Poster Y9 - Elucidating Mechanism in Microfluidics-Enhanced Lipid Nanoparticle Dialysis via Single-Particle Cylindrical Illumination Confocal Spectroscopy

Introduction: Lipid Nanoparticles (LNPs) have emerged as highly efficient carriers for delivering mRNA-based vaccines and therapeutics. After the formulation of aqueous mRNA solution with an alcohol solution of lipids, dialysis is employed as a purification method to remove ethanol from the medium and exchange it with a physiological PBS buffer, rendering LNPs suitable for cell culture and in vivo administration. Current dialysis approaches typically rely on dialysis membranes to enable the diffusion of buffer exchange, which can entail a lengthy process and add the risk of composition drift. Microfluidics as an emerging platform presents a promising alternative for LNP dialysis for precise fluidic exchange. Here we develop a microfluidics-enhanced LNP dialysis process and present a mechanistic understanding of the effect of medium component (salt, pH, and polarity) on LNP quality and transfection activity.

Materials and

Methods: The polydimethylsiloxane (PDMS) microfluidic device is fabricated with soft lithography. The photomask was designed using AutoCAD and fabricated using standard photolithography. LNPs were formulated using a T-junction at controlled flow rates. Dialysis was achieved in a microfluidic device at a flow rate ratio of 1:24 for LNP mixture solution to PBS. Dynamic light Scattering (DLS) measured the LNP size, PDI and zeta potential. Multi-color cylindrical illumination confocal spectroscopy (CICS) (Fig. 1) analyzed single-particle fluorescence burst quantification, enabling particle classification and mRNA payload characterization.

Results, Conclusions, and Discussions: Cy-5 mRNA and TopFluor-PC aided LNP categorization into three groups: empty, loaded, and free mRNA, corresponding to TopFluor-PC signal only, Cy-5 signal only, and coincident signals (Fig. 1). Using a microfluidics system for rapid on-chip dialysis (Fig. 2), we compared its performance with a standard 18-h dialysis. A slightly reduced empty particle fraction and increased average payload per particle were observed using microfluidics, as well as a >10-fold increase in transfection efficiency (Fig. 3). We further fine-tuned the total flow rate, ranging from 0.5, 2, 5, and 50 mL/h. Results have showed the lowest empty particle percentage and highest payload when the total flow rate reached 5 mL/h, where the transfection efficiency showed corresponding trends with a negative correlation between empty particle percentage and transfection efficiency (Fig. 4). Additionally, increasing pH from 4.0 to 7.4, while holding other solvent conditions constant led to larger sizes but lower PDIs (Fig. 5). Our data supports a mechanistic framework suggesting that ionizable lipids remain positively charged in acidic conditions, reducing merging events. At pH 7.4, neutral ionizable lipids have made merging events to happen that lead to increased size and payload, while decreasing empty particles and encapsulation efficiency. Similar approaches were applied to study effects of ionic strength and solvent polarity on LNP quality and bioactivity. This platform offers a unique opportunity to understand the structural features and stability of LNPs and optimize their purification process.

Acknowledgements (Optional):