HTS-BBB: A high-throughput transwell platform for screening mRNA lipid nanoparticle transfection of and transport across the blood-brain barrier

Introduction:: Neurological disorders such as neurodegenerative disease, brain cancer, traumatic brain injury, and stroke affect millions of people in the US, yet lack sufficient treatments. Nucleic acid therapies, including messenger RNA (mRNA) protein replacement and gene editing therapies, have great potential in treating these disorders. Lipid nanoparticles (LNPs) are currently the most clinically advanced delivery vehicle for nucleic acid therapeutics. However, a major hurdle in the systemic delivery of mRNA and their delivery vehicles to the brain is the blood-brain barrier (BBB), a highly selective diffusion barrier made of endothelial cells, basement membrane, pericytes, and astrocytes. Thus, there is an urgent need to identify novel LNPs that cross the BBB. In this work, we designed a high-throughput transwell model of the BBB, specifically optimized for screening mRNA LNPs, henceforth called “HTS-BBB” (Fig. 1A). Unlike most transwells which only assess transport from the apical to the basolateral compartment, the HTS-BBB platform simultaneously captures LNP transport via DiR signal, and mRNA transfection levels in both apical and basolateral cells. Here, we show the creation of HTS-BBB as well as its validation by screening a library of 20 LNPs made with structurally diverse ionizable lipids and reporter mRNA.


Materials and Methods:: 96-well transwells were coated with basement membrane protein collagen, and then seeded with human brain microvascular endothelial (hCMEC/D3) cells. Cell growth was monitored by fluorescence microscopy until a complete monolayer was formed. The monolayer was confirmed functionally by evaluating the transport of FITC-dextran tracers, and structurally by immunofluorescence (IF) of the tight junctions. LNPs containing ionizable lipid, phospholipid, cholesterol, and PEG-lipid were formulated via microfluidic mixing with reporter mRNA. DiR dye for tracking transport was added to the LNPs prior to dialysis for purification. LNP mRNA encapsulation efficiency was measured by RiboGreen assay. Transwells were treated with LNPs for 6 or 24 hours before assessment for LNP transport and transfection. Transport was measured via DiR signal in the basolateral media, signal enhanced by using Triton X-100 to disrupt the LNPs, and normalized using a calibration curve. Transfection was measured via luciferase assay for luciferase mRNA, and by fluorescence for mCherry mRNA.


Results, Conclusions, and Discussions:: To create HTS-BBB, we began by optimizing endothelial monolayer growth on the transwell insert. hCMEC/D3 cells were seeded on collagen-coated transwell inserts at multiple seeding densities and grown for 7 days. A seeding density of 30,000 cells/cm² and 6 days of growth was found to be optimal to grow a complete monolayer with minimal gaps and cell death. Monolayer integrity was confirmed functionally by low transport of tracer FITC-dextran across the monolayer (Fig. 1B), and structurally by IF of tight junction protein ZO-1 (Fig. 1C). Next, luciferase and mCherry mRNA were compared as reporters in a gold standard C12-200 LNP. Luciferase expression had a more sensitive signal, although only with low amounts of DiR loaded into the LNP (Fig. 1D). mCherry signal was much lower, but has the benefit of being able to be visualized under microscopy (Fig. 1E). We proceeded with luciferase mRNA and 1 mol% DiR to maximize the quantitative transfection readout. Having completed the optimization, we next validated HTS-BBB by screening a library of 20 structurally diverse LNPs for transport and transfection (Fig. 1F). By comparing the two measurements, we identified LNP 20 to have low transfection of the endothelial monolayer and high transport across, characteristics of an LNP with potential to cross the BBB. Finally, we confirmed LNPs that crossed the monolayer maintained their transfection ability by seeding additional hCMEC/D3 cells in the basolateral side and measuring luciferase expression.

In this work, we designed and validated HTS-BBB, a high-throughput transwell platform optimized for screening mRNA LNPs. Importantly, this platform captures not only transport across the monolayer, but also transfection of apical and basolateral cells. This platform could easily be re-optimized for other endothelial barriers, such as of the lung, kidney, and bone marrow. It could also be re-optimized to assess delivery of other nucleic acids like siRNA, or other types of nanocarriers like polymeric nanoparticles. Future work for HTS-BBB includes incorporating neurons into the basolateral compartment to assess neuronal transfection after crossing the endothelial monolayer, as well as validating in vitro screening results in vivo, to work towards developing BBB-penetrating mRNA therapeutics.


Acknowledgements (Optional): :

References (Optional): :