Use of Exosomes for Clinic Aims

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Maria Antonietta Di Bella	--	5096	2022-06-13 17:09:42	\|
2	format	Jason Zhu	-4 word(s)	5092	2022-06-14 03:24:27	\|

This entry is adapted from the peer-reviewed paper 10.3390/biology11060804

There has been a rapid growth in the knowledge of cell-secreted extracellular vesicle functions. They are membrane enclosed and loaded with proteins, nucleic acids, lipids, and other biomolecules. After being released into the extracellular environment, some of these vesicles are delivered to recipient cells; consequently, the target cell may undergo physiological or pathological changes. Thus, extracellular vesicles as biological nano-carriers, have a pivotal role in facilitating long-distance intercellular communication. Understanding the mechanisms that mediate this communication process is important not only for basic science but also in medicine. Indeed, extracellular vesicles are currently seen with immense interest in nanomedicine and precision medicine for their potential use in diagnostic, prognostic, and therapeutic applications.

extracellular vesicles exosomes characterization Clinic

1. Introduction

Nanomedicine is a term that was used for the first time in 1991 and is defined as the application of nanotechnologies to medicine for the diagnosis, monitoring, prevention, and treating of many human diseases ^[1].

During recent decades, the applications of nanotechnologies in many biomedical areas have been intensively researched offering excellent results; bio-nanotechnology is an expanding field that requires a multidisciplinary approach including physics, chemistry, material sciences, engineering, informatics, and life sciences. Indeed, nanostructures used for biomarker detection, nano-biochips, nano-electrodes, nano-biosensors, nanostructures for regenerative medicine, nanoparticles (NPs) with antibacterial activities, and NPs for diagnostic applications are currently used ^[2].

The intrinsic, unique, physicochemical, and mechanical characteristics of particles at the nanoscale level enable their application in theranostics, a new area that combines diagnostics and therapy. Nano-systems can work as image agents or as drug delivery systems because they present an increase in the selectivity and efficacy, and, in comparison to traditional therapeutic molecules, by reducing their accumulation in healthy tissues, they show a decrease in the side effects’ incidence and intensity. In cancer therapeutics, they can offer new solutions to overcome the limitations derived from chemotherapy or radiotherapy ^[3]^[4]^[5].

There are different types of synthetic NPs made from various materials including, for example, polymerNPs, solid lipidNPs, crystalNPs, liposomes, micelles, hydrogels, and dendrimers. Nevertheless, each of these formulations, organic or inorganic, presents its advantages and disadvantages for nano-diagnostic or nano-therapeutic applications ^[6]^[7].

2. Exosomes for Clinic Aims

Among the large subpopulation of EVs, ApoBDs are quite variable in size and content; they are typical membrane blebs released by cellular disassembly that are quickly phagocytized by surrounding cells. The apoptotic bodies can harbor different biomolecules: genomic DNA, histones, fragments of the cytoplasm, and intact organelles Their formation represents the expression of both cell clearance and intercellular communication, but little is known about their function compared to the progress in MVs and EXOs investigation ^[8]^[9]^[10]^[11]. Indeed, in the past decade, the scientific interest in MVs and EXOs has rapidly increased; their functional characteristics and the role played by them in human physiology and in the pathogenesis of major human diseases have received extensive attention.

MVs are heterogeneous structures classified as large EVs, incorporating nucleic acids, lipids, and many proteins ^[12]^[13].

Depending on the specific purpose, both MVs and EXOs have merits as potential therapeutic systems.

3. EXOs Characteristics

For many years, researchers have considered EXOs as waste products obtained from the shedding of plasma membranes. After decades, this image of waste bins changed to that of biologically active particles. Later, the term “exosome” was coined ^[14]^[15]^[16]^[17]^[18]^[19]. Since these initial discoveries, the field of EXOs research has enriched considerably as confirmed by the number of publications over the years, and dramatically increased since 2010 by about 50 times according to PubMed.

EXOs are characterized by various types of complex cellular components present both on the surface and packaged inside the lumen after selective mechanisms. To date, omics approaches (proteome analysis, lipidomics, metabolomic analysis, etc.) have identified numerous proteins, nucleic acids, lipids, and metabolites.

Proteins include both membrane and cytosolic components: some surface receptors, adhesion proteins, integrins, cytoskeletal proteins, membrane transport, and fusion-related proteins. The presence of TSG101 and other accessory proteins that are involved in the formation of intraluminal vesicles (ILVs) denotes that the EXOs’ biogenesis pathway depends on the endosomal sorting complexes required for transport (ESCRT). Tetraspanin proteins and phospholipids that play a critical role in membrane biogenesis denote an ESCRT-independent alternative mechanism of biogenesis. Tetraspanins, heat shock proteins (Hsp), and other related proteins involved in MVB production are the most conserved proteins ^[20]^[21]^[22].

Some proteins are specifically related to the nature of the cell and tissue of origin. Different EXOs have specific proteins that reproduce the status of the parental cell. That is one of the reasons for their heterogeneity. For example, T-lymphocyte-derived EXOs have enzymes and perforin on their surface. Antigen-presenting cells (B lymphocytes, dendritic cells) (APCs)-derived EXOs contain major histocompatibility antigen complex (MHC, MHC-I, MHC-II). Tumor-specific proteins are contained in many EXOs from tumor cells ^[23].

EXO cargos also consist of nucleic acids, such as genomic DNA, that can induce phenotype switch, messenger RNA (mRNA) that can be translated in target cells, microRNA that can mediate RNA silencing, circular RNA, long non-coding RNA, and viral RNA. Lipid rafts are present on the surface of EXOs. Based on the specialized EXOCARTA database, exosomes contain 9769 proteins, 3408 mRNAs, 2838 miRNAs, and 1116 lipids; (see references ^[23]^[24]).

Upon their release in the extracellular space, EXOs can be destroyed, or migrate to interact with other cells. The recipient cells may be in proximity or be present at a distant site; the vesicles can travel over large distances being carried via the blood or lymphatic circulation. The lipidic membrane of EXOs acts as a protection barrier to cargo and the signal arrives undiluted and protected from damage bypassing phagocytosis. Molecules such as nucleic acids that could be degraded in the extracellular milieu are protected from enzymatic degradation (for example, RNAases) ^[25].

When EXOs enter target cells, their cargo can be accepted by recipient cells. Once released inside the target cell, the exosome cargo can perform a variety of functions that, depending on the cellular origin, regulate a plethora of physiological activities. EXOs may affect gene expression, regulate metabolism, participate in responses during microbial infection, and facilitate disease progression. Over the past decades, most of the research attention in this field has been focused on exploring the vital roles as long-range messengers of these vesicles ^[26]^[27]^[28]^[29]^[30]^[31]^[32].

The amount of EXO biogenesis in different cells depends on their physiological and/or pathological states. Cellular stress and signals activation can modulate their excretion. Accumulating evidence shows that cancer cells release a larger amount of EXOs compared to normal cells; tumor-derived EXOs can contribute to cancer growth by inducing anti-apoptotic and oncogenic pathways such as invasion, metastasis, and angiogenesis. Some EXOs can promote tumor immune evasion with T-cell apoptosis induction. They are also responsible for epithelial–mesenchymal transition and interconversion to mesenchymal–epithelial transition in several human malignancies. For the above reasons, it is important to take into consideration the function of secreting cells to ensure the safety of these nanocarriers in clinical applications ^[33]^[34]^[35]^[36]^[37].

4. EXOs Source

As mentioned before for EVs, EXOs in vivo are released in many biological fluids (such as synovial fluid, breast milk, urine, and saliva), amniotic liquid, blood serum, and malignant effusions of ascites among others. Neuronal cells, fibroblast cells, adipocytes, intestinal epithelial cells, and tumor cell lines produce EXOs in vitro.

Different studies have suggested that several types of cells seem to be more eligible to produce EXOs for therapeutic purposes and that not all cell-derived vesicles are ideal as drug carriers. Drug capacity and efficient delivery depend on the size, yield, intracavitary composition, and surface proteins that mirror the cell and tissue of origin ^[38].

The major types of used EXOs are derived from:

Dendritic cells have been used due to their low immunogenicity. Interestingly, their EXOs still maintain this immune function. Dendritic cells-derived EXOs can overcome the biological barriers, such as the blood–brain barrier ^[39]^[40]^[41]^[42].
Macrophages are mononuclear phagocytes that have critical roles in innate immunity. Macrophage-derived EXOs are known to express functional immune proteins; they can interact with brain vessel endothelial cells and cross the blood–brain barrier, an ability mediated in part by surface components; they can deliver some factors such as anti-inflammatory cytokines (i.e., IL-4). Moreover, they exhibit strong anti-tumor and anti-inflammatory effects ^[43]^[44].
Mesenchymal stem cells are a popular choice for cell therapy. Indeed, they are easily obtained from different human tissues such as bone marrow, dental pulp, and adipose tissue. Mesenchymal stem cells are capable of self-renewal and are involved in modulating the immune response. EXOs isolated from these cells are extremely beneficial in promoting wound healing and in repairing tissue such as skin and cardiac tissue. Cao et al. ^[45] found that mesenchymal stem cell-derived exosomal miR-125b-5p could promote the repair of renal tubules in acute kidney injury. These vesicles also seem to inhibit cancer progression and have an inflammation melioration capacity. Additionally, these cells are known to secrete relatively high numbers of EXOs ^[46]^[47]^[48]^[49].
Cancer cell lines such as melanoma cells are commonly used to produce EXOs. As reported before, tumor cell-derived EXOs can either block tumor growth or be involved in cancer progression and are capable of converting a normal cell into a transformed one. Thus, more importantly, tumor cells may be a double-edged sword when used for delivering therapeutics agents because their EXOs could show potential risk in aggravating a patient’s malignity instead of improving it or conferring drug resistance ^[50]^[51].
To overcome the risk of horizontal gene transfer when EXOs are recovered from tumor cells or immortalized cells, some researchers have investigated the potential of human Red Blood Cells (RBC) as a source of vesicles. RBCs are abundant in the body, easy to obtain, and available in blood banks. A strategy to generate large-scale amounts of RBC-EXOs for the delivery of RNA and drugs was demonstrated by Usman et al. ^[52].
Plasma exosomes are also derived from Platelets (PLT). These originate from bone marrow megakaryocytes and have no nucleus and a short half-life. PLT-derived EXOs can be obtained from animals, healthy volunteers, and from platelets in disease states. The functions of PLT-EXOs depend mainly on their source as they are rich in a variety of cargos. Platelets in disease states often contain pathogenic factors that can be used as biomarkers for disease diagnosis. EXOs obtained from healthy volunteers or mice can inhibit platelet activation and endothelial inflammation, while human PLT-EXOs have been shown to increase cell proliferation and migration of mesenchymal stromal cells (MSCs) from human bone marrow. PLT-EXOs could present advantageous therapeutic properties, including homologous administration in the clinical setting, thus overcoming the restrictive requirement of other biological products. Although procedures such as high-speed centrifugation of plasma induce the aggregation of PLT-derived EVs more than erythrocytes EVs and washing for preparing ‘washed’ platelets shows that most EXOs will be removed, nowadays isolation protocols with the use of specific commercial kits can avoid this effect ^[53].

A summary of cell lines used to produce EXOs for clinical applications can be found in the research ^[38]^[54].

Recently, vesicles obtained from plant cells or food have gained attention from researchers. Bovine milk EXOs have been studied as a viable alternative of high impact in drug carriers due to their lack of toxicity, on account of their biocompatibility, and stability in an acidic environment so that they can be delivered orally ^[55]^[56].

Exosome-like nanoparticles (ELNs) derived from tissues, organs, apoplastic fluid, and the juice of several plant species such as ginger, lemon, grapefruits, and carrots are characterized by various good properties that make them suitable for clinical applications. ELNs seem to contribute to the plant defense in response to pathogens, and they are also known for their anti-inflammatory and tumor growth-suppression properties. Moreover, they seem to localize in tissue and remain intact inside cells after administration, confirming the possibility that they can be used for intracellular drug delivery ^[57]^[58]^[59]. As regards their composition, they show a lipid bilayer structure comparable to that of mammalian cell-derived EXOs and artificially synthesized liposomes; contrary to those, they lack cholesterol, present a higher percent of phosphatidic acids (a cell-signaling component with many biological activities), phosphatidylcholine, and phosphatidylethanolamine ^[60]. A variable number of proteins and miRNAs have been identified in plant-derived EXOs ^[59]^[61].

However, although the morphology and composition of these ELNs are like mammalian exosomes, despite the several advantages of food and vegetable-derived vesicles, and despite the possibility of their production in large quantities from fresh plant juice, further studies are still required. Information on their biogenesis is still lacking and it is an urgent task to characterize and clearly identify markers of plant EVs. At present, research on the biogenesis of plant EVs is not sufficient and needs to understand all details of their mechanism of action before they can largely be introduced as nano-platforms in medical practice.

5. EXOs Isolation and Storage

Multiple methods and different ways have been established to isolate plant and animal EXOs from biological fluids or in vitro cell culture supernatant. For different purposes and applications, it is very important to select the producer cells, and it is essential to understand the characteristics of the different isolation approaches that enable large-scale bio-manufacturing and assure the efficacy of the strategy. Indeed, it is known that certain procedures may compromise the EVs’ integrity and structure or can be associated with aggregate formation and cargo impurity ^[62].

Among the different isolation methods, the most traditional and commonly used are:

Ultrafiltration: is a method based on the vesicle size, involving the use of fluid pressure to drive the migration through a polymeric membrane with defined pore size; vesicles are separated selectively from the samples with the simultaneous retention of larger molecules. It is simple and fast, but EXOs can be degraded and lost ^[63].
Immunoaffinity: is a capture isolation technique based on the recognition by antibodies or ligands of EXO marker components (antigen) that are exposed on the vesicle surface. The immunoaffinity method has the advantages of rapid isolation, simplicity, and high specificity, and the sample volume can be very small in comparison to ultracentrifugation, but it is very expensive due to the cost of antibodies ^[64].
Size-Exclusion Chromatography (SEC) techniques can isolate EXOs based upon molecular size and density, mainly by means of a column filled with a porous stationary phase with a specific pore size distribution. When the sample enters the gel, small particles with small hydrodynamic radii diffuse into the pores while large molecules with large hydrodynamic radii will not. Hence, the passage of proteins and other smaller contaminating molecules is delayed while larger molecules or larger vesicles (>75 nm) exit the column and will be eluted earlier in the void volume The porous stationary phase contained in the column can be cross-linked dextrans, polyacrylamide, agarose beads (commercially named as Sepharose), and allyldextran in which small particles can penetrate. The primary advantages of this technique are the screening of high-purity EXOs with less protein contamination compared to ultracentrifugation, and the preservation of vesicle integrity, structure, and biological activity as it relies on gravity rather than sheer force for isolation. However, this technique is limited by: (1) the need for dedicated equipment; (2) the accessibility of the chromatography column to contamination; therefore, aseptic working conditions should be ensured especially if the isolated EVs are intended for therapeutic use; (3) an initial large volume is required; (4) low yield; (5) difficulty in scaling up; (6) inability to separate EXOs from vesicles of the same size. Research efforts have been performed to overcome those challenges and enhance SEC efficacy and speed. For instance, the EXO pellet is re-suspended after enrichment by ultracentrifugation in combination with ultrafiltration methods and then further purified using SEC. This combined strategy resulted in improved purity and preserved EXO function. Moreover, commercially available columns and kits based on size-exclusion chromatography were designed to simplify EV isolation; iZON Science produced the qEV Exosome Isolation Kit that, as well as the PURE-EVs kit (Hansa Biomed), allows rapid, high-precision isolation within less than half an hour so the SEC methodology is nowadays relatively easy and fast. However, this combination is not suitable for scale-up production ^[65].
Microfluidics platforms represent emerging isolation methods developed to separate EVs from large cellular debris and protein aggregates. Microfluidics techniques enable the differentiation, capture, enrichment, and isolation of particles of very similar shapes and sizes. Different isolation principles have been designed: size based, immune-affinity based, and dynamic categories that make use of emerging nanomaterials. Size-based exosome separation devices allow the separation of highly pure EXOs driving the plasma inside a channel where nanofilters, nanoporous membranes, or nanoarrays can trap vesicles when fluids flow through them. In another device, an acoustofluidics device, using ultrasound standing waves, in a contact-free continuous flow manner, EXOs are directly isolated from undiluted small blood samples based on their size, density, and compressibility. The result is the formation of clusters of EVs. These clusters are then washed and released upon deactivation of an ultrasound. This device maintains the structures, characteristics, and functions of the EXOs with a purity of about 98%. In addition, it enables the separation time, reagent consumption, and sample volume for isolating EXOs to be significantly reduced with short processing times with decreasing human intervention. In an immunoaffinity-based microfluidic device, the vesicle separation relies on specific biomarkers on the EXOs membrane. A commercial immune-microfluidics chip (ExoChip) allows the isolation of EXOs from mixed cultures because it is functionalized with a commonly expressed antigen, CD63. The specific interactions between CD63 and antibodies immobilized on the chip allow the capture of the vesicles. Unfortunately, to separate them efficiently, the immunoaffinity-based separation microfluidic devices need highly represented antigens (targeted proteins) on the vesicle surface. Other innovative and attractive separation approaches that have the ability to isolate EXOs based on their physical and biochemical properties are being simultaneously developed: some microfluidic isolation methods typically require small starting volumes from serum and cell culture (10 s–100 s of μL), while others can be performed on larger volume samples; they can reduce reagent consumption, are fast, and efficient. However, scalability, validation, sample pretreatments, and standardization are still considered bottlenecks for these devices, which are mainly applied in the field of diagnosis ^[66]^[67].
Ultracentrifugation (100,000× g or greater) is currently the most widely used purification method that mainly depends on vesicle density, size, and shape. It consists of two steps after pelleting down cells: a pre-cleaning and filtering of samples centrifuged at low and intermediate speed centrifugation (500–10,000× g) to remove dead cells and cell debris, followed by the flotation in a density gradient centrifugation to precipitate and enrich EXOs. High-speed centrifugation (40,000–100,000× g) is often combined with a density gradient using commonly iodixanol or sucrose as a medium to remove contaminants such as proteins, protein/RNA aggregates, and lipoproteins. The EXOs can be collected in the density range of 1.1 to 1.2 g/mL Depending on the rotor utilized, this procedure is suitable for large sample processing; it requires little sample pretreatment and has the characteristics of low contamination risk. Moreover, the affordability is high since only one ultracentrifuge is needed for long time use. Apart from the access to expensive equipment, it is of low cost. At the same time, however, the density gradient centrifugation method is time consuming and requires extra care to prevent gradient damage. In addition, damage to EXOs by high-speed centrifugation might occur if used for long times (more than 3–4 h) ^[68].
Co-precipitation is an appealing precipitation-based isolation method thanks to the simple protocol and high yield. Polyethylene glycol (PEG) is generally used as a co-precipitator by decreasing the solubility of EXOs. The method lacks specificity and results in low purity of vesicles ^[69].

Even if various and easy-to-use commercial kits are now available on market, to date, there is still no ideal “gold” EXO isolation method; the low purity and high cost of the preparations restrict their utility. Sometimes the combination of different isolation methods may be better in accordance with the purity required and the sample volume ^[70].

Nevertheless, the storage conditions and preservation of produced EXOs that must be used for therapeutic applications need to be fully elucidated as they can affect the amount and the quality of the final product. Several data suggest that the stability of vesicles from different origins may also be different. To date, storage at −80 °C in phosphate saline buffer is the most used way to preserve EXOs ^[71].

6. EXOs Clinical Applications

The clinical applications of EXOs can be summarized as follows:

Liquid biopsies: because EXOs differ in their composition based on the current state of the secreting cells, being able to isolate them from different body fluids can be considered a potent screening tool. Compared with traditional solid biopsy, liquid biopsy has a number of advantages: firstly, minimal trauma. Thus, EXOs isolated from liquid biopsies can be used as both diagnostic and prognostic non-invasive biomarkers. EXOs released from normal and cancer cell lines have different nucleic acid contents and membrane structures in accordance with their surface proteins, cholesterol contents, and cholesterol/phospholipid ratios. This enables the early detection of many pathological conditions, and their regression or progression in response to therapy. EXOs originating from tumor cells possess active molecules and specific genomic and proteomic features characteristic of a particular tumor type; therefore, their analysis could predict the potential presence of the tumor. For example, human serum exosomal long noncoding RNAs-UCA1 and exosomal miRNAs can be used as diagnostic biomarkers for cancer risk ^[72]^[73]^[74]. Epidermal growth factor receptor (EGFR), placental alkaline phosphatase (PLAP), and leucine-rich alpha-2 glycoproteins (LRG1) are potential biomarkers for non-small cell lung cancer, as they are all overexpressed in patients. Moreover, Grimolizzi et al. found that in both early and advanced-stage non-small cell lung cancer patients, miR-126 was mainly present in EXOs, while in healthy controls, circulating miR-126 was equally distributed between EXOs and exosome-free serum fractions. The detection of prostate cancer can also be achieved, evidencing the presence of exosomal miRNA-141 and miRNA-375 ^[75]^[76]^[77]. EXOs can find application as biomarkers also in cardiovascular diseases, and exosomal miRNAs may be beneficial for diagnosing heart diseases. Another important disease that could benefit from the study and application of EXOs is diabetes mellitus. Recent literature demonstrates that the content of exosomal miRNA is remarkably different in the sera of type I diabetes patients in comparison with that of healthy control. In addition, a pre-clinical study has indicated that exosomes also participate in type 2 diabetes pathogenesis. Certain EXOs biomarkers (P-S396-tau, P-T181-tau, and Ab1–42) seem to predict the development of Alzheimer’s disease up to 10 years in advance; EXOs secreted by various parts of the kidney, contain several biomolecules that might be markers of abnormality present in the kidney ^[78]^[79].
Therapeutic intervention: Several studies have highlighted the therapeutic importance of EXOs. Being able to redirect vesicles to tissues of interest, EXO administration could be used to degrade pathological signals or focus their intrinsic therapeutic activity. EVs regulate various normal physiological and pathological activities; thus they can be used as natural therapeutic agents for treating a variety of common diseases. There are sufficient pre-clinical studies to support the application of dendritic cell-derived EXOs to treat different types of cancer such as metastatic melanoma and non-small cell lung cancer. For example, EXOs derived from mature dendritic cells prevent the production of cancer cells as they contain DHA (C22:16 docosahexaenoic acid, fatty acid), which enhances the antigen-presenting ability of cells and thus inhibits tumor cell proliferation. However, as EXOs participate in the progression of tumors and promote various stages of tumorigenesis, some research aims to regulate the process of EXOs secretion and reduce their release from tumor cells to normal levels or inhibit their uptake by the target cells ^[80]. The results of a preclinical trial indicated that by using dimethylamiloride (DMA), the secretion of EXOs can be repressed in murine tumor models by blocking intracellular Ca²⁺ and Na⁺/Ca²⁺ and Na⁺/H⁺ channels. Indeed, the increase in intracellular Ca²⁺ and reduction in intercellular and intracellular pH values lead to an increase in EXOs secretion, and the consequent uptake by recipient cells. Moreover, in order to remove the metastatic effect of cancer, a biotechnology company named Aethlon Medical has developed an adjuvant therapy called HER2O-some, which decreases HER2-positive EXOs secreted by cancer cells in circulation and thus interrupts the progression of HER2-positive breast cancer. Although the technique based on EXOs removal has achieved great progress, further research is still needed to assess its clinical safety. EXOs derived from bone marrow mesenchymal stem cells could produce protective effects in brain injury models, multiple sclerosis, and other neurological disorders thanks to their ability to enter biological barriers such as the BBB. In epilepsy, the administration of native EXOs can result in a reduction in inflammation, memory preservation, and a decrease in neuronal loss. The regenerative properties of EXOs have been shown after stroke injury in both rat and mouse models. By means of proteomics analysis, EXOs derived from mesenchymal stem cells were found to contain various proteins involved in the process of brain repair function. EXOs may accelerate and stimulate regeneration in several tissues, for instance, kidneys, and also seem to modulate transplant rejection ^[48]^[81]^[82]. Furthermore, EXOs have shown a protective effect on joint damage in a collagenase-induced OA model and in several cardiovascular diseases ^[83]^[84]. EXOs can act as a decoy for virus and bacterial toxins, thus suggesting a potential role as therapeutic agents ^[85]^[86]. These days, well-designed EXOs against COVID-19 may be feasible to prevent initial infection or further internal dissemination of the virus, thus reducing the virus burden and disease severity. Interestingly, EVs can be used in the treatment of COVID-19-associated brain damage due to their unique ability to penetrate the BBB and their potential to be engineered and targeted to a specific part of the CNS ^[87]^[88]^[89]^[90]^[91]. Recently, clinical trials that point to the use of EXOs as therapeutic agents against COVID-19 infection are currently ongoing. Moreover, EXOs are also being explored for their vaccine potential. In order to overcome the shortcomings of existing vaccines and contain escalating cases of COVID-19, several biotechnology companies are focusing on vaccine development using EXOs as a platform against SARS-CoV-2 ^[92]. In the following section, it will be in short reported how the potential use of natural EXOs is largely improved when they are modified and used as carriers of therapeutic agents.
Drug delivery and nanotherapy: any shuttle used for drug delivery must possess several necessary characteristics: (i) can encapsulate an adequate amount of drug to obtain therapeutic effect; (ii) must possess a prolonged inherent stability of size, structure, and bioactivity of the therapeutic agent during circulation before reaching the target organ; (iii) can evade macrophages’ phagocytosis, must have non-toxic properties, be biocompatible with the immune response, and be non-immunogenic. For the past few years, several new nanoscale systems to deliver therapeutic drugs or genes have been designed to improve bioavailability, reduce the toxicity of traditional drugs, and target specific sites. The first clinical success in nanotechnology occurred in 1995 with the approval of Doxil (a formulation of liposomal doxorubicin). Since then, new therapies and biocompatible nanocarriers have been designed (silver nanoparticles, polymer nanoparticles, nanotubes) and used, but until now, an ideal drug delivery system, with long-term safety and biocompatibility, remains to be planned. EXOs are good candidates for delivering vehicles of chemotherapeutics agents to specific cells and tissues and trigger phenotypic changes. EXOs have lower immunogenicity than virus-based delivery systems and liposomes. As aforementioned, the lipid bilayer gives EVs an amphiphilic nature that allows them to store and dissolve both hydrophobic and hydrophilic compounds. Compared with free drugs, exosomes loaded with chemotherapeutic drugs showed a higher efficacy. Examples of such systems are doxorubicin-loaded EXOs, EXO-curcumin, and paclitaxel-loaded EXOs that were shown to exert stronger anti-proliferative activities or cytotoxicity in cancer cells than drugs alone. Curcumin is a polyphenol compound made from turmeric, a flowering plant of the ginger family. Curcumin loaded onto exosomes forms a complex that improves its solubility, stability, and bioavailability enabling to exert its antioxidant, antineoplastic, anti-inflammatory, and chemopreventive properties. Sun et al. treated mice with this complex and found that mice were able to resist lipopolysaccharide-induced septic shock ^[93]. Paclitaxel is a highly hydrophilic molecule used as an antitumor drug, but its clinical application is limited because of dose-dependent toxic side effects. The toxicity resulted in reduced exosomes loaded with this drug. Yang et al. using the zebrafish model demonstrated that exosomes loaded with the doxorubicin drug were able to cross the BBB and inhibit the growth of tumors. Doxorubicin is an amphiphilic drug that inhibits angiogenesis and controls tumor growth ^[94]. In addition, molecules such as catalase with antioxidant properties, anthocyanins with anti-cancer activity against ovarian cancer, and other molecules can exert increased therapeutic effects when loaded on exosomes. EXOs can also deliver DNA and RNA as genetic therapeutic agents. These molecules have sizes that obstacles passive diffusion and they are susceptible to enzyme degradation.

Several methods have been developed to incorporate proteins or nucleic acids into EXOs. Some of them can be performed on donor cells as the composition of the EXOs is highly controlled within cells. Thus, the incorporation of pharmaceutical components can be achieved by:

An active approach: donor cells are co-incubated with small molecular weight drugs or other chemical compounds. Cargos may passively diffuse across the cell membrane and concentrate in the cytoplasm; after appropriate stimulation such as heat or hypoxia, cells release EXOs loaded with the desired cargo. A simple over-expression in the parental cells of desired cargo is most of the time sufficient. The gene transfection approach is used for loading exogenous nucleic acids into donor cells; the cells are transfected with DNA plasmid vectors, noncoding RNAs, etc., that are easily packaged by the natural biomolecular synthesis processes within EXOs. Then, EXOs can be rapidly isolated and purified ^[95]. This approach is simple but can result in poor loading and thus is not suitable for wide applications.
A passive approach: EXOs previously isolated from different sources are incubated with various molecules, preferentially hydrophobic, that can easily penetrate inside and localize in their lumen. To improve EXOs permeabilization, different chemical or physical methods can be used. For example, saponin permeabilization, sonication, and mechanical extrusion over a polycarbonate membrane. A method often used for loading siRNA is electroporation; following the application of high-voltage electricity to the suspension of EXOs and therapeutic agents, temporary pores are created in the membrane through which molecules can pass inside the vesicles ^[96]. Another approach to modifying vesicles and improving their specific targeting ability is surface membrane modification. Using procedures such as chemical modifications to the EXO membrane, click chemistry, etc., target molecules, peptides, and ligand aptamers are allowed to directly anchor on the exosomal membrane ^[97]^[98].

Once the EXOs with the desired cargo are produced, the liberation of their content to the recipient cells occurs by different mechanisms and consequently, the cellular phenotype can be altered. Different methods of modification and functionalization of EXOs have been designed by researchers, but further insights into these reactions need to be acquired to completely understand these strategies and obtain better loading efficiencies ^[99]^[100].

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