{"paper_id":"1fcdabec-75f7-4aee-9190-034227a53ff4","body_text":"Alleviation of Myocardial Infarction by Hydrogen Sulfide-Releasing Nanoparticles: Mechanisms and Therapeutic Effects | 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 Alleviation of Myocardial Infarction by Hydrogen Sulfide-Releasing Nanoparticles: Mechanisms and Therapeutic Effects Yujia Zhan, Xueshan Zhao, Siwei Bi, Ruiqi Liu, Yuxuan Ge, Jun Gu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4987842/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Myocardial infarction (MI), a common and severe disease threatening human health worldwide, results from ischemic and hypoxic-induced necrosis of cardiac tissue due to coronary artery obstruction or rupture. Hydrogen sulfide (H 2 S) is a gasotransmitter involved in various physiological and pathological processes. Exogenous supplementation of H 2 S is significantly beneficial for the treatment of MI. In this study, a novel H 2 S donor - zinc sulfide nanoparticles encapsulated in hyaluronic acid (HA@ZnS NPs), has been developed through a biomimetic mineralization process for the treatment of MI. HA@ZnS NPs can stably release H 2 S at the site of myocardial ischemic injury due to the acidic microenvironment. Compared to the MI group, the NP-treated group significantly improved cardiac function, including increased left ventricular ejection fraction and fractional shortening, as well as reduced end-systolic volume. Furthermore, the NPs significantly reduced the size of the myocardial infarction area, improved left ventricular remodeling, and exerted therapeutic effects by promoting angiogenesis and reducing apoptosis in cardiac tissue. In conclusion, HA@ZnS NPs demonstrate potential for treating MI through precise control of H 2 S release, providing valuable insights into new therapies for MI and laying the groundwork for the clinical application of H 2 S-releasing materials in the future. myocardial infarction hydrogen sulfide nanoparticles myocardial ischemic injury Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Myocardial infarction (MI) occurs due to coronary artery obstruction or rupture, leading to ischemia and hypoxia-induced necrosis in myocardial tissues. It is one of the clinical syndromes of coronary atherosclerotic heart disease (coronary heart disease)( 1 – 4 ), and has become one of the common severe diseases globally threatening human’s health( 2 , 5 ). Coronary heart disease is prevalent in 6.3% of the adults in the United States, and 1 in every 7 deaths is primarily due to coronary heart disease. Moreover, MI is prevalent among 7.9 million adults, with a new case occurring approximately every 40 seconds( 4 , 6 ). Given that the risk of premature cardiovascular diseases in young people is increasing( 7 ), the prevention and treatment of MI have become in a huge need. Recently, emerging fields such as tissue engineering and biomaterials have begun to offer potential therapeutic approaches to address the dilemma( 7 , 8 ). For instance, integrating biomaterials with clinical treatment methods could slow the progression of ventricular remodeling and rebuild cardiac function post-infarction( 8 – 10 ). More recently, gasotransmitters have garnered widespread attention as an emerging therapeutic modality because of their excellent therapeutic efficacy and good biocompatibility. The three recognized gasotransmitters are nitric oxide (NO)( 11 ), carbon monoxide (CO)( 12 ), and hydrogen sulfide (H 2 S)( 13 ). These gases are implicated in a wide range of physiological and pathological processes, such as cardiovascular diseases, oxidative stress, and inflammation. Therefore, developing targeted delivery systems capable of releasing therapeutic doses of gasotransmitters represents an ideal tool in modern medicine( 13 , 14 ). It is proven that exogenous H 2 S plays a vital role in the treatment of MI. Studies have shown that animals treated with H 2 S exhibit reduced myocardial infarction area, improved left ventricular fractional shortening in the risk zone, and enhanced microvascular reactivity. Moreover, both short-term and long-term administration of H 2 S donors can lower blood pressure( 15 , 16 ). Besides, the expression levels of inflammatory factors such as IL-6, IL-8, and TNF-α in myocardial tissue decline following H 2 S treatment( 17 ). These experimental results collectively suggest that supplementing H 2 S is highly meaningful for treating MI. It should be noted that the H 2 S dose-response curve is bell-shaped, with its therapeutic effects limited to a narrow range( 18 ). Therefore, precise dose control is a key issue in the development of H 2 S-based therapies for MI treatment. In the past few years, various types of H 2 S donors responsive to different stimuli including light, biological thiols, pH changes, enzymatic activity, and others have been developed( 19 – 22 ). Despite huge advances have been achieved, most suffered from low water solubility, limited means to modulate release kinetics, and no capacity for targeted delivery, all of which may limit H 2 S-based treatments due to the reactive nature of this signaling molecule( 23 ). To address the aforementioned critical issue, we prepared an H 2 S donor-zinc sulfide nanoparticles (NPs) from hyaluronic acid by biomineralization. It is assumed that the H 2 S could be steadily released from NPs at the lesion site of myocardial ischemic injury, which prevents the injury from further deterioration. The protective effects of NPs on myocardial ischemic injury were carefully examined on rats (Fig. 1A) . And the underlying mechanisms were deciphered both in vitro and in vivo . 2. Materials and methods 2.1. Materials Sodium hyaluronate (HA, mW ≈ 3 KDa) was purchased from Bloomage Biotech (Beijing, China). Sodium sulfide nonahydrate (Na 2 S·9H 2 O), zinc acetate (Zn(CH 3 COO) 2 ), 30 wt % hydrogen peroxide solution (H 2 O 2 ), and paraformaldehyde solution (4 wt% PFA) were purchased from Titan Biotechnology (Shanghai, China). HA@ZnS nanoparticles (NPs) were prepared according to the published procedure with a slight modification( 21 ). Ferric chloride hexahydrate (FeCl 3 • 6H 2 O) and 4-amino- N, N -dimethylaniline sulfate (DMPD) were purchased from Aladdin Biochem Technology (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) medium, trypsin, penicillin, and fetal bovine serum (FBS) were purchased from HyClone (Waltham, USA). Dimethyl sulfoxide (DMSO), Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) kit, and 5% normal goat serum were purchased from Thermo Fisher Scientific (Massachusetts, USA), Dulbecco’s phosphate-buffered saline (DPBS) and streptomycin were purchased from Solarbio (Beijing, China). Bicinchoninic acid (BCA) protein assay kit was purchased from Abcam (Cambridge, UK). CCK-8 reagent was purchased from Solarbio (Beijing China). Antibodies: CD86, IL-6, α-smooth muscle actin (α-SMA), Bcl-2, BAX, and Caspase-3 were purchased from Abcam (Cambridge, UK). Secondary antibodies: Alexa Fluor 488 and 594 were purchased from Molecular Probes (California, USA). 2.2. Animals and cells The procedures of animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Westchina Hospital, Sichuan University (Chengdu, China) (20230228042). Adult male Sprague-Dawley rats (10 weeks old, 200–250 g) were purchased from the Chengdu Dashuo Biotechnology Co., Ltd and housed in standard cages under standard conditions. All animals were acclimatized for one week before use. Rat cardiomyocytes cells (H9C2) and human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC, Manassas, USA), and they were cultured in DMEM with high glucose supplemented with 10% (v/v) FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin at 37°C in a humidified incubator with 95% air and 5% CO 2 . 2.3. In vitro cell studies 2.3.1. In vitro fluorescence imaging of H 2 S release H9C2 cells (2 x 10³/ well in 12-well plates) were incubated with 1.5 mL of glucose-free DMEM containing Na2S or HA@ZnS NPs at 37°C. Subsequently, the pH values were adjusted to 5.5. The incubation lasted for 1 h, and the mixture of WSP-5 (50 µM) and CTAB (200 µM) was added after removing the culture medium. The green fluorescence was then observed using the fluorescence microscope. 2.3.2. Cytocompatibility test To evaluate the cytotoxicity of HA@ZnS NPs, H9C2 cells (8 x 10 3 / well in 96-well plates) were incubated with 200 µL of glucose-free DMEM containing different concentrations of HA@ZnS NPs at 37°C for 24 h, then the culture medium was removed and replaced with serum-free DMEM containing 10 wt% of CCK-8 solution. After incubation for 1 h, the absorbance at 450 nm was collected using the Multi-Detection Reader. 2.3.3. ROS assay test ROS production in H9C2 cells was analyzed using dihydroethidium (DHE). H9C2 cells were incubated with 10 µM of DHE at 37°C for 30 minutes and then washed twice with PBS. DHE staining was then observed using a fluorescence microscope to detect ROS production. Fluorescence was collected at 535 nm excitation and 610 nm emission using a Multi-Detection Reader. 2.3.4. HUVEC tube formation assay Tube formation assay was performed using tanswell chambers (65 mm PET membrane, 0.4 µm pore size) and 24-well plates. The matrix gel was spread evenly in the 24-well plate and incubated at 37°C for 30 min. After co-culture for 7 days, 1.5x10 5 HUVECs/well (3 replicates per group) were inoculated into the 24-well plate and cultured using the complete medium. The control group was incubated at 37°C for 12 h, and the NPs group was incubated with 10 µg/mL of HA@ZnS NPs at 37°C for 12 h. The number of lumen formations was counted in the top, bottom, left, right, and center of view, and the average value was taken. The control data were used as one to calculate the relative formation rate of the number of tubules. 2.4. In vivo studies 2.4.1. Establishment of MI model on rats Adult male Sprague-Dawley rats (SD rats, 10 weeks old, 200–250 g) were used for the studies. Rats were anesthetized with isoflurane, intubated for respiratory support, and maintained under anesthesia using a gas anesthetic machine. The heart was exposed and the left anterior descending coronary artery was ligated to create left ventricular infarction. Then, after ligation for 30 min, NPs or saline (10 injections for 200 µL in total) was injected at the periphery and center of the infarct site, followed by suturing of the surgical wound on the rats' chest. Rats were randomly divided into 5 groups: ( 1 ) Sham group; ( 2 ) MI group (underwent LAD ligation and saline injection); ( 3 ) ZnCl 2 group (underwent LAD ligation and injected with 200 µg/kg of ZnCl 2 ); ( 4 ) Na 2 S group (underwent LAD ligation and injected with 200 µg/kg of Na 2 S); ( 5 ) NPs group (underwent LAD ligation and injected with 200 µg/kg of NPs). 2.4.2. In vivo fluorescence imaging of H2S release To assess the imaging of NPs in the hearts of MI rats, SD rats were divided into three groups: ( 1 ) MI group, untreated MI rats; ( 2 ) Sham + NPs group (200 µg/kg), healthy rats injected with NPs; ( 3 ) MI + NPs group, MI rats injected with NPs (200 µg/kg). 30 min after NPs administration, rats in Sham + NPs group and MI + NPs group were locally injected with 100 µL of 25 µM WSP-5 (dissolved in DMSO) and 10 µL of 100 µM CTAB (dissolved in ddH 2 O) to give the total volume to 200 µL. 30 min after WSP-5 administration, imaging was performed using Spectral Instruments Imaging (Kino, U.S.A) in fluorescence mode (excitation: 500 nm; emission: 525 nm). To study the relationship between NPs dosage and fluorescence intensity, MI rats received a single local injection of different NPs doses (25, 50, 100, 150, 200 µg/kg) 30 min post-ligation. Control rats were injected with saline. 1 h after injection, rats were euthanized. Hearts were harvested, and ex vivo imaging was conducted using Spectral Instruments Imaging in fluorescence mode. To study the metabolism and biodistribution of NPS in MI rats, rats received a single local injection of NPS (200 µg/kg) 30 min post-ligation. At each predetermined time point (0.5, 1, 6, 8, 12, and 24 h post-injection), rats were euthanized, and major organs such as the heart, liver, spleen, and kidney were collected for ex vivo imaging. 2.4.3. Echocardiographic assessment of cardiac function At 7, 14, 21, and 28 days post-surgery, echocardiography was used to evaluate cardiac function in five groups of rats. First, the SD rats were weighed, and chloral hydrate was administered intraperitoneally as an anesthetic at 0.024 g/100 g. Once fully anesthetized and immobilized, the fur at rats' neck and chest was removed using a specialized depilatory cream. The rats were secured on the test table, and ultrasound coupling agent was uniformly applied to the chest for imaging. Echocardiography was conducted using the Vevo 3100 Imaging System (Visual Sonics, Canada). Several parameters including left ventricular ejection fraction (EF), fractional shortening (FS), and end-systolic volume (ESV) were measured and the average values of three separated cardiac cycles were used to represent the cardiac function. 2.4.4. Histology examination Triphenyltetrazolium chloride (TTC) staining was used to assess infarct size 7 days post-myocardial infarction. The heart was sectioned into four horizontal slices (2 mm thick) and incubated in 1% TTC to differentiate between the infarcted and viable myocardium. The infarct sizes were quantified using Image J. 28 days post-surgery, the SD rats were weighed and anesthetized with an intraperitoneal injection of chloral hydrate at a dosage of 0.024 g/100 g. Once the rats were fully immobilized, their abdomens were incised with a surgical scalpel, and blood was withdrawn from the abdominal aorta using a 10 mL syringe. The excised hearts were thoroughly rinsed with saline and then fixed in a 4% paraformaldehyde solution. The hearts were dehydrated in a graded series of 75% ethanol, embedded in paraffin, and serially sectioned (5 µm thick) from apex to base, perpendicular to the longitudinal axis of the heart, ensuring a complete representative cross-section. Slides were stained with Hematoxylin and Eosin (H&E) and Picrosirius Red, then observed under a light microscope for collagen deposition in the myocardial infarction area and morphological changes in surrounding tissues. With Picrosirius Red staining, collagen appears red and normal myocardial tissue appears yellow. Finally, the slides were quantitatively analyzed to measure the infract size, the left ventricular wall thickness and the left ventricular scar size. For the immunohistochemical analysis, slides were pretreated with 3% hydrogen peroxide in PBS for 10 min at room temperature to suppress the endogenous peroxidase activity. Subsequently, the slides were incubated in a blocking solution composed of 5% normal goat serum in DPBS for 1 h and then treated with the primary antibody at 4°C. The antibodies employed included CD86, IL-6, α-SMA. The slides were then treated with Alexa Fluor 488 or 594 secondary antibodies. After washing, antifade mounting medium with DAPI (H-1200-10, Vector Labs Inc., Malvern, PA, USA) was applied to the slides. Fluorescent cells were visualized using an Eclipse Ti2 microscope (Nikon, Japan). For cell apoptosis assays, heart paraffin sections were stained with the TUNEL kit. DAPI was used for nuclear staining. Images were taken by an Olympus Research Slide Scanner VS200. 2.4.5. Western Blot Protein concentrations were measured with the Bicinchoninic Acid (BCA) Protein Assay Kit. Equal volumes of protein samples were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then incubated overnight at 4 ◦ C with antibodies specific for the CD86, IL-6, α-SMA, Bcl-2, BAX, and Caspase-3. On the following day, the membranes were washed and incubated with horseradish peroxidase (HRP)-coupled secondary antibodies. The blots were detected by enhanced chemiluminescence and quantified using the Bio-Rad imaging system. 2.4.6. Flow cytological analysis On one day post-MI, the hearts were perfused with pre-cooled PBS. The injured left ventricular tissues were taken and separated by GentleMACS dissociator. The tissue was digested for 30 min at room temperature at 200 rpm in 5 mL of HBSS buffer containing collagenase II, collagenase IV, and DNase I. The resulting suspension was filtered (70 µm) to produce a single cell suspension. The suspension was centrifuged at 300 ×g for 5 min. Cardiomyocytes were then suspended in DMEM culture medium (with the addition of 10% fetal bovine serum and 1% penicillin-streptomycin) and cultured at 37°C in 5% CO 2 for 2 h. Cells were then washed with PBS and cardiac macrophages were concentrated in adherent cells. Cells were collected and incubated with PerCP-Cy5.5-CD86 and PE-Cy7-CD206 with flow cytometry antibodies for 15 min in the dark at 4°C. After washing with PBS, cardiac macrophage phenotypes were detected by BD FACSCanto II flow cytometry. 2.5. Statistical analysis Data are presented as mean ± standard deviation (SD) (n ≥ 3). Significance was determined by the student’s t-test or one-way analysis of variance (ANOVA) using GraphPad Prism (version 9). The statistical significance was considered when the P value was less than 0.05. 3. Results and discussion 3.1. Synthesis and characterization of HA@ZnS NPs HA@ZnS nanoparticles (NPs) were prepared by the one-step biomineralization. In brief, zinc acetate was dropwise added to the premixed solution of HA and Na2S in anaerobic condition. With stirring, insoluble ZnS gradually formed at the aid of HA, affording HA@ZnS NPs. In infarcted cardiac tissue, the expression of CD44 increased. Given that HA can bind specifically to CD44, it is assumed that NPs could accumulate in infarcted cardiac tissues(24, 25). Thus, in this system HA not only acted as the template for ZnS growth and stabilize HA@ZnS nanoparticles in the solution, but also rendered HA@ZnS NPs targeting to CD44 overexpressed tissues. The average size of HA@ZnS NPs measured by dynamic light scattering (DLS) was around 200 nm (Fig. 1B) . TEM imaging showed that these NPs were spherical with the diameter comparable to that of DLS (Fig. 1C) . Then the H2S release behaviors were investigated in acetate buffer at pH 7.4 and 5.5, respectively. As shown in Fig. 1D , traceable H2S could be detected at pH 7.4, whereas large amount of H2S was liberated in the course of 1 h at acidic pH 5.5, implying that HA@ZnS NPs were pH responsive. Figure 1. A schematic diagram of HA@ZnS NPs. (A) Synthesis of HA@ZnS NPs and their mechanism of treating myocardial infarction. (B) Representative transmission electron microscope (TEM) image of HA@ZnS NPs. (C) Hydrodynamic size distribution of HA@ZnS NPs in deionized water. (D) H 2 S release curves of HA@ZnS NPs in 25 mM acetate buffer (pH 7.4 and 5.5). 3.2. Biocompatibility of HA@ZnS NPs Nanomaterials intended for biomedical applications require low toxicity or nontoxicity. Cell viability assay (CCK8) was employed to study the toxicity of HA@ZnS NPs against rat cardiomyocytes cells (H9C2) and human umbilical vein endothelial cells (HUVECs). Three concentration gradients of the nanomaterials were selected: 1 µg/mL, 5 µg/mL, and 10 µg/mL. As illustrated in Fig. 2 A, no significant cytotoxicity against H9C2 cells or HUVECs was observed within the concentration range of 1–10 µg/mL within 48 hours (p > 0.05). Furthermore, the live/dead staining results on 48 h ( Fig. 2 B ) revealed that most cells (up to 99%) in all groups were stained green and had similar cell morphology, further confirming that HA@ZnS NPs had outstanding cytocompatibility. The migration of endothelial cells plays a crucial role in tissue repair and regeneration( 26 , 27 ). Thus, the effects of HA@ZnS NPs on the migration of HUVECs were also explored in the scratch wound model. As revealed in Fig. 2 C, cell migration in the NPs group was not significantly affected compared to the control group, demonstrating that HA@ZnS NPs do not impair the migration function of endothelial cells, which is the core of angiogenesis. All results together indicated that the concentrations of HA@ZnS NPs used in subsequent studies were safe for H9C2 cells and HUVECs. 3.3. Fluorescence imaging of H 2 S release in vitro and in vivo We conducted further tests on the fluorescence properties of NPs to explore their potential applications in biomedical imaging. WSP-5 is a fluorescent probe containing active disulfides, specifically used for detecting H 2 S in biological samples( 28 ). First, we verified the capacity of NPs and Na 2 S to release H 2 S intracellularly in H9C2 cells ( Fig. 3 A ) . And a single dose of 200 µg/kg HA@ZnS NPs was injected into the hearts of MI rats, followed by the injection of WSP-5 to the same site after 30 min to monitor fluorescence changes ( Fig. 3 B ) . It was found that the green fluorescence representing H 2 S in the heart gradually intensified and peaked at 1-hour postinjection. Then, the fluorescence slowly diminished in the next 23 h ( Fig. 3 C-D ) . Although the intensity become weak at 24-hour postinjection, it was still observable, indicating that NPs could retain at the injection site and ensure steady H 2 S release for a relatively long time ( Fig. 3 C-D ) . In addition, five different doses of NPs were injecting in MI rats, followed by ex vivo imaging, confirming that the fluorescence intensity detected in the heart was highly correlated with the applied dosage ( Fig. 3 E-F ) . Notably, since NPs were locally injected into the myocardium, no fluorescence was observed in other organs after 1 h ( Fig. 3 G ) . This not only demonstrated NPs can accumulate in the heart, but also indicated the metabolism and clearance of NPs within the cardiac tissue. Collectively, these results showcased considerable advantages of NPs in in vivo imaging. 3.4. Therapeutic effects of NPs on repairing cardiac function in MI rats MI refers to the ischemic necrosis of the myocardium, one of severe coronary artery diseases. As the blood flow in the coronary artery is drastically reduced or interrupted, the corresponding myocardium would suffer from severe and persistent acute ischemia, which ultimately leads to the ischemic necrosis of the myocardium( 29 ). When myocardial ischemia occurs, cardiomyocytes undergo necrosis and apoptosis if there is no timely and effective treatment. With the extension of ischemia time, the area of myocardial infarction would continue to expand, ultimately resulting in cardiac insufficiency( 29 , 30 ). Therefore, improving cardiac function is one of the important means for the treatment of MI( 31 ). On day 7, 14, 21, and 28 post-NP injection, the cardiac function of the rats was evaluated using echocardiography, focusing on three indices: left ventricular ejection fraction (EF), fractional shortening (FS), and end-systolic volume (ESV). Figure 4 A and B displayed the echocardiograms and the corresponding quantitative ultrasonic data for each group of myocardial infarction rats. Both the echocardiograms and statistical data reveal that compared with the sham group, the EF and FS values significantly decreased while the ESV values remarkably increased after myocardial infarction. This is primarily due to the extensive death of myocardial cells post-infarction, which induced abnormal cardiac function, thinning of the ventricular walls, and ventricular dilation. These changes in indices indicated the successful construction of the myocardial infarction model in SD rats. The statistical data further implied that over time the cardiac function of the infarct group exhibited a deteriorating trend, i.e., the values of EF and FS continued to decrease, while those of ESV continued to increase. Due to the absence of therapeutic interventions, the condition of the rats in the MI group progressively worsened, ultimately resulting in heart failure. The indices for both Na 2 S group and ZnCl 2 group were almost identical to those of the MI group, i.e., no significant differences were observed. In sharp contrast, the NPs group showed improvements for all indices compared with the MI group, with a surge in EF, suggesting that NPs injection into the infarcted area can ameliorate the function of the damaged myocardium. 3.5. Therapeutic effects of NPs in improving left ventricular remodeling in MI rats MI in rats was induced by ligation of the left anterior descending coronary artery, leading to gradual thinning of the left ventricular wall as the disease progressed, and ultimately replacement of the necrotic tissue with fibrous tissue( 32 ). We assessed the infract size, left ventricular (LV) wall thickness, and LV scar size in MI rats on day 28 post-infarction using TTC staining, HE staining, and Sirius red staining. Compared with the MI group, NPs treatment significantly reduced the size of the myocardial infarction area ( Fig. 4 C and D) . After NPs treatment, fibrosis in the MI area (red) was remarkably reduced and NPs treatment improved the LV wall thickness and LV scar size ( Fig. 4 E and F) . Notably, rats in the NPs group exhibited the lowest degree of fibrosis and the highest degree of normal myocardial recovery in the infarcted area. In contrast, neither the Na 2 S nor ZnCl 2 group showed benefits in reducing infarct size or promoting myocardial recovery. Taken together, these research findings confirm that intramyocardial injection of NPs can effectively lessen the infarct size in myocardial infarction rats and inhibit left ventricular remodeling. 3.6. The mechanism of NPs' therapeutic effects on myocardial ischemic injury IL-6, an important inflammatory factor, is a small molecular protein secreted by macrophages, and involved in the pathological damage of some autoimmune diseases( 33 , 34 ). Following MI, a severe inflammatory response would be induced due to the massive apoptosis of myocardial cells, significantly elevating the expression levels of inflammatory factors. Previous studies have proved that H 2 S can effectively suppress the inflammatory response in the myocardial infarction area( 35 ). After treated with H 2 S, the expression levels of inflammatory factors such as IL-6, IL-8, and TNF-α are significantly declined in animal models with myocardial infarction( 17 ). The primary source of these inflammatory factors is macrophages activated after injury. CD86 is a specific antigen for macrophages, which could be used as the clue for their presence( 36 , 37 ). In this study, we found that the expression of both IL-6 and CD86 cells in the NPs group was much lower than in the MI group ( Fig. 5 A ) . This could be attributed to the anti-inflammatory effect of H 2 S, effectively suppressing the inflammatory response in the myocardial infarction area. α-SMA is a characteristic protein of angiogenesis( 38 ). It was observed that the expression of α-SMA was significantly higher in NPs group than in the MI group ( Fig. 5 A ) , indicating angiogenesis was enhanced in the damaged cardiac tissue upon the intervention of NPs. This effect was also confirmed in the human umbilical vein endothelial cell tubulation assay, where NPs dramatically increased the number of endothelial cell tubules ( Fig. 5 B ) . The anti-inflammatory and angiogenesis-promoting effects of NPs were similarly verified in Western blot, i.e., the expression of the inflammatory indicators IL-6 and CD86 decreased in the NPs group, and the expression of the angiogenesis indicator α-SMA increased in the NPs group. During MI, the hypoxia of cardiac cells in the ischemic area leads to a reduction of ATP production, consequently causing apoptosis of cardiac cells that highly rely on mitochondrial respiration for ATP generation( 39 ). Compared with MI group, as expected, less TUNEL + apoptotic cells were detected in the group treated by NPs ( Fig. 5 C ) , suggesting that NPs can protect ischemic cardiac cells through anti-apoptotic mechanisms. In addition, previous studies have shown that reactive oxygen species (ROS) play a key role in the progression of atherosclerosis and ischaemic heart disease. And excessive ROS leads to sustained oxidative stress and endothelial dysfunction, which in turn leads to disease progression( 40 , 41 ). In the H9C2 hypoxic cell model, we examined the levels of reactive oxygen species using a ROS fluorescence detection probe and showed that NPs were able to significantly weaken ROS levels ( Fig. 5 D ) . The anti-apoptotic effect of NPs was also confirmed in Western blot on rat myocardial tissues ( Fig. 6 ) . The Bcl-2 family of proteins are key regulators of apoptosis, including both pro-apoptotic and pro-survival (anti-apoptotic) members, where Bcl-2 exerts an anti-apoptotic effect, while BAX, a member of the Bcl-2 family, plays a pro-apoptotic role( 42 ). In addition, Caspase s are a specific group of enzymes involved in the process of apoptosis, of which the classical Caspase-3 recognizes and disassembles apoptosis-specific sequences and promotes apoptosis( 43 ). Therefore, the expression of Bcl-2, BAX, and Caspase-3 in myocardial tissues of MI rats was detected by Western blot, and it was found that the expression of Bcl-2 was significantly increased in the NPs group, while the expression of BAX and Caspase-3 was significantly decreased, which further validated the anti-apoptotic effect of NPs. Besides, macrophages in diseased tissues were classified into M1-type (classically activated macrophages) and M2-type (selectively activated macrophages). CD86 is a common marker molecule for M1-type macrophages, which are induced by IFN-γ to produce pro-inflammatory factors. CD206 is a common marker molecule for M2-type macrophages, which are activated by IL-4 to release anti-inflammatory factors( 36 ). By flow cytometric analysis, we examined macrophage polarization in myocardial tissue and found that NPs reduced the proportion of CD86 + M1-type macrophages and increased the proportion of CD206 + M2-type macrophages ( Fig. 7 ) . This further suggests that NPs can improve the function of damaged myocardium through anti-inflammatory effects. 4. Conclusion In this study we reported a novel H 2 S donor, HA@ZnS NPs, for the treatment of myocardial infarction. Releasing study demonstrated that H 2 S release could be triggered by the acidic pH. NPs not only significantly reduced the infarct size but also improved left ventricular remodeling and decreased the levels of inflammatory factors. By promoting angiogenesis in myocardial tissues and reducing cardiomyocyte apoptosis, NPs demonstrated potential in improving cardiac function. These findings provide valuable insights into new therapeutic strategies for myocardial infarction and lay the groundwork for future clinical applications of H 2 S-releasing materials. Declarations Ethics approval and consent to participate The procedures of animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Westchina Hospital, Sichuan University (Chengdu, China) (20230228042). Consent or publication Not applicable. Availability of data and materials Not applicable. Competing interests The authors declare no competing financial interest. Funding This project was supported by National Natural Science Foundation of China (Grant No. 52273090, 22375128, 22105126), the Natural Science Foundation of Sichuan Province (Grant No. 2022NSFSC1395, 2022NSFSC1489), Post-Doctor Research Project, West China Hospital, Sichuan University (Grant No. 20HXBH171, 2021HXBH070), Postdoctoral Science Foundation of China (Grant No. 2021M692282), and the Natural Science Foundation of Shanghai (22ZR1433500). Authors' contributions Y.Z.: Methodology; Validation; Visualization; Writing – original draft; Writing – review & editing. X.Z.: Conceptualization; Investigation; Methodology; Software; Validation; Writing – original draft; Writing – review. S.B.: Supervision; Validation; Writing – review. R.L.: Supervision; Validation; Writing – review. Y.G.: Supervision; Validation; Writing – review. J.G.: Funding acquisition; Project administration; Supervision; Validation; Writing – review & editing. Y.W.: Funding acquisition; Project administration; Supervision; Validation; Writing – review & editing. Acknowledgements Not applicable. Authors' information Y.Z. a , [email protected] ; X.Z. a , [email protected] ; S.B. b , [email protected] ; R.L. b , [email protected] ; Y.G. c , [email protected] ; J.G. a , [email protected] ; Y.W. c , [email protected] a Department of Cardiovascular Surgery, West China Hospital, Sichuan University, Chengdu, 610000, China b Department of Burn and Plastic Surgery, West China Hospital, Sichuan University, Chengdu, 610000, China c Engineering Research Center of Cell & Therapeutic Antibody, Shanghai Frontiers Science Center of Drug Target Identification and Delivery, National Key Laboratory of Innovative Immunotherapy, School of Pharmaceutical Sciences, Shanghai Jiao Tong University, Shanghai 200240, China References Zheng Z, Tan Y, Li Y, Liu Y, Yi GH, Yu CY, et al. Biotherapeutic-loaded injectable hydrogels as a synergistic strategy to support myocardial repair after myocardial infarction. Journal of Controlled Release. 2021;335:216-36. 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DOI: 10.1002/med.21433 Gemici B, Elsheikh W, Feitosa KB, Costa SKP, Muscara MN, Wallace JL. H2S-releasing drugs: Anti-inflammatory, cytoprotective and chemopreventative potential. Nitric Oxide-Biology and Chemistry. 2015;46:25-31. DOI: 10.1016/j.niox.2014.11.010 Calvert JW, Jha S, Gundewar S, Elrod JW, Ramachandran A, Pattillo CB, et al. Hydrogen Sulfide Mediates Cardioprotection Through Nrf2 Signaling. Circulation Research. 2009;105(4):365-U105. DOI: 10.1161/circresaha.109.199919 Elrod JW, Calvert JW, Morrison J, Doeller JE, Kraus DW, Tao L, et al. Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function. Proceedings of the National Academy of Sciences of the United States of America. 2007;104(39):15560-5. DOI: 10.1073/pnas.0705891104 Sodha NR, Clements RT, Feng J, Liu YH, Bianchi C, Horvath EM, et al. Hydrogen sulfide therapy attenuates the inflammatory response in a porcine model of myocardial ischemia/reperfusion injury. Journal of Thoracic and Cardiovascular Surgery. 2009;138(4):977-84. DOI: 10.1016/j.jtcvs.2008.08.074 Rong F, Wang TJ, Zhou Q, Peng HW, Yang JT, Fan QL, et al. Intelligent polymeric hydrogen sulfide delivery systems for therapeutic applications. Bioactive Materials. 2023;19:198-216. DOI: 10.1016/j.bioactmat.2022.03.043 Powell CR, Dillon KM, Matson JB. A review of hydrogen sulfide (H2S) donors: Chemistry and potential therapeutic applications. Biochemical Pharmacology. 2018;149:110-23. DOI: 10.1016/j.bcp.2017.11.014 Zhu YM, Archer WR, Morales KF, Schulz MD, Wang Y, Matson JB. Enzyme-Triggered Chemodynamic Therapy via a Peptide-H2S Donor Conjugate with Complexed Fe2+. Angewandte Chemie-International Edition. 2023;62(22):e202302303. DOI: 10.1002/anie.202302303 Wang ZX, Ge YX, Liu JQ, Shi PYF, Xue RY, Hao B, et al. Integrating a Biomineralized Nanocluster for H2S-Sensitized ROS Bomb against Breast Cancer. Nano Letters. 2024;24(8):2661-70. DOI: 10.1021/acs.nanolett.4c00347 Ge YX, Rong F, Li W, Wang Y. On-demand therapeutic delivery of hydrogen sulfide aided by biomolecules. Journal of Controlled Release. 2022;352:586-99. DOI: 10.1016/j.jconrel.2022.10.055 Szabo C, Papapetropoulos A. International Union of Basic and Clinical Pharmacology. CII: Pharmacological Modulation of H2S Levels: H2S Donors and H2S Biosynthesis Inhibitors. Pharmacological Reviews. 2017;69(4):497-564. DOI: 10.1124/pr.117.014050 Zhang Q, Chen L, Huang LY, Cheng HX, Wang L, Xu L, et al. CD44 promotes angiogenesis in myocardial infarction through regulating plasma exosome uptake and further enhancing FGFR2 signaling transduction. Molecular Medicine. 2022;28(1):145. DOI: 10.1186/s10020-022-00575-5 Teriete P, Banerji S, Noble M, Blundell CD, Wright AJ, Pickford AR, et al. Structure of the regulatory hyaluronan binding domain in the inflammatory leukocyte homing receptor CD44. Molecular Cell. 2004;13(4):483-96. DOI: 10.1016/s1097-2765(04)00080-2 Hasan A, Khattab A, Islam MA, Abou Hweij K, Zeitouny J, Waters R, et al. Injectable Hydrogels for Cardiac Tissue Repair after Myocardial Infarction. Advanced Science. 2015;2(11):1500122. DOI: 10.1002/advs.201500122 Li RT, Liu K, Huang X, Li D, Ding JX, Liu B, et al. Bioactive Materials Promote Wound Healing through Modulation of Cell Behaviors. Advanced Science. 2022;9(10):e2105152. DOI: 10.1002/advs.202105152 Zhou YZ, Mazur F, Liang K, Chandrawati R. Sensitivity and Selectivity Analysis of Fluorescent Probes for Hydrogen Sulfide Detection. Chemistry-an Asian Journal. 2022;17(5):e202101399. DOI: 10.1002/asia.202101399 Frantz S, Hundertmark MJ, Schulz-Menger J, Bengel FM, Bauersachs J. Left ventricular remodelling post-myocardial infarction: pathophysiology, imaging, and novel therapies. European Heart Journal. 2022;43(27):2549-+. DOI: 10.1093/eurheartj/ehac223 Zhang Q, Wang L, Wang SQ, Cheng HX, Xu L, Pei GQ, et al. Signaling pathways and targeted therapy for myocardial infarction. Signal Transduction and Targeted Therapy. 2022;7(1):78. DOI: 10.1038/s41392-022-00925-z Shi HT, Huang ZH, Xu TZ, Sun AJ, Ge JB. New diagnostic and therapeutic strategies for myocardial infarction via nanomaterials. Ebiomedicine. 2022;78:103968. DOI: 10.1016/j.ebiom.2022.103968 Hundahl LA, Tfelt-Hansen J, Jespersen T. Rat Models of Ventricular Fibrillation Following Acute Myocardial Infarction. Journal of Cardiovascular Pharmacology and Therapeutics. 2017;22(6):514-28. DOI: 10.1177/1074248417702894 Broch K, Anstensrud AK, Woxholt S, Sharma K, Tollefsen IM, Bendz B, et al. Randomized Trial of Interleukin-6 Receptor Inhibition in Patients With Acute ST-Segment Elevation Myocardial Infarction. Journal of the American College of Cardiology. 2021;77(15):1845-55. DOI: 10.1016/j.jacc.2021.02.049 Sun JY, Du LJ, Shi XR, Zhang YY, Liu Y, Wang YL, et al. An IL-6/STAT3/MR/FGF21 axis mediates heart-liver cross-talk after myocardial infarction. Science Advances. 2023;9(14):eade4110. DOI: 10.1126/sciadv.ade4110 Wu T, Li H, Wu B, Zhang L, Wu SW, Wang JN, et al. Hydrogen Sulfide Reduces Recruitment of CD11b<SUP>+</SUP>Gr-1<SUP>+</SUP> Cells in Mice With Myocardial Infarction. Cell Transplantation. 2017;26(5):753-64. DOI: 10.3727/096368917x695029 Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. Journal of Clinical Investigation. 2012;122(3):787-95. DOI: 10.1172/jci59643 Ryabov V, Gombozhapova A, Rogovskaya Y, Kzhyshkowska J, Rebenkova M, Karpov R. Cardiac CD68+and stabilin-1+macrophages in wound healing following myocardial infarction: From experiment to clinic. Immunobiology. 2018;223(4-5):413-21. DOI: 10.1016/j.imbio.2017.11.006 Venugopal H, Hanna A, Humeres C, Frangogiannis NG. Properties and Functions of Fibroblasts and Myofibroblasts in Myocardial Infarction. Cells. 2022;11(9):1386. DOI: 10.3390/cells11091386 Wang XW, Guo ZK, Ding ZF, Mehta JL. Inflammation, Autophagy, and Apoptosis After Myocardial Infarction. Journal of the American Heart Association. 2018;7(9):e008024. DOI: 10.1161/jaha.117.008024 Zhang Z, Dalan R, Hu ZY, Wang JW, Chew NW, Poh KK, et al. Reactive Oxygen Species Scavenging Nanomedicine for the Treatment of Ischemic Heart Disease. Advanced Materials. 2022;34(35):e2202169. DOI: 10.1002/adma.202202169 Zhang YX, Murugesan P, Huang K, Cai H. NADPH oxidases and oxidase crosstalk in cardiovascular diseases: novel therapeutic targets. Nature Reviews Cardiology. 2020;17(3):170-94. DOI: 10.1038/s41569-019-0260-8 Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nature Reviews Molecular Cell Biology. 2019;20(3):175-93. DOI: 10.1038/s41580-018-0089-8 Riedl SJ, Shi YG. Molecular mechanisms of caspase regulation during apoptosis. Nature Reviews Molecular Cell Biology. 2004;5(11):897-907. DOI: 10.1038/nrm1496 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4987842\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":362552006,\"identity\":\"33a7a135-9060-4417-94e3-03ca6ae1a643\",\"order_by\":0,\"name\":\"Yujia Zhan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Sichuan University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yujia\",\"middleName\":\"\",\"lastName\":\"Zhan\",\"suffix\":\"\"},{\"id\":362552007,\"identity\":\"d9ab8433-8189-403e-beda-a5845baa47a5\",\"order_by\":1,\"name\":\"Xueshan Zhao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Sichuan University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xueshan\",\"middleName\":\"\",\"lastName\":\"Zhao\",\"suffix\":\"\"},{\"id\":362552008,\"identity\":\"9bdae6dc-7827-4612-9e3b-ee471102bc3e\",\"order_by\":2,\"name\":\"Siwei Bi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Sichuan University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Siwei\",\"middleName\":\"\",\"lastName\":\"Bi\",\"suffix\":\"\"},{\"id\":362552009,\"identity\":\"e7282e71-adda-45e7-a2c4-bda4ce03e753\",\"order_by\":3,\"name\":\"Ruiqi Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Sichuan University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ruiqi\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":362552010,\"identity\":\"f142aab9-4aba-4ded-a503-f107b1bb1dcf\",\"order_by\":4,\"name\":\"Yuxuan Ge\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shanghai Jiao Tong University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yuxuan\",\"middleName\":\"\",\"lastName\":\"Ge\",\"suffix\":\"\"},{\"id\":362552011,\"identity\":\"b7bfc055-e7a1-4cca-b39a-7b82a46737f1\",\"order_by\":5,\"name\":\"Jun Gu\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYHCCBAaGCgYGAxK1nCFRCwMDYxspWuQjEp5JF847LG/Of/iZBEPNHcJaDG8kpEnP3HbYcOeMNDMJhmPPiNAyA6iFd9vtBIMbPGwSjA2HidUyB6jl/BkitchLgLQ0ALUcyCFSiwHPg2RrnmP/DTfcSDO2SDhGjC3tOYm3eWrS5A3OH35440MNMbYc4ElA8BJwKUOxpYH9ADHqRsEoGAWjYCQDAJekOL7UVoVMAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Sichuan University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Jun\",\"middleName\":\"\",\"lastName\":\"Gu\",\"suffix\":\"\"},{\"id\":362552012,\"identity\":\"72c44301-f066-474e-ab36-e8c742f15603\",\"order_by\":6,\"name\":\"Yin Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shanghai Jiao Tong University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yin\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-08-28 03:39:37\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4987842/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4987842/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":66064483,\"identity\":\"91c57ab9-2b8b-4c37-a318-42d69fad593e\",\"added_by\":\"auto\",\"created_at\":\"2024-10-07 11:02:19\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":477712,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA schematic diagram of HA@ZnS NPs. (A) Synthesis of HA@ZnS NPs and their mechanism of treating myocardial infarction. (B) Representative transmission electron microscope (TEM) image of HA@ZnS NPs. (C) Hydrodynamic size distribution of HA@ZnS NPs in deionized water. (D) H\\u003csub\\u003e2\\u003c/sub\\u003eS release curves of HA@ZnS NPs in 25 mM acetate buffer (pH 7.4 and 5.5).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4987842/v1/6888417f4a2549a3fcc5b0f7.png\"},{\"id\":66064476,\"identity\":\"23a53806-92d0-4d19-beaf-970d730778c7\",\"added_by\":\"auto\",\"created_at\":\"2024-10-07 11:02:19\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":671767,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Cell viability of H9C2 cells and HUVECs treated with different concentrations of HA@ZnS NPs (n=6). (B) Live/dead staining (Calcein-AM/PI) of H9C2 cells and HUVECs with different treatments for 48 h (NPs = 10 μg/mL). (C) The cell scratch test for different treatments at different time (0, 24, 48 h) (NPs = 10 μg/mL).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4987842/v1/eba90831c3de4565b2bad99b.png\"},{\"id\":66064480,\"identity\":\"40dc238c-1f16-4cff-b043-cbc539436a03\",\"added_by\":\"auto\",\"created_at\":\"2024-10-07 11:02:19\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":749444,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFluorescence imaging of H\\u003csub\\u003e2\\u003c/sub\\u003eS release from HA@ZnS NPs \\u003cem\\u003ein vitro\\u003c/em\\u003e and \\u003cem\\u003ein vivo\\u003c/em\\u003e. (A) \\u003cem\\u003eIn vitro\\u003c/em\\u003e fluorescence images indicate H\\u003csub\\u003e2\\u003c/sub\\u003eS release in H9C2 cells (Na\\u003csub\\u003e2\\u003c/sub\\u003eS = 20 μM, NPs = 20 μM). (B) \\u003cem\\u003eIn vivo\\u003c/em\\u003e fluorescence images indicate H\\u003csub\\u003e2\\u003c/sub\\u003eS release in MI rats after different treatments. MI, untreated MI rats; Sham+NPs, healthy rats injected with NPs; MI+NPs, MI rats treated with NPs. (C-D) Ex vivo fluorescence images (C) and quantification (D) of H\\u003csub\\u003e2\\u003c/sub\\u003eS release in hearts collected from MI rats at different time points after treatment with the same dose of NPs. (E-F) Ex vivo fluorescence images (E) and quantification analysis (F) of H\\u003csub\\u003e2\\u003c/sub\\u003eS release in hearts of MI rats treated with different doses of NPs. (G) Ex vivo image shows the distribution of NPs in different organs at 1 h post NPs administration. Data in (D, F) are expressed as means ± SD (n = 5).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4987842/v1/48e412c6b9a5e66ef023dd93.png\"},{\"id\":66064478,\"identity\":\"7fccd2aa-0613-4f59-b3d7-f3fe32acea37\",\"added_by\":\"auto\",\"created_at\":\"2024-10-07 11:02:19\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":513495,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eHeart function and structure evaluations in MI rats treated with different groups. (A) Representative echocardiographic images of different treatment groups after 28 days. (B) Quantified values of EF, FS, and ESV at varied time points during different treatments. (C-D) Illustrative digital photographs of TTC-stained myocardial sections (C) and measured infarct sizes (D) across various groups (7 days post-myocardial infarction). After TTC staining, the infarcted myocardial tissue appears white, in contrast to the non-infarcted myocardium, which is red. (E) Representative heart horizontal panoramic views and microscopic images of myocardial sections stained with H\\u0026amp;E or Sirius red. (F) The LV wall thickness and LV scar size quantified by the midline method based on H\\u0026amp;E and Masson sections. Sham, with thoracotomy but without LAD ligation; MI, with LAD ligation and saline injection; ZnCl\\u003csub\\u003e2\\u003c/sub\\u003e, underwent LAD ligation, injected with 200 μg/kg of ZnCl\\u003csub\\u003e2\\u003c/sub\\u003e); Na\\u003csub\\u003e2\\u003c/sub\\u003eS, underwent LAD ligation and injected with 200 μg/kg of Na\\u003csub\\u003e2\\u003c/sub\\u003eS; NPs, underwent LAD ligation and injected with 200 μg/kg of NPs. Data are expressed as the mean ± standard deviation from at least three independent replicates (n = 6). **P \\u0026lt; 0.01, ***P \\u0026lt; 0.001; ns, no significance.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4987842/v1/358f59d9f42ccf2ff41b61b1.png\"},{\"id\":66065110,\"identity\":\"8814f97a-6278-4f8a-ba50-1d0fdba33000\",\"added_by\":\"auto\",\"created_at\":\"2024-10-07 11:10:19\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":704061,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eIn vivo \\u003c/em\\u003eanti-inflammatory effects, angiogenic effects, and anti-apoptotic effects of NPs (NPs = 200 μg/kg). (A) immunohistochemical staining and quantification of IL- 6, CD86, and α-SMA. The brown part represents positive cells. (B) Tube-forming experiments in HUVEC. Data are expressed as mean ± SD of 3 independent replicates. **P \\u0026lt; 0.01, ***P \\u0026lt; 0.001. (C) Representative fluorescent micrographs showing TUNEL+ apoptotic cells (green) in the hearts on day 7 after different treatments. Data are expressed as means ± SD from three independent replicates. **P \\u0026lt; 0.01, ***P \\u0026lt;0.001. (D) Detection of ROS relative fluorescence intensity using DHE fluorescent probe in H9C2 hypoxic cell model.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4987842/v1/78bd21b8be2bd45415e9e492.png\"},{\"id\":66065332,\"identity\":\"b396b066-84fd-4baa-bbed-7d8417134945\",\"added_by\":\"auto\",\"created_at\":\"2024-10-07 11:18:19\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":139336,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIn vivo anti-inflammatory effects, angiogenic effects, and anti-apoptotic effects of NPs (NPs = 200 μg/kg). The expression of IL-6, CD86, α-SMA, Bcl-2, BAX, and Caspase-3 in myocardial tissues of MI rats measured by Western blot. Data are expressed as mean ± SD of 3 independent replicates. **P \\u0026lt; 0.01, ***P \\u0026lt; 0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4987842/v1/52b3eb4ebe7be47ebcdd76a4.png\"},{\"id\":66065111,\"identity\":\"e12c7e7d-4cbb-4fc7-923e-8bfb0c83d4db\",\"added_by\":\"auto\",\"created_at\":\"2024-10-07 11:10:19\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":230608,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Polarization of cardiac macrophages determined using flow cytometric analysis. (B) Percentage of CD86\\u003csup\\u003e+\\u003c/sup\\u003e M1-type macrophages in the heart. (C) Percentage of CD206\\u003csup\\u003e+\\u003c/sup\\u003e M2 type macrophages in the heart. *P \\u0026lt; 0.05\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4987842/v1/a752a20f325357bf4558bb9f.png\"},{\"id\":70403950,\"identity\":\"62e1c041-e275-43ab-942d-b3a033b8405d\",\"added_by\":\"auto\",\"created_at\":\"2024-12-02 22:53:45\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3980365,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4987842/v1/cec3e6ed-3885-482c-9be6-fb6d2a3e6cfb.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Alleviation of Myocardial Infarction by Hydrogen Sulfide-Releasing Nanoparticles: Mechanisms and Therapeutic Effects\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eMyocardial infarction (MI) occurs due to coronary artery obstruction or rupture, leading to ischemia and hypoxia-induced necrosis in myocardial tissues. It is one of the clinical syndromes of coronary atherosclerotic heart disease (coronary heart disease)(\\u003cspan additionalcitationids=\\\"CR2 CR3\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e), and has become one of the common severe diseases globally threatening human\\u0026rsquo;s health(\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e). Coronary heart disease is prevalent in 6.3% of the adults in the United States, and 1 in every 7 deaths is primarily due to coronary heart disease. Moreover, MI is prevalent among 7.9\\u0026nbsp;million adults, with a new case occurring approximately every 40 seconds(\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e). Given that the risk of premature cardiovascular diseases in young people is increasing(\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e), the prevention and treatment of MI have become in a huge need.\\u003c/p\\u003e \\u003cp\\u003eRecently, emerging fields such as tissue engineering and biomaterials have begun to offer potential therapeutic approaches to address the dilemma(\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e). For instance, integrating biomaterials with clinical treatment methods could slow the progression of ventricular remodeling and rebuild cardiac function post-infarction(\\u003cspan additionalcitationids=\\\"CR9\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e). More recently, gasotransmitters have garnered widespread attention as an emerging therapeutic modality because of their excellent therapeutic efficacy and good biocompatibility. The three recognized gasotransmitters are nitric oxide (NO)(\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e), carbon monoxide (CO)(\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e), and hydrogen sulfide (H\\u003csub\\u003e2\\u003c/sub\\u003eS)(\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e). These gases are implicated in a wide range of physiological and pathological processes, such as cardiovascular diseases, oxidative stress, and inflammation. Therefore, developing targeted delivery systems capable of releasing therapeutic doses of gasotransmitters represents an ideal tool in modern medicine(\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e). It is proven that exogenous H\\u003csub\\u003e2\\u003c/sub\\u003eS plays a vital role in the treatment of MI. Studies have shown that animals treated with H\\u003csub\\u003e2\\u003c/sub\\u003eS exhibit reduced myocardial infarction area, improved left ventricular fractional shortening in the risk zone, and enhanced microvascular reactivity. Moreover, both short-term and long-term administration of H\\u003csub\\u003e2\\u003c/sub\\u003eS donors can lower blood pressure(\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e). Besides, the expression levels of inflammatory factors such as IL-6, IL-8, and TNF-α in myocardial tissue decline following H\\u003csub\\u003e2\\u003c/sub\\u003eS treatment(\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). These experimental results collectively suggest that supplementing H\\u003csub\\u003e2\\u003c/sub\\u003eS is highly meaningful for treating MI. It should be noted that the H\\u003csub\\u003e2\\u003c/sub\\u003eS dose-response curve is bell-shaped, with its therapeutic effects limited to a narrow range(\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e). Therefore, precise dose control is a key issue in the development of H\\u003csub\\u003e2\\u003c/sub\\u003eS-based therapies for MI treatment.\\u003c/p\\u003e \\u003cp\\u003eIn the past few years, various types of H\\u003csub\\u003e2\\u003c/sub\\u003eS donors responsive to different stimuli including light, biological thiols, pH changes, enzymatic activity, and others have been developed(\\u003cspan additionalcitationids=\\\"CR20 CR21\\\" citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). Despite huge advances have been achieved, most suffered from low water solubility, limited means to modulate release kinetics, and no capacity for targeted delivery, all of which may limit H\\u003csub\\u003e2\\u003c/sub\\u003eS-based treatments due to the reactive nature of this signaling molecule(\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTo address the aforementioned critical issue, we prepared an H\\u003csub\\u003e2\\u003c/sub\\u003eS donor-zinc sulfide nanoparticles (NPs) from hyaluronic acid by biomineralization. It is assumed that the H\\u003csub\\u003e2\\u003c/sub\\u003eS could be steadily released from NPs at the lesion site of myocardial ischemic injury, which prevents the injury from further deterioration. The protective effects of NPs on myocardial ischemic injury were carefully examined on rats \\u003cb\\u003e(Fig.\\u0026nbsp;1A)\\u003c/b\\u003e. And the underlying mechanisms were deciphered both \\u003cem\\u003ein vitro\\u003c/em\\u003e and \\u003cem\\u003ein vivo\\u003c/em\\u003e.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1. Materials\\u003c/h2\\u003e \\u003cp\\u003eSodium hyaluronate (HA, mW\\u0026thinsp;\\u0026asymp;\\u0026thinsp;3 KDa) was purchased from Bloomage Biotech (Beijing, China). Sodium sulfide nonahydrate (Na\\u003csub\\u003e2\\u003c/sub\\u003eS\\u0026middot;9H\\u003csub\\u003e2\\u003c/sub\\u003eO), zinc acetate (Zn(CH\\u003csub\\u003e3\\u003c/sub\\u003eCOO)\\u003csub\\u003e2\\u003c/sub\\u003e), 30 wt % hydrogen peroxide solution (H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e), and paraformaldehyde solution (4 wt% PFA) were purchased from Titan Biotechnology (Shanghai, China). HA@ZnS nanoparticles (NPs) were prepared according to the published procedure with a slight modification(\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e). Ferric chloride hexahydrate (FeCl\\u003csub\\u003e3\\u003c/sub\\u003e \\u0026bull; 6H\\u003csub\\u003e2\\u003c/sub\\u003eO) and 4-amino-\\u003cem\\u003eN, N\\u003c/em\\u003e-dimethylaniline sulfate (DMPD) were purchased from Aladdin Biochem Technology (Shanghai, China). Dulbecco\\u0026rsquo;s modified Eagle\\u0026rsquo;s medium (DMEM) medium, trypsin, penicillin, and fetal bovine serum (FBS) were purchased from HyClone (Waltham, USA). Dimethyl sulfoxide (DMSO), Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) kit, and 5% normal goat serum were purchased from Thermo Fisher Scientific (Massachusetts, USA), Dulbecco\\u0026rsquo;s phosphate-buffered saline (DPBS) and streptomycin were purchased from Solarbio (Beijing, China). Bicinchoninic acid (BCA) protein assay kit was purchased from Abcam (Cambridge, UK). CCK-8 reagent was purchased from Solarbio (Beijing China). Antibodies: CD86, IL-6, α-smooth muscle actin (α-SMA), Bcl-2, BAX, and Caspase-3 were purchased from Abcam (Cambridge, UK). Secondary antibodies: Alexa Fluor 488 and 594 were purchased from Molecular Probes (California, USA).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. Animals and cells\\u003c/h2\\u003e \\u003cp\\u003e The procedures of animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Westchina Hospital, Sichuan University (Chengdu, China) (20230228042). Adult male Sprague-Dawley rats (10 weeks old, 200\\u0026ndash;250 g) were purchased from the Chengdu Dashuo Biotechnology Co., Ltd and housed in standard cages under standard conditions. All animals were acclimatized for one week before use. Rat cardiomyocytes cells (H9C2) and human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC, Manassas, USA), and they were cultured in DMEM with high glucose supplemented with 10% (v/v) FBS, 100 U/mL of penicillin, and 100 mg/mL of streptomycin at 37\\u0026deg;C in a humidified incubator with 95% air and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. In vitro cell studies\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.1. In vitro fluorescence imaging of H\\u003csub\\u003e2\\u003c/sub\\u003eS release\\u003c/h2\\u003e \\u003cp\\u003eH9C2 cells (2 x 10\\u0026sup3;/ well in 12-well plates) were incubated with 1.5 mL of glucose-free DMEM containing Na2S or HA@ZnS NPs at 37\\u0026deg;C. Subsequently, the pH values were adjusted to 5.5. The incubation lasted for 1 h, and the mixture of WSP-5 (50 \\u0026micro;M) and CTAB (200 \\u0026micro;M) was added after removing the culture medium. The green fluorescence was then observed using the fluorescence microscope.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.2. Cytocompatibility test\\u003c/h2\\u003e \\u003cp\\u003eTo evaluate the cytotoxicity of HA@ZnS NPs, H9C2 cells (8 x 10\\u003csup\\u003e3\\u003c/sup\\u003e/ well in 96-well plates) were incubated with 200 \\u0026micro;L of glucose-free DMEM containing different concentrations of HA@ZnS NPs at 37\\u0026deg;C for 24 h, then the culture medium was removed and replaced with serum-free DMEM containing 10 wt% of CCK-8 solution. After incubation for 1 h, the absorbance at 450 nm was collected using the Multi-Detection Reader.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.3. ROS assay test\\u003c/h2\\u003e \\u003cp\\u003eROS production in H9C2 cells was analyzed using dihydroethidium (DHE). H9C2 cells were incubated with 10 \\u0026micro;M of DHE at 37\\u0026deg;C for 30 minutes and then washed twice with PBS. DHE staining was then observed using a fluorescence microscope to detect ROS production. Fluorescence was collected at 535 nm excitation and 610 nm emission using a Multi-Detection Reader.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.3.4. HUVEC tube formation assay\\u003c/h2\\u003e \\u003cp\\u003eTube formation assay was performed using tanswell chambers (65 mm PET membrane, 0.4 \\u0026micro;m pore size) and 24-well plates. The matrix gel was spread evenly in the 24-well plate and incubated at 37\\u0026deg;C for 30 min. After co-culture for 7 days, 1.5x10\\u003csup\\u003e5\\u003c/sup\\u003e HUVECs/well (3 replicates per group) were inoculated into the 24-well plate and cultured using the complete medium. The control group was incubated at 37\\u0026deg;C for 12 h, and the NPs group was incubated with 10 \\u0026micro;g/mL of HA@ZnS NPs at 37\\u0026deg;C for 12 h. The number of lumen formations was counted in the top, bottom, left, right, and center of view, and the average value was taken. The control data were used as one to calculate the relative formation rate of the number of tubules.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. In vivo studies\\u003c/h2\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.1. Establishment of MI model on rats\\u003c/h2\\u003e \\u003cp\\u003eAdult male Sprague-Dawley rats (SD rats, 10 weeks old, 200\\u0026ndash;250 g) were used for the studies. Rats were anesthetized with isoflurane, intubated for respiratory support, and maintained under anesthesia using a gas anesthetic machine. The heart was exposed and the left anterior descending coronary artery was ligated to create left ventricular infarction. Then, after ligation for 30 min, NPs or saline (10 injections for 200 \\u0026micro;L in total) was injected at the periphery and center of the infarct site, followed by suturing of the surgical wound on the rats' chest. Rats were randomly divided into 5 groups: (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e) Sham group; (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e) MI group (underwent LAD ligation and saline injection); (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e) ZnCl\\u003csub\\u003e2\\u003c/sub\\u003e group (underwent LAD ligation and injected with 200 \\u0026micro;g/kg of ZnCl\\u003csub\\u003e2\\u003c/sub\\u003e); (\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e) Na\\u003csub\\u003e2\\u003c/sub\\u003eS group (underwent LAD ligation and injected with 200 \\u0026micro;g/kg of Na\\u003csub\\u003e2\\u003c/sub\\u003eS); (\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e) NPs group (underwent LAD ligation and injected with 200 \\u0026micro;g/kg of NPs).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.2. In vivo fluorescence imaging of H2S release\\u003c/h2\\u003e \\u003cp\\u003eTo assess the imaging of NPs in the hearts of MI rats, SD rats were divided into three groups: (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e) MI group, untreated MI rats; (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e) Sham\\u0026thinsp;+\\u0026thinsp;NPs group (200 \\u0026micro;g/kg), healthy rats injected with NPs; (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e) MI\\u0026thinsp;+\\u0026thinsp;NPs group, MI rats injected with NPs (200 \\u0026micro;g/kg). 30 min after NPs administration, rats in Sham\\u0026thinsp;+\\u0026thinsp;NPs group and MI\\u0026thinsp;+\\u0026thinsp;NPs group were locally injected with 100 \\u0026micro;L of 25 \\u0026micro;M WSP-5 (dissolved in DMSO) and 10 \\u0026micro;L of 100 \\u0026micro;M CTAB (dissolved in ddH\\u003csub\\u003e2\\u003c/sub\\u003eO) to give the total volume to 200 \\u0026micro;L. 30 min after WSP-5 administration, imaging was performed using Spectral Instruments Imaging (Kino, U.S.A) in fluorescence mode (excitation: 500 nm; emission: 525 nm).\\u003c/p\\u003e \\u003cp\\u003eTo study the relationship between NPs dosage and fluorescence intensity, MI rats received a single local injection of different NPs doses (25, 50, 100, 150, 200 \\u0026micro;g/kg) 30 min post-ligation. Control rats were injected with saline. 1 h after injection, rats were euthanized. Hearts were harvested, and ex vivo imaging was conducted using Spectral Instruments Imaging in fluorescence mode.\\u003c/p\\u003e \\u003cp\\u003eTo study the metabolism and biodistribution of NPS in MI rats, rats received a single local injection of NPS (200 \\u0026micro;g/kg) 30 min post-ligation. At each predetermined time point (0.5, 1, 6, 8, 12, and 24 h post-injection), rats were euthanized, and major organs such as the heart, liver, spleen, and kidney were collected for ex vivo imaging.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.3. Echocardiographic assessment of cardiac function\\u003c/h2\\u003e \\u003cp\\u003eAt 7, 14, 21, and 28 days post-surgery, echocardiography was used to evaluate cardiac function in five groups of rats. First, the SD rats were weighed, and chloral hydrate was administered intraperitoneally as an anesthetic at 0.024 g/100 g. Once fully anesthetized and immobilized, the fur at rats' neck and chest was removed using a specialized depilatory cream. The rats were secured on the test table, and ultrasound coupling agent was uniformly applied to the chest for imaging. Echocardiography was conducted using the Vevo 3100 Imaging System (Visual Sonics, Canada). Several parameters including left ventricular ejection fraction (EF), fractional shortening (FS), and end-systolic volume (ESV) were measured and the average values of three separated cardiac cycles were used to represent the cardiac function.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.4. Histology examination\\u003c/h2\\u003e \\u003cp\\u003eTriphenyltetrazolium chloride (TTC) staining was used to assess infarct size 7 days post-myocardial infarction. The heart was sectioned into four horizontal slices (2 mm thick) and incubated in 1% TTC to differentiate between the infarcted and viable myocardium. The infarct sizes were quantified using Image J.\\u003c/p\\u003e \\u003cp\\u003e28 days post-surgery, the SD rats were weighed and anesthetized with an intraperitoneal injection of chloral hydrate at a dosage of 0.024 g/100 g. Once the rats were fully immobilized, their abdomens were incised with a surgical scalpel, and blood was withdrawn from the abdominal aorta using a 10 mL syringe. The excised hearts were thoroughly rinsed with saline and then fixed in a 4% paraformaldehyde solution. The hearts were dehydrated in a graded series of 75% ethanol, embedded in paraffin, and serially sectioned (5 \\u0026micro;m thick) from apex to base, perpendicular to the longitudinal axis of the heart, ensuring a complete representative cross-section. Slides were stained with Hematoxylin and Eosin (H\\u0026amp;E) and Picrosirius Red, then observed under a light microscope for collagen deposition in the myocardial infarction area and morphological changes in surrounding tissues. With Picrosirius Red staining, collagen appears red and normal myocardial tissue appears yellow. Finally, the slides were quantitatively analyzed to measure the infract size, the left ventricular wall thickness and the left ventricular scar size.\\u003c/p\\u003e \\u003cp\\u003eFor the immunohistochemical analysis, slides were pretreated with 3% hydrogen peroxide in PBS for 10 min at room temperature to suppress the endogenous peroxidase activity. Subsequently, the slides were incubated in a blocking solution composed of 5% normal goat serum in DPBS for 1 h and then treated with the primary antibody at 4\\u0026deg;C. The antibodies employed included CD86, IL-6, α-SMA. The slides were then treated with Alexa Fluor 488 or 594 secondary antibodies. After washing, antifade mounting medium with DAPI (H-1200-10, Vector Labs Inc., Malvern, PA, USA) was applied to the slides. Fluorescent cells were visualized using an Eclipse Ti2 microscope (Nikon, Japan).\\u003c/p\\u003e \\u003cp\\u003eFor cell apoptosis assays, heart paraffin sections were stained with the TUNEL kit. DAPI was used for nuclear staining. Images were taken by an Olympus Research Slide Scanner VS200.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.5. Western Blot\\u003c/h2\\u003e \\u003cp\\u003eProtein concentrations were measured with the Bicinchoninic Acid (BCA) Protein Assay Kit. Equal volumes of protein samples were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then incubated overnight at 4 \\u003csup\\u003e◦\\u003c/sup\\u003eC with antibodies specific for the CD86, IL-6, α-SMA, Bcl-2, BAX, and Caspase-3. On the following day, the membranes were washed and incubated with horseradish peroxidase (HRP)-coupled secondary antibodies. The blots were detected by enhanced chemiluminescence and quantified using the Bio-Rad imaging system.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e2.4.6. Flow cytological analysis\\u003c/h2\\u003e \\u003cp\\u003eOn one day post-MI, the hearts were perfused with pre-cooled PBS. The injured left ventricular tissues were taken and separated by GentleMACS dissociator. The tissue was digested for 30 min at room temperature at 200 rpm in 5 mL of HBSS buffer containing collagenase II, collagenase IV, and DNase I. The resulting suspension was filtered (70 \\u0026micro;m) to produce a single cell suspension. The suspension was centrifuged at 300 \\u0026times;g for 5 min. Cardiomyocytes were then suspended in DMEM culture medium (with the addition of 10% fetal bovine serum and 1% penicillin-streptomycin) and cultured at 37\\u0026deg;C in 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e for 2 h. Cells were then washed with PBS and cardiac macrophages were concentrated in adherent cells. Cells were collected and incubated with PerCP-Cy5.5-CD86 and PE-Cy7-CD206 with flow cytometry antibodies for 15 min in the dark at 4\\u0026deg;C. After washing with PBS, cardiac macrophage phenotypes were detected by BD FACSCanto II flow cytometry.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5. Statistical analysis\\u003c/h2\\u003e \\u003cp\\u003eData are presented as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation (SD) (n\\u0026thinsp;\\u0026ge;\\u0026thinsp;3). Significance was determined by the student\\u0026rsquo;s t-test or one-way analysis of variance (ANOVA) using GraphPad Prism (version 9). The statistical significance was considered when the P value was less than 0.05.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results and discussion\",\"content\":\"\\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1. Synthesis and characterization of HA@ZnS NPs\\u003c/h2\\u003e \\u003cp\\u003eHA@ZnS nanoparticles (NPs) were prepared by the one-step biomineralization. In brief, zinc acetate was dropwise added to the premixed solution of HA and Na2S in anaerobic condition. With stirring, insoluble ZnS gradually formed at the aid of HA, affording HA@ZnS NPs. In infarcted cardiac tissue, the expression of CD44 increased. Given that HA can bind specifically to CD44, it is assumed that NPs could accumulate in infarcted cardiac tissues(24, 25). Thus, in this system HA not only acted as the template for ZnS growth and stabilize HA@ZnS nanoparticles in the solution, but also rendered HA@ZnS NPs targeting to CD44 overexpressed tissues. The average size of HA@ZnS NPs measured by dynamic light scattering (DLS) was around 200 nm \\u003cb\\u003e(Fig.\\u0026nbsp;1B)\\u003c/b\\u003e. TEM imaging showed that these NPs were spherical with the diameter comparable to that of DLS \\u003cb\\u003e(Fig.\\u0026nbsp;1C)\\u003c/b\\u003e. Then the H2S release behaviors were investigated in acetate buffer at pH 7.4 and 5.5, respectively. As shown in \\u003cb\\u003eFig.\\u0026nbsp;1D\\u003c/b\\u003e, traceable H2S could be detected at pH 7.4, whereas large amount of H2S was liberated in the course of 1 h at acidic pH 5.5, implying that HA@ZnS NPs were pH responsive.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eFigure 1.\\u003c/b\\u003e A schematic diagram of HA@ZnS NPs. (A) Synthesis of HA@ZnS NPs and their mechanism of treating myocardial infarction. (B) Representative transmission electron microscope (TEM) image of HA@ZnS NPs. (C) Hydrodynamic size distribution of HA@ZnS NPs in deionized water. (D) H\\u003csub\\u003e2\\u003c/sub\\u003eS release curves of HA@ZnS NPs in 25 mM acetate buffer (pH 7.4 and 5.5).\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec20\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2. Biocompatibility of HA@ZnS NPs\\u003c/h2\\u003e \\u003cp\\u003eNanomaterials intended for biomedical applications require low toxicity or nontoxicity. Cell viability assay (CCK8) was employed to study the toxicity of HA@ZnS NPs against rat cardiomyocytes cells (H9C2) and human umbilical vein endothelial cells (HUVECs). Three concentration gradients of the nanomaterials were selected: 1 \\u0026micro;g/mL, 5 \\u0026micro;g/mL, and 10 \\u0026micro;g/mL. As illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, no significant cytotoxicity against H9C2 cells or HUVECs was observed within the concentration range of 1\\u0026ndash;10 \\u0026micro;g/mL within 48 hours (p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05). Furthermore, the live/dead staining results on 48 h \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB\\u003cb\\u003e)\\u003c/b\\u003e revealed that most cells (up to 99%) in all groups were stained green and had similar cell morphology, further confirming that HA@ZnS NPs had outstanding cytocompatibility. The migration of endothelial cells plays a crucial role in tissue repair and regeneration(\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e). Thus, the effects of HA@ZnS NPs on the migration of HUVECs were also explored in the scratch wound model. As revealed in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, cell migration in the NPs group was not significantly affected compared to the control group, demonstrating that HA@ZnS NPs do not impair the migration function of endothelial cells, which is the core of angiogenesis. All results together indicated that the concentrations of HA@ZnS NPs used in subsequent studies were safe for H9C2 cells and HUVECs.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec21\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.3. Fluorescence imaging of H\\u003csub\\u003e2\\u003c/sub\\u003eS release \\u003cem\\u003ein vitro\\u003c/em\\u003e and \\u003cem\\u003ein vivo\\u003c/em\\u003e\\u003c/h2\\u003e \\u003cp\\u003eWe conducted further tests on the fluorescence properties of NPs to explore their potential applications in biomedical imaging. WSP-5 is a fluorescent probe containing active disulfides, specifically used for detecting H\\u003csub\\u003e2\\u003c/sub\\u003eS in biological samples(\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e). First, we verified the capacity of NPs and Na\\u003csub\\u003e2\\u003c/sub\\u003eS to release H\\u003csub\\u003e2\\u003c/sub\\u003eS intracellularly in H9C2 cells \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e. And a single dose of 200 \\u0026micro;g/kg HA@ZnS NPs was injected into the hearts of MI rats, followed by the injection of WSP-5 to the same site after 30 min to monitor fluorescence changes \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB\\u003cb\\u003e)\\u003c/b\\u003e. It was found that the green fluorescence representing H\\u003csub\\u003e2\\u003c/sub\\u003eS in the heart gradually intensified and peaked at 1-hour postinjection. Then, the fluorescence slowly diminished in the next 23 h \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC-D\\u003cb\\u003e)\\u003c/b\\u003e. Although the intensity become weak at 24-hour postinjection, it was still observable, indicating that NPs could retain at the injection site and ensure steady H\\u003csub\\u003e2\\u003c/sub\\u003eS release for a relatively long time \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC-D\\u003cb\\u003e)\\u003c/b\\u003e. In addition, five different doses of NPs were injecting in MI rats, followed by ex vivo imaging, confirming that the fluorescence intensity detected in the heart was highly correlated with the applied dosage \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE-F\\u003cb\\u003e)\\u003c/b\\u003e. Notably, since NPs were locally injected into the myocardium, no fluorescence was observed in other organs after 1 h \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eG\\u003cb\\u003e)\\u003c/b\\u003e. This not only demonstrated NPs can accumulate in the heart, but also indicated the metabolism and clearance of NPs within the cardiac tissue. Collectively, these results showcased considerable advantages of NPs in \\u003cem\\u003ein vivo\\u003c/em\\u003e imaging.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec22\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.4. Therapeutic effects of NPs on repairing cardiac function in MI rats\\u003c/h2\\u003e \\u003cp\\u003eMI refers to the ischemic necrosis of the myocardium, one of severe coronary artery diseases. As the blood flow in the coronary artery is drastically reduced or interrupted, the corresponding myocardium would suffer from severe and persistent acute ischemia, which ultimately leads to the ischemic necrosis of the myocardium(\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e). When myocardial ischemia occurs, cardiomyocytes undergo necrosis and apoptosis if there is no timely and effective treatment. With the extension of ischemia time, the area of myocardial infarction would continue to expand, ultimately resulting in cardiac insufficiency(\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e). Therefore, improving cardiac function is one of the important means for the treatment of MI(\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e). On day 7, 14, 21, and 28 post-NP injection, the cardiac function of the rats was evaluated using echocardiography, focusing on three indices: left ventricular ejection fraction (EF), fractional shortening (FS), and end-systolic volume (ESV). Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA \\u003cb\\u003eand B\\u003c/b\\u003e displayed the echocardiograms and the corresponding quantitative ultrasonic data for each group of myocardial infarction rats. Both the echocardiograms and statistical data reveal that compared with the sham group, the EF and FS values significantly decreased while the ESV values remarkably increased after myocardial infarction. This is primarily due to the extensive death of myocardial cells post-infarction, which induced abnormal cardiac function, thinning of the ventricular walls, and ventricular dilation. These changes in indices indicated the successful construction of the myocardial infarction model in SD rats. The statistical data further implied that over time the cardiac function of the infarct group exhibited a deteriorating trend, i.e., the values of EF and FS continued to decrease, while those of ESV continued to increase. Due to the absence of therapeutic interventions, the condition of the rats in the MI group progressively worsened, ultimately resulting in heart failure. The indices for both Na\\u003csub\\u003e2\\u003c/sub\\u003eS group and ZnCl\\u003csub\\u003e2\\u003c/sub\\u003e group were almost identical to those of the MI group, i.e., no significant differences were observed. In sharp contrast, the NPs group showed improvements for all indices compared with the MI group, with a surge in EF, suggesting that NPs injection into the infarcted area can ameliorate the function of the damaged myocardium.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec23\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.5. Therapeutic effects of NPs in improving left ventricular remodeling in MI rats\\u003c/h2\\u003e \\u003cp\\u003eMI in rats was induced by ligation of the left anterior descending coronary artery, leading to gradual thinning of the left ventricular wall as the disease progressed, and ultimately replacement of the necrotic tissue with fibrous tissue(\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e). We assessed the infract size, left ventricular (LV) wall thickness, and LV scar size in MI rats on day 28 post-infarction using TTC staining, HE staining, and Sirius red staining. Compared with the MI group, NPs treatment significantly reduced the size of the myocardial infarction area \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC \\u003cb\\u003eand D)\\u003c/b\\u003e. After NPs treatment, fibrosis in the MI area (red) was remarkably reduced and NPs treatment improved the LV wall thickness and LV scar size \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE \\u003cb\\u003eand F)\\u003c/b\\u003e. Notably, rats in the NPs group exhibited the lowest degree of fibrosis and the highest degree of normal myocardial recovery in the infarcted area. In contrast, neither the Na\\u003csub\\u003e2\\u003c/sub\\u003eS nor ZnCl\\u003csub\\u003e2\\u003c/sub\\u003e group showed benefits in reducing infarct size or promoting myocardial recovery. Taken together, these research findings confirm that intramyocardial injection of NPs can effectively lessen the infarct size in myocardial infarction rats and inhibit left ventricular remodeling.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec24\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.6. The mechanism of NPs' therapeutic effects on myocardial ischemic injury\\u003c/h2\\u003e \\u003cp\\u003eIL-6, an important inflammatory factor, is a small molecular protein secreted by macrophages, and involved in the pathological damage of some autoimmune diseases(\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e). Following MI, a severe inflammatory response would be induced due to the massive apoptosis of myocardial cells, significantly elevating the expression levels of inflammatory factors. Previous studies have proved that H\\u003csub\\u003e2\\u003c/sub\\u003eS can effectively suppress the inflammatory response in the myocardial infarction area(\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e). After treated with H\\u003csub\\u003e2\\u003c/sub\\u003eS, the expression levels of inflammatory factors such as IL-6, IL-8, and TNF-α are significantly declined in animal models with myocardial infarction(\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). The primary source of these inflammatory factors is macrophages activated after injury. CD86 is a specific antigen for macrophages, which could be used as the clue for their presence(\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). In this study, we found that the expression of both IL-6 and CD86 cells in the NPs group was much lower than in the MI group \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e. This could be attributed to the anti-inflammatory effect of H\\u003csub\\u003e2\\u003c/sub\\u003eS, effectively suppressing the inflammatory response in the myocardial infarction area.\\u003c/p\\u003e \\u003cp\\u003eα-SMA is a characteristic protein of angiogenesis(\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e). It was observed that the expression of α-SMA was significantly higher in NPs group than in the MI group \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA\\u003cb\\u003e)\\u003c/b\\u003e, indicating angiogenesis was enhanced in the damaged cardiac tissue upon the intervention of NPs. This effect was also confirmed in the human umbilical vein endothelial cell tubulation assay, where NPs dramatically increased the number of endothelial cell tubules \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB\\u003cb\\u003e)\\u003c/b\\u003e. The anti-inflammatory and angiogenesis-promoting effects of NPs were similarly verified in Western blot, i.e., the expression of the inflammatory indicators IL-6 and CD86 decreased in the NPs group, and the expression of the angiogenesis indicator α-SMA increased in the NPs group.\\u003c/p\\u003e \\u003cp\\u003eDuring MI, the hypoxia of cardiac cells in the ischemic area leads to a reduction of ATP production, consequently causing apoptosis of cardiac cells that highly rely on mitochondrial respiration for ATP generation(\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e). Compared with MI group, as expected, less TUNEL\\u0026thinsp;+\\u0026thinsp;apoptotic cells were detected in the group treated by NPs \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC\\u003cb\\u003e)\\u003c/b\\u003e, suggesting that NPs can protect ischemic cardiac cells through anti-apoptotic mechanisms.\\u003c/p\\u003e \\u003cp\\u003eIn addition, previous studies have shown that reactive oxygen species (ROS) play a key role in the progression of atherosclerosis and ischaemic heart disease. And excessive ROS leads to sustained oxidative stress and endothelial dysfunction, which in turn leads to disease progression(\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e). In the H9C2 hypoxic cell model, we examined the levels of reactive oxygen species using a ROS fluorescence detection probe and showed that NPs were able to significantly weaken ROS levels \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eD\\u003cb\\u003e)\\u003c/b\\u003e.\\u003c/p\\u003e \\u003cp\\u003eThe anti-apoptotic effect of NPs was also confirmed in Western blot on rat myocardial tissues \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e\\u003cb\\u003e)\\u003c/b\\u003e. The Bcl-2 family of proteins are key regulators of apoptosis, including both pro-apoptotic and pro-survival (anti-apoptotic) members, where Bcl-2 exerts an anti-apoptotic effect, while BAX, a member of the Bcl-2 family, plays a pro-apoptotic role(\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e). In addition, Caspase s are a specific group of enzymes involved in the process of apoptosis, of which the classical Caspase-3 recognizes and disassembles apoptosis-specific sequences and promotes apoptosis(\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e). Therefore, the expression of Bcl-2, BAX, and Caspase-3 in myocardial tissues of MI rats was detected by Western blot, and it was found that the expression of Bcl-2 was significantly increased in the NPs group, while the expression of BAX and Caspase-3 was significantly decreased, which further validated the anti-apoptotic effect of NPs.\\u003c/p\\u003e \\u003cp\\u003eBesides, macrophages in diseased tissues were classified into M1-type (classically activated macrophages) and M2-type (selectively activated macrophages). CD86 is a common marker molecule for M1-type macrophages, which are induced by IFN-γ to produce pro-inflammatory factors. CD206 is a common marker molecule for M2-type macrophages, which are activated by IL-4 to release anti-inflammatory factors(\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e). By flow cytometric analysis, we examined macrophage polarization in myocardial tissue and found that NPs reduced the proportion of CD86\\u003csup\\u003e+\\u003c/sup\\u003e M1-type macrophages and increased the proportion of CD206\\u003csup\\u003e+\\u003c/sup\\u003e M2-type macrophages \\u003cb\\u003e(\\u003c/b\\u003eFig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e\\u003cb\\u003e)\\u003c/b\\u003e. This further suggests that NPs can improve the function of damaged myocardium through anti-inflammatory effects.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Conclusion\",\"content\":\"\\u003cp\\u003eIn this study we reported a novel H\\u003csub\\u003e2\\u003c/sub\\u003eS donor, HA@ZnS NPs, for the treatment of myocardial infarction. Releasing study demonstrated that H\\u003csub\\u003e2\\u003c/sub\\u003eS release could be triggered by the acidic pH. NPs not only significantly reduced the infarct size but also improved left ventricular remodeling and decreased the levels of inflammatory factors. By promoting angiogenesis in myocardial tissues and reducing cardiomyocyte apoptosis, NPs demonstrated potential in improving cardiac function. These findings provide valuable insights into new therapeutic strategies for myocardial infarction and lay the groundwork for future clinical applications of H\\u003csub\\u003e2\\u003c/sub\\u003eS-releasing materials.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe procedures of animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the Westchina Hospital, Sichuan University (Chengdu, China) (20230228042).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent or publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing financial interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis project was supported by National Natural Science Foundation of China (Grant No. 52273090, 22375128, 22105126), the Natural Science Foundation of Sichuan Province (Grant No. 2022NSFSC1395, 2022NSFSC1489), Post-Doctor Research Project, West China Hospital, Sichuan University (Grant No. 20HXBH171, 2021HXBH070), Postdoctoral Science Foundation of China (Grant No. 2021M692282), and the Natural Science Foundation of Shanghai (22ZR1433500).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026apos; contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eY.Z.: Methodology; Validation; Visualization; Writing\\u0026nbsp;\\u0026ndash;\\u0026nbsp;original draft; Writing\\u0026nbsp;\\u0026ndash;\\u0026nbsp;review \\u0026amp; editing. X.Z.: Conceptualization; Investigation; Methodology; Software; Validation; Writing\\u0026nbsp;\\u0026ndash;\\u0026nbsp;original draft; Writing\\u0026nbsp;\\u0026ndash;\\u0026nbsp;review. S.B.: Supervision; Validation; Writing\\u0026nbsp;\\u0026ndash;\\u0026nbsp;review. R.L.: Supervision; Validation; Writing\\u0026nbsp;\\u0026ndash;\\u0026nbsp;review. Y.G.: Supervision; Validation; Writing\\u0026nbsp;\\u0026ndash;\\u0026nbsp;review. J.G.: Funding acquisition; Project administration; Supervision; Validation; Writing\\u0026nbsp;\\u0026ndash;\\u0026nbsp;review \\u0026amp; editing. Y.W.: Funding acquisition; Project administration; Supervision; Validation; Writing\\u0026nbsp;\\u0026ndash;\\u0026nbsp;review \\u0026amp; editing.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors\\u0026apos; information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eY.Z.\\u003csup\\u003ea\\u003c/sup\\u003e,\\u0026nbsp;yujia_zhan@163.com;\\u0026nbsp;X.Z.\\u003csup\\u003ea\\u003c/sup\\u003e, zhaoxueshan95@wchscu.cn;\\u0026nbsp;S.B.\\u003csup\\u003eb\\u003c/sup\\u003e, bisiweihx2022@wchscu.cn;\\u0026nbsp;R.L.\\u003csup\\u003eb\\u003c/sup\\u003e, rachaelliu@wchscu.cn;\\u0026nbsp;Y.G.\\u003csup\\u003ec\\u003c/sup\\u003e, geyuxuan_lh@outlook.com;\\u0026nbsp;J.G.\\u003csup\\u003ea\\u003c/sup\\u003e, gujun@wchscu.cn;\\u0026nbsp;Y.W.\\u003csup\\u003ec\\u003c/sup\\u003e, yinwang@sjtu.edu.cn\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003csup\\u003ea\\u003c/sup\\u003e Department of Cardiovascular Surgery, West China Hospital, Sichuan University, Chengdu, 610000, China\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003csup\\u003eb\\u003c/sup\\u003e Department of Burn and Plastic Surgery, West China Hospital, Sichuan University, Chengdu, 610000, China\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003csup\\u003ec\\u0026nbsp;\\u003c/sup\\u003eEngineering Research Center of Cell \\u0026amp; Therapeutic Antibody, Shanghai Frontiers Science Center of Drug Target Identification and Delivery, National Key Laboratory of Innovative Immunotherapy, School of Pharmaceutical Sciences, Shanghai Jiao Tong University, Shanghai 200240, China\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eZheng Z, Tan Y, Li Y, Liu Y, Yi GH, Yu CY, et al. 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DOI: 10.1038/nrm1496\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"myocardial infarction, hydrogen sulfide, nanoparticles, myocardial ischemic injury \",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4987842/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4987842/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eMyocardial infarction (MI), a common and severe disease threatening human health worldwide, results from ischemic and hypoxic-induced necrosis of cardiac tissue due to coronary artery obstruction or rupture. Hydrogen sulfide (H\\u003csub\\u003e2\\u003c/sub\\u003eS) is a gasotransmitter involved in various physiological and pathological processes. Exogenous supplementation of H\\u003csub\\u003e2\\u003c/sub\\u003eS is significantly beneficial for the treatment of MI. In this study, a novel H\\u003csub\\u003e2\\u003c/sub\\u003eS donor - zinc sulfide nanoparticles encapsulated in hyaluronic acid (HA@ZnS NPs), has been developed through a biomimetic mineralization process for the treatment of MI. HA@ZnS NPs can stably release H\\u003csub\\u003e2\\u003c/sub\\u003eS at the site of myocardial ischemic injury due to the acidic microenvironment. Compared to the MI group, the NP-treated group significantly improved cardiac function, including increased left ventricular ejection fraction and fractional shortening, as well as reduced end-systolic volume. Furthermore, the NPs significantly reduced the size of the myocardial infarction area, improved left ventricular remodeling, and exerted therapeutic effects by promoting angiogenesis and reducing apoptosis in cardiac tissue. In conclusion, HA@ZnS NPs demonstrate potential for treating MI through precise control of H\\u003csub\\u003e2\\u003c/sub\\u003eS release, providing valuable insights into new therapies for MI and laying the groundwork for the clinical application of H\\u003csub\\u003e2\\u003c/sub\\u003eS-releasing materials in the future.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Alleviation of Myocardial Infarction by Hydrogen Sulfide-Releasing Nanoparticles: Mechanisms and Therapeutic Effects\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-10-07 11:02:14\",\"doi\":\"10.21203/rs.3.rs-4987842/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"680124f7-e7f9-41e3-8adb-d33339a93450\",\"owner\":[],\"postedDate\":\"October 7th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-12-02T22:53:28+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-10-07 11:02:14\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4987842\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4987842\",\"identity\":\"rs-4987842\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}