Microfluidic post-insertion preparation and evaluation of RGD- and PEG-modified macrophage-derived small extracellular vesicles.

Various functionalization methods have been explored to enhance the targeted delivery of small extracellular vesicles (sEVs). We recently developed a microfluidic post-insertion method that enables the simultaneous incorporation of polyethylene glycol (PEG) lipids and high-functionality and high-quality (HFQ) lipids into bovine milk-derived sEVs. Since sEVs from different sources differ in surface composition and properties that significantly impact modifications, it is crucial to determine whether this method is applicable to sEVs from other origins. In this study, we used a PEG-lipid and a peptide consisting of arginine-glycine-aspartic acid (RGD) linked to a serine-glycine repeat (SG)5 to modify sEVs derived from mouse macrophage cells (RAW264.7) and compared the results with those obtained using the conventional bulk mixing method in vitro. The RGD-PEG sEVs prepared via microfluidic post-insertion and bulk mixing exhibited similar physicochemical properties and cellular binding functions. Successful incorporation of both lipids was confirmed by changes in physicochemical properties, cellular binding capacity, and endocytosis pathways. The results indicated that PEG reduced overall uptake, while RGD facilitated cellular association of RGD-PEG sEVs compared to PEG sEVs alone. Our approach for the simultaneous incorporation of PEG and HFQ lipids may open new avenues for the functional application of sEVs derived from diverse origins.