Opportunities and challenges of bacterial extracellular vesicles in regenerative medicine

Abstract

Extracellular vesicles (EVs) are membrane-bound vesicles that are shed or secreted from the cell membrane and enveloped by a lipid bilayer. They possess stability, low immunogenicity, and non-cytotoxicity, exhibiting extensive prospects in regenerative medicine (RM). However, natural EVs pose challenges, such as insufficient targeting capabilities, potential biosafety concerns, and limited acquisition pathways. Although engineered EVs demonstrate excellent therapeutic efficacy, challenges such as low production yield and the complexity of engineering modifications constrain their further clinical applications. Bacteria have advantages such as rapid proliferation, diverse gene editing methods, mature cultivation techniques, and relatively easy preparation of bacterial EVs (BEVs), which can be used to effectively address the challenges currently encountered in the field of EVs. This review provides a description of the biogenesis and pathophysiological functions of BEVs, and strategies for optimizing BEVs preparation to attain efficiency and safety are discussed. An analysis of natural characteristics of BEVs is also conducted to explore how to leverage their advantages or mitigate their limitations, thereby overcoming constraints on the application of BEVs in RM. In summary, engineered BEVs possess characteristics such as high production yield, excellent stability, and high drug-delivering capabilities, laying the foundation for their application in RM.

Keywords: Bacterial extracellular vesicles; Drug delivery; Engineering strategy; Outer membrane vesicles; Regenerative medicine.

Fig. 1

Biogenesis of Bacterial Extracellular Vesicles (BEVs). Gram negative bacteria exhibit two distinct pathways for BEVs biogenesis: a Explosive cell lysis corresponds to the generation of Outer-Inner Membrane Vesicles (OIMVs) and Explosive Outer Membrane Vesicles (EOMVs); b Outer membrane blebbing corresponds to the generation of Outer Membrane Vesicles (OMVs). Gram positive bacteria exhibit one pathway for BEVs biogenesis: c Bubbling cell lysis corresponds to the generation of Cytoplasmic Membrane Vesicles (CMVs)

Fig. 2

The secretion of Bacterial Extracellular Vesicles (BEVs) from B. thetaiotaomicron is divided into three stages: in the first stage, cell size increases, BEVs are formed non-lytically, and are accompanied by the release of formate. In the second stage, cell size decreases, BEV production decreases and is accompanied by the release of lactate. In the final stage, the number of cells decreases, BEVs are mainly formed lytically, and are accompanied by the release of DNA [76]. Reproduced with permission from Wiley Publications, copyright 2024

Fig. 3

The preparation of Bacterial Extracellular Vesicles (BEVs). a Cultivating bacteria under optimal conditions. b Clarifying bacterial culture medium by low-speed centrifugation and retain supernatant. c Filtration of the supernatant to preliminary remove bacteria and other impurities. d Select an appropriately sized tangential flow filtration (TFF) device to concentrate BEVs. e Size-exclusion chromatography (SEC) is used to increase the purity of EVs and remove non-vesicular protein. f Protein detection methods such as WB and ELISA are used to detect the expression of specific proteins in BEVs. g Transmission electron microscopy (TEM) is utilized for examining the morphological characteristics of BEVs. h Finally, BEVs are stored under appropriate conditions. (Created with biorender.com)

Fig. 4

Advantages and main challenges of bacterial extracellular vesicles (created with biorender.com)

Fig. 5

The engineered Escherichia coli Nissle 1917-pET28a-ClyA-BMP-2-CXCR4, generated through genetic recombination techniques, is capable of displaying BMP-2 and CXCR4 on the surface of its produced Bacterial Extracellular Vesicles (BEVs), exhibiting excellent bone-targeting and osteogenic properties [137]. Reproduced with permission from Wiley Publications, copyright 2024

Fig. 6

A method for enhancing the yield and purity of bacterial extracellular vesicles (BEVs) through co-cultivation of E. coli with magnetic iron oxide nanoparticles (MNPs) and utilizing magnetic force for harvesting [134]. Reproduced with permission from Wiley Publications, copyright 2022

Fig. 7

Clinical prospects of engineered Bacterial Extracellular Vesicles (BEVs) in regenerative medicine. a BEVs possess the ability to traverse the blood–brain barrier, serving as effective carriers to transport drugs. b BEVs exhibit anti-inflammatory properties and can ameliorate the local inflammatory microenvironment. c BEVs have the capability to directly promote tissue regeneration. d BEVs possess antibacterial properties, capable of inhibiting bacterial adhesion and proliferation during tissue regeneration processes. (created with biorender.com)

Fig. 8

By employing genetic editing techniques, bacterial extracellular vesicles were obtained with targeting functionality towards A. baumannii, and further combination with metal–organic frameworks materials exhibited excellent in vivo targeting and bactericidal properties [139]. Copyright © 2024, The American Association for the Advancement of Science