{"paper_id":"4a807545-b610-4f72-b2a5-ebbc9f2bb46d","body_text":"Milk-derived haem scavenging microsponges protect heart against ferroptosis-induced reperfusion injury | 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 Article Milk-derived haem scavenging microsponges protect heart against ferroptosis-induced reperfusion injury Yang Zhu, Liwen Zhang, Jun Wen, Jiawei Zhang, Ziyang Sun, Liyin Shen, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4467590/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 ischemia/reperfusion injury with a high incidence of intramyocardial haemorrhage (IMH) contributes to enlarged infarct size by inducing additional cell death and predisposes to risk of heart failure. However, the risk factor in blood remains unverified and unaddressed. Here, we report that haem burstly released from IMH, is the key iron source for ferroptosis, and correspondingly propose the treatment strategy of blocking the cellular uptake of exogenous haem. Unfortunately, there is no existing haem-scavenging materials. We discover that methacryloyl modification of lysine residues on apo-lactoferrin (Apo-Lf), a milk-derived protein screen from natural haem-binding candidates, surprisingly increased the number of haem-binding sites by 86% and binding affinity by one order of magnitude. In animal models, intramyocardially implanted ferroptosis-inhibiting lactoferrin microsponges (FILMS) fabricated from the modified Apo-Lf achieved desirable anti-ferroptosis effects by rapid haem scavenging. Transcatheter FILMS implantation in pigs further demonstrated its safety and translational potential. These results provide deeper mechanistic understanding of ferroptosis-induced I/R injury, and may aid the development of other biomaterial-based therapies. Physical sciences/Materials science/Biomaterials/Biomaterials &#x2013; proteins Biological sciences/Biotechnology/Biomaterials/Biomaterials &#x2013; proteins protein biomaterials ferroptosis hydrogel ischemia/reperfusion injury protein modification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Majority of patients with acute myocardial infarction receive reperfusion therapy in order to promptly reestablish blood flow to ischemic myocardium, consequently minimizing myocardial necrosis and enhancing patient outcomes 1,2 . However, returning of blood can paradoxically induce additional cardiac damage, a phenomenon known as myocardial ischemia/reperfusion (I/R) injury, which affects millions per year 3 . I/R injury diminishes therapeutic efficacy and predisposes to risk of heart failure 4 . Recent evidences and our previous works revealed that ferroptosis, an iron-dependent cell death program that differs from apoptosis and necrosis, is a major cause of I/R injury 5–8 . To date, treatments for cardiac ferroptosis are not clinically available. Seventy percent I/R patients experience IMH following cardiac reperfusion 4,9,10 , which induces iron deposition in the reperfused myocardium 11,12 as red blood cells are lysed to release iron into interstitial space. The released iron needs enter the cells to trigger the ferroptotic storm which occurred in organelles, especially mitochondria 13 . However, the transmembrane transportation efficiency of iron ions is limited 13 . We also found that H9C2 CMs in vitro can tolerate iron ion at concentrations far higher than that in patient myocardium (Supplementary Fig. 1). Thus, there should be an invasive iron carrier which accounts for cellular iron uptake. With in vivo experiments, we narrowed down from whole blood from IMH to haem released from lysed red blood cells as the major iron carrier which smuggles iron into cardiac cells. It is reported that haem oxygenase-1 (HO-1) mediated haem degradation frees iron ions from haem to catalyze Fenton reaction and subsequent lipid peroxidation, a landmark event in ferroptosis 14 . In contrast to iron in ion form, haem causes severe cell damage in vitro at pathologically relevant low concentrations (Supplementary Fig. 1). Based on these understandings, we proposed a treatment strategy of intercepting free haem outside the cardiac cells with intramyocardial implantation of haem-scavenging biomaterial, to block the iron from trespassing the cell membrane. We chose local haem-scavenging over systemic scavenging to lower the risks of 1. inhibiting normal iron-dependent physiological activities in other organs, and 2. metabolically transferring haem from IMH and circulation to kidney and spleen hence causing secondary damages. To the best of our knowledge, no implantable haem-binding biomaterials for ferroptosis inhibition have been developed. Molecules recognized for their ability to bind to haem are predominantly localized within proteins and peptides 15,16 . Here, we designed a milk-derived injectable haem-sequestering microsponge system to rapidly capture free haem released from IMH before they enter cardiac cells and activate ferroptosis. We first screened out a milk-derived apo-metalloprotein, Apo-Lactoferrin (Apo-Lf), from a group of protein candidates for its high haem-binding affinity and capacity. Subsequently, double bonds are modified onto the surface of Apo-Lf, to allow fabrication of ferroptosis-inhibiting lactoferrin micro-sponges (FILMS). Surprisingly, this engineered Apo-Lf gained significantly higher haem-binding affinity and capacity compared to its natural form. Intramyocardially implanted FILMS quickly drained excessive extracellular haem from IMH in reperfused animal hearts before haem molecules were uptaken by cardiac cells. The clinically relevant porcine model demonstrated the compatibility of FILMS with minimally invasive percutaneous transcatheter injection, and the capability of FILMS in preventing I/R injury in large volume of reperfused myocardium. Results Haem released from IMH causes myocardial ferroptosis in I/R injury IMH and iron deposition were observed via cardiac magnetic resonance (CMR) in the left ventricular (LV) wall of patients who received reperfusion one day post MI (Extended Data Fig. 1 a). As a result of reperfusion, cardiac injury markers including NT-proBNP, CK-MB, CK and AST surged in the first 6 h (Extended Data Fig. 1 b). Different compounds of blood were administered into infarcted rat myocardium to identify the iron carrier primarily responsible for IMH-induced ferroptosis and reperfusion injury (Fig. 1 a). Infarcted myocardium which received whole blood or red blood cells (RBCs) showed similar increase of CK-MB, CK and cell death comparable to I/R controls, significantly higher than MI and sham controls, and MI rats received intramyocardial plasma injection (Fig. 1 b, Extended Data Fig. 1 c). The spatiotemporal couplings of RBCs, haemoglobin (HBB) and dead cells further confirmed that RBCs flooding into the interstitial space preluded the I/R injury surge (Fig. 1 c-e, Extended Data Fig. 1 d). We hypothesized that haem was released upon hemolysis of RBCs and acted as the main transmembrane iron carrier. In fact, free haem rapidly accumulated in the reperfused myocardium (Fig. 1 f). In addition, haem injection into infarcted myocardium led to exacerbated injury, same as I/R (Fig. 1 g, Extended Data Fig. 1 c). In vitro cultured H9C2 showed high sensitivity to haem, (IC50 = 5.82 µM), while IC50 of ferric ions is 4.50 mM, almost 3 orders of magnitudes higher than the lethal concentration of haem (Supplementary Fig. 1). Hence, we confirmed that haem is the major risk factor in I/R injury with IMH. Myoglobin level in reperfused myocardium was not significantly different compared to sham, indicating that haem accumulation was only attributed to IMH (Extended Data Fig. 1 e). Exogenous haem was transported across cell membrane and degraded by upregulated haem oxygenase-1 (HO-1) into ferrous ions, which accumulated in either the labile iron pool (LIP) or ferritin proteins (Fig. 1 h, i). Labile iron overload was shown to initiate ferroptosis 17 . RNA-seq comparison between sham and I/R samples revealed a significant enrichment in “ferroptosis” and haem binding terms (Fig. 1 j, k, Extended Data Fig. 1 e). 4-hydroxynonenal (4-HNE), a pivotal marker of lipid peroxidation, exhibited a higher expression level in I/R group (Fig. 1 k). Additionally, a low dose of exogenous haem caused lipid peroxide accumulation (a ferroptosis indicator) and severe death of iPSC-CMs in vitro (Extended Data Fig. 1 f, g, Fig. 1 k), which could only be inhibited by ferroptosis inhibitor deferoxamine (DFO), but not apoptosis inhibitor Z-VAD-FMK (ZVF) or necroptosis inhibitor Gsk-872 (Fig. 1 l). In a word, exogenous haem released from IMH induced ferroptosis via elevating cellular iron level. Therefore, we proposed a strategy to prevent I/R injury by blocking “Trojan horse”, haem, from entering cardiac cells. Circulating haem-binding proteins reduced myocardial injury but brought side effects Some proteins have been reported to bind haem, including hemopexin (Hx), serum albumin (SA), lipopolysaccharide-binding protein (LBP), casein, alpha-1 microglobulin, alpha-1 antitrypsin, lactoferrin (Lf), apo-lactoferrin (Apo-Lf) and transferrin (Tf) 11,15,18,19 . We measured their haem-binding capacities, (Fig. 1 n, Extended Data Fig. 2 a-h), and found that among all tested candidates, Apo-Lf and Hx have competitive haem-binding affinity (~ 0.12–0.13 nM) and the former exhibited the highest haem-binding capacity (55.21 µg/mgprot). Intravenous injection of Apo-Lf or Hx (Extended Data Fig. 2 i) reduced cardiac injuries, featuring lower levels of cell death post IMH and iron overload (Fig. 1 n, Extended Data Fig. 2 j-k). However, the circulating haem-binding proteins brought excessive iron and associated damages to remote organs, revealed by iron deposition in spleen and HBB accumulation in kidney (Fig. 1 o). Hence, an implantable medical device for local haem scavenging is more desirable. The design and fabrication of local haem-sequestering FILMS We proposed to implant injectable protein hydrogel microspheres to form in situ haem-sequestering sponges in the myocardium. Apo-Lf (iron-free form of lactoferrin) was chosen for miscroshpere fabrication for its highest affinity and binding capacity among the candidates, and its cost-effectiveness (Fig. 1 m, Extended Data Fig. 3 a-c). To increase intermolecular interactions for hydrogel formation, we methacrylated 30% surface lysine residues on Apo-Lf to add 13–14 double bonds to each Apo-Lf molecule, obtaining UV-curable Apo-LfMA (Fig. 2 a, Extended Data Fig. 3 a, d-h). Only lysine residues among all amino acid residues participated the methacrylation reaction. Fourteen lysine sites were identified with a modification probability of over 50%, including K46, K47, K71, K288, K296, K301, K320, K332, K348, K358, K460, K539, K627 and K652 (Fig. 2 a, Extended Data Fig. 4 ). Protein secondary structure did not change significantly after iron removal and methacrylation (Extended Data Fig. 3 i). Surprisingly, both haem affinity and binding capacity of the engineered protein dramatically increased compared to pristine Apo-Lf. SPR showed that the haem-binding constant of Apo-LfMA is one order of magnitude lower compared to that of Apo-Lf 0.13 nM to 0.015 nM (thus higher affinity) (Fig. 2 b,c). Molecular docking and molecular dynamic simulation were employed to analyze the protein/haem complex (Fig. 2 d, Extended Data Fig. 5 a). Apo-LfMA bound 13 haems, which was 6 more than the 7 haem-binding sites in Apo-Lf (Extended Data Fig. 5 b). This capacity increase was consistent with experimental quantification results of haem and iron content in the two protein/haem complexes (Fig. 2 e). Methacryloyl modification of the 14 lysine residues changed the intramolecular physical interaction, hence altered the conformation of existing haem binding sites and could theoretically cause structural perturbations at distal regions 20,21 in the protein, which explains the observed enhancement of haem-binding affinity and capacity. Methacryloyl groups increased the hydrophobic area of Apo-LfMA to 956.29 Å 2 from 889.60 Å 2 of Apo-Lf, generally strengthening the hydrophobic interaction between the hydrophobic porphyrin ring of haem and protein (Extended Data Fig. 5 c). Moreover, methacryloyl groups on lysine altered the nearby conformation and involved more amino acids into the existing haem-binding sites, increasing the number of salt bridges, hydrogen bonds and stronger metal coordination in Apo-LfMA/haem binding pockets (Fig. 2 f). For example, Site 1 in the Apo-LfMA/haem complex showed the formation of new metal coordination between SER304 and haem-Fe(Ⅱ) center compared to its counterpart in the Apo-Lf/haem complex (Fig. 2 g). These added interactions contributed to the leap in haem affinity of the original binding sites (Extended Data Fig. 5 b). The new haem-binding pockets could be attributed to the increase in the accessibility of potential binding sites. For example, the distance between HIS-272 and TYR-211 is 6.8 Å in Apo-Lf, too narrow to form a haem-binding site. After modification, the distance between HIS-272 and TYR-211 increases to 8.2 Å in Apo-LfMA, wide enough for haem to accommodate in (Fig. 2 h,i). Decreased radius of gyration (Rg), root mean square deviation (RMSD), solvent accessible surface area (SASA), and root mean square fluctuation (RMSF) also confirmed a more compact structure and higher stability of the Apo-LfMA/haem complex in comparison with the Apo-Lf/haem complex (Extended Data Fig. 5 d-g), which is a result of the strong haem-amino acids interaction in the pockets. Rapid accumulation of haem in myocardium post reperfusion demands highly efficient haem scavenging. Therefore, porous structure was generated in FILMS via ice crystal template technology to enhance haem diffusion and drainage by increasing the specific surface area and accessibility of internal volumes (Fig. 2 j-l). The polymerization of metharyloyl groups reduced the absorbance of unsaturated C = C stretching vibration at 3291 cm − 1 peak and increased the saturated C-C stretching vibration (Extended Data Fig. 6a). The crosslinking process did not significantly alter the relative contents of second structure in Apo-LfMA and FILMS (Extended Data Fig. 6b, c). FILMS (892.7 nm average pore diameter and 88.96% porosity) was found significantly more effective in scavenging haem compared to FILMG without pores (Fig. 2 l,m, Extended Data Fig. 6d,e). In haem buffer solution, FILMS reached haem-absorbing balance within 1 hour, shorter than the time required for exogenous haem to cause significant damages in the myocardium (1 ~ 2 h post reperfusion) (Fig. 2 n). In contrast, non-porous counterpart FILMG requires about 4 hours to reach the haem-absorbing balance, which is theoretically slow considering the time window indicated by Fig. 1 f. Quantification of bound haem on FILMS showed that photo-crosslinking of methacryloyl groups did not decrease the haem-binding capacity of Apo-LfMA (Extended Data Fig. 6f). It is worth noting that the dissociation equilibrium constant of Apo-LfMA is as low as 9.22 x 10 ^−10 , indicating that the desorption of haem from Apo-LfMA is thermodynamically unfavorable. Therefore, FILMS have a desirable haem scavenging rate and total amount, and could significantly decrease its accessibility for cardiac cells. FILMS trapped exogeneous haem and protected CMs from ferroptosis In a high-haem concentration in vitro environment simulating IMH, FILMS salvaged rat CMs and preserved over 90% cell viability (Fig. 3 a,c). Fe 2+ fluorescent signals, revealing the intracellular iron level, demonstrated that FILMS prevented haem from entering the cells, whereas FILMG had a significantly lower effect, and gelatin microgel (GMG) without haem-binding capacity failed to inhibit the increase of intracellular iron level compared to free haem control (Fig. 3 b,d). CMs upregulated iron metabolism indicators including Hmox1 , Ftl and Fth , which are signs of active degradation of haem into labile iron (Fig. 3 e-g). This further induced the expression of iron efflux gene ( Fpn ) and downregulated Tfrc associated with iron transportation (Fig. 3 h,i). These alterations in mRNA transcription indicated the activation of ferroptosis pathway. When extracellular haem was sequestered in the protein hydrogel network of FILMS, cellular iron imbalance was inhibited and ferroptosis was alleviated. Intracellular iron enrichment in haem-treated CMs led to an ROS surge from Fenton reaction (Extended Data Fig. 6g-i), and elevated membrane lipid peroxidation (Fig. 3 j, k). This trend was inhibited by FILMS, while FILMG showed less effect and GMG showed no inhibitory effect. Acsl4 has an important regulatory role in ferroptosis by facilitating the conversion of long-chain fatty acids into acyl-CoA derivatives, whose mRNA expression increased in both blank and GMG groups but not in FILMG and FILMS groups 22 (Fig. 3 l). As the powerhouse of CMs, mitochondria were subject to ferroptosis. Mitochondrial iron levels across the groups exhibited a changing pattern similar to that of the cellular iron levels (Fig. 3 m). Correspondingly, FILMS prevented the mitochondrial membrane potential disruption observed in haem treated CMs (Fig. 3 n), while FILMG and GMG only partially mediated or fail to mediate mitochondrial damage. To demonstrate the clinical relevance of the results above obtained from rat CMs, we validated that FILMS could inhibit ferroptosis of human iPSC-CMs by extracellularly scavenging haem (Fig. 3 o-r). Together, FILMS protected CMs from suffering ferroptosis through scavenging free haem more rapidly than FILMG, while GMG did not protect the CMs in high-haem environment. FILMS inhibited IMH-dependent myocardial ferroptosis in rat I/R model. Through pathological and transmission electron microscopy (TEM) observations, hearts of I/R rats with IMH phenotype were included for analysis (Fig. 4 a, Extended Data Fig. 7a). In rat I/R model, IMH leads to a rapid accumulation of free haem in LV myocardium in the first 24 h to 34.74 ± 5.70 nmol/mgprot, which lead to an 89% increase in myocardial iron content (Fig. 4 b, c). Local implantation of FILMS reduced the amount of free haem in the myocardium to 16.69 ± 1.87 nmol/mgprot, showing no significant difference compared to that of sham baseline. This curtails in haem concentration resulted in an iron content decrease by 81% (Fig. 4 b, c). It is worth noting that such haem content equals to the haem content at 1.49 h post reperfusion in the I/R control (Fig. 1 f), the percentage of dead cardiac cells is theoretically less than 3.6% at this time point (Fig. 1 e), which is confirmed by the measure percentage, 5.7%, (Extended Data Fig. 7b). Without haem drainage into the implanted microspheres, GMG group failed to lower haem concentration and cardiac cell loss (Fig. 4 b, Extended Data. 7b). In both GMG and I/R groups, HBB-positive red blood cells were spatially coupled with TUNEL signals (Fig. 4 d, e), while TUNEL signals were sparse and weak in HGB-positive areas in FILMS treated rats, spatially demonstrating the contribution of IMH to I/R injury and the rescuing effect of FILMS (Fig. 4 d, e, Extended Data Fig. 7b). Both GMG and FILMS microspheres aggregated in the injection sites (Fig. 4 f), but only FILMS served as drainage sites for exogenous haem from IMH. Iron signal concentrated at the FILMS injection sites, rather than in the surrounding tissue, while severe iron deposition was evident in the tissue surrounding the implanted GMG (Fig. 4 f). As transmembrane transport of haem was blocked by FILMS, upregulation of Hmox1 and ferritin in cardiac cells was inhibited (Extended Data Fig. 7c-d, f-g). Elevated iron level in the GMG group led to increased lipid peroxidation and morphological changes of mitochondria including swelling and membrane rupture, whereas these ferroptosis features were mediated by FILMS (Fig. 4 g, Extended Data Fig. 7e, h). To strengthen the mechanism of exogenous haem induced I/R injury and FILMS treatment, we conducted transcriptomic analysis on I/R myocardial tissues treated with FILMS, GMG, and saline, in comparison to sham group. Differential gene expression and principal component analysis (PCA) indicated that FILMS group was most similar to sham group and distinct from GMG and I/R groups (Fig. 4 h-i). GO analysis revealed significant changes in iron metabolism, haem binding, and lipid metabolism in the myocardium after I/R, accompanied by changes at the cellular membrane and mitochondrial organelle levels (Extended Data Fig. 8a, b). These transcriptional changes were suppressed by FILMS. According to KEGG enrichment analysis, ferroptosis was the primary pathway through which FILMS attenuated I/R injury (Extended Data Fig. 8c). Compared to GMG group (Fig. 4 j, Extended Data Fig. 8c), FILMS group downregulated haem metabolism genes (including Hmox1 , Cyba , Hebp1 and Cybb ) and ferroptosis genes ( Ftl and Acsl4 ), and upregulated lipid metabolism and cardiac function recovery genes ( Me3 , Slc4a3 , Tnnt2 and Ascl6 ). Expression of these genes, including ferroptosis genes, was not significantly different between the GMG and I/R groups (Extended Data Fig. 8d-f). HPLC-MS based redox lipidomics revealed that oxidized lipids (oxPLs) as metabolic hallmarks of ferroptosis were upregulated in I/R group compared to the sham group, and downregulated to sham level in FILMS group (Fig. 4 k-m). These results confirmed that FILMS inhibited ferroptosis in I/R rats. Consequently, serum levels of cardiac injury markers in FILMS group including CK-MB, CK, AST and LDH were maintained at those of sham group, confirming that alleviating haem-induced ferroptosis by FILMS reduced I/R injury (Fig. 4 n-q). Long-term echocardiography evaluation 35 days post-surgery demonstrated that FILMS attenuated LV remodeling in terms of both cardiac function (LVEF and LVFS) and geometry (LVIDS and LVESV) compared to GMG and I/R groups (Fig. 4 r, t and u, Extended Data Fig. 9a). Consistently, FILMS reduced fibrotic infarct size by 17.3% compared to I/R group and maintained LV wall thickness (Fig. 4 s, v and w). In addition, FILMS reduced the ratio between type I collagen (Col I) and type III collagen (Col III) in the infarct, which is a sign of less severe fibrosis, while I/R and GMG groups showed high Col I/Col III ratios (Extended Data Fig. 9b-d). Additionally, vessel density and Cx43 expression in FILMS group were significantly higher than those in I/R and GMG groups (Extended Data Fig. 9e-j). Importantly, no adverse effects caused by FILMS were observed in spleen or kidneys (Extended Data Fig. 9k). Minimally invasive trans-endocardial FILMS implantation in a preclinical pig I/R model. Safety and efficacy of FILMS in treating ferroptosis and I/R injury were further demonstrated in a porcine cardiac I/R model. FILMS was compatible with a minimally invasive transcatheter injection system (TEIS01, Dinova Medtech), which was employed in transendocardial hydrogel injection in heart failure patients 23 (Extended Data Fig. 10a). Under the guidance of digital subtraction angiography (DSA), the steerable dual-lumen needle catheter was inserted through the femoral artery and through the aortic valve to reach LV endocardium (Fig. 5 a (i-iii), Extended Data Fig. 10a). The injection sites were positioned using both transthoracic echocardiogram and DSA (Fig. 5 a (ii-iv)). FILMS were injected into the LV wall without leakage, bleeding or wall perforation (Fig. 5 a (v)). Depots of aggregated FILMS were found in the myocardium (Fig. 5 a (vi)). All pigs survived the surgery. In the efficacy study, FILMS were injected into 9 points uniformly distributed in the infarcted LV, followed by immediate reperfusion (Extended Data Fig. 10b, c). Three days post-surgery, TTC staining showed large volume of necrotic myocardium across the LV anterior wall and interventricular septum (Fig. 5 b). Notably, the volume of necrotic myocardium was significantly smaller in FILMS group compared to I/R control, and the color of myocardium surrounding the injected FILMS was closer to healthy myocardium (Fig. 5 b). Size of necrotic myocardium in FILMS group was reduced by 70.4% compared to I/R group (Fig. 5 c). The elevations in myocardial enzyme markers (CK-MB, CK, LDH and AST) and cTnI in serum post I/R injury were also mediated by implanting FILMS (Fig. 5 d-h). H&E staining confirmed IMH in both I/R and FILMS groups (Fig. 5 k). Free haem level in the I/R group increased to 2.02 times that of a healthy myocardium (Fig. 5 i). FILMS reduced the amount of haem to a healthy level by trapping the exogenous haem. In I/R group, transcription and expression levels of HMOX1 and FTL were significantly upregulated, indicating that haem had been internalized into cellular iron (Fig. 5 j,m,n). Both DAB-enhanced Prussian blue staining and tissue quantitative testing confirmed severe iron deposition in the I/R tissue, while FILMS scavenged haem and downregulated HMOX1, thereby significantly alleviated iron overload (Fig. 5 l,o). Downregulation of iron level directly led to a decrease in lipid peroxidation (ACSL4 mRNA and tissue MDA) in myocardium, bringing lipid peroxidation back to healthy level. Therefore, FILMS inhibited IMH induced ferroptosis and I/R injury in clinically relevant large animal model. H&E analyses of major organs confirmed the biosafety of FILMS treatment in vivo (Extended Data Fig. 10d). Discussion In recent years, understanding of the mechanism of ferroptosis, including cardiac ferroptosis, has become more comprehensive and profound 6,7,14,22,24–29 . In contrast, treatments for cardiac ferroptosis are still not clinically available, of which some have been validated in small animal models 13,24 . In this study, instead of inhibiting processes in the middle of the ferroptosis cascade, FILMS targets at the initiator, haem, for cardiac ferroptosis. To the best of our knowledge, FILMS is the first haem-binding, implantable biomaterial. We expect that FILMS could serve as a reference for the design of more potent haem-scavenging biomaterials for treating I/R injury and other ferroptosis-induced diseases, particularly with the reported mechanism of simultaneously improving both binding affinity and capacity of Apo-Lf. Homeostasis of metal ions is essential in vital physiological processes 30 . Various implantable biomaterials have been developed to deliver and supplement metal ions including magnesium, strontium and etc. to injured tissues in which the concentration of metal ions was low due to diseases 31,32 . However, for diseases like ferroptosis in which metal ion concentrations are pathologically high (usually iron, copper, zinc etc. as they are normally low in normal tissue), few biomaterials are available for metal chelating therapy 33 . FILMS represent a biomaterial-based regulatory strategy to temporarily trap excessive harmful ion species from local environments by scavenging their small molecular carriers 34 . In fact, many metal ions incorporate into corresponding organic molecules (e.g. porphyrins) to form metal complexes before binding to proteins to function, e.g. iron and haem, magnesium and chlorophyl Ⅱ 35,36 . The pathological stimuli release these cytotoxic metal complexes from proteins, markedly disturbing the health status of patients 37 . Hence, direct catch of metal complexes is an attractive strategy to deal with metal cytotoxicity. FILMS inhibiting ferroptosis in IMH-induced I/R injury by scavenging haem serves as a demonstration of the feasibility and efficacy of such strategy. This study also demonstrates the potential of medical implants based on milk-derived proteins. Only a few types of natural proteins, such as collagen, fibrinogen, and silk fibroin have been used as major components for clinically approved implants and devices. The application of milk-derived proteins in medical implants is still rare despite of their enormous yield and high biocompatibility. A significant limiting factor is that the processability of milk-derived proteins is hindered by insufficient intermolecular interactions, thereby necessitating the incorporation of additional intermolecular forces or bonds. By introducing crosslinkable methacryloyl groups or white-light sensitive crosslinking catalyst, the most abundant proteins in milk, whey protein and casein, have recently been manufactured into structural materials 38,39 . FILMS demonstrate the advantages of using milk-derived proteins with unique bioactivities as raw materials for medical devices. Methacryloyl modification of Apo-Lf can be chemically considered as mutating the amino acids, similar to site-directed mutagenesis widely used in study the changes of protein activitie 40 . It is worth noting that photo-crosslinking, microfluidic emulsification and freeze-thaw as representative processing methods did not significantly inactivate haem-binding capability of Apo-LfMA. In addition, the changes in the structure-activity relationship of Apo-Lf demonstrates the functional plasticity of milk-derived proteins. Therefore, the development of novel high-activity medical devices based on milk-derived protein for treating diseases including I/R injury has considerable feasibility and application potential. Statistics and reproducibility Statistical analysis was performed using Prism. Results are shown as mean ± SD unless stated otherwise. T-tests were used to compare result from two groups. One-way analysis of variance (ANOVA) was used for multiple comparisons unless stated otherwise. p value of < 0.05 was considered statistically significant. All in vitro and in vivo results are representative of two to six independents. Declarations Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Data availability All relevant data related to this manuscript are available on request from the authors on reasonable request. The remaining data are available within the Article or Supplementary Information. Source data are provided with this paper. Acknowledgements The MALDI-TOF data were collected at Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University with the technical support from Huiwen Wang. We thanked Qiong Huang, Jingyao Chen, Chengcheng Zhang and Yajun Yu from the core facility platform of Zhejiang University School of Medicine for their technical support of pathological staining. We gratefully acknowledge Yungin Li for technical assistance on laser confocal microscopy. We thank Yinping Lv in the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University for her technical assistance on TEM. We would also like to extend our thanks to Zhiwei Ge and Jingqun Yuan at the Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University for their help on UPLC-QTOF/MS and ICP analysis, respectively. This study is financially supported by “Pioneer” and “Leading Goose” R&D Program of Zhejiang Province, China (No. 2024C03074, 2023SDXHDX0004), National Natural Science Foundation of China (No. 82202328, 82170644), State Key Laboratory of Transvascular Implantation Devices, China (No. 012024018), Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (No. SN-ZJU-SIAS-004), the Natural Science Foundation of Zhejiang Province, China (LR23H020001). Author contribution Y. Zhu conceived the idea of FILMS. Y. Zhu, X. Fang, C. Guo, T. Ren and L. Zhang designed the overall project. L. Zhang, Z. Sun, X. Jiang and B. Dai. L fabricated and characterized Apo-LfMA and FILMS, simulated molecular docking and molecular dynamics. L. Zhang conducted in vitro cell experiments with Y. Hua’s assistance under the guidance of X. Fang, T. Ren and C. Gao. L. Shen and JY. Zhang performed the small animal study. J. Wen, WY. Wang and X. Deng collected the clinical data. J. Wen performed the minimally invasive FILMS implantation. JW. Zhang and L. Zhang conducted the biochemical analysis of animal samples. L. Zhang, Y. Gao and Q. Jin performed RNA-seq and lipid metabonomics analysis. WZ. Wang, M. Wang and F. Feng prepared and characterized iPSC-CMs. F. Xu collected SPR data. C. Guo designed the protein study. X. Fang designed the cell experiments. L. Zhang, X. Fang, T. Ren and Y. Zhu designed the small animal studies. Y. Tang, J. Wen and Y. Zhu designed the large animal study. Y. Zhu, L. Zhang, J. Wen, T. Ren, X. Fang, and C. Guo wrote the manuscript. Competing interests : The authors declare no competing interests. References Tsao, C. W. et al. Heart Disease and Stroke Statistics—2023 Update: A Report From the American Heart Association. Circulation 147 , e93–e621 (2023). Timmis, A. et al. European Society of Cardiology: cardiovascular disease statistics 2021: executive summary. Eur. Hear. J. - Qual. Care Clin. Outcomes 8 , 377–382 (2022). Heusch, G. Myocardial ischaemia–reperfusion injury and cardioprotection in perspective. Nat. Rev. Cardiol. 17 , 773–789 (2020). Liu, T. et al. Intramyocardial hemorrhage and the “Wave front” of reperfusion injury compromising myocardial salvage. J. Am. Coll. Cardiol. 79 , 35–48 (2022). Moon, B. F. et al. Iron imaging in myocardial infarction reperfusion injury. Nat. Commun. 11 , 3273 (2020). Fang, X. et al. Ferroptosis as a target for protection against cardiomyopathy. Proc. Natl. Acad. Sci. U. S. A. 116 , 2672–2680 (2019). Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149 , 1060–1072 (2012). Wang, X. D. & Kang, S. Ferroptosis in myocardial infarction: Not a marker but a maker. Open Biol. 11 , 0–3 (2021). Fröhlich, G. M., Meier, P., White, S. K., Yellon, D. M. & Hausenloy, D. J. Myocardial reperfusion injury: looking beyond primary PCI. Eur. Heart J. 34 , 1714–1724 (2013). Moeckel, D. et al. Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk. Sci. Transl. Med. 6 , 1–10 (2014). Menon, A. V. et al. Excess heme upregulates heme oxygenase 1 and promotes cardiac ferroptosis in mice with sickle cell disease. Blood 139 , 936–941 (2022). Maus, M. et al. Iron accumulation drives fibrosis, senescence and the senescence-associated secretory phenotype. Nat. Metab. 5 , 2111–2130 (2023). Fang, X., Ardehali, H., Min, J. & Wang, F. The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease. Nat. Rev. Cardiol. 20 , 7–23 (2023). Mishima, E. et al. A non-canonical vitamin K cycle is a potent ferroptosis suppressor. Nature 608 , 778–783 (2022). Belcher, J. D. et al. Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. Blood 123 , 377–390 (2014). Sankari, S. et al. A haem-sequestering plant peptide promotes iron uptake in symbiotic bacteria. Nat. Microbiol. 7 , 1453–1465 (2022). Fang, X., Ardehali, H., Min, J. & Wang, F. The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease. Nat. Rev. Cardiol. 20 , 7–23 (2023). Madyaningrana, K. et al. Alpha 1-antitrypsin counteracts heme-induced endothelial cell inflammatory activation, autophagy dysfunction and death. Redox Biol. 46 , 102060 (2021). Allhorn, M., Berggard, T., Nordberg, J., Olsson, M. L. & Akerström, B. Processing of the lipocalin α 1-microglobulin by hemoglobin induces heme-binding and heme-degradation properties. Blood 99 , 1894–1901 (2002). Rajasekaran, N. & Naganathan, A. N. A self-consistent structural perturbation approach for determining the magnitude and extent of allosteric coupling in proteins. Biochem. J. 474 , 2379–2388 (2017). Rajasekaran, N., Sekhar, A. & Naganathan, A. N. A universal pattern in the percolation and dissipation of protein structural perturbations. J. Phys. Chem. Lett. 8 , 4779–4784 (2017). Stockwell, B. R. et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell 171 , 273–285 (2017). Wang, B., Lee, R. J. & Tao, L. First-in-human transcatheter endocardial alginate-hydrogel implantation for the treatment of heart failure. Eur. Heart J. 44 , 326 (2023). Stockwell, B. R. Ferroptosis turns 10: emerging mechanisms, physiological functions, and therapeutic applications. Cell 185 , 2401–2421 (2022). Mao, C. et al. DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer. Nature 593 , 586–590 (2021). Bersuker, K. et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 575 , 688–692 (2019). Badgley, M. A. et al. Cysteine depletion induces pancreatic tumor ferroptosis in mice. Science. 368 , 85–89 (2020). Doll, S. et al. FSP1 is a glutathione-independent ferroptosis suppressor. Nature 575 , 693–698 (2019). Mishima, E. et al. DHODH inhibitors sensitize to ferroptosis by FSP1 inhibition. Nature 619 , E9–E18 (2023). Waldron, K. J., Rutherford, J. C., Ford, D. & Robinson, N. J. Metalloproteins and metal sensing. Nature 460 , 823–830 (2009). Bosch-Rué, È., Díez-Tercero, L., Buitrago, J. O., Castro, E. & Pérez, R. A. Angiogenic and immunomodulation role of ions for initial stages of bone tissue regeneration. Acta Biomater. 166 , 14–41 (2023). Vasconcelos, D. M., Santos, S. G., Lamghari, M. & Barbosa, M. A. The two faces of metal ions: From implants rejection to tissue repair/regeneration. Biomaterials 84 , 262–275 (2016). Kim, J.-J., Kim, Y.-S. & Kumar, V. Heavy metal toxicity: An update of chelating therapeutic strategies. J. Trace Elem. Med. Biol. 54 , 226–231 (2019). Crisponi, G. et al. Kill or cure: Misuse of chelation therapy for human diseases. Coord. Chem. Rev. 284 , 278–285 (2015). Mendel, R. R., Smith, A. G., Marquet, A. & Warren, M. J. Metal and cofactor insertion. Nat. Prod. Rep. 24 , 963 (2007). Chalkley, M. J., Mann, S. I. & DeGrado, W. F. De novo metalloprotein design. Nat. Rev. Chem. 6 , 31–50 (2021). Dutra, F. F. & Bozza, M. T. Heme on innate immunity and inflammation. Front. Pharmacol. 5 , 1–20 (2014). Hu, Z. et al. Scalable milk-derived whey protein hydrogel as an implantable biomaterial. ACS Appl. Mater. Interfaces 14 , 28501–28513 (2022). Zhu, Q. et al. White-light crosslinkable milk protein bioadhesive with ultrafast gelation for first-aid wound treatment. Biomater. Res. 27 , 6 (2023). Fu, Y. et al. Engineered P450 atom-transfer radical cyclases are bifunctional biocatalysts: reaction mechanism and origin of enantioselectivity. J. Am. Chem. Soc. 144 , 13344–13355 (2022). Sinclair, P. R., Gorman, N. & Jacobs, J. M. Measurement of heme concentration. Curr. Protoc. Toxicol. 00 , 8.3.1–8.3.7 (1999). Zhang, L. et al. Multileveled hierarchical hydrogel with continuous biophysical and biochemical gradients for enhanced repair of full-thickness osteochondral defect. Adv. Mater. 35 , 2209565 (2023). Zhao, M. et al. Enhancing the engraftment of human induced pluripotent stem cell-derived cardiomyocytes via a transient inhibition of rho kinase activity. J. Vis. Exp. 10 , e59452 (2019). Yang, Y. et al. Elastic 3D-printed hybrid polymeric scaffold improves cardiac remodeling after myocardial infarction. Adv. Healthc. Mater. 8 , e1900065 (2019). Wang, L. et al. Injectable and conductive cardiac patches repair infarcted myocardium in rats and minipigs. Nat. Biomed. Eng. 5 , 1157–1173 (2021). Methods Materials. Lactoferrin (Bega, Australia), methacrylic anhydride (Sigma-Aldrich, USA), oxalic acid (Sigma-Aldrich, USA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (Aladdin, China), gelatin (Sigma-Aldrich, USA). Phosphate buffered saline (PBS, Invitrogen, USA), Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin, and penicillin/streptomycin were bought from Gibco (USA). Cell counting kit-8 (CCK-8, Dojindo, Japan), dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Beyotime Biotechnology, China), FerrOrange and Mito-FerroGreen (Dojindo, Japan), C11 Bodipy (581/591) and Live/dead Kit (Thermo Fisher, USA), Mitotracker red (Yeasen Biotechnology, China), One-Step TUNEL apoptosis assay kit (Beyotime Biotechnology, China), Anti-Hemoglobin (Acbam, UK, ab92492, Polyclonal, 1:500 dilution), Anti-Cardiac Troponin T antibody (Acbam, UK, ab209813, Monoclonal, 1:4000 dilution), Anti-Ferritin Light chain antibody (Abcam, UK, ab69090, Polyclonal, 1:200 dilution), Anti- 4 Hydroxynonenal antibody (Abcam, UK, ab48506, Monoclonal, 1:1000 dilution), Anti-Heme Oxygenase 1 antibody (Abcam, UK, ab52947, Monoclonal, 1:1000 dilution) Anti-Connexin 43 antibody (Acbam, UK, ab11370, Polyclonal, 1:2000 dilution), Anti-CD31 antibody (Abcam, UK, ab182981, Monoclonal, 1:2000 dilution) and Anti-αSMA antibody (Boster, China, BM0002, Monoclonal, 1:400 dilution) were used according to corresponding protocols.H9C2 cell line (GNR 5) was purchased from ATCC and Cell Bank of Typical Culture Collection of Chinese Academy of Science. Clinical data collection. Three patients with acute myocardial infarction admitted to the Department of Cardiology of the Peking University Third Hospital and receiving cardiac magnetic resonance were included. Clinical data on patients' treatment during hospitalization, laboratory indicators and surgical treatment were collected through the electronic medical record system. This study complied with the Declaration of Helsinki and was approved by the Ethics Review Committee of Peking University Third Hospital (M2022577). Methods of cardiac magnetic resonance examination. The CMR examination in the acute phase of myocardial infarction was performed using an MR750w 3.0 T magnetic resonance machine and a 32-pass surface phased-array coil from GE Healthcare, USA. The examination sequences included a movie sequence, a delayed enhancement sequence (performed 10 min after injection of gadolinium contrast agent), and a black-blood T2-weighted sequence. The cine sequence was performed using a steady state free-feeding sequence to acquire two-chamber, three-chamber, and four-chamber long-axis images as well as multilayered short-axis images of the right and left ventricular base toward the apical portion of the ventricle.The T2 pressure-lipid images were acquired by a T2-weighted fast spin-echo sequence. Delayed gadolinium contrast enhancement (LGE) was scanned using an inversion recovery sequence. Cardiac magnetic resonance image postprocessing. CMR images were analyzed by experienced cardiovascular imaging physicians using the postprocessing software C VI42 5.13 (Circle Cardiovascular Imaging, Canada). Edematous areas (signal intensity exceeding 2 standard deviations from the infarcted distal myocardium) within the blood-supplying region of the offender vessel were quantified by T2-weighted image sequences and defined as the infarct at risk area (AAR). Low-signal areas within the edematous region were defined as acute-phase intramyocardial hemorrhage and recovery-phase iron deposition. High-signal areas (signal intensity exceeding 5 standard deviations above the infarcted distal myocardium) in the LGE images were defined as infarcted area. Apo-LfMA preparation and characterization. Apo-Lf was obtained by removing iron from lactoferrin. Lactoferrin was dissolved in PBS (0.2 mol/L, pH 2.0~4.0) with stirring. The pH was adjusted by adding HCl and NaOH (5 mol/L), as indicated by a color change from red to colorless. EDTA was added before dialysis against PBS (50 mmol/L) for 48h with 6-8h buffer exchanges. Iron-free lactoferrin, apo-Lf, was obtained by lyophilization of half the dialyzed solution. Next, we designed a scheme to attach double bonds to the iron-free lactoferrin so that it can be polymerized. Methacrylic anhydride was used to graft double bonds to amino groups of Apo-Lf the remaining half of the iron-free lactoferrin solution from Step 1. The pH was adjusted to neutral and the reaction proceeded for 4 h. The product was dialyzed against deionized water with stirring, with 5-6 h exchanges for 48h total. Lyophilization yielded methacrylated Apo-Lf (apo-LfMA). For NMR characterization, 15 mg of Apo-Lf and Apo-LfMA were dissolved in DMSO- d 6 with 1.0 M LiCl. One-dimensional (1D) and two-dimensional (2D) NMR spectra were collected on Bruker AVANCE NEO 600 MHz NMR spectrometers equipped with cryo-probe. 1D 1 H NMR was performed with a spectral width of 20 ppm, 32 scans, and a relaxation delay of 3.0 s. Correlation spectroscopy (COSY) for 1 H- 1 H was performed with a spectral width of 12 ppm in both the t1 and t2 dimensions, 160 and 2048 complex points in the t1 and t2 dimensions, respectively, 16 scans, and a relaxation delay of 1.0 s. Heteronuclear single quantum coherence (HSQC) spectra were collected with a spectral width of 170 ppm in the t1 and a spectral width of 12 ppm in the t2 dimensions, 160 and 2048 complex points in t1 and t2 dimensions, respectively, and 16 scans. The molecular weights of proteins were measured on a Bruker Rapiflex matrix-assisted laser desorption ionization-time of flight (UltrafleXtreme, MALDI-TOF) mass spectrometer. Samples were dissolved in deionized water before becoming combined with an equal volume of the sinapic acid matrix reconstituted in 0.1% trifluroacetic acid and 30% acetonitrile. The chemical structure of Lf, Apo-Lf and Apo-LfMA was characterized by FT-IR spectroscopy (Nicolet 6700, USA). For HPLC-MS characterization of proteins. Apo-Lf and Apo-LfMA proteins were lysed in trypsin solution at 60℃ for 4h to get peptide samples. The peptide samples were loaded onto a ACQUITY UPLC CSH C18 column connected to an UPLC-Triple-TOF/MS system (Waters, USA). Peptides were separated and eluted with a gradient of 5% to 95% HPLC buffer B (0.1% formic acid in acetonitrile, v/v) in buffer A (0.1% formic acid in water, v/v) at a flow rate of 300 nl min −1 . The eluted peptides were then ionized and analysed by an AB TripleTOF 6600 plus System mass spectrometer (AB SCIEX, Framingham, USA). The pressure of Curtain Gas (N2) was set to 35 psi. Maximum allowed error was set to ± 5 ppm. Declustering potential (DP), 80 V; collision energy (CE), 10 V. For MS/MS acquisition mode, the parameters were almost the same except that the collision energy (CE) was set at ± 50 ± 20 V, ion release delay (IRD) at 67, ion release width (IRW) at 25. The IDA-based auto-MS2 was performed on the 8 most intense metabolite ionsin a cycle of full scan (1 s). The scan range of m/z of precursor ion and product ion were set as 100-2000 Da and 50-2000 Da. The exact mass calibration was performed automatically before each analysis employing the Automated Calibration Delivery System. SPR analysis. SPR measurements were performed on the Biacore 8K Plus (Cytiva) system with the running buffer (0.01M PBS). Proteins as ligands (20 ug/ml) were immobilized on the CM5 sensor chip (GE Healthcare) through amine coupling. Then, ethanolamine-HCl flowed over the chip surface for 7 min, blocking the unreacted carboxyl groups. The flow rate was 30 µl min -1 for a contact time of 120 s followed by 800 s dissociation time. After each injection, the surface was regenerated using 3 M magnesium chloride (for PD-L1) or 10 mM glycine, pH 3.0 (for RBD). Data were fit with a 1:1 Langmuir binding model within the Biacore 8K analysis software (Cytiva, v.4.0.8.19879). Molecular docking and molecular dynamics simulation. From the PDB database, we got the 3D structure of lactoferrin protein (PDB: 1BLF) as a template and removed the ferric irons from the structure to obtained the 3D model of Apo-Lf for subsequent molecular docking. Using the 3D structure file of the modeled Apo-Lf as the initial structure, the amino groups on the 14 Lysine amino acid side chains of the protein were modified and combined with methacrylic anhydride to gain the structure of Apo-LfMA for subsequent molecular docking. The initial structures of proteins were processed using AutoDock Tools 1.5.6 to preserve the original protein charge and generate a pdbqt file for docking. UCSF Chimera was used to remove waters and non-protein atoms. AMBER99SB charges were assigned and pKa values calculated with H + + 3 at pH 7. Haem topology was generated with RDKit, minimized with MMFF94, and AM1-BCC charges assigned with Chimera. The protein-ligand complexes were constructed with Packmol. The Apo-Lf model was prepared in Autodock Tools by adding charges and saving as a pdbqt file. The haem ligand topology was generated with MOPAC semiempirical calculations and PM3 charges assigned. Docking was performed with Autodock 4.2.6 using a 100x100x100 grid box centered on the protein. 100 docking runs were calculated. The top binding modes were optimized using the Amber14 force field, first by 1000 steps of steepest descent, followed by 500 steps of conjugate gradient. GROMACS 5.1.5 was used for the simulations. The temperature was 300K, pH 7, and pressure 1 bar. The protein was centered in the box with a 0.1 nm minimum distance to the edges. Topologies were generated with pdb2gmx (AMBER99SB force field for protein) and AmberTools (GAFF for ligand). TIP3P water and NaCl ions neutralized the systems. Energy minimization was done by steepest descent, followed by 1 ns NVT and 1 ns NPT equilibrations. 50 ns production MD was performed with 2 fs time steps. Analysis included hydrogen bonds, salt bridges, RMSD, RMSF, Radius of Gyrate and SASA. Haem extraction and detection. Protein-haem complexes were lysed with 1 M HCl and haem was extracted with equal volume of 2-butanone. Following evaporation of 2-butanone, the haem was resuspended in DMSO. Haem was extracted from the heart tissues by homogenization in RIPA buffer containing protease inhibitors. Haem from the equal amount of proteins was quantified by fluorescence porphyrin assays with oxalic acid method 41 . In brief, 2 M oxalic acid was added to each sample, and the sample was split into 2 equivalents. One half was heated to 95 °C for 30 min to deprive iron from haem, while the other half was kept at 25 °C set as a subtraction for the corresponding heated sample. The suspensions were centrifuged at 25 °C to remove precipitates prior to measurement of porphyrin fluorescence on a Tecan Infinite M200Pro plate reader (excitation: 400 nm; emission: 608 nm). Haem concentration in each well was calculated against a standard curve prepared with series dilutions of haemin chloride. For heart tissue samples, the concentrations of haem were normalized to protein concentrations which were measured by using BCA protein assay kit (Beyotime). Iron qualification via ICP-MS. Protein-haem complexes were lysed with nitric acid. Tissue samples of infarcted myocardium were primarily homogenized in RIPA buffer containing protease inhibitors and analyzed by BCA method to normalized the data of protein amount in each sample; and then the supernatants were lysed with nitric acid. Agilent ICP-MS instrumentation with MassHunter 4.4 was used to collect data. FILMS, FILMG and GMG preparation. The aqueous phase contained 12% w/v Apo-LfMA and 3% w/v methacrylated gelatin (GelMA) in FILMG and FILMS samples or 12% GelMA in GMG samples with 0.3% LAP photo-sensitive initiator, and the oil phase was liquid paraffin with 5% w/v Span 80 emulsifier. The synthesis of GelMA according to a previously described method 42 . These two phases drove by separately syringe pumps, went through pipes at the speed of 3 mL/min in oil and 100 μL/min in protein solutions, and emulsified into droplets. For FILMG and GMG, emulsion droplets were collected in a 10 cm petri dish at 4°C for 10 min to maintain morphology, then photocrosslinked at 405 nm UV light for 3 min. For FILMS, the collected droplets were frozen at -80°C for 1 min followed by freezing in liquid nitrogen prior to UV-initiated crosslink. Blank Gelatin hydrogel microspheres were prepared via a microfluidic device. For FILMS, the collected droplets were frozen at -80°C for 1 min followed by freezing in liquid nitrogen prior to UV-initiated crosslink. Microspheres were washed 3 times each with petroleum ether, -80°C acetone, and deionized water before lyophilization and storage at 4°C. The characterizations of FILMG and FILMS. The morphology of FILMG and FILMS is observed under an optical microscope and SEM evaluation analysis after lyophilization. X-ray energy-dispersive spectroscopy (EDX, Inca X-Max, UK) was conducted to reveal the typical N and S elements in protein hydrogel (FILMS) in associated with SEM observation. Mercury intrusion porosimeter (Micromeritic AutoPore IV 9510, USA) was used to measure the porosity and specific surface area of FILMG and FILMS. FILMG and FILMS were incubated with 250 μM haem solution at pH=7.2, and the aqueous samples were collected and evaluated about the haem content through oxalic acid method and ICP at 30min, 1h, 2h, 4h, 8h and 24h to determine their haem absorption capacity. Young's effective moduli were measured in static mode using the Piuma nanoindenter (Optics11 life, Netherlands). Cell culture. 1) H9C2 CMs were cultured in Dulbecco's Modified Eagle Medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin, and incubated in a 37°C incubator equilibrated with 95% air and 5% CO 2 . 2) Human induced pluripotent stem cells (iPSCs) were generated and supplied by Cardiovascular Regenerative Medicine Laboratory (Frontier Innovation Center, School of Basic Medical Sciences, Fudan University). The hiPSCs were initially cultured on Matrigel-coated 6-well plate in mTeSR ™ 1 as the manufacturer’s directions. Cardiac differentiation was achieved using small molecules targeting the Wnt pathways as described in a previously reported protocol 43 . Briefly, confluent hiPSCs (>85%) maintained in mTeSR ™ 1 were dissociated with Versene for 5-9 min at 37 °C and then replated onto Matrigel-coated 6-well plate at 1 × 10 6 cells for each well. After culturing for 3 days in mTeSR™1, cells reached almost 100% confluency and then were treated with CHIR99021 at 12 μM in serum-free medium RPMI/B27 containing 1 μg/mL insulin for 24 h (day 0 for differentiation). Then the cultures were washed with PBS and replaced with serum-free medium RPMI/insulin-free B27 for 48 h. On day 3, cells were treated with 10 μM Wnt inhibitor IWR1 in serum-free medium RPMI/insulin-free B27. On day 5, the medium was changed to serum-free medium RPMI/insulin-free B27. Starting from day 7, cells were maintained in the serum-free medium RPMI/B27 containing insulin and the medium was changed every 3 days. Spontaneous beating of cardiomyocytes should first be visible from day 8 to day 10. The purification process of cardiomyocytes could be conducted when most of the cardiomyocytes had started beating. The medium was replaced with glucose-free medium RPMI/B27 containing 4 mM DL-lactate, and the incubation continued for 3-5 days without changing medium. When observing under the microscope, more than 90% of the cells in the field of view showed beating, which was defined as successful purification. Purified cardiomyocytes were used for subsequent experiments. Cell viability. H9C2 or hiPSCs were seeded in 96-well plates at a density of 8000 cell per well. After 12h culture, cells were supplemented with haem solutions of gradient concentrations and cell death inhibitions including DFO (100 μM, T1637, TargetMol), Gsk-872 (10 μM, T4074, TargetMol) and ZVF (50 μM, T7020, TargetMol) without FBS to confirm that haem induced ferroptosis of cardiomyocytes and its working curves. Otherwise, after 12h culture, cells were supplemented with haem solution and materials (GMG, FILMG and FILMS) without FBS to verify the anti-ferroptotic function of materials. After treatment for 24h, MTT (Abcam) stock solution was added to each well at a final concentration of 500 μg·ml -1 and incubated in the dark for 4h at 37 °C. The absorbance at 570 nm was measured in a Tecan plate reader. In addition, the survival of cells was visualized by using Live/Dead kit (Invitrogen TM ) and LSM 880 confocal microscope (Carl Zeiss). Measurements of ROS and lipid peroxidation by using DCFDA and C11-Bodipy 581/591. H9C2 or hiPSCs were seeded in 96-well plates at a density of 8000 cell per well. After 12h culture, cells were supplemented with haem solution and materials (GMG, FILMG and FILMS) without FBS. After treatment for 24h, cells were incubated with 1 µM of DCFDA (Beyotime) and BODIPY 581/591 C11 (Thermo Fisher) for 30 min at 37 °C. Subsequently, cells were visualized by LSM or were trypsinized, resuspended in 300 µl of Hanks’ balanced salt solution (HBSS, Gibco), and then analysed using a flow cytometer (CytoFLEX and CytExpert 2.4, Beckman Coulter) with a 488-nm laser paired with a 530/30 nm bandpass filter. Data were analysed using FlowJo Software 10 (Treestar). Fluorescence imaging for cellular detection. H9C2 cells were seeded in 24-well plates at a density of 50000 cell per well. After 12h culture, cells were supplemented with haem solution and materials (GMG, FILMG and FILMS) without FBS. After treatment for 24h, cellular labile iron was stained by FerroOrange Dye (F374, Dojindo). The Mito-FerroGreen (M489, Dojindo) fluorescent probe with the mitochondrial probe (40743ES50, Yeasen) was utilized to assess mitochondrial ferrous ions (Fe 2+ ) in live cells. JC-10 dye (Solabio) was used to determine mitochondrial membrane potential in cardiomyocytes. All procedures were according to the manufacturer's instructions. Animal study ethics. Adult male SD rats weighing 180-200 g (from the Laboratory Animal Care Facility of Shanghai Jiao Tong University School of Medicine) were housed at a constant temperature (22 ± 2 °C) under a 12-h light/dark cycle and given standard lab chow and water ad libitum. Bama miniature pigs weighing 30-35 kg were used. All investigations in this study conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 8023, revised 1978). Small-animal experiments (SD rats) were approved by Zhejiang Academy of Medical Sciences (ZJCLA-IACUC-20010691). Larger-animal experiments (Bama miniature pigs) were approved by the Experimental Animal Ethics Committee of Peking University Third Hospital (A2023072). In order to avoid experimental differences caused by animal sex, animals of the same sex were used in the same experiment. Study design in vivo . The in vivo study was divided into three parts. The first part aimed to exploring the pathophysiological process of intramyocardial haemorrhage in I/R model. In this part, we built both I/R model and MI model with the administration of blood components. I/R samples were sacrificed after reperfusion of 30min, 1h, 2h, 4h, 8h and 24h (n=4); and the blood, plasma, serum, RBCs, haemoglobin and haem were individually administered in the infarcted area (n=4). The second part focused on the validation of drugs or materials in rat I/R model, including the sham group, the intravenous injection of saline (I/R), Hx and Apo-Lf (n=4) and the intramyocardial injection of saline (I/R), GMG and FILMS (n=9). The third part was a preclinical trial in mini-pig I/R model including the sham group and the intramyocardial injection of saline (I/R) and FILMS (n=3). Rat model of MI with the administration of blood components . Before surgery, fresh blood of male SD rats was individually collected in anticoagulant tubes to obtain the plasma and RBCs samples or in promoting coagulating tubes to acquire the serum samples. The blood was isolated by three rounds of centrifugation at 600g and washed twice to separate the plasma samples and the RBCs. RBCs samples were than resuspended in PBS to obtain an equal volume to the original blood. The concentration of haemoglobin (R05236, BSZH Scientific LLC.) and haem (51280-5G, Sigma-Aldrich) was based on the delta value of haem level in between the sham and I/R infarcted myocardium. The MI model was constructed as previously described 44 . Briefly, animals were anaesthetized with 2% isoflurane inhalation and then intubated and ventilated with a respirator with extra oxygen. The rats were placed in the supine position, followed by a left thoracotomy and pericardiectomy to expose the hearts. Then the left anterior descending (LAD) coronary artery was ligated with a 6-0 silk suture at approximately 2-3 mm from its origin between the left atrium and the pulmonary artery conus to create LV infarction. Blood samples were then evenly injected into infarcted myocardium. The same procedure without ligation of the LAD coronary artery was conducted to sham-operated group. Rat model of I/R and treatments . Male SD rats were anaesthetized with 2% isoflurane inhalation and then intubated and ventilated with a respirator with extra oxygen. The rats were placed in the supine position, followed by a left thoracotomy and pericardiectomy to expose the hearts. Reversible ligation on the left anterior descending (LAD) coronary artery was performed utilizing sterile 6-0 silk suture with a slipknot subsequently following a left thoracotomy around the third intercostal space. Appropriate ligation was confirmed by visual observation of the left ventricle wall turning pale. After 60 min of regional ischemia, the heart was reperfused, resulting in loss of the discoloration of the myocardium distal to the ligation. The treatments with Hx (4 mg/kg) or Apo-Lf (4 mg/kg) were administered through the tail vein at 30 min before surgery. Therapeutic hydrogel microspheres were homogeneously injected into the infarcted myocardium. The same procedure without ligation of the LAD coronary artery was conducted to sham-operated group. Rats were anesthetized and sacrificed at 1 and 35 d post MI to harvest short-term and long-term treated hearts, respectively. Transcatheter endocardial FILMS implantation in mini-pig . In brief, the Chinese giant white pig (Alligator sinensis) (65kg) was anesthetized with tiletamine hydrochloride (4 mg/kg) and zolazepam hydrochloride (4 mg/kg). To evaluate the feasibility and safety of transcatheter endocardial hydrogel implantation for treating IR，minimally invasive intervention was used for hydrogel delivery. As previously described 23 , an 18-F guiding catheter was inserted via the femoral artery and guided crossing the aortic valve under DSA. Injections were performed with a steerable, dual-lumen needle catheter. Transthoracic echocardiography was used to localize the injection site. The tip of the catheter reached the endocardium, and contrast was injected into the LV wall to identify no leakage or perforation. The FILMS was injected into the myocardium at the mid-LV free wall. Pig was euthanized immediately after surgery and fresh heart tissue was obtained. The tissue was cut to observe the hydrogel retention and the presence of thrombosis at the injection site. Mini-pig model of I/R and treatments . The Bama minipigs () were anesthetized with tiletamine hydrochloride (4 mg/kg) and zolazepam hydrochloride (4 mg/kg). To establish the pig ischemia/reperfusion (I/R) model, a transthoracic 2D echocardiographic (ECG) measurement by Simpson’s method was performed to ensure that the animal was healthy before IR induction. Following the baseline ECG measurements, light anesthesia was maintained by continuous intravenous infusion of propofol (30 to 40 μg kg −1 ·min −1 ). An incision was made in the 4th intercostal space at the left edge of the sternum of the pig, and the skin, subcutaneous tissue, and muscle were incised layer by layer, and finally the pericardium was cut to expose the heart. Myocardial ischemia was caused by clamping the vessel below the second angular branch of the anterior descending branch and blocking blood flow with a coronary clip. The ECG changes in the animals were observed to confirm myocardial ischemia. The ECG, heart rate, and arterial pressure were continuously monitored. Defibrillation may be performed if ventricular fibrillation (VF) occurs, and IV epinephrine or atropine may be administered as needed for asystole/bradycardia/hypotension. After 60 minutes of ischemia, the coronary clamp was released to restore blood flow and reperfusion was achieved. The animals were observed for ECG changes to confirm myocardial reperfusion. The hydrogel was injected into the myocardium of the infarct margin and infarct area immediately after myocardial reperfusion (6-8 injection sites, 100 μl per site). Ultimately, the incision is sutured to ensure good hemostasis. Intravenous antibiotics were administered for three days postoperatively. Minipigs were anesthetized and sacrificed at 3 d post MI to harvest short-term treated hearts. Transmission electron microscopy. The rat myocardium tissues were surgically collected and subsequently fixed in 2.5% glutaraldehyde overnight. After washed three times with 0.1 M PBS, the tissues were fixed in 1% osmic acid for 1 h. The tissue segments were then rinsed with pure water, stained in 2% uranyl acetate for 30 min and dehydrated in ethanol of increasing concentrations from 50% to 100% and 100% acetone. After appropriate embedment according to standard procedures, the stained tissues were made into serial ultrathin section with an ultramicrotome (Leica UC7), and observed with a transmission electron microscope. Measurement of cardiac enzymes. Serum enzymes, including AST, CK, CK-MB, and LDH, were measured using an automatic biochemical analyzer (Sysmex). The levels of serum enzymes were assayed according to the instructions provided with the corresponding kits. Measurement of MDA levels. Cardiac MDA levels were measured using thiobarbituric acid method by a commercial kit (#A003-4-1, Jiancheng) according to the manufacturer's instructions. TTC staining. Pigs were euthanized intravenously with high potassium solution and fresh heart tissue samples were obtained. The fixed cardiac tissue samples were cut into consecutive 5-mm slices. The porcine heart slices were immersed in 2% tetraphenyl tetrazolium chloride (TTC) (Solarbio, G3005) at 37 °C for 30 min. The infarcted area (TTC negative, white) was then observed. The larger the white area, the more infarcted zone the hearts would be. Statistical analysis was performed based on the recorded staining results. Quantitative real time PCR. Total RNA was isolated from tissues or cells using Trizol (Pufei), and RNA concentration and purity were measured using a spectrophotometer. RNA was reverse transcribed using the PrimeScript RT reagent Kit (Takara) in accordance with the manufacturer’s instructions, and quantitative PCR was performed using a CFX96 Real Time Syst m (Bio-Rad) with SYBR Green Supermix (Bio-Rad) in accordance with the manufacturer s instructions. The recommended thermal protocol consisted of an initial denaturation at 95°C for 3 min, followed by 39 cycles of denaturation at 95°C for 15s, annealing at 60°C for 20 s and extension at 72°C for 30s. The fold difference in gene expression was calculated using the 2-ΔΔCt method and is presented relative to Gapdh mRNA. All reactions were performed in triplicate, and specificity was monitored using melting curve analysis. The primers are listed in Table 1 of Supplemental materials . Western blot analysis. Total proteins were extracted from the tissues by homogenization in RIPA buffer containing protease inhibitors. The homogenate was cleared by centrifugation at 4°C for 30 min at 12,000 rpm, and the supernatant (containing the protein fraction) was collected. Protein concentration in the supernatant was measured using the BCA Protein Assay Kit (Beyotime). A total of 20 mg protein per sample was resolved in a 10-12% SDS PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% (w/v) BSA in Tris buffered saline containing 0.2% ween 20, and then incubated at 4°C overnight with the following antibodies: anti-Ftl (1:1000; Abcam, ab69090) anti-Hmox1 (1:1000; Abcam, ab13243) and anti-Gapdh (1:10000; 60004-1, Proteintech). The membranes were then washed and probed with the appropriate horseradish peroxidase‒conjugated secondary antibodies (1:4000; Proteintech) and detected using the Pierce ECL System (Thermo Scientific). Transcriptomics and lipid metabonomics study. For the transcriptomics study, total RNA was extracted using Trizol and quality checked with Bioanalyzer 2100. mRNA was purified with two rounds of Dynabeads Oligo(dT). Fragmentation was performed using Magnesium RNA Fragmentation Module at 94°C. Reverse transcription and second strand synthesis prepared cDNA, which was A-tailed and ligated to dual index adapters. Size selection utilized AMPureXP beads. UDG enzyme treatment preceded PCR amplification and 2×150bp PE150 sequencing with Illumina Novaseq 6000. Genes with P < 0.05 and absolute fold changes ≥ 1.5 were identified as differentially expressed genes. Principal component analysis (PCA) was performed with princomp function of R (http://www.r-project.org/) in this experience. GO enrichment analysis of differentially expressed genes and KEGG pathway enrichment analysis were performed by using the cluster Profiler R package. For the lipid metabonomics study, LV tissues were collected according to the manufacturer’s instructions, and the sample extracts were analysed using an ultra-high performance liquid chromatography high-resolution tandem mass spectrometry (ThermoFisher Ultimate 3000 UHPLC; ThermoFisher Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometry). Metabolite quantification and further analysis were performed using a multiple reaction monitoring method. Lipid metabolites with p< 0.05 and fold change > 1.5 were deemed to be significant. Histological assessments and immunostaining. The hearts were fixed in 4% paraformaldehyde overnight at room temperature, embedded in paraffin and consecutively sectioned into 18–20 sections on the short axis at a 500-μm interval. DAB-enhanced Perl’s prussian blue, masson trichrome and picrosirius red staining were performed in accordance with standard procedures. The serial sections were then examined with a digital serial section scanner (VS200, Olympus). After Masson’s trichrome staining, fibrotic tissue (%) was calculated by the following formula: (total fibrotic area/total LV circumference area) × 100%, and the wall thickness of the scarred tissue at the apical and middle slices was measured as well. Data were analyzed using Fiji (ImageJ). For assessment of the fibrillar collagen subtype by picrosirius red staining, the sections were imaged under polarized light. Immunohistochemistry was performed to assess HBB and 4-HNE levels. Immunofluorescence was performed to assess HBB, HMOX1, Ferritin-L, CD31, α-SMA, cTNT and Cx43 levels; the images were captured and analyzed by LCM and Fiji. TUNEL assay. The TUNEL assay was performed with a TUNEL Apoptosis Assay Kit (Beyotime, China) in accordance with the instructions of the manufacturer. Echocardiography. Cardiac functions at 35 d post I/R were assessed using echocardiography (VisualSonics, Canada), M-mode echocardiographic and two-dimensional images in a parasternal short and long axis were recorded. Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic volume (EDV), left ventricular end-systolic volume (ESV) were calculated as previously described 45 . In vivo systemic toxicity experiments. After the rat and mini-pigs were killed, the other main organs (liver, kidney, lung and spleen) were collected for H&E staining, Perl’s prussian blue and immunohistochemical staining of HBB to evaluate systematic pathological changes. The serum was extracted to assess other serum enzymes, including ALT, CRE-J, UREA and UA, were measured using an automatic biochemical analyzer (Sysmex). The levels of serum enzymes were assayed according to the instructions provided with the corresponding kits. Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedDataFigs.docx Supplementarymaterials.docx 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4467590\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":311871917,\"identity\":\"fa6b0c98-16f2-4e0b-a16d-b77942b46ed5\",\"order_by\":0,\"name\":\"Yang 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University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xuexian\",\"middleName\":\"\",\"lastName\":\"Fang\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-05-23 14:40:32\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4467590/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4467590/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":58735307,\"identity\":\"2ad7f8a9-4bc3-4338-bd21-be5f192c30e5\",\"added_by\":\"auto\",\"created_at\":\"2024-06-20 12:34:51\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1282225,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eHaem released from IMH is the cause of ferroptosis in I/R injury. a,\\u003c/strong\\u003e Schematic illustration of inducing IMH in rat MI model through intramyocardial injection of autogenous blood, erythrocytes or plasma. \\u003cstrong\\u003eb\\u003c/strong\\u003e, Serum CK-MB and CK of rats from Sham, I/R, MI, and IMH (MI administered with autogenous blood, erythrocytes or plasma) groups. \\u003cstrong\\u003ec\\u003c/strong\\u003e, Immunofluorescent images of rat heart sections stained with HBB for IMH and TUNEL for cell death. \\u003cstrong\\u003ed\\u003c/strong\\u003e, Intensity profiles along the dashed lines in the fluorescent images. Scale bar = 20 μm. \\u003cstrong\\u003ee\\u003c/strong\\u003e, Correlation between HBB, DAB-Perls’ blue (ferric iron) and TUNEL levels in infarcted LV reperfused for 0 h, 1 h, 4 h, 8 h, and 23 h. \\u003cstrong\\u003ef\\u003c/strong\\u003e, Quantification of labile haem levels in LV tissues of I/R rats after reperfusion for 0 h, 1 h, 4 h, 8 h, and 23 h. \\u003cstrong\\u003eg\\u003c/strong\\u003e, Serum CK-MB and CK levels in I/R, MI rats, and MI rats administered with haem saline solution through intramyocardial injection (2.7 μmol/kg, matching I/R haem level at 24 h post infarction). Immunofluorescent images of HBB and Hmox1 (\\u003cstrong\\u003eh\\u003c/strong\\u003e), cTNT and Ferritin-L (\\u003cstrong\\u003ei\\u003c/strong\\u003e) in Sham and I/R-24h groups. Scale bar = 20 μm. \\u003cstrong\\u003ej\\u003c/strong\\u003e, Heatmap of differentially expressed ferroptosis genes in LV myocardium of Sham and I/R-24h, identified by RNA-seq. \\u003cstrong\\u003ek\\u003c/strong\\u003e, Immunohistochemical images of 4-HNE (a ferroptosis marker) in Sham and I/R-24h rat hearts. \\u003cstrong\\u003el\\u003c/strong\\u003e, C11 BODIPY stained H9C2 cardiomyocytes with or without 24 h 10 μM haem treatment. \\u003cstrong\\u003em\\u003c/strong\\u003e, Normalized cell viability of H9C2 cardiomyocytes treated with different concentrations of haem, in the presence of vehicle (Veh, DMSO), DFO (100 μM, iron chelator), ZVF (50 μM, pan-caspase inhibitor) or Gsk-872 (10 μM, RIPK3 inhibitor). Viability of each group at 0 h was as 100%. Statistics were cells treated with indicated inhibitors compared to the Veh group 24 h (n = 4 biological replicates). \\u003cstrong\\u003en\\u003c/strong\\u003e, Haem-binding capacity and haem-binding K\\u003csub\\u003eD\\u003c/sub\\u003e comparison of haem-binding proteins reported in literatures, including hemopexin (Hx), Serum albumin, Lipopolysaccharide-binding protein, Casein, Alpha 1-Microglobulin, Alpha-1-Antitrypsin, lactoferrin (Lf), and transferrin (Tf). Dotted box: proteins investigated in this work. \\u003cstrong\\u003eo\\u003c/strong\\u003e, Serum CK-MB and CK levels of I/R rats and I/R rats received intravenous administration of Hx or Apo-Lf solution (4 mg/kg). Samples were collected 24 h after surgery. \\u003cstrong\\u003ep\\u003c/strong\\u003e, Iron deposition (Perl’s blue) in spleen and HBB levels in kidney of I/R rats treated with Hx or Apo-Lf. IMH = Intramyocardial hemorrhage; I/R = myocardial infarction and reperfusion; MI = myocardial infarction; CK-MB = creatine kinase, MB form; CK = creatine kinase; HBB = hemoglobin; LV = left ventricle; DFO = deferoxamine; ZVF = Z-VAD-FMK; HNE = 4-hydroxynonenal. \\u0026nbsp;\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4467590/v1/9b6e0847255e97247b785114.png\"},{\"id\":58734982,\"identity\":\"c1cc4f0d-8110-476e-b08d-2d19ab34e05c\",\"added_by\":\"auto\",\"created_at\":\"2024-06-20 12:26:51\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1730987,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eDesign and fabrication of haem-sequestering FILMS. a, \\u003c/strong\\u003ePDB scheme of reaction between Apo-Lf methacrylic anhydride, and methacylated lysine (K) residues on Apo-LfMA. \\u003cstrong\\u003eb\\u003c/strong\\u003e,\\u003cstrong\\u003ec\\u003c/strong\\u003e, Single-cycle kinetic sensorgrams (RU vs. time) for Apo-Lf-haem and Apo-LfMA-haem interactions with haem = 168.07, 240.1, 343, 490, 700, 1000 (nM) flowing over a CM5 chip immobilized Apo-Lf or Apo-LfMA, revealing the affinity between proteins and haem.. \\u003cstrong\\u003ed\\u003c/strong\\u003e MD-simulated haem-binding structural images of Apo-Lf and Apo-LfMA in three visions and the locations of interaction site 1 and site 8. \\u003cstrong\\u003ee\\u003c/strong\\u003e, Haem and iron content in Apo-Lf/haem and Apo-LfMA/haem complexes detected using both oxalic acid method and ICP. \\u003cstrong\\u003ef\\u003c/strong\\u003e, Changes of hydrogen bonds and salt bridge of Apo-Lf and Apo-LfMA before and after haem/protein interaction visualized by 50 ns MD. \\u003cstrong\\u003eg\\u003c/strong\\u003e, 3D and 2D diagrams of pictures of the representative interaction site 1 between haem and Apo-Lf or Apo-LfMA. \\u003cstrong\\u003eh,i\\u003c/strong\\u003e, The comparison of the original environment of a potential haem-binding site between Apo-Lf and Apo-LfMA, and its successful interaction in Apo-LfMA (Site 8). \\u003cstrong\\u003ej\\u003c/strong\\u003e, Schematic illustration of microfluidic fabrication FILMS. \\u003cstrong\\u003ek,l\\u003c/strong\\u003e, Micro-droplet formation in the microfluidics channel and images of obtained FILMG (non-porous ferroptosis inhibiting lactoferrin microgels ) and FILMS. \\u003cstrong\\u003em\\u003c/strong\\u003e, Mercury pressure curve of FILMG and FILMS revealing pore diameter distribution. \\u003cstrong\\u003en\\u003c/strong\\u003e, Haem absorbing performance of FILMG and FILMS in 250 μM haem solution. PDB = protein data bank; MD = molecular dynamics.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4467590/v1/73b7dad4046d637a99b37602.png\"},{\"id\":58734985,\"identity\":\"9c6a0139-484c-43a0-9eb7-d9741d19e6ee\",\"added_by\":\"auto\",\"created_at\":\"2024-06-20 12:26:51\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1622587,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFILMS suppress cell ferroptosis via haem scavenging. a\\u003c/strong\\u003e,\\u003cstrong\\u003eb \\u003c/strong\\u003eLive/dead staining andintracellular Fe\\u003csup\\u003e2+\\u003c/sup\\u003e ions (FerroOrange) of cardiomyocytes treated haem for 24 h in the presence of GMG (gelatin microgels), FILMG or FILMS. \\u003cstrong\\u003ec\\u003c/strong\\u003e, Cardiomyocyte (H9C2) viability after being treated with haem for 24 h in the presence of GMG, FILMG or FILMS (n=6). \\u003cstrong\\u003ed\\u003c/strong\\u003e, Mean Fe\\u003csup\\u003e2+\\u003c/sup\\u003e fluorescent intensities of (\\u003cstrong\\u003eb\\u003c/strong\\u003e) (n=6).\\u003cstrong\\u003e e-i\\u003c/strong\\u003e and \\u003cstrong\\u003el\\u003c/strong\\u003e, RT-PCR of \\u003cem\\u003eHmox1\\u003c/em\\u003e, \\u003cem\\u003eFtl\\u003c/em\\u003e, \\u003cem\\u003eTfrc\\u003c/em\\u003e, \\u003cem\\u003eFth\\u003c/em\\u003e, \\u003cem\\u003eFpn\\u003c/em\\u003e and \\u003cem\\u003eAcsl4\\u003c/em\\u003e expression in H9C2 treated haem for 24 h in the presence of GMG, FILMG or FILMS (n=3). \\u003cstrong\\u003ej\\u003c/strong\\u003e, Representative fluorescent images of C11 BODIPY 581/591 dye stained the lipid peroxidation level of H9C2 cells. \\u003cstrong\\u003ek\\u003c/strong\\u003e, Flow cytometric histogram of oxidized BODIPY and the ratio of oxidized BODIPY/C11 BODIPY (according to mean intensity of flow cytometric histogram) of H9C2 treated with haem for 24h in the presence of GMG, FILMG or FILMS (n=3). \\u003cstrong\\u003em\\u003c/strong\\u003e,\\u003cstrong\\u003en\\u003c/strong\\u003e, Representative fluorescent images of mitochondrial Fe\\u003csup\\u003e2+\\u003c/sup\\u003e ions (Mito-Ferrogreen, pink) with colocalized mitochondria (Mito-Traker, cyan) and JC-10 to visualize the membrane potential of mitochondria of H9C2 treated with haem for 24 h in presence of GMG, FILMG or FILMS. \\u003cstrong\\u003eo\\u003c/strong\\u003e, Scheme of extraction of iPSCs and their differentiation into iPSC-CMs. \\u003cstrong\\u003ep\\u003c/strong\\u003e, Viability of iPSC-CMs treated with haem for 24 h in presence of GMG, FILMG or FILMS (n=5). \\u003cstrong\\u003eq\\u003c/strong\\u003e,\\u003cstrong\\u003er\\u003c/strong\\u003e, Representative fluorescent images of C11 BODIPY 581/591 dye stained the lipid peroxidation level of iPSC-CMs treated with haem for 24 h in presence of GMG, FILMG or FILMS (n=3). iPSCs: induced pluripotent stem cells; iPSC-CMs: induced pluripotent stem cell-differentiated cardiomyocytes. Control: media without haem; blank: haem treatment only.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4467590/v1/40676e5fd3dce119444bbcb6.png\"},{\"id\":58735308,\"identity\":\"7ef3739d-e0ea-4993-9209-bbf9062234e5\",\"added_by\":\"auto\",\"created_at\":\"2024-06-20 12:34:51\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1426359,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFILMS inhibited IMH-induced ferroptosis in rat I/R model. a\\u003c/strong\\u003e, TEM images of the ultrastructure of microvasculature in myocardium of rats subjected to Sham surgery or I/R injury treated with saline, GMG or FILMS. Red triangles: endothelium, yellow stars: red blood cells in the myocardial interstitium. \\u003cstrong\\u003eb\\u003c/strong\\u003e,\\u003cstrong\\u003e \\u003c/strong\\u003eCardiac haem level in Sham rats and I/R rats treated with saline, GMG or FILMS treatment (n=4). \\u003cstrong\\u003ec\\u003c/strong\\u003e, Cardiac iron level was measured in Sham rats and I/R rats with saline, GMG or FILMS treatment (n=4). \\u003cstrong\\u003ed\\u003c/strong\\u003e, Immunofluorescent staining images and of HBB and Hmox1. \\u003cstrong\\u003ee\\u003c/strong\\u003e, Immunofluorescent staining images of HBB and TUNEL with the intensity profiles along the dashed lines in fluorescent images in rats subjected to I/R injury treated with GMG or FILMS. Scale bar = 20 μm. \\u003cstrong\\u003ef\\u003c/strong\\u003e,\\u003cstrong\\u003eg\\u003c/strong\\u003e, DAB Perl’s blue and 4-HNE of sectioned rat heart treated with GMG or FILMS treatment. \\u003cstrong\\u003ei\\u003c/strong\\u003e, Immunofluorescent staining images of HBB and TUNEL with the intensity profiles (\\u003cstrong\\u003ej\\u003c/strong\\u003e) along the dashed lines in fluorescent images in the heart slice of rats subjected to I/R injury treated with GMG or FILMS. Scale bar = 20 μm. \\u003cstrong\\u003ek\\u003c/strong\\u003e, The ratio of TUNEL versus DAPI in \\u003cstrong\\u003ei\\u003c/strong\\u003e was then counted in Sham rats and I/R rats with saline, GMG or FILMS treatment (n=5). \\u003cstrong\\u003el\\u003c/strong\\u003e, \\u003cem\\u003eHmox1 \\u003c/em\\u003eand \\u003cem\\u003eFtl\\u003c/em\\u003e mRNA level were evaluated in Sham rats and I/R rats with saline, GMG or FILMS treatment (n=3). \\u003cstrong\\u003em\\u003c/strong\\u003e, Images of 4-HNE immunohistochemical staining and TEM mitochondrion in cardiomyocytes in the heart slice of Sham rats and I/R rats with saline, GMG or FILMS treatment. \\u003cstrong\\u003en\\u003c/strong\\u003e, \\u003cem\\u003eAcsl4\\u003c/em\\u003e, \\u003cem\\u003eTnnt2\\u003c/em\\u003e and \\u003cem\\u003eSlc4a3\\u003c/em\\u003emRNA measurement in myocardium of Sham rats and I/R rats with saline, GMG or FILMS treatment (n=3). \\u003cstrong\\u003eo\\u003c/strong\\u003e, Serum CK-MB, CK, LDH and AST level measured in rats subjected to Sham surgery or I/R injury treated with saline, GMG or FILMS. \\u003cstrong\\u003ep\\u003c/strong\\u003e, A prognostic evaluation containing echocardiograms, Masson staining and Sirius red of rats subjected to Sham surgery or I/R injury treated with saline, GMG or FILMS in 5-week post-surgery. \\u003cstrong\\u003eq\\u003c/strong\\u003e, LVEF, LVFS, LVESV and LVIDS were assessed by echocardiography on days 35 (n=5). \\u003cstrong\\u003er\\u003c/strong\\u003e, Infarct size and LV wall thickness were measured according to Masson staining images (n=5). 4-HNE = 4-hydroxynonenal; LVEF = left ventricular ejection fraction; LVFS = left ventricular fractional shortening; LVESV = left ventricular end-systolic volume; LVIDS = left ventricular internal dimension in systole.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4467590/v1/b3f7f0ddd06b6d647daed25b.png\"},{\"id\":58734986,\"identity\":\"dc30506e-ae57-4861-9a70-4386dfc9a153\",\"added_by\":\"auto\",\"created_at\":\"2024-06-20 12:26:51\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1373847,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMinimally invasive transendocardial FILMS implantation in mini-pig model alleviated I/R injury. a, \\u003c/strong\\u003e(i) Scheme of a minimally-invasive transcatheter endocardial FILMS implantation procedure monitored by (ii-iii) digital subtraction angiography (DSA) using iohexol as contrast agent and (iv-v) transthoracic echocardiography (TTE) to track hydrogel injection; (vi) injected FILMS embedded in the myocardium. \\u003cstrong\\u003eb\\u003c/strong\\u003e,\\u003cstrong\\u003e \\u003c/strong\\u003eRepresentative images and \\u003cstrong\\u003ec\\u003c/strong\\u003e, quantitative data of triphenyltetrazolium chloride (TTC) staining for infarct size in heart sections of minipigs. Sham: thoracic surgery only; I/R: ischemia 1 h and reperfusion for 72 h, treated with saline injection; FILMS: I/R injury treated with FILMS. Black arrows point to injected FILMS sites. Yellow dash box: zoom-in on the injected FILMS in the apical site. n=3 per group.\\u003cstrong\\u003ed-h\\u003c/strong\\u003e, Serum CK-MB, CK, LDH, AST, and Tnl levels measured in minipigs subjected to Sham surgery or I/R injury treated with saline or FILMS. \\u003cstrong\\u003ei\\u003c/strong\\u003eand \\u003cstrong\\u003ej\\u003c/strong\\u003e, Cardiac haem and \\u003cem\\u003eHMOX1\\u003c/em\\u003e mRNA level in Sham and I/R minipigs with or without FILMS treatment. \\u003cstrong\\u003ek\\u003c/strong\\u003e, Representative images of H\\u0026amp;E staining and (\\u003cstrong\\u003el\\u003c/strong\\u003e) DAB Perl’s blue staining of heart sections obtained from minipigs subjected to Sham surgery or I/R injury treated with saline or FILMS. \\u003cstrong\\u003em\\u003c/strong\\u003e, \\u003cem\\u003eFTL\\u003c/em\\u003e mRNA level in Sham and I/R minipigs with or without FILMS treatment. \\u003cstrong\\u003en\\u003c/strong\\u003e, Western blots of the ferroptosis markers HMOX1 and FTL in minipigs subjected to Sham surgery or I/R injury treated with saline or FILMS. \\u003cstrong\\u003eo\\u003c/strong\\u003e, Cardiac total iron content, (\\u003cstrong\\u003ep\\u003c/strong\\u003e) \\u003cem\\u003eACSL4\\u003c/em\\u003e mRNA level, and (\\u003cstrong\\u003eq\\u003c/strong\\u003e) MDA level was measured in minipigs subjected to Sham surgery or I/R injury treated with saline or FILMS. Tnl = cardiac troponin I.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"image5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4467590/v1/c9a66a25a9210dbccb7cd477.png\"},{\"id\":58736380,\"identity\":\"f28b32a7-d5cb-458e-83ac-d890f44f8d2a\",\"added_by\":\"auto\",\"created_at\":\"2024-06-20 12:50:57\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":8974118,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4467590/v1/f567d771-8c0f-4a62-9d2c-bc21d450b2a2.pdf\"},{\"id\":58734988,\"identity\":\"afc376a6-d504-485e-963b-83797f2c772c\",\"added_by\":\"auto\",\"created_at\":\"2024-06-20 12:26:51\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":10520917,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"ExtendedDataFigs.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4467590/v1/16aa668955710185337720e4.docx\"},{\"id\":58734984,\"identity\":\"10fa0338-d999-48f6-b54b-4f82d4d57e1c\",\"added_by\":\"auto\",\"created_at\":\"2024-06-20 12:26:51\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":68924,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarymaterials.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4467590/v1/0f2772740ee021f38f689554.docx\"}],\"financialInterests\":\"There is \\u003cb\\u003eNO\\u003c/b\\u003e Competing Interest.\",\"formattedTitle\":\"Milk-derived haem scavenging microsponges protect heart against ferroptosis-induced reperfusion injury\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eMajority of patients with acute myocardial infarction receive reperfusion therapy in order to promptly reestablish blood flow to ischemic myocardium, consequently minimizing myocardial necrosis and enhancing patient outcomes\\u003csup\\u003e1,2\\u003c/sup\\u003e. However, returning of blood can paradoxically induce additional cardiac damage, a phenomenon known as myocardial ischemia/reperfusion (I/R) injury, which affects millions per year\\u003csup\\u003e3\\u003c/sup\\u003e. I/R injury diminishes therapeutic efficacy and predisposes to risk of heart failure\\u003csup\\u003e4\\u003c/sup\\u003e. Recent evidences and our previous works revealed that ferroptosis, an iron-dependent cell death program that differs from apoptosis and necrosis, is a major cause of I/R injury\\u003csup\\u003e5\\u0026ndash;8\\u003c/sup\\u003e. To date, treatments for cardiac ferroptosis are not clinically available.\\u003c/p\\u003e \\u003cp\\u003eSeventy percent I/R patients experience IMH following cardiac reperfusion\\u003csup\\u003e4,9,10\\u003c/sup\\u003e, which induces iron deposition in the reperfused myocardium\\u003csup\\u003e11,12\\u003c/sup\\u003e as red blood cells are lysed to release iron into interstitial space. The released iron needs enter the cells to trigger the ferroptotic storm which occurred in organelles, especially mitochondria\\u003csup\\u003e13\\u003c/sup\\u003e. However, the transmembrane transportation efficiency of iron ions is limited\\u003csup\\u003e13\\u003c/sup\\u003e. We also found that H9C2 CMs \\u003cem\\u003ein vitro\\u003c/em\\u003e can tolerate iron ion at concentrations far higher than that in patient myocardium (Supplementary Fig.\\u0026nbsp;1). Thus, there should be an invasive iron carrier which accounts for cellular iron uptake. With \\u003cem\\u003ein vivo\\u003c/em\\u003e experiments, we narrowed down from whole blood from IMH to haem released from lysed red blood cells as the major iron carrier which smuggles iron into cardiac cells. It is reported that haem oxygenase-1 (HO-1) mediated haem degradation frees iron ions from haem to catalyze Fenton reaction and subsequent lipid peroxidation, a landmark event in ferroptosis\\u003csup\\u003e14\\u003c/sup\\u003e. In contrast to iron in ion form, haem causes severe cell damage \\u003cem\\u003ein vitro\\u003c/em\\u003e at pathologically relevant low concentrations (Supplementary Fig.\\u0026nbsp;1).\\u003c/p\\u003e \\u003cp\\u003eBased on these understandings, we proposed a treatment strategy of intercepting free haem outside the cardiac cells with intramyocardial implantation of haem-scavenging biomaterial, to block the iron from trespassing the cell membrane. We chose local haem-scavenging over systemic scavenging to lower the risks of 1. inhibiting normal iron-dependent physiological activities in other organs, and 2. metabolically transferring haem from IMH and circulation to kidney and spleen hence causing secondary damages. To the best of our knowledge, no implantable haem-binding biomaterials for ferroptosis inhibition have been developed. Molecules recognized for their ability to bind to haem are predominantly localized within proteins and peptides\\u003csup\\u003e15,16\\u003c/sup\\u003e. Here, we designed a milk-derived injectable haem-sequestering microsponge system to rapidly capture free haem released from IMH before they enter cardiac cells and activate ferroptosis.\\u003c/p\\u003e \\u003cp\\u003eWe first screened out a milk-derived apo-metalloprotein, Apo-Lactoferrin (Apo-Lf), from a group of protein candidates for its high haem-binding affinity and capacity. Subsequently, double bonds are modified onto the surface of Apo-Lf, to allow fabrication of ferroptosis-inhibiting lactoferrin micro-sponges (FILMS). Surprisingly, this engineered Apo-Lf gained significantly higher haem-binding affinity and capacity compared to its natural form. Intramyocardially implanted FILMS quickly drained excessive extracellular haem from IMH in reperfused animal hearts before haem molecules were uptaken by cardiac cells. The clinically relevant porcine model demonstrated the compatibility of FILMS with minimally invasive percutaneous transcatheter injection, and the capability of FILMS in preventing I/R injury in large volume of reperfused myocardium.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eHaem released from IMH causes myocardial ferroptosis in I/R injury\\u003c/h2\\u003e \\u003cp\\u003eIMH and iron deposition were observed via cardiac magnetic resonance (CMR) in the left ventricular (LV) wall of patients who received reperfusion one day post MI (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea). As a result of reperfusion, cardiac injury markers including NT-proBNP, CK-MB, CK and AST surged in the first 6 h (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb). Different compounds of blood were administered into infarcted rat myocardium to identify the iron carrier primarily responsible for IMH-induced ferroptosis and reperfusion injury (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea). Infarcted myocardium which received whole blood or red blood cells (RBCs) showed similar increase of CK-MB, CK and cell death comparable to I/R controls, significantly higher than MI and sham controls, and MI rats received intramyocardial plasma injection (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec). The spatiotemporal couplings of RBCs, haemoglobin (HBB) and dead cells further confirmed that RBCs flooding into the interstitial space preluded the I/R injury surge (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec-e, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed). We hypothesized that haem was released upon hemolysis of RBCs and acted as the main transmembrane iron carrier. In fact, free haem rapidly accumulated in the reperfused myocardium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef). In addition, haem injection into infarcted myocardium led to exacerbated injury, same as I/R (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eg, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec). \\u003cem\\u003eIn vitro\\u003c/em\\u003e cultured H9C2 showed high sensitivity to haem, (IC50\\u0026thinsp;=\\u0026thinsp;5.82 \\u0026micro;M), while IC50 of ferric ions is 4.50 mM, almost 3 orders of magnitudes higher than the lethal concentration of haem (Supplementary Fig.\\u0026nbsp;1). Hence, we confirmed that haem is the major risk factor in I/R injury with IMH. Myoglobin level in reperfused myocardium was not significantly different compared to sham, indicating that haem accumulation was only attributed to IMH (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ee). Exogenous haem was transported across cell membrane and degraded by upregulated haem oxygenase-1 (HO-1) into ferrous ions, which accumulated in either the labile iron pool (LIP) or ferritin proteins (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eh, i). Labile iron overload was shown to initiate ferroptosis\\u003csup\\u003e17\\u003c/sup\\u003e. RNA-seq comparison between sham and I/R samples revealed a significant enrichment in \\u0026ldquo;ferroptosis\\u0026rdquo; and haem binding terms (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ej, k, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ee). 4-hydroxynonenal (4-HNE), a pivotal marker of lipid peroxidation, exhibited a higher expression level in I/R group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ek). Additionally, a low dose of exogenous haem caused lipid peroxide accumulation (a ferroptosis indicator) and severe death of iPSC-CMs in vitro (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef, g, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ek), which could only be inhibited by ferroptosis inhibitor deferoxamine (DFO), but not apoptosis inhibitor Z-VAD-FMK (ZVF) or necroptosis inhibitor Gsk-872 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003el). In a word, exogenous haem released from IMH induced ferroptosis via elevating cellular iron level. Therefore, we proposed a strategy to prevent I/R injury by blocking \\u0026ldquo;Trojan horse\\u0026rdquo;, haem, from entering cardiac cells.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCirculating haem-binding proteins reduced myocardial injury but brought side effects\\u003c/h2\\u003e \\u003cp\\u003eSome proteins have been reported to bind haem, including hemopexin (Hx), serum albumin (SA), lipopolysaccharide-binding protein (LBP), casein, alpha-1 microglobulin, alpha-1 antitrypsin, lactoferrin (Lf), apo-lactoferrin (Apo-Lf) and transferrin (Tf)\\u003csup\\u003e11,15,18,19\\u003c/sup\\u003e. We measured their haem-binding capacities, (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003en, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea-h), and found that among all tested candidates, Apo-Lf and Hx have competitive haem-binding affinity (~\\u0026thinsp;0.12\\u0026ndash;0.13 nM) and the former exhibited the highest haem-binding capacity (55.21 \\u0026micro;g/mgprot). Intravenous injection of Apo-Lf or Hx (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ei) reduced cardiac injuries, featuring lower levels of cell death post IMH and iron overload (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003en, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ej-k). However, the circulating haem-binding proteins brought excessive iron and associated damages to remote organs, revealed by iron deposition in spleen and HBB accumulation in kidney (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eo). Hence, an implantable medical device for local haem scavenging is more desirable.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eThe design and fabrication of local haem-sequestering FILMS\\u003c/h2\\u003e \\u003cp\\u003eWe proposed to implant injectable protein hydrogel microspheres to form \\u003cem\\u003ein situ\\u003c/em\\u003e haem-sequestering sponges in the myocardium. Apo-Lf (iron-free form of lactoferrin) was chosen for miscroshpere fabrication for its highest affinity and binding capacity among the candidates, and its cost-effectiveness (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003em, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea-c). To increase intermolecular interactions for hydrogel formation, we methacrylated 30% surface lysine residues on Apo-Lf to add 13\\u0026ndash;14 double bonds to each Apo-Lf molecule, obtaining UV-curable Apo-LfMA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea, d-h). Only lysine residues among all amino acid residues participated the methacrylation reaction. Fourteen lysine sites were identified with a modification probability of over 50%, including K46, K47, K71, K288, K296, K301, K320, K332, K348, K358, K460, K539, K627 and K652 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Protein secondary structure did not change significantly after iron removal and methacrylation (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ei).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eSurprisingly, both haem affinity and binding capacity of the engineered protein dramatically increased compared to pristine Apo-Lf. SPR showed that the haem-binding constant of Apo-LfMA is one order of magnitude lower compared to that of Apo-Lf 0.13 nM to 0.015 nM (thus higher affinity) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb,c). Molecular docking and molecular dynamic simulation were employed to analyze the protein/haem complex (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed, Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea). Apo-LfMA bound 13 haems, which was 6 more than the 7 haem-binding sites in Apo-Lf (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). This capacity increase was consistent with experimental quantification results of haem and iron content in the two protein/haem complexes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ee). Methacryloyl modification of the 14 lysine residues changed the intramolecular physical interaction, hence altered the conformation of existing haem binding sites and could theoretically cause structural perturbations at distal regions\\u003csup\\u003e20,21\\u003c/sup\\u003e in the protein, which explains the observed enhancement of haem-binding affinity and capacity. Methacryloyl groups increased the hydrophobic area of Apo-LfMA to 956.29 \\u0026Aring;\\u003csup\\u003e2\\u003c/sup\\u003e from 889.60 \\u0026Aring;\\u003csup\\u003e2\\u003c/sup\\u003e of Apo-Lf, generally strengthening the hydrophobic interaction between the hydrophobic porphyrin ring of haem and protein (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ec). Moreover, methacryloyl groups on lysine altered the nearby conformation and involved more amino acids into the existing haem-binding sites, increasing the number of salt bridges, hydrogen bonds and stronger metal coordination in Apo-LfMA/haem binding pockets (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ef). For example, Site 1 in the Apo-LfMA/haem complex showed the formation of new metal coordination between SER304 and haem-Fe(Ⅱ) center compared to its counterpart in the Apo-Lf/haem complex (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eg). These added interactions contributed to the leap in haem affinity of the original binding sites (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). The new haem-binding pockets could be attributed to the increase in the accessibility of potential binding sites. For example, the distance between HIS-272 and TYR-211 is 6.8 \\u0026Aring; in Apo-Lf, too narrow to form a haem-binding site. After modification, the distance between HIS-272 and TYR-211 increases to 8.2 \\u0026Aring; in Apo-LfMA, wide enough for haem to accommodate in (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eh,i). Decreased radius of gyration (Rg), root mean square deviation (RMSD), solvent accessible surface area (SASA), and root mean square fluctuation (RMSF) also confirmed a more compact structure and higher stability of the Apo-LfMA/haem complex in comparison with the Apo-Lf/haem complex (Extended Data Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ed-g), which is a result of the strong haem-amino acids interaction in the pockets.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eRapid accumulation of haem in myocardium post reperfusion demands highly efficient haem scavenging. Therefore, porous structure was generated in FILMS via ice crystal template technology to enhance haem diffusion and drainage by increasing the specific surface area and accessibility of internal volumes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ej-l). The polymerization of metharyloyl groups reduced the absorbance of unsaturated C\\u0026thinsp;=\\u0026thinsp;C stretching vibration at 3291 cm\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e peak and increased the saturated C-C stretching vibration (Extended Data Fig.\\u0026nbsp;6a). The crosslinking process did not significantly alter the relative contents of second structure in Apo-LfMA and FILMS (Extended Data Fig.\\u0026nbsp;6b, c). FILMS (892.7 nm average pore diameter and 88.96% porosity) was found significantly more effective in scavenging haem compared to FILMG without pores (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003el,m, Extended Data Fig.\\u0026nbsp;6d,e). In haem buffer solution, FILMS reached haem-absorbing balance within 1 hour, shorter than the time required for exogenous haem to cause significant damages in the myocardium (1\\u0026thinsp;~\\u0026thinsp;2 h post reperfusion) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003en). In contrast, non-porous counterpart FILMG requires about 4 hours to reach the haem-absorbing balance, which is theoretically slow considering the time window indicated by Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef. Quantification of bound haem on FILMS showed that photo-crosslinking of methacryloyl groups did not decrease the haem-binding capacity of Apo-LfMA (Extended Data Fig.\\u0026nbsp;6f). It is worth noting that the dissociation equilibrium constant of Apo-LfMA is as low as 9.22 x 10\\u003csup\\u003e^\\u0026minus;10\\u003c/sup\\u003e, indicating that the desorption of haem from Apo-LfMA is thermodynamically unfavorable. Therefore, FILMS have a desirable haem scavenging rate and total amount, and could significantly decrease its accessibility for cardiac cells.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFILMS trapped exogeneous haem and protected CMs from ferroptosis\\u003c/h2\\u003e \\u003cp\\u003eIn a high-haem concentration \\u003cem\\u003ein vitro\\u003c/em\\u003e environment simulating IMH, FILMS salvaged rat CMs and preserved over 90% cell viability (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea,c). Fe\\u003csup\\u003e2+\\u003c/sup\\u003e fluorescent signals, revealing the intracellular iron level, demonstrated that FILMS prevented haem from entering the cells, whereas FILMG had a significantly lower effect, and gelatin microgel (GMG) without haem-binding capacity failed to inhibit the increase of intracellular iron level compared to free haem control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb,d). CMs upregulated iron metabolism indicators including \\u003cem\\u003eHmox1\\u003c/em\\u003e, \\u003cem\\u003eFtl\\u003c/em\\u003e and \\u003cem\\u003eFth\\u003c/em\\u003e, which are signs of active degradation of haem into labile iron (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ee-g). This further induced the expression of iron efflux gene (\\u003cem\\u003eFpn\\u003c/em\\u003e) and downregulated \\u003cem\\u003eTfrc\\u003c/em\\u003e associated with iron transportation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eh,i). These alterations in mRNA transcription indicated the activation of ferroptosis pathway. When extracellular haem was sequestered in the protein hydrogel network of FILMS, cellular iron imbalance was inhibited and ferroptosis was alleviated. Intracellular iron enrichment in haem-treated CMs led to an ROS surge from Fenton reaction (Extended Data Fig.\\u0026nbsp;6g-i), and elevated membrane lipid peroxidation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ej, k). This trend was inhibited by FILMS, while FILMG showed less effect and GMG showed no inhibitory effect. Acsl4 has an important regulatory role in ferroptosis by facilitating the conversion of long-chain fatty acids into acyl-CoA derivatives, whose mRNA expression increased in both blank and GMG groups but not in FILMG and FILMS groups\\u003csup\\u003e22\\u003c/sup\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003el). As the powerhouse of CMs, mitochondria were subject to ferroptosis. Mitochondrial iron levels across the groups exhibited a changing pattern similar to that of the cellular iron levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003em). Correspondingly, FILMS prevented the mitochondrial membrane potential disruption observed in haem treated CMs (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003en), while FILMG and GMG only partially mediated or fail to mediate mitochondrial damage. To demonstrate the clinical relevance of the results above obtained from rat CMs, we validated that FILMS could inhibit ferroptosis of human iPSC-CMs by extracellularly scavenging haem (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eo-r). Together, FILMS protected CMs from suffering ferroptosis through scavenging free haem more rapidly than FILMG, while GMG did not protect the CMs in high-haem environment.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eFILMS inhibited IMH-dependent myocardial ferroptosis in rat I/R model.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eThrough pathological and transmission electron microscopy (TEM) observations, hearts of I/R rats with IMH phenotype were included for analysis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea, Extended Data Fig.\\u0026nbsp;7a). In rat I/R model, IMH leads to a rapid accumulation of free haem in LV myocardium in the first 24 h to 34.74\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.70 nmol/mgprot, which lead to an 89% increase in myocardial iron content (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb, c). Local implantation of FILMS reduced the amount of free haem in the myocardium to 16.69\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.87 nmol/mgprot, showing no significant difference compared to that of sham baseline. This curtails in haem concentration resulted in an iron content decrease by 81% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb, c). It is worth noting that such haem content equals to the haem content at 1.49 h post reperfusion in the I/R control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef), the percentage of dead cardiac cells is theoretically less than 3.6% at this time point (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ee), which is confirmed by the measure percentage, 5.7%, (Extended Data Fig.\\u0026nbsp;7b). Without haem drainage into the implanted microspheres, GMG group failed to lower haem concentration and cardiac cell loss (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eb, Extended Data. 7b).\\u003c/p\\u003e \\u003cp\\u003eIn both GMG and I/R groups, HBB-positive red blood cells were spatially coupled with TUNEL signals (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed, e), while TUNEL signals were sparse and weak in HGB-positive areas in FILMS treated rats, spatially demonstrating the contribution of IMH to I/R injury and the rescuing effect of FILMS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ed, e, Extended Data Fig.\\u0026nbsp;7b). Both GMG and FILMS microspheres aggregated in the injection sites (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef), but only FILMS served as drainage sites for exogenous haem from IMH. Iron signal concentrated at the FILMS injection sites, rather than in the surrounding tissue, while severe iron deposition was evident in the tissue surrounding the implanted GMG (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef). As transmembrane transport of haem was blocked by FILMS, upregulation of Hmox1 and ferritin in cardiac cells was inhibited (Extended Data Fig.\\u0026nbsp;7c-d, f-g). Elevated iron level in the GMG group led to increased lipid peroxidation and morphological changes of mitochondria including swelling and membrane rupture, whereas these ferroptosis features were mediated by FILMS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eg, Extended Data Fig.\\u0026nbsp;7e, h).\\u003c/p\\u003e \\u003cp\\u003eTo strengthen the mechanism of exogenous haem induced I/R injury and FILMS treatment, we conducted transcriptomic analysis on I/R myocardial tissues treated with FILMS, GMG, and saline, in comparison to sham group. Differential gene expression and principal component analysis (PCA) indicated that FILMS group was most similar to sham group and distinct from GMG and I/R groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eh-i). GO analysis revealed significant changes in iron metabolism, haem binding, and lipid metabolism in the myocardium after I/R, accompanied by changes at the cellular membrane and mitochondrial organelle levels (Extended Data Fig.\\u0026nbsp;8a, b). These transcriptional changes were suppressed by FILMS. According to KEGG enrichment analysis, ferroptosis was the primary pathway through which FILMS attenuated I/R injury (Extended Data Fig.\\u0026nbsp;8c). Compared to GMG group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ej, Extended Data Fig.\\u0026nbsp;8c), FILMS group downregulated haem metabolism genes (including \\u003cem\\u003eHmox1\\u003c/em\\u003e, \\u003cem\\u003eCyba\\u003c/em\\u003e, \\u003cem\\u003eHebp1\\u003c/em\\u003e and \\u003cem\\u003eCybb\\u003c/em\\u003e) and ferroptosis genes (\\u003cem\\u003eFtl\\u003c/em\\u003e and \\u003cem\\u003eAcsl4\\u003c/em\\u003e), and upregulated lipid metabolism and cardiac function recovery genes (\\u003cem\\u003eMe3\\u003c/em\\u003e, \\u003cem\\u003eSlc4a3\\u003c/em\\u003e, \\u003cem\\u003eTnnt2\\u003c/em\\u003e and \\u003cem\\u003eAscl6\\u003c/em\\u003e). Expression of these genes, including ferroptosis genes, was not significantly different between the GMG and I/R groups (Extended Data Fig.\\u0026nbsp;8d-f). HPLC-MS based redox lipidomics revealed that oxidized lipids (oxPLs) as metabolic hallmarks of ferroptosis were upregulated in I/R group compared to the sham group, and downregulated to sham level in FILMS group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ek-m). These results confirmed that FILMS inhibited ferroptosis in I/R rats.\\u003c/p\\u003e \\u003cp\\u003eConsequently, serum levels of cardiac injury markers in FILMS group including CK-MB, CK, AST and LDH were maintained at those of sham group, confirming that alleviating haem-induced ferroptosis by FILMS reduced I/R injury (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003en-q). Long-term echocardiography evaluation 35 days post-surgery demonstrated that FILMS attenuated LV remodeling in terms of both cardiac function (LVEF and LVFS) and geometry (LVIDS and LVESV) compared to GMG and I/R groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003er, t and u, Extended Data Fig.\\u0026nbsp;9a). Consistently, FILMS reduced fibrotic infarct size by 17.3% compared to I/R group and maintained LV wall thickness (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003es, v and w). In addition, FILMS reduced the ratio between type I collagen (Col I) and type III collagen (Col III) in the infarct, which is a sign of less severe fibrosis, while I/R and GMG groups showed high Col I/Col III ratios (Extended Data Fig.\\u0026nbsp;9b-d). Additionally, vessel density and Cx43 expression in FILMS group were significantly higher than those in I/R and GMG groups (Extended Data Fig.\\u0026nbsp;9e-j). Importantly, no adverse effects caused by FILMS were observed in spleen or kidneys (Extended Data Fig.\\u0026nbsp;9k).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eMinimally invasive trans-endocardial FILMS implantation in a preclinical pig I/R model.\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eSafety and efficacy of FILMS in treating ferroptosis and I/R injury were further demonstrated in a porcine cardiac I/R model. FILMS was compatible with a minimally invasive transcatheter injection system (TEIS01, Dinova Medtech), which was employed in transendocardial hydrogel injection in heart failure patients\\u003csup\\u003e23\\u003c/sup\\u003e (Extended Data Fig.\\u0026nbsp;10a). Under the guidance of digital subtraction angiography (DSA), the steerable dual-lumen needle catheter was inserted through the femoral artery and through the aortic valve to reach LV endocardium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea (i-iii), Extended Data Fig.\\u0026nbsp;10a). The injection sites were positioned using both transthoracic echocardiogram and DSA (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea (ii-iv)). FILMS were injected into the LV wall without leakage, bleeding or wall perforation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea (v)). Depots of aggregated FILMS were found in the myocardium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea (vi)). All pigs survived the surgery.\\u003c/p\\u003e \\u003cp\\u003eIn the efficacy study, FILMS were injected into 9 points uniformly distributed in the infarcted LV, followed by immediate reperfusion (Extended Data Fig.\\u0026nbsp;10b, c). Three days post-surgery, TTC staining showed large volume of necrotic myocardium across the LV anterior wall and interventricular septum (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). Notably, the volume of necrotic myocardium was significantly smaller in FILMS group compared to I/R control, and the color of myocardium surrounding the injected FILMS was closer to healthy myocardium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). Size of necrotic myocardium in FILMS group was reduced by 70.4% compared to I/R group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ec). The elevations in myocardial enzyme markers (CK-MB, CK, LDH and AST) and cTnI in serum post I/R injury were also mediated by implanting FILMS (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ed-h). H\\u0026amp;E staining confirmed IMH in both I/R and FILMS groups (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ek). Free haem level in the I/R group increased to 2.02 times that of a healthy myocardium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ei). FILMS reduced the amount of haem to a healthy level by trapping the exogenous haem. In I/R group, transcription and expression levels of HMOX1 and FTL were significantly upregulated, indicating that haem had been internalized into cellular iron (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ej,m,n). Both DAB-enhanced Prussian blue staining and tissue quantitative testing confirmed severe iron deposition in the I/R tissue, while FILMS scavenged haem and downregulated HMOX1, thereby significantly alleviated iron overload (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003el,o). Downregulation of iron level directly led to a decrease in lipid peroxidation (ACSL4 mRNA and tissue MDA) in myocardium, bringing lipid peroxidation back to healthy level. Therefore, FILMS inhibited IMH induced ferroptosis and I/R injury in clinically relevant large animal model. H\\u0026amp;E analyses of major organs confirmed the biosafety of FILMS treatment \\u003cem\\u003ein vivo\\u003c/em\\u003e (Extended Data Fig.\\u0026nbsp;10d).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIn recent years, understanding of the mechanism of ferroptosis, including cardiac ferroptosis, has become more comprehensive and profound\\u003csup\\u003e6,7,14,22,24\\u0026ndash;29\\u003c/sup\\u003e. In contrast, treatments for cardiac ferroptosis are still not clinically available, of which some have been validated in small animal models\\u003csup\\u003e13,24\\u003c/sup\\u003e. In this study, instead of inhibiting processes in the middle of the ferroptosis cascade, FILMS targets at the initiator, haem, for cardiac ferroptosis. To the best of our knowledge, FILMS is the first haem-binding, implantable biomaterial. We expect that FILMS could serve as a reference for the design of more potent haem-scavenging biomaterials for treating I/R injury and other ferroptosis-induced diseases, particularly with the reported mechanism of simultaneously improving both binding affinity and capacity of Apo-Lf.\\u003c/p\\u003e \\u003cp\\u003eHomeostasis of metal ions is essential in vital physiological processes\\u003csup\\u003e30\\u003c/sup\\u003e. Various implantable biomaterials have been developed to deliver and supplement metal ions including magnesium, strontium and etc. to injured tissues in which the concentration of metal ions was low due to diseases\\u003csup\\u003e31,32\\u003c/sup\\u003e. However, for diseases like ferroptosis in which metal ion concentrations are pathologically high (usually iron, copper, zinc etc. as they are normally low in normal tissue), few biomaterials are available for metal chelating therapy\\u003csup\\u003e33\\u003c/sup\\u003e. FILMS represent a biomaterial-based regulatory strategy to temporarily trap excessive harmful ion species from local environments by scavenging their small molecular carriers\\u003csup\\u003e34\\u003c/sup\\u003e. In fact, many metal ions incorporate into corresponding organic molecules (e.g. porphyrins) to form metal complexes before binding to proteins to function, e.g. iron and haem, magnesium and chlorophyl Ⅱ\\u003csup\\u003e35,36\\u003c/sup\\u003e. The pathological stimuli release these cytotoxic metal complexes from proteins, markedly disturbing the health status of patients\\u003csup\\u003e37\\u003c/sup\\u003e. Hence, direct catch of metal complexes is an attractive strategy to deal with metal cytotoxicity. FILMS inhibiting ferroptosis in IMH-induced I/R injury by scavenging haem serves as a demonstration of the feasibility and efficacy of such strategy.\\u003c/p\\u003e \\u003cp\\u003eThis study also demonstrates the potential of medical implants based on milk-derived proteins. Only a few types of natural proteins, such as collagen, fibrinogen, and silk fibroin have been used as major components for clinically approved implants and devices. The application of milk-derived proteins in medical implants is still rare despite of their enormous yield and high biocompatibility. A significant limiting factor is that the processability of milk-derived proteins is hindered by insufficient intermolecular interactions, thereby necessitating the incorporation of additional intermolecular forces or bonds. By introducing crosslinkable methacryloyl groups or white-light sensitive crosslinking catalyst, the most abundant proteins in milk, whey protein and casein, have recently been manufactured into structural materials\\u003csup\\u003e38,39\\u003c/sup\\u003e. FILMS demonstrate the advantages of using milk-derived proteins with unique bioactivities as raw materials for medical devices. Methacryloyl modification of Apo-Lf can be chemically considered as mutating the amino acids, similar to site-directed mutagenesis widely used in study the changes of protein activitie\\u003csup\\u003e40\\u003c/sup\\u003e. It is worth noting that photo-crosslinking, microfluidic emulsification and freeze-thaw as representative processing methods did not significantly inactivate haem-binding capability of Apo-LfMA. In addition, the changes in the structure-activity relationship of Apo-Lf demonstrates the functional plasticity of milk-derived proteins. Therefore, the development of novel high-activity medical devices based on milk-derived protein for treating diseases including I/R injury has considerable feasibility and application potential.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistics and reproducibility\\u003c/h2\\u003e \\u003cp\\u003eStatistical analysis was performed using Prism. Results are shown as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SD unless stated otherwise. T-tests were used to compare result from two groups. One-way analysis of variance (ANOVA) was used for multiple comparisons unless stated otherwise. p value of \\u0026lt;\\u0026thinsp;0.05 was considered statistically significant. All in vitro and in vivo results are representative of two to six independents.\\u003c/p\\u003e \\u003c/div\\u003e \"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eReporting summary\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll relevant data related to this manuscript are available on request from the authors on reasonable request. The remaining data are available within the Article or Supplementary Information. Source data are provided with this paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe MALDI-TOF data were collected at Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University with the technical support from Huiwen Wang. We thanked Qiong Huang, Jingyao Chen, Chengcheng Zhang and Yajun Yu from the core facility platform of Zhejiang University School of Medicine for their technical support of pathological staining. We gratefully acknowledge Yungin Li for technical assistance on laser confocal microscopy. We thank Yinping Lv in the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University for her technical assistance on TEM. We would also like to extend our thanks to Zhiwei Ge and Jingqun Yuan at the Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University for their help on UPLC-QTOF/MS and ICP analysis, respectively.\\u003c/p\\u003e\\n\\u003cp\\u003eThis study is financially supported by\\u0026nbsp;\\u0026ldquo;Pioneer\\u0026rdquo; and \\u0026ldquo;Leading Goose\\u0026rdquo; R\\u0026amp;D Program of Zhejiang Province, China (No. 2024C03074, 2023SDXHDX0004), National Natural Science Foundation of China (No. 82202328, 82170644), State Key Laboratory of Transvascular Implantation Devices, China (No. 012024018), Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (No. SN-ZJU-SIAS-004), the Natural Science Foundation of Zhejiang Province, China (LR23H020001).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contribution\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eY. Zhu conceived the idea of FILMS. Y. Zhu, X. Fang, C. Guo, T. Ren and L. Zhang designed the overall project. L. Zhang, Z. Sun, X. Jiang and B. Dai. L fabricated and characterized Apo-LfMA and FILMS, simulated molecular docking and molecular dynamics. L. Zhang conducted in vitro cell experiments with Y. Hua\\u0026rsquo;s assistance under the guidance of X. Fang, T. Ren and C. Gao. L. Shen and JY. Zhang performed the small animal study. J. Wen, WY. Wang and X. Deng collected the clinical data. J. Wen performed the minimally invasive FILMS implantation. JW. Zhang and L. Zhang conducted the biochemical analysis of animal samples. L. Zhang, Y. Gao and Q. Jin performed RNA-seq and lipid metabonomics analysis. WZ. Wang, M. Wang and F. Feng prepared and characterized iPSC-CMs. F. Xu collected SPR data. C. Guo designed the protein study. X. Fang designed the cell experiments. L. Zhang, X. Fang, T. Ren and Y. Zhu designed the small animal studies. Y. Tang, J. Wen and Y. Zhu designed the large animal study. \\u0026nbsp;Y. Zhu, L. Zhang, J. Wen, T. Ren, X. Fang, and C. Guo wrote the manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e: The authors declare no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eTsao, C. W. \\u003cem\\u003eet al.\\u003c/em\\u003e Heart Disease and Stroke Statistics\\u0026mdash;2023 Update: A Report From the American Heart Association. \\u003cem\\u003eCirculation\\u003c/em\\u003e \\u003cstrong\\u003e147\\u003c/strong\\u003e, e93\\u0026ndash;e621 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eTimmis, A. \\u003cem\\u003eet al.\\u003c/em\\u003e European Society of Cardiology: cardiovascular disease statistics 2021: executive summary. \\u003cem\\u003eEur. Hear. J. - Qual. Care Clin. Outcomes\\u003c/em\\u003e \\u003cstrong\\u003e8\\u003c/strong\\u003e, 377\\u0026ndash;382 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eHeusch, G. Myocardial ischaemia\\u0026ndash;reperfusion injury and cardioprotection in perspective. \\u003cem\\u003eNat. Rev. Cardiol.\\u003c/em\\u003e \\u003cstrong\\u003e17\\u003c/strong\\u003e, 773\\u0026ndash;789 (2020).\\u003c/li\\u003e\\n\\u003cli\\u003eLiu, T. \\u003cem\\u003eet al.\\u003c/em\\u003e Intramyocardial hemorrhage and the \\u0026ldquo;Wave front\\u0026rdquo; of reperfusion injury compromising myocardial salvage. \\u003cem\\u003eJ. Am. Coll. Cardiol.\\u003c/em\\u003e \\u003cstrong\\u003e79\\u003c/strong\\u003e, 35\\u0026ndash;48 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eMoon, B. F. \\u003cem\\u003eet al.\\u003c/em\\u003e Iron imaging in myocardial infarction reperfusion injury. \\u003cem\\u003eNat. Commun.\\u003c/em\\u003e \\u003cstrong\\u003e11\\u003c/strong\\u003e, 3273 (2020).\\u003c/li\\u003e\\n\\u003cli\\u003eFang, X. \\u003cem\\u003eet al.\\u003c/em\\u003e Ferroptosis as a target for protection against cardiomyopathy. \\u003cem\\u003eProc. Natl. Acad. Sci. U. S. A.\\u003c/em\\u003e \\u003cstrong\\u003e116\\u003c/strong\\u003e, 2672\\u0026ndash;2680 (2019).\\u003c/li\\u003e\\n\\u003cli\\u003eDixon, S. J. \\u003cem\\u003eet al.\\u003c/em\\u003e Ferroptosis: an iron-dependent form of nonapoptotic cell death. \\u003cem\\u003eCell\\u003c/em\\u003e \\u003cstrong\\u003e149\\u003c/strong\\u003e, 1060\\u0026ndash;1072 (2012).\\u003c/li\\u003e\\n\\u003cli\\u003eWang, X. D. \\u0026amp; Kang, S. Ferroptosis in myocardial infarction: Not a marker but a maker. \\u003cem\\u003eOpen Biol.\\u003c/em\\u003e \\u003cstrong\\u003e11\\u003c/strong\\u003e, 0\\u0026ndash;3 (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eFr\\u0026ouml;hlich, G. M., Meier, P., White, S. K., Yellon, D. M. \\u0026amp; Hausenloy, D. J. Myocardial reperfusion injury: looking beyond primary PCI. \\u003cem\\u003eEur. Heart J.\\u003c/em\\u003e \\u003cstrong\\u003e34\\u003c/strong\\u003e, 1714\\u0026ndash;1724 (2013).\\u003c/li\\u003e\\n\\u003cli\\u003eMoeckel, D. \\u003cem\\u003eet al.\\u003c/em\\u003e Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk. \\u003cem\\u003eSci. Transl. Med.\\u003c/em\\u003e \\u003cstrong\\u003e6\\u003c/strong\\u003e, 1\\u0026ndash;10 (2014).\\u003c/li\\u003e\\n\\u003cli\\u003eMenon, A. V. \\u003cem\\u003eet al.\\u003c/em\\u003e Excess heme upregulates heme oxygenase 1 and promotes cardiac ferroptosis in mice with sickle cell disease. \\u003cem\\u003eBlood\\u003c/em\\u003e \\u003cstrong\\u003e139\\u003c/strong\\u003e, 936\\u0026ndash;941 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eMaus, M. \\u003cem\\u003eet al.\\u003c/em\\u003e Iron accumulation drives fibrosis, senescence and the senescence-associated secretory phenotype. \\u003cem\\u003eNat. Metab.\\u003c/em\\u003e \\u003cstrong\\u003e5\\u003c/strong\\u003e, 2111\\u0026ndash;2130 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eFang, X., Ardehali, H., Min, J. \\u0026amp; Wang, F. The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease. \\u003cem\\u003eNat. Rev. Cardiol.\\u003c/em\\u003e \\u003cstrong\\u003e20\\u003c/strong\\u003e, 7\\u0026ndash;23 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eMishima, E. \\u003cem\\u003eet al.\\u003c/em\\u003e A non-canonical vitamin K cycle is a potent ferroptosis suppressor. \\u003cem\\u003eNature\\u003c/em\\u003e \\u003cstrong\\u003e608\\u003c/strong\\u003e, 778\\u0026ndash;783 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eBelcher, J. D. \\u003cem\\u003eet al.\\u003c/em\\u003e Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease. \\u003cem\\u003eBlood\\u003c/em\\u003e \\u003cstrong\\u003e123\\u003c/strong\\u003e, 377\\u0026ndash;390 (2014).\\u003c/li\\u003e\\n\\u003cli\\u003eSankari, S. \\u003cem\\u003eet al.\\u003c/em\\u003e A haem-sequestering plant peptide promotes iron uptake in symbiotic bacteria. \\u003cem\\u003eNat. Microbiol.\\u003c/em\\u003e \\u003cstrong\\u003e7\\u003c/strong\\u003e, 1453\\u0026ndash;1465 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eFang, X., Ardehali, H., Min, J. \\u0026amp; Wang, F. The molecular and metabolic landscape of iron and ferroptosis in cardiovascular disease. \\u003cem\\u003eNat. Rev. Cardiol.\\u003c/em\\u003e \\u003cstrong\\u003e20\\u003c/strong\\u003e, 7\\u0026ndash;23 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eMadyaningrana, K. \\u003cem\\u003eet al.\\u003c/em\\u003e Alpha 1-antitrypsin counteracts heme-induced endothelial cell inflammatory activation, autophagy dysfunction and death. \\u003cem\\u003eRedox Biol.\\u003c/em\\u003e \\u003cstrong\\u003e46\\u003c/strong\\u003e, 102060 (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eAllhorn, M., Berggard, T., Nordberg, J., Olsson, M. L. \\u0026amp; Akerström, B. Processing of the lipocalin \\u0026alpha; 1-microglobulin by hemoglobin induces heme-binding and heme-degradation properties. \\u003cem\\u003eBlood\\u003c/em\\u003e \\u003cstrong\\u003e99\\u003c/strong\\u003e, 1894\\u0026ndash;1901 (2002).\\u003c/li\\u003e\\n\\u003cli\\u003eRajasekaran, N. \\u0026amp; Naganathan, A. N. A self-consistent structural perturbation approach for determining the magnitude and extent of allosteric coupling in proteins. \\u003cem\\u003eBiochem. J.\\u003c/em\\u003e \\u003cstrong\\u003e474\\u003c/strong\\u003e, 2379\\u0026ndash;2388 (2017).\\u003c/li\\u003e\\n\\u003cli\\u003eRajasekaran, N., Sekhar, A. \\u0026amp; Naganathan, A. N. A universal pattern in the percolation and dissipation of protein structural perturbations. \\u003cem\\u003eJ. Phys. Chem. Lett.\\u003c/em\\u003e \\u003cstrong\\u003e8\\u003c/strong\\u003e, 4779\\u0026ndash;4784 (2017).\\u003c/li\\u003e\\n\\u003cli\\u003eStockwell, B. R. \\u003cem\\u003eet al.\\u003c/em\\u003e Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. \\u003cem\\u003eCell\\u003c/em\\u003e \\u003cstrong\\u003e171\\u003c/strong\\u003e, 273\\u0026ndash;285 (2017).\\u003c/li\\u003e\\n\\u003cli\\u003eWang, B., Lee, R. J. \\u0026amp; Tao, L. First-in-human transcatheter endocardial alginate-hydrogel implantation for the treatment of heart failure. \\u003cem\\u003eEur. Heart J.\\u003c/em\\u003e \\u003cstrong\\u003e44\\u003c/strong\\u003e, 326 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eStockwell, B. R. Ferroptosis turns 10: emerging mechanisms, physiological functions, and therapeutic applications. \\u003cem\\u003eCell\\u003c/em\\u003e \\u003cstrong\\u003e185\\u003c/strong\\u003e, 2401\\u0026ndash;2421 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eMao, C. \\u003cem\\u003eet al.\\u003c/em\\u003e DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer. \\u003cem\\u003eNature\\u003c/em\\u003e \\u003cstrong\\u003e593\\u003c/strong\\u003e, 586\\u0026ndash;590 (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eBersuker, K. \\u003cem\\u003eet al.\\u003c/em\\u003e The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. \\u003cem\\u003eNature\\u003c/em\\u003e \\u003cstrong\\u003e575\\u003c/strong\\u003e, 688\\u0026ndash;692 (2019).\\u003c/li\\u003e\\n\\u003cli\\u003eBadgley, M. A. \\u003cem\\u003eet al.\\u003c/em\\u003e Cysteine depletion induces pancreatic tumor ferroptosis in mice. \\u003cem\\u003eScience.\\u003c/em\\u003e \\u003cstrong\\u003e368\\u003c/strong\\u003e, 85\\u0026ndash;89 (2020).\\u003c/li\\u003e\\n\\u003cli\\u003eDoll, S. \\u003cem\\u003eet al.\\u003c/em\\u003e FSP1 is a glutathione-independent ferroptosis suppressor. \\u003cem\\u003eNature\\u003c/em\\u003e \\u003cstrong\\u003e575\\u003c/strong\\u003e, 693\\u0026ndash;698 (2019).\\u003c/li\\u003e\\n\\u003cli\\u003eMishima, E. \\u003cem\\u003eet al.\\u003c/em\\u003e DHODH inhibitors sensitize to ferroptosis by FSP1 inhibition. \\u003cem\\u003eNature\\u003c/em\\u003e \\u003cstrong\\u003e619\\u003c/strong\\u003e, E9\\u0026ndash;E18 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eWaldron, K. J., Rutherford, J. C., Ford, D. \\u0026amp; Robinson, N. J. Metalloproteins and metal sensing. \\u003cem\\u003eNature\\u003c/em\\u003e \\u003cstrong\\u003e460\\u003c/strong\\u003e, 823\\u0026ndash;830 (2009).\\u003c/li\\u003e\\n\\u003cli\\u003eBosch-Ru\\u0026eacute;, \\u0026Egrave;., D\\u0026iacute;ez-Tercero, L., Buitrago, J. O., Castro, E. \\u0026amp; P\\u0026eacute;rez, R. A. Angiogenic and immunomodulation role of ions for initial stages of bone tissue regeneration. \\u003cem\\u003eActa Biomater.\\u003c/em\\u003e \\u003cstrong\\u003e166\\u003c/strong\\u003e, 14\\u0026ndash;41 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eVasconcelos, D. M., Santos, S. G., Lamghari, M. \\u0026amp; Barbosa, M. A. The two faces of metal ions: From implants rejection to tissue repair/regeneration. \\u003cem\\u003eBiomaterials\\u003c/em\\u003e \\u003cstrong\\u003e84\\u003c/strong\\u003e, 262\\u0026ndash;275 (2016).\\u003c/li\\u003e\\n\\u003cli\\u003eKim, J.-J., Kim, Y.-S. \\u0026amp; Kumar, V. Heavy metal toxicity: An update of chelating therapeutic strategies. \\u003cem\\u003eJ. Trace Elem. Med. Biol.\\u003c/em\\u003e \\u003cstrong\\u003e54\\u003c/strong\\u003e, 226\\u0026ndash;231 (2019).\\u003c/li\\u003e\\n\\u003cli\\u003eCrisponi, G. \\u003cem\\u003eet al.\\u003c/em\\u003e Kill or cure: Misuse of chelation therapy for human diseases. \\u003cem\\u003eCoord. Chem. Rev.\\u003c/em\\u003e \\u003cstrong\\u003e284\\u003c/strong\\u003e, 278\\u0026ndash;285 (2015).\\u003c/li\\u003e\\n\\u003cli\\u003eMendel, R. R., Smith, A. G., Marquet, A. \\u0026amp; Warren, M. J. Metal and cofactor insertion. \\u003cem\\u003eNat. Prod. Rep.\\u003c/em\\u003e \\u003cstrong\\u003e24\\u003c/strong\\u003e, 963 (2007).\\u003c/li\\u003e\\n\\u003cli\\u003eChalkley, M. J., Mann, S. I. \\u0026amp; DeGrado, W. F. De novo metalloprotein design. \\u003cem\\u003eNat. Rev. Chem.\\u003c/em\\u003e \\u003cstrong\\u003e6\\u003c/strong\\u003e, 31\\u0026ndash;50 (2021).\\u003c/li\\u003e\\n\\u003cli\\u003eDutra, F. F. \\u0026amp; Bozza, M. T. Heme on innate immunity and inflammation. \\u003cem\\u003eFront. Pharmacol.\\u003c/em\\u003e \\u003cstrong\\u003e5\\u003c/strong\\u003e, 1\\u0026ndash;20 (2014).\\u003c/li\\u003e\\n\\u003cli\\u003eHu, Z. \\u003cem\\u003eet al.\\u003c/em\\u003e Scalable milk-derived whey protein hydrogel as an implantable biomaterial. \\u003cem\\u003eACS Appl. Mater. Interfaces\\u003c/em\\u003e \\u003cstrong\\u003e14\\u003c/strong\\u003e, 28501\\u0026ndash;28513 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eZhu, Q. \\u003cem\\u003eet al.\\u003c/em\\u003e White-light crosslinkable milk protein bioadhesive with ultrafast gelation for first-aid wound treatment. \\u003cem\\u003eBiomater. Res.\\u003c/em\\u003e \\u003cstrong\\u003e27\\u003c/strong\\u003e, 6 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eFu, Y. \\u003cem\\u003eet al.\\u003c/em\\u003e Engineered P450 atom-transfer radical cyclases are bifunctional biocatalysts: reaction mechanism and origin of enantioselectivity. \\u003cem\\u003eJ. Am. Chem. Soc.\\u003c/em\\u003e \\u003cstrong\\u003e144\\u003c/strong\\u003e, 13344\\u0026ndash;13355 (2022).\\u003c/li\\u003e\\n\\u003cli\\u003eSinclair, P. R., Gorman, N. \\u0026amp; Jacobs, J. M. Measurement of heme concentration. \\u003cem\\u003eCurr. Protoc. Toxicol.\\u003c/em\\u003e \\u003cstrong\\u003e00\\u003c/strong\\u003e, 8.3.1\\u0026ndash;8.3.7 (1999).\\u003c/li\\u003e\\n\\u003cli\\u003eZhang, L. \\u003cem\\u003eet al.\\u003c/em\\u003e Multileveled hierarchical hydrogel with continuous biophysical and biochemical gradients for enhanced repair of full-thickness osteochondral defect. \\u003cem\\u003eAdv. Mater.\\u003c/em\\u003e \\u003cstrong\\u003e35\\u003c/strong\\u003e, 2209565 (2023).\\u003c/li\\u003e\\n\\u003cli\\u003eZhao, M. \\u003cem\\u003eet al.\\u003c/em\\u003e Enhancing the engraftment of human induced pluripotent stem cell-derived cardiomyocytes via a transient inhibition of rho kinase activity. \\u003cem\\u003eJ. Vis. Exp.\\u003c/em\\u003e \\u003cstrong\\u003e10\\u003c/strong\\u003e, e59452 (2019).\\u003c/li\\u003e\\n\\u003cli\\u003eYang, Y. \\u003cem\\u003eet al.\\u003c/em\\u003e Elastic 3D-printed hybrid polymeric scaffold improves cardiac remodeling after myocardial infarction. \\u003cem\\u003eAdv. Healthc. Mater.\\u003c/em\\u003e \\u003cstrong\\u003e8\\u003c/strong\\u003e, e1900065 (2019).\\u003c/li\\u003e\\n\\u003cli\\u003eWang, L. \\u003cem\\u003eet al.\\u003c/em\\u003e Injectable and conductive cardiac patches repair infarcted myocardium in rats and minipigs. \\u003cem\\u003eNat. Biomed. Eng.\\u003c/em\\u003e \\u003cstrong\\u003e5\\u003c/strong\\u003e, 1157\\u0026ndash;1173 (2021).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eMaterials.\\u0026nbsp;\\u003c/strong\\u003eLactoferrin (Bega, Australia), methacrylic anhydride (Sigma-Aldrich, USA), oxalic acid (Sigma-Aldrich, USA), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (Aladdin, China), gelatin (Sigma-Aldrich, USA). Phosphate buffered saline (PBS, Invitrogen, USA), Dulbecco\\u0026rsquo;s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin, and penicillin/streptomycin were bought from Gibco (USA). Cell counting kit-8 (CCK-8, Dojindo, Japan), dichloro-dihydro-fluorescein diacetate (DCFH-DA) (Beyotime Biotechnology, China), FerrOrange and Mito-FerroGreen (Dojindo, Japan), C11 Bodipy (581/591) and Live/dead Kit (Thermo Fisher, USA), Mitotracker red (Yeasen Biotechnology, China), One-Step TUNEL apoptosis assay kit (Beyotime Biotechnology, China), Anti-Hemoglobin (Acbam, UK, ab92492, Polyclonal, 1:500 dilution), Anti-Cardiac Troponin T antibody (Acbam, UK, ab209813, Monoclonal, 1:4000 dilution), Anti-Ferritin Light chain antibody (Abcam, UK, ab69090, Polyclonal, 1:200 dilution), Anti- 4 Hydroxynonenal antibody (Abcam, UK, ab48506, Monoclonal, 1:1000 dilution), Anti-Heme Oxygenase 1 antibody (Abcam, UK, ab52947, Monoclonal, 1:1000 dilution) Anti-Connexin 43 antibody (Acbam, UK, ab11370, Polyclonal, 1:2000 dilution), Anti-CD31 antibody (Abcam, UK, ab182981, Monoclonal, 1:2000 dilution) and Anti-\\u0026alpha;SMA antibody (Boster, China, BM0002, Monoclonal, 1:400 dilution) were used according to corresponding protocols.H9C2 cell line (GNR 5) was purchased from ATCC and Cell Bank of Typical Culture Collection of Chinese Academy of Science.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eClinical data collection.\\u0026nbsp;\\u003c/strong\\u003eThree patients with acute myocardial infarction admitted to the Department of Cardiology of the Peking University Third Hospital and receiving cardiac magnetic resonance were included. Clinical data on patients\\u0026apos; treatment during hospitalization, laboratory indicators and surgical treatment were collected through the electronic medical record system. This study complied with the Declaration of Helsinki and was approved by the Ethics Review Committee of Peking University Third Hospital (M2022577).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMethods of cardiac magnetic resonance examination.\\u0026nbsp;\\u003c/strong\\u003eThe CMR examination in the acute phase of myocardial infarction was performed using an MR750w 3.0 T magnetic resonance machine and a 32-pass surface phased-array coil from GE Healthcare, USA. The examination sequences included a movie sequence, a delayed enhancement sequence (performed 10 min after injection of gadolinium contrast agent), and a black-blood T2-weighted sequence. The cine sequence was performed using a steady state free-feeding sequence to acquire two-chamber, three-chamber, and four-chamber long-axis images as well as multilayered short-axis images of the right and left ventricular base toward the apical portion of the ventricle.The T2 pressure-lipid images were acquired by a T2-weighted fast spin-echo sequence. Delayed gadolinium contrast enhancement (LGE) was scanned using an inversion recovery sequence.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCardiac magnetic resonance image postprocessing.\\u0026nbsp;\\u003c/strong\\u003eCMR images were analyzed by experienced cardiovascular imaging physicians using the postprocessing software C VI42 5.13 (Circle Cardiovascular Imaging, Canada). Edematous areas (signal intensity exceeding 2 standard deviations from the infarcted distal myocardium) within the blood-supplying region of the offender vessel were quantified by T2-weighted image sequences and defined as the infarct at risk area (AAR). Low-signal areas within the edematous region were defined as acute-phase intramyocardial hemorrhage and recovery-phase iron deposition. High-signal areas (signal intensity exceeding 5 standard deviations above the infarcted distal myocardium) in the LGE images were defined as infarcted area.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eApo-LfMA preparation and characterization.\\u0026nbsp;\\u003c/strong\\u003eApo-Lf was obtained by removing iron from lactoferrin. Lactoferrin was dissolved in PBS (0.2 mol/L, pH 2.0~4.0) with stirring. The pH was adjusted by adding HCl and NaOH (5 mol/L), as indicated by a color change from red to colorless. EDTA was added before dialysis against PBS (50 mmol/L) for 48h with 6-8h buffer exchanges. Iron-free lactoferrin, apo-Lf, was obtained by lyophilization of half the dialyzed solution. Next, we designed a scheme to attach double bonds to the iron-free lactoferrin so that it can be polymerized. Methacrylic anhydride was used to graft double bonds to amino groups of Apo-Lf \\u0026nbsp;the remaining half of the iron-free lactoferrin solution from Step 1. The pH was adjusted to neutral and the reaction proceeded for 4 h. The product was dialyzed against deionized water with stirring, with 5-6 h exchanges for 48h total. Lyophilization yielded methacrylated Apo-Lf (apo-LfMA).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eFor NMR characterization,\\u0026nbsp;15 mg of\\u0026nbsp;Apo-Lf and Apo-LfMA\\u0026nbsp;were\\u0026nbsp;dissolved in DMSO-\\u003cem\\u003ed\\u003c/em\\u003e\\u003csub\\u003e6\\u003c/sub\\u003e with 1.0 M LiCl.\\u0026nbsp;One-dimensional (1D) and two-dimensional (2D) NMR spectra were collected on Bruker AVANCE NEO 600 MHz NMR spectrometers equipped with cryo-probe. 1D \\u003csup\\u003e1\\u003c/sup\\u003eH NMR was performed with a spectral width of 20 ppm, 32 scans, and a relaxation delay of 3.0\\u0026thinsp;s. Correlation spectroscopy (COSY) for \\u003csup\\u003e1\\u003c/sup\\u003eH-\\u003csup\\u003e1\\u003c/sup\\u003eH was performed with a spectral width of 12 ppm in both the \\u003cem\\u003et1\\u003c/em\\u003e and \\u003cem\\u003et2\\u003c/em\\u003e dimensions, 160 and 2048 complex points in the \\u003cem\\u003et1\\u003c/em\\u003e and \\u003cem\\u003et2\\u0026nbsp;\\u003c/em\\u003edimensions, respectively, 16 scans, and a relaxation delay of 1.0\\u0026thinsp;s. Heteronuclear single quantum coherence (HSQC) spectra were collected with a spectral width of 170 ppm in the \\u003cem\\u003et1\\u003c/em\\u003e and a spectral width of 12 ppm in the \\u003cem\\u003et2\\u003c/em\\u003e dimensions, 160 and 2048 complex points in \\u003cem\\u003et1\\u003c/em\\u003e and \\u003cem\\u003et2\\u003c/em\\u003e dimensions, respectively, and 16 scans.\\u0026nbsp;The molecular weights of proteins were measured on a Bruker Rapiflex matrix-assisted laser desorption ionization-time of flight (UltrafleXtreme, MALDI-TOF) mass spectrometer. Samples were dissolved in deionized water before becoming combined with an equal volume of the sinapic acid matrix reconstituted in 0.1% trifluroacetic acid and 30% acetonitrile. The chemical structure of Lf, Apo-Lf and Apo-LfMA was characterized by FT-IR spectroscopy (Nicolet 6700, USA).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eFor HPLC-MS characterization of proteins.\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003eApo-Lf and Apo-LfMA proteins were lysed in trypsin solution at 60℃\\u0026nbsp;for 4h to get peptide samples. The peptide samples were loaded onto a ACQUITY UPLC CSH C18 column connected to an UPLC-Triple-TOF/MS system (Waters, USA). Peptides were separated and eluted with a gradient of\\u0026nbsp;5% to 95% HPLC buffer B (0.1% formic acid in acetonitrile, v/v) in buffer A (0.1% formic acid in water, v/v) at a flow rate of\\u0026nbsp;300 nl\\u0026nbsp;min\\u003csup\\u003e\\u0026minus;1\\u003c/sup\\u003e. The eluted peptides were then ionized and analysed by an AB TripleTOF 6600\\u003csup\\u003eplus\\u003c/sup\\u003e System mass spectrometer (AB SCIEX, Framingham,\\u0026nbsp;USA).\\u0026nbsp;The pressure of Curtain Gas (N2) was set to 35 psi. Maximum allowed error was set to \\u0026plusmn; 5 ppm. Declustering potential (DP), 80 V; collision energy (CE), 10 V. For MS/MS acquisition mode, the parameters were almost the same except that the collision energy (CE) was set at \\u0026plusmn; 50 \\u0026plusmn; 20 V, ion release delay (IRD) at 67, ion release width (IRW) at 25. The IDA-based auto-MS2 was performed on the 8 most intense metabolite ionsin a cycle of full scan (1 s). The scan range of m/z of precursor ion and product ion were set as 100-2000 Da and 50-2000 Da. The exact mass calibration was performed automatically before each analysis employing the Automated Calibration Delivery System.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSPR analysis.\\u0026nbsp;\\u003c/strong\\u003eSPR measurements were performed on the Biacore 8K Plus (Cytiva) system with the running buffer (0.01M PBS). Proteins as ligands (20 ug/ml) were immobilized on the CM5 sensor chip (GE Healthcare) through amine coupling. Then, ethanolamine-HCl flowed over the chip surface for 7 min, blocking the unreacted carboxyl groups. The flow rate was 30\\u0026thinsp;\\u0026micro;l\\u0026thinsp;min\\u003csup\\u003e-1\\u003c/sup\\u003e for a contact time of 120\\u0026thinsp;s followed by 800\\u0026thinsp;s dissociation time. After each injection, the surface was regenerated using 3\\u0026thinsp;M magnesium chloride (for PD-L1) or 10\\u0026thinsp;mM glycine, pH\\u0026thinsp;3.0 (for RBD). Data were fit with a 1:1 Langmuir binding model within the Biacore 8K analysis software (Cytiva, v.4.0.8.19879).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMolecular docking and molecular dynamics simulation.\\u0026nbsp;\\u003c/strong\\u003eFrom the PDB database, we got the 3D structure of lactoferrin protein (PDB: 1BLF) as a template and removed the ferric irons from the structure to obtained the 3D model of Apo-Lf for subsequent molecular docking. Using the 3D structure file of the modeled Apo-Lf as the initial structure, the amino groups on the 14 Lysine amino acid side chains of the protein were modified and combined with methacrylic anhydride to gain the structure of Apo-LfMA for subsequent molecular docking. The initial structures of proteins were processed using AutoDock Tools 1.5.6 to preserve the original protein charge and generate a pdbqt file for docking. UCSF Chimera was used to remove waters and non-protein atoms. AMBER99SB charges were assigned and pKa values calculated with H + + 3 at pH 7. Haem topology was generated with RDKit, minimized with MMFF94, and AM1-BCC charges assigned with Chimera. The protein-ligand complexes were constructed with Packmol. The Apo-Lf model was prepared in Autodock Tools by adding charges and saving as a pdbqt file. The haem ligand topology was generated with MOPAC semiempirical calculations and PM3 charges assigned. Docking was performed with Autodock 4.2.6 using a 100x100x100 grid box centered on the protein. 100 docking runs were calculated. The top binding modes were optimized using the Amber14 force field, first by 1000 steps of steepest descent, followed by 500 steps of conjugate gradient. GROMACS 5.1.5 was used for the simulations. The temperature was 300K, pH 7, and pressure 1 bar. The protein was centered in the box with a 0.1 nm minimum distance to the edges. Topologies were generated with pdb2gmx (AMBER99SB force field for protein) and AmberTools (GAFF for ligand). TIP3P water and NaCl ions neutralized the systems. Energy minimization was done by steepest descent, followed by 1 ns NVT and 1 ns NPT equilibrations. 50 ns production MD was performed with 2 fs time steps. Analysis included hydrogen bonds, salt bridges, RMSD, RMSF, Radius of Gyrate and SASA.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eHaem extraction and detection.\\u0026nbsp;\\u003c/strong\\u003eProtein-haem complexes were lysed with 1\\u0026thinsp;M HCl and haem was extracted with equal volume of 2-butanone. Following evaporation of 2-butanone, the haem was resuspended in DMSO. Haem was extracted from the heart tissues by homogenization in RIPA buffer containing protease inhibitors. Haem from the equal amount of proteins was quantified by fluorescence porphyrin assays with oxalic acid method\\u003csup\\u003e41\\u003c/sup\\u003e. In brief, 2\\u0026thinsp;M oxalic acid was added to each sample, and the sample was split into 2 equivalents. One half was heated to 95\\u0026thinsp;\\u0026deg;C for 30\\u0026thinsp;min to deprive iron from haem, while the other half was kept at 25\\u0026thinsp;\\u0026deg;C set as a subtraction for the corresponding heated sample. The suspensions were centrifuged at 25\\u0026thinsp;\\u0026deg;C to remove precipitates prior to measurement of porphyrin fluorescence on a Tecan Infinite M200Pro plate reader (excitation: 400\\u0026thinsp;nm; emission: 608\\u0026thinsp;nm). Haem concentration in each well was calculated against a standard curve prepared with series dilutions of haemin chloride. For heart tissue samples, the concentrations of haem were normalized to protein concentrations which were measured by using BCA protein assay kit (Beyotime).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eIron qualification via ICP-MS.\\u0026nbsp;\\u003c/strong\\u003eProtein-haem complexes were lysed with nitric acid. Tissue samples of infarcted myocardium were primarily homogenized in RIPA buffer containing protease inhibitors and analyzed by BCA method to normalized the data of protein amount in each sample; and then the supernatants were lysed with nitric acid. Agilent ICP-MS instrumentation with MassHunter 4.4 was used to collect data.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFILMS, FILMG and GMG preparation.\\u0026nbsp;\\u003c/strong\\u003eThe aqueous phase contained 12% w/v Apo-LfMA and 3% w/v methacrylated gelatin (GelMA) in FILMG and FILMS samples or 12% GelMA in GMG samples with 0.3% LAP photo-sensitive initiator, and the oil phase was liquid paraffin with 5% w/v Span 80 emulsifier. The synthesis of GelMA according to a previously described method\\u003csup\\u003e42\\u003c/sup\\u003e. These two phases drove by separately syringe pumps, went through pipes at the speed of 3 mL/min in oil and 100 \\u0026mu;L/min in protein solutions, and emulsified into droplets. For FILMG and GMG, emulsion droplets were collected in a 10 cm petri dish at 4\\u0026deg;C for 10 min to maintain morphology, then photocrosslinked at 405 nm UV light for 3 min. For FILMS, the collected droplets were frozen at -80\\u0026deg;C for 1 min followed by freezing in liquid nitrogen prior to UV-initiated crosslink. Blank Gelatin hydrogel microspheres were prepared via a microfluidic device. For FILMS, the collected droplets were frozen at -80\\u0026deg;C for 1 min followed by freezing in liquid nitrogen prior to UV-initiated crosslink. Microspheres were washed 3 times each with petroleum ether, -80\\u0026deg;C acetone, and deionized water before lyophilization and storage at 4\\u0026deg;C.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eThe characterizations of FILMG and FILMS.\\u0026nbsp;\\u003c/strong\\u003eThe morphology of FILMG and FILMS is observed under an optical microscope and SEM evaluation analysis after lyophilization. X-ray energy-dispersive spectroscopy (EDX, Inca X-Max, UK) was conducted to reveal the typical N and S elements in protein hydrogel (FILMS) in associated with SEM observation. Mercury intrusion porosimeter (Micromeritic AutoPore IV 9510, USA) was used to measure the porosity and specific surface area of FILMG and FILMS. FILMG and FILMS were incubated with 250 \\u0026mu;M haem solution at pH=7.2, and the aqueous samples were collected and evaluated about the haem content through oxalic acid method and ICP at 30min, 1h, 2h, 4h, 8h and 24h to determine their haem absorption capacity. Young\\u0026apos;s effective moduli were measured in static mode using the Piuma nanoindenter (Optics11 life, Netherlands).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCell culture.\\u0026nbsp;\\u003c/strong\\u003e1) H9C2 CMs were cultured in Dulbecco\\u0026apos;s Modified Eagle Medium (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin, and incubated in a 37\\u0026deg;C incubator equilibrated with 95% air and 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. 2) Human induced pluripotent stem cells (iPSCs) were generated and supplied by Cardiovascular Regenerative Medicine Laboratory (Frontier Innovation Center, School of Basic Medical Sciences, Fudan University). The hiPSCs were initially cultured on Matrigel-coated 6-well plate in mTeSR\\u003csup\\u003e\\u0026trade;\\u003c/sup\\u003e1 as the manufacturer\\u0026rsquo;s directions. Cardiac differentiation was achieved using small molecules targeting the Wnt pathways as described in a previously reported protocol\\u003csup\\u003e43\\u003c/sup\\u003e. Briefly, confluent hiPSCs (\\u0026gt;85%) maintained in mTeSR\\u003csup\\u003e\\u0026trade;\\u003c/sup\\u003e1 were dissociated with Versene for 5-9 min at 37 \\u0026deg;C and then replated onto Matrigel-coated 6-well plate at 1 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e cells for each well. After culturing for 3 days in mTeSR\\u0026trade;1, cells reached almost 100% confluency and then were treated with CHIR99021 at 12 \\u0026mu;M in serum-free medium RPMI/B27 containing 1 \\u0026mu;g/mL insulin for 24 h (day 0 for differentiation). Then the cultures were washed with PBS and replaced with serum-free medium RPMI/insulin-free B27 for 48 h. On day 3, cells were treated with 10 \\u0026mu;M Wnt inhibitor IWR1 in serum-free medium RPMI/insulin-free B27. On day 5, the medium was changed to serum-free medium RPMI/insulin-free B27. Starting from day 7, cells were maintained in the serum-free medium RPMI/B27 containing insulin and the medium was changed every 3 days. Spontaneous beating of cardiomyocytes should first be visible from day 8 to day 10. The purification process of cardiomyocytes could be conducted when most of the cardiomyocytes had started beating. The medium was replaced with glucose-free medium RPMI/B27 containing 4 mM DL-lactate, and the incubation continued for 3-5 days without changing medium. When observing under the microscope, more than 90% of the cells in the field of view showed beating, which was defined as successful purification. Purified cardiomyocytes were used for subsequent experiments.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCell viability.\\u0026nbsp;\\u003c/strong\\u003eH9C2 or hiPSCs were seeded in 96-well plates at a density of 8000 cell per well. After 12h culture, cells were supplemented with haem solutions of gradient concentrations and cell death inhibitions including DFO (100 \\u0026mu;M, T1637, TargetMol),\\u003c/p\\u003e\\n\\u003cp\\u003eGsk-872 (10 \\u0026mu;M, T4074, TargetMol) and ZVF (50 \\u0026mu;M, T7020, TargetMol) without FBS to confirm that haem induced ferroptosis of cardiomyocytes and its working curves. Otherwise, after 12h culture, cells were supplemented with haem solution and materials (GMG, FILMG and FILMS) without FBS to verify the anti-ferroptotic function of materials. After treatment for 24h, MTT (Abcam) stock solution was added to each well at a final concentration of 500 \\u0026mu;g\\u0026middot;ml\\u003csup\\u003e-1\\u003c/sup\\u003e and incubated in the dark for 4h at 37 \\u0026deg;C. The absorbance at 570 nm was measured in a Tecan plate reader. In addition, the survival of cells was visualized by using Live/Dead kit (Invitrogen\\u003csup\\u003eTM\\u003c/sup\\u003e) and LSM 880 confocal microscope (Carl Zeiss).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMeasurements of ROS and lipid peroxidation by using DCFDA and C11-Bodipy 581/591.\\u003c/strong\\u003e H9C2 or hiPSCs were seeded in 96-well plates at a density of 8000 cell per well. After 12h culture, cells were supplemented with haem solution and materials (GMG, FILMG and FILMS) without FBS. After treatment for 24h, cells were incubated with 1 \\u0026micro;M of DCFDA (Beyotime) and BODIPY 581/591 C11 (Thermo Fisher) for 30\\u0026thinsp;min at 37\\u0026thinsp;\\u0026deg;C. Subsequently, cells were visualized by LSM or were trypsinized, resuspended in 300\\u0026thinsp;\\u0026micro;l of Hanks\\u0026rsquo; balanced salt solution (HBSS, Gibco), and then analysed using a flow cytometer (CytoFLEX and CytExpert 2.4, Beckman Coulter) with a 488-nm laser paired with a 530/30\\u0026thinsp;nm bandpass filter. Data were analysed using FlowJo Software 10 (Treestar).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFluorescence imaging for cellular detection.\\u003c/strong\\u003e H9C2 cells were seeded in 24-well plates at a density of 50000 cell per well. After 12h culture, cells were supplemented with haem solution and materials (GMG, FILMG and FILMS) without FBS. After treatment for 24h, cellular labile iron was stained by FerroOrange Dye (F374, Dojindo). The Mito-FerroGreen (M489, Dojindo) fluorescent probe with the mitochondrial probe (40743ES50, Yeasen) was utilized to assess mitochondrial ferrous ions (Fe\\u003csup\\u003e2+\\u003c/sup\\u003e) in live cells. JC-10 dye (Solabio) was used to determine mitochondrial membrane potential in cardiomyocytes. All procedures were according to the manufacturer\\u0026apos;s instructions.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAnimal study ethics.\\u0026nbsp;\\u003c/strong\\u003eAdult male SD rats weighing 180-200 g (from the Laboratory Animal Care Facility of Shanghai Jiao Tong University School of Medicine) were housed at a constant temperature (22 \\u0026plusmn; 2 \\u0026deg;C) under a 12-h light/dark cycle and given standard lab chow and water ad libitum. Bama miniature pigs weighing 30-35 kg were used. All investigations in this study conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 8023, revised 1978). Small-animal experiments (SD rats) were approved by Zhejiang Academy of Medical Sciences (ZJCLA-IACUC-20010691). Larger-animal experiments (Bama miniature pigs) were approved by the Experimental Animal Ethics Committee of Peking University Third Hospital (A2023072). In order to avoid experimental differences caused by animal sex, animals of the same sex were used in the same experiment.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eStudy design \\u003cem\\u003ein vivo\\u003c/em\\u003e.\\u0026nbsp;\\u003c/strong\\u003eThe \\u003cem\\u003ein vivo\\u003c/em\\u003e study was divided into three parts. The first part aimed to exploring the pathophysiological process of intramyocardial haemorrhage in I/R model. In this part, we built both I/R model and MI model with the administration of blood components. I/R samples were sacrificed after reperfusion of 30min, 1h, 2h, 4h, 8h and 24h (n=4); and the blood, plasma, serum, RBCs, haemoglobin and haem \\u0026nbsp; \\u0026nbsp;were individually administered in the infarcted area (n=4). The second part focused on the validation of drugs or materials in rat I/R model, including the sham group, the intravenous injection of saline (I/R), Hx and Apo-Lf (n=4) and the intramyocardial injection of saline (I/R), GMG and FILMS (n=9). The third part was a preclinical trial in mini-pig I/R model including the sham group and the intramyocardial injection of saline (I/R) and FILMS (n=3).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRat model of MI with the administration of blood components\\u003c/strong\\u003e. Before surgery, fresh blood of male SD rats was individually collected in anticoagulant tubes to obtain the plasma and RBCs samples or in promoting coagulating tubes to acquire the serum samples. The blood was isolated by three rounds of centrifugation at 600g and washed twice to separate the plasma samples and the RBCs. RBCs samples were than resuspended in PBS to obtain an equal volume to the original blood. The concentration of haemoglobin (R05236, BSZH Scientific LLC.) and haem (51280-5G, Sigma-Aldrich) was based on the delta value of haem level in between the sham and I/R infarcted myocardium.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe MI model was constructed as previously described\\u003csup\\u003e44\\u003c/sup\\u003e. Briefly, animals were anaesthetized with 2% isoflurane inhalation and then intubated and ventilated with a respirator with extra oxygen. The rats were placed in the supine position, followed by a left thoracotomy and pericardiectomy to expose the hearts. Then the left anterior descending (LAD) coronary artery was ligated with a 6-0 silk suture at approximately 2-3 mm from its origin between the left atrium and the pulmonary artery conus to create LV infarction. Blood samples were then evenly injected into infarcted myocardium. The same procedure without ligation of the LAD coronary artery was conducted to sham-operated group.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRat model of I/R and treatments\\u003c/strong\\u003e. Male SD rats were anaesthetized with 2% isoflurane inhalation and then intubated and ventilated with a respirator with extra oxygen. The rats were placed in the supine position, followed by a left thoracotomy and pericardiectomy to expose the hearts. Reversible ligation on the left anterior descending (LAD) coronary artery was performed utilizing sterile 6-0 silk suture with a slipknot subsequently following a left thoracotomy around the third intercostal space. Appropriate ligation was confirmed by visual observation of the left ventricle wall turning pale. After 60 min of regional ischemia, the heart was reperfused, resulting in loss of the discoloration of the myocardium distal to the ligation. The treatments with Hx (4 mg/kg) or Apo-Lf (4 mg/kg) were administered through the tail vein at 30 min before surgery. Therapeutic hydrogel microspheres were homogeneously injected into the infarcted myocardium. The same procedure without ligation of the LAD coronary artery was conducted to sham-operated group. Rats were anesthetized and sacrificed at 1 and 35 d post MI to harvest short-term and long-term treated hearts, respectively.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTranscatheter endocardial FILMS implantation in mini-pig\\u003c/strong\\u003e. In brief, the Chinese giant white pig (Alligator sinensis) (65kg) was anesthetized with tiletamine hydrochloride (4 mg/kg) and zolazepam hydrochloride (4 mg/kg). To evaluate the feasibility and safety of transcatheter endocardial hydrogel implantation for treating IR，minimally invasive intervention was used for hydrogel delivery. As previously described\\u003csup\\u003e23\\u003c/sup\\u003e, an 18-F guiding catheter was inserted via the femoral artery and guided crossing the aortic valve under DSA. Injections were performed with a steerable, dual-lumen needle catheter. Transthoracic echocardiography was used to localize the injection site. The tip of the catheter reached the endocardium, and contrast was injected into the LV wall to identify no leakage or perforation. The FILMS was injected into the myocardium at the mid-LV free wall. Pig was euthanized immediately after surgery and fresh heart tissue was obtained. The tissue was cut to observe the hydrogel retention and the presence of thrombosis at the injection site.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMini-pig model of I/R and treatments\\u003c/strong\\u003e.\\u0026nbsp;The Bama minipigs () were anesthetized with tiletamine hydrochloride (4 mg/kg) and zolazepam hydrochloride (4 mg/kg). To establish the pig ischemia/reperfusion (I/R) model, a transthoracic 2D echocardiographic (ECG) measurement by Simpson\\u0026rsquo;s method was performed to ensure that the animal was healthy before IR induction. Following the baseline ECG measurements, light anesthesia was maintained by continuous intravenous infusion of propofol (30 to 40 \\u0026mu;g kg\\u003csup\\u003e\\u0026minus;1\\u003c/sup\\u003e \\u0026middot;min\\u003csup\\u003e\\u0026minus;1\\u003c/sup\\u003e). An incision was made in the 4th intercostal space at the left edge of the sternum of the pig, and the skin, subcutaneous tissue, and muscle were incised layer by layer, and finally the pericardium was cut to expose the heart. Myocardial ischemia was caused by clamping the vessel below the second angular branch of the anterior descending branch and blocking blood flow with a coronary clip. The ECG changes in the animals were observed to confirm myocardial ischemia. The ECG, heart rate, and arterial pressure were continuously monitored. Defibrillation may be performed if ventricular fibrillation (VF) occurs, and IV epinephrine or atropine may be administered as needed for asystole/bradycardia/hypotension. After 60 minutes of ischemia, the coronary clamp was released to restore blood flow and reperfusion was achieved. The animals were observed for ECG changes to confirm myocardial reperfusion. The hydrogel was injected into the myocardium of the infarct margin and infarct area immediately after myocardial reperfusion (6-8 injection sites, 100 \\u0026mu;l per site). Ultimately, the incision is sutured to ensure good hemostasis. Intravenous antibiotics were administered for three days postoperatively. Minipigs were anesthetized and sacrificed at 3 d post MI to harvest short-term treated hearts.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTransmission electron microscopy.\\u0026nbsp;\\u003c/strong\\u003eThe rat myocardium tissues were surgically collected and subsequently fixed in 2.5% glutaraldehyde overnight. After washed three times with 0.1 M PBS, the tissues were fixed in 1% osmic acid for 1 h. The tissue segments were then rinsed with pure water, stained in 2% uranyl acetate for 30 min and dehydrated in ethanol of increasing concentrations from 50% to 100% and 100% acetone. After appropriate embedment according to standard procedures, the stained tissues were made into serial ultrathin section with an ultramicrotome (Leica UC7), and observed with a transmission electron microscope.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMeasurement of cardiac enzymes.\\u0026nbsp;\\u003c/strong\\u003eSerum enzymes, including AST, CK, CK-MB, and LDH, were measured using an automatic biochemical analyzer (Sysmex). The levels of serum enzymes were assayed according to the instructions provided with the corresponding kits.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eMeasurement of MDA levels.\\u0026nbsp;\\u003c/strong\\u003eCardiac MDA levels were measured using thiobarbituric acid method by a commercial kit (#A003-4-1, Jiancheng) according to the manufacturer\\u0026apos;s instructions.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTTC staining.\\u0026nbsp;\\u003c/strong\\u003ePigs were euthanized intravenously with high potassium solution and fresh heart tissue samples were obtained. The fixed cardiac tissue samples were cut into consecutive 5-mm slices. The porcine heart slices were immersed in 2% tetraphenyl tetrazolium chloride (TTC) (Solarbio, G3005) at 37 \\u0026deg;C for 30 min. The infarcted area (TTC negative, white) was then observed. The larger the white area, the more infarcted zone the hearts would be. Statistical analysis was performed based on the recorded staining results.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eQuantitative real time PCR.\\u003c/strong\\u003e Total RNA was isolated from tissues or cells using Trizol (Pufei), and RNA concentration and purity were measured using a spectrophotometer. RNA was reverse transcribed using the PrimeScript RT reagent Kit (Takara) in accordance with the manufacturer\\u0026rsquo;s instructions, and quantitative PCR was performed using a CFX96 Real Time Syst m (Bio-Rad) with SYBR Green Supermix (Bio-Rad) in accordance with the manufacturer s instructions. The recommended thermal protocol consisted of an initial denaturation at 95\\u0026deg;C for 3 min, followed by 39 cycles of denaturation at 95\\u0026deg;C for 15s, annealing at 60\\u0026deg;C for 20 s and extension at 72\\u0026deg;C for 30s. The fold difference in gene expression was calculated using the 2-\\u0026Delta;\\u0026Delta;Ct method and is presented relative to Gapdh mRNA. All reactions were performed in triplicate, and specificity was monitored using melting curve analysis. The primers are listed in Table 1 of \\u003cem\\u003eSupplemental materials\\u003c/em\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eWestern blot analysis.\\u0026nbsp;\\u003c/strong\\u003eTotal proteins were extracted from the tissues by homogenization in RIPA buffer containing protease inhibitors. The homogenate was cleared by centrifugation at 4\\u0026deg;C for 30 min at 12,000 rpm, and the supernatant (containing the protein fraction) was collected. Protein concentration in the supernatant was measured using the BCA Protein Assay Kit (Beyotime). A total of 20 mg protein per sample was resolved in a 10-12% SDS PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% (w/v) BSA in Tris buffered saline containing 0.2% ween 20, and then incubated at 4\\u0026deg;C overnight with the following antibodies: anti-Ftl (1:1000; Abcam, ab69090) anti-Hmox1 (1:1000; Abcam, ab13243) and anti-Gapdh (1:10000; 60004-1, Proteintech). The membranes were then washed and probed with the appropriate horseradish peroxidase‒conjugated secondary antibodies (1:4000; Proteintech) and detected using the Pierce ECL System (Thermo Scientific).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTranscriptomics and lipid metabonomics study.\\u0026nbsp;\\u003c/strong\\u003eFor the transcriptomics study, total RNA was extracted using Trizol and quality checked with Bioanalyzer 2100. mRNA was purified with two rounds of Dynabeads Oligo(dT). Fragmentation was performed using Magnesium RNA Fragmentation Module at 94\\u0026deg;C. Reverse transcription and second strand synthesis prepared cDNA, which was A-tailed and ligated to dual index adapters. Size selection utilized AMPureXP beads. UDG enzyme treatment preceded PCR amplification and 2\\u0026times;150bp PE150 sequencing with Illumina Novaseq 6000. Genes with P\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 and absolute fold changes\\u0026thinsp;\\u0026ge;\\u0026thinsp;1.5 were identified as differentially expressed genes. Principal component analysis (PCA) was performed with princomp function of R (http://www.r-project.org/) in this experience. GO enrichment analysis of differentially expressed genes and KEGG pathway enrichment analysis were performed by using the cluster Profiler R package.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eFor the lipid metabonomics study, LV tissues were collected according to the manufacturer\\u0026rsquo;s instructions, and the sample extracts were analysed using an ultra-high performance liquid chromatography high-resolution tandem mass spectrometry (ThermoFisher Ultimate 3000 UHPLC; ThermoFisher Q Exactive\\u0026trade; Hybrid Quadrupole-Orbitrap\\u0026trade; Mass Spectrometry). Metabolite quantification and further analysis were performed using a multiple reaction monitoring method. Lipid metabolites with p\\u0026lt;\\u0026thinsp;0.05 and fold change\\u0026thinsp;\\u0026gt;\\u0026thinsp;1.5 were deemed to be significant.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eHistological assessments and immunostaining.\\u0026nbsp;\\u003c/strong\\u003eThe hearts were fixed in 4% paraformaldehyde overnight at room temperature, embedded in paraffin and consecutively sectioned into 18\\u0026ndash;20 sections on the short axis at a 500-\\u0026mu;m interval. DAB-enhanced Perl\\u0026rsquo;s prussian blue, masson trichrome and picrosirius red staining were performed in accordance with standard procedures. The serial sections were then examined with a digital serial section scanner (VS200, Olympus). After Masson\\u0026rsquo;s trichrome staining, fibrotic tissue (%) was calculated by the following formula: (total fibrotic area/total LV circumference area) \\u0026times; 100%, and the wall thickness of the scarred tissue at the apical and middle slices was measured as well. Data were analyzed using Fiji (ImageJ). For assessment of the fibrillar collagen subtype by picrosirius red staining, the sections were imaged under polarized light. Immunohistochemistry was performed to assess HBB and 4-HNE levels. Immunofluorescence was performed to assess HBB, HMOX1, Ferritin-L, CD31, \\u0026alpha;-SMA, cTNT and Cx43 levels; the images were captured and analyzed by LCM and Fiji.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTUNEL assay.\\u0026nbsp;\\u003c/strong\\u003eThe TUNEL assay was performed with a TUNEL Apoptosis Assay Kit (Beyotime, China) in accordance with the instructions of the manufacturer.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEchocardiography.\\u003c/strong\\u003e Cardiac functions at 35 d post I/R were assessed using echocardiography (VisualSonics, Canada), M-mode echocardiographic and two-dimensional images in a parasternal short and long axis were recorded. Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-diastolic volume (EDV), left ventricular end-systolic volume (ESV) were calculated as previously described\\u003csup\\u003e45\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eIn vivo\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;systemic toxicity experiments.\\u0026nbsp;\\u003c/strong\\u003eAfter the rat and mini-pigs were killed, the other main organs (liver, kidney, lung and spleen) were collected for H\\u0026amp;E staining, Perl\\u0026rsquo;s prussian blue and immunohistochemical staining of HBB to evaluate systematic pathological changes. The serum was extracted to assess other serum enzymes, including ALT, CRE-J, UREA and UA, were measured using an automatic biochemical analyzer (Sysmex). The levels of serum enzymes were assayed according to the instructions provided with the corresponding kits.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"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\":\"protein biomaterials, ferroptosis, hydrogel, ischemia/reperfusion injury, protein modification \",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4467590/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4467590/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eMyocardial ischemia/reperfusion injury with a high incidence of intramyocardial haemorrhage (IMH) contributes to enlarged infarct size by inducing additional cell death and predisposes to risk of heart failure. However, the risk factor in blood remains unverified and unaddressed. Here, we report that haem burstly released from IMH, is the key iron source for ferroptosis, and correspondingly propose the treatment strategy of blocking the cellular uptake of exogenous haem. Unfortunately, there is no existing haem-scavenging materials. We discover that methacryloyl modification of lysine residues on apo-lactoferrin (Apo-Lf), a milk-derived protein screen from natural haem-binding candidates, surprisingly increased the number of haem-binding sites by 86% and binding affinity by one order of magnitude. In animal models, intramyocardially implanted ferroptosis-inhibiting lactoferrin microsponges (FILMS) fabricated from the modified Apo-Lf achieved desirable anti-ferroptosis effects by rapid haem scavenging. Transcatheter FILMS implantation in pigs further demonstrated its safety and translational potential. These results provide deeper mechanistic understanding of ferroptosis-induced I/R injury, and may aid the development of other biomaterial-based therapies.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Milk-derived haem scavenging microsponges protect heart against ferroptosis-induced reperfusion injury\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-06-20 12:26:46\",\"doi\":\"10.21203/rs.3.rs-4467590/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\":\"136ef777-013c-451d-8172-37c5ca5aa4fb\",\"owner\":[],\"postedDate\":\"June 20th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":32967516,\"name\":\"Physical sciences/Materials science/Biomaterials/Biomaterials \\u0026#x2013; proteins\"},{\"id\":32967517,\"name\":\"Biological sciences/Biotechnology/Biomaterials/Biomaterials \\u0026#x2013; proteins\"}],\"tags\":[],\"updatedAt\":\"2024-06-20T12:26:48+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-06-20 12:26:46\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4467590\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4467590\",\"identity\":\"rs-4467590\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}