Key Problems in the Translation of Extracellular Vesicles Derived from Eukaryotic Prokaryotic Cells

Haixia Wang¹, Yanhong Gao² & Jin Gao^1*

¹Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, WA 99202, USA
²Shangqiu Municipal First Senior High School, Shangqiu, Henan 476000, RPC

H. Wang and Y. Gao contribute equally to this work

*Correspondence to: Dr. Jin Gao, Department of Pharmaceutical Sciences, College of Pharmacy and Pharmaceutical Sciences, Washington State University, Spokane, WA 99202, USA.

Copyright © 2019 Dr. Jin Gao, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction
According to concept of ISEV (The international Society for Extracellular Vesicles), “extracellular vesicles (EVs) are defined as the particles naturally released from cells that are comprised of a lipid bilayer membrane [1]. Acting as important mediators between cells that regulate both physiological and pathological conditions in the living bodies, EVs are nanosized spherical compartments and contain lipids, proteins and various nucleic acids of their source cells [2,3]. Based on their biogenesis and sizes, EVs are generally categorized into three types, including exosomes, microvesicles and apoptotic bodies. Exosomes are generated from endosomal compartments in the cells. The size of exosomes is around 30 to 100nm in diameter. Microvesicles are directly produced through outward budding and fission of the cell plasma membrane, and their sizes have a wide range between 50-2000nm. When cells are going to die or subject to apoptosis, the cell membrane is disrupted to form apoptotic bodies with the size of 50 to 5000nm [4,5].

Besides EVs derived from eukaryotes, prokaryotes also secrete EVs. It has been reported that Gram-positive and Gram-negative bacteria can both generate EVs [6-8]. Gram-negative bacteria spontaneously release EVs via shedding from outer membrane, so-called outer membrane vesicles (OMVs). Since Gram-positive bacteria do not have “outer membrane” when compared to Gram-negative bacteria, Gram-positive bacteria release OMVs from the inner membrane and the released membrane vesicles go through the cell wall and form so-called OMVs. The size of EVs from Gram-positive bacteria was reported to be ~20-100nm in diameter, which was similar to EVs derived from Gram-negative bacteria [8].

Inspired by the generation of naturally secreted EVs, many researchers are also seeking to prepare biomimetic EVs by physical [9,10], and chemical [11] methods in recent years. In general, the artificial EVs exhibit similar properties to the natural ones. As a supplementary to the natural EVs, biomimetic EVs possess some advantages in some aspects, such as purity, yield, targeting ability, etc.

There is no doubt that the purpose of all above efforts is to translate EVs and utilized their good properties to serve the humankind’s healthcare. Though some application of EVs have been tried in animals, there are only few cases for their clinical use [12-14]. In this review, the most outstanding challenges in each stage of commercialization of EVs will be reviewed and the potential solving direction will also be discussed.

Challenges in Application
Although some clinical trials on the applications of EVs have been reported, there are still some challenges existing in the three necessary stages of commercialization, namely production, quality control and application (Figure 1). Here, three most remarkable problems of each steps will be specified.

Manufacturing Issue-Scalability
First of all, it is necessary and urgent to develop a reproducible and scalable approaches to generate clinic-grade EVs [15]. Some strategies have been applied to improve the generation of natural EV, for example culture conditions, such as hypoxia [16], increased calcium concentrations in medium [17,18] and serum starvation [19] may increase the production of EVs. In addition, the continuous culture system has shown the high yield of EVs by 40 times compared to traditional culture flask settings [20]. To scale up the manufacturing process of EVs, a new method have been developed based on nitrogen cavitation to generate biomimetic nanovesicles, and the result showed that the production of EVs was 16-fold higher than naturally secreted EVs [21]. In addition, Gho et al. [22,23] also utilized extrusion and sonication to achieve the very high production of EVs by over 100 folds compared to natural secreting way. Although it looks like that the selfassembly of phospholipid and membrane proteins can form EVs-like nanoparticles [24] and their scalability is possible, this self-assembly process cannot guarantee the surface orientation of membrane proteins and isolated membrane proteins may not maintain conformations after they leave the cell membrane. To meet the basic requirements of biopharmaceuticals (such as repeatability, efficiency and costs), the bioreactor system [20] may hold the most promising perspectives in manufacture of EVs. Although the generating method is a little bit cost- and labor-consuming, the method can produce EVs efficiently in a continuous way. However, the success of this method is dependent on the physiological conditions, including aging, of cell culture.

Quality Control-Uniformity
Besides the necessary bioactive and targeting ingredients, there are also a lot of components in EVs, including DNA, RNA, proteins, carbohydrates, phospholipids, etc. These components can be affected by a lot of factors, including culture conditions, physiological status (as mentioned above), and manufacturing methods. However, there was rare reports regarding the difference between EVs generated from different methods. We once compared the components of naturally secreted EVs and artificial EVs by nitrogen cavitation, and some differences were found [21]. However, as a biopharmaceutical, or its carrier, the uniformity is critical for translation and an ideal method for this purpose is still lacking. The types and contents of each protein on EVs may be characterized by proteomics way, while types and contents of each phospholipid may be analyzed by phospholipids fatty acid spectrum.

Application Stage-Immunotoxicity
The last concern about the clinical application of EVs is the possible immunotoxicity induced by EVs. In the previous studies, we found the EVs generated from nitrogen cavitation can activate HUVECs, while the function can be mitigated by the loaded anti-inflammatory drug. The following studies also demonstrate that the immunotoxicity may be caused by the exposed phosphatidylserine, originally locating on the inner surface of cell membrane, due to the external forces [21]. To avoid the impairment from immunotoxicity, anti-inflammatory therapy may be necessary and helpful in some cases.

Conclusion
In the past decades, the research on EVs indicates that EVs are transforming the traditional nanoparticlebased drug delivery because EVs have the unique features. EVs have the excellent biocompatibility, long circulation time and low immunogenicity, and most importantly, they maintain their parent cell features that make them excellent drug carriers. Different to the conventional drug carrier, some EVs themselves perform as bioactive drug for disease therapy. Some special tissues, such as brain, can also be targeted by EVs, and no special targeting ligands are required. The above features confer EVs some outstanding advantages to be a drug carrier or disease treating agent.

In summary, this review summarized the current most eminent challenges facing each step of commercialization of EVs. With advances in nanotechnology and immunology and genetic engineering, industrialization of EVs may become more and more realistic and eventually realize, and EVs will eventually turn to be a powerful tool for therapy of many human diseases.

Bibliography

1. Thery, C., et al. (2018). Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles, 7(1), 1535750.
2. Vader, P., Breakefield, X. O. & Wood, M. J. (2014). Extracellular vesicles: emerging targets for cancer therapy. Trends in Molecular Medicine, 20(7), 385-393.
3. Raposo, G. & Stoorvogel, W. (2013). Extracellular vesicles: Exosomes, microvesicles, and friends. J Cell Biol., 200, 373-383.
4. Akers, J. C., Gonda, D., Kim, R., Carter, B. S. & Chen, C. C. (2013). Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neuro-Oncol., 113(1), 1-11.
5. Juan, T. & Furthauer, M. (2018). Biogenesis and function of ESCRT-dependent extracellular vesicles. Semin Cell Dev Biol., 74, 66-77.
6. Brown, L., Wolf, J. M., Prados-Rosales, R. & Casadevall, A. (2015). Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi. Nature Reviews Microbiology, 13(10), 620-630.
7. Ellis, T. N. & Kuehn, M. J. (2010). Virulence and immunomodulatory roles of bacterial outer membrane vesicles. Microbiology and molecular biology reviews: MMBR., 74(1), 81-94.
8. Lee, E. Y., et al. (2009). Gram-positive bacteria produce membrane vesicles: proteomics-based characterization of Staphylococcus aureus-derived membrane vesicles. Proteomics, 9(24), 5425-5436.
9. Wang, S. H., Gao, J., Li, M., Wang, L. G. & Wang, Z. J. (2018). A facile approach for development of a vaccine made of bacterial double-layered membrane vesicles (DMVs). Biomaterials, 187, 28-38.
10. Gao, J., Chu, D. F. & Wang, Z. J. (2016). Cell membrane-formed nanovesicles for disease-targeted delivery. J Control Release, 224, 208-216.
11. Ingato, D., Edson, J. A., Zakharian, M. & Kwon, Y. J. (2018). Cancer Cell-Derived, Drug-Loaded Nanovesicles Induced by Sulfhydryl-Blocking for Effective and Safe Cancer Therapy. ACS nano., 12(9), 9568-9577.
12. Escudier, B., et al. (2005). Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial. J Transl Med., 3(1), 10.
13. Morse, M. A., et al. (2005). A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer. J Transl Med., 3(1), 9.
14. Besse, B., et al. (2016). Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC. Oncoimmunology, 5(4).
15. Jiang, X. C. & Gao, J. Q. (2017). Exosomes as novel bio-carriers for gene and drug delivery. International Journal of Pharmaceutics, 521, 167-175.
16. Kucharzewska, P., et al. (2013). Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. P Natl Acad Sci USA., 110, 7312-7317.
17. Constantinescu, P., et al. (2010). P2X7 receptor activation induces cell death and microparticle release in murine erythroleukemia cells. Bba-Biomembranes, 1798(9), 1797-1804.
18. Qu, Y., et al. (2009). P2X7 Receptor-Stimulated Secretion of MHC Class II-Containing Exosomes Requires the ASC/NLRP3 Inflammasome but Is Independent of Caspase-1. J Immunol., 182(8), 5052-5062.
19. Aharon, A., Tamari, T. & Brenner, B. (2008). Monocyte-derived microparticles and exosomes induce procoagulant and apoptotic effects on endothelial cells. Thromb Haemostasis., 100, 878-885.
20. Watson, D. C., et al. (2016). Efficient production and enhanced tumor delivery of engineered extracellular vesicles. Biomaterials, 105, 195-205.
21. Gao, J., Wang, S. H. & Wang, Z. J. (2017). High yield, scalable and remotely drug-loaded neutrophil-derived extracellular vesicles (EVs) for anti-inflammation therapy. Biomaterials, 135, 62-73.
22. Jang, S. C., et al. (2013). Bioinspired Exosome-Mimetic Nanovesicles for Targeted Delivery of Chemotherapeutics to Malignant Tumors. ACS nano., 7(9), 7698-7710.
23. Go, G., Lee, J., Choi, D. S., Kim, S. S. & Gho, Y. S. (2019). Extracellular Vesicle-Mimetic Ghost Nanovesicles for Delivering Anti-Inflammatory Drugs to Mitigate Gram-Negative Bacterial Outer Membrane Vesicle-Induced Systemic Inflammatory Response Syndrome. Advanced Healthcare Materials, 8, e1801082.
24. Molinaro, R., et al. (2016). Biomimetic proteolipid vesicles for targeting inflamed tissues. Nat Mater., 15(9), 1037-1046.