mRNA is released into the cytoplasm after cellular uptake.

Plant-derived C24-alkyl phytosterols — β-sitosterol, stigmasterol, fucosterol, and campesterol — share the core sterol scaffold but differ at the C24 side chain. Prior work showed these analogs can improve transfection in individual cell lines, yet their broader impact across multiple organ-derived cell types, lyophilization conditions, and in vivo models remained unexplored. Wang, Li, Chen et al. addressed this gap with a systematic evaluation across in vitro, lyophilization, and in vivo conditions — including a therapeutic endpoint in an ocular disease model.


Figure 1. Study overview. mRNA-LNPs incorporating various C24-alkyl cholesterol analogs were formulated, characterized, and screened for transfection efficiency across six human organ-derived cell lines. Top-performing formulations were evaluated in vivo for biodistribution and therapeutic efficacy in a retinal degeneration model. (Adapted from Wang et al., Materials Today Bio, 2026)
Figure 1. Study overview. mRNA-LNPs incorporating various C24-alkyl cholesterol analogs were formulated, characterized, and screened for transfection efficiency across six human organ-derived cell lines. Top-performing formulations were evaluated in vivo for biodistribution and therapeutic efficacy in a retinal degeneration model. (Adapted from Wang et al., Materials Today Bio, 2026)


Methodology

The team formulated mRNA-LNPs by substituting cholesterol with each of four phytosterols at equivalent molar ratios, using two ionizable lipids — ALC-0315 and MC3 — across multiple lipid mole fraction ratios. LNPs were prepared via a microfluidic device (PreciGenome NanoGenerator®, Model PG-SYN-FS) and characterized for particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency. Transfection efficiency was assessed in seven cell lines (HEK-293T, HeLa, HepG2, Colo205, ARPE-19, A549, and PC12) both in freshly prepared formulations and after lyophilization followed by reconstitution. In vivo biodistribution was evaluated in C57BL/6J mice using luciferase (FLuc) mRNA with IVIS bioluminescence imaging. For the therapeutic endpoint, MERTK mRNA was encapsulated in β-sitosterol LNPs and delivered by subretinal injection to Royal College of Surgeons (RCS) rats.


Figure 2. Lipid components and physicochemical properties. (A) Chemical structures of the ionizable lipids and C24-alkyl cholesterol derivatives used. (B-I) Particle size, PDI, zeta potential, and encapsulation efficiency of ALC-0315-based mRNA-LNPs before and after lyophilization across all five sterol formulations. (Adapted from Wang et al., Materials Today Bio, 2026)
Figure 2. Lipid components and physicochemical properties. (A) Chemical structures of the ionizable lipids and C24-alkyl cholesterol derivatives used. (B-I) Particle size, PDI, zeta potential, and encapsulation efficiency of ALC-0315-based mRNA-LNPs before and after lyophilization across all five sterol formulations. (Adapted from Wang et al., Materials Today Bio, 2026)


Results

In vitro transfection (fresh formulations). β-Sitosterol LNPs produced the highest eGFP expression across most tested cell lines, including HEK-293T and HeLa cells, outperforming cholesterol controls. This is consistent with the moderate crystalline defects introduced by the ethyl side chain of β-sitosterol, which enhance membrane fusion and endosomal release.


Figure 3. eGFP expression in HEK-293T cells transfected with ALC-0315-based mRNA-LNPs formulated with each cholesterol analog, comparing freshly prepared (top) versus lyophilized and reconstituted (bottom) formulations. Stigmasterol LNPs maintained higher transfection post-lyophilization. (Adapted from Wang et al., Materials Today Bio, 2026)
Figure 3. eGFP expression in HEK-293T cells transfected with ALC-0315-based mRNA-LNPs formulated with each cholesterol analog, comparing freshly prepared (top) versus lyophilized and reconstituted (bottom) formulations. Stigmasterol LNPs maintained higher transfection post-lyophilization. (Adapted from Wang et al., Materials Today Bio, 2026)

Post-lyophilization performance. After freeze-drying and reconstitution, stigmasterol LNPs maintained superior particle integrity and transfection efficiency relative to all other formulations, including cholesterol and β-sitosterol. Stigmasterol’s Δ 22 unsaturated ethyl side chain appears to preserve LNP bilayer organization during the lyophilization–reconstitution cycle, identifying it as a candidate sterol for cold-chain-independent mRNA products.


In vivo biodistribution. In C57BL/6J mice, β-sitosterol combined with ALC-0315, and fucosterol combined with MC3, both produced significantly elevated bioluminescence in target organs compared to cholesterol controls. This indicates that sterol identity influences organ-level delivery selectivity, not just cellular uptake.


Figure 5. In vivo biodistribution. (A) Schematic overview. (B-C) IVIS bioluminescence images of C57BL/6J mice 6h after i.v. injection of freshly prepared or lyophilized mRNA(FLuc)-LNPs. (D-K) Quantification of radiance in liver, spleen, and lungs for MC3- and ALC-0315-based LNPs across all sterol formulations. Beta-Sitosterol/ALC-0315 and fucosterol/MC3 combinations yielded the highest signals. (Adapted from Wang et al., Materials Today Bio, 2026)
Figure 5. In vivo biodistribution. (A) Schematic overview. (B-C) IVIS bioluminescence images of C57BL/6J mice 6h after i.v. injection of freshly prepared or lyophilized mRNA(FLuc)-LNPs. (D-K) Quantification of radiance in liver, spleen, and lungs for MC3- and ALC-0315-based LNPs across all sterol formulations. Beta-Sitosterol/ALC-0315 and fucosterol/MC3 combinations yielded the highest signals. (Adapted from Wang et al., Materials Today Bio, 2026)


Key Findings at a Glance

  • β-Sitosterol LNPs — highest transfection efficiency in fresh formulations across most cell lines

  • Stigmasterol LNPs — superior stability and transfection after lyophilization (cold-chain-free potential)

  • β-Sitosterol + ALC-0315 and Fucosterol + MC3 — highest in vivo bioluminescence in target organs

  • β-Sitosterol LNPs + MERTK mRNA — restored visual function in RCS rats (inherited retinal degeneration)



Ocular Gene Therapy: MERTK Rescue

MERTK encodes a receptor tyrosine kinase expressed in retinal pigment epithelium (RPE) cells that mediates phagocytosis of photoreceptor outer segments. Loss-of-function mutations cause progressive retinal degeneration. The researchers delivered MERTK mRNA encapsulated in β-sitosterol/ALC-0315 LNPs via subretinal injection to RCS rats — an established model of MERTK-deficient retinal degeneration.

OCT imaging confirmed successful subretinal localization of the LNPs. Three weeks post-injection, scotopic electroretinography (ERG) showed significant recovery of b-wave amplitudes in the treated eyes compared to untreated controls, demonstrating functional visual rescue. Western blot confirmed exogenous MERTK protein expression via a FLAG-tagged construct in both ARPE-19 and HEK-293T cells.


Figure 7. Subretinal delivery of (MERTK)mRNA-LNPs and visual function rescue in RCS rats. (A) Schematic of subretinal injection. (B) OCT confirming successful subretinal bleb formation. (C-E) Scotopic ERG recordings at multiple flash intensities showing significant b-wave recovery in beta-sitosterol LNP-treated eyes versus untreated controls - demonstrating functional restoration of vision in this inherited retinal degeneration model. (Adapted from Wang et al., Materials Today Bio, 2026)
Figure 7. Subretinal delivery of (MERTK)mRNA-LNPs and visual function rescue in RCS rats. (A) Schematic of subretinal injection. (B) OCT confirming successful subretinal bleb formation. (C-E) Scotopic ERG recordings at multiple flash intensities showing significant b-wave recovery in beta-sitosterol LNP-treated eyes versus untreated controls - demonstrating functional restoration of vision in this inherited retinal degeneration model. (Adapted from Wang et al., Materials Today Bio, 2026)


PreciGenome NanoGenerator® — Featured Formulation Tool

The LNPs in this study were prepared using the PreciGenome NanoGenerator® microfluidic device (Model PG-SYN-FS), cited directly by the authors. Its laminar-flow microfluidic mixing produces LNPs with tight size distributions and low PDI across non-standard sterol compositions, enabling researchers to isolate the biological effects of sterol substitution from batch-to-batch formulation variability.



Reference

Wang T, Li W, Chen J, et al. “Cholesterol analogs modulate lipid nanoparticle performance for mRNA delivery after lyophilization and enable ocular disease therapy.” Materials Today Bio, Vol. 38, 2026, Article 103044.

Learn more about NanoGenerator®: www.precigenome.com

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