Microfluidic post-insertion of polyethylene glycol lipids and KK or RGD high functionality and quality lipids in milk-derived extracellular vesicles

To achieve the desired delivery effect, extracellular vesicles (EVs) must bypass rapid clearance from circulation and exhibit affinity for target cells; however, it is difficult to simultaneously incorporate two materials into EVs. Post-insertion is a general modification method that can be performed by simply mixing different solutions. Previously, we have developed a microfluidic post-insertion method that supported fast and upscaled modification of EVs using KK-modified high-functionality and -quality (HFQ) lipids. Here, we used microfluidic post-insertion to achieve simultaneous incorporation of polyethylene glycol (PEG) lipids and KK or RGD-modified HFQ lipids into milk-derived EVs to avoid uptake from the reticuloendothelial system and increase the uptake into target cells. PEG lipid and HFQ lipids were formulated to produce micelles and subsequently mixed with EV solution using a microfluidic device. Compared to bulk mixing, microfluidic post-insertion showed higher cellular association. Altered cellular association capacities and endocytic pathways indicated simultaneous incorporation. The cellular association of modified EVs can be adjusted by altering the ratio of (EK)₄-KK in micelles with slight changes in physicochemical properties. Furthermore, microfluidic post-insertion is also suitable for (SG)₅-RGD, which is insoluble in phosphate-buffered saline (PBS). Our results may be valuable for the development and manufacture of functional EVs as drug delivery systems for clinical applications.