Cytotoxicity assessment of microalgae-derived extracellular vesicle-enriched populations in Jurkat cells

Amira Batrisyia Shariman; Puteri Hanis Nafisah Megat Kamil; Fatin Syahirah Othman; Shaza Eva Mohamad; Nadiah Abu.

Extracellular vesicles (EVs) are nano-sized particles that are naturally released by cells. Recently, increasing attention has been directed toward EVs derived from natural sources such as plants and fruits. This is due to their sustainability and cost-effectiveness compared to mammalian-derived EVs. Microalgae are photosynthetic unicellular organisms that have also been reported to secrete EVs. In this study, we aimed to isolate EVs from the well-known microalga Chlorella vulgaris and evaluate their cytotoxicity in Jurkat T cells. EVs were isolated using a combination of ultracentrifugation and size-exclusion chromatography (SEC). Characterization was conducted using MicroBCA protein assay, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). Subsequently, the EV-enriched population was co-cultured with Jurkat T cells, and cell viability and apoptosis were assessed. Our results demonstrated that the EV-enriched population had a mean size below 200 nm. Moreover, the EVs did not cause any significant reduction in cell viability or induce apoptosis. Overall, our preliminary study has shown that microalgae-derived EVs are non-cytotoxic and are biocompatible in a mammalian system.

KEYWORDS: Microalgae; Nanocarriers; Jurkat; Extracellular Vesicles; Chlorella vulgaris



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REFERENCES

1. Adamo, G., Fierli, D., Romancino, D. P., Picciotto, S., Barone, M. E., Aranyos, A., Božič, D., Morsbach, S., Raccosta, S., Stanly, C., Paganini, C., Gai, M., Cusimano, A., Martorana, V., Noto, R., Carrotta, R., Librizzi, F., Randazzo, L., Parkes, R., Capasso Palmiero, U., Rao, E., Paterna, A., Santonicola, P., Iglič, A., Corcuera, L., Kisslinger, A., Schiavi, E. D., Liguori, G. L., Landfester, K., Kralj-Iglič, V., Arosio, P., Pocsfalvi, G., Touzet, N., Manno, M. & Bongiovanni, A. 2021. Nanoalgosomes: Introducing extracellular vesicles produced by microalgae. Journal of Extracellular Vesicles, 10(12), e12081.
2. Adamo, G., Santonicola, P., Picciotto, S., Gargano, P., Nicosia, A., Longo, V., Aloi, N., Romancino, D. P., Paterna, A., Rao, E., Raccosta, S., Noto, R., Salamone, M., Deidda, I., Costa, S., Di Sano, C., Zampi, G., Morsbach, S., Landfester, K., Colombo, P., Wei, M., Bergese, P., Touzet, N., Manno, M., Di Schiavi, E. & Bongiovanni, A. 2024. Extracellular vesicles from the microalga Tetraselmis chuii are biocompatible and exhibit unique bone tropism along with antioxidant and anti-inflammatory properties. Communications Biology, 7, 941.
3. Anderson, W., Kozak, D., Coleman, V. A., Jämting, Å. K. & Trau, M. 2013. A comparative study of submicron particle sizing platforms: Accuracy, precision and resolution analysis of polydisperse particle size distributions. Journal of Colloid and Interface Science, 405, 322–330.
4. Cao, Y., Hou, L., Li, M., Zhang, J., Wang, L., Liu, C., Luo, T., Yan, L. & Zheng, L. 2024. Broccoli extracellular vesicles enhance the therapeutic effects and restore the chemosensitivity of 5-fluorouracil on colon cancer. Food and Chemical Toxicology, 186, 114563.
5. Du, S., Guan, Y., Xie, A., Yan, Z., Gao, S., Li, W., Rao, L., Chen, X. & Chen, T. 2023. Extracellular vesicles: a rising star for therapeutics and drug delivery. Journal of Nanobiotechnology, 21(1), 231.
6. Garaeva, L., Tolstyko, E., Putevich, E., Kil, Y., Spitsyna, A., Emelianova, S., Solianik, A., Yastremsky, E., Garmay, Y., Komarova, E., Varfolomeeva, E., Ershov, A., Sizova, I., Pichkur, E., Vinnikov, I. A., Kvanchiani, V., Kilasoniya Marfina, A., Konevega, A. L. & Shtam, T. 2025. Microalgae-Derived Vesicles: Natural Nanocarriers of Exogenous and Endogenous Proteins. Plants, 14(17), 2354.
7. Maas, S. L. N., de Vrij, J., van der Vlist, E. J., Geragousian, B., van Bloois, L., Mastrobattista, E., Schiffelers, R. M., Wauben, M. H. M., Broekman, M. L. D. & Nolte-'t Hoen, E. N. M. 2015. Possibilities and limitations of current technologies for quantification of biological extracellular vesicles and synthetic mimics. Journal of Controlled Release, 200, 87–96.
8. Mendes, A. R., Spínola, M. P., Lordelo, M. & Prates, J. A. M. 2024. Chemical Compounds, Bioactivities, and Applications of Chlorella vulgaris in Food, Feed and Medicine. Applied Sciences, 14(22), 10810.
9. Nemati, M., Singh, B., Mir, R. A., Nemati, M., Babaei, A., Ahmadi, M., Rasmi, Y., Golezani, A. G. & Rezaie, J. 2022. Plant-derived extracellular vesicles: a novel nanomedicine approach with advantages and challenges. Cell Communication and Signaling, 20(1), 69.
10. Panahi, Y., Darvishi, B., Jowzi, N., Beiraghdar, F. & Sahebkar, A. 2016. Chlorella vulgaris: A Multifunctional Dietary Supplement with Diverse Medicinal Properties. Current Pharmaceutical Design, 22(2), 164–173.
11. Picciotto, S., Barone, M. E., Fierli, D., Aranyos, A., Adamo, G., Božič, D., Romancino, D. P., Stanly, C., Parkes, R., Morsbach, S., Raccosta, S., Paganini, C., Cusimano, A., Martorana, V., Noto, R., Carrotta, R., Librizzi, F., Capasso Palmiero, U., Santonicola, P., Iglič, A., Gai, M., Corcuera, L., Kisslinger, A., Di Schiavi, E., Landfester, K., Liguori, G. L., Kralj-Iglič, V., Arosio, P., Pocsfalvi, G., Manno, M., Touzet, N. & Bongiovanni, A. 2021. Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds. Biomaterials Science, 9(9), 2917–2930.
12. Picciotto, S., Santonicola, P., Paterna, A., Rao, E., Raccosta, S., Romancino, D. P., Noto, R., Touzet, N., Manno, M., Di Schiavi, E., Bongiovanni, A. & Adamo, G. 2022. Extracellular Vesicles From Microalgae: Uptake Studies in Human Cells and Caenorhabditis elegans. Frontiers in Bioengineering and Biotechnology, 10, 830189.
13. Savvidou, M. G., Georgiopoulou, I., Antoniou, N., Tzima, S., Kontou, M., Louli, V., Fatouros, C., Magoulas, K. & Kolisis, F. N. 2023. Extracts from Chlorella vulgaris Protect Mesenchymal Stromal Cells from Oxidative Stress Induced by Hydrogen Peroxide. Plants (Basel), 12(16), 2911.
14. Welsh, J. A., Goberdhan, D. C. I., O'Driscoll, L., Buzas, E. I., Blenkiron, C., Bussolati, B., Cai, H., Di Vizio, D., Driedonks, T. A. P., Erdbrügger, U., Falcon-Perez, J. M., Fu, Q. L., Hill, A. F., Lenassi, M., Lim, S. K., Mahoney, M. G., Mohanty, S., Möller, A., Nieuwland, R., Ochiya, T., Sahoo, S., Torrecilhas, A. C., Zheng, L., Zijlstra, A., Abuelreich, S., Bagabas, R., Bergese, P., Bridges, E. M., Brucale, M., Burger, D., Carney, R. P., Cocucci, E., Crescitelli, R., Hanser, E., Harris, A. L., Haughey, N. J., Hendrix, A., Ivanov, A. R., Jovanovic-Talisman, T., Kruh-Garcia, N. A., Ku'ulei-Lyn Faustino, V., Kyburz, D., Lässer, C., Lennon, K. M., Lötvall, J., Maddox, A. L., Martens-Uzunova, E. S., Mizenko, R. R., Newman, L. A., Ridolfi, A., Rohde, E., Rojalin, T., Rowland, A., Saftics, A., Sandau, U. S., Saugstad, J. A., Shekari, F., Swift, S., Ter-Ovanesyan, D., Tosar, J. P., Useckaite, Z., Valle, F., Varga, Z., van der Pol, E., van Herwijnen, M. J. C., Wauben, M. H. M., Wehman, A. M., Williams, S., Zendrini, A., Zimmerman, A. J., Théry, C. & Witwer, K. 2024. Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches. Journal of Extracellular Vesicles, 13(2), e12404.