Universal Microfluidic Platform for Multifunctional Surface Modification of Small Extracellular Vesicles.

Small Extracellular vesicles (sEVs) hold great promise as therapeutic delivery vehicles due to their inherent biocompatibility. However, their clinical translation is limited by donor cell source dependency and inadequate targeting capabilities. To overcome these challenges, we introduce a universal surface engineering strategy that integrates lipid membrane anchoring with targeted ligand conjugation. At the core of this approach is the sEV Surface-Engineering microfluidic device (ExoSE), a dual-functional platform combining nanofluidic and microfluidic architectures. ExoSE consists of two interconnected modules: (1) a loading module that employs mechanoporation via nanochannels to transiently generate pores in sEV membranes, enabling highly efficient insertion of functionalized lipids and (2) a mixing module with specialized structures that facilitate rapid, covalent attachment of targeting ligands via optimized chemical reactions. This approach achieved lipid incorporation efficiencies of 97.93% for HEK293T-dervied sEVs and 98.47% for milk-derived sEVs, surpassing conventional co-incubation techniques. NanoFCM analysis revealed a 3- to 6-fold increase in ligand binding per sEV. Functionally, RGE peptide-modified sEVs exhibited a 54.13% increase in transmembrane transport efficiency in the in vitro model and enhanced infiltration into glioma spheroid, while AS1411 aptamer-conjugated sEVs showed 77.8% targeting specificity towards breast cancer cells, compared to 32.5% for normal breast cells. In vivo tracking in BALB/c-nude mice confirmed significantly improved brain accumulation of engineered sEVs, with no detectable hepatic or renal toxicity. Unlike traditional donor-cell-dependent genetic modification approaches, ExoSE enables universal, scalable modification of sEVs from diverse sources, including highly abundant milk-derived sEVs, and accommodates diverse ligand types such as peptides, aptamers and proteins. This device represents a transformative advancement in sEV engineering, establishing a standardized and scalable framework for precision-targeted sEV therapeutics with enhanced clinical potential.