Predictive High-Throughput Platform for Dual Screening of mRNA Lipid Nanoparticle Blood-Brain Barrier Transfection and Crossing

Abstract

Lipid nanoparticle (LNP)-mediated nucleic acid therapies, including mRNA protein replacement and gene editing therapies, hold great potential in treating neurological disorders including neurodegeneration, brain cancer, and stroke. However, delivering LNPs across the blood-brain barrier (BBB) after systemic administration remains underexplored. In this work, we engineered a high-throughput screening transwell platform for the BBB (HTS-BBB), specifically optimized for screening mRNA LNPs. Unlike most transwell assays, which only assess transport across an endothelial monolayer, HTS-BBB simultaneously measures LNP transport and mRNA transfection of the endothelial cells themselves. We then use HTS-BBB to screen a library of 14 LNPs made with structurally diverse ionizable lipids and demonstrate it is predictive of in vivo performance by validating lead candidates for mRNA delivery to the mouse brain after intravenous injection. Going forward, this platform could be used to screen large libraries of brain-targeted LNPs for a range of protein replacement and gene editing applications.

Keywords: blood−brain barrier; brain delivery; lipid nanoparticle; mRNA.

Figure 1.

Schematic of blood–brain barrier (BBB) physiology and the development of the high-throughput screening BBB (HTS-BBB) platform for identifying mRNA lipid nanoparticles (mRNA LNPs) that cross the BBB and also transfect the BBB for neurological disorder applications.

Figure 2.

Optimization of brain endothelial monolayer growth in HTS-BBB. A) Schematic showing endothelial monolayer growth timeline and characterization techniques. B) Live/dead imaging of a monolayer seeded at 30,000 cells/cm², on different days of monolayer growth. Scale bar: 100 μm. C) Average Z projections of confocal microscopy images showing immunofluorescence (IF) staining for tight and adherens junction proteins ZO-1 and VE-cadherin on Day 6 of monolayer growth. Scale bar: 20 μm. D) Transport of FITC-dextran (FD) tracers across the monolayer after 4 h, on different days of monolayer growth. Data are shown as mean ± SD, n = 3.

Figure 3.

Optimization of mRNA LNP transfection and transport reporters in HTS-BBB. A) Schematic showing optimization process for transfection and transport readouts. B) Physicochemical characterization of C12–200 LNPs with varying reporter mRNAs and DiR molar percentages. Size and PDI is shown as mean ± SD, n = 3. Encapsulation efficiency is shown as mean ± SD, n = 2. C) mCherry expression in brain endothelial cells grown in a 96-well plate. Scale bar: 100 μm. D) Effect of DiR molar percentage on luciferase and mCherry expression in brain endothelial cells over time, in a 96-well plate. Cells were treated with 60 ng mRNA/20k cells. Data are shown as mean ± SD, n = 3, and normalized to untreated cells. Two-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance, **p < 0.01, ****p < 0.0001. E) Effect of DiR molar percentage on luciferase and mCherry expression in brain endothelial cells, grown in HTS-BBB. Cells were treated with 60 ng mRNA/20k cells for 24 h. Data are shown as mean ± SD, n = 3, and normalized to untreated cells. One-way ANOVA with Tukey’s multiple comparisons test was used to determine statistical significance, *p < 0.05.

Figure 4.

Screening of an LNP library in HTS-BBB for transfection and transport. A) Schematic showing design of library of 14 LNPs, each with a unique ionizable lipid. “C12–200 PEG” indicates a formulation with C12–200 as the ionizable lipid and a higher molar percentage of PEG-lipid. B) Luciferase expression in brain endothelial cells grown in a 96-well plate, treated with LNPs for 24 h at 60 ng mRNA/20k cells. Data are shown as mean + SD, n = 3 biological replicates each with n = 3 technical replicates, normalized to the C12–200 group. C) Cell viability of brain endothelial cells grown in a 96-well plate, treated with LNPs for 24 h at 60 ng mRNA/20k cells. Data are shown as mean + SD, n = 3 biological replicates each with n = 3 technical replicates, normalized to the untreated group. D) Luciferase expression in the brain endothelial monolayer of HTS-BBB, treated with LNPs for 24 h at 60 ng mRNA/20k cells. Data are shown as mean + SD, n = 3 biological replicates each with n = 4 technical replicates, normalized to the C12–200 group. E) Transport of LNPs across the brain endothelial monolayer of HTS-BBB after 24 h, as measured by DiR fluorescence from the LNPs. Data are shown as mean + SD, n = 3 biological replicates each with n = 4 technical replicates. One-way ANOVA with Dunnett’s multiple comparisons test was used to determine statistical significance compared to the C12–200 group, *p < 0.05, ****p < 0.0001.

Figure 5.

In vivo validation of the HTS-BBB mRNA LNP library screen. A) IVIS images and quantification of luciferase mRNA LNP delivery to the brain in adult C57BL/6 mice. Mice were injected intravenously with mRNA LNPs at a dose of 0.3 mg/kg mRNA or PBS and sacrificed after 6 h. Representative IVIS images are shown from the mouse dissected last per treatment group. Relative radiance was calculated by subtracting PBS luminescence from treated groups and reported as mean + SD with n = 3 biological replicates. One-way ANOVA with Šídák’s multiple comparisons test was used to determine statistical significance. B) Distribution of luminescence across brain, heart, lungs, liver, kidneys, and spleen. Data are shown as % of total luminescence = (organ luminscence)/(total organ luminescnce) × 100. Blue dotted line indicates 1% of total organ delivery.