Lipid nanoparticles for mRNA delivery in brain via systemic administration

Abstract

Efficient delivery of messenger RNA (mRNA) to the brain via systemic administration remains a challenge, primarily due to the blood-brain barrier. To address this challenge, we incorporated SR-57227, a ligand of serotonin [5-hydroxytryptamine type 3 (5-HT₃)] receptor, into the design of ionizable lipids to develop lipid nanoparticles (LNPs) for systemic mRNA delivery to the brain. OS4T LNP was identified as an optimized formulation based on multiple assays. Following systemic administration, OS4T LNP achieved over a 50-fold increase in mRNA translation within brain tissues compared to US Food and Drug Administration-approved Onpattro LNPs (DLin-MC3-DMA). In an orthotopic glioblastoma (GBM) mouse model, engineered interleukin-12 mRNA-loaded OS4T LNPs significantly suppressed tumor growth and improved overall survival. This study demonstrates OS4T LNP as a promising platform for brain mRNA delivery and highlights its potential for treating GBM and other central nervous system disorders.

Fig. 1.. Construction of SLNPs for brain delivery.

(A) The structure of SLs. (B) mRNA delivery efficiency in bEnd.3, astrocytes, and N2a cells. Data are presented as means ± SD. (C) Normalized intensity in the brain of mice treated with MC3, S0, S4, and S6 LNPs (n = 3 biological replicates). The intensity was normalized to MC3 LNP group. Data are presented as means ± SD. (D) Representative images of brain tissues. Statistical significance was analyzed by one-way analysis of variance (ANOVA) with the Tukey’s multiple comparisons test. ***P < 0.001; ****P < 0.0001. (A) was created in BioRender (X. Hou, 2025; https://BioRender.com/mjk1zye).

Fig. 2.. Optimization and evaluation of S4 LNP for brain delivery.

(A) The 16 (4)⁴ orthogonal table (first round) and predicted formulations (second round). (B) Effects of each lipid component at different molar ratios on mRNA delivery efficiency of S4 LNP (n = 4 biological replicates). Data are presented as means ± SEM. (C) Relative luminescence intensity in bEnd.3 and N2a cell lines. The intensity was normalized to formulation 0 group. Data are presented as means ± SD. (D) Normalized luminescence intensity in the brain of mice treated with MC3, SM-102, ALC-0315, S4, or OS4 LNPs (n = 3 biological replicates). The intensity was normalized to MC3 LNP group. Data are presented as means ± SD. (E) Representative images of brain tissues. Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparisons test. **P < 0.01; ****P < 0.0001.

Fig. 3.. The mRNA delivery efficiency and safety of CPP-conjugated OS4 LNPs.

(A) The list of different CPPs. (B) The size and PDI of CPP-conjugated OS4 LNPs. (C) Normalized luminescence intensity in the brain from the mice treated with CPP-conjugated OS4 LNPs (n = 3 biological replicates). The intensity was normalized to unmodified OS4 LNP group. (D) Quantification of luminescence intensity in the brain of mice treated with MC3, MC3T, or OS4T LNPs (n = 3 biological replicates). The intensity was normalized to MC3 LNP group. (E) Representative images of brain tissues. (F) Representative of cryo-TEM image of OS4T LNP. Scale bar, 50 nm. (G) The levels of blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in mice following intravenous administration of PBS, MC3, OS4, or OS4T LNPs (n = 3 biological replicates). Data are presented as means ± SD in (B), (C), (D), and (G). Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4.. OST4 LNP enables effective mRNA delivery into different brain cells.

Flow cytometry analysis of GFP expression (A) or tdTomato expression (B) across different brain cells [neuronal nuclei (NeuN)⁺ for neuron, glial fibrillary acidic protein (GFAP)⁺ for astrocyte, CD11b⁺ for microglia, and CD31⁺ for BCEC]. Data are presented as means ± SD in (A) and (C) (n = 3 biological replicates). Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparisons test. (C) Schematic illustrating the delivery of Cre mRNA to activate tdTomato expression, along with the administration regimen. (D) Representative immunofluorescence sections of brain tissues of Ai14 mice after three administrations with PBS, MC3, or OS4T LNPs (Cre mRNA, 1 mg/kg). Scale bar, 2.5 mm. The tdTomato expression in neurons (E), microglia (F), and astrocytes (G) after three administrations with PBS, MC3, or OS4T LNPs (Cre mRNA, 1 mg/kg). Scale bar, 50 μm. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.. Therapeutic efficacy of OS4T LNPs in an orthotopic mouse model of GBM.

(A) The eIL-12 expressed by OS4T LNPs can be activated by MMP9 within the tumor microenvironment. (B) Western blot analysis of the cleavage of eIL-12 by MMP9. (C) The body weight changes in C3H/HeJ mice (n = 5 biological replicates) following administration of OS4T loaded with IL-12 or eIL-12 mRNAs (mRNA, 1 mg/kg). The pink arrows indicate the time of administration. Data are presented as means ± SD. (D) Schematic of the treatment regimen in the CT-2A-Luc GBM model. (E) Luminescence intensity of orthotopic GBM tumor tissues in the mice intravenously treated with PBS, OS4T-FLuc mRNA, MC3T-eIL-12, OS4-eIL-12, or OS4T-eIL-12 LNPs, respectively (mRNA, 1 mg/kg). Data are presented as means ± SD. Statistical significance was analyzed by two-way ANOVA with Tukey’s multiple comparisons test. (F) Survival proportions over time (n = 7 biological replicates). Statistical significance was analyzed by Gehan-Breslow-Wilcoxon test. (G) Representative IVIS images of tumor-bearing mice. The missing image indicates that the mouse had already succumbed at this time. **P < 0.01; ***P < 0.001; ****P < 0.0001. (A) and (D) were created in BioRender (X. Hou, 2025; https://BioRender.com/mjk1zye).