SEC methods are prone to dilution, contamination, and pressure damage. To date, there are no well-defined methods for isolating exosomes with high efficiency and high yield, seriously hampering the wide applications of exosomes in the biomedical field for large scale production. From the standpoint of purity, this method results in a product that also contains all available filter sizes. There are companies that offer a chromatography filtration method that can be combined with other techniques, such as UC and tangential flow filtration (TFF).).
Finally, while treatment with exosomes is generally considered safe, there is still a risk of adverse reactions, especially if the exosomes are not properly purified or if the patient has an unknown sensitivity to the components. As with any medical treatment, it is essential to consult a qualified specialist who can evaluate risks and benefits in the context of each patient's health and treatment objectives. There is increasing evidence that emphasizes the important role of exosomes in different physiological and pathological conditions. Exosomes, virus-sized extracellular vesicles (EVs), carry a complex molecular cargo that is actively processed in the endocytic compartment of cells parentals.
Exosomes carry and deliver this cargo to recipient cells, serving as an intercellular communication system. Methods for recovering exosomes from cell line supernatants or body fluids are not uniformly established. However, studies on the quality and quantity of exosome loads are the basis for the concept of “liquid biopsy”. Exosomes are becoming a potentially useful diagnostic tool and as predictors of disease progression, the response to treatment and overall survival.
Although many novel approaches have been introduced for the isolation of exosomes and the analysis of their loads, the role of exosomes as diagnostic or prognostic biomarkers of diseases remains unconfirmed. This review considers current challenges for validating exosomes as biomarkers of diseases. Focusing on the advantages and limitations of methods for isolating and characterizing exosomes, approaches are proposed to facilitate greater progress in the development of exosomes as biomarkers in human diseases. Where Medical Press is member of the OAI.
Massive reprints for the pharmaceutical industry. The fields of oncology, immunology and regenerative medicine have always presented challenges and have served as focal points for clinical management research. In particular, targeted therapies have made significant advances in the treatment of these diseases, particularly with advances in nanomedicine. 1-4 Several artificial nanocarriers have been developed such as liposomes, nanotubes, micelles, dendrimers and self-assembled peptides for the targeted delivery of therapeutic agents, 5-9 However, with the clinical translation of nanocarriers, several challenges have arisen, such as cytotoxicity, immunogenicity, complex manufacturing processes, and other drawbacks, from 10 to 12 exosomes are also considered nanoparticles (NPs) because of their nanometric size and some properties similar to those of NPs, including passive targeting capacity and improved permeability and retention effects.13 Exosomes are generally very stable, with low immunogenicity, and can be transported long distances in vivo across physiological barriers.
14 Specific membrane proteins and transporter ligands, such as tetraspanins and integrins on the surface of the exosome membrane, mediate the specific binding of exosomes to target cells with specific cytophilic properties and selectivity, 15 Therefore, exosomes are considered a natural nanocarrier. In addition, exosomes can be secreted by all eukaryotic cells and bacteria, are the main mediators of extracellular communication and carry proteins, nucleic acids, lipids and other active components that can be detected in body fluids such as blood, urine, joint fluid, saliva and breast milk 16. Therefore, the exosome as liquid biopsy method can be used for the diagnosis of diseases. Significantly, the emergence of techniques for analyzing the intestinal microbiome has led to the identification of extracellular vesicles (EVs) derived from the intestinal microbiota, which have been detected in blood and urine samples from healthy individuals and patients who have characteristics strongly associated with the intestinal microbiome. These electric vehicles may offer a novel approach to the diagnosis and prognosis of gut-related disorders.17 The properties of natural exosomes make them an alternative to cell therapies with increasing applications in anti-tumor, immunomodulatory and regenerative medicine.
To further improve efficacy, genetically engineered exosomes have recently gained popularity, thanks to loading schemes for different therapeutic agents, genetically engineered membrane modifications, and genetically engineered hybridization with exosome-based artificial NPs (figure). These designed strategies have achieved remarkable results in terms of better drug utilization, better targeting, controlled release and bioimaging, 18-20 However, several studies have demonstrated that exosomes tend to accumulate in the liver, lungs and spleen and are rapidly eliminated by the mononuclear phagocytic system (MPS).On the contrary, orally administered exosomes, such as those derived from plants, have anti-inflammatory and protective characteristics of intestinal tissues and can also contribute to the restructuring of the intestinal microbiota. These exosomes are considered to be optimal delivery vehicles for treating ailments associated with intestinal and microbiota dysbiosis, 38 Figure 1 An overview of the main sources and applications of natural exosomes, as well as the main delivery strategies and engineering modification strategies for exosomes. Figure 2 Exosome biogenesis and absorption mechanisms and the main structure and composition of exosomes.
Exosomes originate in the endosomal system (Figure. Extracellular components, including proteins and lipids, can enter the cell along with cell surface proteins through endocytosis and invagination of the plasma membrane to form early endosomes, which mature further into late endosomes.45 The endosomal membrane sprouts inward to form luminal vesicles (ILV), and late endosomes containing a large number of small vesicles are referred to as multivesicular bodies (MVB), during this process the derived components of cytoplasmic proteins, nucleic acids and lipids are classified in these vesicles, 46 These MVB fuse with lysosomes and are degraded or transported to the plasma membrane through the cytoskeletal network and microtubules to release ILVs extracellularly in the form of exosomes, 47 It has been demonstrated that the fate of MVB is related to their internal cholesterol level. This means that MVB containing high cholesterol concentrations escape lysosome degradation more easily and are released by plasma membrane fusion, 48 The mechanism of exosome biogenesis is extremely complex and involves biological processes such as cargo classification, vesicle formation, vesicle translocation and exosome release. The current view is that the formation and release of MVB and exosomes are primarily related to mechanisms mediated by the endosomal sorting complex required for transport (ESCRT) and related proteins (e.g., VPS4, VTA1, ALIX).
Ubiquitinated structural domains are also present in ESCRT-I and ESCRT-II, which can work together with ESCRT-0 and produce a high affinity for the ubiquitinated cargo at the ILV formation site by sorting the domains, 49,53 ESCRT-I then recruits ESCRT-III through ESCRT-II or Alix and, finally, ESCRT is mechanically dissociated by the action of the AAA-ATPase VPS4,54 However, the depletion of ESCRT components only reduces exosome secretion and does not completely blocks their secretion, so it is speculated that mechanisms not dependent on ESCRT, 55 Often, exosomal membranes are enriched in tetraspanins, which are known to be exosome markers. These proteins have been found to be associated with exosome release and the independent ESCRT pathway. For example, it was discovered that an oncoprotein associated with Epstein Barr virus (latent membrane protein) binds to tetraspanin CD63, can be classified into exosomes, prevent lysosomal degradation and promote the release of exosomes 56. Another study found that in HEK 293T cells, the CD9 and CD82 tetraspanins promote the secretion of beta-linked proteins through a ceramide-dependent exosomal pathway, 57 In particular, sphingomyelin ceramide has been shown to play a role in exosome biogenesis and to inhibit neutral sphingomyelinase, a key enzyme that facilitates the breakdown of sphingomyelin for phosphorylcholine and ceramide, which reduce exosome secretion, 58 In addition, it has been demonstrated that some small GTPases from the family of Rab-related proteins (Rabs) play an important role in intracellular vesicle transport, affecting exosome secretion, 59 Rab35 regulates the coupling of MVB to the plasma membrane in oligodendrocytes and affects exosome secretion, 60 Ostrowski and others used RNA interference analysis to discover that Rab27a and Rab27b mediate the coupling of MVB to the plasma membrane, while the inactivation of Rab27a inhibits the coupling of MVB to the plasma membrane and two Rab27 effector proteins , Slp4 and Slac2b, were also identified, which are associated with the exosomal secretion of Rab27a and Rab27b, 61 In addition, genomic DNA (GDNA) is found primarily in the nucleus, while exosomes are found in the cytoplasm, and the DNA loading mechanism remains unclear. A recent study found that nuclear contents, including gDNA, are loaded into exosomes with the help of tetraspanin CD63 after the collapse of the cytoplasmic structures enclosed by the nuclear membrane (micronucleus). The classic design consists of non-exosomal components: large vesicles, including apoptotic vesicles, are filtered by a 200 nm membrane, while the remaining vesicles 20 to 200 nm in diameter are left behind and smaller particles (proteins) are further filtered through a 20 nm microfilter, eventually leaving exosomes, 74 Compared to ultracentrifugation, ultrafiltration takes less time and is easier to operate. However, some disadvantages have also been described, such as clogging of membrane pores, leading to low capture efficiency or membrane damage.
75 The shear stress caused by membrane pores to exosomes is also a problem that must be taken into account, 74 In addition, some NPs that are similar in size to exosomes cannot be filtered and may affect their purity 75. The exosome membrane contains a large number of tetraspanins that are considered exosome markers (for example, CD9, CD63, CD8), as well as the presence of MHC I and II molecules and heat shock proteins. Immunoaffinity capture techniques have been developed to isolate exosomes taking advantage of the immunological interaction between these proteins (antigens) and their antibodies. A recent study successfully captured exosomes from the plasma of patients with COVID-19 using the immunoaffinity of the CD81 antibody with the exosomal surface antigenic protein CD81,76 In another study, researchers used magnetic beads coated with antibodies against MHC II to isolate exosomes from cell culture supernatants, 77 Usually, the purity of exosomes isolated using immunoaffinity techniques is higher compared to other exosome isolation techniques, 78 However, this method of highly specific isolation can result in yield and cannot be used for high-performance isolation of exosomes. Polymer precipitation methods typically utilize the interaction between highly hydrophilic polymers and the water molecules surrounding exosomes to form a hydrophobic microenvironment that reduces the solubility of exosomes and isolates them under centrifugal conditions.
79 Polyethylene glycol (PEG) is a widely accepted non-toxic polymer that is commonly used in polymer precipitation methods, 80 The polymer precipitation method is relatively simple to perform and has high performance. While it is possible to precipitate exosomes, this can cause high contamination rates and low purity of isolated exosomes. 81,82 Size Exclusion Chromatography (SEC) is also a technique for isolating exosomes based on molecular size. Specifically, as the sample passes through a stationary phase consisting of porous resin particles, molecules smaller than the pores of the stationary phase enter the pores preferentially, resulting in longer distances and relatively slower speeds, and larger molecules that cannot enter the pores are forced to surround the porous particles and elute from the column.
Commonly used materials, such as dextran, agarose and polyacrylamide polymers, 83 Hong et al successfully isolated high-purity exosomes of approximately 50 to 200 nm in size from plasma from acute myeloid leukemia using a small-sized exclusion chromatography column with agarose 2B, 84 Although this method does not affect the structure and activity of exosomes and is inexpensive, the disadvantage is that it is time-consuming and difficult to use for high-performance isolation, 85 In addition, the size of the exosomes can overlap with some contaminants, which is a common drawback of size-based methods of isolating exosomes. To improve this problem, it can be solved by combining different separation methods. Previous studies have demonstrated that the combination of ultrafiltration and SEC to separate exosomes significantly improves the purity of exosomes compared to SEC or ultrafiltration alone, and does not affect their functionality. 86 Microfluidic technology allows the integration of multiple assays, sensors and other components on the same chip to manipulate small volumes of fluid in microchannels tens to hundreds of microns in size, exploiting the biological or physical properties of exosomes for isolation and detection, which demonstrates high efficiency, speed and low number of sample requirements, 87 A commonly used microfluidic technique is based on immunoaffinity.
For example, researchers recently developed a microfluidic device made of polydimethylsiloxane (PDMS) and functionalized with a CD63 antibody that specifically binds to the CD63 antigen on the surface of exosomes to isolate them. The source of the exosomes immobilized with ExoChip was confirmed by immunoelectron microscopy and protein transfer, 88 Another more common technique is the acoustic nanofilter, in which a matrix containing exosomes and other extracellular components is injected into a chamber exposed to ultrasound, using acoustic waves to isolate extracellular vesicles according to their size and density, larger particles are subjected to stronger radiation forces and, therefore, migrate more quickly to the pressure node, and the ultrasound is can adjust to separate particles of The desired size, 89 It has also been demonstrated that the viscoelastic microfluidic separation techniques developed in recent years isolate exosomes with high efficiency and purity by exploiting the different elastic support forces of particles of different sizes through visco-elastic media such as polyoxyethylene, 90 In conclusion, a variety of microfluidic-based isolation techniques have been developed for the acquisition of high-performance exosomes as a result of advances in microfluidics. Figure 3 Exosomes mediate communication between intestinal microbes and host cells and together they maintain the balance of the intestinal microenvironment. The exosomes of healthy intestinal microbes promote food metabolism, protect the integrity of the intestinal barrier and promote the maturation of intestinal immune cells, while the exosomes of intestinal host cells regulate the abundance and diversity of intestinal microbes. Conversely, exosomes from dysregulated intestinal microbiota negatively affect food metabolism, alter the integrity of the intestinal barrier and cause immune dysfunction, while exosomes from intestinal host cells cause deregulation of intestinal microbiota.
Both exosomes derived from intestinal microbes that enter the circulation through the altered intestinal barrier and exosomes derived from microbes present in faeces can be used in liquid biopsies. Table 2 Selected clinical trials with exosomes for the treatment of diseases The different methods affect the carrying capacity, which is related to the properties of the cargo being charged, such as hydrophilicity, hydrophobicity and molecular weight. A previous study that compared the load of hydrophobic paclitaxel (PTX) molecules in exosomes using incubation and ultrasound methods showed higher ultrasound loading rates due to the greater diffusion of PTX molecules in lipid bilayers using ultrasound, 182 Another study evaluated different techniques for loading catalase into exosomes, incubation, freeze-thaw cycle, sonication or extrusion for drug delivery for Parkinson's disease, and demonstrated that efficient loading of cargo by sonication was the most efficient, 179 Ultrasonication and extrusion caused peroxidase to diffuse into the lipid bilayer, resulting in a high loading efficiency of exosome carriers, while saponin selectively removes cholesterol from exosome membranes and forms pores in the lipid bilayer of exosome membranes, which promotes peroxidase binding and improves loading efficiency, 179 In addition, loading methods can affect the stability of exosomes, such as electroporation, electroporation or permeants can alter the integrity of electric vehicle membranes and reduce stability, 179 Electroporation produces siRNA aggregation effects during the charging process and can affect the actual charging rate. To avoid this phenomenon, the researchers suggested adding ethylenediaminetetraacetic acid to electroporation buffer 183. However, relying solely on the administration of the exosome-laden drug is not the main factor in increasing drug accumulation in the injured region, but rather depends more on the selective modification of the exosomes.
Since some specific structures of the exosome membrane surface mediate the targeting function and localization properties of exosomes, appropriate membrane surface modifications can further improve delivery efficiency, including direct modification of the exosome membrane surface and indirectly modified exosome-derived parental cells (Figures 5A and B). Genetic modification of exosome-derived cells is another important method of functionalizing the surface of exosomes. When the receptor genes from target cells are transferred into parental cells, these receptor proteins fuse with exosomal transmembrane proteins during exosome biogenesis to form the target portion expressed on the surface of the exosome. Wang et al used the ischaemic myocardium-targeting peptide plasmid CSTSMLKAC (IMTP) to transcribe MSC with a fused exosomal membrane protein (LAMP2b), and the isolated exosomes expressed the LAMP2B-IMTP complex on the surface and found that injured cardiomyocytes more efficiently captured IMTP exosomes than unmodified exosomes, accumulated more in the ischemic region of the heart, and showed an improved therapeutic effect in acute myocardial infarction, 185 (Figure 5E) In another study, researchers designed genetically the DCs to produce LAMP2b exosomes fused to the RVG peptide, directed to neurons, loaded with exogenous siRNA by electroporation and effectively delivered to the plasma brain through the BBB for targeted therapy of AD, 169 Similarly, the anti-epidermal growth factor receptor (EGFR), a tumor cell receptor, was expressed on the surface of electric vehicles by transducing EV-producing cells that encode EGFR antibodies and fused with the glycosylphosphatidylinositol (GPI) anchor signaling peptides, which significantly improves the bond between exosome and tumor cells and depends on EGFR density, 194 Using tetraspanins expressed on the surface of exosomes, Liang et al designed apo-A1-CD63 complexes on the surface of exosomes and charged miR-26a by electroporation to attack the scavenging receptor class B (SR-B) on the surface of hepatocellular carcinoma cells and successfully administered the Antitumor RNA, 195 Tian et al.
they transferred the peptide directed to RGD-4C fused with the structural domain of lactadherin plasma C1C2 into the membrane of the EV. the average transcription significantly improved the ability of electric vehicles to attack ischemic regions of the brain and significantly decreased the level of inflammation compared to unmodified electric vehicles, 196 In addition, the interaction of CD47 with the immunosuppressive receptor SIRPα on the surface of macrophages can stimulate the immune escape system, 197,198 Since macrophages are primarily responsible for the elimination of circulating exosomes, the CD47-SIRPα signaling axis has been used to design exosomes to escape macrophage phagocytosis, 199 By transferring CD47 into donor cells, overexpressed exosomes effectively escaped MPS phagocytosis, prolonged the circulation time of exosomes and increased distribution in tumor tissues, 200 Meanwhile, CD47 can attack CD47 receptors on the surface of tumor cells and improve the antitumor effect. 200 Advances in nanotechnology greatly contributed to the development of designed exosomes. By incorporating the advantages of liposomes and exosomes, hybrid exosomes based on membrane fusion have become a novel nanopharmaceutical delivery system.
For example, lipid load affects the absorption of target cells and, depending on the properties of the target cell, customized liposomes can be selected to hybridize with exosomes and improve the absorption rate, 201,202 A previous study that used freeze-thaw cycle techniques to hybridize exosomes and lipids found that neutral and anionic lipids did not affect the cellular uptake of hybrid exosomes, while hybridizing cationic lipids with exosomes decreased cellular absorption efficiency. 203 In contrast, cellular uptake of PEG-DOPS hybrid exosomes was significantly increased by nearly twofold compared to unmodified exosomes, 203 In addition, liposomes sensitive to pH, optics and temperature provide controlled release properties to the drug delivery system, further improving drug delivery efficiency and reducing drug side effects. For example, exosomes recently developed from macrophages were hybridized with pH-sensitive liposomes and loaded with adriamycin. Under acidic conditions, the drug was released, which showed greater toxicity to tumor cells, suggesting that this hybrid exosome is a possible drug delivery system for the acidic tumor environment. 204 On the other hand, approaches based on membrane fusion can alter the integrity of membrane proteins and increase the size of exosomes, affecting the stability and targeting capacity of exosomes, 205 From the perspective of synthetic NPs, exosome membranes can be used for synthetic nanoparticle membrane coating technology, which is beneficial for reducing their immunogenicity.
The combination of some biofluorescent materials or NPs with the ability to image exosomes is beneficial for monitoring exosomes in vivo, which is used to determine the biodistribution and pharmacokinetics of exosomes. For example, Gaussian luciferase (Gluc), 228, and Renilla luciferase (Rluc), 229, can be loaded into exosomes by transfection to produce bioluminescence effects after the addition of enzyme substrates, and bioluminescence imaging is then used to show the distribution of exosomes in different organs of the body. Radioisotopes, such as 99mTc, 131I, and 111 in oxine to label exosomes, and single-photon emission computed tomography (SPECT) or positron emission tomography (PET) are the most commonly used non-invasive techniques for imaging radiolabeled exosomes, 230—232 The unique properties of nanomaterials are ideal imaging agents for bioimaging exosomes. For example, researchers have loaded inorganic NPs (superparamagnetic iron oxide) into exosomes through electroporation or coincubation, offering excellent image depth using magnetic resonance imaging (MRI). These basic studies on exosomes are essential for our comprehensive understanding of exosomes and for further research on their applications.
Current techniques for isolating exosomes do not allow for both high-performance and high-purity analyses; greater optimization of isolation protocols can help overcome these deficiencies and accelerate exosome research for both basic and clinical applications. In particular, nanocomposites combined with nanomaterial-modified microfluidic channels are expected to achieve high capture rates and high-performance exosome screening, which may be a new direction for the future. Figure 7 Strategies for the development of multifunctional nanotransmission platforms based on exosomes. Multifunctional nanodelivery platforms mainly include the active loading of therapeutic agents, specific functional modifications, evasion of MPS removal (CD47-SIRPα axis, PEG membrane coating); controlled release through sensitive chemical bonds or hybridized liposomes (pH-sensitive, light-sensitive, heat-sensitive), inorganic nanoparticles (heat-sensitive, light-sensitive) and in vivo bioimaging; the use of adjuvant therapies, such as magnetothermic therapy and therapy photothermic to further improve efficiency.
Exosomes, being natural nanocarriers, have significant clinical potential for translation and application in the diagnosis and treatment of diseases. In the near future, exosomes, as natural nanocarriers, will emerge as a novel therapeutic alternative in the fields of oncology, immunology and regenerative medicine. All authors made a significant contribution to the work reported, whether in the conception, design of the study, execution, data acquisition, analysis and interpretation, or in all of these areas; they participated in the writing, revision or critical review of the article; they gave final approval of the version to be published; they have agreed on the journal to which the article has been submitted; and they accept to be responsible for all aspects of the work. The authors do not report any conflicts of interest in this work. The opinions expressed in all articles published here belong to the specific authors and do not necessarily reflect the views of Dove Medical Press Ltd or any of its employees.
The advantage of this method is the higher charging efficiency and the disadvantage is that electroporation causes the aggregation of exosomes and destabilizes the exosome membrane. This review summarizes current strategies for optimizing exosomes in clinical applications and compares their advantages and disadvantages. However, the disadvantage is that exosomes can block filter holes, shortening membrane life and reducing separation efficiency. Although there are drawbacks, if properly understood and understood, the attractive benefit of rejuvenation at the cellular level can be expected.
Its advantage is that it maintains the integrity and functionality of the exosomes, but its disadvantage is that the loading efficiency of the drug is relatively low. One of the disadvantages of intravenous infusion in general, not just exosomes, is that, in some cases, needles are inserted repeatedly. However, its disadvantages are also evident due to the lack of adequate mechanical properties and low levels of cell adhesion. We summarize and analyze the current charging techniques of the different loads and the advantages and disadvantages of the different loading techniques, and we suggest that the appropriate charging technique should be considered according to the chemical nature and molecular weight of the charge.
Below is a summary chart comparing the purification methods and some advantages and disadvantages of each. The main disadvantage of this method is that the captured exosomes cannot detach from the captured molecules. While exosome treatment is promising as a regenerative therapy, there are some drawbacks and limitations to be taken into account.
