Cutting-Edge Progress in the Acquisition, Modification and Therapeutic Applications of Exosomes for Drug Delivery

Introduction

Exosomes, nano-scale vesicles secreted by cells into their extracellular compartments, play a crucial role in various biological processes such as cell recognition and signal transduction through paracrine and autocrine secretion. They carry an abundant cargo of DNA, RNA, proteins, among other molecules, making them a novel cellular communication tool.¹ Exosomes possess excellent biocompatibility, stability, and targeting capabilities,² making them a promising candidate for drug delivery systems. Moreover, their versatility allows for engineering and modification in various ways to enhance their therapeutic potential. Consequently, exosomes have emerged as a highly effective drug delivery system that has found widespread applications in the treatment of cardiovascular and cerebrovascular diseases, neurological disorders, tumors, and other medical conditions.³ With the further advancement of diagnostic and treatment technologies for exosome-related diseases, the two technical hurdles that constrain the fundamental and clinical application of exosomes, namely the separation, purification, and preservation of exosomes, demand greater attention from us.⁴ Currently, based on exosome particle size, density, surface markers, etc., researchers have developed a variety of separation and purification techniques, including centrifugation, ultrafiltration, immunoaffinity, microfluidics.⁵ To address the preservation challenges, researchers have proposed methods such as freezing, lyophilization, and other storage techniques to maintain the integrity and stability of exosomes to the greatest extent possible.⁶

In this review, we will describe the biogenesis of exosomes and their advantages as drug delivery carriers, summarize isolation and preservation methods along with engineering modification strategies, illustrate the application of exosome-mediated targeted drug delivery in clinical disease models, and highlight key translational challenges and opportunities for future exosome development.

Exosome Biogenesis and Composition

Exosomes are small vesicles secreted by cells, typically ranging from 30 to 150 nm in diameter,⁷ and are widely found in various biological fluids. Exosome biogenesis is a complex and orderly dynamic process that includes the formation of early endosomes, their maturation into multivesicular bodies (MVBs), and the fusion of MVBs with the cell membrane. Specifically, the cell membrane invaginates inward to form a cup-shaped structure, which further develops into early endosomes. These early endosomes then undergo invagination and protrusion through the vesicle membrane, forming multiple small intraluminal vesicles (ILVs) that selectively encapsulate nucleic acids, proteins, and lipids from the cytoplasm. The resulting multivesicular bodies can either fuse with lysosomes or autophagosomes for degradation, or with the cell membrane to release the internal vesicles, known as exosomes^4,8,9 (Figure 1).

Figure 1 Biogenesis of exosomes. Firstly, the cell membrane invaginates to form a cup-shaped structure, which further forms an early nucleosome; Then, the membrane of the early nucleosome invaginates to encapsulate DNA, RNA, or protein in cytoplasm and buds into vesicles to form the late nucleosome, and then the late nucleosome matures into polycystic bodies; Finally, the cyst fuses with the cell membrane and releases the vesicles to form exosomes Some polyethylene can also be fused with lysosomes and degraded. Created in BioRender. Hu, B. (2025) https://BioRender.com/y22x389.

Exosome production is a complex chain of molecular events that requires the collaboration of various protein complexes, this includes both ESCRT-dependent and ESCRT-independent pathways.¹⁰ Together, these factors play a role in ensuring that cells can selectively load molecules into exosomes and regulate the role they play in the exchange of information between cells. It is currently believed that exosome formation is mainly regulated through the ESCRT-dependent pathway, which consists of four complexes: ESCRT-0 (Hrs), ESCRT-I (TSG101, Vps28, Vps37), ESCRT-II (Vps22, Vps36, Vps25), and ESCRT-III (Alix, Vps2).¹¹ In vivo, ubiquitin markers are recognized by the first ESCRT complex, ESCRT-0, which is enriched at the endosomal membrane and delivers ubiquitinated material to ESCRT-I and ESCRT-II. Tsg101 in ESCRT-I recognizes the disulfide bond and induces the endosomal membrane to be pitted, and then ESCRT-III shears the bud neck to form MVBs.¹² The ESCRT non-dependent pathways mainly include the lipid raft-dependent pathway and the four-membrane transprotein family. The lipid raft-dependent pathway mainly involves ceramide. Ceramide induces the formation of luminal vesicles, facilitates the entry of biomolecules rich in proteins, RNA and lipid rafts into luminal vesicles, and promotes the fusion of luminal vesicles with the cell membrane to release exosome.¹³ The tetraspanin family, which includes proteins such as CD63, CD81, and CD9, plays a crucial role in exosome biogenesis and selective cargo loading. Tetraspanins maintain the membrane integrity of multivesicular bodies (MVBs) by forming a meshwork with other membrane proteins.^4,13,14

The structure of exosome vesicles primarily consists of a phospholipid bilayer encapsulating a variety of biologically active molecules.¹⁵ The phospholipid bilayer, which forms the outer shell of the exosome, is mainly composed of phosphatidylcholine and other phospholipids, with a variety of membrane proteins embedded within it, including tetraspanins (eg, CD63, CD9, CD81), heat-shock proteins (HSPs), and other exosome-specific proteins. These proteins play a crucial role in exosome isolation and signaling.¹⁶ The loaded components of lipid vesicles include a variety of bioactive molecules such as proteins, nucleic acids, metabolites.¹⁷ These molecules interact with the phospholipid bilayer to form a stable structure and endow the exosome with rich biological information, which can be transmitted to other cells to exert specific biological effects.

Isolation and Extraction of Exosomes

Exosomes are nanoscale extracellular vesicles present in complex body fluids and serve as critical mediators of intercellular communication. The isolation and purification of exosomes are essential for subsequent research and clinical applications.⁹ Currently, reported methods for exosome isolation and purification include ultracentrifugation, density gradient centrifugation, ultrafiltration, size-exclusion chromatography, polymer precipitation, affinity separation, and microfluidic techniques. Each method possesses distinct advantages and limitations. In this paper, we comprehensively analyze the principles, advantages, disadvantages, separation efficiencies, and applications of each technique (Table 1).

Table 1 Current Methods of Exosome Isolation

Ultracentrifugation

Ultracentrifugation is a prevalent technique for the isolation of exosomes. By progressively increasing the centrifugal force, exosomes with varying densities, sizes, and morphologies can be effectively separated. Initially, low-speed centrifugation removes debris such as dead cells and large biological particles. Subsequently, ultrahigh-speed centrifugation (>100,000 g) is employed to isolate exosomes, resulting in the collection of exosome pellets,¹⁸ Exosomes can achieve higher purity through the addition of PBS followed by ultrahigh-speed centrifugation to eliminate apoptotic vesicles, proteins, and other contaminants.¹⁹ Due to its advantages of yielding a large volume of exosomes, employing a relatively mature methodology, simplicity in operation, and absence of reagent contamination, this method has been extensively utilized over the past few decades. Specifically, it has been widely applied in the isolation of exosomes from urine, serum, cerebrospinal fluid, and cell culture medium. However, this technique also exhibits certain limitations, including poor specificity, potential contamination with proteins of similar molecular weight, high equipment costs, time-consuming procedures, and damage to exosomes caused by repeated centrifugation.^20–23 In order to further improve the separation efficiency of this classical separation technique by exploring the different physical properties of the objects, several types of centrifugal strategies have now been developed, among which density gradient centrifugation is widely used.^24,25

Density Gradient Centrifugation

Differences in sedimentation coefficients among biological particles result in their separation within specific inert gradient media. Under the influence of centrifugal force, these particles will be distributed into distinct zones according to their density.²⁶ This encompasses both rate-zonal centrifugation and isopycnic centrifugation. Presently, these two techniques are utilized in our procedures. Prior to centrifugation, it is necessary to prepare a gradient with progressively increasing density from the top to the bottom of the centrifuge tube. Commonly employed media for this purpose include sucrose and iodixanol.²⁷ The typical procedure is as follows: after preparing the density gradient medium, the ultracentrifuged sample is carefully layered on top of the gradient. Upon centrifugation, the sample components will migrate from the top to the bottom according to their densities. Consequently, exosomes, cellular debris, apoptotic bodies, and proteins will stratify into corresponding density layers within the medium.^28,29 Exosomes, on the other hand, reside in their iso-density zone (1.12–1.19 g/mL),³⁰ without sedimenting at the bottom of the tube. Purified exosomes can be obtained by collecting this specific zone. Exosomes isolated through density gradient centrifugation are nearly devoid of contaminating proteins,³¹ purity is comparatively high; however, these methods suffer from several drawbacks including costly equipment, time-consuming processes, and potential damage to exosomes caused by repeated centrifugation. Consequently, they are not suitable for large-scale preparations.^32,33 In view of the prolonged ultracentrifugal force, which may affect the structure and biological function of the isolated exosomes, other size-based separation methods have been introduced, such as ultrafiltration.

Ultrafiltration (Separation Technique Based on Differences in Particle Size)

Ultrafiltration holds greater potential for industrial-scale exosome preparation compared to ultracentrifugation. The underlying principle involves passing the sample through one or more membranes with varying pore sizes or molecular weight cut-offs. Impurities smaller than the membrane’s pore size are filtered into the filtrate, whereas particles exceeding the pore size in diameter are retained on the membrane surface.²⁷ The most commonly employed media for membrane filtration are cellulose and its derivatives. Ultrafiltration is driven by electrical charge, low-speed centrifugation, and pressure differentials.³⁴ Currently, the conventional centrifugation step in ultrafiltration is conducted as follows: as the sample traverses the membrane, a 200 nm pore-size membrane selectively retains larger particles, such as large proteins and apoptotic vesicles. Meanwhile, a 20 nm pore-size membrane effectively intercepts vesicles, including exosomes within the size range of 20–200 nm, while smaller molecules like small proteins pass through and are collected at the bottom. Over the past decade, ultrafiltration has gained increasing popularity due to its capacity to process large sample volumes, operational simplicity, minimal equipment requirements, absence of chemical additives, time efficiency, high enrichment efficacy, and superior purity.^5,35 Nevertheless, several limitations exist, including the occlusion of membrane pores by large particles, mechanical damage to exosomes, and the absence of an effective mechanism for extracting vesicles and proteins.^36,37 A combination of two or more methods, such as ultrafiltration and density gradient centrifugation, can be a good solution to the problem of exosome clogging.³⁸

Separation Techniques Based on Differences in Particle Size (Size Exclusion Chromatography SEC)

A physical separation principle based on molecular size differences, commonly referred to as gel filtration chromatography, is utilized. This technique comprises two main components: the stationary phase and the mobile phase. Commonly employed stationary phase media include dextran polymers, agarose, and polyacrylamide.³⁹ A physical separation principle based on molecular size differences, commonly referred to as gel filtration chromatography, is utilized. This technique comprises two main components: the stationary phase and the mobile phase. Commonly employed stationary phase media include dextran polymers, agarose, and polyacrylamide.^5,35,40 SEC has the advantages of being moved only by gravity, higher purity, higher recovery, low cost, no need for sample pretreatment, and shorter time consuming, but the most appealing feature is that it is capable of retaining the bioactivity of exosomes.^41–44 In recent years, SEC-based exosome isolation techniques have gained increasing popularity owing to their advantages. Nevertheless, the SEC method also encounters certain challenges, such as potential contamination from similarly sized impurities and the dilution effect of the eluent on exosomes.²⁰ To address the problem that SEC can be doped with impurities of similar size, Gardiner proposed a separation strategy that combines ultrafiltration and SEC, a combination that preserves exosome functionality while greatly improving exosome purity.⁴⁵

Solubility-Based Polymer Precipitation Techniques

The polymer precipitation technique, analogous to ethanol-mediated nucleic acid precipitation, employs highly hydrophilic polymers to interact with water molecules surrounding exosomes. This interaction creates a hydrophobic microenvironment that reduces the solubility of exosomes, leading to their precipitation. Subsequently, centrifugation is utilized to isolate the exosomes.^5,46 Commonly available hydrophilic polymers include ichthyosperm, acetic acid, and organic solvents. Among these, polyethylene glycol (PEG), which is water-soluble and non-toxic, has become the most widely utilized medium for the polymerization and precipitation of exosome.^47–49 Exosomes are initially subjected to pre-treatment to eliminate larger contaminating particles, including cellular debris and apoptotic vesicles. Subsequently, they are incubated with PEG in a 4 °C solution overnight. Finally, exosomes are harvested from the precipitates through low-speed centrifugation.⁵⁰ This method has been extensively utilized for the isolation of exosomes from various biological samples, including urine, serum, cerebrospinal fluid, and cell culture medium.^46,51 The advantages of this technique include its operational simplicity, capability to process large sample volumes, and high throughput. However, it also has limitations, such as potential contamination with non-exosomal proteins and relatively low purity.^52–54 To solve the problem of protein contamination, hydrophobic PEG and hydrophilic dextran can be mixed together to form an aqueous two-phase system, with PEG forming the upper phase and dextran forming the lower phase. By adding PEG and dextran to the exosome solution and centrifuging at low speed, exosomes preferentially aggregate in the dextran phase, while some proteins, macromolecular complexes, etc. preferentially aggregate in the PEG phase, thus improving the recovery of exosomes.⁴⁸

Affinity Separation Techniques Based on Immunoreactivity

Immunoaffinity capture facilitates the isolation of highly purified exosomes through the specific binding of surface antigens on the exosome membrane to their corresponding antibodies.²⁰ In recent decades, a comprehensive identification and documentation of surface markers on exosomes have been conducted. These markers include the tetraspanin family of proteins (CD9, CD63, CD81, CD82), membrane-associated proteins such as Annexin V, epithelial cell adhesion molecules (EpCAM), lysosome-associated membrane protein-2B (LAMP2B), heat shock proteins, and phospholipases.^55–57 During the process of exosome isolation, antibodies specific to certain markers must be immobilized on carriers such as magnetic beads, silica, or resins.^35,48,58 When exosomes traverse the carriers, they are captured by specific antibodies through antigen-antibody interactions. Following this, an elution step is performed to isolate the exosomes from the carrier-exosome complexes. Immunoaffinity capture offers several advantages, including high specificity and purity, preservation of exosomal structural integrity and biological activity, minimal equipment requirements, and straightforward operation, making it an ideal method for isolating subpopulations of exosomes from specific origins.^59,60 However, this approach also exhibits certain limitations, including time-consuming procedures, high antibody costs, stringent elution conditions, and unsuitability for large-scale isolation.⁶¹ When using immunoaffinity capture methods to isolate exosomes, pretreatment can be combined with polymer precipitation techniques, thus avoiding the use of excessively expensive antibodies.⁶²

Microfluidics Based on Fluid Properties

Microfluidics, leveraging its unique fluidic properties, demonstrates significant potential for the separation and purification of exosomes. This technology enables the investigation of the physicochemical characteristics of exosomes at the microscopic level.⁶³ Microfluidics refers to a system comprising a network of fluidic channels with diameters ranging from tens to hundreds of micrometers. This technology is integrated with fluidic control systems and analytical instrumentation to automate the processes of extracting, detecting, separating, and analyzing biomaterials.⁶⁴ Microfluidics is frequently integrated with traditional separation techniques, such as immunoaffinity-based microfluidics for the isolation of highly specific exosomes and size-based microfluidics to enhance the purity of exosome separation.^65,66 Microfluidics leverages the physical (size, density) and chemical (surface antigens) properties of exosomes for their separation. This technology offers significant advantages including rapid and efficient processing, automation, integration, and high throughput, which are revolutionizing the field of exosome isolation and diagnosis. These advancements are particularly valuable for non-invasive disease detection, such as early cancer screening.^67–69 Exosome isolation using microfluidic technology also faces challenges due to the complexity of fabrication techniques and a lack of standardization. These shortcomings necessitate further exploration to develop more efficient and standardized methods.

At present, there are also many emerging and efficient exosome purification systems on the market, for example, EXODUS fully automated exosome extraction system from Shenzhen Huixin Biomedical Technology Company, which utilizes negative pressure oscillation system (NPO) combined with dual-coupled harmonic oscillation system (HO) acting on the nano-ultra-filtration chip to enable the free nucleic acids and proteins and other impurities in the sample to be rapidly removed through the nanopore and sequester the exosomes, thus purifying and enriching the exosomes. Thus, purifying and enriching exosomes. In the future, the integrated and automated design of the equipment will be another new direction of exosome purification, which can quickly process a large number of samples, improve the experimental efficiency, reduce the time and labor costs, and solve the current stage of the exosome preparation of large-scale building problems.⁵

Preservation of Exosomes

The stability of exosomes is critically dependent on their bilayer membrane structure, which safeguards their contents against degradation and denaturation in the extracellular environment.⁷⁰ Differences in storage methods can significantly influence multiple characteristics of exosomes, such as the stability of their biological and physical properties, recovery efficiency, diameter distribution, and functional integrity. Presently, common preservation methods for exosomes include cryopreservation and lyophilization, which will be detailed subsequently.

Cryopreservation is a conventional method for preserving exosomes; however, this technique may result in alterations to the morphology and physical properties of exosomes, the formation and aggregation of multilayered vesicles, as well as changes in molecular bioactivity, content, and surface markers due to the freeze-thaw process.⁷¹ Currently, cryopreservation temperatures of 4°C, −20°C, and −80°C are commonly utilized, with −80°C being the most prevalent. At 4°C, exosomes remain unfrozen and do not undergo morphological damage; however, prolonged storage at this temperature results in a decrease in exosome count as well as reductions in the proteins and nucleic acids they contain, leading to diminished biological activity. For instance, plasma-derived exosomes stored at 4°C experience RNA degradation and loss of proteins and nucleic acids,⁷² and serum-derived exosomes, which contain miR-122 and miR-14, undergo degradation during preservation at 4°C,⁷³ Therefore, 4°C is more appropriate for short-term preservation. Storing exosomes at −80°C may affect their morphology due to the process of freezing and thawing; however, the proteins and nucleic acids they contain remain relatively stable, as do their biological activities.⁷⁴ It has been demonstrated that milk-derived exosomes stored at −80°C for approximately one month retain their physical and biological properties without alteration.⁷⁵ In addition, storage at −80°C has demonstrated a significant effect on the activity of milk-derived extracellular vehicles (EVs).⁷⁶ In contrast, plasma-derived exosomal miRNAs remained stable under frozen conditions (−20°C and −80°C) and did not exhibit significant changes over a period of two years.⁷² Furthermore, preservation at −80°C is considered the optimal storage environment for semen, urine, milk, blood, and bronchoalveolar lavage fluid.^71,77

Freeze-drying represents an innovative approach to exosome preservation, offering a cost-effective strategy that extends the shelf life of exosomes while minimally impacting their morphology and content at ambient temperatures. Protective agents such as alginate and mannitol are commonly incorporated during the freeze-drying process. The inclusion of alginate as a bioprotectant enhances protein, cell membrane, and liposome stability, mitigates ice crystal formation, and preserves exosome integrity, stability, and functionality.^70,78 Charoenviriyakul et al found that the absence of a cryoprotectant led to aggregation of B16BL6 melanoma-derived exosomes. The addition of alginate as a cryoprotectant, however, prevented such aggregation during lyophilization. Furthermore, PAGE analysis demonstrated that the proteins and RNAs within the exosomes were effectively protected by the inclusion of alginate as a cryoprotectant.⁷⁰ In addition, mannitol can serve as a cryoprotectant to safeguard exosomes during lyophilization.⁷⁹ While lyophilization offers numerous preservation advantages, the duration of exosome stability and its applicability to different types of exosomes remain unclear. This field is still in its preliminary stages and lacks standardized protocols.

Heterogeneity is observed among exosomes derived from different sources, and their stability exhibits considerable variation under identical preservation conditions. For instance, exosomes isolated from sperm can be stored at −80°C for up to 30 years without significant alterations in morphology, physical properties, or nucleic acid and protein composition,⁷⁷ While bronchoalveolar lavage fluid-derived exosomes exhibit significant alterations in protein abundance and volume when frozen compared to fresh samples.⁷¹ Furthermore, varying levels of acidity and alkalinity significantly influence the preservation and functionality of exosomes. Acidic conditions result in a higher yield of isolated proteins and nucleic acids, as well as enhanced cellular uptake of exosomes. Specifically, HEK 293T cells produce exosomes with protein and nucleic acid concentrations that are fivefold higher in acidic environments compared to neutral conditions,⁸⁰ In alkaline environments, however, the concentrations are significantly lower. Exosomes derived from human urine are isolated under acidic conditions, which facilitates the maintenance of their stability and integrity, resulting in a higher yield of total RNA and minimized protein degradation.⁸¹

Based on the current evidence, storage at −80°C appears to be the most favorable condition for preserving biological fluids and isolated exosomes. For short-term storage of exosomes, a temperature of 4°C is recommended. Additionally, the addition of cryoprotectants or adjustment of pH can help maintain the integrity and biological activities of exosomes. Further research is warranted to better understand how storage conditions affect exosome particles, functions, and activities, which will contribute to the development of improved exosome storage strategies and comprehensive cryopreservation methods.

Advantages of Exosomes as Drug Delivery Systems

Biocompatibility

Exosomes, as endogenous substances, are not recognized, captured, or cleared by the mononuclear phagocyte system in the body.⁸² Usman et al introduced antisense oligonucleotides into erythrocyte-derived exosomes via electroporation and demonstrated that these exosome-loaded antisense oligonucleotides significantly inhibited breast cancer cell growth and were non-immunogenic in vivo.⁸³ In contrast, synthetic nanocarriers can induce an immune response in the body after multiple drug administrations, leading to rapid clearance by the immune system and potential adverse immune reactions such as allergic responses.^84–86

Biostability

Exosomes exhibit excellent biostability, enabling prolonged circulation in vivo, even under inflammatory conditions, where they offer robust protection for therapeutic agents.⁸⁷ Moreover, the expression of CD47 on the exosomal membrane effectively prevents clearance by the mononuclear phagocyte system, thereby extending their circulation time in vivo. Additionally, surface modification of exosomes can further enhance their in vivo circulation duration.⁸⁸ Exosomes derived from macrophages were functionalized with aminoethyl anisamide polyethylene glycol (AA-PEG), which significantly diminishes their recognition and internalization by the monocyte-macrophage system (MPS), thereby preventing rapid clearance and markedly extending their circulation time in vivo.^89,90 Asparagus phylum-extracted nanovesicles (ACNV) have been shown to inhibit hepatocellular carcinoma proliferation through activation of the apoptotic pathway; however, the cytosolic effects mediated by the mononuclear phagocyte system result in rapid exosome clearance from the bloodstream. Modification of ACNV with distearoyl-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) altered its pharmacokinetic profile, leading to enhanced in vivo circulation time.^91,92

Targeted

Exosomes are specifically recognized by signaling molecules, such as receptors on the cell membrane surface or associated proteins in the extracellular matrix, and subsequently bind to the surface of target cells. This recognition facilitates directed migration of exosomes to specific tissues or organs, enabling the delivery of therapeutic molecules (eg, miRNAs) to their intended sites while minimizing degradation.⁹³ Moreover, exosomes derived from certain cellular sources possess intrinsic targeting capabilities. For instance, exosomes from central nervous system (CNS) cells can traverse the blood-brain barrier and target specific neurons, whereas exosomes from hypoxic tumor cells are preferentially recruited to hypoxic tumor tissues.^94–96 Furthermore, surface modification of exosomes can be employed to enhance their targeting capabilities. For instance, the conjugation of cyclic RGD peptides to natural exosomes significantly improves their tumor-targeting ability. This functionalization facilitates the enhanced accumulation of chemotherapeutic agents, such as DOX, within the tumor microenvironment.⁹⁷ Macrophage-derived exosomes engineered with the AS1411 aptamer exhibit efficient blood-brain barrier (BBB) penetration and precise cancer cell targeting capabilities.⁹⁸

In addition to exhibiting excellent biocompatibility, biostability, and targeting capabilities, exosomes also possess superior permeability. As lipid-coated vesicles, they can effectively traverse the blood-brain barrier, cytoplasmic membranes, and other biological barriers, thereby achieving efficient drug delivery.⁹⁶ Exosomes are distinguished by their remarkable modifiability, which can be influenced by the characteristics of their source cells, thereby modulating their properties and functions. By manipulating the gene expression profiles of exosome-producing cells, it is possible to regulate both the secretion and composition of exosomes. For instance, certain studies have demonstrated that overexpression of genes encoding specific membrane proteins or drug-carrying proteins can enhance the cargo capacity of exosomes for therapeutic agents or specific molecules. Furthermore, exosomes can undergo modifications through chemical, biological, immunological, and physical approaches, such as chemical or enzymatic alterations of functional molecules on their surface to impart targeted biological effects. This versatility in modification enhances the flexibility and controllability of exosome-based drug delivery systems, facilitating the implementation of personalized medicine.^90,99–101

In addition to exosomes, there are several carriers that can be used for drug delivery. Lipid nanoparticles (LNP) is a synthetic liposome. Although it can efficiently encapsulate nucleic acids and achieve rapid delivery (eg, mRNA vaccines), its cationic lipid and PEG lipid components may trigger immune reactions or hepatotoxicity, in addition to its organ targeting and penetration limitations.^102,103 In contrast, exosomes, as natural vesicles secreted by cells, have low immunogenicity, natural targeting, and excellent biocompatibility, which can cross complex barriers and reduce the risk of off-targeting.⁹⁶ In addition, exosomes can carry a variety of endogenous active molecules, and will become a safer and more precise next-generation delivery system after further optimization of drug-carrying efficiency through engineering modification.

Engineered Exosomes

Engineered exosomes are vesicles that have undergone surface modifications and encapsulation of therapeutic molecules. As an innovative drug delivery platform, engineered exosomes have shown considerable promise in diverse fields such as oncology, cardiology, stomatology, neurology, and tissue regeneration and repair.¹⁰⁴

Engineered Exosome Preparation

Despite the widespread application of natural exosomes in systemic disease therapy, their inherent limitations, including low yield, impurities, and inadequate targeting capabilities, have constrained their therapeutic efficacy. Conversely, advancements in engineered exosome technology offer a more comprehensive approach to disease diagnosis and treatment. This technology leverages the inherent properties of exosomes, such as biocompatibility, natural carriage of bioinformatic molecules, and low immunogenicity, and enhances their functionalities through engineering techniques. These enhancements include specific targeting, improved drug loading capacity, and resistance to organismal clearance. Commonly employed methods for preparing engineered exosomes can be categorized into two main types: cell-based engineering and direct exosome modification. Cell-based engineering encompasses genetic manipulation, alteration of cell culture conditions, and pharmacological treatments, while direct exosome modification includes biological, immunological, physical, chemical modifications, and the conjugation of biomaterials. These approaches refine and specify the functional capabilities of exosomes.

In the field of cellular engineering, researchers have concentrated on genetically modifying parental cells of exosomes to enhance the expression of specific proteins or signaling molecules. This approach aims to improve the targeting accuracy and therapeutic efficacy of exosomes. Modified exosomes can be engineered during their biosynthesis to incorporate functional components such as surface peptides or targeting ligands, thereby increasing their affinity for target cells.¹⁰⁵ Alvarez-Erviti et al were the first to demonstrate that exosomes secreted by plasmid-transfected dendritic cells, when fused with neuron-specific rabies virus glycoprotein and exosomal lysosome-associated membrane protein 2b (Lamp2b), could deliver siRNA specifically to the brain. The fusion of a target protein with Lamp2b facilitates its localization on the exosome surface, setting a precedent in this field.¹⁰⁶ Modifying cell culture conditions represents an effective engineering strategy that can indirectly influence the yield and characteristics of exosomes by adjusting parameters such as the composition of the culture medium, cell density, or oxygen concentration.¹⁰⁷ This method not only enhances the purity of exosome isolation but also improves the stability and biological activity of the encapsulated therapeutic agents. Under hypoxic conditions, Patton et al demonstrated that culturing pancreatic cancer cells resulted in an increased release of exosomes with a reduced average particle size, thereby providing favorable conditions for the adaptive growth of these cells.¹⁰⁸ The strategy of co-incubating parental cells with drugs facilitates the natural encapsulation of drug components into the exosome interior during exosome biogenesis, thereby eliminating the need for additional drug loading steps and preserving the bioactivity and biocompatibility of the drug. In inflammation-related autoimmune and inflammatory diseases as well as cancer, co-incubation of curcumin with EL-4 (mouse lymphoma cell)-derived exosomes has been shown to enhance the therapeutic efficacy of exosomes.¹⁰⁹ From this point of view, genetic engineering technology can not only successfully construct highly targeted engineered exosomes, but also significantly increase the drug loading capacity of engineered exosomes to achieve precision therapy, but the biosafety of genetic engineering still needs more research to examine. However, the biological safety of genetic engineering still needs more research. Although changing the cell growth conditions is an effective method to increase the production of exosomes, the degree of increase in the production is not enough to become a major method for mass production of exosomes.

The advancement of physical processing techniques has opened new avenues for engineering exosomes, enabling more precise therapeutic targeting and enhanced safety in the presence of external physical stimuli such as magnetic fields, laser irradiation, ultrasound, and electroporation. For instance, exosomes derived from human umbilical cord mesenchymal stem cells (HUCMSC) have been successfully ultrasonicated to encapsulate silver nanoparticles (AgNPs), thereby acquiring antimicrobial properties and promoting wound healing.¹¹⁰ Additionally, neutrophil-derived exosomes modified with superparamagnetic iron oxide nanoparticles (SPIONs) have demonstrated selective accumulation at tumor sites when subjected to external magnetic fields.¹¹¹ Furthermore, the loading of tumor necrosis factor (TNF-α) and SPION-modified exosomes markedly enhances cancer-targeting efficacy under external magnetic fields while reducing in vitro and in vivo toxicity.¹¹² Additionally, Wan et al demonstrated that electroporation-mediated loading of Cas9 ribonucleoprotein (RNP) into exosomes purified from hepatic stellate cells facilitated efficient cytoplasmic delivery of RNP, promoting specific accumulation in hepatic tissues and enabling tissue-specific gene therapy in the liver.¹¹³ Engineered exosomes prepared by physical methods have better biosafety by reducing the incorporation of intermediate mediators other than drugs, but they are overly dependent on preparation methods and equipment.

In addition, biological, immunological, and chemical modifications are employed to enhance the spatial targeting capabilities of exosomes. TLyp-1 peptide-modified exosomes exhibit high transfection efficiency in lung cancer treatment due to the selective binding of TLyp-1 peptide to neuropilin-1 (NRP1) and neuropilin-2 (NRP2).^114–116 Anti-CD3 and anti-EGFR (epidermal growth factor receptor) antibody-engineered exosomes facilitate the binding of T cells to cancer cells, thereby enabling precise therapy.¹¹⁷ The tumor microenvironment (TME) is characterized by relative acidity, likely resulting from elevated glycolysis rates and increased lactate production, which facilitates tumor-targeted modification of engineered exosomes. Paclitaxel (PTX), a common chemotherapeutic agent, can be encapsulated with NaHCO3 in exosomes. Upon endocytosis by cancer cells, this combination rapidly generates carbon dioxide bubbles, creating an acidic environment that promotes effective drug release.¹¹⁸ By surface modification, the engineered exosomes are more targeted, but the receptor-ligand reaction binding is weaker and the drug adhesion is not very strong during drug delivery.

Furthermore, researchers in the fields of biology, immunology, and chemical modification have achieved significant advancements in the biomaterial loading of exosomes. These developments facilitate disease treatment by utilizing various types of biomaterials, such as hydrogel scaffolds or dressings, to provide structural support and enhance exosome delivery.^119,120 Specifically, a bio-responsive polyethylene glycol (PEG)/DNA hybrid hydrogel system was developed and loaded with exosomes derived from human apical papillary stem cells (SCAP-Exo). This system has demonstrated efficacy in promoting bone regeneration under both normal and diabetic conditions.¹²¹ Biomaterials loaded with exosomes have better targeting, slow drug release, and can be combined with other therapeutic means to realize multifunctional collection, but the safety and high cost of biomaterials limit their large-scale clinical application. Comparison of advantages and disadvantages regarding engineered exosome preparation methods (Table 2).

Table 2 Comparison of Advantages and Disadvantages of Engineered Exosome Preparation Methods

Engineered Exosome Applications and Advances

Exosomes have demonstrated extensive potential as drug delivery vehicles in the treatment of specific diseases. Through targeted engineering and modification, exosomes can facilitate efficient and precise drug delivery. Research has indicated that exosomes can serve as carriers for anticancer agents, anti-inflammatory compounds, and genetic materials, thereby enabling the treatment of tumors, inflammatory conditions, cardiovascular disorders, neurological ailments, and other diseases (Figure 2).

Figure 2 Clinical application of extracellular vesicle therapy. Created in BioRender. Hu, B. (2025) https://BioRender.com/b12c371.

Tumor Related Diseases

Engineered exosomes, as an innovative drug delivery platform and an ideal modality for clinical therapeutic applications, can significantly enhance anti-tumor efficacy in cancer treatment and augment the effectiveness of conventional therapies such as radiotherapy, photothermal therapy, photodynamic therapy, and gene therapy. In preclinical studies, engineered exosomes are typically administered intravenously to target tumor sites and deliver their cargo, including chemotherapeutic agents, non-coding RNAs, and immune molecules, directly into the tumor microenvironment, thereby inducing tumor cell apoptosis.

Chemotherapy usually leads to systemic adverse effects such as myelosuppression.^122,123 Through the exosome-mediated delivery of chemotherapeutic agents, enhanced anti-tumor efficacy can be achieved while minimizing systemic toxic effects.¹¹¹ In breast cancer, the encapsulation of paclitaxel (PTX) within exosomes effectively prevents recurrence and metastasis, and significantly inhibits tumor growth in murine models of breast cancer.^118,124 Macrophage-derived exosomes loaded with paclitaxel (PTX) and conjugated with aminoethyl anisamide polyethylene glycol (AA-PEG) were evaluated in a murine lung cancer metastasis model.⁸⁹ The findings indicated that the presence of PEG hindered the recognition and internalization of exosomes by the mononuclear phagocyte system (MPS), consequently prolonging the circulation time of exosomes in vivo.⁹⁰

Gene therapy for oncology involves the modification of DNA or RNA to treat cancer. One common approach is to load non-coding RNA into engineered exosomes, which are then delivered to the tumor site to exert therapeutic effects.¹²⁵ In gliomas, exosomes loaded with miR-29a-3p have been shown to inhibit tumor migration and angiogenesis by targeting ROBO1.¹²⁶ In hepatocellular carcinoma, engineered exosomes modified with miR-199a effectively deliver this microRNA to hepatocellular carcinoma cells, thereby significantly enhancing their sensitivity to Adriamycin in vitro and promoting the antitumor effects of Adriamycin on hepatocellular carcinoma in vivo.¹²⁷

Photodynamic therapy (PDT) is a minimally invasive treatment modality for cancer that primarily employs photosensitizers.¹²⁸ Recent studies have demonstrated that engineered exosomes can effectively deliver Rose Bengal, a photosensitizer¹²⁹ in conjunction with Erastin, an inducer of ferroptosis¹³⁰ to a murine model of hepatocellular carcinoma, thereby inducing iron-dependent cell death in tumor cells. Acoustic kinetic therapy (AKT), another ROS-mediated cancer treatment, utilizes ultrasound and acoustic sensitizers to target and destroy tumors.¹³¹ When combined with engineered exosomes, AKT enables more controlled drug release and enhanced precision in tumor targeting. The presence of the blood-brain barrier (BBB) and hypoxic tumor microenvironment poses significant challenges to conventional AKT efficacy. However, macrophage-derived exosomes modified with the AS1411 aptamer exhibit robust BBB penetration and targeting capabilities, thereby enhancing therapeutic outcomes,¹³² and the exosomes loaded with acoustic sensitizers (ICGs) and catalase-loaded silica nanoparticles (CATSiO2) can effectively treat tumors.¹³³ Additionally, the oxygen generated from the catalase-catalyzed decomposition of hydrogen peroxide significantly improves the hypoxic tumor microenvironment.

Cardiovascular Disease

Cardiovascular diseases (CVD) is a collective term for heart diseases and cardiovascular diseases, and CVD has always been the leading cause of mortality, with 40% of deaths in the population being attributed to CVD.¹³⁴ Consequently, enhancing the therapeutic efficacy for CVD is of paramount importance. Current treatment modalities for CVD, including pharmacological interventions, interventional procedures, and open surgeries, have significantly improved patient outcomes. However, these treatments are associated with certain complications. Recent advancements in exosome research have revealed their potential in mitigating inflammation, promoting ventricular remodeling, and facilitating neovascularization, thereby offering promising therapeutic avenues for CVD.^135,136

Myocardial Infarction

Acute myocardial infarction (AMI) is a severe cardiovascular condition characterized by sustained ischemia and hypoxia in the coronary arteries. Emerging evidence indicates that exosomes play a crucial role in both the diagnosis and therapeutic approaches for AMI.¹³⁷ Su et al conducted a comprehensive analysis of miRNA expression profiles in exosomes derived from 6 patients with acute myocardial infarction (AMI) and 6 patients with stable coronary artery disease (SCAD). They observed that the expression levels of exosomal miR-3, miR-4, miR-507, miR-656, and miR-1915-3p were significantly reduced in AMI specimens compared to SCAD specimens. This finding suggests that these specific exosomal miRNAs may serve as potential biomarkers for the diagnosis of AMI.¹³⁸ A growing body of evidence indicates that exosomes derived from various tissues can suppress cardiac inflammatory responses, exert immunomodulatory effects, and mitigate myocardial injury via distinct molecular mechanisms. For instance, Pan et al demonstrated that miR-146a, contained within exosomes derived from adipose stem cells, mitigates inflammatory responses and enhances the local microenvironment by inhibiting the release of interleukin 6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α). Furthermore, it alleviates myocardial cell injury induced by acute myocardial infarction through the downregulation of early growth response factor 1 (EGR1).¹³⁹

Atherosclerosis

Atherosclerosis (AS) is characterized by a chronic inflammatory response, with macrophages serving as key mediators in the formation and progression of atherosclerotic plaques.¹⁴⁰ Macrophages, which are versatile immune cells, can be categorized into classically activated macrophages (M1-type) and alternatively activated macrophages (M2-type) based on their functional properties and activation states. Research has demonstrated that the stimulation of both murine and human macrophages with oxidized low-density lipoprotein (oxLDL), followed by the isolation and characterization of exosomes, reveals that exosomal miR-146a contributes to the development of AS by inhibiting cell migration, promoting macrophage retention within the vascular wall, and inducing oxidative stress.^141,142 MSC-derived exosomes can also be activated via the miR-let7/HMGA2/NF-κB pathway, which induces macrophage polarization from the M1 to the M2 phenotype, exerts anti-inflammatory effects, and regulates the progression of atherosclerosis.¹⁴³

Diseases of the Nervous System

Low intracerebral drug concentrations have posed a significant challenge in the advancement of therapeutic strategies for central nervous system (CNS) diseases. The existence of the blood-brain barrier (BBB) and its selective permeability restricts the effective entry of pharmacological agents into the brain.¹⁴⁴ In recent years, a growing body of research has indicated that exosomes may serve as promising natural drug delivery systems (DDSs). Firstly, exosomes exhibit minimal immunogenicity, thereby enhancing drug efficacy and minimizing adverse reactions;¹⁴⁵ Secondly, their unique phospholipid-membrane-protein structure facilitates efficient intracellular delivery of therapeutic agents into target cells.¹⁴⁶ Additionally, exosomes demonstrate remarkable stability, which prevents phagocytosis by macrophages and extends the half-life of the drug;¹⁴⁷ Most importantly, the lipid bilayer of exosomes can assist in crossing natural barriers such as the blood-brain barrier (BBB), thereby improving the bioavailability of central nervous system (CNS) therapeutic drugs.^148,149

Alzheimer’s Disease

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder, characterized by progressive cognitive decline and memory impairment.¹⁵⁰ Silymarin (silibinin, Slb) has demonstrated therapeutic potential in AD by inhibiting amyloid-β (Aβ) aggregation and suppressing astrocyte activation; however, its clinical application is limited due to low bioavailability.¹⁵¹ In this study, Huo et al investigated the efficacy of exosome-encapsulated silymarin (Exo-Slb) in treating Alzheimer’s disease. Specifically, Huo et al encapsulated Slb within macrophage-derived exosomes (Exo-Slb), which upon entering the brain, selectively bind to Aβ1-42 and inhibit the aggregation of Aβ1-42. Additionally, Exo-Slb is internalized into astrocytes, where it regulates the nuclear factor κB (NF-κB) pathway to suppress astrocyte activation, thereby mitigating astrocyte-mediated neuronal damage and effectively alleviating cognitive deficits in AD mice.¹⁵² Lee et al demonstrated that exosomes derived from adipose stem cells significantly reduced the expression levels of Aβ42 and Aβ40, as well as the Aβ42/Aβ40 ratio in neural stem cells of Alzheimer’s disease mice. Furthermore, these exosomes were capable of modulating the Aβ42/Aβ40 ratio. Additionally, exosomes from adipose stem cells of Alzheimer’s disease mice could inhibit apoptosis in neural stem cells, effectively repair synapses damaged by Aβ in the brain, and consequently regulate neural function. These findings suggest that exosomes can potentially intervene in Alzheimer’s disease progression by modulating apoptosis and promoting synaptic growth.¹⁵³

Parkinson’s Disease (PD)

Parkinson’s disease is the second most common neurodegenerative disease.¹⁵⁴ In addition to conventional diagnostic criteria such as patient history, bradykinesia, and muscle tone assessment, the measurement of exosome levels can serve as an auxiliary tool for diagnosis and identification of therapeutic targets. Alpha-synuclein (α-syn), a primary component of Lewy bodies (LBs), contributes to mitochondrial dysfunction, increased oxidative stress, and degeneration of dopaminergic neurons through its aggregation. Consequently, strategies aimed at reducing α-syn expression represent promising therapeutic approaches to mitigate the progression of Parkinson’s disease (PD).¹⁵⁵ Stuendl et al investigated exosomes in the cerebrospinal fluid (CSF) of patients with Parkinson’s disease. Their findings demonstrated that CSF-derived exosomes from these patients could induce the oligomerization of soluble α-Syn in target cells, leading to further disease progression. This suggests that interventions targeting CSF-derived exosomes-induced α-Syn oligomerization may effectively modulate disease development.¹⁵⁶ Clinically, Parkinson’s disease (PD) is commonly managed through dopamine injections. However, the blood-brain barrier (BBB) significantly hinders the efficient delivery of dopamine to the brain. To address this issue, drug delivery systems (DDS) that can directly encapsulate dopamine are required for effective treatment.¹⁵⁷ While antisense oligonucleotides (ASOs) have shown promise in various clinical applications, including the treatment and management of PD, their ability to reduce the expression of α-synuclein (α-syn) remains limited by challenges in safely and effectively delivering ASOs to neurons. Yang J et al developed a safe and efficient delivery method for ASOs by utilizing exosomes derived from bone marrow mesenchymal stem cells (hbmMSC) to encapsulate ASO4 (Exo-ASO4). It was demonstrated that Exo-ASO4 can effectively penetrate the brain parenchyma of Parkinson’s disease (PD) model mice, leading to a significant reduction in the expression levels of α-synuclein. Consequently, the use of exosomes to deliver exogenous ASO4 into the brain markedly ameliorates dopaminergic neuronal degeneration and motor dysfunction in PD model mice.¹⁵⁸

Other Diseases

Stem cell exosomes have demonstrated significant potential in disease-specific therapies. For instance, in the treatment of diabetes, stem cell exosomes can markedly enhance blood glucose regulation and mitigate tissue damage by modulating insulin secretion and alleviating insulin resistance.¹⁵⁹ In skin photoaging repair, stem cell exosomes can promote collagen synthesis and cell proliferation, reducing wrinkles and hyperpigmentation.^160,161 In oral and maxillofacial disorders, stem cell exosomes can assist in the management of conditions such as periodontitis, endodontitis, and oral cancer, contributing to both diagnosis and treatment¹⁶² (Table 3).

Table 3 Engineered Exosome Applications

Summary and Outlook

Traditional drug delivery systems predominantly rely on exogenous carriers. Although nanoscale delivery vehicles have emerged, practical applications continue to face multiple challenges, including concerns over carrier safety, in vivo heterogeneity, and low bioavailability.⁸⁵ Exosomes exhibit excellent biocompatibility and minimal immunogenicity. Through engineered modifications, they can target specific therapeutic sites and load designated drugs, achieving targeted transport and controlled release. This not only enhances drug bioavailability and therapeutic efficacy but also offers broad prospects for development.⁹⁶ Especially for the engineering modification of exosomes, compared with the inefficiency of cell engineering, direct modification of purified natural exosomes can directly, efficiently and rapidly obtain a large number of engineered exosomes, and reduce the uncertainty in the process of isolation and purification of exosomes, so as to make the engineered exosomes obtained more homogeneous. Engineered exosomes have been used in the research of tumours, cardiovascular diseases, neurological diseases and other fields, and have demonstrated superior therapeutic effects. Moreover, the application of exosomes in clinical medicine, bioengineering, regenerative medicine, cosmetics, and medical aesthetics is currently under active exploration.

Nevertheless, exosomes continue to encounter numerous challenges in practical applications. For instance, establishing a standardized protocol for exosome isolation, production, and preservation remains a critical issue. Additionally, addressing the in vivo heterogeneity of exosomes is essential to ensure consistent activity and therapeutic efficacy during application. Improving drug loading efficiency is another significant challenge. Furthermore, effective clinical translation while ensuring the safety of translational applications is crucial.¹⁰⁴ These challenges not only affect the yield of exosomes, but also limit the homogeneity of their quality. In the future, we may be able to realize the efficient separation of exosomes by developing new exosome separation technologies, such as the fully automated exosome extraction system based on microfluidic chip EXODUS of Shenzhen Huixin Biomedical Technology Company, and the automated and integrated exosome purification purifier of Wuhan JAU Biotechnology Company; and to maintain the stability and long-term activity of exosomes by exploring the appropriate cryopreservation additives. In summary, despite the multitude of challenges in exploring exosomes, the opportunities are undoubtedly greater. With deeper insights into exosome biology, innovative discoveries in preparation and modification techniques, and further optimization of targeting, stability, and drug delivery systems, the potential of exosomes in biomedicine will be increasingly realized.