Introduction

Malignant tumors remain a major global health burden, with high incidence and mortality rates.1 Traditional treatments are often limited by tumor heterogeneity, immunosuppressive microenvironments, and biological barriers. As a result, only about 0.7% of intravenously administered drugs typically reach tumor sites, while off-target toxicity compromises therapeutic outcomes and patient quality of life.2 Tumor-targeted therapies, designed to maximize drug accumulation in tumors while sparing normal tissues, have thus become a key focus in oncology. Among these approaches, nanodrug delivery systems stand out for their ability to improve targeting efficiency.3 More than 90 nanocarrier systems have already reached clinical use, highlighting the rapid progress of the field.4

Although the nanocarrier systems, represented by liposomes and polymer nanoparticles, have been applied in clinical practice, they still exhibited limitations: Firstly, their material composition may trigger immune responses or cause unexpected toxicity; secondly, they are easily recognized and cleared by the immune system in the complex in vivo environment, resulting in a short circulation time; more importantly, they usually lack intrinsic biological targeting ability and mainly rely on passive targeting or simple ligands on the surface for active targeting, but this targeting is often not precise enough and difficult to overcome multiple biological barriers in the tumor microenvironment.5,6 In addition, the “cargo” encapsulation and release control of these nanocarrier systems still face challenges, such as premature leakage or insufficient release, which affects therapeutic efficacy and increases off-target toxicity.7 These deficiencies have prompted researchers to seek more biologically intelligent delivery platforms.

Exosomes—naturally secreted lipid bilayer vesicles (30–150 nm in diameter)—have emerged as particularly promising nanocarriers8 Derived from diverse sources (eg, animals, plants, and microorganisms),9 they exhibit superior biocompatibility compared to synthetic carriers such as liposomes or polymers. Their natural phospholipid membrane confers structural stability and reduces risks of systemic toxicity,10,11 while their small size facilitates blood–brain barrier penetration, offering unique advantages for brain tumor therapy.12 Exosomes also exhibit intrinsic targeting capacity and can be engineered for enhanced functionality. Beyond therapeutic delivery, their abundance in bodily fluids and stability make them attractive diagnostic biomarkers,13 enabling sensitive monitoring of disease progression and treatment response.14 A defining feature of exosomes is their biological plasticity: they can be readily modified with antibodies, peptides, or fluorescent probes to enable personalized delivery of bioactive agents.15 Furthermore, the specific protein profiles on the exosome membrane and target cell surface enable precise tissue or cell type homing through specific recognition.16,17 This facilitates the efficient delivery of their “cargo” into target cells, thereby regulating target cell functions and influencing physiological processes, such as immune responses and signal transduction. These properties provide a strong molecular basis for exosome-mediated cancer therapy.18–20

Animal-derived exosomes, particularly those from tumor cells, mesenchymal stem cells (MSCs), immune cells, endothelial progenitors, platelets, and fibroblasts, are most widely used in tumor-targeted applications. They are extractable from common bodily fluids including blood, urine, or milk, supporting large-scale preparation.21,22 Compared to animal-derived exosomes, plant-derived exosomes from sources such as grapes, ginger, and carrots exhibit superior immunotolerance and enhanced biocompatibility.23 For instance, ginseng-derived exosomes suppress tumor growth by modulating macrophage polarization through the toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88) pathway. Besides, ginger-derived exosome-like nanoparticles enhance anti-programmed cell death ligand 1 (PD-L1) therapy in melanoma by suppressing bacterial phospholipase C expression and increasing docosahexaenoic acid accumulation, underscoring the potential of plant-derived exosomes in reshaping tumor immunotherapy via host–microbiota interactions.24 Microbial exosomes, particularly those from gut microbiota, also play key roles in modulating intestinal immunity. They hold promise not only for intestinal disorders but also for systemic cancer therapy.25 For example, Kyong-Su Park et al combined E. coli-derived exosomes (SyBV) with tumor-derived exosomes for treatment. The results showed that SyBV activated dendritic cells (DCs) and exhibited reduced toxicity compared to native E. coli outer membrane vesicles (OMVs). This positions SyBV as a safe candidate for tumor immunotherapy that significantly enhances the efficacy of melanoma immunotherapy.26

Despite these advantages, exosomes face limitations in drug-loading capacity and targeting efficiency. To overcome the limitations of natural exosomes, engineering strategies (such as physical manipulation, chemical coupling, and genetic modification) provide effective solutions. Among them, exosome mimics and in vivo exosome biogenesis engineering are the two most promising directions at present.

Exosome mimics are artificial carriers prepared through artificial methods such as cell squeezing, liposome fusion, and membrane reorganization, which have the advantages of controllable yield, precise composition, drug loading increase and simple preparation, reducing the clinical transformation cost. Exosome mimics have to some extent solved the problem of large-scale production of natural exosomes, but the artificial carriers are easily recognized and cleared by immune cells in the body and lack the endogenous targeting property of natural exosomes.27,28

To further improve the precision and effectiveness of tumor treatment, in vivo exosome biogenesis engineering is a hot research direction. Exosome biogenesis engineering as an optimization strategy, its core principle is to regulate the exosome biogenesis process of tumor cells, stem cells, and other host cells in vivo through small molecule inhibitors, gene editing, and microenvironmental regulation (such as regulating the ESCRT pathway, HD-PTP, etc.)29 to achieve “on-demand generation of therapeutic exosomes”. The prominent advantages of this technology include: no need for an in vitro preparation and reinfusion process, significantly simplifying the treatment process. Endogenous exosomes retain the complete natural membrane structure and targeting proteins, and have better stability and targeting in the body. Using patient’s own cells as exosome parent cells can avoid the risk of immune rejection from heterologous carriers, and is particularly suitable for precise treatment of tumor heterogeneity. Currently, research in this field has achieved key breakthroughs, such as exosomes derived from malignant ascites of advanced gastric cancer patients containing abundant MET genes, which can promote enhanced tumor angiogenesis. By blocking the secretion of these exosomes, tumor growth can be slowed down.30 Exosome targeting may enhance the therapeutic effect of GC and provide inspiration for developing similar methods for other primary tumor types.

In summary, from the limitations of traditional drug delivery systems, such as poor targeting and strong toxicity, to the advantages of natural exosomes, such as superior biocompatibility and strong targeting, and to the technological iterations of exosome mimics (solving the problem of large-scale production) and in vitro/in vivo exosome biogenesis engineering (achieving in situ precise regulation), exosome-related delivery systems, with their core characteristics of precise targeting, low toxicity and high efficiency, and multi-functional synergy, gradually surpass traditional synthetic carriers and become the core carrier for tumor-specific treatment, promoting the advancement of tumor treatment towards precision and low toxicity. This review summarizes the current methods for exosome isolation and characterization, studies the engineering strategies for tumor-targeted exosomes, and summarizes the latest progress in exosome-based cancer treatment.

Preparation and Characterization of Exosomes

Isolation, purification, and characterization of exosomes are essential prerequisites for both basic research and clinical translation. Multiple methods have been developed based on different separation principles. Traditional approaches such as differential and density gradient centrifugation are widely used for preliminary separation, while more advanced techniques—including immunocapture and size-exclusion chromatography—enable higher purity and specificity. The choice of method depends on the research objective, sample type, and intended application. Characterization typically combines complementary techniques: transmission electron microscopy (TEM) is employed to analyze morphology, nanoparticle tracking analysis (NTA) to determine size distribution and concentration, and Western blotting to detect marker proteins like CD63 and CD81 (Figure 1). Optimizing and standardizing these processes is crucial to ensure reproducibility, support functional studies of exosomal cargo, and accelerate clinical application as diagnostic markers and therapeutic carriers. Moreover, the selection of source cells, preparation and isolation, surface modification, drug loading, characterization and quality control are the core steps in exosome engineering. The following systematically elaborates the technical key points, applicable scenarios and optimization strategies of each critical step, providing technical support for the application of exosomes in tumor treatment.

Figure 1 Exosome isolation and characterization. (a) Isolation Methods. (b) Increase Production Methods. (c) Characterization Methods: i) Physical and chemical characterization; ii) Molecular component analysis; iii) Functional activity analysis. Created with Biorender.

Selection of Source Cells

The type of the source cell directly determines the secretion quantity, biological compatibility, targeting potential and safety of exosomes, which is the fundamental prerequisite for exosome engineering. The selection should follow the three principles of “high secretion, low toxicity, and targeted adaptation”. Common source cells include:

Mesenchymal Stem Cells (MSCs)

The most widely used source cells in clinical research, which can be isolated from easily accessible tissues such as bone marrow, fat, and umbilical cord. They have strong proliferation ability and efficient exosome secretion efficiency. Their derived exosomes have excellent biological compatibility, low immunogenicity, and inherent tumor chemotaxis, and can migrate directionally through the tumor microenvironment (TME) based on inflammatory signals.31 However, it should be noted that MSCs from different donors may have batch differences, and the aging during in vitro expansion may affect the stability of exosome functions.32

Tumor Cells

Tumor cell-derived exosomes have a unique “homologous targeting property”, with tumor-specific antigens such as HER2 and CA125 retained on the surface, and can specifically bind to homologous tumor cells, significantly improving targeting accuracy.33 However, a core limitation is that they may carry oncogenic factors, such as metastasis-promoting miRNAs, which exert a facilitative effect on tumor growth.34

Immune Cells

Dendritic cells (DCs), macrophages, and natural killer (NK) cells are common choices. DC-derived exosomes carry MHC-peptide, CD80, and CD86, which can activate anti-tumor immune responses and are suitable for immunotherapy;35 M1-type macrophage-derived exosomes secrete inflammatory factors such as TNF-α and IL-6, which can reshape the immunosuppressive TME.36 However, immune cell culture is costly, and the secretion quantity of exosomes is limited, making it difficult to meet the requirements of large-scale production.

Plant/Microbial Cells

Exosomes derived from plants such as ginseng and ginger, and microorganisms such as Escherichia coli and Lactobacillus, have advantages such as low cost, easy large-scale extraction, and strong immune tolerance. For example, exosome-like nanoparticles derived from ginger can enhance the anti-PD-L1 treatment effect by regulating the intestinal microbiota, but their natural targeting property is weak and require subsequent surface modification to improve tumor targeting accuracy.37

Engineering Cell Lines

Through genetic modification to transform HEK293T and CHO cells, stable exosome-producing cell lines can be constructed. For example, by genetic modification, HEK-293T cells can be made to express the Lamp2b-iRGD - tyrosine fragment fusion membrane protein, allowing the exosomes secreted to carry the iRGD targeting peptide, and achieving precise targeting through binding to the αv integrin of target cells.38 Such cells can make up for the deficiencies of natural source cells, but the safety of genetic modification needs to be strictly evaluated.

Isolation and Purification of Exosomes

The isolation and purification of exosomes is the primary step in their applications. Although exosomes are abundant in bodily fluids, the high complexity of these fluids poses significant challenges for their separation.39 Currently, six primary methods for isolating and extracting exosomes exist: ultracentrifugation, density gradient centrifugation, filtration, differential centrifugation, immunomagnetic bead separation, and size exclusion chromatography.40 Here, each method possesses distinct advantages and limitations, often requiring a tradeoff between product purity and yield in practical applications (Table 1).

Table 1 Comparison of Common Methods for Isolating Exosomes

Ultracentrifugation technology, based on high-speed centrifugation (100,000–150,000×g), enables the separation of exosomes across different size and density ranges.49 It currently serves as the gold standard for exosome isolation, with density gradient ultracentrifugation and differential speed ultracentrifugation being the two most common methods.50 Ultracentrifugation is applicable to cell culture media and various bodily fluids, enabling the acquisition of high-concentration exosomes. For instance, Yahang Liang et al successfully extracted exosome miR-106a-5p from highly metastatic colorectal cancer cells via ultracentrifugation and demonstrated its pivotal role in promoting liver metastasis.51 However, ultracentrifugation relies on expensive equipment, and high centrifugal forces may potentially damage exosome membranes.

Differential ultracentrifugation separates components based on sedimentation rates by progressively increasing centrifugal speeds: 500 × g to pellet contaminating cells, 2000 × g for cell debris, 10,000 × g for apoptotic bodies and macrovesicles, and finally 100,000 × g for exosomes.52,53 This approach is well-suited for large-volume samples and is cost-effective but requires careful control of speed and time to ensure proper separation, and it yields relatively low purity.

Density gradient centrifugation involves adding a concentrated sample to sucrose or iodixanol to form a density gradient.54 Through ultracentrifugation, exosomes (typically accumulating at densities of 1.13–1.19 g/mL) can be separated from other vesicles, reducing contamination from proteins and larger extracellular vesicles, thereby further purifying exosomes.55 However, this method has low throughput and is highly sensitive to centrifugation time.

Ultrafiltration uses pressure differences across a membrane to separate large particles like dead cells and proteins based on molecular weight cut-off, resulting in a concentrated sample that is further purified using membranes of different pore sizes for exosome isolation.56 This method is straightforward, economical, and applicable to large-scale preparation but may lead to losses due to membrane clogging, and shear stress may damage exosomes.

The immunomagnetic bead method relies on specific binding between exosome markers (eg, CD63, CD81) and antibodies, enabling the isolation of exosomes from specific sources with high purity.57,58 However, it is costly, and elution steps may damage exosomes.

Through gel filtration, SEC separates exosomes based on the differences in their particle sizes. Gels with pores of different sizes allow larger particles to elute first than smaller ones, separating components by elution time.59 Improved SEC protocols, such as the two-dimensional size-exclusion liquid chromatography (2D-SELC) platform with fully automated sampling and fraction collection, avoid human error.60 SEC maintains the natural state of exosomes with high purity and reproducibility, applicable to various samples, but equipment costs are high, and factors like pore size, packing, and flow rate affect separation.

Beyond these six approaches, alternative techniques such as polymer precipitation61 and microfluidics62 are being explored. Given the diversity of exosome sources and intended applications, no single method is universally applicable. Achieving uniform, homogeneous exosome populations will likely require hybrid strategies tailored to physicochemical properties and biological context.

Besides optimizing purification methods, strategies to boost exosome secretion are critical for large-scale applications. Modulating cell culture conditions can effectively promote exosome release; for example, subjecting cells to stress or hypoxia often induces more exosome secretion.63 Studies have confirmed that medium pH significantly affects exosome yield, with acidic conditions favoring production.64 In MSC culture systems, adding substances like fenoterol, norepinephrine or N-methyldopamine can increase exosome yield by up to threefold.64 Similarly, rapamycin and other small molecules (calcium phosphate particles and adiponectin) modulate signaling pathways such as mammalian target of rapamycin and autophagy to promote release.65–67 Advances in cell culture formats further support scalable production. Compared with conventional two-dimensional systems, three-dimensional (3D) cultures more closely mimic the in vivo microenvironment, promoting cell–cell and cell–matrix interactions. For example, umbilical cord-derived MSCs grown in microcarrier-based 3D systems secreted up to 20 times more exosomes than in 2D culture; combined with tangential flow filtration, yield increased up to 140-fold that of 2D culture, demonstrating strong potential for industrial-scale preparation.68 Additionally, exogenous physical stimulation is an effective means to promote exosome generation, including radiation and electrical stimulation. For instance, cells cultured with magnetic nanoparticles under magnetic force showed enhanced exosome generation by upregulating exosome transport and secretion pathways.69 Shanguo Zhang et al significantly increased MSC exosome secretion by applying electrical stimulation via a self-powered, portable, and efficient triboelectric nanogenerator.70

The isolation of exosomes requires multidimensional characterization to verify their physical properties, molecular composition, and functional activity, ensuring quality and application potential.

Method of Improving the Drug Delivery of Exosomes

The core bottlenecks of exosomes as drug delivery carriers include insufficient targeting accuracy, low drug loading efficiency, and short in vivo circulation half-life. These issues need to be optimized from multiple aspects such as carrier modification, drug loading optimization, and delivery assistance.

Enhancement of Targeting

By screening based on cell sources and surface engineering modification, the recognition and binding ability of exosomes to target tissues can be enhanced. Cells with natural homing properties naturally carry molecules related to the recognition of target tissues, which can significantly enhance targeting. For example, mesenchymal stem cells (MSCs) and tumor-derived cells can achieve targeted delivery without additional modification. In the experimental verification of the HT1080 cell nude mouse tumor transplantation model, exosomes derived from HeLa or HT1080 cell sources all demonstrated the ability to target and homing to the same tumor tissue.71 The membrane surface of mesenchymal stem cells is richly distributed with receptor molecules (CD29, CD90, CD105, etc.), covering various types of chemokine receptors and growth factor receptors. The signaling molecules released at the tumor site will precisely match the surface receptors of MSCs, thereby guiding MSCs to migrate towards the tumor microenvironment. Therefore, MSCs and their secreted exosomes have the potential to actively target tumor tissues and exert anti-tumor effects.72 To improve the targeting treatment effect of CD24-expressing tumors (such as ovarian cancer ID8 cells, triple-negative breast cancer 4T1 cells), Li et al transfected M0-type macrophages (Raw264.7) with Siglec-10 by lentivirus, induced polarization, and prepared targeted exosomes SM1Aexo by compression method. In vitro experiments confirmed that SM1Aexo had a significantly better CD24-blocking effect on ID8 cells and 4T1 cells than wild-type M1 exosomes (M1Aexo), and could increase the phagocytosis rate of bone marrow-derived macrophages (BMDMs) for EGFP-labeled tumor cells by 3.47 times. In vivo studies further demonstrated that when this targeted exosome was coupled with alginate oxide (OSA) to form a hydrogel delivery system, it could significantly inhibit ovarian cancer peritoneal metastasis and the growth of triple-negative breast cancer, providing a typical example for enhancing the efficiency of tumor delivery by targeted modification of exosomes.73

Optimization of Drug Loading Efficiency

Exogenous drugs can be introduced into exosomes through passive and active methods, such as co-incubation, ultrasound treatment, electroporation, and freeze-thaw cycles, and these have been well reviewed (Figure 2). 74 There are two types of passive loading. One is to incubate the drug with the source cells, and then the exosomes released by the source cells will contain the target drug (Figure 2ai). The other is to adopt a direct co-incubation strategy, where the exosomes and the drug are incubated in the same system, and the drug molecules can penetrate the membrane structure of the exosomes in a concentration-dependent manner,75 thereby completing the delivery into the interior of the vesicles (Figure 2aii). Previous studies have shown that co-incubating exosomes with drug-loaded liposomes can also result in higher drug loading and better targeting ability for hybrid exosomes.76 The electroporation technique can cause the exosome membrane to form micropore channels, creating conditions for the inward flow of drug molecules, and this method is currently regarded as the preferred strategy for loading nucleic acid substances (Figure 2bi). The ultrasonic mechanical shear force can cause the exosome membrane to deform,77 enabling effective loading of drugs (Figure 2bii). Detergents can remove cholesterol from the exosome membrane, reduce the membrane barrier, and facilitate the loading of macromolecular drugs, and are commonly used methods for loading hemolytic drugs (Figure 2biii). When loading exosomes with drugs using the compression method, lipid bilayer fragments can achieve self-assembly into spherical structures, and through treatment with filters of different pore diameters,78 it is conducive to preparing exosomes with uniform particle size (Figure 2biv). The freeze-thaw cycle is characterized by 3–5 cycles of room temperature and −80°C alternation,79 forming pores on the exosome membrane surface to help drugs enter the interior, but this method may damage the structure and properties of the exosomes (Figure 2bv).

Figure 2 Various technologies for drug loading into Extracellular vesicles (EVs). (a) Passive Loading: i) Direct co-culture of drugs and cells: Achieved through varying drug concentration gradients, suitable for hydrophobic drugs and small-dose medications; ii) Incubation: Directly mix hydrophobic drugs and small molecule drugs with purified exosomes. (b) Active Loading: i) Electroporation: Membrane electroporation under electric current, which creates temporary pores, is suitable for the loading of hydrophilic drugs and is considered the preferred method for loading nucleic acid molecular. ii) Sonication: Capable of loading hydrophilic molecules, though some small molecules may reside on the exterior of EVs. iii) Detergent treatment: Detergents remove cholesterol from membranes, allowing for the loading of large molecules, typically used for hemolytic drugs. iv) Extrusion: Fusing drugs with the membranes of extruded and fragmented EVs. v) Freeze-thaw cycles: This method can load drugs into exosomes, but it may alter the properties of the EVs. Reprinted with permission from,74 Copyright 2024 MDPI.

Extension of Intra-Body Circulation

Extending the in-body circulation time of exosomes is one of the key steps to enhance their drug delivery efficiency. It can effectively reduce the clearance by the reticuloendothelial system and provide sufficient time for exosomes to target and accumulate at the lesion site. In recent years, several studies have achieved significant extension of exosome in-body circulation through surface modification or functional fragment integration strategies. For instance, researchers constructed an “immune invisible” barrier by surface modification with biocompatible polymers. Using polyoxazoline (POx) as a substitute for traditional PEG, they modified extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs). This modification strategy successfully reduced the recognition and clearance by the immune system while retaining the inherent immune regulatory function of MSC-EVs, increasing the blood half-life of EVs after intravenous injection by 6 times.80 At the same time, the extension of circulation time significantly improved the targeting accumulation efficiency of EVs in tumor tissues, laying the foundation for the subsequent drug action. Integrating functional fragments to inhibit macrophage phagocytosis is beneficial for extending the retention time of the exosome system in the in-body environment. Exosomes are easily cleared by the liver and spleen mononuclear phagocyte system (MPS) in the body, with limited circulation time, which affects the targeted delivery efficiency. To solve this problem, Justin F. Creeden et al constructed CD9-engineered intelligent exosomes (ExoSmart), which were co-modified with CD47p110–130 functional fragments and RGD targeting peptides. CD47p110–130 can specifically bind to SIRPα on the surface of macrophages and inhibit phagocytosis by transmitting the “don’t eat me” signal, significantly reducing the clearance rate of exosomes in RES organs.81 The RGD peptide integrates with the αvβ3 integrin highly expressed in pancreatic ductal adenocarcinoma (PDAC) cells to achieve active targeting, converting the advantage of extended circulation into efficient enrichment at the lesion site. This study confirmed that through the anti-clearance modification mediated by the CD47-SIRPα axis, it is possible to maintain the natural carrier advantage of exosomes while optimizing their circulation stability, and when combined with targeted modification, it can significantly enhance the therapeutic effect of PDAC treatment, providing a reference for the improvement of the in-body circulation of the exosome drug delivery system.

Characterization of Exosomes

The reliable use of exosomes requires rigorous, multidimensional characterization to confirm their physical properties, molecular composition, and functional activity. Together, these analyses ensure sample quality, reproducibility, and application potential.

Physical Characterization

Imaging techniques provide direct insights into exosome morphology and size. TEM typically depicts the cup-shaped appearance of exosomes, while cryoelectron microscopy (cryo-EM) captures their native spherical form.82 Complementary techniques such as dynamic light scattering (DLS) and NTA assess size distribution and concentration, confirming the characteristic 30–150 nm range.83 Zeta potential analysis further evaluates surface charge, with the naturally negative membrane potential contributing to exosome stability during circulation.84

Molecular Composition Analysis

Detecting specific molecular markers is essential for confirming exosome origin and integrity. At the protein level, this includes transmembrane proteins such as CD63, CD82, and CD9, transport and fusion-related proteins such as annexins and heat shock proteins, and adhesion-related proteins such as integrins.85 Techniques including Western blotting, flow cytometry, and immunofluorescence allow both qualitative and quantitative assessment.86 At the nucleic acid level, exosomes carry diverse cargo including messenger RNA (mRNA) and other nucleic acid molecules. Reverse transcription-polymerase chain reaction, sequencing, and related methods identify and quantify these molecules, with expression profiles often reflecting the physiological state of source cells.87 Emerging omics approaches, particularly proteomics, are increasingly used to comprehensively map protein composition and abundance, supporting the establishment of quality control standards.

Functional Activity Analysis

Since therapeutic efficacy depends on biological function, functional assays are critical. These typically include evaluating cellular uptake efficiency, in vivo biodistribution, and therapeutic outcomes in relevant models. For example, the binding affinity between the modified exosomes and tumor cells was verified by using in vitro fluorescence microscopy and flow cytometry to confirm the targeting ability; in vivo, the enrichment effect in tumor tissues was verified through near-infrared fluorescence imaging and bioluminescence imaging. In vitro, the anti-tumor effect of the drug-loaded exosomes was evaluated by the CCK-8 cell viability assay; in vivo, the tumor suppression rate was verified through a tumor xenograft mo for immunotherapy-related exosomes, immune activation indicators such as DC maturation and CD8+T cell proliferation ability were detected by flow cytometry.88,89 Such studies verify whether exosomes not only retain structural integrity but also deliver their cargo effectively to target cells, thereby validating their use as drug carriers or diagnostic tools.

Comprehensive characterization of exosomes is an important part of exosome quality control. To ensure the therapeutic activity and homogeneity of exosomes, a systematic assessment of their purity, safety, and consistency is necessary. Among them, the purity of exosomes can be ensured by detecting the positive expression levels of typical exosome markers such as CD9 and CD63, and screening for the presence of plasma contaminants such as apolipoprotein A (ApoA), thereby eliminating the interference of impurities and ensuring the high purity of the samples. Through quality control, contaminants such as viruses and impurities in the culture medium can be avoided from contaminating exosomes, thus meeting the requirements of clinical applications.90 The batch stability of exosomes is easily affected by various factors, such as the type of source cells, the number of cell passages, and the method of drug loading.91 There may be differences between different batches. Therefore, the consistency check needs to be throughout the entire process of exosome quality control. By strictly standardizing the cultivation requirements of source cells, optimizing the steps of separation and purification, and other key operations, ensure that the exosomes from different batches maintain stable consistency in core indicators such as particle size, drug loading rate, and expression levels of characteristic markers, to ensure the reliability of therapeutic effects.

Engineering Methods to Enhance Exosome Tumor Targeting

Natural exosomes, as endogenous nanovesicles, although having low immunogenicity, good biocompatibility and the ability to cross biological barriers, have limited targeting ability, low drug loading efficiency, poor process controllability, lack of responsiveness to pathological microenvironments or the intelligence for collaborative diagnosis and treatment, their inherent limitations seriously limit the potential for clinical application. In addition, natural exosomes also exhibit significant heterogeneity and bottlenecks in large-scale production, making quality control and standardized application challenging. This highlights the necessity of engineering exosomes.

Engineered exosomes are nanovesicles modified to optimize the structure, composition, or function of natural exosomes. Through precise design, they can be endowed with improved targeting ability, greater drug-loading capacity, and enhanced bioactivity.92 Compared with their natural counterparts, engineered exosomes overcome key limitations such as low intrinsic targeting and limited loading efficiency, making them more suitable for therapeutic and diagnostic applications. Strategies for engineering exosomes generally fall into two categories: drug loading and surface modification. This section focuses on surface modifications, which are designed to enhance tumor-targeting efficiency. Approaches include chemical modification, physical modification, and genetic engineering of exosome-producing cells (Figure 3). These strategies enable the addition of targeting ligands, functional molecules, or responsive elements to exosome membranes, thereby improving their precision in recognizing and interacting with tumor cells.

Figure 3 Engineering modifications for enhancing exosome tumor-targeted therapy. Created with Biorender.

Chemical Modification

Chemical modification introduces targeting molecules onto the exosome surface through well-defined reactions, thereby enhancing their ability to selectively recognize tumor cells. A major advantage of this approach is the broad choice of targeting ligands—including antibodies, peptides, and aptamers—that can be tailored to different tumor types. For instance, human epidermal growth factor receptor2 (HER2) antibodies have been used to target HER2-overexpressing breast cancer,93 RGD peptides bind integrins commonly found on tumor cells,94 and the AP-9R aptamer specifically recognizes annexin A2 on lung cancer cells, supporting applications in diagnosis and therapy.95 Chemical modification methods are generally mild, controllable, and versatile, making them a cornerstone of exosome engineering. Two major strategies are used: covalent binding and non-covalent binding.

Covalent binding: Exosome membranes contain abundant reactive groups, such as thiols and amines, that allow covalent attachment of ligands via amidation reactions, thiol-maleimide reactions, and click chemistry. Amidation reactions link activated carboxyl groups on targeting ligands with amines on exosomal proteins, forming stable amide bonds. Jun Yeong Kim et al quantified the amount of amine groups in exosomal proteins using fluorescamine reagent, then activated the amines on exosomes at an amine/NHS molar ratio of 1:2 for 3 hours, facilitating easier binding to i-motif targeting heads.96 This method offers high modification efficiency and stable linkage between targeting molecules and exosomes, effectively improving exosome recognition and binding to tumor cells. Maleimide groups specifically react with thiols to form stable thioether bonds.97 This reaction is highly specific and selective, proceeding efficiently under mild physiological pH conditions. Chaoxiang Chen et al used TCEP (a mild reducing agent) to reduce disulfide bonds on milk-derived exosomes to thiols, then conjugated ligands to the exosome surface via thiol-maleimide reactions, demonstrating the feasibility of ligand modification without damaging vesicle structure.98 Common click chemistry reactions, including copper-catalyzed azide-alkyne cycloaddition and strain-promoted copper-free click reactions (eg, between azides and dibenzocyclooctyne (DBCO)), form stable triazole rings through azide-alkyne reactions.99 These reactions are efficient, highly specific, and occur under mild conditions, widely used in chemical biology. For example, Miao Deng et al labeled tumor cells and tumor-derived exosomes with azide groups while modifying nanoparticles with alkyne groups (DBCO); specific binding via bioorthogonal click reaction formed stable covalent bonds.100 This study tracked and downregulated exosomal macrophage migration inhibitory factor, inhibiting the role of tumor-derived exosomes in inducing pre-metastatic niche formation in the liver and promoting tumor metastasis, showing good therapeutic effects against pancreatic cancer. Dumei Ma et al successfully distinguished breast cancer patients from healthy individuals by detecting serum exosomes via click reaction.101

Non-covalent binding: Non-covalent approaches rely on electrostatic attraction and hydrophobic interactions. Positively charged ligands can associate with the negatively charged exosome membrane through electrostatic forces, while hydrophobic ligands insert into the lipid bilayer.102,103 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Polyethylene Glycol (DSPE-PEG), a lipid-like material, integrates functional molecules into the exosome surface through hydrophobic interactions with membrane lipids and renders exosomes stealthy, reducing protein adsorption and enhancing tumor targeting.104 For instance, Rui Deng et al first reacted DSPE-PEG2000-maleimid (DSPE-PEG2000-Mal) with a thiol group of a hepatocyte-targeting cell-penetrating peptide (HTPP) to form DSPE-PEG2000-HTPP, then utilized the hydrophobicity of DSPE to fuse into the exosome lipid bilayer, ultimately modifying HTPP onto the exosome surface via a linker.105 This study showed that HTPP-modified exosomes significantly enhanced targeting to liver cancer. This method is relatively simple and has minimal impact on exosome bioactivity, but binding stability is poorer than covalent binding. Examples of exosome systems based on chemical modification for tumor therapy are summarized in Table 2.

Table 2 Examples of Exosome Systems Based on Chemical Modification for Tumor Therapy

Physical Modification

Physical modification employs external stimuli or mechanical processes to alter exosome membranes, enabling functional enhancement or hybridization with other nanostructures. Common approaches include freeze–thaw cycles, membrane extrusion, sonication, and magnetic guidance.111 The fusion with lipid nanoparticle (LNP) is a typical direction. Compared to LNPs, the endogenous lipid membrane structure of exosomes can significantly reduce immunogenicity and liver toxicity. The inherent tumor-targeting property of exosomes combined with surface engineering modifications (such as targeting peptide conjugation) can precisely target tumors, overcoming the problem of LNPs’ tendency to accumulate non-targetedly in the liver and spleen.112 At the delivery level, exosomes can simultaneously carry various bioactive molecules such as proteins, nucleic acids, and lipids, having better compatibility than the single loading characteristic of LNPs. However, there are also some issues with exosomes, such as the reliance on complex processes like ultracentrifugation for separation and purification, which are time-consuming and difficult to control in terms of purity, and the greater difficulty in large-scale production compared to LNPs.113 Exosomes are prone to aggregation during storage, while LNPs have an advantage in improving the stability of the formulation (such as PEG modification and optimization of lipid components).114 Based on this, the hybrid exosome - LNP can achieve complementary advantages through physical fusion (such as ultrasound-mediated membrane fusion or forced assembly by extrusion). By leveraging the structural stability and mature preparation process of LNPs, the problems of exosome storage and large-scale production can be improved. The low immunogenicity, targeting ability, and multi-load capacity carried by exosomes are conducive to the construction of an efficient delivery system with both biocompatibility and formulation stability. Zhuo et al’s cholesterol-enriched milk-derived exosomes (MEs) exemplify this complementary strategy. Modulating cholesterol content in the exosomal membrane enhances fusion with cancer cell membranes, preserving exosomes’ inherent low toxicity and precise targeting while enabling efficient cytosolic siRNA delivery.115 Xu et al incorporated macrophage-derived exosomes into ROS-responsive cationic liposomes to construct a biomimetic nanocomplex (E-cLip-DTX/si) for the co-delivery of DTX and Bcl-2 siRNA. In the mouse model of breast cancer, this nanosystem exhibited potent tumor growth inhibition and extended in vivo circulation time.116 Compared with chemical modification, these methods are simpler, avoid introducing reactive chemicals, and preserve most of the intrinsic properties of exosomes.

Freeze–Thaw Cycles

Repeated freezing and thawing destabilize lipid bilayers, promoting fusion between exosomes and other vesicles. Yifan Gao et al applied this method to fuse liposomes with γδ T cell–derived exosomes, achieving targeted photoimmunotherapy for melanoma while retaining the exosomes’ antiviral activity against Epstein–Barr virus–related tumors.117 Similarly, Lili Cheng et al reported a fusion efficiency of 97.4% when combining thermosensitive liposomes with genetically engineered exosomes through three freeze–thaw cycles.118

Membrane Extrusion

This widely used technique forces exosomes and other lipid vesicles (eg, liposomes) through polycarbonate membranes under mechanical pressure, inducing membrane remodeling and fusion.119 The resulting hybrid exosomes display uniform size and preserved biological function. Yang Yang et al demonstrated this by hybridizing Raw 264.7 cell–derived exosomes with liposomes, then loading thalidomide to regulate Treg cells for tumor immunotherapy.120 In another example, autologous breast cancer cell–derived exosomes were fused with liposomes via incubation–extrusion, yielding a hybrid system for combined chemo-, immuno-, and photothermal therapy.121 Beyond mammalian systems, Rufan Huang et al fused grapefruit-derived exosomes with engineered mesenchymal stem cell–derived exosomes, producing hybrid vesicles with strong inflammatory tissue tropism.122

Sonication

Ultrasound disrupts and reassembles membranes, enabling the fusion of exosomes with other vesicles. Yong-Yu Jhan et al sonicated liposome–exosome mixtures at different ratios, followed by extrusion, producing hybrid exosomes of ~100 nm. This method increased vesicle yield by 6- to 43-fold compared with conventional approaches.123

Magnetic Guidance

Incorporating magnetic nanoparticles into or onto exosomes enables external magnetic fields to direct their accumulation at tumor sites. Mengran Li et al coated exosome membranes onto magnetic mesoporous silica nanoparticles.124 These constructs not only evaded immune clearance and exhibited tumor tropism but also achieved rapid, magnetically controlled accumulation at tumor sites. Capture efficiency for homologous tumor cells reached 94.37% ± 1.3%.

Overall, physical modification offers a straightforward way to enhance exosome function while preserving their biochemical composition. However, to achieve optimal tumor targeting, these methods are often combined with chemical or genetic modifications. Representative examples of exosome systems based on physical modification for tumor therapy are summarized in Table 3.

Table 3 Examples of Exosome Systems Based on Physical Modification for Tumor Therapy

Genetic Modification

Genetic modification enhances tumor targeting by engineering the parent cells to express specific ligands or proteins on the exosome surface. These exosomes can then bind selectively to receptors on tumor cells or within the tumor microenvironment, improving delivery precision and therapeutic efficacy.

For example, Lingmin Zhang et al used CRISPR/Cas9 knock-in technology to fuse a tumor-associated macrophage (TAM)-targeting peptide (CRV) to the lysosome-associated membrane glycoprotein 2 gene.128 The resulting exosomes displayed high levels of CRV on their surface, effectively reprogramming M2-type TAMs into tumoricidal M1-type macrophages and suppressing metastasis. Similarly, Jung Hyun Park et al genetically modified adipose-derived stem cells with a pDisplay vector encoding the DE532 peptide, which binds gastric cancer MKN45 cells. The resulting DE532 exosomes, when loaded with alvespimycin hydrochloride (17-DMAG), achieved precise targeting and reduced drug-associated toxicity.129 In another study, Peishan Li et al transfected HEK293T cells with a constructed PD1 high-expression plasmid, selected stably transfected cell lines (PD1-HEK 293T cells) with puromycin, and obtained recombinant PD1 protein exosomes (PD1 Exo) after purification and isolation.130 PD1 Exo helps address key issues of immune exhaustion and escape in tumor immune checkpoint therapy.

The key advantage of genetic modification lies in the natural expression and assembly of targeting molecules during exosome biogenesis, ensuring high compatibility and preserving vesicle integrity. However, this strategy remains technically demanding, with challenges including high cost, complex procedures, and variable transfection efficiency.131 Representative exosome systems based on genetic modification for tumor therapy are summarized in Table 4.

Table 4 Examples of Exosome Systems Based on Gene Modification for Tumor Therapy

Applications of Exosomes in Tumor-Targeted Therapy

Exosome-based nanosystems, as natural vesicles, offer distinct advantages including excellent biocompatibility, minimal immunogenicity, and effective delivery of therapeutic cargo. These features make them highly attractive for tumor-targeted applications. By harnessing these properties, exosome platforms not only overcome limitations of conventional single-agent therapies but also create opportunities for synergistic effects in combination strategies. We have summarized and organized the advantages of the exosome delivery system compared to the traditional delivery system in different treatment methods in the form of a table (Table 5). Building on these strengths, exosome systems are increasingly being investigated across diverse therapeutic modalities. The following sections detail their specific roles in various treatment modalities from two dimensions: single therapy and combined immunotherapy (Figure 4).

Table 5 The Advantages of Exosome-Based Delivery Systems in Different Tumor Treatment

Figure 4 Exosome-based nanosystem strategies for tumor therapy. Created with Biorender.

Single Therapy Modalities Chemotherapy

Exosomes can efficiently encapsulate chemotherapeutic drugs and deliver them to tumor tissues, thereby enhancing antitumor activity while minimizing systemic toxicity. This makes them highly suitable for chemotherapy across different cancer types. For instance, Tooba Soudi et al developed a co-delivery system using exosomes loaded with carvedilol and 5-fluorouracil, which synergistically improved treatment outcomes in breast cancer.136 Exosomes derived from healthy epithelial breast cells, loaded with lapatinib via electroporation, inhibited the proliferation and promoted apoptosis of SKBR3 breast cancer cells at low doses.137 This result confirms that exosomes as drug carriers can achieve low-dose breast cancer treatment, improve drug efficacy and chemotherapeutic index, and enhance cancer treatment effects.

Exosomes possess natural tumor tissue targeting tropism. Biomimetic artificial exosomes constructed based on this biological characteristic can enhance the accumulation of chemotherapeutic drugs in homotypic tumors. Artificial exosomes prepared by modifying phospholipid liposomes with cancer cell membrane proteins not only have high yield and are easy to prepare but also specifically deliver loaded doxorubicin and vorinostat, effectively inhibiting non-small cell lung cancer growth with no significant adverse reactions.138 Engineering modifications to add targeting molecules to the exosome surface can further improve the efficacy of tumor-targeted therapy. Pratiksha Tiwari et al modified exosomes with the laminin receptor-binding peptide YIGSR (YIGSR-Exo) and formed hybrid-Exo with lipid-polymer nanoparticles (LPNP) via freeze-thaw method, then loaded dasatinib (DST) to form DST-FuNP@YIGSR-Exo (Figure 5a). Confocal microscopy revealed that DST-FuNP@YIGSR-Exo selectively accumulated in cancer cells highly expressing laminin receptors via receptor-mediated endocytosis, ensuring effective drug delivery (Figure 5b–d).139

Figure 5 (a) Schematic of Exosome fusion with LPNPs; (b) Confocal images of Nile red labeled localization after 2 and 6 hours of treatment in MDA-MB-231 cells; (c) Quantitative histogram data of Nile red labeled confocal images located in MDA-MB-231 cells after 2 and 6 hours of treatment; (d) Flow cytometry provided quantitative evaluation of cell uptake after 2 and 6 hours of incubation in MDA-MB-231 cells. Reprinted with permission from.139 Copyright 2025 John Wiley and Sons; (e) Schematic diagram of preparation of exosomes loaded with DOX and Boron clusters, and their application in treating DOX-Resistant breast cancer cells and promoting cell apoptosis. Reprinted with permission from.140 Copyright 2024 John Wiley and Sons.

Notably, exosomes can cross the blood-brain barrier (BBB) through mechanisms such as receptor-mediated endocytosis and lipid raft-mediated micropinocytosis via specific proteins.141,142 Therefore, compared with traditional nanodelivery systems, exosomes have a natural advantage in drug delivery for brain tumors. Sana Shaikh et al demonstrated through in vivo computed tomography (CT) imaging experiments that loading temozolomide (TMZ) and gold nanoparticles (AuNPs) via M1 macrophage exosomes could deliver them to the brain, overcoming the BBB, and efficiently inhibit glioblastoma growth.143 Additionally, resistance induced by traditional chemotherapy often leads to tumor recurrence, a major cause of cancer death. Using exosome drug delivery systems can effectively overcome chemotherapy resistance. The mechanism of chemotherapy resistance is mainly related to the overexpression of P-glycoprotein (P-gp), which actively pumps drugs out of cells using ATP energy, leading to chemotherapy resistance.144 When exosomes are used as the delivery system, their endocytosis process also consumes ATP, which can competitively inhibit P-gp-mediated drug efflux, thereby alleviating resistance.145 Yi-Ru Bao et al formed a supramolecular complex of boron clusters and doxorubicin (DOX), increasing the drug loading capacity of DOX in exosomes, and utilized the ATP consumption effect of exosomes to alleviate DOX resistance in breast cancer cells (Figure 5e). 140

Immunotherapy

The core of immunotherapy is to utilize the human immune system to activate immune responses (especially CD4+/CD8+ T cells) to eliminate tumors.146 Some types of exosomes carry information that helps activate immune responses.147 Current exosome-based immunotherapies are mainly divided into cancer vaccines, immune checkpoint therapies, and adoptive cell therapies. Xinyi Zhang et al have previously reviewed this in detail.148 Tumor cell-derived exosome vaccines contain various tumor antigens and are promising vaccine development carriers. Yan Zou et al prepared a tumor nanovaccine platform (Exo-CpG) by loading CpG oligodeoxynucleotides (CpG) onto homologous exosomes derived from glioblastoma multiforme (GBM), which can effectively target lymph nodes, activate immune responses, and induce long-term immune memory, achieving a 100% survival rate in tumor-bearing mice within 5 months and effectively preventing metastasis.149 Similarly, Shanshan Li et al constructed a GBM tumor-derived exosome nanosystem loaded with CpG adjuvant (Exo-CpG) (Figure 6a). This system can effectively cross the BBB and accumulate in the brain (Figure 6b and c), activate the body’s innate immunity through facilitating the maturation of antigen-presenting DCs in lymph nodes and eliciting potent T-cell res