Formation of Protein Nanoparticles in Microdroplet Flow Reactors

Nanoparticlesare increasingly being used for biological applications,such as drug delivery and gene transfection. Different biologicaland bioinspired building blocks have been used for generating suchparticles, including lipids and synthetic polymers. Proteins are anattractive class of material for such applications due to their excellentbiocompatibility, low immunogenicity, and self-assembly characteristics.Stable, controllable, and homogeneous formation of protein nanoparticles,which is key to successfully delivering cargo intracellularly, hasbeen challenging to achieve using conventional methods. In order toaddress this issue, we employed droplet microfluidics and utilizedthe characteristic of rapid and continuous mixing within microdropletsin order to produce highly monodisperse protein nanoparticles. Weexploit the naturally occurring vortex flows within microdropletsto prevent nanoparticle aggregation following nucleation, resultingin systematic control over the particle size and monodispersity. Throughcombination of simulation and experiment, we find that the internalvortex velocity within microdroplets determines the uniformity ofthe protein nanoparticles, and by varying parameters such as proteinconcentration and flow rates, we are able to finely tune nanoparticledimensional properties. Finally, we show that our nanoparticles arehighly biocompatible with HEK-293 cells, and through confocal microscopy,we determine that the nanoparticles fully enter into the cell withalmost all cells containing them. Due to the high throughput of themethod of production and the level of control afforded, we believethat the approach described in this study for generating monodisperseprotein-based nanoparticles has the potential for intracellular drugdelivery or for gene transfection in the future.

Automated summary

Nanoparticles have become increasingly important in biological applications, but obtaining stable and controlled protein nanoparticles has been challenging. In this study, we used droplet microfluidics to produce highly monodisperse protein nanoparticles by utilizing the characteristic of rapid and continuous mixing in microdroplets. The internal vortex flows within microdroplets were found to determine the uniformity of the nanoparticles, and by adjusting parameters such as protein concentration and flow rates, we were able to control the nanoparticle size. These protein nanoparticles showed high biocompatibility with HEK-293 cells and were able to fully enter the cells. Due to the high throughput and level of control, this approach has the potential for future intracellular drug delivery or gene transfection.