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

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 via 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 via 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 in vitro performance. This approach enables practical, long-duration microfluidic precision manufacturing of RNA-LNPs while preserving formulation quality and biological performance.