A Novel Ultrasound‐Responsive Biomimetic Nanoparticle for Targeted Delivery and Controlled Release of Nitric Oxide to Attenuate Myocardial Ischemia Reperfusion Injury

Targeted and controlled nitric oxide (NO) release is critical due to an extremely short half life and low bioavailability for treating cardiovascular diseases. To address this challenge, various sustained‐release precursors and NO donors activated by light, enzyme, or pH are developed. However, their efficacy is limited by the deep location and rapid blood flow of the heart. Herein, a platelet membrane‐coated nanoparticle (B‐P@PLT) is designed with a polymeric core loaded with BNN6, an ultrasound‐responsive NO donor, for the targeted treatment of myocardial ischemia reperfusion injury (MIRI). B‐P@PLT can specifically target the ischemic myocardium and release NO during ultrasound (US) irradiation, thereby increasing the local concentration of NO. B‐P@PLT + US shows promising results in promoting angiogenesis, reducing reactive oxygen species production, protecting cardiomyocytes both in vitro and in vivo, and ultimately decreasing cardiac injury and improving heart function in MIRI mice. These findings demonstrate a simple and noninvasive strategy for targeted delivery and controlled release of NO, highlighting its potential therapeutic application in MIRI.

challenges due to its short half life, limited diffusion distances, high reactivity with oxygen species, and concentration-dependent therapeutic outcomes. [11] Although significant progress has been achieved by designing NO sustained-release precursors or NO donors stimulated by light, enzyme, and pH changes, [12] some issues still need to be addressed, such as poor penetration, insufficient controllability, and inadequate targeting. Particularly for MIRI, where rapid blood flow in the heart is a major concern, efficient targeting and controlled local release of NO are critical for successful treatment. [13] Cell membrane biomimetic nanotechnology has been extensively used in drug delivery systems over the last decades. This approach involves camouflaging synthetic nanoparticles with natural cell membranes, which confers advantages such as prolonged circulation time in vivo, high efficiency of specific targeting, and reduction in clearance by the immune system. [14] Different types of cell membranes have been exploited to develop novel biomimetic nanoparticles for imaging and therapy, including red blood cells, white blood cells, platelets, and cancer cells. [15] These biomimetic nanoparticles not only retain the physicochemical features of the synthetic vehicles but also inherit the intrinsic functionalities of the original cell membranes, which have also emerged with promising results in drug delivery and molecular imaging in cardiovascular disease. [16,17] During the early stages of myocardial ischemia reperfusion injury (MIRI), platelets heavily accumulate in the injured myocardial area. [18][19][20] Platelets play a crucial role in the process of myocardial damage in MIRI by mediating acute microvessel closure and causing inflammation in the ischemic/reperfused myocardium. [21] They can secrete or expose adhesion proteins such as fibrinogen, fibronectin, von Willebrand factor, thrombospondin, and P-selectin, which are involved in cell-cell interactions during inflammation. [22,23] Additionally, our previous study found that ultrasound imaging with platelet membrane-coated PLGA microbubbles can detect early MIRI noninvasively. [24] Based on the ability of targeting and immune escape of platelets, mimicking the inherent adhesive function of platelets can be a powerful approach for targeting the injured endothelium of MIRI.
In addition, N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine (BNN6) has been identified as an ultrasound-sensitive NO donor. [25,26] The release of NO radicals from BNN6 can be induced by US irradiation, because of sonoluminescence and the generation of high temperature and pressure under the US. [27] Thus, BNN6 can be used as a precursor drug to achieve the artificial and controlled release of NO. This novel approach offers a promising strategy for precisely regulating NO release in the treatment of MIRI, which is crucial for achieving optimal therapeutic outcomes.
Based on those concepts, we designed a platelet membranecoated nanoparticle that has a PLGA core loaded with BNN6 (B-P@PLT), which allows for the controlled release of NO in response to US irradiation. PLGA has been widely used in drug delivery due to its ability to protect drugs from rapid degradation while allowing for sustained release. Moreover, PLGA has received regulatory approval by the US Food and Drug Administration for clinical applications. [28] The platelet membrane coating of B-P@PLT serves to enhance targeting to the damaged endothelium of MIRI while also avoiding clearance by the monocyte-macrophage system. The amount of NO released from B-P@PLT in the MIRI region can be controlled by varying the US parameters and irradiation time.

Synthesis and Characterization of B-P@PLT
The construction of B-P@PLT was briefly described as follows. The synthesized BNN6 was a beige powder ( Figure S1A, Supporting Information) with light yellow color when dissolved in DMSO ( Figure S1B, Supporting Information), which turned red after ultrasonic irradiation ( Figure S1C, Supporting Information) due to the product after the release of NO. The chemical formula of the synthetic method of BNN6 and its ultrasonic decomposition to produce NO are shown in Figure S1D,E, Supporting Information. In theory, one BNN6 molecule could release two NO molecules under ultrasonic irradiation. We obtained the 1 H-NMR spectrum of the raw compound BPA ( Figure S2A, Supporting Information) and the product BNN6 ( Figure S2B, Supporting Information) and the peaks were found to be consistent with the published 1 HNMR spectra. [29] Mass spectrometry indicated a molecular weight of 279 Da, identical to the theoretical molecular weight of BNN6 calculated from the chemical formula ( Figure S3, Supporting Information). These results demonstrated the successful synthesis of BNN6. The emulsion strategy has been widely applied to produce nanoparticles to load hydrophobic drugs or other molecules with a high loading efficiency. [30] B-P was prepared using a single emulsion solvent evaporation method. The B-P was coated by platelet membrane through a sonication procedure, and then B-P@PLT was acquired ( Figure 1A). The loading efficiency of BNN6 in B-P was 15.6 %. The entrapment efficiency of BNN6 was 92.2%, suggesting that most of the added BNN6 was effectively encapsulated into the B-P. It can be found that the BNN6 loading capacity of B-P@PLT was higher compared with other BNN6 carriers. [26,31,32] Such a high BNN6 loading capacity will be greatly favorable for reducing the administration dose and enhancing the efficacy of NO. The appearance of B-P after lyophilization was white powder ( Figure S4C, Supporting Information) and the scanning electron microscope (SEM) results showed it was spherical nanoparticles with round, smooth, equal shapes ( Figure S4A,B, Supporting Information). B-P@PLT appeared as a core-shell structure under transmission electron microscope (TEM), indicating that the platelet membrane coated on the B-P successfully ( Figure 2A). Dynamic light scattering (DLS) analysis indicated that the B-P@PLT had an average diameter of about 206.6 AE 8.1 nm and a narrow hydrated particle size distribution (PDI = 0.266), basically consistent with the results of TEM and SEM ( Figure 2B). The average diameter of PLGA and B-P was 205.4 AE 6.6 and 202.6 AE 6.5 nm, respectively ( Figure 2C). The zeta potentials of PLGA, B-P, B-P@PLT, and PLT were À19.5 AE 1.1, À17.0 AE 0.1, À10.4 AE 0.6, and À10.7 AE 0.3 mV, respectively ( Figure 2D). After cloaking with platelet membrane, the size of B-P@PLT did not change significantly compared to the bare B-P or PLGA, while the zeta potential of B-P@PLT increased by about 8 mV, approaching that of PLT. This phenomenon is consistent with the previous analyses of nanoparticles after platelet membrane coating, which can be ascribed to the veiling of the highly negative PLGA core with the less negatively charged platelet membrane, [33] further confirming the successful platelet membrane coating onto B-P@PLT. To determine the stability in solution over time, B-P and B-P@PLT were stored in water at room temperature and they exhibited stable size over 3 days ( Figure S4D, Supporting Information). The Fouriertransform infrared (FTIR) data obtained in this study provided further evidence that BNN6 was successfully loaded into B-P. Specifically, the characteristic peaks at 1021 cm À1 (C-N) and 1511 cm À1 (-N-N═O) exhibited a noticeable increase following BNN6 loading, while these characteristic peaks did not appear in the PLGA spectra ( Figure 2E). The protein compositions of platelet (PLT), platelet membranes (PM), B-P, and B-P@PLT were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( Figure 2F). As expected, the protein profiles of B-P@PLT were similar to that of the PLT and PM. PM did not lose much protein during extraction from PLT. Western blot analysis further revealed that the B-P@PLT contained the same protein as that of the platelet membrane, including integrin α2, CD41, CD47, and CD42c, which are important for platelet adhesion ( Figure 2G). CD47 has been implicated as a marker of self-recognized protein and provides a "do-not-eat-me" signal. [34] B-P@PLT could effectively shield with platelet membrane and prevent from being engulfed by the reticuloendothelial system. Taken together, B-P@PLT was coated with the platelet membrane successfully, and the B-P@PLT inherited the characteristic proteins of platelets.

US-Triggered NO Release from B-P@PLT
The US parameters for excitation NO release from BNN6, B-P, and B-P@PLT were evaluated in water. The concentration of released NO was quantitatively monitored by a commercial Griess assay kit. The standard curve for NO detection is shown in Figure S5A, Supporting Information. In Figure S5B,  Supporting Information, BNN6 was stable in water without US irradiation. Under a weak US irradiation (0.5 W cm À2 ), NO was released from BNN6 and increased significantly with the increase of ultrasonic power density and the concentration of BNN6. The sonoluminescence and the local transient high temperature and pressure induced by the ultrasonic cavitation can cause sonochemical reactions and result in the generation of free radicals, which was considered to be the possible mechanism for the release of NO from BNN6 under US irradiation. [29] Based on the above experimental results and combined with the previous findings, [26,29] we used the following US parameters for the next in vitro experiments: 1 W cm À2 , 1 MHz, and 50% duty cycle. As indicated in Figure 2H, the concentration of NO released from BNN6 (100 μM), B-P, and B-P@PLT (equal to 100 μM of BNN6) increased with the accumulation of intermittent US irradiation time. At 5 min of US irradiation, about 22.78 μM of NO was released by BNN6 and 12.9 μM by B-P. Compared to BNN6, NO concentration was reduced by 43.4% for B-P, and %56.6% of NO was released. After 60 min of US irradiation, the final cumulative NO concentration was similar and may be more than 60 μM due to incomplete conversion of NO to NO 2À and some NO volatilization in the solution. Probably because the BNN6 was loaded into the PLGA nanoparticles, the NO release rate was slowed down. This was beneficial for the treatment of cardiovascular diseases, as high concentrations of NO are toxic. NO release behavior of continuous US irradiation was similar to that of intermittent US treatment ( Figure S6, Supporting Information). These results revealed that the dose-controllable NO release from B-P@PLT can be achieved by manipulating the US irradiation time or power density.

In Vitro Biosafety
After incubation with different concentrations of BNN6, B-P, and B-P@PLT for 24 or 48 h in vitro, the viability of H9c2 and human umbilical vein endothelial cells (HUVECs) was evaluated by a CCK-8 assay. When the concentration of BNN6 was higher than 200 μM, the cell viability decreased significantly. But the cell viability was still relatively high even though the concentration of B-P or B-P@PLT was higher than 200 μM ( Figure S7A-F, Supporting Information), which suggested that loading BNN6 into PLGA nanoparticles could reduce the cytotoxicity of BNN6. Platelet aggregation assay and hemolytic test were used to evaluate the hemocompatibility of B-P and B-P@PLT. The natural platelet was prone to be easily activated by thrombin. However, neither B-P nor B-P@PLT activated platelets ( Figure S8, Supporting Information). B-P or B-P@PLT did not cause hemolytic even at concentrations up to 5 mg mL À1 ( Figure S9A,B, Supporting Information). These results suggested that the B-P and B-P@PLT were biocompatible and could be used in subsequent cell experiments.

Endothelial Tropism of B-P@PLT In Vitro
To explore the targeting of B-P@PLT to injured endothelium, HUVECs were pretreated with hypoxia reoxygenation (H/R) and then incubated with B-P and B-P@PLT. HUVECs under normoxia were used as control groups. Fluorescence images showed that the amount of B-P@PLT binding to HUVECs was significantly higher than that of B-P under hypoxia (P < 0.001). However, the binding number of B-P and B-P@PLT was not statistically different under normoxia, reflecting that B-P@PLT mainly binds to injured endothelial cells ( Figure 3A,B). The 96-well plate was coated with type IV collagen and incubated with different particles. The number of B-P@PLT adhering to collagen-coated plates was significantly higher than that of B-P  (P < 0.001) and also higher than that of the uncoated plates (P < 0.001) ( Figure 3C). Since type IV collagen is one of the major components exposed by damaged vascular endothelium after MIRI, B-P@PLT could efficiently bind to MIRI in theory. [35] CD47 was acknowledged as a "don't-eat-me" signal, [34] if armed on nanoparticles, could prevent B-P@PLT from being recognized by the mononuclear phagocyte system and subsequently elongated its circulation time. As mentioned above, the presence of CD47 on B-P@PLT had been proven by western blot. After incubating with Raw 264.7 cells for 2 h, the internalization of B-P and B-P@PLT was analyzed by flow cytometry. It showed that B-P@PLT was taken up significantly less than B-P (P < 0.001) ( Figure 3D,E), suggesting its ability to escape immune surveillance.

Proangiogenic Capacity of B-P@PLT and B-P after US Irradiation In Vitro
NO has been defined as a master regulator of angiogenesis and an important relaxing factor of cardiovascular health. [36,37] We evaluated the proangiogenic capacity of B-P@PLT and B-P after US irradiation by assessing their effects on the proliferation, migration, and capillary-like tube formation of HUVECs. After H/R, HUVECs were pretreated with H/R and then treated with PBS, B-P, B-P@PLT, B-P þ US, and B-P@PLT þ US (US parameter: 1.0 W cm À2 , 50% duty cycle, 1 MHz, 5 min). HUVECs under normoxia were used as negative control groups. HUVEC proliferation was detected using an EdU kit and CCK-8 assay. As shown in Figure 4A,B, a higher proliferation rate was found in the B-P þ US and B-P@PLT þ US groups compared with the PBS groups (P < 0.05). There is no significant difference between the B-P, B-P@PLT, and PBS groups (P > 0.05). CCK-8 assay revealed that B-P and B-P@PLT after US irradiation could significantly promote cell proliferation in HUVECs (P < 0.001) ( Figure S10B, Supporting Information). However the proproliferative effect on H9c2 cells was not significant (P > 0.05) (Figure S10A, Supporting Information). Next, the migration of HUVECs was evaluated using a scratch assay ( Figure 4C) and a transwell migration assay ( Figure 4E,F), B-P þ US and B-P@PLT þ US groups significantly increased HUVECs migration compared with PBS ( Figure 4D,G). Finally, a tube formation assay was performed to examine whether B-P þ US and B-P@PLT þ US groups could enhance the angiogenesis of HUVECs ( Figure S11A,B, Supporting Information). The total junctions and the total branching length were increased in HUVECs treated with B-P þ US and B-P@PLT þ US compared to those treated with PBS (P < 0.001), while no significant increase was observed in B-P and B-P@PLT group (P > 0.05), indicating that B-P þ US and B-P@PLT þ US could enhance the proangiogenic effects.
We then evaluated the effects of B-P þ US and B-P@PLT þ US on endothelial permeability. It was reported that 70 kDa molecules had limited penetration across the normal endothelium. [38] We then used a transwell model to measure endothelial permeability ( Figure S12A, Supporting Information). HUVECs treated with B-P þ US and B-P@PLT þ US showed lower permeability, as shown by more 70 kDa dextran-FITC diffusing from the upper to lower chamber compared with PBS groups (P < 0.01) ( Figure S12B, Supporting Information). There was no obvious difference between the B-P, B-P þ US, and PBS groups (P > 0.05). The results indicated that B-P or B-P@PLT injection combined with US irradiation could reduce endothelial permeability and protect the integrity of the endothelium in vitro.

Protection of Cardiomyocyte
Cardiomyocyte death secondary to MIRI has been associated with the increased level of ROS. [39] The elevation of ROS can trigger the opening of the mitochondrial permeability transition pore (mPTP), which in turn amplifies ROS production, resulting in detrimental consequences for the affected myocardial cells. [40] H9c2 cells were pretreated with H/R as above. We evaluated the cardiomyocyte protection of B-P@PLT after US irradiation by assessing their effects on reducing the opening of mPTP, ROS, and apoptosis of cardiomyocytes. The opening of mPTP in H9c2 cells was assessed using calcein-AM assay ( Figure 5C,D). The green fluorescence of calcein was maintained at a significantly higher level in cells of B-P þ US and B-P@PLT þ US groups than that of B-P and B-P@PLT groups (P < 0.001), indicating that B-P þ US and B-P@PLT þ US inhibited the opening of mPTP. Compared with the PBS group, significantly less ROS were produced in the B-P þ US and B-P@PLT þ US groups (P < 0.05) ( Figure 5A,B). Apoptosis of H9c2 was detected using a TUNEL assay. As shown in Figure 5E,F, apoptosis in the B-P þ US and B-P@PLT þ US groups was significantly reduced compared with the PBS groups (P < 0.001). Mitochondria have been viewed as one of the important targets in the cardioprotection provided by NO. [6,41] The protective mechanism has been further elucidated by the fact that NO inhibits the opening of mPTP, thereby limiting the generation of more mitochondrial ROS. [42] Taken together, our results suggested that NO delivery kept the protective effect of cardiomyocytes by maintaining the function of mitochondria and subsequently reducing the production of ROS.

Screening Optimal Injection Dose and Frequency
To determine the optimal injection dose of B-P@PLT in vivo, MIRI mice were injected with different doses of DiR-loaded B-P or B-P@PLT (5, 20, and 40 mg kg À1 ) via the tail vein after reperfusion. Ex vivo fluorescence imaging at 24 h revealed that the hearts of the B-P@PLT group with the dose of 5 mg kg À1 exhibited no significant fluorescence signal increases than those of the PBS group ( Figure S13A,B, Supporting Information). In contrast, the B-P@PLT group at doses of 20 and 40 mg kg À1 exhibited stronger fluorescence signals than the B-P group ( Figure 6A and S13C, Supporting Information). Semiquantitative analysis confirmed that the fluorescence intensity of the B-P@PLT group was 2.1-fold at doses of 20 mg kg À1 and 2.5-fold at doses of 40 mg kg À1 higher than that of the B-P group (P < 0.01) ( Figure 6B and S13D, Supporting Information). Myocardial enzyme assays (CKMB and LDH-1) were used to evaluate the efficacy of different doses. The efficacy between the 5 mg kg À1 group and the PBS group was not significantly different (P > 0.05). However, there was a significant difference between the 20 and 40 mg kg À1 groups compared to the PBS group. Both the 20 and 40 mg kg À1 groups exhibited significant nanoparticle enrichment in MIRI hearts. Nevertheless, the level of CKMB and LDH-1 showed no significant difference between the two groups (P > 0.5) ( Figure S13E,F, Supporting Information).
To explore the optimal injection frequency of B-P@PLT in vivo, MIRI mice were injected with 20 mg kg À1 of B-P@PLT via the tail vein after reperfusion. Treatment was conducted according to the injection protocol in Figure S14A, Supporting Information, (one injection) and S14B, Supporting Information (two injections). The left-ventricular ejection fraction (EF) and left-ventricular fractional shortening (FS) were used as indicators of treatment efficacy. The results showed a www.advancedsciencenews.com www.small-structures.com significant improvement in cardiac function with both one and two injections. However the EF and FS between one and two injections were not significantly different. This observation may be due to the wide therapeutic range of NO. After a certain level of efficacy, increasing the dose may not be effective for treatment. [43,44] Therefore, for safety reasons, we finally used a single injection (20 mg kg À1 ) combined with multiple US irradiation treatments for the following in vivo studies.

Targeting Ability and Biodistribution In Vivo
Ex vivo fluorescence imaging was also used to explore targeting ability in vivo. The hearts of the B-P@PLT group were cut into three sections from the base to the apex. We found that the B-P@PLT mainly accumulated in the anterior wall of the left ventricle, specifically the location of the ischemic and necrotic areas ( Figure 6A). As to other major organs ( Figure S15A,B, www.advancedsciencenews.com www.small-structures.com Supporting Information), B-P@PLT exhibited a trend of lower accumulation in the liver (P < 0.05), which was probably attributed to the reduced macrophage cell uptake caused by platelet membrane decoration. In other organs, no significant difference was found between B-P and B-P@PLT groups. We further explored the capacity of B-P@PLT to bind to endothelium in MIRI at the histological level. The adhesion of DiI-loaded B-P and B-P@PLT in myocardial microcirculation was validated by immunofluorescence ( Figure 6C). The blood vessels were simultaneously marked by CD31. We found that DiI-loaded B-P@PLT remained in line with endothelium in the lumen. By semiquantitative analysis of immunofluorescence intensity in the ischemic area of the left ventricle, the fluorescence intensity of B-P@PLT in the hearts of MIRI rats was significantly higher than that of B-P and the sham-operated group ( Figure 6D), which was consistent with the ex vivo fluorescence analysis. Similarly, we found that B-P@PLT remained adherent to the vascular endothelium 3 days after injection (Figure S16A,  B, Supporting Information), which provided enough time for B-P@PLT to release NO. Combining the results from in vivo therapy, our treatment adopted the regimen of US irradiation once a day for 3 consecutive days. Moreover, to test the influence of platelet membrane decoration on the pharmacokinetics, the blood was collected at predetermined time points after injection of DiR-loaded B-P or B-P@PLT. The results showed that B-P@PLT had a longer circulation time than B-P ( Figure 6E), which was probably due to the presence of CD47 on the surface of B-P@PLT. These results demonstrated that platelet membrane decoration of B-P@PLT significantly facilitated their accumulation in the ischemic heart and targeted the endothelium. It has been reported that NO has both protective and detrimental effects on MIRI, which mainly depend on its concentration. In low concentrations (10 À12 -10 À7 M), NO regulates vasodilation, angiogenesis, and cytoprotection. In high concentrations (>10 À6 M) NO may produce a killing effect. [11,12] Therefore, it was necessary to accurately control the release of NO in the treatment of MIRI. To observe whether NO concentration was also increased specifically in the infarct area after US irradiation, we examined NO concentrations in the infarcted myocardium and the distal myocardium of the MIRI heart 24 h after B-P@PLT þ US treatment. In Figure 6F,G, NO concentrations were moderately elevated in the MIRI groups compared to the sham-operated group due to endogenous NO production by activated macrophages. [8] In the infarcted myocardium, NO level was markedly elevated after US irradiation treatment in the B-P@PLT group, which was significantly higher than that of the B-P þ US group (P < 0.01), suggesting higher exogenous NO generated from B-P@PLT in infarcted myocardium due to the targeted accumulation. In B-P@PLT þ US group, the NO level in the infarcted myocardium was significantly higher than that in the distal myocardium. The results above firmly proved that NO could be targeted delivered and released locally in MIRI by platelet membrane-decorated nanoparticles combined with US irradiation.

The Protective Effect against MIRI at the Early Stage
According to the above results, B-P@PLT combined with US irradiation can artificially increase NO concentration in MIRI. Next, we further verified whether B-P@PLT þ US had a therapeutic effect in mice with MIRI. The treatment regimen is shown in Figure 7A. Nagar-Olsen staining was performed to detect cardiac injury at an early stage, by which the damaged myocardium was stained red. After B-P@PLT þ US treatment, the staining area was significantly diminished compared with the PBS group (P < 0.001), suggesting a relieved cardiac injury ( Figure 7B,C). In addition, B-P@PLT þ US also reduced oxidative stress in the risk area compared with the PBS group (P < 0.01) ( Figure 7D,E), indicating a reduced ROS generation after treatment. Myocardial zymograms were recognized as effective indicators of cardiac function. The levels of LDH, LDH-1, CK, and CKMB in serum were significantly higher in the MIRI groups than in the sham group ( Figure S17, Supporting Information). After treatment, the activity of LDH, LDH-1, CK, and CKMB in the B-P@PLT þ US group was significantly decreased (P < 0.01), while not different in the B-P and B-P@PLT groups, compared with the PBS group (P > 0.01). Cell apoptosis at the border of the risk area was also decreased, as illustrated by a TUNEL assay ( Figure 7F,G). The activity of LDH-1 and cell apoptosis in the B-P þ US group was also decreased compared with the PBS group (P < 0.05). Although B-P had no active targeting capability, it also produced a small amount of NO by US irradiation, which might play a role.

Angiogenesis and the Cardiac Function Improvement
A properly coordinated angiogenic response is associated with favorable outcomes in animal models of acute myocardial infarction as evidenced by smaller infarct scars, less remodeling, and a better-preserved cardiac function. [10] Thus, the angiogenesis potency and cardiac function protection of B-P@PLT þ US was determined by histochemical stain at 7 days after treatment. The treatment regimen is shown in Figure 8A. Capillary and arteriole density levels were significantly increased in the MIRI groups compared to the sham group because of the angiogenesis response during wound healing after MIRI. [36] Immunofluorescence staining for CD31 showed that B-P þ US and B-P@PLT þ US significantly improved the density of capillaries at the infarcted border zone compared to the PBS group (P < 0.01). B-P@PLT þ US group was significantly more effective than B-P þ US in this regard (P < 0.001) ( Figure 8B,C). Neovascularization was further evaluated by immunostaining targeting of α-SMA, a biomarker of functional arterioles. Similar trends were observed in arteriole density. The mean fluorescence intensity of α-SMA per field at the infarcted border zone in the B-P@PLT þ US group was significantly higher than that of the PBS group (P < 0.01) ( Figure 8D,E). These results indicated that local NO delivery effectively promoted angiogenesis with markedly enhanced capillary density. Then we measured the vascular permeability by Evens blue injection to evaluate the vascular integrity of the heart after treatment ( Figure S18, Supporting Information). 4 h after Evans blue injection, hearts with various treatments were perfused and harvested. The amount of Evans blue was normalized by heart weight. We found that the amount of Evans blue in the B-P@PLT þ US group was significantly lower than that in the PBS group (P < 0.01), suggesting a strong rescue of vascular integrity by local NO delivery treatment.
The EF and FS of mice of different groups were measured by echocardiography. The B-P@PLT þ US group had the best cardiac function ( Figure 9A-C). The cardiac remodeling after MIRI of different groups was further investigated by histological analysis. Masson trichrome staining showed that the treatment of B-P@PLT þ US significantly reduced the infarct and fibrosis size (P < 0.01). Concurrently, increased thickness of the infarcted left ventricular wall was observed in sections distal to the apex due to increased cardiomyocyte survival in the infarcted zone ( Figure 9D,E).

The Safety and Immunogenicity In Vivo
To assess the clinical translational potential, the safety and immunogenicity of B-P@PLT were evaluated in C57BL/6 mice. B-P and B-P@PLT were intravenously injected with or without US irradiation. The blood and major organs were collected after 7 days. As shown in Figure S19, Supporting Information, histological analysis of major organs, including the heart, liver, spleen, lung, kidney, and brain, revealed no obvious difference compared to healthy mice with PBS injection. In addition, ALT, AST,BUN,CRE, IgG, and IgM in serum didn't elevate after B-P, B-P@PLT, B-P þ US, and B-P@PLT þ US treatment, indicating  (Table S1, Supporting Information). These results indicated that B-P@PLT with US irradiation did not initiate a significant immunological response or an inflammatory response. It had good safety in the treatment of cardiovascular diseases. www.advancedsciencenews.com www.small-structures.com

Conclusion
In this study, we successfully developed a platelet membranecoated nanoparticle loaded with BNN6, which showed a specific binding ability to the injured myocardium of mice suffering from MIRI. We demonstrated that the release of NO from the nanoparticles could be controlled by adjusting the duration and power intensity of US irradiation. B-P@PLT þ US exhibited the ability to promote angiogenesis, reduce ROS, and protect cardiomyocytes in vitro. Furthermore, in a mouse model of MIRI, the injection of B-P@PLT combined with US irradiation led to optimized therapeutic efficacy, including reduced cardiac injury at an early stage, promoted angiogenesis, and improved heart function. PLGA nanoparticles coated with cell membranes are highly biocompatible, and the safe intensity of US irradiation poses no harm to humans. Therefore, this strategy of targeted delivery and locally controlled release of NO holds great clinical translational potential and may provide an effective option for the treatment of related diseases in the future.

Experimental Section
Materials and Animals: Polylactic-co-glycolic acid (PLGA, a lactide/ glycolide ratio of 50:50, inherent viscosity: 0.67 dL g À1 , Mw: 17 000 Da) was obtained from Macklin (Shanghai, China). Polyvinyl alcohol (PVA) was purchased from Aladdin Chemistry (Shanghai, China). Dichloromethane was obtained from Sinopharm Group Chemical Reagent Company (Shanghai, China). Prostaglandin E 1 (PGE 1 ) was purchased from MedChemExpress (New Jersey, USA). All the other chemicals used in this study were of analytical grade. BNN6 was synthesized in our lab. Fluorescent dye DiR (1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindotricarbocyanine iodide) and DiI (1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindocarbocyanine perchlorate) were bought from Yesen Biotech (Shanghai, China). www.advancedsciencenews.com www.small-structures.com The male C57BL/6 (8-10 weeks) mice used in this study were purchased from Vital River Laboratory (Beijing, China). Animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology. Animals were used in accordance with the experimental animal care guidelines of the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology.
Synthesis and Characterization of BNN6: BNN6 was synthesized as reported previously. [31] 2.34 mL (10 mmol) of N,N'-bis-sec-butylaminop-phenylenediame (BPA, TCI America) was added to 18 mL of ethanol, and then 20 mL of a 6 M degassed aqueous solution of NaNO 2 (Sigma-Aldrich) was added under stirring and nitrogen protection. After 30 min, 20 mL of a 6 M aqueous solution of HCl was added dropwise through a separating funnel. The reaction solution color gradually changed from orange to red, and the reaction yielded a beige precipitate. After being stirred for 4 h, the solid product was collected by centrifugation at 1000 rpm for 15 min. The collected precipitate was washed with water and 50% v/v ethanol/water, in turn, three times to remove residual reactants and then dried under a freezing vacuum in the dark. The structure of the synthesized BNN6 was tested by 1 H-NMR (Bruker AV400) and mass spectrometry (Thermo Fisher, Orbitrap LC/MS) measurements.
Isolation of Platelet Membrane: Platelet membrane was obtained by a repeated freeze-thawing process as previously described. [24,45] Fresh whole blood from male C57BL/6 mice was collected into an EDTA tube and then centrifuged at 200 g for 20 min at room temperature to separate red blood cells and white blood cells. The supernatant was collected containing platelets (namely platelet-rich plasma or PRP) and further centrifuged at 200 g for 20 min again to remove the remaining blood cells. Then, PBS containing 1 mM EDTA and 2 μM PGE 1 was added to prevent platelet activation. Platelets were collected by centrifugation at 800 g for 20 min at room temperature. To prevent protease activation, 1 mL of PBS containing 1 mM EDTA and protease inhibitor tablets (Pierce, 1 tablet for 10 mL) was added to the platelet precipitates. The platelets were frozen at À80°C and thawed at room temperature; this freeze-thaw cycle was repeated three times to yield platelet membranes.
Preparation and Characterization of B-P and B-P@PLT: PLGA nanoparticle loaded with BNN6 (B-P) was prepared by O/W solvent evaporation method as previously reported. [45,46] Briefly, 2 mg BNN6 and 10 mg PLGA were dissolved in 500 μL dichloromethane. This mixture was added to 3 mL of PVA solution (0.5% w/v) and was sonicated for 5 min with a probe sonicator (LC1000N, Ultrasonic processor, China) to generate an oil-inwater emulsion. The final emulsion was added to 10 mL of water and the solvent was evaporated in a fume hood under gentle stirring for 3 h. The excess BNN6 was collected and its absorption at 380 nm was measured to calculate the entrapment and loading efficiency of the carriers. The particles were collected via centrifugation at 20 000 g for 20 min. For fluorescently loaded nanoformulations, DiI-loaded or DiRloaded nanoparticles were loaded into the polymeric cores at 0.1 wt% in the same method.
Upon preparation of B-P, cell membrane coating was performed by adding the appropriate surface area equivalent of either platelet membrane followed by 5 min of sonication in a bath sonicator (KH-160TDB, KUNSHAN, China). 1.5 mL aliquots of platelet solution containing 3 Â 10 9 platelets were prepared and used to cloak 1 mg of PLGA nanoparticles. [44] Particle size, polydispersity (PDI), and surface zeta potential were characterized using DLS (NanoBrook 90 Plus PALS, USA). The surface morphology was investigated by SEM (Hitachi SU8010, Japan). Membrane coating was confirmed by TEM (Hitachi HT7700, Japan) at 100 kV. FTIR spectra with wavenumbers ranging from 400 to 4000 cm À1 were performed by a Nicolet iS50R spectrometer (Thermo Scientific, USA).
Examination of Platelet Membrane Proteins: SDS-PAGE was performed under reducing conditions in 10% acrylamide to examine whether the compositions of the platelet, platelet membrane (PM), B-P, and B-P@PLT proteins were similar. Platelet, platelet membrane, and B-P@PLT (50 μL, 15 μg mL À1 in protein content) were centrifuged and redispersed with the loading buffer (20 μL) to solubilize the membrane-associated proteins. Finally, the resulting protein containing loading buffer (5 μL) was loaded for electrophoresis (VE 680, Tanon) at 100 V for 100 min, followed by silver staining to visualize the protein bands on SDS-polyacrylamide gels. Western blotting was performed to assess the presence of specific platelet membrane markers using mouse anti-CD47 antibody (Proteintech, 66 304-1-Ig), mouse anti-CD42c antibody (Abclonal, A10113), mouse anti-CD41 antibody (Abcam, ab181582), and mouse anti-integrin alpha 2 antibody (Abcam, ab133557), respectively.
In Vitro US Parameters Exploration and NO Detection: The concentration of NO was measured by the Total Nitric Oxide Assay Kit (Beyotime). Briefly, 1 mL of different concentrations of BNN6 dispersion were exposed to US irradiation using the ultrasonic gene transfection instrument (Nepa Gene, Japan) for 5 min at room temperature with different power densities (0, 0.5, 1.5, 2.5 W cm À2 . The duty cycle was set to 50% and the frequency was set to 1 MHz according to the previous report. [29] After sonication, 50 μL of the sample was taken out and placed in a 96-well plate, and then Griess Reagent I (50 μL) and Griess Reagent II (50 μL) were added to the wells. After incubating for 10 min in the dark, the absorbance was recorded at 540 nm using a microplate reader (TECAN). NaNO 2 was used as a standard.
Similarly, 1 mL BNN6 (100 μM), B-P, and B-P@PLT (nanoparticles concentration equal to 100 μM BNN6) dispersion were exposed to US over different irradiation periods (US parameters: 1.0 W cm À2 , 50% duty cycle, 1 MHz). The control without sonication was also executed for comparison. At each interval, the amount of released NO was quantified using the Griess assay method.
Platelet Aggregation Assay: The aggregation of platelets in the presence of B-P and B-P@PLT was assessed using a spectrophotometric method. PRP was obtained from C57BL/6 mice (using sodium citrate as the anticoagulant). PBS was used to regulate the platelet concentration in PRP to 1 Â 10 9 per mL. PRP (1 mL) was then loaded into a cuvette, followed by the addition of 100 μL B-P or B-P@PLT (2 mg mL À1 ) in PBS solution. As negative and positive controls, PRP was mixed with PBS or thrombin (0.5 IU/mL, Sigma Aldrich). Transmittance changes were recorded by a microplate reader (TECAN) to measure the change in absorbance at 650 nm every 200 s for a total of 2000 s, and platelet aggregation was demonstrated by measuring the decrease in absorbance based on the reduction in turbidity. Each sample was assayed in triplicate (n = 3).
Haemocompatibility Assay: The hemolysis test was performed according to previous reports. [47] Erythrocytes were obtained from healthy C57BL/6 mice, washed three times with normal saline, and suspended in normal physiological saline. Next, erythrocyte suspension and nanoparticles solution were added to a centrifuge tube. The final concentrations of B-P and B-P@PLT were 0.5, 1, 2, and 5 mg mL À1 . The negative and positive controls were PBS and Triton X-100, respectively. The samples were incubated at 37°C for 1 h, and centrifugation was performed at 5000 rpm for 10 min. Then, 100 μL of the supernatant from each tube was carefully collected and added to each well of 96-well plates in triplicate. The absorbance of each well was obtained at 540 nm in a microplate reader (TECAN), and the percentage of RBC hemolysis was calculated (percentage of lysis% = (OD test -OD nagative )/OD positive Â 100) (n = 3).
Cell Viability: Cell viability was measured using cell counting kit-8 as previously described (CCK-8, Dojindo, Japan). [48] H9c2 and HUVECs were seeded into 96-well plates at a density of 5 Â 10 3 cells per well and exposed to 0.05, 5, 50, 100, 200, 400, 800 μM of different BNN6, B-P, and B-P@PLT (equal to the concentration of BNN6) diluted in serum-free DMEM for 24 or 48 h. Similarly, B-P and B-P@PLT (nanoparticles concentration equal to 100 μM BNN6) diluted in serum-free DMEM that were preirradiated with US for 5 min (US parameters: 1.0 W cm À2 , 50% duty cycle, 1 MHz) (named B-P þ US group, B-P@PLT þ US group), no US groups were set as control (named B-P group, B-P@PLT group). Then incubated with H9c2 and HUVEC cells for 24 or 48 h. Subsequently, the cells were washed with PBS and further incubated with CCK-8 solution for 2 h. Cell proliferation was determined by measuring the absorbance at 450 nm with a microplate reader (TECAN).
Endothelial Tropism of B-P@PLT In Vitro: HUVECs' hypoxic culture was performed using the AnaeroPack system (AnaeroPack-Anaero 5%, Mitsubishi Gas Chemical, Tokyo, Japan). The oxygen concentration www.advancedsciencenews.com www.small-structures.com decreased to <0.1% within 1 h, and the carbon dioxide concentration was maintained at about 5%. [49] HUVECs were cultured in a hypoxia container for 10 h, transferred in a CO 2 incubator at 37°C for the next 6 h, and then incubated with DiI-loaded B-P or DiI-loaded B-P@PLT for 1 h. HUVECs under normoxia were used as a control. Nuclei were loaded with DAPI. The cells were washed with PBS and then photographed by fluorescence microscope (Olympus, Japan). Fluorescence intensity analysis was performed using ImageJ software. 100 μL 0.5 mg mL À1 collagen type IV (Sigma Aldrich) in 0.25% sterile acetic acid was added in a 96-well plate and incubated overnight at 4°C. Collagen-coated or noncoated plates were first blocked with 3% BSA for 30 min and then incubated with 100 μL DiIloaded B-P and DiI-loaded B-P@PLT. Noncoated plates were used as a control. After 1 h of incubation, the collagen layer was washed and then dissolved with 100 μL DMSO. Fluorescence intensity was measured using a microplate reader (TECAN). Nanoparticle Clearance In Vitro: Raw 264.7 macrophages were used for in vitro cellular uptake test. The macrophages were seeded in a 6-well plate and incubated with DiI-loaded B-P and DiI-loaded B-P@PLT for 2 h, followed by washing with PBS. Flow cytometry was performed to investigate the positive uptake cell ratio.
Proangiogenic capacity of B-P@PLT In Vitro: As mentioned before, HUVECs were cultured in a hypoxia airtight container for 10 h to induce cell injury as a pretreatment. The cells medium was then replaced with DMEM containing B-P or B-P@PLT (nanoparticles concentration equal to 100 μM BNN6) that was preirradiated with US for 5 min (US parameters: 1.0 W cm À2 , 50% duty cycle, 1 MHz), cells incubated with DMEM containing B-P or B-P@PLT without US irradiation were the control groups. The cells were then placed in the CO 2 incubator at 37°C for 6 h reoxygenation. No hypoxia/reoxygenation (H/R) treatment served as the negative control.
For HUVECs proliferation analysis, EdU Cell Proliferation Kit (Beyotime) was used according to the manufacturer's instructions. For the scratch wound assay, confluent cells were starved for 12 h, and a linear scratch was made in the cell monolayer using a 200 μL sterile pipette tip. PBS was used to wash the cells three times, after which the different groups of nanoparticles were added to a serum-free medium. After 0 and 24 h of incubation, cell migration was observed using a microscope (Olympus, Japan). A transwell migration assay was also applied to evaluate migration ability. HUVECs pretreated with B-P, or B-P@PLT, B-P þ US, and B-P@PLT þ US were plated in serum-free medium in the upper chamber of a transwell, into which was inserted an 8.0 μm pore size membrane, while the lower well contained DMEM with 10% FBS. After culture for 12 h, cells on the upper surface of the membrane were removed, and migrant cells on the lower surface were fixed with paraformaldehyde and stained with 0.1% crystal violet. After photographing, crystal violet was extracted with 33% acetic acid and absorbance was detected at 595 nm.
To assess tube formation, 48-well plates were coated with Matrigel (Corning) for 30 min at 37°C. Then, HUVECs at passages 3-5 were incubated with B-P, B-P@PLT, B-P þ US, and B-P@PLT þ US during reoxygenation after hypoxic treatment of cells for 6 h and imaged using a microscope (Olympus, Japan). Images of vascular networks were analyzed with ImageJ's tool "Angiogenesis Analyzer." Dextran Diffusion Assay: The endothelial permeability assay was performed in vitro by measuring the diffusion of FITC dextran (Sigma-Aldrich) across a monolayer of confluent HUVECs. To prevent cell migration, a 0.4 μm 12-well hanging cell culture insert (Corning) was used for HUVECs culture to mimic the endothelial layer. When reached 100% confluency, cells were treated with hypoxia for 10 h. Complete DMEM containing B-P or B-P@PLT with or without US irradiation was added to the upper chamber during the reoxygenation period. Then the upper chamber culture media was replaced with 1 mg mL À1 FITC Dextran culture media and incubated for the next 60 min. 100 μL culture media of the upper and lower chamber were extracted respectively to detect the fluorescence intensity (Ex 490 nm, Em 520 nm). The permeability index was calculated as lower chamber intensity divided by upper chamber intensity.
Reduction of Cardiomyocyte Damage In Vitro: H/R treatment of H9c2 was performed as previously described in Section 2.12. To measure apoptosis of H9c2, the One Step TUNEL Apoptosis Assay Kit (Beyotime) was used according to the manufacturer's instructions. Mitochondrial permeability transition pore formation was measured using the mitochondrial Transition Pore (mPTP) Assay Kit (Beyotime) according to the manufacturer's instructions. H9c2 cells were cultured in a confocal dish. After 10 h of hypoxia, B-P, and B-P@PLT, with or without US irradiation, were added during the reoxygenation process. The mPTP opening was measured by monitoring calcein AM fluorescence in the absence and presence of CoCl 2 . [50] Cells were detected with a confocal laser scanning microscope (ZEISS, LSM780). Quantitative fluorescence analysis was performed using Image J. Intracellular ROS levels were detected using a Reactive Oxygen Species Assay Kit (Beyotime). Same as before, H9c2 cells were treated with hypoxia and reoxygenation and then loaded with DCFH-DA for 30 min at 37°C in the dark. Finally, the cells were observed under a fluorescence microscope (Leica DMi8), and fluorescence intensity was quantified by ImageJ.
Safety Assessment of B-P@PLT: To evaluate biosafety after the administration of B-P and B-P@PLT with or without US irradiation, healthy C57BL/6 mice were randomly divided into four groups (n = 4) and given 20 mg kg À1 B-P, B-P@PLT, B-P þ US, and B-P@PLT þ US. PBS was used as a control. The whole blood and organs were collected 7 d after administration. The blood samples were centrifuged at 3000 rpm for 10 min at 4°C to obtain serum. The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and urea nitrogen (BUN) were determined using an automated Chemray 240 clinical analyzer (Rayto, Shenzhen, China). Routine blood was tested using a mindray BC-30vet hematologic analyzer (Mindray Corporation, China). Total IgG and IgM concentrations in serum were determined using ELISA Kit (Enzyme-linked Biotechnology, Shanghai, China) based on the manufacturer's instructions. The heart, lung, liver, spleen, and kidney were harvested for histological assessment by hematoxylin eosin (H&E) staining.
Mice Myocardial Ischemia/Reperfusion Injury (MIRI) Model: 8-10 weeks old C57BL/6 mice were anesthetized with pentobarbital sodium (60 mg kg À1 ) and then ventilated after tracheal intubation. The body temperature was maintained at 37°C with a heating pad. The heart was exposed through a thoracotomy performed in the fourth or fifth intercostal space and the left anterior descending (LAD) coronary artery was transiently ligated for 60 min with an 8-0 silk suture followed by reperfusion. The successful MIRI model was confirmed by the presence of ST elevation on the electrocardiogram.
MIRI mice were randomly divided into five groups that underwent different treatments: 1) PBS; 2) B-P; 3) B-P@PLT; 4) B-P þ US; and 5) B-P@PLT þ US. The sham operation group was used as a negative control. Nanoparticles injection was completed within 15 min after reperfusion, followed by US irradiation (5 min) of the chest in some groups. The B-P þ US and B-P@PLT þ US groups were irradiated by US once a day for 3 d after the operation.
Pharmacokinetics Studies: The experiments were performed on healthy male C57BL/6 mice as described previously. [51] To evaluate the circulation half-life of B-P@PLT, 20 mg kg À1 of DiR-loaded nanoparticles were injected into the tail vein of the mice. 20 μL of blood were collected at 5 min, 1, 6, 24, and 48 h following the injection. The same dose DiR-loaded was also tested in parallel as controls. The collected blood samples were diluted with 30 μL PBS in a 96-well plate before fluorescence measurement (TECAN) (n = 4).
In Vivo Targeting Ability to the Injured Endothelium: MIRI mice were injected via the tail vein with DiR-loaded B-P or DiR-loaded B-P@PLT (50, 20, and 40 mg kg À1 ) respectively after reperfusion. Heart, lung, liver, spleen, and kidney were harvested at 24 h after injection for ex vivo fluorescence imaging using an in vivo imaging system (IVIS; PerkinElmer, Inc., Waltham) (Ex 750 nm, Em 790 nm). Then, the hearts of each group were cut into three sections from the base to the apex to display the cross sections.
For histological analysis, MIRI mice were injected via the tail vein with DiI-loaded B-P or DiI-loaded B-P@PLT, respectively, after reperfusion. The heart was harvested 24 h or 3 d after injection and fixed in formaldehyde. Immunofluorescence staining with anti-CD31 primary antibody (Abcam, ab182981) was performed with the manufacturer's instructions to identify the vascular endothelium in the hearts.
In vivo NO Release Detection: NO production in myocardial tissue after injection and sonication was assessed via a Griess assay (Beyotime) with the manufacturer's instructions. Briefly, 1 d after the injection and single-US treatment of different groups, the mice hearts were collected and immediately dissected into the infarcted part and distal part and weighed. Subsequently, the heart tissues were crumbled into small pieces in homogenizer tubes and extracted with cell and tissue lysis buffer for NO assay immediately. After centrifugation, supernatant (50 μL) was mixed with Griess reagent I (50 μL) and Griess reagent II (50 μL) at room temperature for 10 min. The concentrations of NO were determined using a microplate reader (TECAN) at a wavelength of 540 nm and calculated based on a sodium nitrite (NaNO 2 ) standard curve.
Myocardial Enzyme Assays: At 24 h after MIRI surgery and treatment, serum was separated from the blood by centrifugation at 3000 rpm for 15 min at 4°C. The myocardial enzymes including lactate dehydrogenase (LDH), lactic dehydrogenase 1 (LDH-1), creatine kinase (CK), and MB isoenzyme of creatine kinase (CKMB) were measured in accordance with the manufacturer's instructions (Changchun HuiLi Biotech, China).
Modified Miles Assay: Quantification of in vivo permeability was performed using the modified Miles assay as described previously. [52] 7 d after nanoparticles injection and US irradiation, 200 μL Evans blue (1%) was injected 4 h before euthanization. Hearts were weighed and ground fully with 1 mL trichloroacetic acid to extract the leaked dye. The supernatant was retained by centrifugation at 12 000 rpm for 20 min; then three times the volume of anhydrous ethanol was added and mixed. The fluorescence intensity of each sample was measured using a microplate reader (TECAN).
Histological Analysis: At treatment of day 1 or day 7, mice were anesthetized with pentobarbital sodium (60 mg kg À1 ). Hearts were harvested and perfused with saline and then fixed in paraformaldehyde for 24 h. The heart samples were embedded into paraffin blocks and cut into 4 μm-thick sections. For frozen sections, heart samples were immersed in 30% glucose over 48 h after fixation with paraformaldehyde, then cut into 8 μm, and embedded with OCT. Nagar-Olsen staining was performed with paraffin sections following a standard protocol, the ischemic myocardium was colored red, and the normal myocardium was colored yellow. ROS generation in myocardial tissue was determined using the redox dye dihydroethidium (Beyotime). The heart sections were also stained using the One Step TUNEL Apoptosis Assay Kit (Roche) to detect cell apoptosis. Angiogenesis was evaluated by staining sections with an anti-CD31 antibody (Abcam, ab182981) and an anti-alpha smooth muscle actin (α-SMA) antibody (Abcam, ab32575). Cardiac cells were identified by staining sections with anti-Cardiac Troponin T antibody (ab8295, Abcam). Masson trichrome staining was performed with paraffin sections following a standard protocol. Micrographs were acquired using Pannoramic Scan (3D HISTECH, Hungary) or confocal microscope (ZEISS, LSM780). Quantification was performed using Image J analysis.
Cardiac Function Measurement: The transthoracic echocardiograph was performed by using the Vevo 1100 Imaging System (VisualSonics, Toronto, Canada). When measuring the cardiac function, all animals were anesthetized by isoflurane inhalation mixed with oxygen, and M-mode images were acquired. Then the left-ventricular EF and leftventricular FS were calculated as indicators of left ventricular function and structure. All measurements were recorded from five continued cardiac cycles.
Statistical Analysis: Graphs were plotted and appropriate statistical analyses were conducted using GraphPad Prism 9 and OriginPro 9. All quantitative results were expressed as the mean AE SD. Statistical analysis was performed by sStudent's t-test (α = 0.05) or one-way analysis of variance (ANOVA). Statistical significance was set at *P < 0.05, **P < 0.01, ***P < 0.001.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.