Simultaneous Loading and Purification of Curcumin-Loaded Milk Whey Extracellular Vesicles by Tangential Flow Filtration for Neuroblastoma Therapy

Extracellular vesicles (EVs) are nanoparticles released by cells and potential drug carriers. However, their translation is limited by the absence of standardized, scalable purification methods and low yields. Drug loading creates an additional downstream challenge, namely the removal of unbound drug. Here, we demonstrate that a single tangential flow filtration process can simultaneously isolate whey-derived EVs (wEVs) and remove residual unbound drugs from the whey matrix. Bovine milk whey was used as the EV source, and curcumin (Cur) as the model drug. Passive loading, followed by constant-volume diafiltration and concentration in one ultrafiltration module, removed 98.9 ± 0.0% of the whey impurities, together with most of the residual free Cur. The process recovered 1.1 × 1013 particles per mL of processed whey and 10.9 ± 0.1% of the added Cur, about one third of which was associated with the vesicles. wEVs and Cur-wEVs showed comparable particle concentration, size, and ζ potential, indicating preserved vesicle integrity. At an equal total amount of Cur, Cur-wEVs exhibited higher intracellular Cur accumulation and greater inhibition of SK-N-AS neuroblastoma cell proliferation than free Cur (IC50 16.34 μM vs. 31.14 μM). Combining loading and purification in a single membrane operation addresses a major bottleneck in EV-based drug delivery.