Plant-based extracellular vesicles (EVs) are emerging as alternatives to mammalian cells for nanomedicine drug delivery applications. Large-scale processing and characterization for any EV, however, has been challenging.

Researchers Cristiana Boi, PhD, associate professor, University of Bologna, and her PhD student Sara Giancaterino, first author, discussed the options and challenges in a recent paper.

“Plant-based EVs have several advantages,” Boi tells GEN. “They do not require the use of cell cultures and can be isolated directly from plant or fruit extracts, which avoids the costs of upstream processing. They also are widely available and inexpensive sources particularly when compared to EVs isolated from biofluids and human cells.”

Boi and Giancaterino are developing a membrane process to purify EVs from lemons. The goal is to “develop a drug delivery system for encapsulating bioactive molecules useful for human health. We also plan to purify EVs from milk using the same process.” Milk, they point out, is rich in nanolipidic EVs and is inherently biocompatible.

Quality control is a crucial step

In terms of the at-scale processing and characterizing EVs, which are heterogenous, “quality control of EVs is a crucial step, and lack of analytics is the main hurdle to EV processing,” notes Boi. “Co-isolates and different subpopulations of EVs must be correctly identified, and the mode of action of the final product must be fully understood. The most crucial aspects to be addressed in the future are storage and dosing strategies, identifying critical quality attributes, and defining a viral inactivation step.”

Boi and Giancaterino recommend membrane-based processes for EV isolation because they are flexible, gentle, reproducible, and inexpensive. Unlike ultracentrifugation, membrane separation allows high throughput and continuous production, making it suitable for commercial operations. The membrane can be chosen for molecular size and then functionalized to increase product yield and/or obtain specific populations of intact, bioactive EVs. “In this sense, tangential flow filtration is considered the best operative mode, as it allows constant permeate flux with limited membrane fouling,” Boi explains.

EVs derived from plants and other non-traditional sources are still in their development stages so, she points out, “commercialization is slowed by regulatory requirements and lack of knowledge about specific biomarkers.”

Membrane processes and tangential flow filtration, however, already are standard in large-scale manufacturing, Boi continues. “The knowledge can be transferred easily to the field of EV production.”

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