(93e) Scaling Curcumin Loading into Milk Whey Extracellular Vesicles (EVs) for Neuroblastoma Therapy

Extracellular Vesicles (EVs) are membrane-bound nanoparticles released by cells that carry proteins, lipids and nucleic acids from the parent cells. Due to their natural biocompatibility, ability to cross biological barriers, and cell-targeting capabilities, they hold immense potential in various medical applications, particularly in drug and gene delivery [1]. However, despite their therapeutic potential, the clinical and large-scale application of EVs is limited by their intrinsic heterogeneity, difficult upstream processing that mostly relies on human cell culture, and the lack of standardized and high-throughput processing methods. Alternative biological sources of EVs, such as milk, plants, algae, and bacteria, are gaining increasing attention, due to their cost-effectiveness, ease of isolation from large volumes of fluid, and improved yields [2].

The aim of this research is to isolate EVs from alternative biological sources through a fully scalable membrane-based process, to develop a drug delivery system to target neuroblastoma (NB), an aggressive form of pediatric cancer. We decided to use bovine milk whey, a by-product of Parmesan cheese production, as a source of EVs. Milk whey, although rich in valuable compounds, causes significant environmental issues due to its extremely high volume production. In our study, pretreated milk whey is loaded with curcumin (Cur), a potent anticancer and anti-inflammatory compound, whose therapeutic potential is hampered by low bioavailability and stability [3]. After loading by passive incubation, curcumin-loaded whey-derived EVs (Cur-EVs) were purified with an integrated ultrafiltration/diafiltration membrane process, using hollow fiber modules operated in cross-flow. Pure whey EVs and Cur-EVs were isolated with the same process and characterized by different techniques, such as Western blot, NTA, DLS and SEM microscopy, to compare their dimensions and morphology. Finally, the therapeutic potential of Cur-EVs was evaluated in vitro on SK-N-AS, a NB cell line, through cell viability assays and internalization studies using confocal microscopy.

Western blot analysis of whey EVs confirmed the presence of the classical tetraspanin EVs marker CD81, and the absence of the negative markers Cytochrome C and VEGF. In addition, we identified a uniform population of EVs with a mean size of approximately 180 nm, an average concentration of around 3∙10¹³ particles/mL, and a z-potential of -12 mV. SEM microscopy imaging revealed a similar morphology of whey EVs and Cur-EVs, while NTA and DLS analysis of Cur-EVs showed a slight decrease in size to 165 nm, with a consistent average concentration of approximately 2∙10¹³ particles/mL, and a similar z-potential of -13 mV. The efficacy of the purification process was evaluated by SEC-HPLC analysis, showing a remarkable impurity removal of up to 98 %, after diafiltration and final concentration. The loading efficiency of curcumin, determined via C18-HPLC analysis, was approximately 34 %. Confocal microscopy images of SK-N-AS cells treated with Cur-EVs confirmed the internalization of curcumin into NB cancer cells. Finally, the effect of Cur-EVs on the viability of NB cells was evaluated. Cur-EVs treatment more efficiently inhibited SK-N-AS proliferation compared to free Cur treatment. This observation was supported by the significantly lower IC₅₀ at 48 hours for Cur-EVs (16.34 μM) compared to the IC₅₀ of free Cur (31.14 μM). Overall, our results highlight the promising potential of membrane-based EV isolation, especially when combined with innovative sources such as dairy whey, in the development of effective cancer therapies that are readily translatable to clinical practice.

References

[1] Piunti C, Cimetta E. Microfluidic approaches for producing lipid-based nanoparticles for drug delivery applications. Biophys Rev (Melville). 2023 Sep 18;4(3):031304. doi: 10.1063/5.0150345. PMID: 38505779; PMCID: PMC10903496

[2] Giancaterino S, Boi C. Alternative biological sources for extracellular vesicles production and purification strategies for process scale-up. Biotechnol Adv. 2023 Mar-Apr; 63:108092. doi: 10.1016/j.biotechadv.2022.108092. Epub 2023 Jan 3. PMID: 36608746.

[3] Wilken, R., Veena, M.S., Wang, M.B. et al. Curcumin: A review of anti-cancer properties and therapeutic activity in head and neck squamous cell carcinoma. Mol Cancer 10, 12 (2011). https://doi.org/10.1186/1476-4598-10-12