Mechanisms for determining the load capacity and distribution of mRNA LNPs by PEG content. a, b The putative aggregation processes and characteristics of the LNP formulation at a high concentration of PEG mol% (a); or a low concentration of PEG mol% (b) and the synthesis drift during dialysis from pH 4.0 (left) to pH 7.4 (right). The labeled population fractions are real data from the formula with PEG mol% = 1.5% (a) or 0.5% (b). a Each number classification represents mass behavior during hemodialysis: 1, subdividing empty LNPs; 2, install empty LNPs; 3, splitting of lipophilic complexes with initially high mRNA load; 4, the same mRNA load remains for lipophilic complexes with an initially low or intermediate load; 5, fusion of empty LNPs with mRNA complexes; 6, incorporation of non-lipophilic complexes. The cross sign represents the finding that the mRNA load of lipophilic complexes does not increase during dialysis due to non-integration under this condition. B labels are: 1, a combination of lipophilic complexes; 2, fusion of empty LNPs with mRNA complexes; 3, the incorporation of non-lipophilic complexes; 4, Split the empty LNPs. attributed to him: Nature Connections (2022). DOI: 10.1038 / s41467-022-33157-4
An important component of vaccines that protect people from SARS-CoV-2 and its variants are lipid nanoparticles, or LNPs. These circular particles carry therapeutic mRNA payloads, which are extracts of genetic material that stimulates our immune systems to defend against COVID-19.
Even with its success, some properties about the particles, such as the load distribution, are unknown. Researchers and the U.S. Food and Drug Administration want more insights into these properties to improve metrology reporting in pharmaceutical manufacturing.
A new molecular detection platform developed by two professors from the Whiting School of Engineering responds to the FDA’s call. Hai-Quan Mao and Tza-Huei (Jeff) Wang want to manipulate how many mRNA particles the LNP can carry and whether the mRNA is uniformly packaged within the particle to help researchers design more efficient and effective treatments and vaccines.
“Our platform processes molecules at the level of single nanoparticles, but unlike current imaging methods for mRNA LNPs, our approach is based on fluorescence spectroscopy and gives us the ability to see the particles,” said Wang, a professor in the mechanics departments. Engineering and Biomedical Engineering at The Whiting School, and Principal Investigator at the Institute of Bionanotechnology.
The ability to isotope inside the nanoparticles allows researchers to distinguish and quantify empty LNPs that do not contain mRNA and LNPs with mRNA and free-floating mRNA in a sample.
Their platform, called Cylindrical Illumination Spectroscopy, or CISC, works by labeling mRNA and LNP components with fluorescent signals of up to three colors and passing the sample through a detection plane. The detection level reads the fluorescent signals and measures their intensity before comparing the strength of the intensity with that of a single mRNA molecule.
Analyzing the data using an algorithm called deconvolution tells the team how many mRNA copies are present within the LNP – if any – and their distribution in the sample. The team’s platform overcomes the limitations of contrast and increases the throughput of sample analysis, which is demonstrated in cryo-transmission electron microscopy, the current gold standard for imaging mRNA LNPs.
Tests using this sensing platform revealed that from a standard solution of mRNA LNP used in academic research studies, no more than 50% of LNPs were loaded with mRNA particles, and of mRNA-filled LNPs, most contained two to three mRNA particles per particle.
“The ability to quantitatively analyze the payload properties of mRNA LNPs at the single-particle level has not been performed before. We are fascinated by the large presence of null LNPs, and by changing the formulation conditions, a single nanoparticle can load as little as one to the like,” said Mao, a professor in the Departments of Science Materials, Engineering, and Biomedical Engineering at The Whiting School and Director of the Bio Nanotechnology Institute, “Up to ten mRNA molecules.”
The team’s results have been published in Nature Connections.
“There are a lot of groups doing research on LNP,” Wang said. “However, when they discover a formula that might work well, it has been difficult to relate these discoveries to the composition and payload distribution of nanoparticles. By using this platform, we can provide a more comprehensive understanding of what is happening at the single particle level.”
More research is needed to find out how many mRNA molecules per LNP capsule are optimal for the most effective treatment. However, the null LNPs revealed by the new platform show that there is a need to improve mRNA encapsulation methods within the LNPs.
Mao and Wang say their platform shows that it has the potential not only to be used throughout LNP-related research and development, but also in the development of other drug delivery systems and quality control procedures in the manufacturing stage. The team submitted a file patent application She covers the technology and works with collaborators on using the platform to analyze other types of therapeutic charges in diverse nanoparticle systems to treat different diseases.
“The Food and Drug Administration recently addressed the need for better quality metrics in nanoparticle design in Pharmaceutical industryMichael J. said: Mitchell, a prominent scientist in the field of LNP research and the 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 to new therapies administered into the bloodstream, which have very stringent requirements. The new detection platform developed by the doctors. Mao and Wang’s team is a potentially important step forward. in addressing needs in the research and regulatory phase, and could help advance mRNA LNP technology beyond vaccines.”
Sixuan Li et al, Load distribution and capacity of mRNA lipid nanoparticles, Nature Connections (2022). DOI: 10.1038 / s41467-022-33157-4
Johns Hopkins University
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