Tregs Biomimetic Nanoparticle to Reprogram In ammatory and Redox Microenvironment in Infarct Tissue to Inhibit the Remodeling of the Left Ventricle of Myocardial Ischemia Reperfusion Injury in Mice


 Background

At present, patients with myocardial infarction remain an increased risk for myocardial ischemia/reperfusion injury (MI/RI), which currently lacks an effective therapeutic method. It is still a bottleneck that effectively deliver drug to ischemic myocardium to treat MI/RI. Inspired by the protective effect of regulatory T cells (Tregs) on MI/RI and natural role of platelets in adhesion with damaged blood vessel in heart during myocardial infarct, a Tregs biomimetic nanoparticle (CsA@PPTK) was prepared by camouflaging a cyclosporin A (CsA)-loaded and reactive oxygen species (ROS)-sensitive nanoparticle with platelet membrane.
Results

In MI/RI mice, CsA@PPTK could be preferentially delivered to ischemic myocardium. CsA@PPTK significantly scavenged ROS in ischemic myocardium, while it also markedly increased the generation of Tregs and the ratio of M2 type macrophage to M1 type macrophage in ischemic myocardium. Furthermore, CsA@PPTK significantly attenuated apoptosis of cardiomyocytes in ischemic myocardium. At the same time, CsA@PPTK obviously reduced the infarct size, fibrosis area and the protein expression of MMP-9, while increased the protein expression of CX43. Subsequently, the remodeling of the left ventricle was significant alleviated. Finally, heart function of MI/RI mice was markedly improved.
Conclusion

CsA@PPTK has great potential in the treatment of MI/RI. This study provides a novel class of heart protective biomimetic platform that is beneficial for treatment of MI/RI.


Results
In MI/RI mice, CsA@PPTK could be preferentially delivered to ischemic myocardium. CsA@PPTK signi cantly scavenged ROS in ischemic myocardium, while it also markedly increased the generation of Tregs and the ratio of M2 type macrophage to M1 type macrophage in ischemic myocardium.
Furthermore, CsA@PPTK signi cantly attenuated apoptosis of cardiomyocytes in ischemic myocardium. At the same time, CsA@PPTK obviously reduced the infarct size, brosis area and the protein expression of MMP-9, while increased the protein expression of CX43. Subsequently, the remodeling of the left ventricle was signi cant alleviated. Finally, heart function of MI/RI mice was markedly improved.

Conclusion
CsA@PPTK has great potential in the treatment of MI/RI. This study provides a novel class of heart protective biomimetic platform that is bene cial for treatment of MI/RI.

Background
Coronary heart disease such as blockage of coronary artery, acute myocardial infarction (AMI) and cardiac failure has become one of the leading causes of death in the world, accounting for over 17.3 million deaths every year [1]. Current therapy for AMI mainly concentrates on restoration coronary blood ow (reperfusion) through medications and/or revascularization procedures including via percutaneous coronary intervention or coronary artery bypass grafting. Although reperfusion strategy is vital for survival [2], patients with myocardial infarction remain an increased risk for myocardial ischemia/reperfusion injury (MI/RI), which currently lacks an effective therapeutic method [3][4][5]. So, exploring new strategies to treat MI/RI shows important clinical signi cance and social bene ts.
MI/RI is the result of multiple factors, including apoptosis of cardiomyocytes, reactive oxygen species (ROS) burst production, and in ammatory cell recruitment. A single target based therapy is di cult to effectively inhibit reperfusion injury. During reperfusion, ROS bursts and Ca 2+ overload occur in cardiomyocytes, leading to the opening of mitochondrial permeability transition pore (mPTP). The opening of mPTP further induces more ROS release from mitochondria and apoptosis of cardiomyocytes [6]. Apoptotic cardiomyocytes release damage associated molecular patterns (DAMP) and subsequently activate the in ammatory cascade reaction of endothelial cells. Then in ammatory cells were recruited to damaged myocardium tissue [7] and release ROS and cytokine in damaged myocardium tissue, which further aggravate apoptosis of cardiomyocytes and damage of myocardium. Finally, there forms a vicious cycle between burst release of ROS and apoptosis of cardiomyocytes in ischemic myocardium tissue.
Immunosuppressive regulatory T cells (Tregs) account for about 5-10% of peripheral CD4 + T cells. Tregs participates in regulation of immune response in pathological or physiological states in an "active" manner, and plays an important role in maintaining immune tolerance and immune response homeostasis. Immunological studies have found that Tregs has protective effect on MI/RI [8]. Tregs can actively target to ischemic myocardium and play the role of anti-apoptosis, anti-in ammatory, antioxidant and reducing the remodeling of the left ventricle [9][10][11][12]. Therefore, many researchers hope to use adoptive input of Tregs or increase of Tregs to treat MI/RI [13]. However, the preparation of Tregs is di cult, and there are problems such as immune rejection if adoptive transfusion is carried out in clinical practice, which makes it di cult for Tregs to be applied in clinical practice. Nevertheless, Tregs internal effects suggest that scavenging ROS, reducing the opening of mPTP and alleviating cardiac in ammatory reaction are considered effective methods to improve myocardial function after MI/RI. Cyclosporine A (CsA), an immunosuppressant, can inhibit the opening of mPTP through binding with cyclophilic protein D on the mitochondrial membrane [14]. However, the solubility of CsA in water is poor [15] and its distribution in vivo lacks ischemic myocardium-targeted characteristic. Previously, we reported that nanoparticle modi ed with SS-31 peptide increased the distribution of CsA in ischemic myocardium, and this nanoparticle inhibited apoptosis of cardiomyocytes through mitochondrial pathway [16]. Moreover, ROS is an important factor causing reperfusion injury, and it can damage a variety of cells in myocardial tissue through lipid peroxidation, DNA damage, etc. At the same time, ROS can also induce the recruitment of in ammatory cells, resulting in MI/RI [6]. Antioxidant drugs or bioactive materials can exert therapeutic effects on MI/RI by eliminating ROS [17]. In our previous study, we demonstrated that antioxidant drugs can play a role in the treatment of MI/RI through the targeted delivery of resveratrol to ischemic myocardium. ROS-responsive materials can be reduced at high ROS levels, which can eliminate ROS in vivo while releasing drugs, consequently displayed antioxidant effect [18]. For example, polymer materials containing thioketal [19] and phenylboric acid [20] can play ROS scavenging role after being prepared as nanoparticles. However, most of drug delivery system is consisted of exogenous materials. When it enters the bloodstream, the mononuclear phagocytic system (MPS) can recognize these exogenous substances and quickly eliminate them from body, subsequently shortening their circulation time [21]. Thus, long-term retention in the blood and precise accumulation in ischemic myocardium are two major bottlenecks that ischemic myocardium targeting drug delivery system need to overcome.
In recent years, because biomimetic drug delivery system can not only simulate the function of cells or organelles in vivo, but also avoid the complex steps of cell or organelle preparation in vivo. Thus, biomimetic drug delivery system has achieved great development. Moreover, the composition of biomimetic drug delivery system is clear, and it has the prospect of clinical transformation. For example, some researchers have carried out biomimetic design of neutrophils [22], macrophages [23] and mitochondria [24] to treat diseases such as tumors or myocardial ischemia.
There are some biomacromolecule such as GPIB-IX-V, αIIBβ3, intercellular cell adhesion molecule-1, pselectin and α2β1 integrin on the surface of platelet, which can bind with their corresponding receptors or collagens on vascular endothelial surface including von willebrand factor, αVβ3 integrin, p-selectin glycoprotein ligand (PSGL-1), collagen, etc. Besides, von willebrand factor is highly expressed on the endothelial cell surface of damaged blood vessels in ischemic myocardial tissue. Therefore, platelet displays damaged vascular endothelial targeting effect with the characteristic of multiple sites binding.
Actually, the cardiac blood ow velocity is fast, the a nity of multiple sites binding of platelet with damaged blood vessels in ischemic myocardia is stronger than that of a single site binding drug delivery system [25,26]. This subsequently results in platelet recruitment in AMI area in vivo [1]. Thus, platelet and its membranes can be used as ischemic myocardium targeting material to guide nanoparticle to concentrate at ischemic myocardium [1,27,28]. Most importantly, when platelet membrane is coated on the surface of drug delivery system, due to CD47 on the membrane, it can not only speci cally accumulate ischemic myocardium but also can effectively reduce the non-speci c phagocytosis and prolong the circulation time of drug delivery system in vivo [29,30]. Finally, the targeting effect to ischemic myocardium and biocompatibility of drug delivery system are greatly improved.
In this paper, poly (5,5-dimethyl-4,6-dithio-propylene glycol azelate) (PTK) was prepared. PTK contains a large amount of thioketal, which is not only sensitive to various types of ROS such as O 2− , ClO − and H 2 O 2 [31] but can also scavenge these ROS. CsA was encapsulated by PTK polymer to form CsA@PTK. The platelet membrane has the ischemic myocardium targeting effect, which can simulate the ischemic myocardium targeting effect of Tregs when platelet membrane is coated with nanoparticles. By using CsA@PTK as core and platelet membrane as shell, a Tregs biomimetic nanoparticle (CsA@PPTK) was prepared. CsA@PPTK could simulate Tregs to play a series of roles of Tregs such as ischemic myocardium targeting, anti-in ammatory, anti-apoptotic, scavenging of reactive oxygen species, and ultimately reducing the remodeling of the left ventricle and protecting ischemic myocardium (Scheme 1).
To set up hypoxia re-oxygenation (H/R) injured H9c2 cells model, H9c2 cells were incubated in a hypoxic environment (95% N 2 and 5% CO 2 ) for 3 h at 37 °C by using hypoxic culture medium. Next, the culture medium was replaced with DMEM solution containing CsA, CsA@PTK and CsA@PPTK, and H9c2 cells were cultured in a standard incubator with 5% CO 2 in normal atmosphere for 4 h at 37 ℃. Then, the cell viability, intracellular ROS, mitochondrial ROS, the opening of mPTP and mitochondrial membrane potential of H9c2 cells were investigated.
The effect of CsA@PPTK on the viability of H/R damaged H9c2 cells H9c2 cells were treated with MTT (20 μL/well, 5 mg/mL) for 4 h. The cell culture medium was replaced with dimethylsulfoxide (DMSO) solution. By using a microplate reader (Bio-Rad Laboratories, Richmond, CA, USA), the absorbance of DMSO solution was detected at 490 nm. The control cells were cultured in normoxic conditions with FBS-free DMEM solution for 7 h.

The effect of CsA@PPTK on the intracellular ROS of H/R damaged H9c2 cells
The intracellular ROS was detected by using 2',7'-dichlorodihydro uorescin diacetate (DCFH-DA). DCFH-DA probe was diluted with serum-free medium at the ratio of 1:1000. Then, DCFH-DA solution was cultured with H/R damaged H9c2 cells for 20 min at 37 ℃. After DCFH-DA solution was removed, and the cells were washed with serum-free culture medium for 3 times. DAPI was used for nucleus staining. The cells were observed under uorescent microscope (Olympus, Japan). The uorescence intensity was semi-quanti ed with Image Pro software. Fluorescence intensity indicated ROS level in the cell. The stronger the uorescence intensity was, the higher the ROS level was.
The effect of CsA@PPTK on the mitochondrial ROS of H/R damaged H9c2 cells MitoSOX Indicator was diluted to 5 μM with HBSS. Then, the diluted MitoSOX Indicator solution was cultured with H/R damaged H9c2 cells for 10 min at 37 ℃. DAPI was used for nucleus staining. The cells were observed by using confocal laser scanning microscopy (CLSM, Olympus, Japan). The uorescence intensity was semi-quanti ed with Image Pro software. Fluorescence intensity indicated ROS level in mitochondria. The stronger the uorescence intensity was, the higher the ROS level was.
The effect of CsA@PPTK on the opening of mPTP of H/R damaged H9c2 cells H/R damaged H9c2 cells were incubated with 5 μL Calcein AM solution (1 mmol/L) and 5 μL CoCl 2 solution (80 mM) for 15 min at 37 °C. Next, HBSS/Ca (3.5 mL) was added into cell mixture. The cells were collected and re-suspended in buffer solution (400 μL) to carry out ow cytometric analysis. The stronger the uorescence intensity was, the less the opening of mPTP was.
The effect of CsA@PPTK on the mitochondrial membrane potential of H/R damaged H9c2 cells JC-1 solution (5 μg/mL) was cultured with H/R damaged H9c2 cells for 15 min at 37 °C. Then, the H9c2 cells were slightly rinsed 2 times with assay buffer. The uorescent intensity of H9c2 cell solution were respectively detected at 530/590 nm (excitation/emission wavelength, red uorescence) and 485/530 nm (green uorescence) by uorescence spectrophotometer. The ratio between red uorescent intensity and green uorescent intensity was calculated, which indicated the mitochondrial membrane potential. The greater the ratio value was, the higher the mitochondrial membrane potential was. Cellular uptake of CsA@PPTK on H/R damaged H9c2 cells H9c2 cells were seeded into 6 well plates (1×10 5 cells/well). After hypoxia for 3 h, H9c2 cells were incubated with DMEM solution containing CsA, CsA@PTK or CsA@PPTK (30 μg CsA/mL) in a standard incubator with 5% CO 2 in normal atmosphere for 0.5 h and 2 h. H9c2 cells were washed for 3 times with PBS (pH 7.4) and lysed by 100 μL RIPA lysis buffer. CsA in cell lysis was determined by HPLC. The protein content in cell lysis was determined by coomassie brilliant blue. The content of CsA in cell lysis was normalized by protein content in cell lysis.
To investigate the endocytic pathway of CsA@PPTK, H9c2 cells were seeded into 6 well plates (1×10 5 cells/well). After hypoxia for 2 h, 2-deoxy-D-glucosesucrose (ATP depletion agent, 1 mg/mL), sucrose (inhibitor of clathrin-mediated uptake, 150 mg/mL), methyl-β-cyclodextrin (inhibitor of caveolae-mediated uptake, 0.005 mg/mL), colchicine (inhibitor of macropinocytosis, 0.8 mg/mL) were respectively added to H9c2 cells, and H9c2 cells were incubated for 1 h at 37 °C in hypoxic culture medium. Then, the hypoxic culture medium was replaced with DMEM solution containing CsA, CsA@PTK or CsA@PPTK (30 μg CsA/mL), and H9c2 cells were incubated in a standard incubator with 5% CO 2 in normal atmosphere for 2 h. The cells were collected and lysed. Finally, CsA in cell lysis was determined by using the same method as described above.

MI/RI mice model
Kunming mice were anesthetized with 2% iso urane inhalation. About 2 mm long vertical incision was cut at 2-3 mm away from the left sternal border. The chest wall muscle was separated. A small hole was made at intercostal space with a small hemostatic forceps to open the pleural membrane and pericardium.
With the slightly open of hemostatic forceps, the heart was smoothly exposed out of the hole. By using a 6/0 silk suture, the left anterior descending coronary artery (LDA) was ligated at a site 3 mm from its origin. Only when the anterior wall of left ventricle (LV) became pale, the ligation was regarded as success. Heart was put back into chest immediately after ligation. The air in chest was extruded out, and the chest was closed with 4/0 suture. 30 min later, the 6/0 suture was carefully pulled out to restore the blood ow of LDA. The sham group performed the same surgical procedure except that LDA was not ligated.

Biodistribution of CsA@PPTK in MI/RI mice
Acute myocardial ischemic Kunming mice were intravenously injected a single dose of Cy7.5 labeled CsA@PPTK (Cy7.5@PPTK) 5 min before reperfusion. 24 h after injection of Cy7.5@PPTK, various organs (heart, liver, lung, spleen, and kidney) were collected and imaged using Caliper IVIS Lumina (Siemens, Germany). The heart tissue was then cut into ve or six transverse sections and imaged again.

Cardiac function of MI/RI mice
The cardiac function of MI/RI mice was assessed by transthoracic echocardiogram (Vevo 2100, VisualSonics, Canada). Animals were anesthetized using a mixture of iso urane and oxygen before undergoing transthoracic echocardiogram procedure at the 4 week and 10 week after reperfusion. Two dimensional images of LV were collected. Then, LV functional parameters such as left ventricular shortening fraction (FS %) and left ventricular ejection fraction (LVEF %) were calculated by using Vevo 2100 softwar.
Histological and immunohistochemical analysis MI/RI mice were sacri ced by intraperitoneal injection of over-dose pentobarbital sodium. Hearts and blood were harvested. The contents of in ammatory cytokines such as IL-1β and TGF-β in plasma were determined by ELISA kit. After being xed in 4% paraformaldehyde, heart tissue was dehydrated by using gradient concentration of alcohol and embedded into para n. Then, heart tissue was cut into 4 μm thick sections for histological and immunohistological staining. For infarct size and brosis evaluation, masson trichrome staining was performed and total sections were scanned to acquire the whole images of heart horizontal planes. The brotic area was identi ed using ImageJ software. The brosis was calculated as brotic area/total area in images. Tregs cell in section of heart tissues were marked by FoxP3 antibody. M1-type macrophage and M2-type macrophage in section of heart tissues were tracked by CD86 antibody and CD206 antibody, respective. Then, uorescence of FoxP3, CD206 and CD86 in section of heart tissues was observed by using CLSM. The expression of MMP-9, CX43 and α-SMA in section of heart tissues was observed by immuno uorescence method. TUNEL staining was used to track the apoptotic cardiomyocyte.

Statistical analysis
All results are expressed as means±SD. Comparison between two groups was performed with two-tailed Student's t test. The difference was considered statistically signi cant at the value of p < 0.05, 0.01 and 0.001.

Characterization of TK and PTK
The mass spectrum of 5,5-dimethyl-4,6-dithio-azelaic acid (TK) is showing in Figure.S1A, which was consistent with the theoretic molecular weight of TK. In addition, 1 H NMR spectroscopy of TK showed peaks at 1.59, 2.90 and 2.68 ppm, it was assigned to Ha, Hb and Hc of TK, respectively (Figure.S1B).
Compared with TK, there was two new peak appeared at 4.18 and 1.98 ppm in 1 H NMR spectroscopy of poly(5,5-dimethyl-4,6-dithio-propylene glycol azelate) (PTK), which were assigned to Hd and He of propanediol ( Figure.

Characterization of CsA@PPTK
Transmission electron microscopy showed that the appearance of CsA@PTK and CsA@PPTK was spherical with good dispersion (Figure.1A). Besides, CsA@PTK displayed a uniform structure from inside to outside, while CsA@PPTK showed obvious core-shell structure. This indicated that platelet membrane was coated on the surface of CsA@PTK. As compared to CsA@PTK, the size of CsA@PPTK did not show signi cant change. The zeta potential of CsA@PPTK obviously decreased to -25 Mv, which was similar to zeta potential of platelet ( Figure.1B). The co-localization of the DiR-labeled platelet membrane (red) and coumarin 6 labeled CsA@PTK (green) further substantiated the successful coating of platelet membrane on the surface of CsA@PTK ( Figure.1C). SDS-PAGE electrophoresis revealed that the majority protein of platelet membrane was retained in CsA@PPTK (Figure.1D). All above results indicated that the platelet membrane was coated on PTK nanoparticles successfully. The drug loading of CsA in CsA@PPTK was 4.97%. As shown in Figure. The protective effectof CsA@PPTK on H/R injured H9c2 cells At rst, the cytotoxicity of PPTK on cardiomyocytes was assessed by MTT method. As shown in Figure.S5A, 0.2~1 mg/mL @PTK increased the activity of normal H9c2 cells after incubation for 24 h. In addition, 0.5 mg/mL, 1 mg/mL and 2 mg/mL @PTK improved the activity of hypoxia for 3 h and reoxygenation for 4 h (H/R) injured H9c2 cells ( Figure.S5B). It was reported that the optimized concentration of CsA to protect H/R injured H9c2 cells was 15 and 30 μg/mL [16]. The drug loading of CsA@PPTK was 4.97%. Therefore, 1 mg/mL @PTK was much higher than the maximum dose of @PTK that needed in the experiment, indicating that under the therapeutic doses, CsA@PTK exhibited no cytotoxicity on cardiomyocytes.
As shown in Figure The effect of CsA@PPTK on mitochondrial ROS is showing in Figure.3A-B, compared with control group, red uorescence intensity was much stronger in H/R injured H9c2 cells, while red uorescence intensity was much weaker in CsA, @PPTK, CsA@PTK and CsA@PPTK treated H/R injured H9c2 cells. The statistical results of red uorescence intensity in H/R injured H9c2 cells are showing in Figure.3C. The ratio of red to blue uorescence intensity in H/R model group was signi cantly higher than that in control group, indicating that mitochondria produced a large amount of ROS after H9c2 cells was injured by H/R. Compared with H/R model group, the ratio of red to blue uorescence intensity in CsA, @PPTK, CsA@PTK and CsA@PPTK treated group was signi cantly reduced. Meanwhile, CsA@PPTK treated H/R injured H9c2 cells showed the lowest ratio of red to blue uorescence intensity, indicating CsA, @PPTK, CsA@PTK and CsA@PPTK reduced mitochondrial ROS level in H/R injured H9c2 cells, and CsA@PPTK exhibited the strongest effect on reducing mitochondrial ROS level in H/R injured H9c2 cells.
The effect of CsA@PPTK on the opening of mPTP in H/R injured H9c2 cells was shown in Figure.3D-E.
Compared with the normal group, the calcein uorescence intensity in mitochondria in H/R injured H9c2 cells was signi cantly decreased, indicating that H/R could signi cantly increase the opening of mPTP in H9c2 cells. Compared with the H/R model group, the calcein uorescence intensity in mitochondria was signi cantly increased in CsA, CsA@PTK and CsA@PPTK treated H/R injured H9c2 cells, indicating that CsA, CsA@PTK and CsA@PPTK played a protective role against H/R injury by reducing the opening of mPTP in H/R injured H9c2 cells.
Next, JC-1 staining was used to detect the mitochondrial membrane potential, and the results are showing in Figure.3F. Compared with normal cultured H9c2 cells, the red uorescence was obviously decreased and green uorescence was obviously increased in H/R injured H9c2 cells, suggesting mitochondrial membrane potential was damaged during H/R in H9c2 cells. However, compared with H/R model group, the ratio of red to green uorescence was signi cantly increased in CsA, CsA@PTK and CsA@PPTK treated H/R injured H9c2 cells, indicating that CsA, CsA@PTK and CsA@PPTK recovered the damaged mitochondrial membrane potential.

Cellular uptake of CsA@PPTK by H/R injured H9c2 cells
The speci c cellular uptake behavior of H9c2 cells towards CsA@PPTKwas studied by HPLC. The uptake of CsA@PPTK by H9c2 cells is shown in Figure.S7. The uptake of CsA@PTK and CsA@PPTK by normal and H/R injured H9c2 cells exhibited time-dependent manner. The uptake of CsA@PPTK in H/R injured H9c2 cells was much higher than that in normal H9c2 cells at 2 h. Compared with CsA@PTK, a large amount of CsA@PPTK was taken up by H/R injured H9c2 cells.
The effects of different uptake pathway inhibitors on the uptake of CsA@PPTK in H/R injured H9c2 cells were observed. As shown in Figure.S8, sucrose, colchicine, 2-deoxy-D-glucose and methyl-β-cyclodextrin showed no signi cant effect on the uptake of CsA@PPTK by H/R injured H9c2 cells. This suggested that the receptor-mediated endocytosis pathway did not involve in the uptake of CsA@PPTK by H/R injured H9c2 cells. Compared with the control group, the uptake of CsA@PPTK in H/R injured H9c2 cells signi cantly reduced at 4 ℃. In theory, adsorptive endocytosis is temperature dependent. The platelet membrane coated on the surface of CsA@PPTK is very similar with membrane of H/R injured H9c2 cells, which resulted in the strong adsorptive effect between CsA@PPTK and H/R injured H9c2 cells. Therefore, CsA@PPTK was taken up by H/R injured H9c2 cells mainly through adsorption endocytosis [35].

The ex-vivo targeted of CsA@PPTK
The speci c binding of Coumarin 6@PPTK on aortic vessels with endothelia injured is showing in Figure.4A-B. The green uorescence intensity of Coumarin 6@PPTK in normal aortic vessels was very weak, indicating little amount of Coumarin 6@PPTK was bound with normal aortic vessels. Compared with Coumarin 6@PTK, the green uorescence intensity of Coumarin 6@PPTK in aortic vessels with endothelia injured was much stronger, suggesting a large amount of Coumarin 6@PPTK was bound with endothelia injured aortic vessels. The above data implied that CsA@PPTK could speci cally bind with endothelia injured vessels in ischemic area.

In vivo targeting of CsA@PPTK
As shown in Figure.S9, after acute myocardial ischemic mice were intravenously injected a single dose of Cy7.5 labeled CsA@PPTK (Cy7.5@PPTK) 5 min before reperfusion, Cy7.5 labeled CsA@PTK (Cy7.5@PTK) was mainly distributed in the liver and spleen, and little amount of Cy7.5@PTK was distributed in heart. However, compared with Cy7.5@PTK, a large amount of Cy7.5@PPTK was distributed in the heart, while little amount of Cy7.5@PPTK was distributed in the liver of MI/RI mice. As shown in Figure.4C, Cy7.5@PPTK was distributed in the whole heart when it was intravenously injected to sham mice. However, Cy7.5@PPTK was mainly distributed in downstream areas of the occluded coronary artery heart when it was intravenously injected to MI/RI mice. Cy7.5@PTK distributed in the whole heart of MI/RI mice. Next, the heart was cut into six or seven transverse sections from apex to atrium, and the distribution of Cy7.5@PPTK in transverse sections is showing in Figure.4D. Cy7.5@PPTK distributed in all heart transverse sections of sham mice. Cy7.5@PTK also appeared in all heart transverse sections of MI/RI mice. However, Cy7.5@PPTK was mainly distributed in transverse sections of ischemic area especial in transverse section of apex of MI/RI mice heart. This indicated that Cy7.5@PPTK exhibited obvious ischemic myocardium targeting characteristic.
In vivo therapeutic effect of CsA@PPTK on MI/RI mice On 28th day after reperfusion, echocardiography was applied to reveal the recovery of overall cardiac functions of MI/RI mice, and results are showing in Figure.5. The mice in sham group showed regular and stable heart beats along with a large amplitude. The amplitude of heart beats was reduced and ventricular cavity volume was increased in normal saline treated MI/RI mice ( Figure. CsA@PPTK. Compared with normal saline, @PPTK obviously increased the heart function of MI/RI mice, indicating that ROS scavenging was an effective and necessary treatment method for MI/RI mice. CsA@PPTK treated mice showed the highest EF and FS in all group, signifying that ROS scavenging and anti-apoptosis could improve heart function signi cantly. The echocardiography was also applied to reveal the recovery of overall cardiac functions on 70th day post MI/RI, and results showed that CsA@PPTK performed the strongest effect on the recovery of heart function at dose of 2.5mg/kg ( Figure.S10).This suggested that early reduction of reperfusion injury increased cardiac function for a longer period of time.
Figure.S11 shows the ROS level in infarcted heart area 1 day after reperfusion, red uorescence represents ROS. The results indicated that compared with sham group, the ROS level in ischemic myocardium in normal saline treated group (MI/RI model group) was signi cantly increased. CsA, @PPTK, CsA@PTK and CsA@PPTK markedly decreased ROS level in ischemic myocardium. CsA@PPTK treated group showed the lowest ROS level in ischemic myocardium among 4 groups, which resulted from the following two reasons. Firstly, CsA inhibited the production of ROS in mitochondria. Secondly, PTK strongly scavenged ROS in ischemic cardiomyocytes and ischemic myocardium.
The phenotype of macrophage in the infarcted heart tissue was detected on 4th day post reperfusion. The M1 and M2 type macrophages were stained by their surface markers of CD86 (red) and CD206 (green), respectively. As shown in Figure.6A-B, the heart tissue exhibited strong green uorescence in CsA@PPTK treated group, while heart tissue displayed weak green uorescence in normal saline treated group. As compared with normal saline treated group, the ratio of M2 type macrophages to M1 type macrophages obviously increased in heart tissue in CsA, CsA@PTK, @PPTK and CsA@PPTK treated groups. CsA@PPTK treated group exhibited the highest ratio of M2 type macrophages to M1 type macrophages in heart tissue among 4 groups. Besides, as compared with normal saline treated group, the number of M2 type macrophages and M1 type macrophages signi cantly reduced in heart tissue in CsA, CsA@PTK, @PPTK and CsA@PPTK treated groups. CsA@PPTK treated groups showed a minimum of M2 type macrophages and M1 type macrophages in all groups. In addition, acute in ammatory reaction usually occurs after reperfusion, which is re ected by the secretion of in ammatory factors such as TNF-α from the activated immune cells [36]. The experimental results showed that compared with normal saline treated group, IL-1β and TGF-β concentration in serum was signi cantly decreased in CsA@PPTK treated MI/RI mice on 4th day after reperfusion ( Figure.6C-D). Immuno uorescence staining results showed that compared with the sham group, Tregs in myocardial tissue signi cantly increased in the model group, and Tregs in myocardial tissue decreased after the treatment of CsA. Compared with model group, CsA@PPTK signi cantly increased the number of Tregs in the infarct area ( Figure.6E-F). The above data demonstrated that CsA@PPTK alleviated MI/RI and recovered the heart function through regulating the phenotype of macrophage and reducing systemic in ammatory reaction.
TUNEL staining images of infarcted heart area on 7th day after reperfusion is showing in Figure.7A-B.
The positive staining represents the apoptotic cardiomyocytes. Compared with normal saline treatment, CsA, @PPTK, CsA@PTK and CsA@PPTK signi cantly reduced the apoptosis of cardiomyocytes in ischemic area. CsA@PPTK treated group displayed a minimum number of apoptotic cardiomyocytes in ischemic area among all groups, which resulted from inhibiting the production of mitochondrial ROS and the recovery of damaged mitochondrial membrane potential of cardiomyocytes.
Masson trichrome staining and H&E staining of heart tissue are showing in Figure.7C. In masson trichrome staining section, normal heart muscle is represented in red and brotic areas are represented in blue. Compared with sham group, the blue areas were increased in normal saline treatment group. Meanwhile, the ventricles were signi cantly dilated, and the walls of the ventricles were signi cantly thinner in the model group than that in sham group. The percentage of myocardial brosis area in myocardial section area of mice is showing in Figure.7D. The area of myocardial brosis in normal saline group was signi cant greater than that in sham group. CsA, @PPTK, CsA@PTK and CsA@PPTK signi cantly reduced myocardial brosis as compared with normal saline. As compared with the same dose of CsA, CsA@PPTK signi cantly reduced myocardial brosis at the dose of 2.5 mg/kg. The percentage of left ventricular area to myocardial section area is showing in Figure.7E. The percentage of left ventricular area to myocardial section area in normal saline group was marked smaller than that in sham group. CsA@PPTK signi cantly increased the percentage of left ventricular area to myocardial section area as compared with normal saline. As compared with the same dose of CsA, CsA@PPTK signi cantly increased the percentage of left ventricular area to myocardial section area at the dose of 2.5 mg/kg. The above results demonstrated that CsA@PPTK strongly inhibited the cardiac remodeling and brosis, subsequently markedly improved the cardiac function of MI/RI mice. These results were also consistent with the echocardiography data. In addition, a large number of in ammatory cells were in ltrated, and the structure of cardiomyocytes was unclear in normal saline group. The cardiomyocytes in sham group were orderly arranged and the cell structure was intact. In 2.5 mg/kg CsA@PPTK treatment group, the arrangement of cardiomyocytes was regular, and the in ammatory cell in ltration was decreased. These results indicated that CsA@PPTK exhibited anti-in ammatory effect and protected myocardium through anti-in ammatory effect.
The CX43 is the main protein that constitutes the intercellular gap junction of ventricular myocytes [37]. The effect of CsA@PPTK on the expression of CX43 in myocardium of MI/RI mice is showing in Figure. Interestingly, the arteriole density in the infarct area was also increased (Figure.8E) in CsA@PPTK treatment group, indicating that blood vessels were preserved in infarct area by using CsA@PPTK. This demonstrated that CsA@PPTK reduced apoptosis of endothelial cells of heart arteries, and subsequently improved blood supply in infarct area and cardiac function of MI/RI mice.

Discussion
Currently, there is no ideal treatment method for MI/RI. Many studies suggest that MI/RI is actually the result of interaction of multiple factors including cardiomyocytes apoptosis, oxidative stress and in ammatory reaction. Increased ROS leads to apoptosis of cardiomyocytes, in ammatory cell recruitment and in ammatory reaction [38,39] . High level of ROS can cause cardiomyocytes apoptosis by affecting mitochondrial membrane potential. ROS can also lead to oxidation of cardiomyocytes membrane and recruitment of in ammatory factors [6] . Thus, ROS is an important factor in reperfusion injury. Scavenging ROS has been proved to have protective effect on MI/RI. In recent years, ROS-responsive materials have been paid more attention in drug delivery system. This kind of material can not only be used as a carrier, but also can degrade and release drug at sites with high ROS level. Consequently, they display ROS active targeted drug delivery effect. At present, ROS-responsive materials are mainly based on phenylboric acid and thioketal. Thioketal bond can be broken by various ROS such as potassium superoxide (KO 2 ), H 2 O 2 , hydroxyl radical (•OH), hypochlorite (ClO − ) and peroxynitrite (ONOO − ) at low concentrations [40]. In this study, based on thioketal, a ROS-responsive material PTK was designed and synthesized. PTK was highly biocompatible and contained a large amount of thioketal bonds. In vitro experiments showed that PTK could eliminate ROS and played a protective role against H/R damaged cardiomyocytes through eliminating ROS. PTK could also scavenge ROS in ischemic myocardium in vivo. In addition, PTK released the contained drugs while scavenging ROS, and displayed characteristics of ROS-sensitive drug release. Therefore, PTK was a multifunctional bioactive material with both ROS-responsive drug release and ROS elimination properties.
The cell experiment showed that CsA, CsA@PTK and CsA@PPTK reduced the opening of mPTP and restored the damaged mitochondrial membrane potential of H9c2 cells induced by H/R. Besides, the ROS level in H/R damaged H9c2 cells and its mitochondria was decreased by using @PPTK, CsA@PTK and CsA@PPTK. The cellular uptake experiment indicated that more amount of CsA@PPTK was accumulated in H/R damaged H9c2 cells than CsA and CsA@PTK. Therefore, as compared with @PPTK and CsA@PTK, the scavenging effect of CsA@PPTK on ROS in H/R damaged H9c2 cells and its mitochondria was stronger. The above results demonstrated that CsA@PPTK protected H9c2 cells from H/R injury by strongly blocking the opening of mPTP and eliminating ROS in H/R damaged H9c2 cells.
The key point for targeted drug delivery system to play effective role in MI/RI treatment is that it can target to the area of ischemic myocardium and release drugs rapidly. After being intravenously injected, as compared with CsA@PTK, the amount of CsA@PPTK accumulated in liver of MI/RI mice was signi cantly reduced. This demonstrated that platelet membrane coated on the surface of nanoparticle markedly reduced the phagocytosis of nanoparticle by MPS. This was because the CD47 on the platelet membrane could inhibit the clearance of CsA@PPTK by MPS [30]. The ex vivo experiment results indicated CsA@PPTK could speci cally bind to the endothelial-damaged blood vessel wall. After being intravenously injected to MI/RI mice, CsA@PPTK mainly distributed in apical tissue that was the most severe cardiac ischemia area. This indicated that CsA@PPTK could actively target to ischemic myocardium of MI/RI mice. This was resulted from that transmembrane protein GPIV and GPIX on the platelet membrane could target and bind with the damaged blood vessels, while the integrin-related proteins CD9 and CD81 on the platelet membrane could increase the uptake of CsA@PPTK by cardiomyocytes [41,42]. The in vitro experimental results showed that CsA@PPTK quickly released CsA at the present of ROS. Thus, CsA@PPTK signi cantly increased the viability of H/R damaged H9c2 cells.
There was a large amount of ROS in ischemic myocardium tissue after reperfusion. Therefore, CsA@PPTK could fast release CsA and reduced the opening of mPTP of cardiomyocytes in cardiac ischemia area. At the same time, CsA@PPTK strongly scavenged ROS in ischemic myocardium tissue.
Finally, CsA@PPTK alleviated apoptosis of cardiomyocytes and decreased myocardial infarction size in MI/RI mice.
Phase one in ammatory reaction induced by M1 type macrophages is closely related to the generation of ROS, which is known as activation of in ammatory macrophages and facilitating recruitment of in ammatory cell to infarct area [43,44]. The phenotype change of macrophages plays a key role in the progress of tissue repair. In the subsequent stages of MI/RI, it is essential for an ideal tissue repair process that pro-in ammatory M1 type macrophage polarizes to reparative M2 type macrophage. The increased ratio of M2 type macrophage to M1 type macrophage is of bene ts to reducing chronic in ammation and brosis in cardiac ischemia tissue. Some studies have also found that timely transformation of M1-type macrophages into M2-type macrophages can effectively increase the repair of infarcted myocardial tissue [45,46]. In vivo experimental data indicated that CsA@PPTK obviously increased the ratio of M2 type macrophage to M1 type macrophage in heart tissue. At the same time, in vivo experimental results also showed that in CsA@PPTK treated group, the number of M1 type macrophages and M2 type macrophages markedly decreased, indicating the number of macrophages recruited to ischemic myocardial tissue decreased. This was resulted from the decrease in heart injury of MI/RI mice by CsA@PPTK, which further resulted in the decrease in the number of recruited macrophages. Besides, CsA@PPTK signi cantly reduced myocardial brosis area as compared with CsA, @PPTK and CsA@PTK. CsA@PPTK greatly decreased the systemic in ammatory reaction by reducing IL-1β and TGF-β concentration in serum of MI/RI mice. This was resulted from following three reasons.
Firstly, CsA@PPTK improved the oxidized microenvironment of MI/RI by scavenging ROS in cardiac ischemia area. Secondly, CsA@PPTK reduced apoptosis of cardiomyocytes in ischemic myocardium tissue by releasing CsA to inhibit the opening of mPTP. The vicious cycle between the burst release of ROS and apoptosis of cardiomyocytes in ischemic myocardium tissue was blocked. Finally, CsA can inhibit the immune response mediated by T lymphocytes, and reduce the release of various lymphocytes such as IL-2, IL-3, IFN-γ and the expression of IL-2R. Consequently, recruitment of in ammatory cells to damaged myocardium tissue was weakened and in ammatory reaction was alleviated.
IL-1β is a pro-in ammatory factor. Our study showed that MI/RI signi cantly increased the level of IL-1β in serum, while CsA, @PPTK, CsA@PTK and CsA@PPTK reduced the level of IL-1β to a certain extent.
CsA@PPTK displayed the most signi cant effect on the reduction of IL-1β. These results indicated that CsA@PPTK displayed anti-in ammatory effect, which was consistent with the results of immuno uorescence staining. TGF-β, secreted by anti-in ammatory macrophages, has been shown to play a protective role in the myocardial matrix by inhibiting the synthesis of metalloproteinases and inducing the synthesis of protease inhibitors [47,48]. However, TGF-β overexpression can also induce the transformation of cardiac broblasts into myo broblasts, leading to myocardial brosis and cardiac remodeling [49] . Our study showed that TGF-β level increased to a certain extent after MI/RI, which resulted from compensation after myocardial injury. There was no signi cant change in the level of TGF-β in the CsA@PPTK group compared with the sham group, which suggested that CsA@PPTK could better maintain the homeostasis of injured myocardial tissue, and subsequently reduced cardiac remodeling.
The results showed that the number of Tregs in the myocardial infarction area increased signi cantly 3 days after reperfusion, which was due to the fact that circulating Tregs in ltrated into the myocardial tissue through vascular wall in the early stage of myocardial injury to antagonize excessive in ammatory response. CsA displayed an inhibitory effect on Tregs. Thus, a signi cant decrease in Tregs in cardiac tissue was observed after free CsA was administered. Studies have found that ROS scavenger can increase the number of Tregs. Therefore, the increase of Tregs by CsA@PPTK might be due to the ROS scavenging effect of PTK, which counteracted the inhibitory effect of CsA on Tregs, and this counteraction effect was much stronger at higher doses.
It was reported that Tregs increased in myocardial tissue after reperfusion in mice. Tregs alleviated heart damage of MI/RI mice by inhibiting apoptosis of cardiomyocytes and neutrophil in ltration and reducing infarct size [11,12]. Besides, Tregs inhibited the activity of cytotoxic T cells and pro-in ammatory M1 type macrophage, and then suppressed local in ammatory response and systemic cytotoxic response induced by myocardial infarction, thereby reducing ventricular remodeling and improving cardiac function [50,51]. The experiment results indicated that CsA@PPTK augmented Tregs in ischemic myocardium tissue. In a word, by reprogram Tregs generation, the ratio of M2 type macrophage to M1 type macrophage and redox microenvironment, CsA@PPTK alleviated in ammatory reaction and subsequently reduced myocardial brosis and the remodeling of left ventricle. In general, CsA@PPTK could not only simulate the effect of Tregs through anti-apoptotic, anti-in ammatory and anti-oxidant effects, but also played a synergistic protective role on MI/RI by directly increasing the number of Tregs in myocardial infarction area.
MMP-9 increased sharply after myocardial infarction, and it was closely related to the remodeling of left ventricle. Elevated MMP-9 levels have been found in ventricular remodeling tissue after AMI [52,53]. In MI/RI animal models, the expression of MMP-9 was increased with the prolongation of heart injury. The activity of MMP-9 is positively correlated with the severity of AMI, and the mortality of MI/RI animals was signi cantly increased with the increase of MMP-9 expression. In the process of infarction, in ammatory reaction plays an important role in expression of MMP-9. CsA@PPTK signi cantly alleviated in ammatory reaction in MI/RI mice, which resulted in the decrease of MMP-9 expression in left ventricle [54]. Furthermore, the experimental data also demonstrated that CsA@PPTK markedly attenuated left ventricular remodeling by reducing MMP-9 expression in left ventricle anterior wall of MI/RI mice.
The CX43 is the most important protein in the gap junction of the left ventricular muscle [37]. The cardiac electrochemical impulse is mainly transmitted to the cardiomyocytes of the left ventricle through CX43 to maintain the rhythmic contraction of the left ventricle. Under normal circumstances, CX43 mainly exists as a phosphorylated status. When myocardial ischemia occurs, the expression of CX43 will be reduced, and CX43 protein is dephosphorylated, which eventually induces arrhythmias. The experimental results showed that CsA@PPTK signi cantly increased the expression of CX43 as compared with free CsA and CsA@PTK, indicating CsA@PPTK greatly increased the activity of cardiomyocytes and cardiac function in the late repair period of MI/RI.

Conclusion
Platelet membrane-based Tregs biomimetic nanoparticle (CsA@PPTK) is suitable for ischemia myocardium targeting drug delivery and ROS-triggered drug release. CsA@PPTK signi cantly reduced the remodeling of the left ventricle and enhanced heart protective effect through reprograming in ammatory and redox microenvironment and attenuating apoptosis of cardiomyocytes at infarct tissue.

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Competing interests
There is no competing interest.