Engineered ATP-Loaded Extracellular Vesicles: A Dual-Functional Strategy for Improving Myocardial Infarction Therapy

Engineered ATP-Loaded Extracellular Vesicles: A Dual-Functional Strategy for Improving Myocardial Infarction Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Engineered ATP-Loaded Extracellular Vesicles: A Dual-Functional Strategy for Improving Myocardial Infarction Therapy Farshid Jaberi Ansari, Javad Behroozi, Mohsen Chamanara, Mostafa Shahrezaee, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6422821/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Myocardial infarction (MI) is a major component of cardiovascular disease. The primary challenge in MI therapy is the lack of energy in the affected tissue. Extracellular vesicles (EVs) areengineered with adenosine triphosphate (ATP) and targeted with an anti-myosin antibody (T-EV) to effectively deliver energy to damaged myocardial tissue and mitigate the effects of myocardial infarction (MI). Furthermore, using an in vivo MI rat model to evaluate cardiac repair, we compared cardiac function, infarct size, and the expression of troponin and α-actin four weeks after MI with those in a healthy group. The results indicate that, compared with no treatment, the use of Target-ATP-EVs (T-ATP-EVs) enhances the viability of hypoxic cells by 46% and reduces apoptosis by 28%. In the animal study, there was a 26% increase in the left ventricular ejection fraction (LVEF) and a 28% decrease in the infarct size compared with those of the MI group when this treatment was applied. Additionally, the expression levels of troponin and α-actin increased approximately two-fold in vivo with the use of these engineered EVs. In this study, engineered EVs were investigated as a strategy to deliver ATP directly to cardiomyocytes and heart tissue in both in vitro and in vivo models. The system described here enhances cardiomyocyte survival and function, marking a significant advancement in the treatment of myocardial infarction. Myocardial Infarction Exosome Engineered Extracellular Vehicles Targeted Delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights 1. Engineered ATP-loaded EVs specifically deliver energy to ischemic myocardial tissue, enhancing cardiac recovery. 2. ATP-loaded EVs increase cardiomyocyte viability by 46% and reduce apoptosis by 28% under hypoxic conditions. 3. Treatment with ATP-loaded EVs increased the left ventricular ejection fraction by 26% and decreased the infarct size by 28% in a rat model of myocardial infarction. 4. The use of anti-myosin antibody-tagged EVs for precise, noninvasive delivery offers a novel treatment approach for myocardial infarction. Introduction Myocardial infarction (MI) is the most common cause of mortality and morbidity worldwide [ 1 ]. The main cause of MI is often atherosclerosis, where the principal coronary artery that provides blood to the heart is suddenly obstructed [ 2 ]. Sedimentation of cholesterol in the membrane of arteries is the main cause of atherosclerosis [ 3 ]. Traditional MI treatments, including coronary artery bypass grafting (CABG), angioplasty, atherectomy, valve surgery, stenting, and transmyocardial revascularization (TMR), are effective at restoring blood flow and relieving symptoms but have significant limitations and risks. These methods are often invasive, require complex surgeries that carry the potential for complications, have extended recovery times, and, in some cases, do not address the underlying damage to heart tissue. Emerging treatments, such as stem cells, tissue engineering, 3D cardiac patches, and cell-free therapies, focus on tissue regeneration, making them sustainable solutions for MI patients [ 4 – 6 ]. Despite their potential, cell-based therapies face several limitations, including low engraftment efficiency, insufficient differentiation into mature cardiomyocytes, and inadequate anchorage within the myocardial tissue. The advantages of cell-free therapy include its nonaggressive, biocompatible, nonimmunogenic, and lack of need for cell engraftment or differentiation [ 7 – 9 ]. An excellent example of cell-free therapy is the use of extracellular vesicles (EVs), particularly exosomes. Exosomes are nanosized vesicles ranging from 30 to 150 nm in diameter that are secreted by various cell types in the body. The method of isolation significantly influences the size distribution and properties of these exosomes [ 10 , 11 ]. Exosome therapy is an alternative method that replaces traditional cell therapy. It offers the advantages of cell therapy while avoiding its associated adverse effects. They facilitate intercellular communication by transporting biomolecules such as proteins, lipids, and RNAs, influencing physiological and pathological processes in recipient cells [ 12 ]. One of the critical challenges of systemic exosome delivery is the competition between therapeutic exosomes and endogenous exosomes in body fluids [ 11 ]. Exosomes derived from heart tissue, such as those derived from cardiosphere cells, exhibit cardioprotective effects by regulating key processes such as apoptosis, hypertrophy, angiogenesis, and cell proliferation [ 13 ]. During MI, the level of ATP decreases to 80%; thus, ATP production via the respiratory pathway ceases, which results in the production of only a small amount of ATP through the anaerobic glycolysis pathway [ 14 ]. A reduction in the ATP concentration results in the production of lactic acid under anaerobic conditions and reduces the pH of cardiomyocytes. A reduction in pH causes Na + /H + exchange, increasing the concentration of Na + in the cells; subsequently, the Na + /Ca 2+ balance is disturbed, and the Ca 2+ concentration increases in the cells [ 15 ]. An increasing Ca 2+ concentration in cells causes myocardial contracture and activates protease enzymes in mitochondria, which lead cardiomyocytes toward apoptosis or necrosis, depending on the time and severity of ischemia [ 16 ]. Administering ATP to damaged cardiomyocytes offers a straightforward approach to reduce ischemic injury. The solubility of ATP in blood is very high, and the half-life of ATP is very short. Therefore, the use of appropriate carriers is necessary to prevent ATP degradation and ensure efficient delivery of ATP to cells. EVs are nonimmune and noninvasive carriers for the delivery of ATP into cells. Additionally, ischemic preconditioning (IPC)-EVs constitute a well-recognized protective strategy that strengthens cellular resilience to subsequent ischemic events. This effect is achieved by modulating the composition of EVs released by various cells. These EVs, particularly those derived from endothelial cells following IPC, play a critical role in cardioprotection by transmitting survival signals to cardiomyocytes [ 17 ]. Additionally, MSC-derived EVs under IPC reduce inflammation and apoptosis, enhancing cardiac function and survival in myocardial infarction models [ 18 ]. Furthermore, EVs generated under IPC conditions in normal hearts have been shown to reduce infarct size and trigger prosurvival pathways in heart failure models. These findings highlight their significant potential as therapeutic agents for ischemic injury recovery. IPC appears to play a crucial role in enhancing EV cargo for cardioprotection, establishing a foundation for EV-based therapies for ischemic heart diseases [ 19 ]. Recently, inhalable stem cell-derived EVs were shown to promote heart repair after myocardial infarction [ 20 ]. A critical problem in the systemic delivery of EVs is the competition of therapeutic EVs with an enormous number of natural EVs in body fluids and a lack of targeting [ 11 ]. To increase the drug delivery and circulation time of EVs into the injured myocardium, since the membranes of cardiomyocytes are disrupted during MI and exposed to an intracellular matrix such as myosin, we used an anti-myosin antibody conjugated to the EVs. [ 21 ]. In a previous study, a liposome targeted with an anti-myosin antibody was reported [ 22 ]. Additionally, iRGD-EVs were used to target tumors or engineer cell donor EVs with plasmid carriers to express cardiac-homing peptides on the membrane of EVs for targeted myocardial infarcted tissue [ 23 ]. In this study, we examined anti-myosin antibody-conjugated ATP-loaded EVs (T-ATP-EVs) as engineered EVs to target cardiomyocytes and heart tissue both in vitro and in vivo. We explored ATP delivery to ischemic cardiac tissue via EVs with increased cardioprotective potential through IPC. Direct ATP administration, although beneficial in theory, is limited by rapid dissolution in blood, reducing its efficacy. Moreover, the exposure of myosin in injured cardiac tissue during myocardial infarction provides a distinct opportunity for targeted therapeutic delivery. To overcome this challenge and capitalize on therapeutic opportunities, we developed a dual-functional strategy in which ATP-loaded EVs (ATP-EVs) are combined with T-ATP-EVs. This approach delivers ATP at suitable concentrations into cells and utilizes the cardioprotective properties of IPC-modulated EVs to enable precise targeting of the injured myocardium. This system improves cardiomyocyte survival and function, representing a significant advancement in therapeutic approaches for myocardial infarction. Materials and methods Ischaemic preconditioning and EV isolation H9c2 cells were obtained from ATCC and cultured in high-glucose (4.5 g/L) DMEM supplemented with 15% FBS. At 37°C, the cells were grown in a humidified atmosphere containing 95% air and 5% CO 2 . The cells were seeded in a 10 6 cells/T 75 cell culture flask and passaged when confluent. The cells were subjected to hypoxic conditions upon reaching 80% confluency. After three washes with PBS, the medium was replaced with 15% EV-depleted FBS. A hypoxic chamber (< 0.1 mmHg, O₂ < 1%, CO₂ = 5%) was used to simulate hypoxic conditions in vitro for 30 minutes, followed by intermittent reoxygenation for 10 minutes in an anoxic chamber. This cycle was repeated three times to induce ischemic preconditioning. The cells were then returned to normal conditions for two days to produce EVs. Two days after ischemic preconditioning, the supernatant of the cell culture was collected for EV isolation via an EXOCIB kit. For EV isolation via the EXOCIB method, cell culture supernatants were processed according to the manufacturer’s protocol. Briefly, the supernatants were centrifuged at 3,000 × g for 30 minutes to remove debris and dead cells. The conditioned medium was then filtered through a 0.22 µm filter to remove any cell particulates or apoptotic bodies. In a sterile vessel, the supernatant was transferred, EXOCIB EV precipitation solution was added, and the mixture was refrigerated overnight. Afterward, the mixture was centrifuged at 1,500 × g for 30 minutes. Following aspiration of the supernatant, the tube was centrifuged again at 1,500 × g for 5 minutes to remove any residual EXOCIB solution. Characterization of Ischemic Preconditioning-Derived EVs 10 µl of 1 mg/ml isolated EVs were added to the cover and diluted in 1 ml of PBS. The size and polydispersity index (PI) of intact EVs and targeted EVs were determined via dynamic light scattering (Malvern, UK) with a 405 nm laser beam. In DLS, the paths of particles undergoing Brownian motion are tracked from the scattered light. The size and morphology of the isolated EVs (10 µL, 1 mg/ml) were observed via field emission electron microscopy. After they were allowed to air dry and dehydrated with a graded series of alcohol, they were fixed with 2% glutaraldehyde, and a gold coating was used for better contrast (JEM-2000EXTEM, Japan). The alamarBlue cell viability reagent is a resazurin-based solution that measures the reducing power of living cells to determine viability quantitatively. Resazurin is reduced to resorufin in viable cells, changing the blue dye to red. This color change and increased fluorescence can be detected by absorbance (detected at 570 and 600 nm) or fluorescence (excitation between 530–560) and emission at 590 nm. The cells were cultured in 96-well plates at 5000 cells/well and 37°C. After treatment with different types of EVs, 10% v/v alamarBlue was added to the medium, and the mixture was incubated for 6–8 h and then analysed with a plate reader. Rat model of myocardial infarction Male Wistar rats (n = 5 for each group, approximately 300 g in weight) were used to establish the MI model. The rats were given CO 2 for 2 min until they became dizzy and then given general anaesthesia with a mixture of 120 mg/kg body weight ketamine, 5 mg/kg midazolam (Dormicum, Roche) and 0.5 mg/kg medetomidine through an intraperitoneal injection. Surgical operation was performed under artificial ventilation with 100% oxygen-induced myocardial infarction. The heart was exposed via a left-sided limited thoracotomy. The left anterior descending artery (LAD) was ligated with a 5–0 polyester suture 1 mm from the tip of the normally positioned left auricle. Through a 15 mm opening in the 4th intercostal space, a 10 cm length of 5–0 silk suture was used to ligate the left coronary artery (LAD). After surgery, the rats were randomized into three groups (n = 5): the PBS control group, the intact ATP-EV group and the T-ATP-EV group. All the groups were injected intravenously (200 µL of 1 mg/ml EVs or with only PBS as a control). The impact of EVs on H9c2 cell proliferation and migration H9c2 cells were seeded at a density of 5×10 3 cells/well in 96-well plates overnight and then treated with different concentrations of 0.1, 1, or 10 µg/ml EVs after the medium was removed for two days. The H9c2 cells were subsequently washed 2 times with PBS, fixed with 2% glutaraldehyde and then stained with 0.1 mM Dapi for 5 min, after which microscopy with a fluorescence microscope was performed at 40x magnification [ 24 ]. A monolayer of 10 5 H9c2 cells was grown in DMEM supplemented with 15% FBS for at least 48 hours at 37°C and 5% CO 2 to allow for cell adhesion and migration. The confluent monolayer was then scored with a 100 µL pipette, and the medium was immediately exchanged with fresh medium containing 10 µg/mL EVs (free-EV FBS). To inhibit cell proliferation, the cells were treated with fresh serum-free culture medium containing 10 mg/ml mitomycin [ 25 ]. ATP loading and antibody tagging of EVs The preparation of T-ATP-EVs involved some modifications to the ultrasound bath. The EVs were ultrasonicated with various concentrations of ATP at a protein concentration of 1 mg/ml (3 cycles). A 1 mM ATP solution was prepared by dissolving 552 µg of ATP in 1 mL of PBS (ATP molar weight of 552 g/L) as a stock solution, and then different concentrations of ATP (100, 10, 1 and 0.1 molar) were prepared with diluted stock solution in PBS. Subsequently, the EVs were washed two times with PBS and isolated on a 100 kDa MWCO ultrafiltration column (Millipore) at 5,000 × g for 20 min to remove the unincorporated free ATP. All the experiments were carried out at 4°C. The EVs were tagged with an anti-myosin antibody (Abcam, USA), which targets the infarcted heart via myosin exposure during myocardial infarction. The antibody was conjugated to the EVs with some modifications. Using a 100-fold molar excess of antibody, DIPE-NHS (dioleoyl phosphatidyl ethanol amine N-hydroxyl succinimide) and the anti-myosin antibody were combined for 1 hour to produce the DOPE-antibody. The DOPE antibody was incubated with the EVs at a lipid:EV ratio of 1000:1 (1000 molecules of DOPE antigen for each EV). To remove excess reagents, an ultrafiltration column with a 100 kDa MWCO ultrafiltration column (Millipore) 5000 g was used for 20 min. Echocardiography/cardiac function assessment The rats were anaesthetized with 3% isoflurane and underwent transthoracic echocardiography on Day 0 (before the creation of the MI model), Day 3 (after MI model creation), and Day 30 (after MI model creation). The left ventricular ejection fraction (LVEF) and fractional shortening (FS) of the left ventricle were analysed. Rats with a normal EF after Day 3 were excluded from the following studies. The investigators were blinded to the identities of the rats and groups. Determination of Infarct Size Using Masson & Trichrome (M&T) Staining After the rats were anaesthetized as described above (n = 5 for each group) and sacrificed on day 30, the hearts were removed, rinsed with saline and soaked in 4% (v/v) paraformaldehyde until analysis. The specimens were prepared via transverse dissection across the infarct area. Heart section samples were rinsed with running water for 30 min, dehydrated in a graded ethanol series, cleared, embedded in paraffin, and then cut into sections with a thickness of 5 µm for Masson's Trichrome staining. This staining was used to quantify fibrosis in the left ventricle in various treatment groups. The heart sections were also subjected to immunohistochemistry (IHC) at a thickness of 10 µm. Calculation of the fibrotic area The left ventricular (LV) fibrotic area induced by myocardial infarction after 30 days was calculated via the following equation: Specimens stained with Masson’s trichrome were used to quantify the fibrosis area in the left ventricle in different groups [ 26 ]. $$\:\text{I}\text{n}\text{f}\text{a}\text{r}\text{c}\text{t}\:\text{s}\text{i}\text{z}\text{e}=\frac{\text{F}\text{i}\text{b}\text{r}\text{o}\text{t}\text{i}\text{c}\:\text{a}\text{r}\text{e}\text{a}\:\text{o}\text{f}\:\text{L}\text{V}}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{L}\text{V}\:\text{a}\text{r}\text{e}\text{a}\:(\text{F}\text{i}\text{b}\text{r}\text{o}\text{t}\text{i}\text{c}\:\text{a}\text{r}\text{e}\text{a}\:\text{o}\text{f}\:\text{L}\text{V}+\text{N}\text{o}\text{n}\:\text{f}\text{i}\text{b}\text{r}\text{o}\text{t}\text{i}\text{c}\:\text{a}\text{r}\text{e}\text{a}\:\text{o}\text{f}\:\text{L}\text{V})}\times\:100$$ Immunohistochemistry (IHC) For immunohistochemistry (IHC), after the heart samples were prepared, the sections were fixed in 4% (v/v) paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.01% saponin, and blocked with protein blocking solution (DAKO) for 1 h. Each paraffin section stained for actin with primary antibodies (1:30) was incubated overnight at 4°C, and subsequently, secondary antibodies (1:100) were added at room temperature for 1.5 h, followed by a 30 min incubation with Power vision poly-HRP-conjugated anti-rabbit IgG. Images were captured with a Zeiss microscope. Data analysis was performed with the Allred score on the basis of the semiquantitative estimation of the percentage abundance of positive biomarkers in the cells and the staining intensity. Flow cytometry A direct flow cytometric method was used to investigate the effects of different intact EVs and targeted EVs on hypoxia-induced apoptosis and necrosis. Analysis with a marker (FL1H: Annexin, V FITC) and (FL3H: PI) was performed to identify live cells, early apoptotic bodies, late apoptotic bodies and necrotic cells. Statistical analysis Statistical analyses were conducted via GraphPad Prism 9. The data are expressed as the means ± standard errors of the means (SEMs). For comparisons between two groups, nonparametric t tests were utilized, whereas one-way ANOVA was employed for multiple group comparisons. A p value of less than 0.05 was considered statistically significant. Compared with the control treatment, the T-ATP-EV treatment significantly increased cell viability and reduced apoptosis in vitro. In vivo, both ATP-EVs and T-ATP-EVs significantly improved the left ventricular ejection fraction and reduced the infarct size compared with those of the control group, underscoring the efficacy of the targeted delivery system in ameliorating cardiac function postmyocardial infarction. Results and Discussion Characterization of EVs This study aimed to use ATP-EVs as an efficient energy source for cardiomyocytes. Following cell culture and EV isolation, the EVs were conjugated with anti-myosin antibodies for targeting, as described earlier, and enriched with ATP via ultrasonic power (Fig. 1 A). The size distribution of EVs was analysed via dynamic light scattering (DLS), which revealed a uniform and homogeneous size distribution for both ATP-EVs and T-ATP-EVs (Fig. 1 A). The results revealed a significant size difference, with ATP-EVs averaging 81.43 ± 4.27 nm and T-ATP-EVs averaging 126.66 ± 4.04 nm. These findings are consistent with previous studies, which reported an antibody size of approximately 10–15 nm, further validating the observed size increase upon antibody conjugation [ 27 , 28 ]. Our findings agree with their reported antibody size of approximately 10–15 nm[ 29 ] and revealed a significant difference between ATP-EVs and T-ATP-EVs. Additionally, field emission scanning electron microscopy (FESEM) was employed to examine the morphology of the EVs. The images indicated that both ATP-EVs and T-ATP-EVs retained a spherical to oval shape, with antibody conjugation causing a size increase but not altering the overall morphology (Fig. 1 A). These observations align with previous studies reporting that EVs typically exhibit round to oval shapes, with size and morphology influenced by external modifications. Western blot analysis confirmed that the isolated vesicles were indeed EVs, ruling out contamination by other extracellular vesicle types (Fig. 1 B). Effect of EVs on Proliferation EVs contain factors for mitigation and proliferation, such as c-myc, cyclin and cytokines [ 30 – 32 ]. Our results of nuclei stained with Dapi revealed that the number of nuclei/slides was 64 ± 5 for the control group after treatment with different concentrations of EVs; this number increased to 92 ± 6, 131 ± 7, and 172 ± 7 for the 0, 1 and 10 µg/ml EV groups, respectively. All the EV treatment groups were significantly different from the control group (Fig. 1 c). These results show that EVs increase the proliferation of myoblasts. These results are consistent with those of Li et al., who reported that cardiac progenitor cell-derived EVs promote H9c2 cell proliferation via Akt/mTOR activation. They reported that the effects of EVs on the proliferation of H9c2 cells are dose- and time dependent. In this study, after treatment with 200 µg/ml EVs, an optimal effect of EVs for 48 h was observed [ 33 ]. A previous study revealed the effect of MSC-derived EVs on the proliferation of wound-healing cells via the expression of many genes involved in cell cycle control, such as C-myc, Cyclin A1, and Cyclin D2. In this study, cells treated with MSC-derived EVs expressed more than two times more genes than control cells did [ 34 ]. miR-21-3p/5p isolated from cardiac telocytes and MSCs stimulates the proliferation of endothelial cells via the activation of ERK, Akt and HIF-1α [ 35 , 36 ]. This study revealed that EVs contain metabolites and microRNAs that can increase the viability and proliferation of cells and that the effects of EVs on recipient cells are mostly dose dependent. Some reports have also shown that EVs suppress proliferation. Wang et al. reported that EVs containing miR-155 suppress fibroblast proliferation and promote fibroblast inflammation during cardiac injury[ 37 ]. This study revealed that the further suppression of fibroblast proliferation by miR-155 can indirectly inhibit fibrosis formation after MI. Similar studies have shown that the effects of EVs can be altered by their metabolite content. Effect of EVs on migration Furthermore, the viability, proliferation, and migration of myoblasts are important for heart therapy, although myoblast cells have less potential for migration, and mostly endothelial cells, such as HUVECs, are considered for migration and tube formation; however, in this study, we used a scratch test on myoblasts to investigate the potential of EVs. Our results revealed that the scratch size for the control group was 0.65 + 0.1 mm. On Day 1, this size decreased to 0.6 + 0.1 mm without EV treatment after treatment with 10 µg/ml EVs, and this size decreased to 0.55 + 0.08 mm. On Day 2, this size decreased to 0.46 + 0.07 mm without EV treatment, and after treatment with 10 µg/ml EVs, this size decreased to 0.28 + 0.07 mm. The results revealed that only the EV-treated group after Day 2 was significantly different from the other groups ( Fig. 1 D ) . MSC-derived EVs promote HUVEC migration and tube formation by upregulating VEGFR-2 and Ang-1[ 38 ]. Another study revealed that carbonic anhydrase 9-enriched EVs from renal cell carcinoma (RCC) cells promote HUVEC migration and tube formation [ 39 ]. Additionally, MSC-derived EVs enhanced the migration of normal and diabetic wound fibroblasts. Compared with depleted conditioned medium, MSC-derived EVs significantly increased migration by more than 30% [ 34 ]. In bone marrow mesenchymal stem cell-derived EVs, the PI3K/Akt/eNOS signalling pathway is activated by miR-126, which promotes EC survival, proliferation, and migration in ECs that are subjected to I/R injury [ 40 ]. Effects of T-ATP-EVs on Hypoxic Cell Viability and Apoptosis After the cells reached 80% confluency, we used the alamarBlue assay to investigate the effects of ATP-EVs and T-ATP-EVs on the viability of hypoxic cells. The results of this analysis revealed that the viability of hypoxic cells in the control group was 33.91 ± 4.2%. This percentage increased to 93.97% after treatment with ATP-EVs and to 79.38 ± 0.9% after treatment with T-ATP-EVs ( Fig. 2 A ) . To confirm the effects of ATP-EVs and T-ATP-EVs on cell viability, we used flow cytometry analysis (annexin-PI). Representative images of flow cytometry data from different EV treatments are shown in Fig. 2Ba . The results of the flow cytometry analysis of live cells revealed that after hypoxia, the percentage of live cells was 42.8 ± 2.76%. This percentage increased to 91.33 ± 4.5% after treatment with ATP-EVs and to 80.36 ± 2.55% after treatment with T-ATP-EVs (Fig. 2Bb) . The results of the flow cytometry analysis, which measured the degree of apoptosis induced by hypoxic conditions, revealed that hypoxia caused 38.95 ± 4.19% of the total number of apoptotic cells in the control group. This percentage decreased to 3.91 ± 2.03% after the application of ATP-EVs and to 10.43 ± 1.46% after the administration of T-ATP-EVs. These results revealed the strong effects of both ATP-EVs and T-ATP-EVs in preventing the initiation of the apoptotic cascade (Fig. 2Bc). The results of the flow cytometry analysis revealed that hypoxia caused 5.23 ± 2.80% necrosis in the control group. After treatment with ATP-EVs, this percentage decreased to 4.33 ± 3.37%, and after applying T-ATP-EVs, it reached 3.53 ± 1.7% (Fig. 2Bd). These data revealed a significant difference between ATP-EVs and T-ATP-EVs compared with the control group in terms of increased viability and decreased apoptosis induced by hypoxia. However, there was no significant difference in the percentage of necrosis among the treatment groups. The results of this study revealed that ATP-EVs increased the survival rate of hypoxic cells by 60%, whereas T-ATP-EVs increased the survival rate by 46%. This was mainly due to 1) the reduced effect of the antibody under in vitro conditions and 2) the negative effect of the antibody on cellular metabolism. In a previous report, Nejatollahi et al. noted that antibodies bound to anti-RTFscFv decreased the viability of prostate cancer cells[ 41 ], similar to the results of our flow cytometry analysis. These results revealed that the percentage of viable cells increased by 49% after treatment with ATP-EVs, whereas it increased by 38% after treatment with T-ATP-EVs. Additionally, the flow cytometry results revealed that ATP-EVs reduced apoptosis caused by hypoxia by 35% and T-ATP-EVs promoted apoptosis by 28%. In other words, although the anti-myosin antibody increases the targeting of damaged heart tissue postinfarction, cell tests in an in vitro environment revealed that antibody attachment to EVs slightly decreases cell viability. This study revealed that both types of EVs increased cell survival and metabolism. These findings are consistent with other researchers' results on the effects of EVs on enhancing cell survival and metabolism. EVs contain various miRNAs, metabolites, proteins, and growth factors, such as SDF1, IGF1, NGF, and HGF30, which increase the survival, metabolism, proliferation, and growth of target cells [ 42 ]. Previous studies have shown that EVs significantly impact cell survival under different conditions. EVs have been reported to reduce ROS levels in cells treated with H2O2 [ 43 ]. They also demonstrated that EVs derived from cardiomyocytes produce many miRNAs; the four miRNAs specific to or highly expressed in heart tissue are miR-133a/b, miR-208a (with antifibrotic properties), miR-499 (with antiapoptotic properties), and miR-1 (antioxidants). These miRNAs are involved in regulating cardiogenesis and cardiac functions such as contractility and message conduction. Additionally, miR-499 and miR-208 are involved in the expression of cardiac sarcomere genes such as miR-133a and miR-1. Moreover, EVs derived from mesenchymal stem cells contain the transcription factor CXCR4, which increases VEGF expression and blood vessel formation [ 44 ]. Previous reports indicate that ischemic preconditioning is a powerful approach for increasing the survival and regeneration of cells under ischemic conditions [ 26 ]. The creation of ischemic conditions for EV production increases the resistance of the heart to hypoxia [ 45 ]. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that provides various physiological responses to ischaemic conditions[ 46 ]. EVs isolated from endothelial cells express more HIF-1, miR-210, and miR-126, which play crucial roles in heart regeneration [ 47 ]. miR-210 is known as a regulator of HIF-1, increasing its expression. HIF-1 shifts the metabolism of cardiac progenitor cells to a glycolytic state, reducing oxygen usage during heart infarction[ 48 ]. Compared with those in normal cells, the expression levels and numbers of miRNAs such as miR-210, miR-199a-3p, miR-24, miR-22, and miR-21 in EVs secreted from cells under IPC conditions increased. This increase in miRNAs enhances the potential of damaged cells against hypoxia and protects heart tissue. miR-26 and miR-223 also play essential roles in cardiac tissue regeneration, and their amounts in EVs are greater under IPC conditions than under normal conditions. In an ischemia‒reperfusion injury model in rats, treatment with EVs prepared from hypoxic cells improved heart function and prevented fibrosis development[ 49 ]. Cardiac endothelial cells under hypoxic conditions increase the expression of miR-210 and miR-126, both of which have proangiogenic properties[ 47 ]. These studies indicate that EVs contain metabolites and compounds that increase cell metabolism and survival. Moreover, reports have shown that EVs produced under IPC conditions contain greater amounts of miRNAs and metabolites, providing greater protection than those produced under normal conditions. These features increase the resistance of IPC-condition-produced EVs, safeguarding cells from hypoxia and infarction-induced apoptosis and necrosis. If the energy demand of a cell persists during hypoxia, cardiomyocytes may undergo apoptosis. Reports have shown that the intracellular ATP concentration ranges from 1–10 mM depending on the cell type[ 50 ]. The ATP concentration outside cells is approximately µM, and in distant areas such as blood, it is approximately nM. Since the extracellular ATP concentration is much lower than the intracellular ATP concentration, cells cannot meet their energy needs externally[ 51 ]. Previous reports have shown that ATP has a very short half-life when injected intravenously because it quickly dissolves in the blood, preventing it from reaching target tissue at appropriate concentrations[ 52 ]. These limitations make the use of ATP as a therapeutic agent impractical without a suitable carrier. Additionally, high ATP concentrations can be toxic. The mechanisms of ATP toxicity include metabolic and oxidative stress. Increased ATP levels can increase the intracellular calcium concentration and disrupt the mitochondrial membrane potential, leading to apoptosis or necrosis. Previous reports have shown that elevated intracellular calcium disturbs muscle cell contractile activities and rhythm, affecting heart pacemaker cells. Therefore, a suitable carrier is necessary to transport ATP effectively to the MI area. Effects of targeted ATP-EVs on heart function To investigate the effects of ATP-EVs and T-ATP-EVs on the left ventricular ejection fraction (LVEF), we used echocardiography analysis. The results revealed that the LVEF decreased from 86 ± 2% in the sham group to 54.33 ± 2.08% in the MI group. After ATP-EV treatment, the LVEF increased to 73.66 ± 1.52%, and applying T-ATP-EVs increased the LVEF to 80 ± 1%. Schematic images of the echocardiography results are presented in Fig. 3 A, and a quantitative plot of the LVEF for different treatment groups is shown in Fig. 3 B. These results demonstrated a significant improvement in LVEF with both ATP-EVs and T-ATP-EVs after MI and indicated a significant difference between the effects of ATP-EVs and T-ATP-EVs. After myocardial infarction, cardiomyocytes experience a severe decrease in energy due to disruption of the aerobic metabolic pathway caused by a lack of oxygen, leading to reliance on the anaerobic pathway. ATP is a crucial molecule for the cell. The transfer rate of ATP across the membrane is very low because of its high anionic charge, so ATP is usually transported by carriers in the membrane[ 53 ]. Recent studies have reported the therapeutic applications of ATP. In one study, a group treated with liposomes containing ATP exhibited improved wound healing compared with a group treated with only liposomes without ATP[ 54 ]. There are also several reports on the use of ATP-containing liposomes for treating ischaemia‒reperfusion models, which show that ATP-containing liposomes improve heart function compared with that of the control group[ 55 ]. Another study demonstrated that ATP-containing liposomes were used in cerebral ischemia, improving brain tolerance to ischemia-related injuries. In another report, ATP-containing liposomes were used to treat liver failure and were shown to exert a protective effect on the liver[ 56 ]. The ATP concentration is very precisely controlled in the cell because ATP plays a key role as an energy source for many intracellular activities. One of the main challenges in the therapeutic use of EVs is the competition between natural EVs in the serum and therapeutic EVs[ 57 ]. Previous studies have shown that the concentration of serum EVs is approximately 10¹¹ EVs per milliliter, whereas the concentration of therapeutic EVs is less than 10⁹ EVs per milliliter. In many studies, EVs are targeted to specific tissues via various methods, such as platelet membranes[ 58 ] or homing peptides[ 59 ]. H9c2-derived EVs were used in this research because myoblast cells are closer precursors to cardiomyocytes, making them similar to cardiomyocytes in terms of origin and differentiation, and their EVs likely have a similar membrane structure to cardiomyocytes. These EVs can outperform other types of natural EVs in the serum and blood in terms of their ability to target cardiomyocytes[ 13 ]. These findings suggest that myoblasts may be good candidates as sources of EVs for cardiac treatment[ 60 ]. The results of this research revealed that the intravenous use of both ATP-EVs and T-ATP-EVs increased cardiac output after MI and improved overall heart function. In other words, both types of EVs target the heart: ATP-EVs passively and T-ATP-EVs actively targeted the damaged heart tissue. This study revealed that ATP-EVs increased the cardiac LVEF by 19%, whereas targeted T-ATP-EVs increased it by an additional 7%, increasing the total LVEF to 26%. Previous studies have shown that EVs effectively reduce the size of the infarct area[ 61 ]. This study also revealed that ATP-EVs reduced the size of the infarct area by 22%, and T-ATP-EVs further reduced it by another 6%, resulting in a reduction of 28%. Zhuo Wan et al. reported that the use of EVs increased the LVEF by 10% after MI[ 61 ]. Kai Kang et al. reported that EVs secreted from CXCR4-overexpressing mesenchymal stem cells increased the LVEF by 25%, from 33–58%[ 44 ]. Another study by Chunxiao Wang et al. reported that the use of EVs containing miR-155 increased the LVEF from 25–50% after MI[ 62 ]. Effect of T-ATP-EVs on infarct size To investigate the effects of ATP-EVs and T-ATP-EVs on the size of the infarct area after MI, we used Masson's trichrome staining to calculate the percentage of collagen deposition in the infarct area. The results revealed that the size of the infarcted area decreased from 42.33 ± 1.15% in the MI group to 20.66 ± 1.52% in the ATP-EV group. Additionally, treatment with T-ATP-EVs reduced the infarct area to 14%. Cross-sectional images of heart sections stained with Masson's trichrome are shown in Fig. 4 A, and a quantitative diagram of the infarct area size via ImageJ software is shown in Fig. 4 B. These results indicate that, compared with those in the MI group, the infarcted area in both the ATP-EV and T-ATP-EV groups was significantly smaller. Moreover, the data revealed that compared with ATP-EVs, T-ATP-EVs significantly reduced the infarct area. Previous studies have shown that EVs reduce the infarct area induced by MI [ 63 ]. Shiqi Hu et al. combined platelet membranes with EVs (P-XOs) to target injured hearts, and their data revealed that, compared with MI alone, P-XOs decreased the infarcted area by 19%, from 27–18%, whereas XOs alone decreased the infarcted area by approximately 10%[ 64 ]. Kai Kang et al. reported that EVs secreted from CXCR4-overexpressing mesenchymal stem cells increased vWF + angiogenesis markers more than sixfold compared with those in the MI group and decreased the infarct area by 28%, from 50–22%, after MI[ 44 ]. Another study by Yuliang Feng et al. reported that the infarct area decreased from 40–15% when EVs enriched with miR-22 were used[ 26 ]. Additionally, Daya D. Verma et al. reported that treating an MI heart with ATP-loaded liposomes decreased the infarcted area by 40%, from 70–30%[ 65 ]. Effect of T-ATP-EVs on troponin and α-actin To investigate the effects of ATP-EVs and T-ATP-EVs on maintaining heart structure, we used immunohistochemistry analysis of the cardiomyocyte biomarkers troponin and α-actin[ 66 , 67 ]. Both troponin and α-actin are key specific biomarkers of cardiomyocytes. Representative images of IHC troponin biomarkers are shown in Fig. 7A, 7B , and representative images of IHC α-actin biomarkers are shown in Fig. 7C . Figure 7A shows the relative expression levels of troponin across various treatment groups. In the MI (control) group, troponin expression was notably low. The application of EVs results in a moderate increase in expression, whereas ATP-EVs significantly increase expression. Notably, treatment with T-ATP-EVs further increased troponin levels, approaching those observed in the sham group, which presented the highest expression. Statistical analysis confirmed significant differences, with ATP-EVs (P = 0.0005) and T-ATP-EVs (P = 0.0125) showing superior efficacy compared with the MI control. These findings underscore the potential effectiveness of targeted EV therapies in improving cardiac biochemical markers postmyocardial infarction. This study revealed significant variations in α-actin expression levels among the different treatment groups. In the sham group, the α-actin level was 4.8 ± 0.44, which decreased to 2.2 ± 0.44 in the MI control group. Following treatment, the level increased to 3.4 ± 0.54 with ATP-EVs and further increased to 4.1 with T-ATP-EVs, indicating a recovery trend. Additionally, the differences in baseline expression were statistically significant, with ATP-XOs yielding a P value of 0.0024 compared with the MI control and sham treatments showing P values of 0.0010 and 0.0079 compared with the MI control and ATP-XOs, respectively. These findings suggest that both ATP-EVs and T-ATP-EVs effectively increase α-actin expression, underscoring their potential as therapeutic interventions for MI ( Fig. 7B ). The quantitative results for troponin and α-actin are shown in Fig. 7C . These IHC results indicated a significant difference between the ATP-EV and T-ATP-EV groups and the MI group. Additionally, there was a significant difference between ATP-EVs and T-ATP-EVs, suggesting that T-ATP-EVs have greater affinity for heart tissues and better prevent the destruction of cardiomyocytes. Jia Huang et al. reported that EVs derived from cancer cells improve the viability of target cells by delivering alpha-smooth muscle actin[ 68 ]. Shiqi Hu et al. combined platelet membranes with EVs (P-XOs) to target the injured heart; their data revealed that P-XOs accumulated 1.8 times more than XOs did, and the expression of α-sarcomeric actin was greater after treatment with P-XOs[ 64 ]. Kyle I. Mentkowski et al. reported that EVs fused to a cardiomyocyte-specific peptide (CMP) accumulate twice as much in heart tissues than do bare EVs after intravenous injection[ 57 ]. Another study by Adam Vandergriff et al. compared two types of peptides, a cardiac-homing peptide (CHP) and a scramble peptide (Scr), for targeting the infarcted heart after conjugation with EVs. This study revealed that EF changes for the CHP peptide after 21 days were three times greater than those for the Scr peptide, and the infarct area decreased to 5% for CHP-EVs, whereas it decreased to 10% for Scr-EVs, indicating that CHP-EVs better targeted heart tissues after MI[ 69 ]. Robert C. Scott et al. reported that treating an MI heart with targeted VEGF-loaded liposomes increased vessel density by two-fold compared with that in the MI group[ 70 ]. Conclusions Cardiovascular diseases, with myocardial infarction (MI) as a leading cause, remain a major global health challenge because of the high morbidity and mortality associated with cardiac tissue damage. This study introduces a novel dual-functional therapeutic approach that leverages ischemic preconditioning (IPC)-derived EVs loaded with ATP conjugated with anti-myosin antibodies (T-ATP-EVs) to address the limitations of conventional and emerging MI treatments. By combining the natural cardioprotective properties of EVs with targeted delivery, this method aims to optimize energy replenishment in hypoxic cardiomyocytes and enhance cardiac repair mechanisms. The findings of this research highlight the significant efficacy of T-ATP-EVs in improving both cellular and cardiac outcomes. In vitro, these EVs increased cell viability by 46% and reduced apoptosis by 28% compared with those in the control groups, demonstrating their ability to preserve cardiomyocyte health under hypoxic conditions. In vivo results further supported these benefits, with a 26% improvement in the left ventricular ejection fraction (LVEF) and a 28% reduction in infarct size. Additionally, the T-ATP-EVs effectively restored structural integrity markers, including troponin and α-actin, with troponin preservation reaching 78% and α-actin levels increasing to 85% of baseline values, highlighting their role in maintaining the functional and structural framework of cardiac tissue. This study also addressed key challenges associated with ATP delivery and systemic EV use. The rapid degradation of ATP and competition with natural serum EVs have historically limited therapeutic effectiveness. By incorporating anti-myosin antibodies, targeted EVs achieve selective accumulation in infarcted myocardial regions, increasing delivery precision and minimizing off-target effects. This targeted delivery mechanism, coupled with the inherent resilience of IPC-derived EVs, represents a promising strategy for overcoming these barriers. The implications of this research extend beyond myocardial infarction therapy. The dual-function approach presented here has the potential for adaptation to other ischemic conditions, where targeted energy delivery and cellular protection are critical. However, translating these findings into clinical applications requires further investigation, including studies in larger animal models, evaluations of long-term safety and efficacy, and potential modifications to scale up production for human use. In conclusion, T-ATP-EVs represent an innovative and effective therapeutic strategy for enhancing cardiac repair post-MI. By addressing the critical limitations of existing treatments and introducing targeted, cell-free therapies, this approach provides a solid foundation for future advancements in cardiovascular medicine, with the ultimate goal of improving patient outcomes and quality of life. Declarations Authorship contribution Farshid Jaberi Ansari: Investigation, Methodology, Writing – original draft, Writing – review & editing; Javad Behroozi: Data curation, Formal analysis, Methodology, Validation, Writing – original draft, Writing – review & editing; Mohsen Chamanara: Methodology, Validation, Writing – original draft, Writing – review & editing; Mostafa Shahrezaee: Methodology, Validation, Writing – original draft, Writing – review & editing; Ali Shakermoghaddam: Methodology, Validation, Writing – original draft, Writing – review & editing; Seyed Hossein Mousavi: Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing; Hossein Ahmadi Tafti: original draft, Writing - review & editing; Mohammad Ali shokrgozar: Methodology, Writing – original draft; Amir Amanzadeh: Methodology, Writing – original draft; Mahdi Ghorbani: Methodology, Validation, Writing – original draft; David W. Greening: Supervision, Visualization, Writing – original draft, Writing – review & editing; Reza Heidari: Conceptualization, Methodology, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing. Ethical approval Approval for animal ethics was conducted according to guidelines provided by the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Aja University of Medical Sciences (IR.AJAUMS.REC.1401.167). Declaration of Competing Interest The authors declare that they have no known financial or personal conflicts of interest that could have influenced the work reported in this paper. Consent for publication Not applicable since study did not include humans. Competing interests The authors have no relevant financial or non-financial interests to disclose. Funding There is no funding for this article. 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Mononuclear phagocyte system blockade improves therapeutic exosome delivery to the myocardium. Theranostics. 2020;10(1):218. Wang C, et al. Macrophage-derived mir-155-containing exosomes suppress fibroblast proliferation and promote fibroblast inflammation during cardiac injury. Mol Ther. 2017;25(1):192–204. Petrelli A, et al. Education inequalities in cardiovascular and coronary heart disease in Italy and the role of behavioral and biological risk factors. Nutr Metab Cardiovasc Dis. 2022;32(4):918–28. Hu S, et al. Platelet membrane and stem cell exosome hybrids enhance cellular uptake and targeting to heart injury. Nano Today. 2021;39:101210. Verma DD, et al. ATP-loaded liposomes effectively protect the myocardium in rabbits with an acute experimental myocardial infarction. Pharm Res. 2005;22:2115–20. Chaulin AM. Cardiac Troponins as Biomarkers of Cardiac Myocytes Damage in Case of Arterial Hypertension: From Pathological Mechanisms to Predictive Significance. Life. 2022;12(9):1448. Clément S, Chaponnier C, Gabbiani G. A subpopulation of cardiomyocytes expressing α-skeletal actin is identified by a specific polyclonal antibody. Circ Res. 1999;85(10):e51–8. Huang J, et al. Cancer cell-derived exosomes promote cell proliferation and inhibit cell apoptosis of both normal lung fibroblasts and non-small cell lung cancer cell through delivering alpha-smooth muscle actin. Am J Transl Res. 2019;11(3):1711. Vandergriff A, et al. Targeting regenerative exosomes to myocardial infarction using cardiac homing peptide. Theranostics. 2018;8(7):1869. Scott RC, et al. Targeting VEGF-encapsulated immunoliposomes to MI heart improves vascularity and cardiac function. FASEB J. 2009;23(10):3361–7. Supplementary Files Graphicalabstract.png Graphical Abstract The graphical abstract illustrates the creation and function of ATP-loaded extracellular vesicles (T-ATP-EVs) engineered with anti-myosin antibodies for targeted delivery to damaged heart tissue. These findings reveal how these specialized EVs increase the ATP supply to ischemic cardiomyocytes, resulting in increased cell survival, reduced infarct size, and improved cardiac function in myocardial infarction models. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revisions Needed 28 Sep, 2025 Reviewers agreed at journal 06 May, 2025 Reviewers invited by journal 01 May, 2025 Editor assigned by journal 11 Apr, 2025 First submitted to journal 10 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6422821","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":450696920,"identity":"e988167f-3de2-4274-849a-8088ba7dee32","order_by":0,"name":"Farshid Jaberi Ansari","email":"","orcid":"","institution":"Aja University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Farshid","middleName":"Jaberi","lastName":"Ansari","suffix":""},{"id":450696921,"identity":"42280243-6072-49ad-a9ce-d1fe73ef3cf1","order_by":1,"name":"Javad Behroozi","email":"","orcid":"","institution":"Aja University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Javad","middleName":"","lastName":"Behroozi","suffix":""},{"id":450696922,"identity":"cf34e604-76f8-4b63-999e-e835384d1832","order_by":2,"name":"Mohsen Chamanara","email":"","orcid":"","institution":"Aja University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mohsen","middleName":"","lastName":"Chamanara","suffix":""},{"id":450696923,"identity":"5de33d96-7d5c-4658-b011-b542f18e47fd","order_by":3,"name":"Mostafa Shahrezaee","email":"","orcid":"","institution":"Aja University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mostafa","middleName":"","lastName":"Shahrezaee","suffix":""},{"id":450696924,"identity":"8597c56f-52a6-407f-9f62-19c29e95cb5b","order_by":4,"name":"Ali Shakerimoghaddam","email":"","orcid":"","institution":"Aja University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Shakerimoghaddam","suffix":""},{"id":450696925,"identity":"3c5109cd-0d78-4b6c-8048-d1043d1dcbc0","order_by":5,"name":"Seyed Hossein Mousavi","email":"","orcid":"","institution":"Aja University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Hossein","lastName":"Mousavi","suffix":""},{"id":450696926,"identity":"18ee0ad4-3e1a-4401-872a-50549a084150","order_by":6,"name":"Amir Amanzadeh","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Amir","middleName":"","lastName":"Amanzadeh","suffix":""},{"id":450696927,"identity":"1fc143d5-f49a-4abc-b1f6-0236456d4662","order_by":7,"name":"Mohammad Ali Shokrgozar","email":"","orcid":"","institution":"Pasteur Institute of Iran","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Ali","lastName":"Shokrgozar","suffix":""},{"id":450696928,"identity":"9f8147c0-4635-4b6b-a2b9-43bdd9e2edf6","order_by":8,"name":"Hossein Ahmadi Tafti","email":"","orcid":"","institution":"Tehran Heart Center","correspondingAuthor":false,"prefix":"","firstName":"Hossein","middleName":"Ahmadi","lastName":"Tafti","suffix":""},{"id":450696929,"identity":"44176048-5cc7-4a35-9fb7-7787daece936","order_by":9,"name":"Mahdi Ghorbani","email":"","orcid":"","institution":"Aja University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahdi","middleName":"","lastName":"Ghorbani","suffix":""},{"id":450696930,"identity":"feed154b-0e4c-48a6-9690-e5e24ab5f286","order_by":10,"name":"David W. Greening","email":"","orcid":"","institution":"Baker Heart and Diabetes Institute","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"W.","lastName":"Greening","suffix":""},{"id":450696931,"identity":"d3855f53-c27d-4fb0-938e-bdf759d0a2c9","order_by":11,"name":"Reza Heidari","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-2985-8396","institution":"Aja University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Reza","middleName":"","lastName":"Heidari","suffix":""}],"badges":[],"createdAt":"2025-04-10 19:34:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6422821/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6422821/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82087505,"identity":"98882411-e64c-442a-b4ba-eba8afb03b1e","added_by":"auto","created_at":"2025-05-06 15:26:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":696112,"visible":true,"origin":"","legend":"\u003cp\u003eComprehensive characterization of exosomes and their effects on cell behavior. \u003cstrong\u003e(A)\u003c/strong\u003e Characterization of exosomes via dynamic light scattering (DLS) and field emission scanning electron microscopy (FESEM). DLS analysis revealed the size distribution of the exosomes before ATP loading and targeting (76.5 nm), after ATP loading (83.9 nm), and after ATP loading with targeting modifications (122 nm), revealing stable vesicle sizes throughout the process. FESEM images revealed the uniform, spherical morphology of the exosomes across all conditions (scale bar = 100 nm). \u003cstrong\u003e(B)\u003c/strong\u003e Western blot analysis of the exosomal markers CD9 and CD81 confirmed the identity and consistent presence of the exosomes across all the experimental conditions. \u003cstrong\u003e(C)\u003c/strong\u003e Effects of various exosome concentrations (0.1, 1, and 10 µg/ml) on the proliferation of hypoxia-treated H9c2 cells. Representative images of DAPI-stained nuclei showingincreased nuclear density with increasing exosome concentrations. Quantitative analysis revealed a dose-dependent increase in the number of nuclei per slide: the number of nuclei in the control (64 ± 5), 0.1 µg/ml (92 ± 6), 1 µg/ml (131 ± 7), and 10 µg/ml (172 ± 7) groups was significantly greater than that in the control group (p \u0026lt; 0.05). \u003cstrong\u003e(D)\u003c/strong\u003e Effects of exosomes on myoblast cell migration were assessed via scratch assays. Images at Days 0, 1, and 2 revealed enhanced scratch closure in the exosome-treated group (10 µg/ml) than in the control group. Quantitative analysis revealed a significant reduction in scratch size over time, especially on Day 2 (control: 0.46 ± 0.07 mm; treated: 0.28 ± 0.07 mm; p \u0026lt; 0.05), indicating improved cell migration upon exosome treatment.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6422821/v1/83e2a0b0b9be1fd01dbe9a97.png"},{"id":82086102,"identity":"b64beac1-4f4d-4d62-b9c1-d09b39e689fe","added_by":"auto","created_at":"2025-05-06 15:10:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1080410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 2A.\u003c/strong\u003eFlow cytometry analysis of hypoxic cells treated with ATP-EVs and T-ATP-EVs. Live cells: Flow cytometry results revealed a significant increase in the percentage of live cells in the ATP-EV and T-ATP-EV groups compared with the control group and the untreated EV group. ATP-EVs presented the highest percentage of live cells, indicating their strong viability-enhancing properties under hypoxic conditions. Apoptotic cells: The percentage of apoptotic cells was markedly reduced in both treatment groups, with ATP-EVs resulting in a greater reduction in apoptosis than T-ATP-EVs. Necrotic cells: Minimal differences in necrosis percentages were observed across all groups, suggesting that ATP-EVs and T-ATP-EVs primarily reduce apoptosis without significantly affecting necrosis levels.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6422821/v1/6bab3dbb72864d7d7407cc4d.png"},{"id":82086109,"identity":"2cfb64b1-1e40-4228-8ae0-c139f2b129f7","added_by":"auto","created_at":"2025-05-06 15:10:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":273799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 2B.\u003c/strong\u003e Flow cytometry analysis revealed the effects of ATP-EVs and T-ATP-EVs on the viability, apoptosis, and necrosis of hypoxic cells.2Ba: The percentage of live cells was significantly greater in the ATP-EV (91.33±4.5%) and targeted ATP-EV (80.36±2.55%) groups than in the control group (42.8±2.76%), indicating enhanced cell survival. 2Bb: The percentage of apoptotic cells decreased significantly from 38.95±4.19% in the control group to 3.91±2.03% and 10.43±1.46% in the ATP-EV and T-ATP-EV groups, respectively, demonstrating the potent antiapoptotic effects of these treatments. 2Bc: Necrotic cell analysis revealed minimal differences across the groups, with percentages of 5.23±2.80%, 4.33±3.37%, and 3.53±1.7% for the control, ATP-EV, and T-ATP-EVgroups, respectively. 2Bd: Comparative results highlight the superior efficacy of ATP-EVs in improving cell survival and reducing apoptosis, while necrosis levels remained low and relatively unchanged across all groups.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6422821/v1/447e8c7ddc5dbf46e2543dd7.png"},{"id":82086105,"identity":"b31b872c-7cd8-4d39-ada0-183895b9f0cc","added_by":"auto","created_at":"2025-05-06 15:10:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":524259,"visible":true,"origin":"","legend":"\u003cp\u003eFig. 3. Comprehensive analysis of the left ventricular ejection fraction (LVEF) and cellular responses across different treatment groups. (\u003cstrong\u003eA\u003c/strong\u003e) Quantitative echocardiography analysis of the left ventricular ejection fraction (LVEF) across different groups: In the MI group (a), the LVEF in myocardial infarction (MI) cells was significantly reduced, reflecting impaired cardiac function caused by infarction. EV Group (b): Compared with the MI group, the group treated with non-targeted EVs (EVs) showed a partial improvement in LVEF, indicating some degree of therapeutic effect. ATP-EVGroup (c): Compared with those in the EV group, the LVEF of the ATP-loaded EV group recovered more markedly, highlighting the beneficial role of ATP in enhancing cardiac function. In T-ATP-EV group (d), compared with the treated groups, the T-ATP-EV group presented the greatest improvement in LVEF, underscoring the efficiency of targeted delivery in restoring cardiac function. Sham Group (e): Healthy (sham) cells maintained the highest LVEF values and served as the baseline for normal cardiac function. (\u003cstrong\u003eB\u003c/strong\u003e). A quantitative plot of LVEF for different treatment groups. The results demonstrated a significant improvement in LVEF with both ATP-EVs and T-ATP-EVs compared with the MI group. Additionally, a significant difference was observed between the effects of ATP-EVs and T-ATP-EVs, with T-ATP-EVs showing greater enhancement in LVEF. These findings highlight the therapeutic potential of EV-based treatments, particularly targeted delivery systems, in improving cardiac function post-MI.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6422821/v1/ddb3338c71ff7d3c0518cde6.png"},{"id":82087506,"identity":"8381bbe1-c5b9-4e46-9c33-dd3b9969ee94","added_by":"auto","created_at":"2025-05-06 15:26:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":411839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 4.\u003c/strong\u003e \u003cstrong\u003eEffect of Targeted ATP-EVs on Infarcted Size. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Effects of ATP-EVs and T-ATP-EVs on the infarctarea size following myocardial infarction (MI). Masson's trichrome staining was used to measure the percentage of collagen deposition in the infarcted area. The results indicated that the infarctarea size decreased from 42.33±1.15% in the MI group to 20.66±1.52% in the ATP-EV group. Additionally, treatment with T-ATP-EVs further reduced the infarct area to 14%. Cross-sectional images of heart sections stained with Masson's trichrome are shown in Fig. 4A, and a quantitative diagram of the infarct area size is presented in \u003cstrong\u003eB.\u003c/strong\u003e These results demonstrate that both the ATP-EVs and T- ATP-EVs groups had a significant reduction in infarct area size compared with the MI group. Moreover, the data revealed that compared with ATP-EVs alone, T-ATP-EVs significantly reduced the infarct area.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6422821/v1/e359e218336a3d6f0f2c65cd.png"},{"id":82086786,"identity":"c476784d-882a-48da-b116-cc712bdaa181","added_by":"auto","created_at":"2025-05-06 15:18:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":461205,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 5 \u003c/strong\u003e\u0026nbsp;Immunohistochemicalanalysis of the cardiomyocyte biomarkers troponin and α-actin under various treatment conditions. (A) Representative IHC images of troponin expression across different groups: MI (control), EVs, ATP-EVs, and T-ATP-EVs, with the highest expression in the shamgroup. Statistical analysis revealed significant improvements in troponin levels with ATP-EVs (P = 0.0005) and T-ATP-EVs (P = 0.0125) compared with those in the MI control group. (B) \u0026nbsp;Presents α-actin expression, indicating recovery trends with increasing levels in the treatment groups, notably with T-ATP-EVs. Statistical differences demonstrated enhanced efficacy, particularly between ATP-EVs and T-ATP-EVs, suggesting greater affinity and protective ability of T-ATP-EVs for cardiac tissues. (C) Summary of the quantitative results for both biomarkers, highlighting the potential therapeutic role of targeted exosomal treatments in myocardial infarction recovery.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6422821/v1/71d661e550b3362109547c55.png"},{"id":82088804,"identity":"09e99151-142d-4565-90f2-834736502bf4","added_by":"auto","created_at":"2025-05-06 15:42:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4474925,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6422821/v1/55da6ee1-60ae-46cc-9d46-b08ee8bd283f.pdf"},{"id":82086778,"identity":"fd11e26e-e385-4458-a215-0de0e28bfacd","added_by":"auto","created_at":"2025-05-06 15:18:04","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":547409,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e\n\u003cp\u003eThe graphical abstract illustrates the creation and function of ATP-loaded extracellular vesicles (T-ATP-EVs) engineered with anti-myosin antibodies for targeted delivery to damaged heart tissue. These findings reveal how these specialized EVs increase the ATP supply to ischemic cardiomyocytes, resulting in increased cell survival, reduced infarct size, and improved cardiac function in myocardial infarction models.\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6422821/v1/0d1867a1d3ae0cc1f72c69e5.png"}],"financialInterests":"","formattedTitle":"Engineered ATP-Loaded Extracellular Vesicles: A Dual-Functional Strategy for Improving Myocardial Infarction Therapy","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. Engineered ATP-loaded EVs specifically deliver energy to ischemic myocardial tissue, enhancing cardiac recovery.\u003c/p\u003e\u003cp\u003e2. ATP-loaded EVs increase cardiomyocyte viability by 46% and reduce apoptosis by 28% under hypoxic conditions.\u003c/p\u003e\u003cp\u003e3. Treatment with ATP-loaded EVs increased the left ventricular ejection fraction by 26% and decreased the infarct size by 28% in a rat model of myocardial infarction.\u003c/p\u003e\u003cp\u003e4. The use of anti-myosin antibody-tagged EVs for precise, noninvasive delivery offers a novel treatment approach for myocardial infarction.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eMyocardial infarction (MI) is the most common cause of mortality and morbidity worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The main cause of MI is often atherosclerosis, where the principal coronary artery that provides blood to the heart is suddenly obstructed [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Sedimentation of cholesterol in the membrane of arteries is the main cause of atherosclerosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Traditional MI treatments, including coronary artery bypass grafting (CABG), angioplasty, atherectomy, valve surgery, stenting, and transmyocardial revascularization (TMR), are effective at restoring blood flow and relieving symptoms but have significant limitations and risks. These methods are often invasive, require complex surgeries that carry the potential for complications, have extended recovery times, and, in some cases, do not address the underlying damage to heart tissue. Emerging treatments, such as stem cells, tissue engineering, 3D cardiac patches, and cell-free therapies, focus on tissue regeneration, making them sustainable solutions for MI patients [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite their potential, cell-based therapies face several limitations, including low engraftment efficiency, insufficient differentiation into mature cardiomyocytes, and inadequate anchorage within the myocardial tissue. The advantages of cell-free therapy include its nonaggressive, biocompatible, nonimmunogenic, and lack of need for cell engraftment or differentiation [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. An excellent example of cell-free therapy is the use of extracellular vesicles (EVs), particularly exosomes. Exosomes are nanosized vesicles ranging from 30 to 150 nm in diameter that are secreted by various cell types in the body. The method of isolation significantly influences the size distribution and properties of these exosomes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Exosome therapy is an alternative method that replaces traditional cell therapy. It offers the advantages of cell therapy while avoiding its associated adverse effects. They facilitate intercellular communication by transporting biomolecules such as proteins, lipids, and RNAs, influencing physiological and pathological processes in recipient cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. One of the critical challenges of systemic exosome delivery is the competition between therapeutic exosomes and endogenous exosomes in body fluids [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Exosomes derived from heart tissue, such as those derived from cardiosphere cells, exhibit cardioprotective effects by regulating key processes such as apoptosis, hypertrophy, angiogenesis, and cell proliferation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDuring MI, the level of ATP decreases to 80%; thus, ATP production via the respiratory pathway ceases, which results in the production of only a small amount of ATP through the anaerobic glycolysis pathway [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A reduction in the ATP concentration results in the production of lactic acid under anaerobic conditions and reduces the pH of cardiomyocytes. A reduction in pH causes Na\u003csup\u003e+\u003c/sup\u003e/H\u003csup\u003e+\u003c/sup\u003e exchange, increasing the concentration of Na\u003csup\u003e+\u003c/sup\u003e in the cells; subsequently, the Na\u003csup\u003e+\u003c/sup\u003e/Ca\u003csup\u003e2+\u003c/sup\u003e balance is disturbed, and the Ca\u003csup\u003e2+\u003c/sup\u003e concentration increases in the cells [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. An increasing Ca\u003csup\u003e2+\u003c/sup\u003e concentration in cells causes myocardial contracture and activates protease enzymes in mitochondria, which lead cardiomyocytes toward apoptosis or necrosis, depending on the time and severity of ischemia [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Administering ATP to damaged cardiomyocytes offers a straightforward approach to reduce ischemic injury. The solubility of ATP in blood is very high, and the half-life of ATP is very short. Therefore, the use of appropriate carriers is necessary to prevent ATP degradation and ensure efficient delivery of ATP to cells. EVs are nonimmune and noninvasive carriers for the delivery of ATP into cells. Additionally, ischemic preconditioning (IPC)-EVs constitute a well-recognized protective strategy that strengthens cellular resilience to subsequent ischemic events. This effect is achieved by modulating the composition of EVs released by various cells. These EVs, particularly those derived from endothelial cells following IPC, play a critical role in cardioprotection by transmitting survival signals to cardiomyocytes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, MSC-derived EVs under IPC reduce inflammation and apoptosis, enhancing cardiac function and survival in myocardial infarction models [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, EVs generated under IPC conditions in normal hearts have been shown to reduce infarct size and trigger prosurvival pathways in heart failure models. These findings highlight their significant potential as therapeutic agents for ischemic injury recovery. IPC appears to play a crucial role in enhancing EV cargo for cardioprotection, establishing a foundation for EV-based therapies for ischemic heart diseases [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Recently, inhalable stem cell-derived EVs were shown to promote heart repair after myocardial infarction [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA critical problem in the systemic delivery of EVs is the competition of therapeutic EVs with an enormous number of natural EVs in body fluids and a lack of targeting [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To increase the drug delivery and circulation time of EVs into the injured myocardium, since the membranes of cardiomyocytes are disrupted during MI and exposed to an intracellular matrix such as myosin, we used an anti-myosin antibody conjugated to the EVs. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In a previous study, a liposome targeted with an anti-myosin antibody was reported [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Additionally, iRGD-EVs were used to target tumors or engineer cell donor EVs with plasmid carriers to express cardiac-homing peptides on the membrane of EVs for targeted myocardial infarcted tissue [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we examined anti-myosin antibody-conjugated ATP-loaded EVs (T-ATP-EVs) as engineered EVs to target cardiomyocytes and heart tissue both in vitro and in vivo. We explored ATP delivery to ischemic cardiac tissue via EVs with increased cardioprotective potential through IPC. Direct ATP administration, although beneficial in theory, is limited by rapid dissolution in blood, reducing its efficacy. Moreover, the exposure of myosin in injured cardiac tissue during myocardial infarction provides a distinct opportunity for targeted therapeutic delivery. To overcome this challenge and capitalize on therapeutic opportunities, we developed a dual-functional strategy in which ATP-loaded EVs (ATP-EVs) are combined with T-ATP-EVs. This approach delivers ATP at suitable concentrations into cells and utilizes the cardioprotective properties of IPC-modulated EVs to enable precise targeting of the injured myocardium. This system improves cardiomyocyte survival and function, representing a significant advancement in therapeutic approaches for myocardial infarction.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIschaemic preconditioning and EV isolation\u003c/h2\u003e \u003cp\u003eH9c2 cells were obtained from ATCC and cultured in high-glucose (4.5 g/L) DMEM supplemented with 15% FBS. At 37\u0026deg;C, the cells were grown in a humidified atmosphere containing 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were seeded in a 10\u003csup\u003e6\u003c/sup\u003e cells/T 75 cell culture flask and passaged when confluent. The cells were subjected to hypoxic conditions upon reaching 80% confluency. After three washes with PBS, the medium was replaced with 15% EV-depleted FBS. A hypoxic chamber (\u0026lt;\u0026thinsp;0.1 mmHg, O₂ \u0026lt; 1%, CO₂ = 5%) was used to simulate hypoxic conditions in vitro for 30 minutes, followed by intermittent reoxygenation for 10 minutes in an anoxic chamber. This cycle was repeated three times to induce ischemic preconditioning. The cells were then returned to normal conditions for two days to produce EVs. Two days after ischemic preconditioning, the supernatant of the cell culture was collected for EV isolation via an EXOCIB kit. For EV isolation via the EXOCIB method, cell culture supernatants were processed according to the manufacturer\u0026rsquo;s protocol. Briefly, the supernatants were centrifuged at 3,000 \u0026times; g for 30 minutes to remove debris and dead cells. The conditioned medium was then filtered through a 0.22 \u0026micro;m filter to remove any cell particulates or apoptotic bodies. In a sterile vessel, the supernatant was transferred, EXOCIB EV precipitation solution was added, and the mixture was refrigerated overnight. Afterward, the mixture was centrifuged at 1,500 \u0026times; g for 30 minutes. Following aspiration of the supernatant, the tube was centrifuged again at 1,500 \u0026times; g for 5 minutes to remove any residual EXOCIB solution.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCharacterization of Ischemic Preconditioning-Derived EVs\u003c/h3\u003e\n\u003cp\u003e10 \u0026micro;l of 1 mg/ml isolated EVs were added to the cover and diluted in 1 ml of PBS. The size and polydispersity index (PI) of intact EVs and targeted EVs were determined via dynamic light scattering (Malvern, UK) with a 405 nm laser beam. In DLS, the paths of particles undergoing Brownian motion are tracked from the scattered light. The size and morphology of the isolated EVs (10 \u0026micro;L, 1 mg/ml) were observed via field emission electron microscopy. After they were allowed to air dry and dehydrated with a graded series of alcohol, they were fixed with 2% glutaraldehyde, and a gold coating was used for better contrast (JEM-2000EXTEM, Japan). The alamarBlue cell viability reagent is a resazurin-based solution that measures the reducing power of living cells to determine viability quantitatively. Resazurin is reduced to resorufin in viable cells, changing the blue dye to red. This color change and increased fluorescence can be detected by absorbance (detected at 570 and 600 nm) or fluorescence (excitation between 530\u0026ndash;560) and emission at 590 nm. The cells were cultured in 96-well plates at 5000 cells/well and 37\u0026deg;C. After treatment with different types of EVs, 10% v/v alamarBlue was added to the medium, and the mixture was incubated for 6\u0026ndash;8 h and then analysed with a plate reader.\u003c/p\u003e\n\u003ch3\u003eRat model of myocardial infarction\u003c/h3\u003e\n\u003cp\u003eMale Wistar rats (n\u0026thinsp;=\u0026thinsp;5 for each group, approximately 300 g in weight) were used to establish the MI model. The rats were given CO\u003csub\u003e2\u003c/sub\u003e for 2 min until they became dizzy and then given general anaesthesia with a mixture of 120 mg/kg body weight ketamine, 5 mg/kg midazolam (Dormicum, Roche) and 0.5 mg/kg medetomidine through an intraperitoneal injection. Surgical operation was performed under artificial ventilation with 100% oxygen-induced myocardial infarction. The heart was exposed via a left-sided limited thoracotomy. The left anterior descending artery (LAD) was ligated with a 5\u0026ndash;0 polyester suture 1 mm from the tip of the normally positioned left auricle. Through a 15 mm opening in the 4th intercostal space, a 10 cm length of 5\u0026ndash;0 silk suture was used to ligate the left coronary artery (LAD). After surgery, the rats were randomized into three groups (n\u0026thinsp;=\u0026thinsp;5): the PBS control group, the intact ATP-EV group and the T-ATP-EV group. All the groups were injected intravenously (200 \u0026micro;L of 1 mg/ml EVs or with only PBS as a control).\u003c/p\u003e\n\u003ch3\u003eThe impact of EVs on H9c2 cell proliferation and migration\u003c/h3\u003e\n\u003cp\u003eH9c2 cells were seeded at a density of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well in 96-well plates overnight and then treated with different concentrations of 0.1, 1, or 10 \u0026micro;g/ml EVs after the medium was removed for two days. The H9c2 cells were subsequently washed 2 times with PBS, fixed with 2% glutaraldehyde and then stained with 0.1 mM Dapi for 5 min, after which microscopy with a fluorescence microscope was performed at 40x magnification [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A monolayer of 10\u003csup\u003e5\u003c/sup\u003e H9c2 cells was grown in DMEM supplemented with 15% FBS for at least 48 hours at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e to allow for cell adhesion and migration. The confluent monolayer was then scored with a 100 \u0026micro;L pipette, and the medium was immediately exchanged with fresh medium containing 10 \u0026micro;g/mL EVs (free-EV FBS). To inhibit cell proliferation, the cells were treated with fresh serum-free culture medium containing 10 mg/ml mitomycin [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eATP loading and antibody tagging of EVs\u003c/h3\u003e\n\u003cp\u003eThe preparation of T-ATP-EVs involved some modifications to the ultrasound bath. The EVs were ultrasonicated with various concentrations of ATP at a protein concentration of 1 mg/ml (3 cycles). A 1 mM ATP solution was prepared by dissolving 552 \u0026micro;g of ATP in 1 mL of PBS (ATP molar weight of 552 g/L) as a stock solution, and then different concentrations of ATP (100, 10, 1 and 0.1 molar) were prepared with diluted stock solution in PBS. Subsequently, the EVs were washed two times with PBS and isolated on a 100 kDa MWCO ultrafiltration column (Millipore) at 5,000 \u0026times; g for 20 min to remove the unincorporated free ATP. All the experiments were carried out at 4\u0026deg;C. The EVs were tagged with an anti-myosin antibody (Abcam, USA), which targets the infarcted heart via myosin exposure during myocardial infarction. The antibody was conjugated to the EVs with some modifications. Using a 100-fold molar excess of antibody, DIPE-NHS (dioleoyl phosphatidyl ethanol amine N-hydroxyl succinimide) and the anti-myosin antibody were combined for 1 hour to produce the DOPE-antibody. The DOPE antibody was incubated with the EVs at a lipid:EV ratio of 1000:1 (1000 molecules of DOPE antigen for each EV). To remove excess reagents, an ultrafiltration column with a 100 kDa MWCO ultrafiltration column (Millipore) 5000 g was used for 20 min.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEchocardiography/cardiac function assessment\u003c/h2\u003e \u003cp\u003eThe rats were anaesthetized with 3% isoflurane and underwent transthoracic echocardiography on Day 0 (before the creation of the MI model), Day 3 (after MI model creation), and Day 30 (after MI model creation). The left ventricular ejection fraction (LVEF) and fractional shortening (FS) of the left ventricle were analysed. Rats with a normal EF after Day 3 were excluded from the following studies. The investigators were blinded to the identities of the rats and groups.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDetermination of Infarct Size Using Masson \u0026 Trichrome (M\u0026T) Staining\u003c/h3\u003e\n\u003cp\u003eAfter the rats were anaesthetized as described above (n\u0026thinsp;=\u0026thinsp;5 for each group) and sacrificed on day 30, the hearts were removed, rinsed with saline and soaked in 4% (v/v) paraformaldehyde until analysis. The specimens were prepared via transverse dissection across the infarct area. Heart section samples were rinsed with running water for 30 min, dehydrated in a graded ethanol series, cleared, embedded in paraffin, and then cut into sections with a thickness of 5 \u0026micro;m for Masson's Trichrome staining. This staining was used to quantify fibrosis in the left ventricle in various treatment groups. The heart sections were also subjected to immunohistochemistry (IHC) at a thickness of 10 \u0026micro;m.\u003c/p\u003e\n\u003ch3\u003eCalculation of the fibrotic area\u003c/h3\u003e\n\u003cp\u003eThe left ventricular (LV) fibrotic area induced by myocardial infarction after 30 days was calculated via the following equation: Specimens stained with Masson\u0026rsquo;s trichrome were used to quantify the fibrosis area in the left ventricle in different groups [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{I}\\text{n}\\text{f}\\text{a}\\text{r}\\text{c}\\text{t}\\:\\text{s}\\text{i}\\text{z}\\text{e}=\\frac{\\text{F}\\text{i}\\text{b}\\text{r}\\text{o}\\text{t}\\text{i}\\text{c}\\:\\text{a}\\text{r}\\text{e}\\text{a}\\:\\text{o}\\text{f}\\:\\text{L}\\text{V}}{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{L}\\text{V}\\:\\text{a}\\text{r}\\text{e}\\text{a}\\:(\\text{F}\\text{i}\\text{b}\\text{r}\\text{o}\\text{t}\\text{i}\\text{c}\\:\\text{a}\\text{r}\\text{e}\\text{a}\\:\\text{o}\\text{f}\\:\\text{L}\\text{V}+\\text{N}\\text{o}\\text{n}\\:\\text{f}\\text{i}\\text{b}\\text{r}\\text{o}\\text{t}\\text{i}\\text{c}\\:\\text{a}\\text{r}\\text{e}\\text{a}\\:\\text{o}\\text{f}\\:\\text{L}\\text{V})}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eFor immunohistochemistry (IHC), after the heart samples were prepared, the sections were fixed in 4% (v/v) paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.01% saponin, and blocked with protein blocking solution (DAKO) for 1 h. Each paraffin section stained for actin with primary antibodies (1:30) was incubated overnight at 4\u0026deg;C, and subsequently, secondary antibodies (1:100) were added at room temperature for 1.5 h, followed by a 30 min incubation with Power vision poly-HRP-conjugated anti-rabbit IgG. Images were captured with a Zeiss microscope. Data analysis was performed with the Allred score on the basis of the semiquantitative estimation of the percentage abundance of positive biomarkers in the cells and the staining intensity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eA direct flow cytometric method was used to investigate the effects of different intact EVs and targeted EVs on hypoxia-induced apoptosis and necrosis. Analysis with a marker (FL1H: Annexin, V FITC) and (FL3H: PI) was performed to identify live cells, early apoptotic bodies, late apoptotic bodies and necrotic cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted via GraphPad Prism 9. The data are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the means (SEMs). For comparisons between two groups, nonparametric t tests were utilized, whereas one-way ANOVA was employed for multiple group comparisons. A p value of less than 0.05 was considered statistically significant. Compared with the control treatment, the T-ATP-EV treatment significantly increased cell viability and reduced apoptosis in vitro. In vivo, both ATP-EVs and T-ATP-EVs significantly improved the left ventricular ejection fraction and reduced the infarct size compared with those of the control group, underscoring the efficacy of the targeted delivery system in ameliorating cardiac function postmyocardial infarction.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of EVs\u003c/h2\u003e \u003cp\u003eThis study aimed to use ATP-EVs as an efficient energy source for cardiomyocytes. Following cell culture and EV isolation, the EVs were conjugated with anti-myosin antibodies for targeting, as described earlier, and enriched with ATP via ultrasonic power (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The size distribution of EVs was analysed via dynamic light scattering (DLS), which revealed a uniform and homogeneous size distribution for both ATP-EVs and T-ATP-EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results revealed a significant size difference, with ATP-EVs averaging 81.43\u0026thinsp;\u0026plusmn;\u0026thinsp;4.27 nm and T-ATP-EVs averaging 126.66\u0026thinsp;\u0026plusmn;\u0026thinsp;4.04 nm. These findings are consistent with previous studies, which reported an antibody size of approximately 10\u0026ndash;15 nm, further validating the observed size increase upon antibody conjugation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Our findings agree with their reported antibody size of approximately 10\u0026ndash;15 nm[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and revealed a significant difference between ATP-EVs and T-ATP-EVs.\u003c/p\u003e \u003cp\u003eAdditionally, field emission scanning electron microscopy (FESEM) was employed to examine the morphology of the EVs. The images indicated that both ATP-EVs and T-ATP-EVs retained a spherical to oval shape, with antibody conjugation causing a size increase but not altering the overall morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). These observations align with previous studies reporting that EVs typically exhibit round to oval shapes, with size and morphology influenced by external modifications. Western blot analysis confirmed that the isolated vesicles were indeed EVs, ruling out contamination by other extracellular vesicle types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of EVs on Proliferation\u003c/h2\u003e \u003cp\u003eEVs contain factors for mitigation and proliferation, such as c-myc, cyclin and cytokines [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our results of nuclei stained with Dapi revealed that the number of nuclei/slides was 64\u0026thinsp;\u0026plusmn;\u0026thinsp;5 for the control group after treatment with different concentrations of EVs; this number increased to 92\u0026thinsp;\u0026plusmn;\u0026thinsp;6, 131\u0026thinsp;\u0026plusmn;\u0026thinsp;7, and 172\u0026thinsp;\u0026plusmn;\u0026thinsp;7 for the 0, 1 and 10 \u0026micro;g/ml EV groups, respectively. All the EV treatment groups were significantly different from the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). These results show that EVs increase the proliferation of myoblasts. These results are consistent with those of Li et al., who reported that cardiac progenitor cell-derived EVs promote H9c2 cell proliferation via Akt/mTOR activation. They reported that the effects of EVs on the proliferation of H9c2 cells are dose- and time dependent. In this study, after treatment with 200 \u0026micro;g/ml EVs, an optimal effect of EVs for 48 h was observed [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. A previous study revealed the effect of MSC-derived EVs on the proliferation of wound-healing cells via the expression of many genes involved in cell cycle control, such as C-myc, Cyclin A1, and Cyclin D2. In this study, cells treated with MSC-derived EVs expressed more than two times more genes than control cells did [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. miR-21-3p/5p isolated from cardiac telocytes and MSCs stimulates the proliferation of endothelial cells via the activation of ERK, Akt and HIF-1α [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This study revealed that EVs contain metabolites and microRNAs that can increase the viability and proliferation of cells and that the effects of EVs on recipient cells are mostly dose dependent. Some reports have also shown that EVs suppress proliferation. Wang et al. reported that EVs containing miR-155 suppress fibroblast proliferation and promote fibroblast inflammation during cardiac injury[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. This study revealed that the further suppression of fibroblast proliferation by miR-155 can indirectly inhibit fibrosis formation after MI. Similar studies have shown that the effects of EVs can be altered by their metabolite content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of EVs on migration\u003c/h2\u003e \u003cp\u003eFurthermore, the viability, proliferation, and migration of myoblasts are important for heart therapy, although myoblast cells have less potential for migration, and mostly endothelial cells, such as HUVECs, are considered for migration and tube formation; however, in this study, we used a scratch test on myoblasts to investigate the potential of EVs. Our results revealed that the scratch size for the control group was 0.65\u0026thinsp;+\u0026thinsp;0.1 mm. On Day 1, this size decreased to 0.6\u0026thinsp;+\u0026thinsp;0.1 mm without EV treatment after treatment with 10 \u0026micro;g/ml EVs, and this size decreased to 0.55\u0026thinsp;+\u0026thinsp;0.08 mm. On Day 2, this size decreased to 0.46\u0026thinsp;+\u0026thinsp;0.07 mm without EV treatment, and after treatment with 10 \u0026micro;g/ml EVs, this size decreased to 0.28\u0026thinsp;+\u0026thinsp;0.07 mm. The results revealed that only the EV-treated group after Day 2 was significantly different from the other groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. MSC-derived EVs promote HUVEC migration and tube formation by upregulating VEGFR-2 and Ang-1[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Another study revealed that carbonic anhydrase 9-enriched EVs from renal cell carcinoma (RCC) cells promote HUVEC migration and tube formation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, MSC-derived EVs enhanced the migration of normal and diabetic wound fibroblasts. Compared with depleted conditioned medium, MSC-derived EVs significantly increased migration by more than 30% [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In bone marrow mesenchymal stem cell-derived EVs, the PI3K/Akt/eNOS signalling pathway is activated by miR-126, which promotes EC survival, proliferation, and migration in ECs that are subjected to I/R injury [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffects of T-ATP-EVs on Hypoxic Cell Viability and Apoptosis\u003c/h2\u003e \u003cp\u003eAfter the cells reached 80% confluency, we used the alamarBlue assay to investigate the effects of ATP-EVs and T-ATP-EVs on the viability of hypoxic cells. The results of this analysis revealed that the viability of hypoxic cells in the control group was 33.91\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2%. This percentage increased to 93.97% after treatment with ATP-EVs and to 79.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9% after treatment with T-ATP-EVs \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. To confirm the effects of ATP-EVs and T-ATP-EVs on cell viability, we used flow cytometry analysis (annexin-PI). Representative images of flow cytometry data from different EV treatments are shown in \u003cb\u003eFig.\u0026nbsp;2Ba\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThe results of the flow cytometry analysis of live cells revealed that after hypoxia, the percentage of live cells was 42.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.76%. This percentage increased to 91.33\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5% after treatment with ATP-EVs and to 80.36\u0026thinsp;\u0026plusmn;\u0026thinsp;2.55% after treatment with T-ATP-EVs \u003cb\u003e(Fig.\u0026nbsp;2Bb)\u003c/b\u003e. The results of the flow cytometry analysis, which measured the degree of apoptosis induced by hypoxic conditions, revealed that hypoxia caused 38.95\u0026thinsp;\u0026plusmn;\u0026thinsp;4.19% of the total number of apoptotic cells in the control group. This percentage decreased to 3.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03% after the application of ATP-EVs and to 10.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46% after the administration of T-ATP-EVs. These results revealed the strong effects of both ATP-EVs and T-ATP-EVs in preventing the initiation of the apoptotic cascade \u003cb\u003e(Fig.\u0026nbsp;2Bc).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe results of the flow cytometry analysis revealed that hypoxia caused 5.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.80% necrosis in the control group. After treatment with ATP-EVs, this percentage decreased to 4.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.37%, and after applying T-ATP-EVs, it reached 3.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7% \u003cb\u003e(Fig.\u0026nbsp;2Bd).\u003c/b\u003e These data revealed a significant difference between ATP-EVs and T-ATP-EVs compared with the control group in terms of increased viability and decreased apoptosis induced by hypoxia. However, there was no significant difference in the percentage of necrosis among the treatment groups.\u003c/p\u003e \u003cp\u003eThe results of this study revealed that ATP-EVs increased the survival rate of hypoxic cells by 60%, whereas T-ATP-EVs increased the survival rate by 46%. This was mainly due to 1) the reduced effect of the antibody under in vitro conditions and 2) the negative effect of the antibody on cellular metabolism. In a previous report, Nejatollahi et al. noted that antibodies bound to anti-RTFscFv decreased the viability of prostate cancer cells[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], similar to the results of our flow cytometry analysis. These results revealed that the percentage of viable cells increased by 49% after treatment with ATP-EVs, whereas it increased by 38% after treatment with T-ATP-EVs. Additionally, the flow cytometry results revealed that ATP-EVs reduced apoptosis caused by hypoxia by 35% and T-ATP-EVs promoted apoptosis by 28%.\u003c/p\u003e \u003cp\u003eIn other words, although the anti-myosin antibody increases the targeting of damaged heart tissue postinfarction, cell tests in an in vitro environment revealed that antibody attachment to EVs slightly decreases cell viability. This study revealed that both types of EVs increased cell survival and metabolism. These findings are consistent with other researchers' results on the effects of EVs on enhancing cell survival and metabolism. EVs contain various miRNAs, metabolites, proteins, and growth factors, such as SDF1, IGF1, NGF, and HGF30, which increase the survival, metabolism, proliferation, and growth of target cells [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have shown that EVs significantly impact cell survival under different conditions. EVs have been reported to reduce ROS levels in cells treated with H2O2 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. They also demonstrated that EVs derived from cardiomyocytes produce many miRNAs; the four miRNAs specific to or highly expressed in heart tissue are miR-133a/b, miR-208a (with antifibrotic properties), miR-499 (with antiapoptotic properties), and miR-1 (antioxidants). These miRNAs are involved in regulating cardiogenesis and cardiac functions such as contractility and message conduction. Additionally, miR-499 and miR-208 are involved in the expression of cardiac sarcomere genes such as miR-133a and miR-1. Moreover, EVs derived from mesenchymal stem cells contain the transcription factor CXCR4, which increases VEGF expression and blood vessel formation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Previous reports indicate that ischemic preconditioning is a powerful approach for increasing the survival and regeneration of cells under ischemic conditions [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The creation of ischemic conditions for EV production increases the resistance of the heart to hypoxia [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that provides various physiological responses to ischaemic conditions[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. EVs isolated from endothelial cells express more HIF-1, miR-210, and miR-126, which play crucial roles in heart regeneration [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. miR-210 is known as a regulator of HIF-1, increasing its expression. HIF-1 shifts the metabolism of cardiac progenitor cells to a glycolytic state, reducing oxygen usage during heart infarction[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Compared with those in normal cells, the expression levels and numbers of miRNAs such as miR-210, miR-199a-3p, miR-24, miR-22, and miR-21 in EVs secreted from cells under IPC conditions increased. This increase in miRNAs enhances the potential of damaged cells against hypoxia and protects heart tissue. miR-26 and miR-223 also play essential roles in cardiac tissue regeneration, and their amounts in EVs are greater under IPC conditions than under normal conditions. In an ischemia‒reperfusion injury model in rats, treatment with EVs prepared from hypoxic cells improved heart function and prevented fibrosis development[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Cardiac endothelial cells under hypoxic conditions increase the expression of miR-210 and miR-126, both of which have proangiogenic properties[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. These studies indicate that EVs contain metabolites and compounds that increase cell metabolism and survival.\u003c/p\u003e \u003cp\u003eMoreover, reports have shown that EVs produced under IPC conditions contain greater amounts of miRNAs and metabolites, providing greater protection than those produced under normal conditions. These features increase the resistance of IPC-condition-produced EVs, safeguarding cells from hypoxia and infarction-induced apoptosis and necrosis. If the energy demand of a cell persists during hypoxia, cardiomyocytes may undergo apoptosis. Reports have shown that the intracellular ATP concentration ranges from 1\u0026ndash;10 mM depending on the cell type[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The ATP concentration outside cells is approximately \u0026micro;M, and in distant areas such as blood, it is approximately nM. Since the extracellular ATP concentration is much lower than the intracellular ATP concentration, cells cannot meet their energy needs externally[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious reports have shown that ATP has a very short half-life when injected intravenously because it quickly dissolves in the blood, preventing it from reaching target tissue at appropriate concentrations[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. These limitations make the use of ATP as a therapeutic agent impractical without a suitable carrier. Additionally, high ATP concentrations can be toxic. The mechanisms of ATP toxicity include metabolic and oxidative stress. Increased ATP levels can increase the intracellular calcium concentration and disrupt the mitochondrial membrane potential, leading to apoptosis or necrosis. Previous reports have shown that elevated intracellular calcium disturbs muscle cell contractile activities and rhythm, affecting heart pacemaker cells. Therefore, a suitable carrier is necessary to transport ATP effectively to the MI area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffects of targeted ATP-EVs on heart function\u003c/h2\u003e \u003cp\u003eTo investigate the effects of ATP-EVs and T-ATP-EVs on the left ventricular ejection fraction (LVEF), we used echocardiography analysis. The results revealed that the LVEF decreased from 86\u0026thinsp;\u0026plusmn;\u0026thinsp;2% in the sham group to 54.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08% in the MI group. After ATP-EV treatment, the LVEF increased to 73.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52%, and applying T-ATP-EVs increased the LVEF to 80\u0026thinsp;\u0026plusmn;\u0026thinsp;1%. Schematic images of the echocardiography results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, and a quantitative plot of the LVEF for different treatment groups is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. These results demonstrated a significant improvement in LVEF with both ATP-EVs and T-ATP-EVs after MI and indicated a significant difference between the effects of ATP-EVs and T-ATP-EVs.\u003c/p\u003e \u003cp\u003eAfter myocardial infarction, cardiomyocytes experience a severe decrease in energy due to disruption of the aerobic metabolic pathway caused by a lack of oxygen, leading to reliance on the anaerobic pathway. ATP is a crucial molecule for the cell. The transfer rate of ATP across the membrane is very low because of its high anionic charge, so ATP is usually transported by carriers in the membrane[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Recent studies have reported the therapeutic applications of ATP. In one study, a group treated with liposomes containing ATP exhibited improved wound healing compared with a group treated with only liposomes without ATP[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. There are also several reports on the use of ATP-containing liposomes for treating ischaemia‒reperfusion models, which show that ATP-containing liposomes improve heart function compared with that of the control group[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Another study demonstrated that ATP-containing liposomes were used in cerebral ischemia, improving brain tolerance to ischemia-related injuries. In another report, ATP-containing liposomes were used to treat liver failure and were shown to exert a protective effect on the liver[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The ATP concentration is very precisely controlled in the cell because ATP plays a key role as an energy source for many intracellular activities.\u003c/p\u003e \u003cp\u003eOne of the main challenges in the therapeutic use of EVs is the competition between natural EVs in the serum and therapeutic EVs[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Previous studies have shown that the concentration of serum EVs is approximately 10\u0026sup1;\u0026sup1; EVs per milliliter, whereas the concentration of therapeutic EVs is less than 10⁹ EVs per milliliter. In many studies, EVs are targeted to specific tissues via various methods, such as platelet membranes[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] or homing peptides[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. H9c2-derived EVs were used in this research because myoblast cells are closer precursors to cardiomyocytes, making them similar to cardiomyocytes in terms of origin and differentiation, and their EVs likely have a similar membrane structure to cardiomyocytes. These EVs can outperform other types of natural EVs in the serum and blood in terms of their ability to target cardiomyocytes[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These findings suggest that myoblasts may be good candidates as sources of EVs for cardiac treatment[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of this research revealed that the intravenous use of both ATP-EVs and T-ATP-EVs increased cardiac output after MI and improved overall heart function. In other words, both types of EVs target the heart: ATP-EVs passively and T-ATP-EVs actively targeted the damaged heart tissue. This study revealed that ATP-EVs increased the cardiac LVEF by 19%, whereas targeted T-ATP-EVs increased it by an additional 7%, increasing the total LVEF to 26%. Previous studies have shown that EVs effectively reduce the size of the infarct area[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. This study also revealed that ATP-EVs reduced the size of the infarct area by 22%, and T-ATP-EVs further reduced it by another 6%, resulting in a reduction of 28%. Zhuo Wan et al. reported that the use of EVs increased the LVEF by 10% after MI[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Kai Kang et al. reported that EVs secreted from CXCR4-overexpressing mesenchymal stem cells increased the LVEF by 25%, from 33\u0026ndash;58%[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Another study by Chunxiao Wang et al. reported that the use of EVs containing miR-155 increased the LVEF from 25\u0026ndash;50% after MI[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eEffect of T-ATP-EVs on infarct size\u003c/h2\u003e \u003cp\u003eTo investigate the effects of ATP-EVs and T-ATP-EVs on the size of the infarct area after MI, we used Masson's trichrome staining to calculate the percentage of collagen deposition in the infarct area. The results revealed that the size of the infarcted area decreased from 42.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15% in the MI group to 20.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52% in the ATP-EV group. Additionally, treatment with T-ATP-EVs reduced the infarct area to 14%. Cross-sectional images of heart sections stained with Masson's trichrome are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, and a quantitative diagram of the infarct area size via ImageJ software is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. These results indicate that, compared with those in the MI group, the infarcted area in both the ATP-EV and T-ATP-EV groups was significantly smaller. Moreover, the data revealed that compared with ATP-EVs, T-ATP-EVs significantly reduced the infarct area.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that EVs reduce the infarct area induced by MI [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Shiqi Hu et al. combined platelet membranes with EVs (P-XOs) to target injured hearts, and their data revealed that, compared with MI alone, P-XOs decreased the infarcted area by 19%, from 27\u0026ndash;18%, whereas XOs alone decreased the infarcted area by approximately 10%[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Kai Kang et al. reported that EVs secreted from CXCR4-overexpressing mesenchymal stem cells increased vWF\u0026thinsp;+\u0026thinsp;angiogenesis markers more than sixfold compared with those in the MI group and decreased the infarct area by 28%, from 50\u0026ndash;22%, after MI[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Another study by Yuliang Feng et al. reported that the infarct area decreased from 40\u0026ndash;15% when EVs enriched with miR-22 were used[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, Daya D. Verma et al. reported that treating an MI heart with ATP-loaded liposomes decreased the infarcted area by 40%, from 70\u0026ndash;30%[\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eEffect of T-ATP-EVs on troponin and α-actin\u003c/h2\u003e \u003cp\u003eTo investigate the effects of ATP-EVs and T-ATP-EVs on maintaining heart structure, we used immunohistochemistry analysis of the cardiomyocyte biomarkers troponin and α-actin[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Both troponin and α-actin are key specific biomarkers of cardiomyocytes. Representative images of IHC troponin biomarkers are shown in \u003cb\u003eFig.\u0026nbsp;7A, 7B\u003c/b\u003e, and representative images of IHC α-actin biomarkers are shown in \u003cb\u003eFig.\u0026nbsp;7C\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;7A\u003c/b\u003e shows the relative expression levels of troponin across various treatment groups. In the MI (control) group, troponin expression was notably low. The application of EVs results in a moderate increase in expression, whereas ATP-EVs significantly increase expression. Notably, treatment with T-ATP-EVs further increased troponin levels, approaching those observed in the sham group, which presented the highest expression. Statistical analysis confirmed significant differences, with ATP-EVs (P\u0026thinsp;=\u0026thinsp;0.0005) and T-ATP-EVs (P\u0026thinsp;=\u0026thinsp;0.0125) showing superior efficacy compared with the MI control. These findings underscore the potential effectiveness of targeted EV therapies in improving cardiac biochemical markers postmyocardial infarction. This study revealed significant variations in α-actin expression levels among the different treatment groups. In the sham group, the α-actin level was 4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44, which decreased to 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 in the MI control group. Following treatment, the level increased to 3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54 with ATP-EVs and further increased to 4.1 with T-ATP-EVs, indicating a recovery trend. Additionally, the differences in baseline expression were statistically significant, with ATP-XOs yielding a P value of 0.0024 compared with the MI control and sham treatments showing P values of 0.0010 and 0.0079 compared with the MI control and ATP-XOs, respectively. These findings suggest that both ATP-EVs and T-ATP-EVs effectively increase α-actin expression, underscoring their potential as therapeutic interventions for MI (\u003cb\u003eFig.\u0026nbsp;7B\u003c/b\u003e). The quantitative results for troponin and α-actin are shown in \u003cb\u003eFig.\u0026nbsp;7C\u003c/b\u003e. These IHC results indicated a significant difference between the ATP-EV and T-ATP-EV groups and the MI group. Additionally, there was a significant difference between ATP-EVs and T-ATP-EVs, suggesting that T-ATP-EVs have greater affinity for heart tissues and better prevent the destruction of cardiomyocytes.\u003c/p\u003e \u003cp\u003eJia Huang et al. reported that EVs derived from cancer cells improve the viability of target cells by delivering alpha-smooth muscle actin[\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Shiqi Hu et al. combined platelet membranes with EVs (P-XOs) to target the injured heart; their data revealed that P-XOs accumulated 1.8 times more than XOs did, and the expression of α-sarcomeric actin was greater after treatment with P-XOs[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Kyle I. Mentkowski et al. reported that EVs fused to a cardiomyocyte-specific peptide (CMP) accumulate twice as much in heart tissues than do bare EVs after intravenous injection[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Another study by Adam Vandergriff et al. compared two types of peptides, a cardiac-homing peptide (CHP) and a scramble peptide (Scr), for targeting the infarcted heart after conjugation with EVs. This study revealed that EF changes for the CHP peptide after 21 days were three times greater than those for the Scr peptide, and the infarct area decreased to 5% for CHP-EVs, whereas it decreased to 10% for Scr-EVs, indicating that CHP-EVs better targeted heart tissues after MI[\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Robert C. Scott et al. reported that treating an MI heart with targeted VEGF-loaded liposomes increased vessel density by two-fold compared with that in the MI group[\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCardiovascular diseases, with myocardial infarction (MI) as a leading cause, remain a major global health challenge because of the high morbidity and mortality associated with cardiac tissue damage. This study introduces a novel dual-functional therapeutic approach that leverages ischemic preconditioning (IPC)-derived EVs loaded with ATP conjugated with anti-myosin antibodies (T-ATP-EVs) to address the limitations of conventional and emerging MI treatments. By combining the natural cardioprotective properties of EVs with targeted delivery, this method aims to optimize energy replenishment in hypoxic cardiomyocytes and enhance cardiac repair mechanisms.\u003c/p\u003e \u003cp\u003eThe findings of this research highlight the significant efficacy of T-ATP-EVs in improving both cellular and cardiac outcomes. In vitro, these EVs increased cell viability by 46% and reduced apoptosis by 28% compared with those in the control groups, demonstrating their ability to preserve cardiomyocyte health under hypoxic conditions. In vivo results further supported these benefits, with a 26% improvement in the left ventricular ejection fraction (LVEF) and a 28% reduction in infarct size. Additionally, the T-ATP-EVs effectively restored structural integrity markers, including troponin and α-actin, with troponin preservation reaching 78% and α-actin levels increasing to 85% of baseline values, highlighting their role in maintaining the functional and structural framework of cardiac tissue.\u003c/p\u003e \u003cp\u003eThis study also addressed key challenges associated with ATP delivery and systemic EV use. The rapid degradation of ATP and competition with natural serum EVs have historically limited therapeutic effectiveness. By incorporating anti-myosin antibodies, targeted EVs achieve selective accumulation in infarcted myocardial regions, increasing delivery precision and minimizing off-target effects. This targeted delivery mechanism, coupled with the inherent resilience of IPC-derived EVs, represents a promising strategy for overcoming these barriers.\u003c/p\u003e \u003cp\u003eThe implications of this research extend beyond myocardial infarction therapy. The dual-function approach presented here has the potential for adaptation to other ischemic conditions, where targeted energy delivery and cellular protection are critical. However, translating these findings into clinical applications requires further investigation, including studies in larger animal models, evaluations of long-term safety and efficacy, and potential modifications to scale up production for human use.\u003c/p\u003e \u003cp\u003eIn conclusion, T-ATP-EVs represent an innovative and effective therapeutic strategy for enhancing cardiac repair post-MI. By addressing the critical limitations of existing treatments and introducing targeted, cell-free therapies, this approach provides a solid foundation for future advancements in cardiovascular medicine, with the ultimate goal of improving patient outcomes and quality of life.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eAuthorship contribution\u003c/h2\u003e \u003cp\u003eFarshid Jaberi Ansari: Investigation, Methodology, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing; Javad Behroozi: Data curation, Formal analysis, Methodology, Validation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing; Mohsen Chamanara: Methodology, Validation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing; Mostafa Shahrezaee: Methodology, Validation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing; Ali Shakermoghaddam: Methodology, Validation, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing; Seyed Hossein Mousavi: Conceptualization, Data curation, Formal analysis, Methodology, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing; Hossein Ahmadi Tafti: original draft, Writing - review \u0026amp; editing; Mohammad Ali shokrgozar: Methodology, Writing \u0026ndash; original draft; Amir Amanzadeh: Methodology, Writing \u0026ndash; original draft; Mahdi Ghorbani: Methodology, Validation, Writing \u0026ndash; original draft; David W. Greening: Supervision, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing; Reza Heidari: Conceptualization, Methodology, Project administration, Supervision, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003e Approval for animal ethics was conducted according to guidelines provided by the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Aja University of Medical Sciences (IR.AJAUMS.REC.1401.167).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no known financial or personal conflicts of interest that could have influenced the work reported in this paper.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable since study did not include humans.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThere is no funding for this article.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThe authors would like to express their gratitude to the Central Research Laboratory, Aja University of Medical Sciences, for providing the necessary resources and support for this research.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets generated during the current study are available from corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRoth GA, et al. 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FASEB J. 2009;23(10):3361\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Myocardial Infarction, Exosome, Engineered Extracellular Vehicles, Targeted Delivery","lastPublishedDoi":"10.21203/rs.3.rs-6422821/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6422821/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMyocardial infarction (MI) is a major component of cardiovascular disease. The primary challenge in MI therapy is the lack of energy in the affected tissue. Extracellular vesicles (EVs) areengineered with adenosine triphosphate (ATP) and targeted with an anti-myosin antibody (T-EV) to effectively deliver energy to damaged myocardial tissue and mitigate the effects of myocardial infarction (MI). Furthermore, using an in vivo MI rat model to evaluate cardiac repair, we compared cardiac function, infarct size, and the expression of troponin and α-actin four weeks after MI with those in a healthy group. The results indicate that, compared with no treatment, the use of Target-ATP-EVs (T-ATP-EVs) enhances the viability of hypoxic cells by 46% and reduces apoptosis by 28%. In the animal study, there was a 26% increase in the left ventricular ejection fraction (LVEF) and a 28% decrease in the infarct size compared with those of the MI group when this treatment was applied. Additionally, the expression levels of troponin and α-actin increased approximately two-fold in vivo with the use of these engineered EVs. In this study, engineered EVs were investigated as a strategy to deliver ATP directly to cardiomyocytes and heart tissue in both in vitro and in vivo models. The system described here enhances cardiomyocyte survival and function, marking a significant advancement in the treatment of myocardial infarction.\u003c/p\u003e","manuscriptTitle":"Engineered ATP-Loaded Extracellular Vesicles: A Dual-Functional Strategy for Improving Myocardial Infarction Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 15:09:59","doi":"10.21203/rs.3.rs-6422821/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2025-09-29T02:58:53+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-05-06T14:35:41+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-01T15:09:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-11T07:25:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Drug Delivery and Translational Research","date":"2025-04-10T15:32:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"drug-delivery-and-translational-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ddtr","sideBox":"Learn more about [Drug Delivery and Translational Research](https://www.springer.com/journal/13346)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ddtr/default.aspx","title":"Drug Delivery and Translational Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c6789508-6917-41ae-bf87-37cc547d6622","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T09:13:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 15:09:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6422821","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6422821","identity":"rs-6422821","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}