The World Health Organization officially named coronavirus infectious disease 2019 (COVID-19) as an infectious disease caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). The occurrence of this disease was first registered at the end of the year 2019, and it has since become a global public health concern ^[1][2][1,2]. In addition to causing a disruption to health care systems, COVID-19 had a significant economic effect worldwide. Clinically, COVID-19 affects the lungs, with most cases exhibiting asymptomatic or mild symptoms; however, in some cases, particularly among the elderly or those with underlying conditions, interstitial pneumonia (IP) or acute respiratory distress syndrome (ARDS) can occur, resulting in a need for mechanical ventilation in intensive care units [3]. Additionally, COVID-19 has been observed to present systemic manifestations, including cardiovascular, gastrointestinal, hematopoietic, renal, and immune system dysfunction, sepsis, multi organ failure, and, in some cases, death ^[4][5][4,5].

SARS-CoV-2 is the seventh human coronavirus discovered to date. It is structurally similar to SARS-CoV and MERS-CoV, both of which lead to acute respiratory diseases [6]. Morphologically, SARS-CoV-2 is a pleomorphic or spherically enveloped virus particle with a diameter range of 80–120 nm, containing a 30 kB positive single-stranded RNA within its membrane and a variety of viral proteins, including its spike (S) protein. Studies have found that the spike protein of SARS-CoV-2 is highly structurally homologous to that of SARS-CoV. In addition, SARS-CoV-2 has been shown to have an increased affinity for the host cell’s angiotensin converting enzyme 2 (ACE2) receptor, which likely contributed to the enhanced infection and transmission potential of the virus ^[7][8][9][7,8,9]. Alongside SARS-CoV-2, there are four other known human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1), which can cause mild common cold symptoms [10].

Recent reports suggest that the viral infections of the lungs and respiratory tract and the inflammation and injury involve the intracellular communication via extracellular vesicles (EVs). These EVs are composed of different types of vesicles, namely exosomes, microvesicles (MVs), and apoptotic bodies (ABs), based on the size, biogenesis pathway, and markers ^[11][12][11,12]. Exosomes are approximately 30–200 nm in diameter and are released via the endosomal pathway; MVs are generated via budding off from the membrane, and ABs are released from the cells undergoing apoptosis. In general, exosomes and MVs are collectively referred to as EVs ^[13][14][15][13,14,15]. Furthermore, EVs have the capability to transfer viral particles from infected cells to healthy cells, as well as to modify the body’s immune responses [16]. On the other hand, the plasma of COVID-19 patients was found to contain exosomes enriched with fragments of the SARS-CoV-2 virus, including its spike protein. This suggests that exosomes extracted from the plasma of COVID-19 patients could potentially elicit an immune response to the infection.

2. Role of EVs in COVID-19 Infection and Associated Pathogenesis

Viruses access host cells via cell-surface receptors and can even transfer these receptors to receptor-null cells, making them more infectious ^[17][18][58,59]. Integrins, in particular, are suitable for attaching and/or entering both enveloped and non-enveloped viruses. For example, SARS-CoV has a binding motif of Arg-Gly-Asp (RGD) that it uses to bind to integrins, allowing for an alternate route of viral transmission ^[19][60]. Exosomes, which are known to have various adhesion molecules, are believed to bind to target cell membranes and enter via tetraspanin-enriched microdomains (TEMs). Studies on CoV proteolytic priming suggest that blocking the tetraspanin function could be a potential way to prevent infection. SARS-CoV-2 is believed to bind to respiratory epithelial cells and replicate in the airways and alveolar epithelial cells, inciting an immune response. Infected cells are thought to produce exosomes containing viral antigens, self-antigens, and 20S proteasomes ^[16][20][21][16,61,62]. Exosomes can carry the ACE2 receptor to recipient cells, enabling viral entry, and viruses can use them as a pathway for intra-host spreading. It has been observed that human lung epithelial cells, which are susceptible to SARS-CoV-2 infection, can release exosomes with viral components that can facilitate the transmission of SARS-CoV-2 RNA into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), resulting in increased expression of inflammation-related genes in the hiPSC-CMs. Furthermore, exosomes carrying the ACE2 receptor from both healthy donors and recovered COVID-19 patients have been found to reduce SARS-CoV-2 infection by blocking the binding of the viral S protein to its receptor ^[22][63]. This phenomenon suggests that ACE2+ exosomes can act as an inhibitory decoy, which could potentially be used as a therapeutic for treating COVID-19. This is akin to the manner in which HIV packages its proteins and RNA into vesicles to spread to non-infected cells, and further investigation is necessary in the context of SARS-CoV-2 ^[23][24][64,65]. During viral infection, EVs are internalized by recipient cells and play an important role in activating the innate immune system, modulating host defense, and evading immune detection ^[25][26][66,67]. The EVs emitted by infected cells are distinct from those of healthy cells and include biomolecules such as RNA, lipids, and proteins. SARS-CoV-2 is one such virus which spreads through exosomes and has been found in vacuoles inside host cells. Its assembly is similar to that of SARS-CoV and close to the rough endoplasmic reticulum ^[25][26][27][66,67,68]. Bioinformatics analysis has uncovered the presence of proteins involved in coagulation, transport, complement, protease inhibitor, and defense/immunity activities in exosomes. Research on exosomes derived from cells infected with respiratory syncytial virus has discovered that these exosomes activate an innate immune response, stimulating the release of cytokines and chemokines from monocytes and airway epithelial cells ^[28][29][69,70]. Patients suffering from coronavirus infections, such as SARS and MERS, experience high levels of pro-inflammatory cytokines and chemokines, leading to pulmonary inflammation. Severe COVID-19 and mortality have been linked to platelet degranulation, low platelet count, and increased levels of IL-6. The presence of cytokines such as TNF, IL-1β, and IL-6 in SARS-CoV-2 infection may be associated with exosomes, potentially contributing to a cytokine storm and tissue damage ^[30][71].

3. EVs as Tool for Diagnosis of COVID-19

Exosomes and extracellular RNAs (exRNAs) are involved in numerous pathological processes. ExRNAs, containing molecules such as mRNAs, miRNAs, small nuclear RNAs, transfer RNAs, and long non-coding RNAs (lncRNAs), are released during anti-viral responses and play a role in regulating the innate immune system of the host organism ^[31][72]. For COVID-19, early biomarkers, such as those linked to EV proteins, blood coagulation related markers, and liver damage, have been identified, with EV coatomer protein complex subunit beta 2 (COPB2) having the greatest predictive value for severe disease ^[32][33][73,74]. Exosomes are advantageous biomarkers for detecting infection from limited sample sources as they can be easily isolated and stored. Exosomal miRNA is used to monitor chronic infections of HBV and HCV, the latter of which alters the miRNA cargo of exosomes ^[34][75]. Culturing SARS-CoV-2 sub-genomic RNAs in Vero cells revealed high levels of specific proteins, including S, Orf3a, E, M, Orf6, Orf7a, and N, and low levels of Orf7b, which can be used as biomarkers for the disease ^[35][76]. Additionally, Wölfel et al. found that the presence of the E gene sub-genomic RNA indicates active viral infection and transcription ^[36][77]. On the other hand, Alexandersen et al. detected SARS-CoV-2 sub-genomic RNAs in diagnostic samples, which may not necessarily point to active virus replication or infection ^[37][78]. Moreover, SARS-CoV-2-derived EVs have been shown to increase the levels of circulating tissue factor (TF)-positive EVs, thus potentially contributing to thrombosis in patients with COVID-19 ^[32][73]. Additionally, electron microscopy studies have uncovered the early formation and accumulation of EVs containing viral replication complexes associated with SARS-CoV-2 ^[38][79]. Exosomes isolated from patient samples contain various proteins which may be used as potential biomarkers, such as fibrinogen, fibronectin, complement C1r subcomponent, and serum amyloid P-component. It has also been suggested that circulating exosomes may be involved in inflammatory, coagulant, and immunomodulatory processes in COVID-19 ^[39][80].

4. Therapeutic Applications of EVs for COVID-19

Exosomes, while believed to be involved in the transmission of SARS-CoV-2, may conversely be an advantage for the treatment of COVID-19. To curb the spread of the virus, the uptake of exosomes by neighboring cells could be impeded. Consequently, various treatments for COVID-19 have been proposed, including mesenchymal stem cells (MSCs) and their resulting exosomes. MSCs can generate a variety of cytokines and paracrine factors which can interact with immune cells such as T cells, B cells, dendritic cells, macrophages, and natural killer cells, which could help to reduce the cytokine storm associated with COVID-19. Clinical research has indicated that human umbilical cord-derived MSCs (HUMSCs) could be of benefit in improving the pulmonary function of those with SARS-CoV-2-related pneumonia ^[40][41][81,82]. An alternative approach to MSCs to treat COVID-19 is through secretome-based therapy and exosomes isolated from cell secretome, with exosomes being deemed more effective ^[42][43][44][83,84,85]. Studies have revealed that exosomes derived from MSCs are non-toxic and can have a similar effect to their parental cells in models of acute and chronic lung injury, sepsis, and ARDS, suggesting that they could be more successful than MSCs in constraining the inflammatory response of COVID-19 ^[45][46][86,87]. Investigations have demonstrated that utilizing exosomes in a clinical environment can be advantageous, with some research indicating that exosomes can be more effective than parent cells in diminishing lung injury in ARDS ^[47][88]. Moreover, exosomes can reduce the cytokine storm related to ARDS and increment the anti-inflammatory signaling mediators, thereby lessening the seriousness of lung injury. The use of exosomes has additionally been analyzed for treating COVID-19, where convalescent plasma containing exosomes is used as an immunomodulator ^[48][89]. Numerous restorative techniques have been created to treat COVID-19, such as antivirals, anti-infection agents, biologics, immunizations, and convalescent plasma. The adequacy of these medicines is constrained by the high rate of mutation of SARS-CoV-2, which impedes the body’s normal immune response. Plasma treatment has been exhibited to be powerful in seriously sick patients and focusing on the SARS-CoV-2 S protein utilizing neutralizing antibodies is another conceivable treatment. Cytokine treatment, interferon-α2b, and small interfering RNAs have likewise been utilized to repress viral replication ^[49][90]. Exosomes are of great interest for therapeutic applications due to their high bioavailability, biocompatibility, and low immunogenicity. This has accelerated research into the use of EVs for the treatment of COVID-19, with a particular focus on early detection and novel therapies. Additionally, exosomes are composed of double lipid bilayers and are capable of transporting various biomolecules which are involved in both physiological and pathological processes, including those of the host immune response. Several studies have highlighted the importance of exRNAs as biomarkers and potential therapeutic targets for COVID-19, emphasizing the necessity of identifying exRNAs for the development of effective treatments ^[46][87]. Exosomes, small vesicles containing microRNA, proteins, and cytokines, have been proposed as a promising therapeutic approach to reduce inflammation and improve the outcomes of severe COVID-19 patients ^[48][89]. Bone marrow-derived exosomes have been demonstrated to reduce alveolar inflammation and support edema clearance, as well as restore the permeability of epithelial membranes in various animal models of acute lung injury, ARDS, asthma, and other inflammatory diseases ^[50][91]. Additionally, miRNA-155 mimic-loaded exosomes were found to increase miRNA-155 levels in primary mouse hepatocytes and the liver of miRNA-155 knockout mice, while significantly reducing and preventing the production of TNFα and the expression of SOCS1 mRNA in RAW macrophages, respectively ^[51][92]. MSC-derived exosomes have been demonstrated to protect lung epithelial cells against oxidative stress-induced cell death ^[52][53][93,94]. Animal studies have also shown that MSC-derived exosomes can increase the proliferation of lung epithelial cells and modulate the phenotype and function of lung-infiltrating dendritic cells. Systemic administration of MSC-derived exosomes has been found to decrease Escherichia coli endotoxin-induced acute lung injury in a mouse model and to guard the brain against sepsis-induced injury in rats ^[54][55][95,96]. Moreover, these exosomes have been observed to reduce endothelial cell apoptosis, attenuate the cytokine storm caused by SARS-CoV-2, and decrease IL-6 production, while increasing IL-10 production ^[47][56][88,97].

5. Targeting Exosomal Pathways Pharmacologically as COVID-19 Therapy

Pharmacological inhibitors of exosomes are a potentially effective treatment to combat viral infections such as SARS-CoV-2. Examples of these inhibitors include GW4869, Dynasore, calpeptin, Manumycin A, Y27632, Imipramine, and pantethine ^[57][98]. The primary way these inhibitors work is by blocking the release of viral extracellular vesicles and microvesicles, as well as by preventing the EVs’ movement or lipid metabolism. Experiments have revealed these inhibitors to be capable of decreasing the production of exosomes and microvesicles and inhibiting SARS-CoV-2 replication in vitro. As a result, they could have therapeutic potential in the case of COVID-19 ^[17][58].

6. Utilizing EVs as a Delivery Tool for COVID-19 Therapy

EVs have been demonstrated to be a reliable delivery vector for drugs due to their ability to reach target tissues, cross biological barriers, and protect their cargo from the immune system and degradation. Compared to synthetic delivery systems, exosomes have several advantages, including a more natural presence in bodily fluids, enhanced stability under physiological conditions, and less toxicity and immunogenicity [15]. Exosomes are also able to deliver specific cargo to recipient cells and cross the blood–brain barrier to deliver materials to the brain ^[58][59][99,100]. miRNAs and various drugs have been encapsulated into exosomes and used to target molecules within infected cells to reduce local inflammation or prevent apoptosis. One study found that pre-treating mesenchymal stem cells with IL-1β augmented their immunomodulatory effects, enabling their exosomes to transfer miR-146a to target cells. Exosomes derived from MSCs, being equipped with immunomodulatory components, have been proposed as a potential treatment option for COVID-19 in combination with antiviral drugs ^[60][61][101,102]. It has been found that MSC-derived exosomes delivered via inhalation had beneficial effects on lung repair in patients affected by the virus ^[62][103]. Moreover, Gunasekaran et al. highlighted the role of exosomes released from infected cells in the infection and spread of SARS-CoV-2 ^[63][104]. Considering their drug delivery capabilities and anti-inflammatory effect and their capacity to regenerate damaged tissues, exosomes may be used as therapeutics for COVID-19, although further research and clinical trials are needed to assess their efficacy and safety ^[64][65][105,106].