Engineering Biomimetic Nanoparticle Performance Through Fabrication Method Selection: Turbulent Jet Mixing, Microfluidics, and Extrusion.

Nanoparticle (NP) fabrication has advanced rapidly, driven by the growing role of nanomedicine in targeted drug delivery. Each fabrication strategy offers unique advantages and limitations. Here, we conduct a comparative evaluation of three prominent methods: turbulent jet mixing, microfluidic mixing, and extrusion, for producing biomimetic nanoparticles (BNPs). BNPs are emerging as next-generation drug delivery platforms, combining liposomal biocompatibility with enhanced cellular uptake, prolonged circulation, and selective targeting, achieved by incorporating membrane proteins from source cells into synthetic lipid bilayers to confer cell-mimicking functionality. Using neuron-derived BNPs ("Neurosomes") as a model, we systematically assess physicochemical and biological properties across fabrication methods and their impact on BNP function. Turbulent jet and microfluidic mixing produce BNPs with superior stability, higher membrane protein incorporation, improved batch-to-batch reproducibility, and enhanced targeting, whereas extrusion leads to diminished performance due to shear-induced protein loss. Notably, this study presents the first application of high-resolution LC-MS/MS proteomics to quantitatively compare membrane-associated protein profiles across fabrication methods. These results highlight the critical influence of fabrication techniques on BNP structure and function and provide actionable insights for optimizing production strategies, facilitating the scalable development of targeted nanotherapeutics.