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Abstract

Extracellular vesicles (EVs) have emerged as a promising strategy for treating a wide spectrum of pathologies, as they can deliver their cargo to recipient cells and regulate the signaling pathway of these cells to modulate their fate. Despite the great potential of EVs in clinical applications, their low yield and the challenges of cargo loading remain significant obstacles, hindering their transition from experimental research to clinical practice. Therefore, promoting EV release and enhancing EV cargo-loading are promising fields with substantial research potential and broad application prospects. In this review, we summarize the clinical applications of EVs, the methods and technologies for their large-scale production, engineering, and modification, as well as the challenges that must be addressed during their development. We also discuss the future perspectives of this exciting field of research to facilitate its transformation from bench to clinical reality.

Keywords

Clinical application large-scale extracellular vesicles

Introduction

Advances in nanotechnology have significantly impacted the biomedical field.¹ Over the past four decades, various nanomaterial-based drug delivery systems have been developed.² Traditional nanoparticles, such as carbon nanotubes, graphene oxide (GO), noble metal nanoparticles (NMNPs), and mesoporous silicon nanoparticles (MSNs), are characterized by controllable particle size, diverse morphologies, and ease of large-scale production. However, these traditional nanoparticles suffer from poor biocompatibility and high immunogenicity.¹ Extracellular vesicles (EVs) have emerged as a promising focus in nanotherapy, potentially addressing the limitations of traditional nanotechnology. EVs are nanoscale lipid bilayer membrane capsules secreted by cells, which can be taken up by recipient cells.³ They play critical roles in cell communication, including signaling and the delivery of biological cargos.⁴ EVs also exhibit immune tolerance, circulatory system stability, and the ability to traverse biological barriers, such as the blood-brain barrier, blood-CSF barrier, and blood-tumor barriers.^5
–10 Furthermore, EVs derived from different donor cells inherit unique characteristics and functions from their source cells, making them ideal vectors for diagnosing and treating various diseases.^11
–13 Consequently, interest in EVs has grown rapidly, as evidenced by the exponential increase in PubMed publications on EVs every year (Figure 1).

Figure 1.

Number of PubMed entries using “EV” OR “exosome” OR “extracellular vesicles” as search terms. Search performed December 31, 2024.

EVs contain a variety of bioactive components, including proteins,¹⁴ lipids,¹⁵ and nucleic acids,¹⁶ which exert biological functions by delivering specific biomolecules.¹⁷ To date, EV-based therapies and vaccines have emerged.^18,19 For instance, in July 2023, the first human clinical trial of platelet EVs for wound healing treatment was conducted.²⁰ EVs were decorated with a recombinant SARS-CoV-2 receptor-binding domain as an inhalable COVID-19 vaccine. Plasma EVs expressing ACE2 increased in patients with COVID-19, and EVs carrying ACE2 on their surfaces can inhibit SARS-CoV-2 infection.²¹ The design of artificially engineered small EVs displaying ACE2 variants on their surfaces can protect against SARS-CoV-2 infection.²² Additionally, EVs can encapsulate nucleic acids, proteins, lipids, and chemical drugs, showing positive effects in various aspects of cancer and metabolic diseases.^23
–27 This indicates that the active molecules loaded into EVs play a crucial role in their clinical application, and loading specific cargo into EVs or onto their surfaces is key to achieving their therapeutic effect. It has been reported that this process of loading specific cargo into EVs or modifying their surfaces is also called EV modification or engineering, enabling the delivery of specific molecules to recipient cells.²⁸ The methods of producing engineered EVs mainly include cell modification to produce specially modified EVs and specific modification after EVs isolation. Although EVs have diverse biological functions, they still face challenges, such as low production, which is one of the key issues limiting their clinical application and a topic we will discuss in this review.

As per previous research, achieving biological outcomes typically requires a dose of 10⁶–10¹¹ EVs per treatment in mice, which may necessitate liters of conditioned media and several months of collection.^5,29
–31 In clinical experiments, the inhaled dose of EVs for mice is 10⁵ particles/g, and for a mouse weighing 20–40 g, it requires approximately 2–4 × 10⁶ particles per mouse.³¹ For the regulatory effect of injecting EVs through the tail vein to treat liver disease, the injection amount was 10⁹ particles per mouse.³² Furthermore, mice were treated by intraperitoneal injection of EVs loaded with siRNA at a dose of 10⁹ particles per time, three times a week.³³ Studies have shown that the optimal therapeutic dose of EV aerosol inhalation is 10⁵ particles/g in a mouse lung injury model. Further clinical trials have been conducted in healthy human volunteers using doses ranging from 2 to 16 × 10⁸.³¹ It is evident that different clinical treatments require a large amount of EVs, and low yield is a major drawback of EV production at present. Therefore, increasing EV yield is essential. Recently, many methods for promoting EVs secretion and loading foreign cargo into EVs have been reported, such as the development of large-scale cell culture platforms,³⁴ cell stimulation to increase EV yield per cell,³⁵ and the production of EV-like nanovesicles.³⁶ These large-scale production methods bring hope for the clinical application of EVs.

In this review, we summarize the clinical application prospects of EVs, methods to enhance EV production, and the advancements in engineering EVs to create functional EVs. Additionally, we discuss the challenges of transitioning EVs from the laboratory to clinical application. This review offers new perspectives and insights into EVs-based nanotherapy and diagnosis.

Clinical applications of EVs

The understanding of the role of EVs in disease biology is still evolving, with a substantial increase in studies exploring their potential utility in diagnosing and treating various pathologies.³⁷ EVs carry distinct cargoes under different pathological conditions, enabling disease detection and monitoring through changes in specific cargoes. The unique properties of EVs in delivering functional cargo to cells also make them promising therapeutic vehicles for disease treatment and prevention. EVs are transitioning from academic research to biotechnology and advancing toward clinical applications. In this section, we will discuss the versatility of EVs in translational medicine, including their applications in disease treatment, prevention, and diagnosis (Figure 2).

Figure 2.

Application of EVs in disease diagnosis, prevention, and treatment: (a) natural or engineered EVs, administered orally, via injection, inhalation, or topical application, can impact multiple organs, including the liver, intestines, skin, etc., (b) injection or inhalation of EVs carrying viral antigens can induce an immune response in animals, and (c) EVs isolated from body fluids can detect changes in nucleic acid, proteins, and other components and can be utilized for liquid biopsy in diseases.

Therapeutic potential of EVs

EVs exhibit significant potential as drug delivery vehicles due to several key features that enhance their effectiveness. Firstly, their excellent biocompatibility, combined with their inherent ability to transport materials, ensures they can effectively carry therapeutic agents. Additionally, EVs possess low immunogenicity, minimizing adverse immune responses and making them safer for clinical applications. Their intrinsic long-term circulatory capability allows for sustained drug release, ensuring therapeutic effects are maintained over time. Moreover, the good biomembrane penetration capacity of EVs facilitates the delivery of drugs directly to target cells, further enhancing therapeutic efficacy. Collectively, these attributes position EVs as promising candidates in the field of drug delivery systems.^38
–40 They are rich in bioactive substances and have demonstrated therapeutic potential in various applications.⁴¹ Numerous studies have highlighted the significant roles of EVs in treating various diseases, including cancer,⁴² nerve diseases,⁴³ tissue injuries,⁴⁴ and pathogenic infections.⁴⁵ Several EV studies have entered clinical trials, such as EVs in fibrin gel for cartilage repair (NCT06713902), nasal drop therapy with EVs for amyotrophic lateral sclerosis (NCT06598202), and the safety and efficacy of stem cell EVs in patients with retinitis pigmentosa (NCT06242379). Specifically, a large body of research has shown that cell-derived EVs,^46
–48 EVs present in body fluids,^49,50 bacterial outer membrane vesicles (OMVs),^51
–53 and plant-derived EV-like nanoparticles (PELNs)^54
–56 all hold significant potential in disease treatment. Through these advancements, EVs are emerging as a revolutionary approach to drug delivery, warranting further exploration and development in therapeutic applications.

For instance, EVs derived from stem cells can promote cell regeneration or repair^57,58 and serve as substitutes for stem cells to avoid immune rejection and carcinogenic risks associated with stem cell therapy, offering significant application value in regenerative medicine. Mesenchymal stem cells (MSCs) are known for their excellent properties in cell therapy. However, the risk of immune rejection linked to cell transplant therapy limits their use.⁵⁹ MSC derived EVs function as effective therapeutic vehicles and appear to replicate the broad therapeutic effects observed in MSCs themselves,^60,61 giving rise to the novel concept of “cell-free” stem cell therapy.⁶² Additionally, MSC-derived EVs have shown potential therapeutic application in SARS-CoV-2 pneumonia,^63,64 while M2 macrophage-derived EVs have been found to promote diabetic fracture healing by acting as immunomodulators.⁶⁵ In a study on osteoarthritis, EVs from human bone marrow MSCs (hBM-MSCs) were shown to promote extracorporeal cartilage regeneration by stimulating chondrocytes to produce proteoglycans and type II collagen.⁶⁶ Similarly, EVs from human umbilical cord MSC (huc-MSCs) ameliorate bone loss in senile osteoporotic mice.⁶⁷ Furthermore, adipose-derived mesenchymal stem cells (ADSCs)-derived EVs have demonstrated the potential to alleviate neuronal damage, promote neurogenesis, and rescue memory loss in mice with Alzheimer’s disease.⁶⁸ In addition to stem cells, various immune cells-derived EVs have been studied and characterized for their anti-tumor or tumor-promoting functions.⁶⁹ Studies have shown that immune cell-derived EVs possess immune activity.⁷⁰ EVs derived from CD4⁺T cells increase the antitumor response of CD8⁺T cells by enhancing their proliferation and activity without affecting regulatory T cells (Tregs). Moreover, EVs derived from interleukin-2 (IL2)-stimulated CD4⁺T cells induce a stronger antitumor response of CD8⁺T cells compared with that of IL2-unstimulated CD4⁺T cell-derived EVs.⁷¹ Natural killer (NK) cell-derived EVs exert cytotoxic effects on melanoma cells and can inhibit tumor cells by delivering tumor suppressor microRNA (miR-186).^72,73 Furthermore, NK cell-derived EVs carrying miR-1249-3p reduce insulin resistance and inflammation in mouse models of type 2 diabetes.⁷⁴

Vesicles produced by gram-negative bacteria are referred to as OMVs.^75
–78 OMVs, with a structure similar to cell-derived EVs, are natural functional nanomaterials.⁵¹ Additionally, various bacterial-derived OMVs have been reported to exhibit anti-cancer and inflammatory regulatory effects.^79
–82 Research has demonstrated that OMVs possess a significant ability to effectively induce antitumor immune responses and completely eradicate established tumors without notable adverse effects. This antitumor response of OMVs is durable, with secondary and tertiary tumor re-challenges being fully rejected by mice cured from the primary challenge. Furthermore, systematically administered OMVs target and accumulate in tumor tissue, subsequently induce the production of antitumor cytokines CXCL10 and interferon (IFN)-γ.⁸³ Similar studies have demonstrated that E. coli OMVs lead to complete remission in two mouse syngeneic tumor models. Mechanistic investigations revealed that E. coli OMV treatment increases the infiltration and activation of CD8⁺T cells, particularly cancer antigen-specific CD8⁺T cells with high expression of TCF-1 and PD-1.⁸⁴ Additionally, Lactobacillus amylovorus derived OMVs significantly alleviate aflatoxin B1-induced intestinal injury by inhibiting the production of inflammatory factors, reducing intestinal permeability, and increasing the expression of tight junction proteins.⁸⁵ Therefore, OMVs hold great potential for application prospects in disease treatment, particularly in the field of tumor therapy.

Studies on vesicles from natural sources, including edible plants, are gaining momentum due to their biological implications.⁸⁶ Plant-derived vesicles, referred to as EV-like nanoparticles (ELNs),⁸⁷ have emerged as a novel and promising area of research in nanomedicine. These vesicles are structurally and functionally similar to exosomes found in mammalian systems but offer unique advantages, such as easy availability and high yield due to their plant origins.^88,89 In recent years, EVs have been isolated from various plant species and their different parts (e.g. fruits, roots, seeds, leaves) using standardized isolation and purification methods, such as ultracentrifugation.^56,90
–92 ELNs can be engineered to encapsulate various therapeutic agents, including anti-cancer drugs, antibiotics, and RNA molecules. Their inherent stability and biocompatibility make them suitable carriers for targeted drug delivery, potentially reducing side effects and enhancing therapeutic efficacy.^93,94 For instance, ELNs derived from mulberry bark could prevent DSS-induced colitis through the AhR/COPS8 pathway.⁹⁵ Intravenous injection of Momordica charantia L.-derived ELNs has been shown to mitigate doxorubicin cardiotoxicity, improving cardiac function and myocardial structure.⁵⁶ Additionally, modifying cRGD-targeted doxorubicin (DOX) nanoparticles (DN) onto the surface of orange-derived ELNs significantly enhances tumor accumulation and penetration, efficiently inhibiting ovarian cancer growth.⁹⁶

EVs are present in almost all body fluids, including blood, milk, urine, and others.^97
–101 Among these, milk EVs, in addition to the advantages of traditional EVs such as low immunogenicity and high biocompatibility, also exhibit high yield and partial resistance to digestion.^102
–105 Based on these characteristics, we believe milk EVs are highly suitable for disease treatment. Studies have shown that the abundant proteins and miRNAs in milk EVs play a role in regulating immune and inflammatory pathways. Oral administration of milk EVs has been shown to prevent colon shortening, reduce intestinal epithelium disruption, and inhibit inflammatory cell infiltration and tissue fibrosis in a mouse model of ulcerative colitis. Mechanistically, milk EVs attenuate inflammatory responses by inhibiting the TLR4-NF-κB signaling pathway and NLRP3 inflammasome activation.^106
–108 Additionally, milk EVs miR-148a protects against necrotizing enterocolitis by regulating p53 and sirtuin 1 .^109,110 Beyond natural milk EVs, engineered milk EVs have also demonstrated significant therapeutic potential. For instance, miR-31-5p mimics were successfully encapsulated into milk EVs via electroporation, enhancing cellular uptake of miR-31-5p and providing resistance to degradation. These engineered EVs carrying miR-31-5p promoted angiogenesis and accelerated diabetic wound healing.¹¹¹ Similarly, studies have shown that encapsulating TNF-α siRNA into milk EVs via electroporation and orally administering these engineered EVs reduced colonic TNF-α expression and intestinal inflammation in mice.¹¹² Additionally, previous studies from our research group have demonstrated the potential therapeutic effects of oral milk EVs, including altering the miRNA expression profile in the serum of piglets,¹¹³ promoting the level of intestinal secreted immunoglobulin A (sIgA) in mice, increasing the expression of intestinal polymeric immunoglobulin receptor (pIgR) in IPEC-J2 cells to regulate intestinal immune function,¹¹⁴ and alleviating DON- and LPS-induced intestinal injury in piglets through oral administration of milk EVs in vivo.^115,116 Furthermore, miRNA-loaded milk EVs have shown antiviral effects in vivo and in intestinal organoids,⁴⁵ while pituitary EVs have demonstrated potential in alleviating liver damage caused by carbon tetrachloride through a novel endocrine regulatory system.^117,118 These studies highlight the versatility of EVs, with EVs from various sources exhibiting therapeutic effects on different diseases, underscoring their significant potential for clinical application.

In addition to natural EVs, specifically modified EVs, such as those with protein modifications, nucleic acid alterations,^111,119 and chemical drug loading,¹²⁰ have also demonstrated therapeutic potential. For example, inhalable cell-derived EVs used as paclitaxel carriers for lung cancer treatment have shown enhanced targeted antitumor effects while reducing adverse effects on the liver, kidney, and intestine.¹²¹ Furthermore, injecting EVs loaded with siRNA has demonstrated the ability to cross the blood-brain barrier and knock down specific mRNA in the brains of mice, with increased knockdown efficiency.^122,123 Topical application of EVs containing sodium alginate precursors or miR-21 has been found to accelerate skin wound healing.^124,125 Additionally, the immune response and targeting can be modulated by designing genetically engineered EVs with surface-displayed antibody targeting groups and immunomodulatory proteins.^126,127

EVs are actively being explored as therapeutic agents, with both natural and engineered EVs demonstrating therapeutic potential across various diseases. Furthermore, due to their low immunogenicity and ability to cross barriers,^8,128 EVs hold significant promise for development and application in the field of disease therapy. However, the large-scale production of EVs remains a critical challenge for their clinical use, which will be discussed later.

Vaccines

Nanoparticle vaccine formulations have emerged as an innovative antigen delivery system capable of activating both local and systemic cell-mediated and antibody responses, while ensuring the gradual release of immunogens.¹²⁹ EVs represent a new and essential source of immune response antigens and molecules that can be utilized for animal vaccination.^57,130,131 EVs can be engineered to display viral antigens, inducing strong and specific CD8(+) T cell and B cell reactions.¹³² For instance, EVs were decorated with a recombinant SARS-CoV-2 receptor-binding domain as an inhalable COVID-19 vaccine, which, after lyophilization, can be stably stored at room temperature for over 3 months.¹⁸ EVs loaded with mRNAs encoding immunogenic forms of the SARS-CoV-2 Spike and Nucleocapsid proteins elicited long-lasting cellular and humoral responses to both.¹³³ Furthermore, EVs were genetically engineered to express either the SARS-CoV-2 delta spike or the more conserved nucleocapsid protein on their surface. In two different animal models, including rabbits and mice, these engineered EVs were injected to produce potent antibodies and a robust T-cell response with nanogram quantities of proteins without any adjuvant.¹³⁴ These studies indicate that EVs modified through the engineering of viral domains can effectively generate immune responses, offering a promising new direction for the development of novel genetically engineered vaccines.

EVs derived from cancer cells are particularly appealing for cancer immunotherapy as they can present tumor-specific antigens to the immune system, potentially triggering robust anti-tumor immune responses.^135
–137 They hold promise as a cancer vaccine. Several studies have emphasized the potential of EVs as cancer vaccines in preclinical models and early-phase clinical trials. EVs loaded with tumor antigens have been demonstrated to elicit strong anti-tumor immunity in mouse models. For instance, EVs derived from melanoma cells carrying antigens successfully activated CTLs and inhibited tumor growth.¹³⁸ A phase I clinical trial using dendritic cell-derived EVs loaded with tumor antigens in patients with advanced melanoma demonstrated safety and feasibility, with some patients showing immune responses.¹³⁹ These findings highlight the significant potential of EVs for development and application, whether as a viral vaccine or a cancer vaccine.

Diagnostic potential of EVs

Currently, circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and EVs represent the three main branches of liquid biopsy. CtDNA and CTCs have been approved for clinical use by the U.S. Food and Drug Administration (FDA).¹⁴⁰ Compared to ctDNA and CTCs, EVs exhibit greater advantages and unique characteristics in liquid biopsy. First, the abundance of EVs in biofluids makes them relatively easy to obtain. Second, EVs are secreted by living cells and carry a variety of biological information from their parental cells. Third, EVs are inherently stable due to their lipid bilayer structure, allowing them to circulate under physiological conditions, even within the harsh tumor microenvironment. This high biological stability also enables the long-term storage of specimens for EV isolation and detection.¹⁴¹ We conducted a PubMed search using the keywords “Extracellular Vesicle” OR “exosome” and “Plasma” OR “Serum” OR “Urinary” OR “Milk” OR “Sputum” OR “Semen.” Several representative studies on the role of EVs in disease diagnosis were identified and are listed (Table 1).

Table 1.

Research on EVs in disease diagnosis.

Source of EVs	Potential marker	Marker type	Disease type
Plasma	miR-483-3p, let-7d-3p	miRNA	Sepsis¹⁴²
	miR-185-5p	miRNA	Colorectal cancer¹⁴³
	miR-1246, miR-21	miRNA	Breast cancer¹⁴⁴
	tRNA-ValTAC-3, tRNA-GlyTCC-5, tRNA-ValAAC-5, tRNA-GluCTC-5	tsRNA	Liver cancer¹⁴⁵
	BACE1-AS	lncRNA	Alzheimer’s disease¹⁴⁶
	circ_0079439	circRNA	Gastric cancer¹⁴⁷
	CD151	Protein	Lung cancer¹⁴⁸
	NY-ESO-1	Protein	Non-small cell lung cancer¹⁴⁹
Serum	CD8, CD2, CD62P, CD42a, CD44, CD326, CD142, CD31, and CD14	Protein	Transient ischemic attacks¹⁵⁰
	Glypican-1	Protein	Early pancreatic cancer¹⁵¹
	AHSG, ECM1	Protein	Non-small cell lung cancer¹⁵²
	miR-99b-5p, miR-150-5p	miRNA	Colorectal cancer¹⁵³
	TBILA, AGAP2-AS1	lncRNA	Non-small cell lung cancer¹⁵⁴
	piR-15254, piR-1029, novel-piR-32132, novel-piR-43597, novel-piR-35395	piRNA	Hepatocellular carcinoma¹⁵⁵
Urinary	Phosphatidylserine, lactosylceramide	lipid	Prostate cancer¹⁵⁶
	TM256, LAMTOR	Protein	Prostate cancer¹⁵⁷
	miR-375, miR-146a	miRNA	Bladder cancer¹⁵⁸
Milk	miR-378, miR-185, miR-142-5p, miR-223	miRNA	Mastitis^159,160
Sputum	miR-142-3p	miRNA	Idiopathic pulmonary fibrosis¹⁶¹
Semen	PSA, miR-142-3p, miR-142-5p, miR-223-3p	Protein and miRNA	Prostate cancer¹⁶²

One potential clinical application of EVs involves their use as diagnostic and prognostic biomarkers.^163,164 EVs can carry biomolecules from their original cells, which may contain indicators of pathophysiological conditions, and can be detected in various body fluids such as urine,¹⁶⁵ blood,¹⁶⁶ saliva,¹⁶⁷ and cerebrospinal fluid,¹⁶⁸ serving as a liquid biopsy.¹⁶⁹ They have demonstrated effectiveness in diagnosing and treating cancer,^170,171 cardiovascular disease,¹⁷² tuberculosis,¹⁷³ and central nervous system diseases.¹⁷⁴ EVs are structurally stable and rich in content, containing numerous specific cytokines, functional mRNA, growth factors, lipids, and other substances involved in intercellular material transport and information exchange. They are closely associated with the onset and progression of various diseases. Changes in EV composition, including specific proteins, nucleic acids, signaling molecules, and lipids, may reflect the state of parental cells.^175
–177 Markers detected using EVs mainly include changes in their protein and nucleic acid molecules. Research indicates that circulating EV mRNA signatures can assist in the early diagnosis of clear cell renal cell carcinoma and the detection of prostate cancer.^178,179 EV-encapsulated miRNA can serve as a biomarker for breast cancer diagnosis.^144,180 The serum GPC1 content of EVs in early pancreatic cancer patients was significantly higher than in healthy individuals. GPC1(+) circulating EVs were identified in the serum of pancreatic cancer patients with absolute specificity and sensitivity, distinguishing healthy individuals and patients with benign pancreatic disease from those with early- and late-stage pancreatic cancer.¹⁵¹

In recent years, numerous companies have invested in research related to EV diagnosis, including EV Diagnostics, EV Plus, and Exosomics. In May 2023, EV Diagnostics, a molecular diagnostics company, announced interim results from a prospective randomized study involving over 1000 patients to evaluate the clinical utility of its ExoDx™ prostate test over a 5-year period.¹⁸¹ The emergence of innovative technologies is advancing the application of EVs in clinical diagnosis, such as flow cytometry, confocal and non-confocal (fluorescence) microscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, surface plasmon resonance (SPR), mass spectrometry (MS), and micro nuclear magnetic resonance (μNMR).¹⁸² As a novel in vitro diagnostic technology, EVs hold significant potential and value for research and development in the screening and diagnosis of tumors and other chronic diseases.

Large-scale production of EVs

While EVs have shown therapeutic potential in various diseases,^183
–185 their natural production is limited, and generating a substantial quantity of EVs within a short timeframe remains a challenge.¹⁸⁶ This limitation results in high production costs, which currently impede the widespread production and application of EVs. Consequently, it is crucial to explore new methods to enhance EV production and reduce associated costs. Recent studies have proposed various strategies to improve EV production, including the development of large-scale cell culture platforms,³⁴ increasing the quantity of EVs secreted by individual cells through conditional stimulation or genetic modification,^35,187 and producing EVs with nanovesicle-like structures by promoting cell disruption.^188,189 We have summarized these methods for the large-scale production of EVs and EV mimetics (Figure 3).

Figure 3.

Mass production of EVs and EV mimetics: (a) expanding cell culture to collect more EVs, (b) adjusting pH, calcium ion concentration, glucose content, oxygen levels, and other culture conditions, as well as applying physical stimuli such as increased temperature, light exposure, or electric shock, can effectively enhance EV secretion, and (c) using physical forces (such as ultrasound, grinding, or extrusion) to disrupt the cell membrane, followed by the reformation of a large number of vesicles with structures similar to EVs.

Large-scale cell culture platforms

The traditional EV production protocols rely on two-dimensional (2D) flask cultivation, which requires substantial amounts of cell culture plastics, media, and space. In contrast, three-dimensional (3D) cell culture employs gels, microcarriers, hollow fiber bioreactors, or synthetic scaffolds to enable cell growth from a 2D plane to a 3D state.^190
–193 Research has demonstrated that the yield of MSCs EVs from 3D cultures is over 20 times higher than that from 2D cultures.¹⁹⁴ Moreover, applying flow stimulation to dental pulp or adipose-derived stem cells on 3D constructs, or subjecting them to mechanical stretching within 3D-engineered skeletal muscle tissues, has been shown to significantly enhance EV production compared to static conditions.¹⁹⁵ Additionally, 3D bioreactors offer an alternative approach with the benefit of increased scalability, with systems currently ranging from less than 1 L to over 80 L in capacity.¹⁹⁶ A microcarrier-based bioreactor culture system, specifically a scalable S/XF microcarrier bioreactor culture system, has been developed for the efficient production of MSC-EVs using MSCs from three different human tissue sources. This system achieves higher concentrations and productivity of MSC-EVs compared to conventional static culture systems.¹⁹⁷ Furthermore, the use of the CELLine cell culture system increases EV production by tumor cells.¹⁹⁸ It is important to note that this method primarily focuses on increasing EV yield by expanding the cell culture volume, and does not appear to affect the amount of EVs secreted by individual cell. In addition to the availability of larger quantities of EVs, EV function also changes under varying culture conditions. The study demonstrated that EVs derived from 3D cultures of umbilical cord mesenchymal stem cells in a hollow-fiber bioreactor exhibit enhanced osteochondral regeneration activity.¹⁹⁹ Similarly, 3D culture of MSCs generates exosomes with higher yields and improved therapeutic efficacy for cisplatin-induced acute kidney injury.²⁰⁰ Thus, we hypothesize that different culture conditions influence not only EV production but also EV function, potentially by altering EV components.

Cell stimulation or genetic modification to increase EV yield per cell

Several factors can promote EV release, including adjustments to the culture environment, physical stimulation, and molecular interference.²⁰¹ Studies have demonstrated that hydrogen ion concentration^202,203, calcium ion concentration,²⁰⁴ glucose concentration,²⁰⁵ and oxygen content^206,207 can influence EV secretion. Notably, an acidic pH (pH = 4) has been reported to enhance the stability of EVs in vitro, thereby improving their separation and collection rates. In an acidic environment, the concentration of EV proteins and RNA collected has been shown to increase fivefold.²⁰⁸ The transport and fusion of multivesicular bodies (MVBs) with the plasma membrane are regulated by calcium ions.²⁰⁹ Increased intracellular Ca²⁺ levels promote EV secretion²⁰⁴ and affect the expression of proteins associated with EV release, such as members of the Rab protein family.²¹⁰ Additionally, glucose concentration can influence EV secretion, as both insufficient and excessive glucose levels can promote EV release. For instance, under low-glucose conditions, EV secretion from H9C2 cardiomyocytes was increased.²⁰⁵ Similarly, studies have shown that the amount of EVs secreted by trophoblast cells increases in high-glucose environments.²¹¹ Oxygen content is another factor affecting EV secretion. Under hypoxic conditions, the expression level of Rab27, a key regulatory factor involved in EV secretion, increases, leading to enhanced EV release.²¹²

Furthermore, physical stimuli such as temperature, light, and electric shock can also enhance the production of EVs. Temperature, a critical factor in maintaining the normal life activities of all organisms, also regulates cell EV secretion.²¹³ Changes in temperature have been shown to affect the release of EVs by breast cancer cells, with twice as many secreted EVs at 39°C compared to 35°C.²¹³ Light has also been found to impact EV secretion, with studies demonstrating that immune function EV secretion can be increased by over 13 times when dendritic cells are stimulated with light at 365 nm, while maintaining overall quality, biocompatibility, cell internalization ability, and immunogenicity. This method has shown similar effects on other cell types.³⁵ Additionally, low-energy electric fields have been observed to activate intracellular signals, including Rho-GTPase and endocytosis, both involved in EV formation mechanisms.²¹⁴ Studies indicate that low-level electricity can enhance EV secretion by activating Rho-GTPase, without altering EV morphology or surface-labeled proteins, and without inducing cytotoxicity.²¹⁵ In addition to physical stimulation, regulating the expression of proteins related to EV genesis can also affect EV secretion. For instance, regulating the expression of key EV genesis proteins such as Rab (Rab27, Rab37, Rab39) and Phospholipase D (PLD) can influence EV secretion.^{187,216
–218} Activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB-plasma membrane fusion and the release of CD63-enriched EVs.²¹⁹ KIBRA promotes EV secretion by inhibiting the proteasomal degradation of Rab27a.²²⁰ Additionally, inhibition of Sirtuin 1 expression has been shown to increase the number of exosomes secreted by cells.²²¹ PLD has been shown to participate in the biogenesis of EVs, with EV biogenesis and budding into MVBs controlled by ARF6 and PLD2.²²² Our study also demonstrated that inhibiting Rab27 protein expression significantly reduces EV secretion.²²³

Above research shows that moderate pH reduction, adjustment of calcium ion concentration, glucose content, oxygen content, and other culture conditions, along with the exogenous application of physical stimuli such as temperature, light, and electric shock, can effectively enhance the formation and secretion of EVs, serving as a viable approach to increase EV production. Our previous study demonstrated that GHRH stimulation promotes the secretion of pituitary extracellular vesicles and influences the content of its cargo miR-375-3p, thereby regulating the proliferation and IGF-1 expression of hepatocytes.²²³ However, whether these aforementioned stimuli affect the cargo, function, and stability of EV remains to be further studied.

Broken cell membrane assembles into vesicle particles

While populations of EVs can be isolated from EV-secreting cell lines,^1,224 the low yield of EVs and the complexity of their purification have driven an increase in studies using EV mimetics. Recently, artificial EVs have been developed to address the limitations of natural EVs, emerging as new theragnostic biomaterials with potential clinical applications.^225,226 Various methods have been identified to disrupt cell membranes and reassemble them into vesicle particles, including extrusion through micro-sized pore filters,^36,188,227 ultrasonic techniques,²²⁸ nitrogen cavitation,²²⁹ and homogenization grinding.¹⁸⁹ According to the “2023 MISEV guidelines,” EV-like particles produced through direct artificial manipulation are termed EV mimetics.²³⁰ Among these, cell fragmentation by shear force is the most widely used method for producing EV mimetics. Extruded cells can yield EVs that closely resemble naturally secreted EVs in shape and content, with a productivity rate 1000 times higher than that of natural cells. This is achieved by sequentially passing cells through nanofilters with progressively smaller pore sizes, such as 10–5–0.22 μm filters successively.²³¹ Additionally, it has been demonstrated that the amount of EVs produced using 10–1–0.2 μm filtration membranes is three times greater than that of naturally secreted EVs.²³² Vesicles can also be produced by mechanically grinding cells, for example, using homogenizers and filters of 0.8 and 0.22 μm, which produce vesicles similar in morphology and structure to EVs.¹⁸⁹ Similarly, vesicular structures can be formed through membrane fusion induced by ultrasound following hypotonic and mechanical cell disruption.²²⁸ EV mimetics exhibit a vesicular structure comparable to EVs and contain CD63 and TSG101 proteins.²³³ Currently, numerous methods for preparing EV mimetics have been published, all achieving higher production rates compared to natural EVs. Using the keywords “Exosome mimetic,” “Engineered Extracellular Vesicles,” “Cell-derived nanovesicles,” “exosome-like vesicles,” or “Engineering exosomes,” we conducted a PubMed search and screened several representative studies on EV mimetic preparation (Table 2).

Table 2.

Comparison of production of EV mimetics and natural EV by different methods.

Methods	Cell	Particle diameter	Multiplier
Continuous crushing	U937	128 nm	Both total protein and particle count were increased by more than 100 times²³⁴
Freeze-thaw, Continuous crushing	HCAEC	200 nm	Total protein content increased by more than three times²³²
Continuous crushing	NK92-MI	99 nm	The number of particles increased by more than 50 times²³⁵
Continuous crushing	U937	130 nm	Total protein content increased by more than 15 times²³⁶
Slicing living cell membrane	ES-D3	100 and 300 nm	Both total protein and particle count were increased by more than 100 times²³⁷
Continuous crushing	bEnd.3	141 nm	Total protein content increased by more than 30 times. The number of particles increased by more than 15 times²³⁸
Ultrasonication	hucMSCs	133 nm	The number of particles increased 20 times²³⁹
Continuous crushing	RAW264.7	189 nm	Total protein content increased by more than three times. The number of particles increased by more than four times²⁴⁰
Continuous crushing	ES cell	100 nm	The number of particles increased 250 times¹⁸⁸
Homogenizer grinding	MDA-MB-231	93 nm	The number of particles increased by more than 200 times²⁴¹
Extrusion	HEK293	114–124 nm	The number of particles increased by more than 50 times³⁶

EVs by themselves or as vehicles for the delivery of drug payloads are being actively explored as therapeutic agents. Natural EVs from various sources perform diverse biological functions but exhibit batch effects and functional inconsistencies.^242
–244 The core component of EVs is derived from the cell membrane. These methods utilize physical forces to disrupt the cell membrane, and the disrupted cell membrane then reforms into small vesicles containing proteins or drugs. Consequently, engineered EVs may offer improved consistency to ensure batch-to-batch reproducibility of their therapeutic efficacy, presenting greater potential for production and application. This physical method of producing EVs addresses two major limitations of natural EV production, including low yields and inconsistency in production. Loading specific proteins or nucleic acid drugs and while leveraging the rapid mass production of EV mimetics is another promising approach to complement natural EVs.

Production of engineered EVs

The composition of EVs, including proteins, lipids, non-coding RNA (ncRNA), and other components, can vary depending on the tissue source or physiological state. In recent years, there has been growing interest in engineering EVs or modifying them to specifically alter their cargo. When protein or nucleic acid drugs are introduced directly into the body, they are susceptible to elimination by the immune response. However, the vesicle structure of EVs can protect the drug molecules from the internal environment. By loading specific nucleic acids or proteins for different purposes, the components of EVs can be altered to perform specific biological functions.^22,245 Additionally, the engineering modification of EVs can enhance targeting efficiency and specificity.^246
–248 For example, EVs conjugated with anti-HER2 antibodies have demonstrated selective delivery to HER2-positive breast cancer cells.²⁴⁹ Similarly, EVs derived from cells engineered to express Lamp2b fused with an RVG peptide (targeting the acetylcholine receptor) have been shown to deliver siRNA specifically to neurons.¹²² EV engineering has been extensively researched in fields such as targeted tumor therapy,^250,251 bone injury repair,²⁵² gene editing,^28,253 and other areas. Its development will lead to the creation of new technologies and products for human and animal health. Methods for engineering EV modification can be categorized into two groups: endogenous modification and exogenous modification (Figure 4).

Figure 4.

Two modification methods of engineered EVs: (a) genetically modifying the donor cells to achieve EV modification. This can be done by directly overexpressing or fusing the target protein with EV proteins (such as CD9, CD63, Lamp2b) to efficiently load the target protein into EVs. Another approach is to induce cell disruption after overexpression to generate EV mimetics that carry the target molecule and (b) after isolating EVs, exogenous cargo can be loaded into EVs using techniques such as electroporation, co-incubation, ultrasound, or transfection reagents.

Indirect engineering of EVs by modifying EV-producing cells

Endogenous modification involves altering parent cells before isolating EVs so that the secreted EVs carry specific molecules. Available data suggest that specifically modified EVs can be generated by modifying donor cells.^254,255 A vector containing the target gene is transfected into donor cells using genetic engineering technology, and the proteins and nucleic acids are loaded into EVs. Proteins or ncRNAs encoded by the inserted genes are produced by donor cells and secreted into EVs through a natural packaging process. EVs enriched with target proteins or nucleic acids can then be obtained by isolating and purifying the cell culture supernatant.^252,256 However, during EV biosynthesis, cells utilize a complex sorting system to determine which substances are packaged into EVs.^257
–261 This system limits specificity when directly expressing target proteins or nucleic acids in donor cells, often resulting in random packaging into EVs and low loading efficiency.³⁸

Reports indicate that different nucleic acid sequences exhibit varying sorting efficiencies in EVs.²⁶² Sequences with high GC content are more likely to be sorted into EVs. For example, miRNAs containing GGNG sequences are reportedly more likely to be sorted into EVs.²⁶³ Additionally, miRNAs with CNGGAG and CGGGNG sequences (both containing the mentioned GGNG) were sorted into EVs 24 and 80 times more than those in AML12 cells and SVEC cells, respectively. Furthermore, introducing the CGGGAG sequence at the end of miRNAs with low GC content can enhance the loading of target miRNAs into EVs by 10–20 times.²⁶⁴ Studies on miRNA sequences have also shown that miRNAs containing GGCU and GGAG sequences are preferentially sorted into EVs. This is primarily due to the RNA-binding proteins SYNCRIP and hnRNPA2B1 in EVs, which bind to GGCU and GGAG sequences.²⁶¹ Similar studies have shown that the pre-miR-451 backbone can efficiently load siRNA into EVs.²⁶⁵ Moreover, target RNA loading can be facilitated by introducing RNA-binding proteins.²⁶⁶ For example, the RNA-binding protein HuR can bind to the specific AUAU sequence on miR-155, and when adsorbed by HuR, this interaction enables the successful generation of miR-155-rich EVs through plasmid transfection.²⁶⁷ These findings on miRNA or siRNA loading highlight that efficient loading can be achieved by incorporating specific short cluster sequences or a simple loading skeleton, as well as enriching target RNA using RNA-binding proteins.

The protein sorting efficiency of EVs varies with different protein types, with membrane-localized proteins being more easily sorted into EVs.²⁴⁵ For non-membrane-localized target proteins, fusing the target protein with the constitutive EV proteins (Lamp2b,^267,268 CD63,^38,269 CD9²⁸) can enhance the abundance of the protein of interest in EVs. This process is mediated by the expression of the constitutive protein, which can also improve the specificity of negative loading of the target protein into EVs. Another method was to use the late-domain (L-domain) pathway to load foreign proteins into EVs. Studies have shown that WW-Cre recombinant enzyme proteins can be recognized by Ndfip1 protein in the L-domain pathway, leading to ubiquitination and subsequent uptake into EVs.²⁷⁰ Furthermore, while genetic modification of donor cells and direct overexpression of the target protein do not always achieve efficient loading, fusion with EV marker proteins can result in significant loading of specific functional proteins. However, it remains to be verified whether the fusion protein affects the function and structure of the target protein, as well as the morphological structure and function of EVs.

Direct engineering of EVs

Direct engineering of EVs, also known as exogenous modification, involves loading target molecules directly onto or into the surface or cavity of isolated and purified EVs through passive or active methods. Current techniques for delivering therapeutic agents into EVs primarily include co-incubation,^120,271 electroporation,²⁷² extrusion,²⁷³ ultrasonic treatment,²⁷⁴ calcium chloride-mediated transfection,²⁷⁵ saponin permeabilization,²⁷⁶ freeze-thaw cycles,²⁷⁷ and the use of the Exo-Fect commercial kit.¹²³ For instance, the isolated and purified curcumin can be successfully loaded into EVs via co-incubation. EVs isolated by ultracentrifugation from cell supernatant were incubated with curcumin for 5 min, resulting in the encapsulation of curcumin within the EVs.¹²⁰ In addition, studies have demonstrated the translocation of target substances into EVs using electroporation. Following EV isolation, Cas9-sgRNA complexes were loaded by electroporation and administered through the mouse tail vein system to treat liver disease by knocking out the target gene.²⁷⁸ Furthermore, comparisons of Exo-Fect, electroporation, heat shock, and saponin methods for miRNA loading into EVs after EVs separation and purification have shown that Exo-Fect improves the efficiency of miRNA loading into EVs.²⁷⁹ These methods generally involve separating and purifying EVs, isolating and purifying substrates, and loading the target substance into EVs, particularly for proteins and miRNAs. However, it is important to note that these physical or chemical methods may cause membrane damage or leave behind chemical residues.^280,281 While these methods are highly feasible, more efficient technologies may need to be further developed for clinical applications.

Challenges in EV industry

EVs are an important class of vesicles in vivo and have garnered significant attention since the field of vesicle trafficking was awarded the Nobel Prize in Physiology or Medicine in 2013. Numerous studies have since highlighted the substantial clinical potential of EVs in diagnostics and therapeutics. Despite these advancements, several challenges remain to be addressed, particularly concerning their immunogenicity, safety for long-term use, mass manufacturing, and purification. While EVs are generally considered biocompatible and less immunogenic than synthetic nanoparticles, their immunogenicity can vary depending on the source, cargo, and modification methods. Engineered EVs, in particular, may pose additional immunogenic risks due to alterations in their surface molecules or cargo, which could trigger unwanted immune responses.²⁸² Likewise, artificial vesicles or synthetic analogs of EVs, while advantageous for scalable production and precise functionalization, may lack the sophisticated immunological compatibility that natural EVs possess, raising concerns regarding biocompatibility and immunotoxicity.^283,284 Long-term safety studies are equally imperative.

As drug carriers and diagnostic tools, EVs face additional challenges related to efficient drug loading and stability. Current physical and chemical loading methods can damage EV membranes or leave chemical residues, while the co-incubation method, though preserving EV integrity, suffers from low efficiency.²⁷³ Genetically engineering parent cells to produce EVs with specific target molecules offers a more robust and scalable alternative, but achieving high-purity EVs remains difficult due to their distribution in complex biological systems. Common isolation methods like ultracentrifugation are cost-effective but time-consuming and yield impure EVs, whereas techniques such as size-exclusion chromatography and immunoaffinity capture achieve higher purity but are costly and labor-intensive.^285
–287 Additionally, high-quality reference specimens with consistent purity and well-characterized markers are essential for research and clinical applications, but batch variability and scalability issues remain unresolved.²⁸⁸ Currently, some companies researching EVs have developed products for the standardized separation of EVs (Hansa BioMed Life Science, Vesiculab, etc.). EV diversity further complicates the identification of universal markers, as commonly used markers like CD9, CD81, and CD63 are not universally present.^289,290 Specifying detection standards for EV from different sources may be the direction of future development. Current detection methods also lack the sensitivity and specificity needed for low-concentration EV markers or disease-specific EVs. Finally, the cost-effectiveness of EV diagnostics compared to traditional methods like ELISA is uncertain, emphasizing the need for innovative approaches to improve efficiency, scalability, and accuracy while reducing costs. Addressing these challenges, particularly those related to immunogenicity and long-term safety, will be critical for the successful clinical translation of EVs and engineered EVs.

Concluding remarks and future perspective

EVs have introduced a promising new approach for disease surveillance, drug delivery, and gene therapy across a wide range of diseases. Over three decades have passed since EVs were first described, and significant progress has been made in understanding their biology and functions. Naturally occurring EVs, engineered EVs, and EV mimetics all demonstrate biological therapeutic effects. From the perspective of production cost and maintaining batch efficiency consistency, endogenous modification and the production of EV mimetics are considered more likely to achieve clinical application. The loading of specific drugs into EVs may reflect their particular functions, with the loading of nucleic acid or protein drugs into EVs potentially being more cost-effective and efficient through endogenous modifications, while the loading of chemical drugs may require exogenous modifications. Despite the significant progress in this field, unanswered questions remain that could guide future research. For instance, current research on the production of EV mimetics lacks investigation into their stability, which is crucial in determining whether EV mimetics exhibit similar stability to EVs. Furthermore, there is a lack of technical standards for the production of EVs, engineered EVs, and EV mimetics, as well as detailed standardized processes for the collection, preservation, separation, and purification of EVs from different samples. Another major challenge is achieving precise and efficient targeting of EVs to specific tissues or cell types. While surface modification with ligands, peptides, or antibodies has shown promise, these approaches often face challenges such as off-target effects, limited binding affinity, and potential immunogenicity of the added targeting moieties. Researchers are encouraged to address these key issues to facilitate the transition of EVs from the laboratory to clinical use.

Footnotes

Data availability

No new data have been generated in this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National key research and development program (2022YFD1300904, 2022YFD1300401); National Natural Science Foundation of China (32372958, 32072812); and Natural Science Foundation of Guangdong Province (2024A1515010510, 2023A1515012127)

ORCID iD

Yuxuan Wang

References

Fan

Jiang

Wang

, et al. Engineered extracellular vesicles as intelligent nanosystems for next-generation nanomedicine. Nanoscale Horizons 2022; 7(7): 682–714.

Antimisiaris

Mourtas

Marazioti

Exosomes and exosome-inspired vesicles for targeted drug delivery. Pharmaceutics 2018; 10(4): 218.

Mulcahy

Pink

Carter

DR.

Routes and mechanisms of extracellular vesicle uptake. J Extracell Vesicles 2014; 3: 10.

Jafari

Malih

Eini

, et al. Improvement, scaling-up, and downstream analysis of exosome production. Crit Rev Biotechnol 2020; 40(8): 1098–1112.

Gupta

Zickler

El Andaloussi

Dosing extracellular vesicles. Adv Drug Deliv Rev 2021; 178: 113961.

Saleh

Lázaro-Ibáñez

Forsgard

, et al. Extracellular vesicles induce minimal hepatotoxicity and immunogenicity. Nanoscale 2019; 11(14): 6990–7001.

Cheng

Sharples

Scicluna

, et al. Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood. J Extracell Vesicles 2014; 3: 10.

Gao

, et al. Neural stem cell-derived extracellular vesicles mitigate Alzheimer’s disease-like phenotypes in a preclinical mouse model. Signal Transduct Target Ther 2023; 8(1): 228.

Krämer-Albers

EM.

Extracellular vesicles at CNS barriers: mode of action. Curr Opin Neurobiol 2022; 75: 102569.

10.

Yang

Peng

Feng

, et al. Immune cell-derived extracellular vesicles - new strategies in cancer immunotherapy. Front Immunol 2021; 12: 771551.

11.

Nie

Zhong

, et al. Membrane vesicles nanotheranostic systems: sources, engineering methods, and challenges. Biomed Mater 2021; 16(2): 022009.

12.

Wang

Qin

Wang

, et al. Young sca-1(+) bone marrow stem cell-derived exosomes preserve visual function via the miR-150-5p/MEKK3/JNK/c-Jun pathway to reduce M1 microglial polarization. J Nanobiotechnol 2023; 21(1): 194.

13.

Wang

Bojmar

, et al. Tumour extracellular vesicles and particles induce liver metabolic dysfunction. Nature 2023; 618(7964): 374–382.

14.

Momenbeitollahi

Aggarwal

Strohle

, et al. Extracellular vesicle (EV) dot blotting for multiplexed EV protein detection in complex biofluids. Anal Chem 2022; 94(20): 7368–7374.

15.

Blandin

Dugail

Hilairet

, et al. Lipidomic analysis of adipose-derived extracellular vesicles reveals specific EV lipid sorting informative of the obesity metabolic state. Cell Rep 2023; 42(3): 112169.

16.

You

Tian

Yang

, et al. Intradermally delivered mRNA-encapsulating extracellular vesicles for collagen-replacement therapy. Nat Biomed Eng 2023; 7(7): 887–900.

17.

Liang

Duan

, et al. Engineering exosomes for targeted drug delivery. Theranostics 2021; 11(7): 3183–3195.

18.

Wang

Popowski

Zhu

, et al. Exosomes decorated with a recombinant SARS-CoV-2 receptor-binding domain as an inhalable COVID-19 vaccine. Nat Biomed Eng 2022; 6(7): 791–805.

19.

Harrell

Jovicic

Djonov

, et al. Mesenchymal stem cell-derived exosomes and other extracellular vesicles as new remedies in the therapy of inflammatory diseases. Cells 2019; 8(12): 1605.

20.

Johnson

Law

SQK

Shojaee

, et al. First-in-human clinical trial of allogeneic, platelet-derived extracellular vesicles as a potential therapeutic for delayed wound healing. J Extracell Vesicles 2023; 12(7): e12332.

21.

El-Shennawy

Hoffmann

Dashzeveg

, et al. Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2. Nat Commun 2022; 13(1): 405.

22.

Kim

Cho

Kim

, et al. Engineered small extracellular vesicles displaying ACE2 variants on the surface protect against SARS-CoV-2 infection. J Extracell Vesicles 2022; 11(1): e12179.

23.

Kadota

Fujita

Araya

, et al. Human bronchial epithelial cell-derived extracellular vesicle therapy for pulmonary fibrosis via inhibition of TGF-β-WNT crosstalk. J Extracell Vesicles 2021; 10(10): e12124.

24.

Hosen

Goody

Zietzer

, et al. Circulating MicroRNA-122-5p is associated with a lack of improvement in left ventricular function after transcatheter aortic valve replacement and regulates viability of cardiomyocytes through extracellular vesicles. Circulation 2022; 146(24): 1836–1854.

25.

Zhang

Dong

Chen

, et al. Extracellular vesicle-mediated delivery of miR-101 inhibits lung metastasis in osteosarcoma. Theranostics 2020; 10(1): 411–425.

26.

Cocks

Martinez-Rodriguez

Del Vecchio

, et al. Diverse roles of EV-RNA in cancer progression. Semin Cancer Biol 2021; 75: 127–135.

27.

Akbar

Azzimato

Choudhury

, et al. Extracellular vesicles in metabolic disease. Diabetologia 2019; 62(12): 2179–2187.

28.

Zhou

Wei

, et al. In vitro and in vivo RNA inhibition by CD9-HuR functionalized exosomes encapsulated with miRNA or CRISPR/dCas9. Nano Lett 2019; 19(1): 19–28.

29.

Hao

Song

Zhou

, et al. Promotion or inhibition of extracellular vesicle release: emerging therapeutic opportunities. J Control Release 2021; 340: 136–148.

30.

Kordelas

Rebmann

Ludwig

, et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. Leukemia 2014; 28(4): 970–973.

31.

Shi

Yang

Monsel

, et al. Preclinical efficacy and clinical safety of clinical-grade nebulized allogenic adipose mesenchymal stromal cells-derived extracellular vesicles. J Extracell Vesicles 2021; 10(10): e12134.

32.

You

Nguyen

, et al. Vitamin A-coupled stem cell-derived extracellular vesicles regulate the fibrotic cascade by targeting activated hepatic stellate cells in vivo. J Control Release 2021; 336: 285–295.

33.

Mendt

Kamerkar

Sugimoto

, et al. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight 2018; 3(8): e99263.

34.

Thippabhotla

Zhong

3D cell culture stimulates the secretion of in vivo like extracellular vesicles. Sci Rep 2019; 9(1): 13012.

35.

Ruan

Erwin

Light-induced high-efficient cellular production of immune functional extracellular vesicles. J Extracell Vesicles 2022; 11(3): e12194.

36.

Wang

Mao

, et al. Bioinspired engineering of fusogen and targeting moiety equipped nanovesicles. Nat Commun 2023; 14(1): 3366.

37.

Kalluri

LeBleu

VS.

The biology, function, and biomedical applications of exosomes. Science 2020; 367(6478): eaau6977.

38.

Liu

Design strategies and application progress of therapeutic exosomes. Theranostics 2019; 9(4): 1015–1028.

39.

Tenchov

Sasso

Wang

, et al. Exosomes─nature’s lipid nanoparticles, a rising star in drug delivery and diagnostics. ACS Nano 2022; 16(11): 17802–17846.

40.

Kibria

Ramos

Wan

, et al. Exosomes as a drug delivery system in cancer therapy: potential and challenges. Mol Pharm 2018; 15(9): 3625–3633.

41.

Lin

Jiang

, et al. Exosomal cargo-loading and synthetic exosome-mimics as potential therapeutic tools. Acta Pharmacol Sin 2018; 39(4): 542–551.

42.

Shimizu

Sawada

Kimura

Pathophysiological role and potential therapeutic exploitation of exosomes in ovarian cancer. Cells 2020; 9(4): 814.

43.

Gorabi

Kiaie

Barreto

, et al. The therapeutic potential of mesenchymal stem cell-derived exosomes in treatment of neurodegenerative diseases. Mol Neurobiol 2019; 56(12): 8157–8167.

44.

Ocansey

DKW

Zhang

Wang

, et al. Exosome-mediated effects and applications in inflammatory bowel disease. Biol Rev Camb Philos Soc 2020; 95(5): 1287–1307.

45.

Liang

Xie

Hou

, et al. miRNAs derived from milk small extracellular vesicles inhibit porcine epidemic diarrhea virus infection. Antiviral Res 2023; 212: 105579.

46.

Zhang

Xiao

Wang

, et al. Human adipose and umbilical cord mesenchymal stem cell-derived extracellular vesicles mitigate photoaging via TIMP1/Notch1. Signal Transduct Target Ther 2024; 9(1): 294.

47.

Wang

Lou

Xie

, et al. Nutrient availability regulates the secretion and function of immune cell-derived extracellular vesicles through metabolic rewiring. Sci Adv 2024; 10(7): eadj1290.

48.

Koo

Cheng

Udani

, et al. Optimizing cell therapy by sorting cells with high extracellular vesicle secretion. Nat Commun 2024; 15(1): 4870.

49.

Jing

Zhang

, et al. Milk-derived extracellular vesicles functionalized with anti-tumour necrosis factor-α nanobody and anti-microbial peptide alleviate ulcerative colitis in mice. J Extracell Vesicles 2024; 13(6): e12462.

50.

Barreiro

Lay

Leparc

, et al. An in vitro approach to understand contribution of kidney cells to human urinary extracellular vesicles. J Extracell Vesicles 2023; 12(2): e12304.

51.

Huang

Meng

Chen

, et al. Bacterial outer membrane vesicles as potential biological nanomaterials for antibacterial therapy. Acta Biomater 2022; 140: 102–115.

52.

Zhou

Yang

, et al. Bacterial outer membrane vesicles as a platform for biomedical applications: an update. J Control Release 2020; 323: 253–268.

53.

Feng

Cheng

, et al. Engineered bacterial outer membrane vesicles as controllable two-way adaptors to activate macrophage phagocytosis for improved tumor immunotherapy. Adv Mater 2022; 34(40): e2206200.

54.

Zhu

Ginger: a representative material of herb-derived exosome-like nanoparticles. Front Nutr 2023; 10: 1223349.

55.

Zhao

Lin

Jiang

, et al. Exosome-like nanoparticles derived from fruits, vegetables, and herbs: innovative strategies of therapeutic and drug delivery. Theranostics 2024; 14(12): 4598–4621.

56.

Yan

Bian

, et al. Momordica charantia L.-derived exosome-like nanovesicles stabilize p62 expression to ameliorate doxorubicin cardiotoxicity. J Nanobiotechnol 2024; 22(1): 464.

57.

Cheng

Zhao

, et al. Bioengineered bacteria-derived outer membrane vesicles as a versatile antigen display platform for tumor vaccination via plug-and-display technology. Nat Commun 2021; 12(1): 2041.

58.

Kim

Lee

, et al. Mesenchymal stem/stromal cell-derived exosomes for immunomodulatory therapeutics and skin regeneration. Cells 2020; 9(5): 1157.

59.

Cai

Wang

, et al. Extracellular vesicles derived from different sources of mesenchymal stem cells: therapeutic effects and translational potential. Cell Biosci 2020; 10(1): 69.

60.

Madrigal

Rao

Riordan

NH.

A review of therapeutic effects of mesenchymal stem cell secretions and induction of secretory modification by different culture methods. J Transl Med 2014; 12: 260.

61.

Yin

Liu

, et al. Engineered mesenchymal stem cell-derived extracellular vesicles: a state-of-the-art multifunctional weapon against Alzheimer’s disease. Theranostics 2023; 13(4): 1264–1285.

62.

Maguire

Stem cell therapy without the cells. Commun Integr Biol 2013; 6(6): e26631.

63.

Sengupta

Lazo

, et al. Exosomes derived from bone marrow mesenchymal stem cells as treatment for severe COVID-19. Stem Cells Dev 2020; 29(12): 747–754.

64.

Akbari

Rezaie

Potential therapeutic application of mesenchymal stem cell-derived exosomes in SARS-CoV-2 pneumonia. Stem Cell Res Ther 2020; 11(1): 356.

65.

Wang

Lin

Zhang

, et al. M2 macrophage-derived exosomes promote diabetic fracture healing by acting as an immunomodulator. Bioact Mater 2023; 28: 273–283.

66.

Vonk

van Dooremalen

SFJ

Liv

, et al. Mesenchymal stromal/stem cell-derived extracellular vesicles promote human cartilage regeneration in vitro. Theranostics 2018; 8(4): 906–920.

67.

Chen

, et al. Extracellular vesicles from human umbilical cord blood ameliorate bone loss in senile osteoporotic mice. Metabolism 2019; 95: 93–101.

68.

Huang

Zheng

, et al. ADSCs-derived extracellular vesicles alleviate neuronal damage, promote neurogenesis and rescue memory loss in mice with Alzheimer’s disease. J Control Release 2020; 327: 688–702.

69.

Shi

Yao

Yin

, et al. Extracellular vesicles derived from immune cells: role in tumor therapy. Int Immunopharmacol 2024; 133: 112150.

70.

Prokopeva

Emene

Gomzikova

MO.

Antitumor immunity: role of NK cells and extracellular vesicles in cancer immunotherapy. Curr Issues Mol Biol 2023; 46(1): 140–152.

71.

Shin

Jung

, et al. Novel antitumor therapeutic strategy using CD4(+) T cell-derived extracellular vesicles. Biomaterials 2022; 289: 121765.

72.

Zhu

Kalimuthu

Gangadaran

, et al. Exosomes derived from natural killer cells exert therapeutic effect in melanoma. Theranostics 2017; 7(10): 2732–2745.

73.

Neviani

Wise

Murtadha

, et al. Natural killer-derived exosomal miR-186 inhibits neuroblastoma growth and immune escape mechanisms. Cancer Res 2019; 79(6): 1151–1164.

74.

Wang

Chen

, et al. Natural killer cell-derived exosomal miR-1249-3p attenuates insulin resistance and inflammation in mouse models of type 2 diabetes. Signal Transduct Target Ther 2021; 6(1): 409.

75.

Sartorio

Pardue

Feldman

, et al. Bacterial outer membrane vesicles: from discovery to applications. Annu Rev Microbiol 2021; 75: 609–630.

76.

Zheng

Feng

, et al. Engineered bacterial outer membrane vesicles: a versatile bacteria-based weapon against gastrointestinal tumors. Theranostics 2024; 14(2): 761–787.

77.

Chen

Luo

, et al. Surface mineralization of engineered bacterial outer membrane vesicles to enhance tumor photothermal/immunotherapy. ACS Nano 2024; 18(2): 1357–1370.

78.

Han

Choi

Lee

, et al. Extracellular RNAs in periodontopathogenic outer membrane vesicles promote TNF-α production in human macrophages and cross the blood-brain barrier in mice. FASEB J 2019; 33(12): 13412–13422.

79.

Qing

Lyu

Zhu

, et al. Biomineralized bacterial outer membrane vesicles potentiate safe and efficient tumor microenvironment reprogramming for anticancer therapy. Adv Mater 2020; 32(47): e2002085.

80.

Soult

Lonergan

Shah

, et al. Outer membrane vesicles from pathogenic bacteria initiate an inflammatory response in human endothelial cells. J Surg Res 2013; 184(1): 458–466.

81.

Gong

Chen

Mao

, et al. Outer membrane vesicles of Porphyromonas gingivalis trigger NLRP3 inflammasome and induce neuroinflammation, tau phosphorylation, and memory dysfunction in mice. Front Cell Infect Microbiol 2022; 12: 925435.

82.

Ban

Sun

Huang

, et al. Engineered bacterial outer membrane vesicles encapsulating oncolytic adenoviruses enhance the efficacy of cancer virotherapy by augmenting tumor cell autophagy. Nat Commun 2023; 14(1): 2933.

83.

Kim

Park

Dinh

NTH

, et al. Bacterial outer membrane vesicles suppress tumor by interferon-γ-mediated antitumor response. Nat Commun 2017; 8(1): 626.

84.

Won

Lee

Bae

, et al. Mass-produced gram-negative bacterial outer membrane vesicles activate cancer antigen-specific stem-like CD8(+) T cells which enables an effective combination immunotherapy with anti-PD-1. J Extracell Vesicles 2023; 12(8): e12357.

85.

Shi

Wang

, et al. Probiotic-derived extracellular vesicles alleviate AFB1-induced intestinal injury by modulating the gut microbiota and AHR activation. J Nanobiotechnol 2024; 22(1): 697.

86.

Lee

Chien

, et al. Tomato-fruit-derived extracellular vesicles inhibit fusobacterium nucleatum via lipid-mediated mechanism. Food Funct 2023; 14(19): 8942–8950.

87.

Yao

Yue

, et al. Advances in bioactivity of MicroRNAs of plant-derived exosome-like nanoparticles and milk-derived extracellular vesicles. J Agric Food Chem 2022; 70(21): 6285–6299.

88.

Nemati

Singh

Mir

, et al. Plant-derived extracellular vesicles: a novel nanomedicine approach with advantages and challenges. Cell Commun Signal 2022; 20(1): 69.

89.

Zhu

Zhao

Ding

, et al. Applications of plant-derived extracellular vesicles in medicine. MedComm 2024; 5(10): e741.

90.

Teng

Ren

Sayed

, et al. Plant-derived exosomal MicroRNAs shape the gut microbiota. Cell Host Microbe 2018; 24(5): 637–652.e8.

91.

Qiu

Wang

Guo

, et al. Rgl-exomiR-7972, a novel plant exosomal microRNA derived from fresh rehmanniae radix, ameliorated lipopolysaccharide-induced acute lung injury and gut dysbiosis. Biomed Pharmacother 2023; 165: 115007.

92.

Feng

Yue

Zhang

, et al. Plant-derived exosome-like nanoparticles: emerging nanosystems for enhanced tissue engineering. Int J Nanomedicine 2024; 19: 1189–1204.

93.

Wei

Zhang

Cheng

, et al. Plant-derived exosome-like nanoparticles - from laboratory to factory, a landscape of application, challenges and prospects. Crit Rev Food Sci Nutr 2024; 11: 1–19.

94.

Shen

Zhang

Liu

, et al. Solanum lycopersicum derived exosome-like nanovesicles alleviate restenosis after vascular injury through the keap1/Nrf2 pathway. Food Funct 2024; 16(2): 539–553.

95.

Sriwastva

Deng

Wang

, et al. Exosome-like nanoparticles from mulberry bark prevent DSS-induced colitis via the AhR/COPS8 pathway. EMBO Rep 2022; 23(3): e53365.

96.

Long

Pan

, et al. Orange-derived extracellular vesicles nanodrugs for efficient treatment of ovarian cancer assisted by transcytosis effect. Acta Pharm Sin B 2023; 13(12): 5121–5134.

97.

Nieuwland

Siljander

PR.

A beginner’s guide to study extracellular vesicles in human blood plasma and serum. J Extracell Vesicles 2024; 13(1): e12400.

98.

Tóth

EÁ

Turiák

Visnovitz

, et al. Formation of a protein corona on the surface of extracellular vesicles in blood plasma. J Extracell Vesicles 2021; 10(11): e12140.

99.

Yung

Zhang

Kuhn

, et al. Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid. J Extracell Vesicles 2024; 13(4): e12422.

100.

Zeng

Chen

Luo

, et al. Biological characteristics and roles of noncoding RNAs in milk-derived extracellular vesicles. Adv Nutr 2021; 12(3): 1006–1019.

101.

Dong

Zieren

Horie

, et al. Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium. J Extracell Vesicles 2020; 10(2): e12044.

102.

Prasadani

Kodithuwakku

Pennarossa

, et al. Therapeutic potential of bovine milk-derived extracellular vesicles. Int J Mol Sci 2024; 25(10): 5543.

103.

Badawy

El-Hofey

Shaban

, et al. Camel milk extracellular vesicles/exosomes: a fascinating frontier in isolation and therapeutic potential. Food Funct 2024; 16(2): 344–365.

104.

Samuel

Fonseka

Sanwlani

, et al. Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis. Nat Commun 2021; 12(1): 3950.

105.

Zhang

Lin

, et al. Milk-derived small extracellular vesicles: a new perspective on dairy nutrition. Crit Rev Food Sci Nutr 2024; 64(33): 13225–13246.

106.

Tong

Hao

Zhang

, et al. Milk-derived extracellular vesicles alleviate ulcerative colitis by regulating the gut immunity and reshaping the gut microbiota. Theranostics 2021; 11(17): 8570–8586.

107.

Zhang

, et al. Comparative study on the immunomodulatory function of extracellular vesicles from different dairy products. Food Funct 2022; 13(5): 2504–2514.

108.

Filler

Yeganeh

, et al. Bovine milk-derived exosomes attenuate NLRP3 inflammasome and NF-κB signaling in the lung during neonatal necrotizing enterocolitis. Pediatr Surg Int 2023; 39(1): 211.

109.

Guo

Zhang

JH.

Human breast milk-derived exosomal miR-148a-3p protects against necrotizing enterocolitis by regulating p53 and sirtuin 1. Inflammation 2022; 45(3): 1254–1268.

110.

Golan-Gerstl

Elbaum Shiff

Moshayoff

, et al. Characterization and biological function of milk-derived miRNAs. Mol Nutr Food Res 2017; 61(10): 7048–7059.

111.

Yan

Chen

Wang

, et al. Milk exosomes-mediated miR-31-5p delivery accelerates diabetic wound healing through promoting angiogenesis. Drug Deliv 2022; 29(1): 214–228.

112.

Han

Kim

Jang

, et al. Oral TNF-α siRNA delivery via milk-derived exosomes for effective treatment of inflammatory bowel disease. Bioact Mater 2024; 34: 138–149.

113.

Lin

Chen

Xie

, et al. Oral administration of bovine and porcine milk exosome alter miRNAs profiles in piglet serum. Sci Rep 2020; 10(1): 6983.

114.

Zeng

Wang

Luo

, et al. Porcine milk-derived small extracellular vesicles promote intestinal immunoglobulin production through pIgR. Animals 2021; 11(6): 1522.

115.

Xie

Chen

, et al. Porcine milk exosome miRNAs protect intestinal epithelial cells against deoxynivalenol-induced damage. Biochem Pharmacol 2020; 175: 113898.

116.

Xie

Hou

Sun

, et al. Porcine milk exosome MiRNAs attenuate LPS-induced apoptosis through inhibiting TLR4/NF-κB and p53 pathways in intestinal epithelial cells. J Agric Food Chem 2019; 67(34): 9477–9491.

117.

Xiong

Fan

Wang

, et al. New signaling kid on the block in the endocrine system: the role of extracellular vesicles. Endocrinology 2023; 164(8): bqad099.

118.

Xiong

Wang

Luo

, et al. Pituitary-derived small extracellular vesicles promote liver repair by its cargo miR-143-3p. Sci Rep 2024; 14(1): 16635.

119.

El-Andaloussi

Lee

Lakhal-Littleton

, et al. Exosome-mediated delivery of siRNA in vitro and in vivo. Nat Protoc 2012; 7(12): 2112–2126.

120.

Sun

Zhuang

Xiang

, et al. A novel nanoparticle drug delivery system: the anti-inflammatory activity of curcumin is enhanced when encapsulated in exosomes. Mol Ther 2010; 18(9): 1606–1614.

121.

Zheng

Zhu

Tang

, et al. Inhalable CAR-T cell-derived exosomes as paclitaxel carriers for treating lung cancer. J Transl Med 2023; 21(1): 383.

122.

Alvarez-Erviti

Seow

Yin

, et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol 2011; 29(4): 341–345.

123.

Aqil

Munagala

Jeyabalan

, et al. Milk exosomes - natural nanoparticles for siRNA delivery. Cancer Lett 2019; 449: 186–195.

124.

Wang

, et al. Paintable bioactive extracellular vesicle ink for wound healing. ACS Appl Mater Interfaces 2023; 15(21): 25427–25436.

125.

Deng

Chen

, et al. Engineered human adipose stem-cell-derived exosomes loaded with miR-21-5p to promote diabetic cutaneous wound healing. Mol Pharm 2020; 17(5): 1723–1733.

126.

Fei

Dai

, et al. Mesenchymal stem cell-derived extracellular vesicles with high PD-L1 expression for autoimmune diseases treatment. Adv Mater 2022; 34(1): e2106265.

127.

Cheng

Dai

Smbatyan

, et al. Eliciting anti-cancer immunity by genetically engineered multifunctional exosomes. Mol Ther 2022; 30(9): 3066–3077.

128.

Song

Yuan

, et al. Multiplexed strategies toward clinical translation of extracellular vesicles. Theranostics 2022; 12(15): 6740–6761.

129.

Montaner-Tarbes

Fraile

Montoya

, et al. Exosome-based vaccines: pros and cons in the world of animal health. Viruses 2021; 13(8): 1499.

130.

Mehanny

Lehr

Fuhrmann

Extracellular vesicles as antigen carriers for novel vaccination avenues. Adv Drug Deliv Rev 2021; 173: 164–180.

131.

Huang

Rong

Tang

, et al. Engineered exosomes as an in situ DC-primed vaccine to boost antitumor immunity in breast cancer. Mol Cancer 2022; 21(1): 45.

132.

Sabanovic

Piva

Cecati

, et al. Promising extracellular vesicle-based vaccines against viruses, including SARS-CoV-2. Biology 2021; 10(2): 94.

133.

Tsai

Atai

Cacciottolo

, et al. Exosome-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity. J Biol Chem 2021; 297(5): 101266.

134.

Cacciottolo

Nice

, et al. Exosome-based multivalent vaccine: achieving potent immunization, broadened reactivity, and strong T-Cell responses with nanograms of proteins. Microbiol Spectr 2023; 11(3): e0050323.

135.

Wolfers

Lozier

Raposo

, et al. Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 2001; 7(3): 297–303.

136.

Chaput

Taïeb

Schartz

, et al. The potential of exosomes in immunotherapy of cancer. Blood Cells Mol Dis 2005; 35(2): 111–115.

137.

Kee

Al-Masawa

, et al. Scalable production of extracellular vesicles and its therapeutic values: a review. Int J Mol Sci 2022; 23(14): 7986.

138.

Escudier

Dorval

Chaput

, et al. Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial. J Transl Med 2005; 3(1): 10.

139.

Morse

Garst

Osada

, et al. A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer. J Transl Med 2005; 3(1): 9.

140.

De Rubis

Rajeev Krishnan

Bebawy

. Liquid biopsies in cancer diagnosis, monitoring, and prognosis. Trends Pharmacol Sci 2019; 40(3): 172–186.

141.

Wang

, et al. Exosomes as a new frontier of cancer liquid biopsy. Mol Cancer 2022; 21(1): 56.

142.

Qiu

Fan

Zheng

, et al. Diagnostic potential of plasma extracellular vesicle miR-483-3p and let-7d-3p for sepsis. Front Mol Biosci 2022; 9: 814240.

143.

Shi

Fang

Tian

, et al. Discovery of extracellular vesicle-delivered miR-185-5p in the plasma of patients as an indicator for advanced adenoma and colorectal cancer. J Transl Med 2023; 21(1): 421.

144.

Hannafon

Trigoso

Calloway

, et al. Plasma exosome microRNAs are indicative of breast cancer. Breast Cancer Res 2016; 18(1): 90.

145.

Zhu

Gong

, et al. Exosomal tRNA-derived small RNA as a promising biomarker for cancer diagnosis. Mol Cancer 2019; 18(1): 74.

146.

Fotuhi

Khalaj-Kondori

Hoseinpour Feizi

, et al. Long non-coding RNA BACE1-AS may serve as an Alzheimer’s disease blood-based biomarker. J Mol Neurosci 2019; 69(3): 351–359.

147.

Lin

Shao

, et al. Plasma exosomal hsa_circ_0079439 as a novel biomarker for early detection of gastric cancer. World J Gastroenterol 2023; 29(22): 3482–3496.

148.

Sandfeld-Paulsen

Jakobsen

Bæk

, et al. Exosomal proteins as diagnostic biomarkers in lung cancer. J Thorac Oncol 2016; 11(10): 1701–1710.

149.

Sandfeld-Paulsen

Aggerholm-Pedersen

Bæk

, et al. Exosomal proteins as prognostic biomarkers in non-small cell lung cancer. Mol Oncol 2016; 10(10): 1595–1602.

150.

Burrello

Bianco

Burrello

, et al. Extracellular vesicle surface markers as a diagnostic tool in transient ischemic attacks. Stroke 2021; 52(10): 3335–3347.

151.

Melo

Luecke

Kahlert

, et al. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 2015; 523(7559): 177–182.

152.

Niu

Song

Wang

, et al. Tumor-derived exosomal proteins as diagnostic biomarkers in non-small cell lung cancer. Cancer Sci 2019; 110(1): 433–442.

153.

Zhao

Song

Niu

, et al. Circulating exosomal miR-150-5p and miR-99b-5p as diagnostic biomarkers for colorectal cancer. Front Oncol 2019; 9: 1129.

154.

Tao

Tang

Yang

, et al. Exploration of serum exosomal LncRNA TBILA and AGAP2-AS1 as promising biomarkers for diagnosis of non-small cell lung cancer. Int J Biol Sci 2020; 16(3): 471–482.

155.

Rui

Wang

Xiang

, et al. Serum exosome-derived piRNAs could Be promising biomarkers for HCC diagnosis. Int J Nanomedicine 2023; 18: 1989–2001.

156.

Skotland

Ekroos

Kauhanen

, et al. Molecular lipid species in urinary exosomes as potential prostate cancer biomarkers. Eur J Cancer 2017; 70: 122–132.

157.

Øverbye

Skotland

Koehler

, et al. Identification of prostate cancer biomarkers in urinary exosomes. Oncotarget 2015; 6(30): 30357–30376.

158.

Andreu

Otta Oshiro

Redruello

, et al. Extracellular vesicles as a source for non-invasive biomarkers in bladder cancer progression. Eur J Pharm Sci 2017; 98: 70–79.

159.

Tong

Ibeagha-Awemu

, et al. Identification and characterization of differentially expressed exosomal microRNAs in bovine milk infected with Staphylococcus aureus. BMC Genomics 2019; 20(1): 934.

160.

Sun

Aswath

Schroeder

, et al. MicroRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection. BMC Genomics 2015; 16: 806.

161.

Njock

Guiot

Henket

, et al. Sputum exosomes: promising biomarkers for idiopathic pulmonary fibrosis. Thorax 2019; 74(3): 309–312.

162.

Barceló

Castells

Bassas

, et al. Semen miRNAs contained in exosomes as non-invasive biomarkers for prostate cancer diagnosis. Sci Rep 2019; 9(1): 13772.

163.

Kodam

Ullah

Diagnostic and therapeutic potential of extracellular vesicles. Technol Cancer Res Treat 2021; 20: 15330338211041203.

164.

Tan

PPS

Hall

Chilian

, et al. Exosomal microRNAs in the development of essential hypertension and its potential as biomarkers. Am J Physiol Heart Circ Physiol 2021; 320(4): H1486–H1497.

165.

Merchant

Rood

Deegens

JKJ

, et al. Isolation and characterization of urinary extracellular vesicles: implications for biomarker discovery. Nat Rev Nephrol 2017; 13(12): 731–749.

166.

Nomura

Extracellular vesicles and blood diseases. Int J Hematol 2017; 105(4): 392–405.

167.

Han

Bartold

Ivanovski

The emerging role of small extracellular vesicles in saliva and gingival crevicular fluid as diagnostics for periodontitis. J Periodontal Res 2022; 57(1): 219–231.

168.

Muraoka

Jedrychowski

Yanamandra

, et al. Proteomic profiling of extracellular vesicles derived from cerebrospinal fluid of Alzheimer’s disease patients: a pilot study. Cells 2020; 14(1): 181.

169.

Zhao

Fan

Hsu

, et al. Extracellular vesicles as cancer liquid biopsies: from discovery, validation, to clinical application. Lab Chip 2019; 19(7): 1114–1140.

170.

Weng

Zhang

, et al. Therapeutic roles of mesenchymal stem cell-derived extracellular vesicles in cancer. J Hematol Oncol 2021; 14(1): 136.

171.

Urabe

Kosaka

Ito

, et al. Extracellular vesicles as biomarkers and therapeutic targets for cancer. Am J Physiol Cell Physiol 2020; 318(1): C29–c39.

172.

Han

Yang

Sun

, et al. Extracellular vesicles in cardiovascular disease: biological functions and therapeutic implications. Pharmacol Ther 2022; 233: 108025.

173.

Zheng

LaCourse

Song

, et al. Using extracellular vesicles in blood to diagnose paediatric tuberculosis. Nat Biomed Eng 2022; 6(8): 979–931.

174.

Gratpain

Mwema

Labrak

, et al. Extracellular vesicles for the treatment of central nervous system diseases. Adv Drug Deliv Rev 2021; 174: 535–552.

175.

Hoshino

Costa-Silva

Shen

, et al. Tumour exosome integrins determine organotropic metastasis. Nature 2015; 527(7578): 329–335.

176.

Kawamura

Iinuma

Wada

, et al. Exosome-encapsulated microRNA-4525, microRNA-451a and microRNA-21 in portal vein blood is a high-sensitive liquid biomarker for the selection of high-risk pancreatic ductal adenocarcinoma patients. J Hepatobiliary Pancreat Sci 2019; 26(2): 63–72.

177.

Elkrief

Ganne-Carrié

Manceau

, et al. Hepatocyte-derived biomarkers predict liver-related events at 2 years in child-Pugh class A alcohol-related cirrhosis. J Hepatol 2023; 79(4): 910–923.

178.

Chen

Zhao

, et al. Circulating exosomal mRNA profiling identifies novel signatures for the detection of prostate cancer. Mol Cancer 2021; 20(1): 58.

179.

Tian

Guo

, et al. Circulating exosomal mRNA signatures for the early diagnosis of clear cell renal cell carcinoma. BMC Med 2022; 20(1): 270.

180.

Joyce

Kerin

Dwyer

RM.

Exosome-encapsulated microRNAs as circulating biomarkers for breast cancer. Int J Cancer 2016; 139(7): 1443–1448.

181.

Tutrone

Lowentritt

Neuman

, et al. ExoDx prostate test as a predictor of outcomes of high-grade prostate cancer - an interim analysis. Prostate Cancer Prostatic Dis 2023; 26(3): 596–601.

182.

Beekman

Enciso-Martinez

Rho

, et al. Immuno-capture of extracellular vesicles for individual multi-modal characterization using AFM, SEM and Raman spectroscopy. Lab Chip 2019; 19(15): 2526–2536.

183.

D’Ascenzo

Femminò

Ravera

, et al. Extracellular vesicles from patients with acute coronary syndrome impact on ischemia-reperfusion injury. Pharmacol Res 2021; 170: 105715.

184.

Gargiulo

Morande

Largeot

, et al. Diagnostic and therapeutic potential of extracellular vesicles in B-cell malignancies. Front Oncol 2020; 10: 580874.

185.

Mazzariol

Camussi

Brizzi

, et al. Extracellular vesicles tune the immune system in renal disease: A focus on systemic lupus erythematosus, antiphospholipid syndrome, thrombotic microangiopathy and ANCA-vasculitis. Int J Mol Sci 2021; 22(8): 4194.

186.

Grangier

Branchu

Volatron

, et al. Technological advances towards extracellular vesicles mass production. Adv Drug Deliv Rev 2021; 176: 113843.

187.

Ostrowski

Carmo

Krumeich

, et al. Rab27a and rab27b control different steps of the exosome secretion pathway. Nat Cell Biol 2010; 12(1): 19–30; sup pp 1–13.

188.

Kim

Yoon

, et al. Large-scale generation of cell-derived nanovesicles. Nanoscale 2014; 6(20): 12056–12064.

189.

Miao

Meng

, et al. Genetic engineering cellular vesicles expressing CD64 as checkpoint antibody carrier for cancer immunotherapy. Theranostics 2021; 11(12): 6033–6043.

190.

Cacciamali

Villa

Dotti

3D cell cultures: evolution of an ancient tool for new applications. Front Physiol 2022; 13: 836480.

191.

Habanjar

Diab-Assaf

Caldefie-Chezet

, et al. 3D cell culture systems: tumor application, advantages, and disadvantages. Int J Mol Sci 2021; 22(22): 12200.

192.

Lee

JW.

3D spheroid cultures of stem cells and exosome applications for cartilage repair. Life 2022; 12(7): 939.

193.

Marchini

Gelain

Synthetic scaffolds for 3D cell cultures and organoids: applications in regenerative medicine. Crit Rev Biotechnol 2022; 42(3): 468–486.

194.

Haraszti

Miller

Stoppato

, et al. Exosomes produced from 3D cultures of MSCs by tangential flow filtration show higher yield and improved activity. Mol Ther 2018; 26(12): 2838–2847.

195.

Guo

Debbi

Zohar

, et al. Stimulating extracellular vesicles production from engineered tissues by mechanical forces. Nano Lett 2021; 21(6): 2497–2504.

196.

Adlerz

Patel

Rowley

, et al. Strategies for scalable manufacturing and translation of MSC-derived extracellular vesicles. Stem Cell Res 2020; 48: 101978.

197.

de Almeida Fuzeta

Bernardes

Oliveira

, et al. Scalable production of human mesenchymal stromal cell-derived extracellular vesicles under serum-/Xeno-free conditions in a microcarrier-based bioreactor culture system. Front Cell Dev Biol 2020; 8: 553444.

198.

Mitchell

Court

Mason

, et al. Increased exosome production from tumour cell cultures using the Integra CELLine culture system. J Immunol Methods 2008; 335(1-2): 98–105.

199.

Yan

Exosomes produced from 3D cultures of umbilical cord mesenchymal stem cells in a hollow-fiber bioreactor show improved osteochondral regeneration activity. Cell Biol Toxicol 2020; 36(2): 165–178.

200.

Cao

Wang

Tang

, et al. Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury. Stem Cell Res Ther 2020; 11(1): 206.

201.

Debbi

Guo

Safina

, et al. Boosting extracellular vesicle secretion. Biotechnol Adv 2022; 59: 107983.

202.

Boussadia

Lamberti

Mattei

, et al. Acidic microenvironment plays a key role in human melanoma progression through a sustained exosome mediated transfer of clinically relevant metastatic molecules. J Exp Clin Cancer Res 2018; 37(1): 245.

203.

Parolini

Federici

Raggi

, et al. Microenvironmental pH is a key factor for exosome traffic in tumor cells. J Biol Chem 2009; 284(49): 34211–34222.

204.

Savina

Furlán

Vidal

, et al. Exosome release is regulated by a calcium-dependent mechanism in K562 cells. J Biol Chem 2003; 278(22): 20083–20090.

205.

Garcia

Ontoria-Oviedo

González-King

, et al. Glucose starvation in cardiomyocytes enhances exosome secretion and promotes angiogenesis in endothelial cells. PLoS One 2015; 10(9): e0138849.

206.

Tse

Tan

Park

, et al. Microenvironmental hypoxia induces dynamic changes in lung cancer synthesis and secretion of extracellular vesicles. Cancers 2020; 12(10): 2917.

207.

Bister

Pistono

Huremagic

, et al. Hypoxia and extracellular vesicles: a review on methods, vesicular cargo and functions. J Extracell Vesicles 2020; 10(1): e12002.

208.

Ban

Lee

, et al. Low pH increases the yield of exosome isolation. Biochem Biophys Res Commun 2015; 461(1): 76–79.

209.

Mayer

Intracellular membrane fusion: SNAREs only?

Curr Opin Cell Biol 1999; 11(4): 447–452.

210.

Savina

Fader

Damiani

, et al. Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner. Traffic 2005; 6(2): 131–143.

211.

Rice

Scholz-Romero

Sweeney

, et al. The effect of glucose on the release and bioactivity of exosomes from first trimester trophoblast cells. J Clin Endocrinol Metab 2015; 100(10): E1280–E1288.

212.

Dorayappan

KDP

Wanner

Wallbillich

, et al. Hypoxia-induced exosomes contribute to a more aggressive and chemoresistant ovarian cancer phenotype: a novel mechanism linking STAT3/Rab proteins. Oncogene 2018; 37(28): 3806–3821.

213.

Otsuka

Yamamoto

Ochiya

Uncovering temperature-dependent extracellular vesicle secretion in breast cancer. J Extracell Vesicles 2020; 10(2): e12049.

214.

Hasan

Hama

Kogure

Low electric treatment activates rho GTPase via heat shock protein 90 and protein kinase C for intracellular delivery of siRNA. Sci Rep 2019; 9(1): 4114.

215.

Fukuta

Nishikawa

Kogure

Low level electricity increases the secretion of extracellular vesicles from cultured cells. Biochem Biophys Rep 2020; 21: 100713.

216.

Matsui

Sakamaki

Nakashima

, et al. Rab39 and its effector UACA regulate basolateral exosome release from polarized epithelial cells. Cell Rep 2022; 39(9): 110875.

217.

Onallah

Mannully

Davidson

, et al. Exosome secretion and epithelial-mesenchymal transition in ovarian cancer are regulated by Phospholipase D. Int J Mol Sci 2022; 23(21): 13286.

218.

Laulagnier

Grand

Dujardin

, et al. PLD2 is enriched on exosomes and its activity is correlated to the release of exosomes. FEBS Lett 2004; 572(1-3): 11–14.

219.

Verweij

Bebelman

Jimenez

, et al. Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling. J Cell Biol 2018; 217(3): 1129–1142.

220.

Song

Tang

Han

, et al. KIBRA controls exosome secretion via inhibiting the proteasomal degradation of rab27a. Nat Commun 2019; 10(1): 1639.

221.

Latifkar

Ling

Hingorani

, et al. Loss of Sirtuin 1 alters the secretome of breast cancer cells by impairing lysosomal integrity. Dev Cell 2019; 49(3): 393–408.e7.

222.

Ghossoub

Lembo

Rubio

, et al. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2. Nat Commun 2014; 5: 3477.

223.

Xiong

Wang

, et al. GHRH-stimulated pituitary small extracellular vesicles inhibit hepatocyte proliferation and IGF-1 expression by its cargo miR-375-3p. J Nanobiotechnol 2024; 22(1): 649.

224.

Corbett

Taatizadeh

, et al. Challenges and opportunities in exosome research-Perspectives from biology, engineering, and cancer therapy. APL bioengineering 2019; 3(1): 011503.

225.

Liu

, et al. Artificial exosomes for translational nanomedicine. J Nanobiotechnol 2021; 19(1): 242.

226.

Zha

Lin

, et al. Exosome-mimetics as an engineered gene-activated matrix induces in-situ vascularized osteogenesis. Biomaterials 2020; 247: 119985.

227.

Lee

Kim

, et al. Brain-targeted exosome-mimetic cell membrane nanovesicles with therapeutic oligonucleotides elicit anti-tumor effects in glioblastoma animal models. Bioeng Transl Med 2023; 8(2): e10426.

228.

Thamphiwatana

Angsantikul

Escajadillo

, et al. Macrophage-like nanoparticles concurrently absorbing endotoxins and proinflammatory cytokines for sepsis management. Proc Natl Acad Sci USA 2017; 114(43): 11488–11493.

229.

Dong

Gao

Zhang

, et al. Neutrophil membrane-derived nanovesicles alleviate inflammation to protect mouse brain injury from ischemic stroke. ACS Nano 2019; 13(2): 1272–1283.

230.

Welsh

Goberdhan

DCI

O’Driscoll

, et al. Minimal information for studies of extracellular vesicles (MISEV2023): from basic to advanced approaches. J Extracell Vesicles 2024; 13(2): e12404.

231.

Han

Jeong

Kim

, et al. Mesenchymal stem cell engineered nanovesicles for accelerated skin wound closure. ACS Biomater Sci Eng 2019; 5(3): 1534–1543.

232.

Yoo

Kim

, et al. Attenuation of tumor necrosis factor-α induced inflammation by umbilical cord-mesenchymal stem cell derived exosome-mimetic nanovesicles in endothelial cells. Tissue Eng Regen Med 2020; 17(2): 155–163.

233.

Liu

Sun

Zeng

, et al. Engineering and characterization of an artificial drug-carrying vesicles nanoplatform for enhanced specifically targeted therapy of glioblastoma. Adv Mater 2023; 35(41): e2303660.

234.

Jang

Kim

Yoon

, et al. Bioinspired exosome-mimetic nanovesicles for targeted delivery of chemotherapeutics to malignant tumors. ACS Nano 2013; 7(9): 7698–7710.

235.

Zhu

Gangadaran

Kalimuthu

, et al. Novel alternatives to extracellular vesicle-based immunotherapy - exosome mimetics derived from natural killer cells. Artif Cells Nanomed Biotechnol 2018; 46(sup3): S166–S179.

236.

Goh

Zou

Ong

, et al. Bioinspired cell-derived nanovesicles versus exosomes as drug delivery systems: a cost-effective alternative. Sci Rep 2017; 7(1): 14322.

237.

Yoon

Jeong

, et al. Generation of nanovesicles with sliced cellular membrane fragments for exogenous material delivery. Biomaterials 2015; 59: 12–20.

238.

, et al. Exosomes and biomimetic nanovesicles-mediated anti-glioblastoma therapy: a head-to-head comparison. J Control Release 2021; 336: 510–521.

239.

Wang

Abhange

Wen

, et al. Preparation of engineered extracellular vesicles derived from human umbilical cord mesenchymal stem cells with ultrasonication for skin rejuvenation. ACS Omega 2019; 4(27): 16317–22645.

240.

Choo

Kang

Kim

, et al. M1 macrophage-derived nanovesicles potentiate the anticancer efficacy of immune checkpoint inhibitors. ACS Nano 2018; 12(9): 8977–8993.

241.

Wen

Soliwoda

, et al. Cell-derived nanovesicles prepared by membrane extrusion are good substitutes for natural extracellular vesicles. Extracellular vesicle 2022; 1: 100004.

242.

Chen

Wang

Wei

, et al. Extracellular vesicles derived from different sources play various roles in diabetic retinopathy. Front Endocrinol 2022; 13: 1064415.

243.

Huang

Feng

, et al. Associations of age and sex with characteristics of extracellular vesicles and protein-enriched fractions of blood plasma. Aging Cell 2024; 24(1): e14356.

244.

Krupova

Leroux

Péchoux

, et al. Comparison of goat and cow milk-derived extracellular vesicle miRNomes. Sci Data 2023; 10(1): 465.

245.

Fordjour

Guo

, et al. A shared, stochastic pathway mediates exosome protein budding along plasma and endosome membranes. J Biol Chem 2022; 298(10): 102394.

246.

Ohno

Takanashi

Sudo

, et al. Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells. Mol Ther 2013; 21(1): 185–191.

247.

Zhang

, et al. Engineered extracellular vesicles for cancer therap. Adv Mater 2021; 33(14): e2005709.

248.

Kang

Mun

Chun

, et al. Engineered small extracellular vesicle-mediated NOX4 siRNA delivery for targeted therapy of cardiac hypertrophy. J Extracell Vesicles 2023; 12(10): e12371.

249.

Kooijmans

SAA

Fliervoet

LAL

van der Meel

, et al. PEGylated and targeted extracellular vesicles display enhanced cell specificity and circulation time. J Control Release 2016; 224: 77–85.

250.

Zhang

, et al. Engineered neutrophil-derived exosome-like vesicles for targeted cancer therapy. Sci Adv 2022; 8(2): eabj8207.

251.

Liu

Wang

, et al. Engineered extracellular vesicles and their mimetics for cancer immunotherapy. J Control Release 2022; 349: 679–698.

252.

Zhang

, et al. Engineering exosomes for bone defect repair. Front Bioeng Biotechnol 2022; 10: 1091360.

253.

Osteikoetxea

Silva

Lázaro-Ibáñez

, et al. Engineered cas9 extracellular vesicles as a novel gene editing tool. J Extracell Vesicles 2022; 11(5): e12225.

254.

Yang

, et al. Engineered extracellular vesicles with SHP2 high expression promote mitophagy for Alzheimer’s disease treatment. Adv Mater 2022; 34(49): e2207107.

255.

Thomas

Eldridge

Nosrati

, et al. WNT3A-loaded exosomes enable cartilage repair. J Extracell Vesicles 2021; 10(7): e12088.

256.

Hall

Prabhakar

Balaj

, et al. Delivery of therapeutic proteins via extracellular vesicles: review and potential treatments for Parkinson’s disease, glioma, and schwannoma. Cell Mol Neurobiol 2016; 36(3): 417–427.

257.

Xie

Zhang

Jiang

Current knowledge on exosome biogenesis, cargo-sorting mechanism and therapeutic implications. Membranes 2022; 12(5): 498.

258.

Ferreira

da Rosa Soares

Ramalho

, et al. LAMP2A regulates the loading of proteins into exosomes. Sci Adv 2022; 8(12): eabm1140.

259.

Villarroya-Beltri

Baixauli

Gutiérrez-Vázquez

, et al. Sorting it out: regulation of exosome loading. Semin Cancer Biol 2014; 28: 3–13.

260.

Yokoi

Villar-Prados

Oliphint

, et al. Mechanisms of nuclear content loading to exosomes. Sci Adv 2019; 5(11): eaax8849.

261.

Santangelo

Giurato

Cicchini

, et al. The RNA-Binding protein SYNCRIP is a component of the hepatocyte exosomal machinery controlling MicroRNA sorting. Cell Rep 2016; 17(3): 799–808.

262.

Ferguson

Nguyen

Exosomes as therapeutics: the implications of molecular composition and exosomal heterogeneity. J Control Release 2016; 228: 179–190.

263.

Villarroya-Beltri

Gutiérrez-Vázquez

Sánchez-Cabo

, et al. Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs. Nat Commun 2013; 4: 2980.

264.

Garcia-Martin

Wang

Brandão

, et al. MicroRNA sequence codes for small extracellular vesicle release and cellular retention. Nature 2022; 601(7893): 446–451.

265.

Reshke

Taylor

Savard

, et al. Reduction of the therapeutic dose of silencing RNA by packaging it in extracellular vesicles via a pre-microRNA backbone. Nat Biomed Eng 2020; 4(1): 52–68.

266.

Wozniak

Adams

King

, et al. The RNA binding protein FMR1 controls selective exosomal miRNA cargo loading during inflammation. J Cell Biol 2020; 219(10): e201912074.

267.

Zhou

Gao

, et al. Fusion protein engineered exosomes for targeted degradation of specific RNAs in lysosomes: a proof-of-concept study. J Extracell Vesicles 2020; 9(1): 1816710.

268.

Wang

Chen

Zhao

, et al. Engineered exosomes with ischemic myocardium-targeting peptide for targeted therapy in myocardial infarction. J Am Heart Assoc 2018; 7(15): e008737.

269.

Yao

Lyu

Yoo

, et al. Engineered extracellular vesicles as versatile ribonucleoprotein delivery vehicles for efficient and safe CRISPR genome editing. J Extracell Vesicles 2021; 10(5): e12076.

270.

Sterzenbach

Putz

Low

, et al. Engineered exosomes as vehicles for biologically active proteins. Mol Ther 2017; 25(6): 1269–1278.

271.

Luan

Sansanaphongpricha

Myers

, et al. Engineering exosomes as refined biological nanoplatforms for drug delivery. Acta Pharmacol Sin 2017; 38(6): 754–763.

272.

Wei

Chen

Zhao

, et al. Mononuclear phagocyte system blockade using extracellular vesicles modified with CD47 on membrane surface for myocardial infarction reperfusion injury treatment. Biomaterials 2021; 275: 121000.

273.

Salarpour

Forootanfar

Pournamdari

, et al. Paclitaxel incorporated exosomes derived from glioblastoma cells: comparative study of two loading techniques. Daru 2019; 27(2): 533–539.

274.

Gao

Zhang

Xia

, et al. Berberine-loaded M2 macrophage-derived exosomes for spinal cord injury therapy. Acta Biomater 2021; 126: 211–223.

275.

Zhang

Lee

Zhu

, et al. Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo. Am J Physiol Lung Cell Mol Physiol 2017; 312(1): L110–l121.

276.

Haney

Klyachko

Zhao

, et al. Exosomes as drug delivery vehicles for Parkinson’s disease therapy. J Control Release 2015; 207: 18–30.

277.

Kalani

Chaturvedi

Kamat

, et al. Curcumin-loaded embryonic stem cell exosomes restored neurovascular unit following ischemia-reperfusion injury. Int J Biochem Cell Biol 2016; 79: 360–369.

278.

Wan

Zhong

Pan

, et al. Exosome-mediated delivery of cas9 ribonucleoprotein complexes for tissue-specific gene therapy of liver diseases. Sci Adv 2022; 8(37): eabp9435.

279.

de Abreu

Ramos

Becher

, et al. Exogenous loading of miRNAs into small extracellular vesicles. J Extracell Vesicles 2021; 10(10): e12111.

280.

Sudji

Subburaj

Frenkel

, et al. Membrane disintegration caused by the steroid saponin digitonin is related to the presence of cholesterol. Molecules 2015; 20(11): 20146–20160.

281.

Orefice

NS.

Development of new strategies using extracellular vesicles loaded with exogenous nucleic acid. Pharmaceutics 2020; 12(8): 705.

282.

Elsharkasy

Nordin

Hagey

, et al. Extracellular vesicles as drug delivery systems: why and how? Adv Drug Deliv Rev 2020; 159: 332–343.

283.

Yáñez-Mó

Siljander

Andreu

, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 2015; 4(1): 27066.

284.

Xia

Zhang

Liu

, et al. Immunogenicity of extracellular vesicles. Adv Mater 2024; 36(33): e2403199. DOI: 10.1002/adma.202403199

285.

Yang

Zhang

, et al. Progress, opportunity, and perspective on exosome isolation - efforts for efficient exosome-based theranostics. Theranostics 2020; 10(8): 3684–3707.

286.

Patil

Sawant

Kunda

NK.

Exosomes as drug delivery systems: a brief overview and progress update. Eur J Pharm Biopharm 2020; 154: 259–269.

287.

Staubach

Bauer

Tertel

, et al. Scaled preparation of extracellular vesicles from conditioned media. Adv Drug Deliv Rev 2021; 177: 113940.

288.

Lai

Chau

Chen

, et al. Exosome processing and characterization approaches for research and technology development. Adv Sci 2022; 9(15): e2103222.

289.

Mathieu

Névo

Jouve

, et al. Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9. Nat Commun 2021; 12(1): 4389.

290.

Spitzberg

Ferguson

Yang

, et al. Multiplexed analysis of EV reveals specific biomarker composition with diagnostic impact. Nat Commun 2023; 14(1): 1239.

Extracellular vesicles: From large-scale production and engineering to clinical applications

Abstract

Keywords

Introduction

Clinical applications of EVs

Therapeutic potential of EVs

Vaccines

Diagnostic potential of EVs

Large-scale production of EVs

Large-scale cell culture platforms

Cell stimulation or genetic modification to increase EV yield per cell

Broken cell membrane assembles into vesicle particles

Production of engineered EVs

Indirect engineering of EVs by modifying EV-producing cells

Direct engineering of EVs

Challenges in EV industry

Concluding remarks and future perspective

Footnotes

Data availability

Declaration of conflicting interests

Funding

ORCID iD

References