Research progress and application prospect of engineered small extracellular vesicles

Indirect sEvs engineering method

Endogenous loading method is to make the target drug appear in the donor cells of the vesicle. This is an engineered loading method based on the donor cells ³⁸ . This method of engineered loading is based on the generation mechanism of sEvs, these donor cells sort these cargoes into the vesicles ³⁹ . Studies have shown that the type and condition of donor cells can impact the yield of sEvs ⁴⁰ , by appropriately nurturing or modifying the donor cell, it is possible to obtain sEvs with the desired characteristics. The main method to achieve this step is to co-incubate the donor cells with drugs; the expression of therapeutic substances required for research in the donor cells of the vesicles requires the introduction of exogenous nucleic acid sequences into the cells, relying on transfection methods ⁴¹ . When sEvs are formed in donor cells, the cells actively sort specific proteins, nucleic acids (such as mRNA, miRNA, and lncRNA), and other molecules into sEvs. By introducing exogenous nucleic acid sequences into donor cells, the cells’ own sorting mechanism can be used to integrate these nucleic acids and expressed proteins into sEvs. This method avoids additional modifications of sEvs after secretion and retains their natural biocompatibility and membrane integrity. The endogenous loading method is an attractive method for creating desirable sEvs. First of all, it relies on the normal process of cells producing sEvs, which means that there is no need to consider the immune reaction when entering the organism, this method is safe^42,43; moreover, the conditions are controllable, and the flexibility is high in controlling the functions of engineered sEvs. The main loaded therapeutic substances include proteins, RNA, small nucleic acids, hydrophobic small molecule drugs, and so on ⁴⁴ .

Transfection

Transfection is a mechanism by which appropriate exogenous sequences are found and transfected into cells to express drugs within cells, and donor cells are used to produce sEvs, thereby collecting drug-loaded sEvs. The transfection process can be accomplished through physical methods [electroporation ⁵⁴ , gene gun ⁵⁵ , sonication ⁵⁶ , chemical methods (calcium phosphate method ⁵⁷ ), biological methods (mainly virus-mediated ⁵⁸ ), and so on].

In general, chemical transfection methods have lower efficiency, and the efficiency is influenced by the cell line ⁵⁹ while physical methods may cause significant damage to the cells ⁶⁰ . Strategies based on viral transfection have become popular in recent years; due to its stable and effective transfection ability, it has been applied in sEvs-based delivery systems ⁶¹ , this transfection method is suitable for efficient loading of a variety of cells ⁶² . Retrovirus, lentivirus ⁶² , adenovirus ⁶³ , and adeno-associated viruses have been widely used as viral vectors for gene delivery ⁶⁴ . After the viral vector is transfected into the donor cells, the expressed therapeutic substances may be loaded into sEvs, which bind to the target cells and play a regulatory role ⁶⁵ . Transfection facilitates integration of non-native genetic constructs into donor cells and makes the cells acquire new phenotypes, and these sEvs produced can be modified ⁶⁶ . With the continuous development of cell biology and the deepening of sEvs modification, this method has become a routine tool for controlling and studying the gene function of donor cells. However, the downside is that it is relatively complicated and cumbersome, and its effectiveness after transfection is uncertain, which may lead to abnormal gene expression in donor cells ⁶⁷ . In addition, the encapsulation of drugs and the negative effects of viral transfection cannot be guaranteed, such as cell malformations, which also need to be studied in depth.

Due to the high biocompatibility and low immunogenicity of sEvs, as well as their ability to efficiently penetrate biological barriers, they have become ideal drug carriers, but their disadvantages are also very obvious. The advantages and disadvantages of the endogenous loading method are shown in Table 1. From the perspective of cytotoxicity, the co-culture method requires high concentrations of drugs to act on donor cells for a long time. The drugs will continuously and directly act on various metabolic links of the donor cells, interfering with their normal life activities. Therefore, the cytotoxicity is usually higher than that of the transfection method and is concentration-dependent. In addition, the drugs will go through two major steps: transmembrane and sorting, which limits the loading efficiency. The cytotoxicity of conventional transfection methods primarily stems from the transfection reagent itself and the interaction between the transfection complex and the cell membrane. With technological breakthroughs, an increasing number of transfection reagents are now demonstrating lower biotoxicity. Furthermore, because drug loading is a passive process, the amount of drug carried by exosomes obtained through co-culture varies greatly. Transfection, on the other hand, involves genetically modified donor cells, whose markers are relatively consistent with the cargo contents.

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Table 1.

The advantages and disadvantages of the endogenous loading method.

Disadvantages	Specific description	Advantages	Specific description
Low Loading Efficiency 23	1. Relying on passive uptake or active sorting mechanisms of parental cells, the proportion of drugs entering sEvs is low (especially hydrophilic molecules) 2. The drug loading capacity is limited by the physicochemical properties of the drug (such as lipid solubility) and the metabolic state of the cell	Low immunogenicity and high biocompatibility 23	1. Non-toxic, suitable for long-term treatment and long-term circulation in the body 2. Natural lipid and protein components can be metabolized and degraded by the body
Cytotoxicity Risk 68	1. Drugs (such as chemotherapeutic agents) may damage parental cells and affect their ability to secrete sEvs 2. Some genetic engineering methods (such as transfection) may cause cell stress or functional abnormalities.	Active targeting and cross-physiological barrier capabilities significantly improve drug delivery efficiency 5	1. It can specifically recognize target cells through membrane surface integrins and chemokine receptors 2. It can penetrate the blood–brain barrier, placental barrier and other physiological barriers that are difficult to break through by traditional delivery systems, and is suitable for brain diseases and fetal treatment.
Complex Production Process 69	It is necessary to culture parent cells on a large scale and optimize culture conditions (such as culture medium composition and bioreactor parameters), and the steps for sEvs isolation and purification are cumbersome (such as ultracentrifugation and size exclusion chromatography) and costly.	Multifunctional drug delivery compatibility 70	The lipid membrane shields drugs such as nucleic acids and proteins from enzymatic degradation or oxidative damage, maintaining drug stability
Safety Concerns 71	1. Parental cells may carry oncogenic proteins or viral contaminants, which are delivered to recipient cells through sEvs 2. Immunogenic risks (such as misfolding of membrane proteins) may be introduced during the gene editing process.	It can be modified and optimized to meet diverse treatment needs.	1. Enhance targeting, drug loading or tracking capabilities through gene editing (such as expression of targeting ligands) or chemical modification (such as connection to antibodies, fluorescent markers). 2. Reduce the risk of immune rejection using exosomes secreted by the patient’s own cells (such as stem cells, immune cells)

Direct sEvs engineering method

Due to the limitations of sEvs’ endogenous loading methods, the development of exogenous loading methods is necessary. Different from endogenous loading method, exogenous loading method loads drugs directly into isolated sEvs ⁷² . Commonly used exogenous loading methods include co-incubation, extrusion, ultrasonic, electroporation, freeze−thaw cycle, and so on ⁷³ . See Table 2 for details.

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Table 2.

The advantages and disadvantages of the exogenous loading method.

Exogenous loading method	Advantage	Disadvantage	Cargos
Co-incubation	The operation process is simple and convenient, and causes minimal damage to the membrane.	The loading efficiency is low and it is difficult to obtain ideal results during experimental operations.	Nucleic Acids: microRNA 74 ; siRNA 75 Proteins: β-glucuronidase 76 ; Catalase 77 Hydrophobic small molecule compounds, such as Curcumin 78 and PTX 79
Ultrasonic	Widely used, high efficiency, no additional modifications required, reducing the risk of biological toxicity.	Due to the influence of mechanical stress, membrane structure and surface proteins may be damaged.	Nucleic Acids: siRNA 80 , MicroRNA 81 Hydrophilic/hydrophobic compounds, DOX 82 Hollow gold nanoparticle—hybrids 83 Proteins: tripeptidyl peptidase 1 (TPP1) 84
Electroporation	High controllability.	The experimental requirements are high, the membrane structure may be damaged, and the load may agglomerate due to the electric field.	Small molecule drugs, such as PTX 85 , DOX 86 Nucleic Acids: siRNA 87 , microRNA 88 Proteins: insulin 89
Freeze−Thaw cycle	Widely used and generally gentle.	Low production efficiency, unstable Evs and limited adaptability.	Proteins and peptides durgs90,91 Nucleic Acids: microRNA 92
Extrusion	The loading efficiency is high, and the particle size is relatively uniform due to passing through the porous membrane with a specific diameter.	May cause damage to extracellular vesicle structure.	Since high pressure passes through the pore membrane, substances with relatively large molecular weight are not suitable. The substances identified in the existing literature include chemical small molecule drugs 93 , Hydrophobic drugs 94 , Catalase 77 .

With the deepening of research, many loading methods have been developed, but unfortunately there is currently no efficient and balanced drug delivery method is an important factor restricting clinical application. Although various drug loading methods differ significantly in principle and operation, they exhibit quantifiable differences in key performance indicators such as loading efficiency, stability, and reproducibility. Loading efficiency is the primary indicator for evaluating drug loading methods. Co-incubation methods typically exhibit stable but low loading efficiency and are often used for loading hydrophilic small molecule drugs. The loading efficiency of small molecule drugs is similar to that of nucleic acid drugs and protein molecule drugs, generally speaking, the load efficiency will not exceed 15%^{74,75,95–97}. Similar to extrusion, ultrasonic methods utilize mechanical shear force to distort the membrane structure, enabling efficient intercalation of small molecule drugs^98,99. Ultrasonic loading supports a wider variety of drug types than extrusion loading, and its loading efficiency is significantly higher. Electroporation loading efficiency fluctuates considerably, varying greatly with different electric field conditions. Electroporation is suitable for loading small molecule drugs, nucleic acid drugs, and protein drugs; however, nucleic acid molecules exhibit the lowest loading efficiency among all loaded drugs^89,100–102. The freeze−thaw cycle method has a relatively narrow application, mainly used for loading protein molecules. Co-incubation, because it does not damage the membrane structure, has a significant advantage in stability. Ultrasonic, electroporation, and extrusion methods cause greater damage to the membrane structure, and therefore their stability is less than satisfactory.

From a practical perspective, among the above drug loading methods, due to the safety of electroporation, this method is only suitable for small-scale preparation under safe conditions and cannot form a certain production scale in reality, and will cause the loading to aggregate ¹⁰³ . The ultrasonic method is likely to disrupt the membrane structure ¹⁰⁴ ; the extrusion method has high requirements on equipment and cost, and the extrusion method is sometimes used with saponin, which has a certain degree of toxicity and is not suitable for large-scale use ¹⁰⁵ . Although the co-incubation method is low in efficiency, overall it is the simplest among all methods. It does not destroy the stability of the membrane, is low cost and highly safe ¹⁰⁶ . In fact, during the co-incubation operation, reasonable temperature and pH can also be adjusted, or gentle stirring can be used to improve loading efficiency. Therefore, the co-incubation method has broad application prospects in clinical applications ¹⁰⁷ .

Genetic engineering

The approach of genetic engineering involves directing the fusion of a protein or polypeptide sequence to a selected membrane protein sequence ¹²³ . This can also be referred to as targeted peptide technology. Targeted peptide technology is currently the most widely used membrane modification strategy ¹²⁴ . Using genetic technology, the target gene is inserted, deleted or modified at specific sites, thereby enhancing the targeted delivery function of sEvs ¹²⁵ . Protein fusion is presented through genetic techniques by editing sequences to construct fusion proteins and fuse the target protein with the membrane protein so as to improve the therapeutic ability of sEvs^123,126,127. Genetic engineering is the basis for modifying the surface of exosomes to enhance their targeting ability. Specific proteins can be transplanted onto the exosome membrane by transfecting plasmid vectors. As shown in Figure 2, current research on modifying membrane structures through genetic engineering usually selects lysosomal membrane proteins represented by Lamp-2b^128–132, and tetraspin proteins represented by CD63, CD9, and CD81^133–136. Among them, since Lamp-2b has a signal peptide, Lamp-2b is often the subject of experiments ¹³⁷ . The N-terminus of Lamp-2b is located on the extracellular side of the membrane and can be connected to the target sequence ¹²³ . Liang et al. ¹²⁸ constructed a plasmid and linked the chondrocyte affinity peptide to the membrane protein Lamp-2b to target the obtained sEvs to chondrocytes. In some studies, a glycosylation motif was added to the N-terminus of the peptide-Lamp-2b fusions to enhance the expression of the fusion protein and protect the target peptide from shearing or degradation^138–140. To inhibit the loss of peptides in vivo, Hung et al. designed a fusion protein between the targeting peptide and Lamp-2b to contain glycosylation sequences at different positions. The introduction of the glycosylation motif both protected the peptide from degradation and resulted in increased overall expression of the Lamp-2b fusion protein in cells and sEvs ¹⁴¹ . In addition to Lamp-2b as the target for protein fusion, tetraspanin protein family is also often chosen as the target for protein fusion^135,136. Based on membrane fusion technology, Li et al. ¹³³ constructed engineered sEvs overexpressing CD63-VEGFC fusion protein, the engineered sEvs can be successfully delivered to lymphatic endothelial cells in mice, significantly improving lymphedema in mice. Liang et al. ¹³⁵ introduced tumor cell targeting function by expressing CD63 transmembrane protein and Apo-A1 sequence in 293T cell hosts and delivered the generated sEvs to HepG2 cells. Genetic engineering is a modification strategy that often uses highly expressed membrane proteins in sEvs as target protein modification sites. In addition to the most commonly used Lamp-2b and tetraspanins, it also includes platelet-derived growth factor receptor, lactadherin, glycosylphosphatidylinositol, epidermal growth factor receptor, human epidermal growth factor receptor 2, and so on^142–145. In addition to enhancing the targeted delivery capability, protein fusion can also enable sEvs to have a certain ability of drug polymerization. Li et al. ¹³⁴ improved the RNA loading efficiency of sEvs by constructing a fusion protein in which the membrane protein CD9 was fused to HuR (an RNA-binding protein that interacts with MiR-155), in donor cells, fused CD9-HuR successfully enriched MiR-155 into sEvs when MiR-155 was overexpressed.

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Figure 2.

Genetic engineering methods improve the targeting and transport function of sEvs. In simple terms, it involves using genetic engineering to splice sequences and transfect them into donor cells, fusing the targeting peptide onto the membrane proteins of vesicles. The Evs produced by the donor cells then possess high targeting capabilities. Figure 2 was created using “biorender” (biorender.com)

Chemical modification

Chemical principles are used to modify membranes to enhance therapeutic capabilities ¹⁴⁶ . Mainly includes covalent modification and non-covalent modification ¹⁴⁷ . Among covalent modifications, the principle of click chemistry is the most commonly used. It was proposed by Nobel Prize winner in Chemistry K. Barry Sharpless in 2001 and refers to a type of highly efficient, highly selective, modular chemical reaction. The core features are mild reaction conditions, high yield, few by-products, and the ability to be completed quickly in a complex system, just like two molecules “clicking” together precisely. In the biomedical field, “click chemistry” can be applied in the fields of biomolecule labeling and imaging, drug development and delivery, diagnosis and biosensing. As shown in Figure 3, the principle of click chemistry which is the most common copper-catalyzed azide-alkyne cycloaddition (CuAAC) in click chemistry, is that the amino groups of membrane proteins can be easily modified with alkyne groups through the condensation reaction of EDC-NHS [1-ethyl-(carbodiimide-n-hydroxysuccinimide)] ¹⁴⁸ . Alkyne-labeled proteins can then be bioorthogonally coupled to azide-containing reagents via a copper-catalyzed azide-alkyne cycloaddition (CuAAC) “click reaction” ¹⁴⁹ .

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Figure 3.

Schematic diagram of chemical modification basics. (a) Covalent modification, using the principle of “click chemistry,” EDC/NHS condenses the alkyne group of the alkyne-containing compound onto the amino group of the protein; then a bioorthogonal reaction with an azide-containing reagent is carried out under copper ion catalysis. (b) Non-covalent modification, which are hydrophobic insertion, aptamer modification, and electrostatic interaction, aim to improve the uptake of vesicles by target cells or tissues. Figure 3(a) and (b) was created using “biorender” (biorender.com).

Treating Ev membranes with covalent modifications allows highly specific binding, meaning drugs or other molecules can be precisely attached to the membrane, thereby enhancing targeting and therapeutic efficacy^20,150,151. To develop engineered exosomes for glioma, Jia et al. ¹⁵⁰ used sEvs produced by RAW264.7 cells to study the NRP-1 transmembrane protein overexpressed in glioma cells, the research team used EDC-NHS to condensate 4-pentynoic acid with phosphatidylethanolamine on the exosome membrane, then, the REG with azide (NRP-1 targeting peptide) and the alkyne group undergo a cycloaddition reaction using the principle of click chemistry to couple to the membrane through chemical bonds, thereby obtaining targeting ability. Considering that high concentrations of copper ions are toxic to cells and cause physiological disturbances to sEvs, a bioorthogonal reaction without copper ion catalysis is a more suitable approach ¹⁵² . Bertozzi et al. developed a metal-free variant, strain-promoted azide-alkyne cycloaddition (SPAAC), by careful design and modification of the alkyne group, which has been used to label glycoproteins on the cell surface in vitro and in vivo without significant cytotoxicity. This also led to bioorthogonal reactions, which use the principles of click chemistry to undergo chemical reactions in vivo without interfering with their own biochemical reactions. However, some chemists were not satisfied with the second-order reaction rate of SPAAC, so Blackman et al. successfully developed the inverse electron demand Diels-Alder (iEDDA) reaction between the cycloaddition of s-tetrazine and trans-cyclooctene (TCO) derivatives, resulting in a copper-free click chemistry reaction that is faster than the SPAAC reaction. Blackman is a chemist, and his achievements were mentioned in the two articles.^153,154 These reaction methods are all covalent binding, which does not lead to changes in the size and function of sEvs ¹⁵⁵ , and also enhanced targeting capacities. “Click” chemistry usually involves fast and efficient reactions that can complete a large number of modifications in a short period of time ¹⁴⁶ . Such efficiency is crucial for future large-scale production and applications. “Click” chemistry is different from general traditional chemistry ¹⁵⁶ . The reaction of traditional chemistry must take into account changes in temperature and pressure, which often leads to the destruction of the Phospholipid bilayer in biomedical experiments ¹⁴⁸ . “Click” chemistry reacts very quickly, and has no negative impact on sEvs size or bioactivity ¹⁰⁴ . This also fully demonstrates that the application of “click” chemical principles is a suitable method for surface modification ¹⁴⁸ .

Inserting Phospholipid molecules into the lipid bilayer (hydrophobic insertion) is not a covalent coupling process, as it does not form a covalent bond with the membrane. Since the vesicle membrane is a Phospholipid bilayer, Phospholipid molecular binding with functional groups can be inserted into Ev membranes by hydrophobic interactions ¹⁵⁷ . It is also a method to enhance targeting and drug delivery efficiency. Kim et al. ¹⁵¹ used DSPE-PEG-AA to modify the surface of sEvs released by autologous macrophages. PEG (polyethylene glycol) prolonged the blood circulation time of drug carriers in vivo to target the sigma receptors overexpressed in lung cancer cells. DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine) is a Phospholipid and is fused to the Phospholipid membrane, thus making a rational modification. Cheng et al. combined the receptor binding domain (RBD) targeting SARS-CoV-2 spike protein to the linking molecule DSPE-PEG-NHS to form RBD-PEG-DSPE, which was inserted onto the surface of Evs derived from lung glomus cells by co-culture, and loaded mRNA vaccine into Evs by electroporation. Develop novel vaccines targeting lung mucosal cells and alveolar cells ¹⁵⁸ . In addition to the hydrophobic insertion method, there are many other non-covalent binding methods. For example, since the sEvs membrane carries a negative charge, a portion of positive charge is added to the sEvs membrane to enhance the targeting efficiency of the biomembrane by utilizing the principle of multivalent electrostatic interactions ¹⁵⁹ , ¹⁶⁰ ; and aptamers are used for surface modification ¹⁶¹ .

Although chemical modification of membranes allows for rapid and efficient enhancement of vesicles with high targeting capabilities and efficient drug delivery, there are still challenges and drawbacks. For example, the surface complexity of vesicles produced by different cells varies, which may affect the efficiency of the reaction and even endanger the structure and function of the carrier^162–164.

In addition, the use of chemically modified vesicles may encounter issues of cytotoxicity in clinical transformation.

To enhance the targeting of therapeutic Evs, in addition to the most commonly used genetic engineering and chemical modification methods such as peptide fusion and hydrophobic insertion, and aptamer modification to obtain engineered exosomes, physical principles can also be used to achieve more precise targeting ¹⁶⁵ .

Physical stimulation

The integration of Engineered EVs with external physical stimuli (such as ultrasound, laser, and magnetic fields), can achieve more precise targeted delivery. Villa et al. ¹¹⁶ used pretreated myo-exosomes, immobilized them on ferromagnetic nanotubes, to achieve controlled and precise delivery of myo-exosomes to skeletal muscle, promoting beneficial muscle responses in muscular dystrophy mice. In addition to using magnetic fields, ultrasound is also a highly efficient method to enhance the targeting of nanovesicles. In myocardial infarction research, adipose stem cells were pretreated to secrete exosomes (ADSC-EXO-SDF-1α) that highly express stromal cell-derived factor 1α (SDF-1α). A dual-modified targeted nanobubbles (TNBCD81-cRGD) were designed—using perfluoropropane (C3F8) as the gas core and phospholipid DSPC as the shell, with a cyclic arginine-glycine-aspartic peptide (cRGD) targeting the myocardium and an exosome-anchored antibody (anti-CD81) simultaneously attached to the surface. Triggered by low-intensity pulsed ultrasound (LIPUS), the nanobubbles directionally burst, releasing exosomes. The greatest advantage of this method is its high spatiotemporal control precision, allowing for non-invasive focusing on tiny deep regions within the body, achieving on-demand, targeted release ¹⁶⁶ . Compared with gene engineering targeted peptide fusion technology and membrane modification technology based on chemical principles, the biggest advantage of physical intervention is that it makes up for the lack of targeting while achieving “spatiotemporally controllable” treatment. The physical signal can be turned on or off at any time, and the timing and duration of treatment can be artificially controlled.

Clinical application

Since there are few discussions on the clinical application of engineered Evs, the translation rate of results needs to be improved. Although the application of engineered exosomes in clinical treatment is still in the early exploration stage, its huge therapeutic potential based on its unique delivery mechanism and combination with new biomedical materials has driven academia and industry to actively plan its clinical transformation path.

As of 2025, there are nearly 560 exosome clinical drugs under development around the world, but most of them are in the early stages, and the field as a whole is still in the exploration and verification stage. There are two main types of Evs used in clinical trials, namely, human tissue/cell exosomes and plant exosomes ¹⁸⁴ . So far, the complete process and results of clinical trials using exosomes from human samples have been reported; however, plant-derived exosomes are still in their infancy, and there is currently no data from clinical trials ¹⁸⁵ . In an analysis of exosomes in human samples under different conditions, a total of 116 trials were recorded, of which 50% were biomarker applications, 28% therapeutic, 5.1% drug delivery studies, and the least related to vaccine research ¹⁸⁴ . The Globaldata database shows that the largest clinical application area of Evs is oncology (54%), followed by central nervous system diseases (13%) and infectious diseases (8%) ¹⁸⁶ . Because it is released under pathological and physiological conditions such as cancer, immune response, neurodegeneration, and cell death, and contains intracellular proteins, nucleic acids and other substances, it can be used as a clinical marker for disease diagnosis and assessment of the severity of the disease ¹⁸⁷ , its internal microRNA is the most commonly used tissue-specific biomarker ¹⁶⁵ , in cardiovascular diseases, the levels of microRNAs related to them are increased. Measuring the levels of microRNAs inside Evs can provide a strong basis for disease diagnosis ¹⁸⁸ . In addition, in the field of extracellular vesicle diagnostic applications, most of the cancer diagnoses that account for the highest proportion adopt liquid biopsy, which is a minimally invasive and rapid method for in vitro diagnosis of tumors. Because the bioactive substances carried by tumor Evs are expressed differently in normal people and tumor patients, the detection of markers can help improve the specificity and sensitivity of early diagnosis^{186,189–191}.

Generally, human exosomes are concentrated using differential centrifugation and ultrafiltration, and then identified through electron microscopy and detection of relevant biomarkers ¹⁸⁵ . To ensure their bioactivity, compliance with Good Manufacturing Practices (GMP) is fundamental to successful clinical trials. In recent years, GMP-grade extracellular vesicle production methods have focused on cell type, culture environment and system, dissociation enzymes, and culture media, and further purification is required after production ¹⁸⁵ . It generally involves three steps: removing cells and cell debris; concentrating the conditioned medium; and purifying the product^185,192.

While most applications rely on biomarkers for diagnosis, a growing number of clinical trials are exploring drugs that utilize Evs as therapeutic agents. A search of clinicaltrials.gov yielded the following Table 4.

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Table 4.

Clinical trial situation.

Types of diseases	Clinical disease	Clinical number	Source	Phase	Intervention/methods	Results
Cancer	Recurrent Malignant Gliomas	NCT01550523	Tumor cells	I	Loaded with IGF1R anti sense molecule	IGF-IR downregulation to < or = 10%
	Lung cancer	NCT01159288	Dendritic Cell	II	Vaccination	No reported
	Pancreatic cancer	NCT03608631	MSCs	I	Loaded with KrasG12D siRNA	Ongoing
	Colon Cancer	NCT01294072	Plant	I	Loaded with curcumin	Ongoing
Neurological diseases	Craniofacial Neuralgia	NCT04202783	Exosome products containing neonatal stem cell	Not Applicable	Ultrasound delivery of intravenously-infused exosomes	Suspended
	Progressive Multiple Sclerosis	NCT07146087	MSCs	I	Intravenous infusions of allogeneic mesenchymal stem cell (MSC)-derived exosomes	Suspended
	Alzheimer’s Disease	NCT04388982	Allogenic Adipose Mesenchymal Stem Cells	I and II	Nasal drip	Cognitive function has been improved but There were no significant differences in altered amyloid or tau deposition
	Trigeminal Neuralgia	NCT05152368	Allogeneic Adult Umbilical Cord Stem Cells	I	Intranasal instillation	Ongoing
	Acute Ischemic Stroke	NCT06138210	Human induced pluripotent stem cell	I	Intravenous injection	Ongoing
Infectious diseases	Pulmonary Infection	NCT04544215	Mesenchymal progenitor cells	I and II	Aerosol inhalation	Suspended
	COVID-19 Infection	NCT04969172	No reported	II	Exosomes overexpressing CD24	No reported
	COVID-19 Infection	NCT05216562	MSCs	II and III	Intravenous injection	No reported
	COVID-19 Infection	NCT04276987	MSCs	I	Aerosol inhalation	Different degrees of resolution of pulmonary lesions
	COVID-19 Infection	NCT04602442	MSCs	II	Aerosol inhalation	No change
	COVID-19 Associated Acute Respiratory Distress Syndrome	NCT04493242	Bone marrow mesenchymal stem cell	II	Infusions	The risk of death has been reduced.

The clinical application and transformation rate of Evs are not ideal, and data on the clinical application of engineered Evs are severely lacking. This is because their reliable effects depend on a suitable microenvironment and a well-controlled separation system. Currently, various cell types have been used for the effective separation and fabrication of Evs, such as HEK293 cells ¹⁹³ , MSCs ¹⁹⁴ , dendritic cells ¹⁹⁵ , and adipose-derived MSCs ¹⁹⁶ . Downstream processing and purification methods for Evs include density centrifugation, precipitation, chromatography, and membrane filtration. These processes all affect its quantity, stability, and purity ¹⁹⁷ , Therefore, with the expansion of clinical applications, the manufacturing processes developed under the background of academic theory need to be re-examined and updated.

Evs are at a crossroads in clinical application. Numerous academic publications and substantial research funding in the field have spurred exploration and research into their applications. Various strategies to optimize their therapeutic effects are under development, and regulatory frameworks are being established to ensure safe and successful clinical trials.