Monodisperse LNPs - from Efficient Microfluidic Production and Loading to in Vitro Testing

Abstract Carrier nanoparticles facilitate the encapsulation of drug or mRNA molecules thereby enhancing their bioavailability. Microfluidic mixers provide a unique environment for the precise and continuous generation of nanoparticles by antisolvent precipitation. A major challenge is to understand the influence of microfluidic channel designs and geometries on the continuous production of small, uniform lipid nanoparticles (LNPs) and to identify conditions that ensure effective and controllable mixing of aqueous and organic phases in laminar flows. Another important challenge is that sufficient quantities for preclinical and clinical studies must be produced within a reasonable period of time. With this dual objective, different versions of a low aspect ratio laminar mixer (LARLM) were produced using two-photon polymerization (2PP). In the LARLM the organic phase forms a thin layer a few micrometers with a uniform velocity distribution in the center of the channel, surrounded by the aqueous phase. This concept has three major advantages: Firstly, it keeps all particles centralized in the channel, thus preventing contamination during prolonged particle generation. Secondly, diffusive mixing in the thin central stream occurs very quickly, and thirdly, the growing nanoparticles move at a homogeneous speed, which enables inline measurement of the particles. In systematic experiments with design versions of varied channel dimensions the operational parameters such as lipid concentrations and flow rates and the capability to produce LNPs with desired properties and loading capacities were explored. An interfacial dispersion model (IDM) could explain the surprising reduction of particle sizes with increased productivity. The latter allowed us to produce nanoparticles in the range of 50 nm to 180 nm (with 0.02 < PDI < 0.1) under stable conditions with a productivity of around one liter of LNP suspensions every three hours. Such performance has never been reached before with microfluidics. Moreover, LNPs loaded with coumarine-6 and various drugs were produced in the LARLM. Moreover, in-vitro experiments could confirm an improved bioavailability of coumarin-6 in cell culture experiments when loaded in LNPs by the LARLM. These results highlight the unique capabilities of LARLM devices and their potential to support nanoparticle formulation studies including preclinical and in further developments also clinical studies as required for approval as a marketable medicine. Competing Interest Statement The authors have declared no competing interest.