An important part of the vaccines that protect people against the SARS-CoV-2 virus and its variants are lipid nanoparticles, or LNPs. These circular particles carry therapeutic mRNA payloads, the fragments of genetic material that prompt our immune system to defend against COVID-19.
Even with their success, certain characteristics of the particles, such as the distribution of the charge, are unknown. Researchers and the Food and Drug Administration want to better understand these characteristics to improve the reporting of statistics in pharmaceutical production.
A new molecular detection platform developed by two professors from the Whiting School of Engineering answers the FDA’s call. Hai-Quan Mao and Tza-Huei (Jeff) Wang want to investigate how many mRNA molecules an LNP can carry and whether the mRNA is packed uniformly in the particle, to help researchers design more efficient and effective treatments and vaccines.
“Our platform processes molecules at the single nanoparticle level, but unlike current imaging methods for mRNA-LNPs, our approach is based on fluorescence spectroscopy and allows us to see through the particles,” said Wang, a professor in the Departments of Mechanical Engineering and Biomedical Engineering at the Whiting School, and a core researcher at the Institute for NanoBioTechnology.
The ability to see inside the nanoparticles allows the researchers to distinguish and measure empty LNPs containing no mRNA, LNPs containing mRNA and free-floating mRNA in a sample.
Their platform, called cylindrical illumination confocal spectroscopy, or CISC, works by tagging mRNA and LNP components with fluorescent signals of up to three colors and passing the sample through a detection plane. The detection plane reads the fluorescence signals and measures their intensity before comparing the strength of the intensities to that of a single mRNA molecule.
The data analysis with an algorithm called deconvolution tells the team how many mRNA copies are in the LNP — if any — and their distribution in the sample. The team’s platform overcomes contrast limitations and increases throughput of sample analysis, which is seen in cryotransmission electron microscopy, the current gold standard for imaging mRNA LNPs.
Tests conducted with this detection platform revealed that of a benchmark solution of mRNA LNP used in academic research studies, more than 50% of the LNPs are unloaded with mRNA molecules, and of the mRNA-filled LNPs, most contain two to three mRNAs. molecules per particle.
“It has never been done before to quantitatively resolve the payload characteristics of mRNA LNPs at the single-particle level. We are intrigued by the substantial presence of empty LNPs, and by changing the formulation conditions, a single nanoparticle can only up to ten mRNA molecules,” said Mao, a professor in the departments of Materials Science and Engineering and Biomedical Engineering at the Whiting School and director of the Institute for NanoBioTechnology.
The team’s results are published in nature communication.
“There are many groups doing LNP research,” Wang said. “However, if they discover a formula that could work well, it was difficult to link those discoveries back to the composition and charge distribution of the nanoparticles. With this platform, we can provide a more comprehensive understanding of what happens when single particle level.”
More research is needed to learn how many mRNA molecules per LNP capsule are optimal for the most effective treatment. However, the empty LNPs revealed by the new platform show that there is a need to improve methods for packaging the mRNA into the LNPs.
Mao and Wang say their platform demonstrates that it has the potential to be used not only in all stages of LNP-related research and development, but also in the development of other drug delivery systems and quality control measures in the manufacturing stage. The team has submitted an application patent application: covering the technique and working with collaborators to use the platform to analyze other types of therapeutic payloads in different nanoparticle systems for the treatment of various diseases.
“The FDA recently addressed the need for better quality measurement data in nanoparticle design in the pharmaceutical industrysaid Michael J. Mitchell, a leading scientist in LNP research and Skirkanich Assistant Professor of Innovation in the Department of Bioengineering at the University of Pennsylvania.
“This will become increasingly important as mRNA-LNP technology expands beyond vaccines into novel therapies that are delivered into the bloodstream and are subject to very stringent requirements. The new detection platform developed by the team of Drs. Mao and Wang is a potentially important step forward in addressing needs at the research and regulatory stage, and could potentially aid the development of mRNA LNP technology beyond vaccines.”
Sixuan Li et al, Payload distribution and capacity of mRNA lipid nanoparticles, nature communication (2022). DOI: 10.1038/s41467-022-33157-4
Johns Hopkins University
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