Ultrasound-assisted antifouling for continuous microfluidic manufacturing of RNA-LNP.

Abstract

Microfluidic platforms produce RNA-loaded lipid nanoparticles (RNA-LNPs) with superior uniformity, encapsulation efficiency, and control over size compared to bulk methods, and unlike bulk approaches, their throughput can be scaled over orders of magnitude <i>via</i> parallelization without altering the formulation. Despite these advantages, continuous manufacturing of RNA-LNPs by microfluidics has been hindered by rapid fouling of microchannels exposed to the complex mixtures of lipids, cholesterol, and RNA in mixed aqueous-organic solvents used for LNP formulation, degrading chip output quality over time. Conventional antifouling coatings have proven ineffective under these conditions, and alternative approaches, such as lubricant-infused coatings, can be difficult to reconcile with pharmaceutical quality requirements related to contamination. Here, we introduce an ultrasound-based strategy that prevents fouling in microfluidics without any chemical surface modification. Acoustic forces, optimized to remain below thresholds for cavitation or RNA degradation, actively suppress deposition on channel walls <i>via</i> boundary-driven acoustic streaming generated at the liquid-solid interface along the channel walls. By integrating a back-pressure regulator, we extend the usable acoustic intensity range by suppressing cavitation, enabling long-term stable operation. To demonstrate feasibility, we integrate piezoelectric actuation and back-pressure regulation into a PDMS staggered herringbone mixing chip with untreated channel surfaces and show that this strategy achieves uninterrupted RNA-LNP production for more than six hours, representing a >36-fold increase in operational lifetime compared to devices without acoustic actuation. We compare RNA-LNPs produced using our chip after >6 hours of use with those generated by the same chips without acoustic actuation, evaluated at short time scales prior to fouling, and show no significant differences in physicochemical properties or <i>in vitro</i> performance. This approach enables practical, long-duration microfluidic precision manufacturing of RNA-LNPs while preserving formulation quality and biological performance.