Lipid Nanoparticles (LNPs) containing mRNA revolutionized the pharmaceutical field by enabling direct delivery of large delicate mRNA molecules so that the expression of proteins could be precisely controlled 1. These mRNA LNP products have helped with combating the SARS-CoV-2 pandemic and offered treatments for genetic diseases caused by the absence of specific proteins needed for biological function in patients 2. However, despite being widely used to suppress the pandemic, these relatively new mRNA LNP products have yet to be fully characterized and optimized. For example, drug developers use ensemble-average measurement techniques which gloss over the important biophysical properties of the mRNA LNPs such as their distribution of sizes and mRNA payloads, and are in need of new high-resolution data to advance the properties of a vast array of therapeutic products 3, 4. Quantifying the mRNA number per LNP and reducing the proportion of empty LNPs in a dose are both important contributions in quantitative biophysical measurements which are necessary to advance mRNA-LNP products 4. In this thesis, we introduce and take first steps towards establishing a new method for quantifying the distribution of mRNA per LNP, with single-particle resolution, and which does not require the mRNA to be labeled with a dye prior to measurement. This method works by first trapping the mRNA-LNPs in the wells of a microfluidic flow cell, and then releasing the mRNA molecules so they can be quantified. For the single-particle imaging, we use ConvexLens Induced Confinement (CLiC) microscopy 5 which serves to isolate and entropically trap particles while enabling reagent-exchange during live imaging. After the particles are trapped and identified in a confined volume, a lipid-like detergent, Triton X-100, is introduced to disperse mRNA payloads contents into the wells; as well as a stain, Ribogreen, to fluorescently label the mRNA and count them using particle-tracking algorithms. We present results for a representative mRNA LNP sample and count an average of 2.57 ± 0.9mRNA/LNP. This is reasonable given the physical considerations made during the data analysis.
Martin Jasinski (Fri,) studied this question.