| Background Myocardial infarction is the irreversible injury with a high mortality rate worldwide,which mainly occurs as a result of inadequate blood supply,initiated by the presence of coronary atherosclerosis.After the occurrence of myocardial infarction,heart fibroblasts supersede the apoptotic and necrotic cardiomyocytes and promote the formation of noncontractile scar tissues,ultimately resulting in heart failure and even death.Early reperfusion is an important approach for saving the ischemic myocardium through timely restoring blood supply.However,reperfusion itself can cause additional injury to myocardium,namely myocardial ischemia/reperfusion(I/R)injury.Currently,various therapeutic approaches have been presented against myocardial I/R injury.Ischemic conditionings,including ischemic preconditioning,ischemic postconditioning and remote ischemic conditioning,have been extensively explored for protection of myocardium against I/R injury.However,the above ischemic conditionings exist some disadvantages,such as unpredictability of ischemic event and invasive interference,leading to the limited values in the clinic application.Although pharmacological interference is non-invasive and easily feasible for patients,none of drugs was found to have satisfactory effects on myocardial I/R injury.In addition,both stem cell transplantation and gene therapy have been confirmed as effective approaches in the repair of the myocardial infarction via promoting neovascularization and cardiomyocyte survival.However,low cell reservation and survival rate,as well as low gene transfection efficiency and ethical concerns severely restrict the application in clinical translation.Many previous studies have proved that the primary mechanism of myocardial I/R injury is abundant amounts of reactive oxygen species(ROS),generated from mitochondrial respiratory chain,cause the irreversible injury to myocardial cells.Recently,a large amount of ceria,palladium,vanadium,copper,and gold-based nanoparticles possess mimicking natural enzyme activities and have been developed to scavenge ROS and cure ROS-related diseases in vivo.However,these nanoparticles are composed of heavy metals,which have potential side effects on living organisms.Naturally occurring components are suitable for biomedical applications because of the negligible side effects and favorable biosafety in vivo.Natural melanin has excellent free radicals scavenging ability and has been widely used in biomedical fields,such as UV irradiation protection,cancer theranostics,acute kidney injury and anti-infective therapy.Inspired by naturally occurring allomelanin derived from fungi,we employed a distinct self-assembly synthesis strategy to engineer allomelanin nanoparticles(AMNPs).And then,the antioxidative and anti-inflammatory properties of AMNPs were investigated.Finally,we evaluated the therapeutic potential of AMNPs in myocardial I/R injury model.Methods In the present study,AMNPs were synthesized by using oxidizing agent NaIO4 to oligomerize and polymerize 1,8-DHN at room temperature.By screening the reaction conditions about the molar ratio of NaIO4 to 1,8-DHN monomer,the optimal AMNPs would be acquired.Then,the surface of optimal AMNPs would be modified by polyethylene glycol(PEG)through the noncovalent bond,resulting in the generation of PEGylated AMNPs(AMNPs@PEG).To substantiate the antioxidative competences of AMNPs and AMNPs@PEG,a series of free radical scavenging experiments were performed,and the scavenging rate of free radicals,such as 2,2-diphenyl-1-picrylhydrazyl(DPPH·)radical,2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate)·+(ABTS·+)radical,and 2-phenyl4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide(PTIO·)radical,as well as hydroxyl radical(·OH)and superoxide anion radical(O2·-)would be calculated.Next,we examined the protective effect of AMNPs@PEG against H2O2-induced oxidative stress on myocardial cells through Cell Counting Kit-8(CCK-8)assay and Calcein acetoxymethyl ester(Calcein-AM)/propidium iodide(PI)double staining assay in vitro.Besides,the intracellular ROS levels of myocardial cells were monitored by a specific fluorescent probe,namely 2’,7’-dichlorofluorodiacetate(DCFHDA).And the fluorescence intensity of tetramethylrhodamine ethyl ester perchlorate(TMRE)was used to reflected the levels of mitochondrial membrane potential(ΔΨm)on myocardial cells after the H2O2 or AMNPs@PEG treatment.The optimal excitation wavelength of AMNPs@PEG was determined by the photoacoustic(PA)imaging system.Then,the PA signals of AMNPs@PEG were collected at different time points and at various concentrations under the optimal excitation wavelength.Subsequently,all the myocardial I/R mice were received the intravenous injection of AMNPs@PEG,following by in vivo PA imaging.And the sham-operated mice were used as controls.The distributions of AMNPs@PEG in myocardial tissues were observed by biological transmission electron microscope(TEM).Transthoracic echocardiography was used to measure the cardiac function of the mice.And the size of myocardial infarct was measured by Evans Blue and triphenyl tetrazolium chloride(TTC)method.Terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)staining was performed to evaluate the severity of cardiomyocyte apoptosis.The scar area in infarct heart was measured by Masson’s trichrome staining.The α-SMA and CD31 immunohistochemical staining were performed to investigate the level of angiogenesis after the treatment of AMNPs@PEG.In addition,the expression level of vascular endothelial growth factor(VEGF)was detected by immunofluorescence staining.In order to elucidate the biological mechanisms of therapy from AMNPs@PEG,transcriptomics was applied to analyze the differentially expressed genes(DEGs)in myocardial tissue between the saline and AMNPs@PEG-treated mice.And the immune cell populations in heart,such as neutrophils,M1 subtype macrophages and M2 subtype macrophages,were analyzed by flow cytometry.Quantitative polymerase chain reaction(qPCR)was used to analyzed the mRNA encoding for cytokines involved in pro-inflammation(such as TNF-α,CCL2,and iNOS)and anti-inflammation(such as Arg-1,IL-10,and MRC1).In the end,the normal C57 BL/6J mice,received a single intravenous injection with AMNPs@PEG,were used to assess the biological security of AMNPs@PEG in vivo.Results AMNPs were generated through oxidative oligomerization and polymerization of 1,8-DHN.As soon as the oxidizing agent NaIO4 was added under vigorous stirring at room temperature,the liquid mixture of 1,8-DHN and NaIO4 rapidly changed to yellow and then gradually turned dark gray.In addition,the morphology and size distribution of AMNPs was altered as the molar ratio of NaIO4 to 1,8-DHN monomer changed.By screening multiple reaction conditions,we found that the optimal molar ratio of NaIO4 to 1,8-DHN monomer was 0.05:1,resulted in ultrasmall sphericity-like nanoparticles.After PEGylated surface modification of AMNPs,the size of nanoparticles was raised from 31.2±4.64 nm to 36.0±5.31 nm,and Zeta-potential was altered from-47.4±1.24 mV to-39.0±0.44 mV,respectively.In addition,compared to AMNPs without PEGylation,AMNPs@PEG possessed the better dispersibility and hydrophilicity.Next,a series of free radical scavenging experiments were performed to substantiate the antioxidative activities of AMNPs and AMNPs@PEG.The scavenging rate of DPPH· radical,ABTS·+radical and PTIO· radical,as well as ·OH and O2·-reached 75.52%,85.58%and 76.03%,as well as 74.31%and 73.59%,corresponding to 12.5 μtg mL-1,15 μg mL-1,and 80 μg mL-1,as well as 80 μg mL-1 and 80 μg mL-1 of AMNPs,respectively.As expected,AMNPs@PEG possessed highly broad anti oxidative capabilities similar to that of AMNPs.The cell viability of myocardial cells remained higher than 90%at various concentrations of AMNPs@PEG,indicating almost no cytotoxicity of AMNPs@PEG.Additionally,live/dead cell staining showed that a lot of dead cells(red fluorescence)could be observed after treatment with H2O2 alone.However,the number of dead cells(red fluorescence)was significantly reduced,accompanied by a dramatical increase in the number of live cells(green fluorescence)via the increase of AMNPs@PEG concentration.And the results of CCK-8 assay showed that the cell viability of myocardial cells fell to 55.09%under oxidative stress,but the cell viability rose to 79.04%with the addition of 10 μg mL-1 of AMNPs@PEG.The strong intracellular ROS level(green fluorescence)could be detected from myocardial cells treated with H2O2 alone.However,under oxidative stress,the intracellular ROS level significantly decreased as the myocardial cells were treated with AMNPs@PEG.Similarly,compared to the normal myocardial cells,oxidative stress could obviously induce the ΔΨm level depolarization of myocardial cells.Whereas,the ΔΨm level could be recovered to 78.26%of that in normal myocardial cells with the addition of 10 μg mL-1 of AMNPs@PEG against the oxidative injury.In vitro PA imaging experiment exhibited that the 680 nm optics wavelength was selected as the optimal excitation wavelength,and the PA imaging signal of AMNPs@PEG was highly stable for a prolonged time.Moreover,the PA imaging signal of AMNPs@PEG displayed a concentration-dependent characteristic.After intravenous injection of AMNPs@PEG,the PA imaging signal in I/R injury mice was significantly enhanced,and the results of biological TEM showed that AMNPs@PEG could target and accumulate at the locations of I/R injury.48 h after reperfusion,the results of Evans Blue and TTC double staining showed that intravenous administration of AMNPs@PEG markedly decrease the myocardial infarct area in I/R mice as compared with those having saline treatment(23.71±10.12%vs.53.79±10.19%,P<0.001).And cardiac function was obviously improved in I/R mice receiving AMNPs@PEG treatment compared with those receiving saline treatment.Besides,compared to saline,administration of AMNPs@PEG markedly decreased the number of myocardial apoptosis in I/R mice.On day 28 post-operation,the results of Masson’s trichrome staining showed that intravenous administration of AMNPs@PEG to myocardial I/R mice resulted in significant decrease of the fibrosis area as compared with those injected with saline.Besides,compared with saline,administration of AMNPs@PEG significantly improved cardiac function in I/R mice.Additionally,AMNPs@PEG treatment markedly increased the number of α-SMA+and CD31+blood vessels in myocardial I/R mice as compared with those receiving saline treatment.And the expression levels of VEGF in the infarct hearts of I/R mice were significantly raised after treated with AMNPs@PEG as compared with those treated with saline.The mRNA-sequencing had identified 592 DEGs between the saline and AMNPs@PEG-treated myocardial I/R mice.Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of these DEGs showed that apoptosis signaling pathway,IL-17 signaling pathway,NOD-like receptor signaling pathway and the expression of genes related to inflammation were significantly inhibited in AMNPs@PEG-treated mice in comparison with saline-treated mice.Flow cytometric analysis confirmed that the number of infiltrating neutrophils was markedly lower at the sites of myocardial I/R injury in AMNPs@PEG-treated mice than saline-treated mice.In addition,the proportion of M1 subtype macrophages was exhibited to be obviously lower and the percentage of M2 subtype macrophages was showed to be significantly higher at the area of myocardial I/R injury in AMNPs@PEG-treated mice than saline-treated mice.And the qPCR analysis revealed that a suppressed expression of the pro-inflammatory cytokines(such as TNF-α,CCL2,and iNOS)and an enhanced expression of the anti-inflammation cytokines(such as Arg-1,IL-10,and MRC1)were confirmed in infarct hearts of AMNPs@PEG-treated mice when compared with saline-treated mice.In vivo biocompatible experiment showed AMNPs@PEG did not cause distinct influence over the growth of mice,as well as hematology indicatrix,liver function indicators and kidney function indicators.Besides,no obvious damage was detected in H&E-stained histological sections of the major organs(such as heart,liver,spleen,lung,and kidney).Conclusions In general,we engineered AMNPs via oxidative oligomerization and polymerization of 1,8-DHN.After PEGylated surface modification,AMNPs@PEG possessed the better dispersibility and hydrophilicity than AMNPs.Importantly,AMNPs@PEG also possessed highly broad antioxidative properties similar to that of AMNPs against multiple reactive oxygen and nitrogen radicals.In vitro cellular experiment confirmed that AMNPs@PEG could protect myocardial cells from oxidative injury through elimination of intracellular free radicals and suppression of ΔΨm depolarization.Moreover,as an excellent PA contrast agent,AMNPs@PEG could be visualized by PA imaging at the myocardial injury sites.Importantly,intravenous administration of AMNPs@PEG was of benefit to alleviation of myocardial injury and improvement of cardiac function against myocardial I/R injury.Meanwhile,AMNPs@PEG could promote the macrophage to switch from M1 subtype towards M2 subtype,and prevent neutrophils infiltration at the myocardial injury sites,accompanied by a suppressed expression of the pro-inflammatory cytokines and an enhanced expression of the anti-inflammation cytokines. |
