EVs are involved in the process of cardiovascular pathology, the communication between cardiomyocytes, fibroblasts, smooth muscle cells and endothelial cells, and the regulation of cardiac regeneration, ventricular remodeling and angiogenesis (22–24). Cardiomyocytes are the main cells of the heart and their EVs play different roles in myocardial repair (25). Myocardial ischemia and I/R injury are the main causes of myocardial injury. And in this part, we mainly describe the effect of cardiomyocyte-derived EVs on injured myocardium.

Myocardial infarction is a common manifestation of ischemic heart disease/coronary artery disease (26). It is characterized by sudden interruption of coronary blood flow and necrosis of supplying cardiomyocytes. Although primary coronary angioplasty and drug therapy repair the impaired cardiac function to some extent, the mortality rate remains high. More and more evidences have shown that paracrine factors play an important role in the process of myocardial infarction, the changes of microRNAs (miRs) in circulation can accurately reflect the myocardial injury in vivo (4), and the nearby living myocardium can protect myocardial cells from hypertrophy by capturing EVs. Circulating miRs released by the damaged myocardium after acute myocardial infarction (AMI) can also be transferred to distal organs through circulation via exosomes and affect the biological activity of recipient cells functionally. Yang et al. (27) found that miR-30 was highly enriched in exosomes isolated from serum of AMI patients or hypoxic cardiomyocyte culture medium, and miR-30a mediated up-regulation of core autophagy regulators Beclin-1, Atg12 and LC3II/LC3I. Regulating autophagy through exosomes is also a promising strategy for the treatment of ischemic heart disease. It has been found that rat cardiomyocytes can promote survival and inhibit apoptosis by releasing a variety of paracrine factors, including insulin-like growth factor-1, vascular endothelial growth factor (VEGF) and transforming growth factor-β, under co-culture conditions (28, 29). Cardiac Progenitor Cells participate in cardiac function recovery by secreting EVs (CPC EVs) through a paracrine mechanism. CPC EVs can reduce scar formation, alleviate undesirable remodeling, and improve cardiac function in acute and chronic myocardial infarction models (28). These findings are supported by other preclinical models of myocardial infarction (26).

The process of myocardial ischemia/reperfusion (I/R) involves injury of endothelial cell function and structure in a number of ways, including mass release of reactive oxygen species (ROS), direct injury of white blood cells, calcium overload, and reduced secretion of NO. EVs derived from cardiomyocytes were initially discovered under hypoxia and re-oxygenation conditions (6–8). Cardiomyocytes secreted exosomes containing circHIPK3 under hypoxic conditions to protect injured cardiomyocytes. In addition, the inflammatory response is involved in all aspects of I/R injury (25, 30). Chen et al. (31) found that eNOS activation in cardiac microvascular endothelial cells (CMECs) required a crosstalk between cardiomyocytes (CMs) and CMECs through the uptake of CM-derived sEVs. Tongxinluo induced CM-sEVs contain increased levels of long Intergenic non-protein coding RNA, regulator of reprogramming (Linc-ROR). Upon uptake into CMECs, linc-ROR downregulates its target miR-145-5p leading to activation of the eNOS pathway by facilitating the expression of p70s6k1 in these cells. The activation of CMEC-derived eNOS works to increase survival in both the CMECs and the CMs themselves.

Immune cells play an important regulatory role in the development of myocardial ischemia, I/R injury, septic cardiomyopathy and chemotherapy-related cardiomyopathy (32–34). EVs derived from immune cells show pleiotropism in pathological states. EVs have therapeutic potential of anti-apoptosis and anti-fibrosis, promoting angiogenesis, inhibiting ventricular remodeling, improving cardiac function and inhibiting local inflammatory response (35).

In the process of myocardial injury, macrophages are recruited to the damaged area to initiate release EVs into the peripheral tissue. Macrophage-derived EVs aggravate myocardial injury, inflammation, and promote myocardial fibrosis. Studies demonstrate that in the process of myocardial ischemia, macrophage derived exosomes deliver miR-155 to cardiac fibroblasts, which inhibit proliferation and promoted inflammation, suggesting that macrophage derived EVs containing miR-155 aggravate myocardial injury (36). Macrophages themselves are also receptors for miR-155, and endothelial cell derived EVs containing miR-155 can promote macrophage polarization (37).

Antigen presenting T cells have the ability to release specific EVs. Treg cells can improve the healing and remodeling after myocardial infarction (38, 39) and delays the progression of atherosclerosis (40). However, compared with cardiovascular diseases, Treg-derived exosomes are more recognized in organ transplantation and have a greater application prospect (41–44). The regulatory effect of DC-based EVs on the heart may depend on Treg activation, but it is still difficult to determine whether Treg exosomes have a cardioprotective effect. Studies have shown that Treg inhibits the effect of other T cells (43, 44), such as Th1 cells dependent on the transfer of exosomal miRNAs to receptor cells (45). Neutrophils are the most abundant white blood cells in human peripheral blood, accounting for about 50 to 70% of the total number of white blood cells. They play an important role in the innate immune system and are the first line of defense for the body to respond to the invasion of pathogens. They can resist the invasion of external pathogens through various ways such as phagocytosis, degranulation and production of reactive oxygen species (46). In the early stage of myocardial infarction, neutrophils and monocytes rapidly infiltrate the infarct region (47), releasing inflammatory EVs and triggering an inflammatory cascade (48). Neutrophil-derived and mast cell-derived EVs play an important role in initiating injury-related molecular patterns (DAMPs) of the innate immune response.

Different types of stem cell (SC) derived EVs can convey different biological information. EVs with cardioprotective function may come from marrow mesenchymal stem cell (MSC), embryonic stem cells, and hematopoietic stem cells. Various strategies have been tried for the treatment of cardiovascular disease with SC transplantation therapy. Studies have found that the paracrine factors of transplanted cells, not the transplanted cells themselves, play a major role in repairing damaged tissue.

The research on EVs delivery of drugs is increasing gradually (69, 70). Some small molecule chemical drugs and gene drugs have been successfully loaded into EVs, showing great potential in the treatment of tumors, cardiovascular diseases and neurological diseases. EVs can accumulate drugs in treated cells, improve the stability and blood circulation time of small molecule drugs, and improve the efficacy of small molecule drugs (Figure 2). Sun et al. (71) showed that curcumin-loaded exosomes can increase the concentration of curcumin in vivo, increase the stability of the drug, and improve its anti-inflammatory and antioxidant effects.

RNA is an unstable macromolecular material making it difficult to achieve effective delivery. Existing deliveries include use of cationic liposomes, dendritic macromolecules, cationic polymer particles, but the carrier is still in the process of clinical application. This technology faces problems with security, stability, and tissue targeting (72). Overexpression of specific microRNAs via transfection in donor cells enables their packaging into EVs. For example, overexpression of miR-125b targeting SIRT7 in bone marrow MSC-derived exosomes down-regulated the levels of Bax, caspase-3 apoptotic proteins and IL-1β, IL-6, TNF-α inflammatory factors, and up-regulated the expression of Bcl-2, in order to repair the myocardial injury of I/R-rats (73). In addition, miR-181a delivered by exosomes of MSCs may inhibit the inflammatory response through c-Fos and promote the polarization of Treg cells to protect myocardial injury caused by miRs (74–77). These results suggest that donor cell engineering allows for directed cargo loading of small nucleotides into EVs, providing an effective therapeutic strategy for ischemia-reperfusion injury.

EVs can also be used as carriers of proteins for myocardial repair. Proteins can be genetically engineered from donor cells or encapsulated directly into EVs. In the former, transfected donor cells with a plasmid carrying the target gene are used to synthesize the protein encoded by the inserted gene. These proteins are loaded into exosomes. The supernatant of the cell culture is collected for isolation and purification of exosomes. In addition, proteins can be directly encapsulated in exosomes. Studies have shown that Yim et al. (78) encapsulated proteins in exosomes through an optically reversible protein-protein interaction, which significantly increased the level of target proteins in the receptor cells in vivo and in vitro, further proving the possibility of EV cargo design for not only nucleotides, but also protein.

EVs are considered as ideal natural drug carriers due to their good histocompatibility and low immunogenicity, but drug loading and targeted delivery are the problems that must be solved when EVs are used for cardiac repair. To complete drug loading and targeted delivery, researchers have paved the way for engineering EVs. For example, gene engineering technology can be used to express targeted proteins or peptides to the surface of EVs, which can improve the targeting effect of the drug and enhance the efficacy. However, the complexity of the genetic modification process has greatly hindered its widespread application. Recently, modification methods based on physical and chemical properties have been developed. In this section, we will summarize the design and modification strategies of EVs as therapeutic platforms. As shown in Table 2, we summarize strategies for the engineering of EVs.

Tian et al. (86) conjugated functional ligand RGDyk cyclic peptide to the surface of EVs through biological orthogonal reaction between the EVs with surface modification of cyclostyle and the azide polypeptide. They encapsulated them with curcumin to target the cerebral lesion area in a cerebral artery occlusion mouse model. This effectively inhibited inflammatory response and apoptosis in the focus region, and developed a novel exon-based targeted drug delivery vector for ischemic brain injury. Ligand fragments or homing peptides discovered by phage display and in vivo biopanning methods fused to the enriched molecules on the external part of exosomes have been exploited to improve the ability of exosomes to target specific tissues or organs carrying cognate receptors.

The homing peptide of 9AA (CSTSMLKAC, 9AA) can specifically target the myocardial cells of MIRI (87). Xu Wang used technology of molecular cloning and lentivirus packaging to engineer exosomal enriched membrane protein (Lamp2b) fused with ischemic myocardium-targeting peptide 9AA. The result found that exosomes engineered by IMTP can specially target ischemic myocardium, and mesenchymal stem cell-derived IMTP-exosomes exert enhanced therapeutic effects on AMI.

In addition to peptides, binding targeted proteins such as nanosomic and signal regulatory protein α to exosome surfaces has also become an effective targeting strategy (88). For cardiovascular diseases, gene modification can also be used to overexpress bioactive substances with cardio protective effects in exosomes, such as miRNA or proteins to enhance exosome mediated cardio protective effects and reverse the effect of the pathological environmental secretome. Ni et al. (89) found that tissue matrix metalloproteinase inhibitor 2 (TIMP2) modified MSCs derived exosomes from human umbilical cord blood activated the Akt/Sfrp2 pathway, inhibiting cardiomyocyte apoptosis and extracellular matrix remodeling while promoting angiogenesis, improving heart function following myocardial ischemia. Compared with normal HUC-EXO, HUC-ExotimP2 increased the number of CD31⁺ and lectin immune-active cells in myocardial infarction rats and promoted angiogenic activity in the infarct area.

Although exosomes have shown promising in the field of drug delivery, it is not easy to achieve specific targeting of exosomes through surface modification, and the reaction conditions need to be strictly controlled to avoid the destruction and aggregation of exosomes caused by improper temperature, pressure and osmotic pressure. At present, the efficiency of exosome targeted drug delivery is not ideal as modifications result in clearing by the immune system.

EVs have favorable biocompatibility, non-immunogenicity and non-tumorigenesis. They are physiologically more stable than graphted cells, circulate throughout the body, and can cross the blood-brain barrier, and are more resistant to freezing and thawing than cells, have the advantage of long-term storage (90), and have natural properties for therapeutic use. In addition, EVs prove suitable for modification to deliver drugs to target cells (91). However, there is still a lack of standardized methods for the collection, separation and purification of EVs, and different separation methods lead to large differences in the purity, size and concentration of EVs (92), hindering the introduction of EVs into clinical practice.

The most common EVs separation technique is differential centrifugation, however, EVs obtained by this technique often contain aggregates of cell culture medium, cell proteins and particles (93). In addition, production of EVs is difficult to scale up due to time-consuming isolation process, low yield, and need for specialized equipment. Another common separation technique is based on monoclonal antibodies to isolate EVs-associated antigens (94). However, the disadvantage of this technique is low specificity: non-EVs materials or cells carrying the antigen can easily bind with the antibody, which greatly reduces the purity of the extracted EVs (95, 96). High performance liquid chromatography can provide highly purified EVs, but this technique also requires expensive equipment and low yields, which limits its widespread use (93). At present, there is still a lack of a consensus, standardized high specificity, high yield EVs isolation and purification method.

The lack of appropriate preservation methods is another major problem that limits the clinical use of EVs. Generally, EVs are stored at −80°C, become unstable under long term storage (97). Studies have shown that the surface and morphological characteristics of EVs change and the protein degrades after 4 days of storage at −80°C (98). The size of EVs decreased when stored at 4 or 37°C, indicating structural change or degradation. These studies show that the storage conditions of EVs are relatively harsh, which limits its clinical use (99).

Before clinical use of EVs in the treatment of cardiovascular diseases, there are still many difficulties to be solved: the criteria for isolation and identification, high yield and more economical protocols are still to be developed. Current storage conditions are harsh, and the in vivo dynamics of EVs have not been studied in detail. EVs have irreplaceable advantages in the treatment of cardiovascular diseases, including those diseases that lack effective drug therapy (100).

Engineered EVs are nano-vesicles made of natural EVs or cell membranes by special methods. Engineered EVs can be divided into multilayer vesicles 50–1,000 nm and single-layer vesicles, in which single-layer vesicles are further divided into small vesicles (SUV) 20–100 nm, large vesicles (LUV) 100–1,000 nm and large vesicle (GUV) 1–200 μm (5, 101). Engineered EVs also need to be given targeted, stimulus-responsive properties. To target cardiomyocytes, EVs need to be modified by genetic engineering or chemistry.

The exosomal donor cells are genetically modified to express the targeted peptide, effectively expressing the targeted peptide on secreted exosomes (63). Kim et al. (102) used lentiviral vectors fused with lysosomal associated membrane glycoprotein 2B and ischemic myocardial targeting peptide to genetically modify bone marrow MSCs to express ischemic myocardial targeting peptide. Fluorescence microscopy imaging showed that the number of exosomes expressing ischemic myocardial targeting peptide was greater than that of natural exosomes. Li et al. (103) reported a programmed exosome that would provide human antigen R with extremely high affinity to RNA. HuR and exosome transmembrane protein CD9 sequence were fused to modify donor cells, and the modified donor cells expressed the fusion protein HuR-CD9. These generated exosomes actively increased RNA loading. Ohno et al. (104) transfected pDisplay vector with GE11 peptide specifically binding to expression of EGFR into HEK293 cells to successfully express G11 peptide on the isolated exosome membrane. In subsequent experiments, they demonstrated the inhibitory effect of EGF-specific exosomes delivered Let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2 ^−/− mice. Gene modification in exosome donor cells can greatly improve the stability of functional exosomes, but it is expensive and time-consuming to perform gene manipulation in donor cells.

At present, there are two chemical modification methods for artificial vesicles: wet chemistry method and microfluidic technology (101). The wet chemical methods grouped with the thin-film hydration method is the most classic (105). This method can be used in the synthesis of multilayer vesicles (106). Hammons et al. (107) used the film hydration method to dissolve dioleyl-phosphatidylcholine and poly-oxyethylene in chloroform, which was dried by rotary evaporation and then vacuum drying. Next, aqueous solution containing carbon nanotube pore protein was used for hydration. Ultrasound was used to remove the film on the inner wall of the container and the sample was extruded through the 200 nm polycarbonate film with a micro extruder to prepare hybrid bionic vesicles (108). Based on the film hydration method, a team replaced the “skeleton” required for self-assembly with block copolymers instead of pure phospholipids which broadened the application scope of the original method (109). In 2018, inkjet printing technology began being used to prepare emulsions (110). Inkjet technology can print amphiphilic molecules directly onto the receiving medium to form vesicles (111), which can quickly encapsulate fragile biomolecules such as proteins or other biological complexes, maintaining some degree of structural integrity of these molecules (112).

Currently, the reassembly of phospholipid membranes with different functions has become a new direction of engineered EVs, which makes it possible for EVs to have specificity, enabling repair and anti-inflammatory functions at the same time. However, there are few related studies, and they are mainly used in cardiac ischemia and ischemia reperfusion injury in cardiovascular diseases.

To achieve precise drug delivery, engineered EVs need to add stimulus-response modules. Drug release can be achieved by temperature, light, ultrasound, magnetic field or electric field stimulation. This greatly improves the controllability of drug delivery, thus increasing drug targeting and reducing drug side effects to achieve more accurate and controllable treatment of diseases.

MSC-EVs have high therapeutic potential for tissue repair. There is evidence of functional engineered EVs isolated from human bone marrow MSCs by introducing lentiviral particles containing BMP2-expressing plasmids. By enhancing the BMP2 signaling cascade in target cells and tissues, the repair of tissues such as myocardium and bone are promoted (115). However, this does not meet all treatment needs. Previous studies on EVs administration have explored the mechanisms of local application (116), systemic administration (117), intrathecal administration (118), vitreous injection (119), and nasal administration (120), but problems such as high demand, low efficiency and strong ectopic effect of EVs have limited its application. Huang et al.'s team encapsulated it in alginate saline gel to promote angiogenesis and regeneration of tissues such as skin, bone, and heart muscle, achieving location or site specificity. These physical wrapping methods resulted in EVs being released within hours of being placed in the desired location (121). Recent studies have used EVs encapsulated in alginate hydrogels for regenerative medicine applications in the treatment of myocardial ischemia or myocardial infarction (122).

The conventional diagnostic methods of heart failure include echocardiography, MRI imaging and identification of myocardial necrosis markers. Exosomes show great diagnostic potential as biomarkers of AMI, and we focus on the diagnostic value of miRNAs and proteins carried by exosomes (124, 125).

miRNAs found in serum and plasma are characterized by stability, time, and tissue specificity. Exosomes, as effective carriers of miRNAs, may provide a new possibility for the diagnosis of AMI. Beg et al. studied the plasma exosome miRNA in patients with heart failure and normal controls with average LVEF of (22.2 ± 7.2) %, and found that the ratio of miR-146a/miR-16 in peripheral blood of patients with heart failure was significantly higher (126). Studies have shown that exosome miR-146a has a cardiomyocyte protective function and has a protective effect on oxidative stress (114). The exosomes isolated from the serum of patients with myocardial infarction contain miRNA-183. Studies have confirmed that the level of miRNA-183 is positively correlated with the degree of myocardial ischemia injury, and classified miRNA-183 as a new biomarker for the diagnosis of myocardial infarction. miRNA-183 is highly enriched in exosomes in patient serum with AMI (127–129). It is worth noting that exosome derived miRNAs can also be detected in urine. In AMI rats, exosome associated miR-1 specifically increased in both blood and urine (91, 130), suggesting that urine derived exosomes may be a new diagnostic method for AMI. The non-invasive extraction of urine may greatly improve the diagnostic rate of myocardial infarction and has a good clinical application prospect. Though miRs is the leading diagnostic marker in the context of exosomes, the potential value of exosomal specific carrier proteins should not be overlooked in the diagnostic evaluation of myocardial infarction.