Photodynamic metabolite-powered zero-waste “ferroptosis amplifier” for enhanced hypertrophic scar therapy

Photodynamic metabolite-powered zero-waste “ferroptosis amplifier” for enhanced hypertrophic scar therapy | 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 Photodynamic metabolite-powered zero-waste “ferroptosis amplifier” for enhanced hypertrophic scar therapy Tao Chen, Yuan Chen, Shan Wang, Xiu Mao, Yao Wen, Xingyu Zhu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4498276/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Hypertrophic scar (HS) is a somatopsychic disease that significantly affects quality of life. 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) shows promise for HS treatment, while challenges like poor transdermal delivery and the accumulation of photodynamic by-products restrict its effectiveness. Inspired by the natural phenomenon that a whale fall brings life to thousands, this study proposes a zero-waste strategy by leveraging the photodynamic metabolite heme to establish a “ferroptosis amplifier”, which allows these metabolic wastes to be transformed into new sources of energy, thereby amplifying ferroptosis response following PDT. This is achieved by encapsulating 5-ALA and baicalin within human H-ferritin (HFn), subsequently incorporated into polyvinylpyrrolidone (PVP) microneedles (FAB@MN). The FAB@MN exhibits excellent targeting towards hypertrophic scar fibroblasts (HSFs) and pH-responsive programmed drug release. The treatment begins with the release of 5-ALA, which is converted into PpIX to activate PDT. Baicalin is then released, which directly triggers ferroptosis while also facilitating the breakdown of photodynamic waste heme into Fe 2+ and CO, thereby amplifying ferroptosis. Unlike conventional PDT only focuses on immediate effects, this approach uses photodynamic waste to fuel a sustained ferroptosis response after PDT, offering a new path for treatment. Biological sciences/Biotechnology/Biomaterials/Bioinspired materials Biological sciences/Biological techniques/Nanobiotechnology/Nanoparticles ferroptosis hypertrophic scars mitophagy photodynamic therapy photodynamic waste recycle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hypertrophic scar (HS), characterized by excessive proliferation of fibroblasts and over-deposition of extracellular matrix, has been regarded as a complex skin disorder, imposing substantial social and financial burdens 1 – 3 . Especially for scars on the face, they not only cause various physical discomforts, such as itching and pain, but also lead to anatomical deformities, severely hindering the patient's mental health and social interactions, affecting the quality of life 4 , 5 . The Food and Drug Administration (FDA) has approved photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) for HS treatment 6 . As a precursor of photosensitizer, 5-ALA is metabolically converted to protoporphyrin IX (PpIX), which generates abundant reactive oxygen species (ROS) for hypertrophic scar fibroblasts (HSFs) killing upon light exposure. However, PpIX further metabolizes into free heme, which not only leads to the loss of photosensitizer but may also induce toxicity to normal tissues 7 . For the past few decades, 5-ALA-generated heme has been considered a useless metabolic waste. Despite attempts to reduce its production, the effects have been minimal 8 – 10 . From the perspective of natural material cycle, even waste materials can provide energy for new life processes under suitable conditions. This is evidenced by the phenomenon of whale falls in nature: when a giant whale dies and sinks to the seabed, its body becomes a rich food source for deep-sea organisms, thus promoting the flourishing development of life in the ecosystem. Inspired by this natural phenomenon of "a whale fall brings life to thousands," we are exploring the possibility of turning these useless heme into potential "auxiliary energy source" to amplify the therapeutic efficacy of PDT. Numerous studies have demonstrated heme can be degrade into Fe 2+ and CO through the action of heme oxygenase-1 (HO-1), thereby inducing iron overload and ferroptosis 11 , 12 . Coincidentally, it has been observed that HSFs were naturally addicted to iron, suggesting ferroptosis as a potential alternative treatment 13 . Inspired by this, 5-ALA-generated heme may become a potential energy source for ferroptosis, amplifying the therapeutic effect for HS. However, due to the limited HO-1 quantity within cells, catalyzing the excessive accumulation of heme to facilitate the process of ferroptosis presents a significant challenge. Therefore, efforts to improve the expression of HO-1 and optimize the utilization of photodynamic waste are critical for establishing an efficient ferroptosis bioreactor post-PDT. Baicalin is a flavonoid extracted from the root of scutellaria baicalensis. As one of the oldest medicinal plants first recorded in Shennong Bencaojing, a Chinese ancient pharmacopeia, baicalin has been widely used to treat inflammation, infection and cancer for centuries 14 – 16 . Recent studies have reported that baicalin can significantly promote the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), which in turn upregulated the expression levels of HO-1 17–18 . These findings suggest the co-delivery of 5-ALA and baicalin could potentially accelerate the biotransformation of useless heme, thereby promoting ferroptosis in HSFs. Moreover, further investigations have revealed baicalin's inherent capacity to induce ferroptosis in cancer treatments 19 . This dual-action of baicalin, coupled with the generated heme, may form a continuous ferroptosis bioreactor within HSFs. Compared to other ferroptosis inducers such as erastin, baicalin is more affordable and exhibits lower toxicity to normal tissues. However, its limited water solubility, and non-specific targeting severely restrict its clinical application 20 , 21 . Recently, nanotechnology has been applied to overcome the poor bioavailability of hydrophobic drugs and enhance their therapeutic efficiency 22 , 23 . Of note, human H-ferritin (HFn) has garnered significant interests, due to its excellent drug loading capacity and intrinsic targeting ability to transferrin receptor 1 (TfR1), which is highly expressed in most tumor cell 24 – 26 . Moreover, TFR1 has been found to be overexpressed on HSFs recently, suggesting that HFn-based delivery strategies may be useful for targeted therapies against HS 13 . Herein, a zero-waste “ferroptosis amplifier” has been constructed by co-encapsulating 5-ALA and baicalin into a HFn nanocage (FAB NPs). To improve transdermal delivery efficacy in vivo , these FAB NPs were further integrated with polyvinylpyrrolidone (PVP) microneedles (FAB@MNs), providing a painless and convenient method of application. On one hand, after traversing the epidermal barrier, FAB@MNs can target HSFs through the TFR1-HFn interaction, thereby enhancing drug delivery and retention within cells. On the other hand, a synergistic therapy for HS can be implemented by PDT and ferroptosis based on multistage cascade metabolites. Specifically, the 5-ALA first biotransformed into PpIX to initiate PDT. Subsequently, the baicalin was released to trigger ferroptosis. Meanwhile, the baicalin could also serve as an HO-1 activator to facilitate the breakdown of photodynamic waste heme into Fe 2+ and CO, which contributed to the amplification of the ferroptosis process. Unlike traditional approaches that solely focus on the immediate effects of PDT, this strategy emphasizes transforming photodynamic waste into an “auxiliary energy source” to trigger the subsequent ferroptosis cascade. Just as every whale fall in the ocean heralds the abundance of new life, each short-term session of PDT powers a continuous ferroptosis bioreaction, thereby allowing FAB@MN to realize significant anti-scarring outcomes following a single laser irradiation. Despite the potential for further optimization, we anticipate more breakthroughs in this field, potentially bringing revolutionary changes to HS treatments. Importantly, such a “waste to energy” strategy not only reflects the spirit of scientific exploration inspired by nature but also introduces a new perspective on utilizing photodynamic waste to enhance the synergistic efficacy of PDT and ferroptosis, opening up a new path for treating other similar diseases. Scheme 1. Schematic illustration of FAB@MN on HS treatment. A Schematic illustration of the fabrication process of a FAB@MN patch. B The targeting of FAB@MN for HSFs. C Scheme illustrating the formation of a ferroptosis amplifier utilizing photodynamic waste. D Schematic illustration of the treatment process of FAB@MN for HS. Results and Discussion Fabrication and characteristics of FAB@MN Herein, we reported a one-pot method to co-encapsulate 5-ALA and baicalin within the hollow core of the HFn nanocage (FAB NP), which was subsequently incorporated into PVP MN patches (Fig. 1 A). Specifically, 5-ALA and baicalin were mixed with a solution of HFn, and then the mixture was heated to 60 ℃. It has been demonstrated that hyperthermia can expand the channels of HFn, thereby facilitating the encapsulation of small molecules without damaging the protein structures 24 , 27 . Transmission electron microscopy (TEM), dynamic light scattering (DLS), and Zeta potential analysis were performed to characterize HFn and the newly synthesized FAB NPs. As shown in Fig. 1 B, HFn exhibited a typical ring-like structure with a hollow core in the center, whereas FAB NPs presented as solid spheres, possibly due to the filling of 5-ALA and baicalin. Notably, the dual encapsulation of 5-ALA and baicalin did not alter the high mono-dispersity and good colloidal stability of HFn, which was crucial for its potential in biomedical treatments (Fig. 1 B, C). Moreover, the solution of FAB NPs exhibited a clear orange-yellow color, indicating successful encapsulation of baicalin and its transformation from a water-insoluble form into a water-soluble one (Fig. 1 C and Figure S1 ). According to the DLS data, the average diameters of HFn and FAB NPs were 10.11 nm and 11.68 nm, respectively (Fig. 1 D). The slight increase in size of FAB NPs compared to HFn may also be attributed to the drug encapsulation within HFn. Additionally, the Zeta potential remained negative both before and after drug loading with no significant differences, suggesting that the drugs were encapsulated within the HFn nanocage rather than being adsorbed onto the shell (Fig. 1 D). The ultraviolet-visible (UV-Vis) absorption spectra confirmed the successful drug encapsulation within HFn, with characteristic absorption of 5-ALA and baicalin respectively (Fig. 1 E). And high-performance liquid chromatography (HPLC) analysis determined the encapsulation efficiencies of 54.92% for 5-ALA and 37.6% for baicalin. To confirm that the drug loading process did not affect the structure of HFn, comprehensive analyses including native polyacrylamide gel electrophoresis (Native-PAGE), circular dichroism spectrum (CD) and size-exclusion chromatography (SEC) were performed. As shown in Fig. 1 F, the FAB NPs appeared as a ≈ 440 kDa protein in Native-PAGE, similar to that of HFn. And the CD spectra and SEC elution profiles of FAB NPs also closely matched those of HFn, indicating the secondary structure and oligomerization states of HFn remained unchanged after drug encapsulation (Fig. 1 G (i-ii)). The stability of FAB NPs in cell culture media was also assessed, revealing that no significant change in size occurred over a 48-hour period under physiological conditions (Figure S2). To facilitate the in-vivo biomedical applications, we developed a percutaneous delivery system for FAB NPs utilizing PVP MNs (Fig. 1 H). Specifically, a 20% PVP solution containing FAB NPs was cast into a poly-dimethylsiloxane (PDMS) mold to dry overnight. The optical images demonstrated that the fabricated MN patch consisted of an array of 225 needles spread over a 14 × 14 mm² area, and the patch turned slightly yellow after loading with FAB NPs (Fig. 1 I). The scanning electron microscopy (SEM) further revealed that each MN was pyramid-shaped, with 340 µm in diameter at the base and 800 µm in height (Fig. 1 K). Additionally, the mechanical hardness of the fabricated FAB@MN was evaluated, showing a force of 0.11N/needle which was sufficient for skin penetration (Fig. 1 J). Intriguingly, the mechanical strength of FAB@MN was found to be higher than that of the blank PVP MN, possibly due to the hydrogen bonding and electrostatic interactions between the proteins and polymers. The penetration ability of FAB@MN was further demonstrated on rabbit ear HS models, which showed regularly spaced perforations in the skin post-application (Fig. 1 L). And the fluorescence staining images revealed these MNs successfully penetrated the epidermal layer, capable of delivering RhB-labeled FAB NPs to the deeper dermal layer (Fig. 1 M (i-ii)). Targeting ability and programmable release behavior of FAB NPs Although various nanodrugs have been explored to eliminate HSFs, the development of their targeting capabilities has often been neglected. Recently, the HFn protein drug delivery system has been widely used in cancer treatments, becoming a hotspot in the field of nanomedicine 24 , 28 . Researches have shown the HFn not only possessed basic characteristics of protein-based nanoparticles, but also displayed notable features including excellent drug loading capacity, high targeting specificity towards TFR1, and acid-responsive release 29 – 31 . In this study, HFn was used to encapsulate 5-ALA and baicalin, aiming to optimize the therapeutic effects of free drugs while simultaneously reducing damage to normal tissues. To gain deeper insights into the therapeutic potential of FAB NPs for HS, their targeting ability and release behavior were meticulously characterized. The strong and stable molecular interaction was crucial to ensuring the high targeting specificity of FAB NPs. In this regard, dual molecular blind docking studies were carried out to delve into the complex molecular interactions of FAB NPs, with 5-ALA and baicalin serving as ligands to co-interact with HFn (PDB ID: 7CK9). As shown in Fig. 2 A, the analysis revealed a significant interaction between 5-ALA, baicalin and HFn, mediated through the formation of fourteen contacts involving five hydrogen bonds (GLN58, LYS143, GLU61, ASN109, GLU116), seven van der waals (ALA144, GLU147, TYR54, GLU140, ASN25, GLN83, SER113) and two pi-alkyl interactions (ARG22, LEU26). And the binding energies for 5-ALA and baicalin with HFn were calculated as -4.4 and − 9.4 kcal·mol − 1 respectively, indicating that the resulting complex structures were stable, laying a solid foundation for FAB NPs to exhibit targeting ability similar to HFn. Numerous studies have utilized HFn to target the overexpressed TFR1 in tumor cells, yielding favorable therapeutic outcomes 24 , 31 . Inspired by this, we examined the expression of TFR1 in HSFs to explore the possibility of HFn-mediated targeted therapy for HS. According to the results from western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR), the TfR1 level in HSFs was significantly higher than that in normal fibroblasts (Fig. 2 B(i-iii) and Figure S3). And the immunohistochemical staining of HS samples also demonstrated the increased TFR1 levels in cells, which potentially offered more binding sites for FAB NPs (Figure S4). To verify the targeting ability of FAB NPs towards HSFs, we constructed RhB-labeled FAB NPs and co-incubated them with normal fibroblasts and HSFs. As shown in Fig. 3B (iv) and Figure S5, HSFs had an eightfold higher fluorescence intensity than normal fibroblasts, highlighting FAB NPs' superior targeting efficacy to HSFs. Flow cytometry also confirmed these findings, with results consistent with the observed fluorescence patterns (Figure S6). As many drugs proven effective in 2D cell cultures lose their efficacy in dense scar tissues in vivo , we further investigated the deep penetration abilities of FAB NPs in 3D scarring spheroid models using confocal laser scanning microscopy (CLSM) (Fig. 2 C (i)). As show in Fig. 2 C (ii), the spheroids treated with free 5-ALA and baicalin remained relative dim, with drugs predominantly accumulating at the periphery. In sharp contrast, spheroids incubated with FAB NPs exhibited widespread fluorescence throughout the entire structure. The fluorescence intensity of treated spheroids was further quantified by 3D surface plots, revealing obvious fluorescence signals even reaching the core of spheroids after incubation with the FAB NPs (Fig. 2 C(iii)). These results suggested that the strong targeting ability of FAB NPs facilitated drug penetration into dense scars, potentially further enhancing the therapeutic efficacy in vivo . Considering the excellent targeting and penetration abilities exhibited by FAB NPs in both 2D and 3D scar models, we proceeded to assess the specific cytotoxicity of FAB NPs against HSFs. For that, normal fibroblasts and HSFs were exposed to various concentrations of FAB NPs, and the CCK8 assay indicated that FAB NPs displayed significantly higher cytotoxicity towards HSFs, further affirming their outstanding selective therapeutic effect on HS (Figure S7). The subcellular location and release behavior of FAB NPs were also explored. As shown in Fig. 2 D (i) and Figure S8, a large portion of FAB NPs colocalized with LysoTracker (a typical marker of lysosome) after internalization. Recent studies have demonstrated the acidic-responsive dissociation property of HFn, which suggested a great potential for FAB NPs to release 5-ALA and baicalin in acidic compartments of lysosomes, thus minimizing premature leakage during administration 24 , 32 . Following this, the release behavior of FAB NPs was examined under lysosome-mimicking conditions in vitro . As expected, a complete release of 5-ALA and baicalin was observed after 24 hours of incubation at pH 5.0, significantly faster than that at pH 7.4. Moreover, notable discrepancies were observed in the release rates of 5-ALA and baicalin, particularly within the initial 4 hours, with approximately 81.41% of 5-ALA and 53.87% of baicalin released (Fig. 2 D (ii)). To gain a deeper insight into the mechanisms behind the differential release behavior of the two drugs, we conducted molecular dynamics simulations and computed the free energy landscape (FEL) and free binding energy for each drug-protein interaction (Fig. 3l). In cases where the protein-ligand interaction was weak or unstable, the FEL exhibited multiple minima clusters. Conversely, strong and stable interactions resulted in nearly single and smooth energy clusters in the potential energy distribution. As shown in Fig. 2 D (iii), the 5-ALA-HFn complex had four small energy clusters, whereas the baicalin-HFn complex exhibited only a single concentrated energy cluster, indicating the binding affinity between HFn and baicalin was stronger. Additionally, the binding free energy of 5-ALA-HFn was calculated as -10.68 kcal·mol − 1 , higher than that of baicalin-HFn at -42.06 kcal·mol − 1 . A lower binding free energy often indicated a stronger binding affinity, which may explain why baicalin was released more slowly than 5-ALA from the FAB NP (Figure S9). The diagram in Fig. 2 E summarized the therapeutic strategies and characteristics of FAB NPs for treating HSFs. Initially, the fabricated FAB NPs can selectively bind to the significantly overexpressed TFR1 on the surface of HSFs, thereby achieving precise drug localization and reducing the side effects on surrounding tissues. Once internalized by the cells, FAB NPs were degraded within the lysosomal acidic environment, initiating a programmable sequential release of encapsulated agents. Specifically, the rapid release of 5-ALA quickly initiated PDT, laying the groundwork for early treatment, whereas the slow release of baicalin provided lasting support for the therapy. This sequential release pattern allowed for a programmable approach to treating HS, significantly improving the precision and efficiency of the therapy. Enhanced anti-scarring effects through FAB@MN-mediated photodynamic waste reutilization 5-ALA served as a precursor of photosensitizer PpIX, demonstrating notable photodynamic activity against HSFs 6 , 33 . However, a significant challenge arose as PpIX was quickly metabolized into non-photosensitive heme, which significantly diminished its ability to produce ROS 7 , 10 . Currently, various strategies have focused on promoting immediate PpIX accumulation during PDT, while overlooking the negative metabolic impacts of PpIX conversion to heme and the potential to reutilize photodynamic waste 8 , 9 , 34 , 35 . Inspired by the energy cycling phenomenon of whale falls, we developed a “waste to energy” strategy by integrating 5-ALA and baicalin into a single nanotherapeutic platform to fully reutilize photodynamic waste heme for enhanced anti-scarring therapy. In this strategy, FAB@MN initially released 5-ALA to biosynthesize PpIX, which can be transformed into heme. And then, the released baicalin boosted the expression of HO-1, significantly accelerating the decomposition of heme into therapeutically beneficial Fe 2+ , CO and biliverdin. Through this method, we transformed the "end" metabolic waste into a new energy against HSFs, thereby enhancing the anti-scarring effects. To explore the effects of FAB@MN on PpIX metabolism regulation and subsequent heme utilization, we compared the main upstream and downstream metabolites within the 5-ALA-PpIX-heme-Fe 2+ /CO/ biliverdin metabolic axis among different groups. By employing Enzyme-Linked Immunosorbent Assay (ELISA) and fluorescence imaging techniques, the levels of PpIX and heme were initially quantified. As shown in Figure S10, both FA@MN and FAB@MN groups displayed a similar elevation in PpIX levels, indicating the successful delivery of 5-ALA and equivalent photodynamic activity of FA@MN and FAB@MN. However, the levels of heme, a downstream metabolite of PpIX, revealed significant differences between the two groups. In the FA@MN group, heme was significantly increased compared to the control group, while changes in the FAB@MN group were minimal, suggesting that FAB@MN may reduce the synthesis of heme or promote its degradation (Fig. 3A). Following this clue, we further tracked the decomposition products of heme, including Fe 2+ , CO and biliverdin, to gain a deeper understanding of the underlying mechanism by which FAB@MN acted upon heme. As depicted in Fig. 3B and 3C, compared to the FA@MN group which displayed almost no fluorescence for Fe 2+ and CO, the FAB@MN group showed pronounced fluorescence signals, confirming the role of baicalin in promoting heme degradation. However, the fluorescence of biliverdin in the FAB@MN group did not differ from other groups, possibly due to the presence of abundant biliverdin reductase enzymes in the human body that facilitated biliverdin’s rapid degradation (Figure S11) 7 , 36 . Additionally, the FB@MN group also exhibited minor Fe 2+ signals but lacked any CO signals, which may result from the independent ferroptosis effect of baicalin, aligning with the findings of prior research 19 . Notably, the introduction of HO-1 inhibitors significantly reversed the observed increase in CO and Fe 2+ within the FAB@MN group, further implying the close correlation between the enhanced heme decomposition activity and HO-1 proteins. As the rate-limiting enzymes catalyzing oxidative degradation of heme, HO-1 has been regarded as an important regulator in the 5-ALA-PpIX-heme- Fe 2+ /CO/ biliverdin metabolic axis 37 , 38 . Extensive mechanistic studies have revealed that HO-1 gene responded to positive regulation by Nrf2, we therefore examined the expression of Nrf2 and HO-1 in HSFs to figure out the exact molecular mechanisms of FAB@MN 39 , 40 . As shown in Fig. 3D and 3E, the FAB@MN significantly increased endogenous Nrf2 expression, while this increase up-reregulated the expression of HO-1 at protein levels. Conversely, there was no obvious change in the expression level of Nrf2 and HO-1 in the FA@MN group, underscoring baicalin as the critical element enhancing the activity of the Nrf2/HO-1 pathway. To better understand the interactions between baicalin and Nrf2/HO-1 signaling axis at the atomic level, molecular docking studies were subsequently conducted. As shown in Fig. 3F, baicalin formed hydrogen bond interactions with multiple residues of Nrf2 such as ARG-456, ASN-507 and GLN-26, showing a robust binding affinity of -8.8 kcal·mol − 1 . These findings suggested that baicalin can directly target the inner cavity of Nrf2, activating the Nrf2/HO-1 signaling pathway and promoting heme degradation. From the results above, it was confirmed that FAB@MN can decompose photodynamic waste heme into an auxiliary energy source under the action of baicalin. This “waste to energy” process was similar to how a whale fall bring life to thousands, suggesting the potential to amplify anti-scarring effects after PDT. We, therefore, sought to validate the hypothesis. As indicated by CCK-8 assay, the relative cell viability in FA@MN and FB@MN groups was reduced to 72.2% and 61.9% respectively, implying poor therapeutic efficacy of PDT and baicalin-mediated killing effects (Fig. 3H). In contrast, only 13.8% of HSFs survived in the FAB@MN group after a single irradiation, which was far greater than the cumulative effect of FA@MN and FB@MN. The flow cytometry analysis also showed the highest apoptosis rate in FAB@MN group, further confirming the enhanced therapeutic effects of FAB@MN (Fig. 3G). Building on these findings, it can be concluded that FAB@MN not only generated the photosensitizer PpIX to initiate PDT under irradiation, but also upregulated HO-1 to convert heme into a significant amount of Fe 2+ and CO (Fig. 3I). This chemical reaction storm has been demonstrated to enhance the anti-scarring effects of PDT, truly achieving the conversion of waste into treasure. Figure 3. Biosynthesis of therapeutic metabolites in HSFs following treatment with FAB@MN. A Quantitative analysis of intracellular heme in different groups by Elisa kits. B,C Fluorescence images and the corresponding 3D surface plot of (B) CO and (C) Fe 2+ in HSFs with various treatments. D Molecular docking visualization results of baicalin with HO-1 protein. E Western blot and F the corresponding quantitative analysis of Nrf2 and HO-1 proteins in different groups (n = 3). G Cell apoptosis after being treated with different materials by flow cytometry. H Statistical analysis of the cell viability of HSFs (n = 3). I Scheme illustrating FAB NPs enhanced their cytotoxic effect by recycling photodynamic waste heme. FAB@MN-induced continuous ferroptosis in HSFs Ferroptosis was widely recognized as a new form of programmed cell death, closely associated with the accumulation of Fe 2+ and lipid peroxidation (LPO) 41 , 42 . Recent studies have shown that the heme decomposition products, specifically Fe 2+ and CO, have the potential to promote ferroptosis 43 , 44 . Based on this, we speculated that the FAB@MN-mediated reutilization of photodynamic waste heme could amplify the ferroptosis process, thereby enhancing killing effects on HSFs. Furthermore, studies have indicated that baicalin not only acted as a promoter of HO-1 to assist in the decomposition of heme but may also exert an independent ferroptosis effect, and this dual-action effect may help FAB@MN to provide dual assurance for continuous ferroptosis (Fig. 4 A). To clarify the role of ferroptosis in the action of FAB@MN, we introduced deferoxamine (DFO) as a ferroptosis inhibitor in cell viability assays. As shown, the cell viability in the FAB@MN group recovered from 14.9–76.2%, which meant ferroptosis played a dominant role in FAB@MN-induced cell death independent of PDT (Figure S12). According to our strategy, FAB@MN transformed photodynamic waste heme into a chemical storm of Fe 2+ and CO. This cascade, in conjunction with baicalin's inherent ferroptosis potential, was anticipated to initiate robust and sustained ferroptosis in HSFs. To demonstrate it, we investigated several ferroptosis biomarkers across different groups. As shown in Figure S13 and S14, the cells treated with FAB@MN had the highest levels of ROS, which was partly due to the PDT induced by PpIX, and partly a result of continuous ferroptosis. Additionally, FAB@MN-treated HSFs exhibited the highest intracellular iron content, which was 2.44-fold, 2.61-fold and 1.84-fold higher than those observed in Control, FA@MN and FB@MN groups (Fig. 4 B). While the level of antioxidants such as GSH dropped to 39.43% of the control group after treatment with FAB@MN, significantly lower than those in other treatment groups (Fig. 4 C). Furthermore, upon evaluating changes in intracellular LPO with the C11-BODIPY591/581 probe, it was found the green fluorescence intensity in HSFs treated with FAB@MN was higher than that in the FB@MN group, indicating increased LPO production through the biotransformation of heme post-PDT (Fig. 5E and Figure S14). And the end product of lethal LPO, intracellular malondialdehyde (MDA), was also increased by 1241.18% in FAB@MN-treated HSFs, far exceeding that in other groups (Fig. 4 D). It was noteworthy that the use of the iron chelator DFO can significantly reverse the aforementioned abnormal ferroptosis biomarkers in the FAB@MN group, further confirming that FAB@MN promoted ferroptosis in HSFs. Besides, the expression levels of two typical negative regulators against ferroptosis, xCT and GPX4, were also evaluated. The dysfunction of xCT led to the inactivation of GPX4, resulting in the legitimate accumulation of LPO and ultimately triggering ferroptosis 32 . As shown in Fig. 4 F- 4 H, the protein and gene expression levels of GPX4 and xCT in FAB@MN group were much lower than those in FB@MN group, evidencing the augmented ferroptosis caused by the reutilization of heme. In addition to the Fe 2+ produced from heme degradation directly promoting ferroptosis, studies have also shown that the generated CO can also induce cellular ferroptosis, which mechanism involved downregulating the activity of GPX4 and increasing the concentration of ROS 45 . Furthermore, mRNA expression of additional ferroptosis-promoting genes, including ACSL4, FTH1, and TFR1, was significantly higher in the FAB@MN group, compared to the control group and others (Fig. 4 H). As previously reported, mitochondrial dysfunction was closely involved in ferroptosis. Therefore, we investigated the mitochondrial structure and mitochondrial membrane potential (MMP) of HSFs treated by different materials. Initially, JC-1 probe was utilized as the fluorescent indictor for MMP detection. As depicted in Fig. 4 I, FAB@MN-treated HSFs exhibited the brightest green fluorescence and weakest red fluorescence, revealing FAB@MN caused the most severe MMP depolarization within cells. In addition, according to the flow cytometry results, the intensity of TMRM fluorescence signal decreased the most in FAB@MN group, further supporting the significant depolarization of MMP induced by FAB@MN (Fig. 4 L). Subsequently, Mito-Tracker staining was employed to evaluate the mitochondrial structure. Unlike the elongated and interconnected mitochondria seen in the control group, the mitochondria of HSFs treated with FAB@MN were abnormally rod-like, spherical and fragmented (Fig. 4 J). Notably, all observed mitochondrial damage observed in the FAB@MN group could be reversed to levels comparable with the control group by co-treatment with DFO, conforming the mitochondrial damage in HSFs was induced by FAB@MN-mediated ferroptosis. Furthermore, alterations in mitochondrial ultrastructure were characterized by TEM. As results shown, the HSFs treated with FAB@MN experienced severe mitochondrial shrinkage with diminished crista, which was consistent with the characteristic mitochondrial structure observed in ferroptosis (Fig. 4 K) 13 . The mitochondrial number and length were further quantified with ImageJ. As expected, the FAB@MN-treated HSFs exhibited significant changes in mitochondrial structure, including an increase in the number of mitochondria and a reduction in branch length. However, these changes were reversed by the iron chelator DFO (Fig. 4 M and Figure S15). Collectively, by deeply delving into the potential of 5-ALA-generated heme in the process of ferroptosis, FAB@MN achieved sustained ferroptotic effects on HSFs, ultimately enhancing the anti-scarring efficacy of PDT. The superiority of this strategy lied in its ability to not only combine PDT and ferroptosis, but also to fully utilize the photodynamic waste as beneficial resources for ferroptosis, thereby amplifying therapeutic effects. Figure 4. FAB@MN-induced continuous ferroptosis in HSFs. A Schematic illustration to reveal the mechanism of photodynamic waste augmented ferroptosis in HSFs. B ICP-OES measurement of intracellular iron. C The GSH and D MDA levels in HSFs with different treatments. Error bars are means ±SD (n = 3). E Fluorescence images of intracellular LPO in different groups by staining with C11-BODIPY581/591 probe (scale bar: 50 μm). F Western blot analysis and G densitometric analysis of the expression of GPX4 and xCT proteins in HSFs treated with different interventions (n = 3). H Heatmap of expressed mRNA in different groups. I Fluorescent assessment of MMP level in HSFs using JC-1 fluorescent probe (scale bar: 50 μm). J Representative fluorescent images of mitochondrial morphology in HSFs using Mito-Tracker Deep Red fluorescent probe. Cell nuclei were stained by blue fluorescent Hoechst 33342 (scale bar: 10 μm). K TEM images of mitochondrial ultrastructure. Structures colored red indicate mitochondria (scale bar:1 μm and 500 nm). L Flow cytometry analysis of MMP in HSFs stained by TMRM probe. M Statistical data of mitochondrial individuals in HSFs based on fluorescent images with Mito-Tracker Deep Red probe (n = 3). Mechanism of FAB@MN-induced ferroptosis in HSFs In HSFs, FAB@MN acted as a persistent inducer of ferroptosis by utilizing photodynamical waste heme and the inherent ferroptosis property of baicalin. To further explore its underlying mechanisms of ferroptosis, mRNA-sequencing analysis was conducted on FAB@MN-treated HSFs. As shown in Fig. 5A, the volcano plot revealed a total of 2314 differentially expressed genes (DEGs) following the treatment with FAB@MN, of which 1364 DEGs were up-regulated and 1049 DEGs were down-regulated. According to gene ontology (GO) enrichment analysis, DEGs were significantly enriched in biological processes such as mitochondrial membrane permeability, stress response to metal ion, fatty acid oxidation and oxidative stress (Fig. 5B). Further, we conducted gene set enrichment analysis (GSEA) based on Kyoto Encyclopedia of Genes and Genomes (KEGG) database to reveal the changes in cellular signaling pathways of HSFs induced by FAB@MN. The results showed that HIF1, mitophagy and ferroptosis pathways were significantly activated, with genes such as HO-1, P62, xCT, and LC3B involved in these processes (Fig. 5C, D). Numerous studies have demonstrated that the activation of the HIF-1 pathway was also closely linked to ferroptosis and mitophagy, emphasizing their critical role in the damage to HSFs caused by FAB@MN 46 , 47 . Recently, as research into the mechanisms of ferroptosis and mitophagy has deepened, people have become increasingly aware of the potential close relationship between the two phenomena. In this study, we conducted protein-protein interaction network analysis (PPI) on DEGs enriched in both ferroptosis and mitophagy. As shown in Fig. 5E, the proteins encoded by genes mainly involved in ferroptosis and mitophagy exhibited extensive interactions, indicating a possible association between FAB@MN-induced ferroptosis and mitophagy. Existing researches have suggested that excessive activation of mitophagy may significantly increase the release of iron ions from the mitochondria 48 , 49 . These iron ions then participated in the LPO reaction chain, further exacerbating cell membrane damage and leading to ferroptosis. Based on this, we hypothesized that FAB@MN not only induced ferroptosis but also triggered mitophagy, and there might be a direct or indirect relationship between them. As shown in Fig. 5F (i), TEM analysis indicated FAB@MN significantly enhanced the formation of mitophagosomes in HSFs. And the fluorescence imaging also revealed a marked increase in colocalization between mitochondria (stained with Mito-Tracker) and lysosomes (stained with Lyso-Tracker), further supporting the enhancement of mitophagy activity (Fig. 5F (ii-iii)). To elucidate the molecular mechanism of FAB@MN-induced mitophagy, the expression level of several mitophagy-related proteins including LC3B, P62, PINK1 and Parkin were subsequently measured by immunoblotting assays (Fig. 5G). As expected, FAB@MN markedly upregulated the protein ratio of LC3B-II and LC3B-I, while decreasing P62 levels, indicating the formation of autophagosomes and occurrence of autophagy. Furthermore, the abundance of PINK1 and Parkin proteins also significantly increased, suggesting that FAB@MN activated mitophagy via classical PINK1/ Parkin pathway. As the key proteins that regulated mitophagy, PINK1 and Parkin interacted with each other to promote the recognition and clearance of damaged mitochondria 47 , 50 , 51 . Under normal conditions, PINK1 was rapidly degraded. However, when mitochondria were damaged, it accumulated and acted as a beacon to recruit Parkin from the cytosol to the surface of mitochondria. Subsequently, Parkin, endowed with E3 ubiquitin ligase activity, can tag these damaged mitochondria, thereby attracting a large amount of P62 to the mitochondrial outer membrane and promoting the ubiquitination of LC3B I. This process has been shown to ultimately lead to autophagosomes engulfing the damaged mitochondria, thus triggering mitophagy. Numerous studies have confirmed that excessive activation of mitophagy can promote the occurrence of ferroptosis 45 , 52 , 53 . To explore the effect of mitophagy on FAB@MN-induced ferroptosis, we employed the autophagy inhibitor chloroquine (CQ) to co-treat with HSFs. Surprisingly, it was found the CQ could significantly restore the cell viability of FAB@MN from 16.7–67.9% (Fig. 5H). Furthermore, CQ treatment notably counteracted the reduction of GPX4 and xCT proteins, and decreased the fluorescence signals of Fe 2+ and LPO in FAB@MN-treated HSFs (Fig. 5I-K). These observations suggested that ferroptosis induced by FAB@MN occurred in a mitophagy-dependent manner, which enhanced our comprehension of the anti-scarring effects of FAB@MN. Currently, the function of mitophagy remained controversial. For example, in neuronal cells, mitophagy cleared damaged mitochondria to maintain normal physiological conditions; however, mitophagy induced by ionizing radiation may promote ferroptosis 53 , 54 . In this study, FAB@MN excessively activated mitophagy through the PINK1/Parkin pathway, leading to the accumulation of Fe 2+ and LPO within cells, which ultimately triggered ferroptosis (Fig. 5L). This finding suggested that further research into the regulation of mitophagy could improve the therapeutic efficacy of FAB@MN for HS, and provided important theoretical support for the future application of FAB@MN in other related therapeutic areas. Figure 5. The mechanism of FAB@MN-induced ferroptosis in HSFs. A Volcano plot of DEGs in HSFs upon FAB@MN treatments. B GO functional analysis of DEGs in FAB@MN-treated HSFs. C KEGG pathway analysis of DEGs in HSFs upon FAB@MN treatments. D Circular visualization of the results of functional enrichment analysis. E PPI network of DEGs in HSFs upon FAB@MN treatments. F (i) TEM image of mitophagosomes in FAB@MN-treated HSFs. (ii) Representative fluorescence images of Mito-Tracker and Lyso-Tracker with Hoechst to show the formation of mitophagosomes in HSFs (scar bar: 5 µm and 1 µm). (iii) Scheme diagram of the formation of lysosome and mitochondria. G Western blot analysis of the expression of P62, LC3B, PINK1 and Parkin proteins in HSFs treated with different interventions. H Statistical analysis of the cell viability of HSFs (n = 3). I Western blot analysis of the expression of xCT and GPX4. J CLSM images of Fe 2+ upon different treatments (scar bar: 25 µm). K Fluorescence images of LPO stained with C11-BODIPY581/591 probe (scale bar: 50 µm). L Schematic of the ferroptosis mechanism induced by mitophagy. In vivo anti-scarring effects of FAB@MN Encouraged by the superior therapeutic efficacy of FAB@MN observed in vitro , we proceeded to monitor its anti-scarring activity in vivo . Specifically, we established rabbit ear HS models, and applied treatments once a week for a month (Fig. 6 A). As depicted in Fig. 6 B, the rabbits receiving PBS, FA@MN and FB@MN still exhibited typical HS features with dark-red surfaces and thick uplift after four-week treatments. However, in FAB@MN group, the HS appeared smooth and closely resembled surrounding normal skin. Ultrasound images further demonstrated that FAB@MN led to the most significant reduction in scar thickness, decreasing from 2.94 ± 0.38 to 0.46 ± 0.13 mm (Fig. 6 C and Figure S16). As predicted, FA@MN and FB@MN had limited therapeutic effects in vivo , indicating that short-term PDT or baicalin-induced ferroptosis alone was ineffective. In contrast, FAB@MN leveraged a series of photodynamic waste to establish a sustained bioreactor integrating PDT and ferroptosis, opening up new avenues to effectively treat and prevent scar recurrence. Histological analyses were further performed to assess the therapeutic outcomes of various treatments. Hematoxylin − eosin (HE) staining showed the HS treated with FAB@MN has returned to the smooth appearance of normal skin, whereas the other three groups still exhibited varying degrees of protuberance, accompanied by densely packed and disorganized muscle fibers (Fig. 6 D). And the results of Masson staining also demonstrated a noticeable reduction in collagen deposition at the HS site following treatment with FAB@MN (Fig. 6 E and Figure S16). Further examination of collagen fiber was conducted via Sirius red staining. Notably, the control, FA@MN and FB@MN groups exhibited abundant red-orange type I collagen densely distributed at the HS sites, while in contrast, the FAB@MN group displayed increased deposition of green-stained type III collagen fibers, suggesting the abnormal collagen was significantly remodeled by FAB@MN (Fig. 6 E). Besides, the GPX4 level in HS tissues was also evaluated to assess the ferroptosis effects via immunohistochemical staining. As shown in Fig. 6 E and Figure S16, FAB@MN notably reduced the area of GPX4-positive cells and exhibited a more effective therapeutic outcome than FB@MN, likely due to the ferroptosis cascade initiated by photodynamic waste. The biosafety of FAB@MN in vivo was verified by the HE staining of vital organs (major organs (heart, liver, spleen, lung, and kidney), as well as hematological and biochemical analyses. It was observed no significant organ damage occurred in FAB@MN group, and all blood parameters were within the normal limits after one-month treatment, demonstrating its negligible systemic toxicity in vivo (Figure S17-S18). Overall, these findings underscored the remarkable anti-scarring ability of FAB@MN with favorable biocompatibility in vivo , and the process was depicted in Fig. 6 F. On one hand, owing to its excellent targeting delivery and programmable release behavior, FAB@MN not only accurately targeted pathological HSFs without damaging normal tissues, but also optimized treatment timing, thereby enhancing treatment specificity and effectiveness. On the other hand, the utilization of photodynamic waste provided auxiliary energy for ferroptosis, further enhancing the synergistic therapeutic effect of PDT and ferroptosis. Based on the significant anti-scarring effects demonstrated by FAB@MN in in vivo experiments, this strategy was expected to provide patients with more efficient treatment options. Figure 6. The therapeutic efficacy of FAB@MN in rabbit ear HS models. A Time arrangement for animal experiments. B Representative photographs of the scars with four rounds of treatments. C Ultrasound images of scars after predetermined treatments (scale bars: 2 mm). D HE, E Masson, Sirus red and GPX4 immunohistochemical staining of scar tissues after various treatments. F Scheme diagram of efficient anti-scarring efficacy of FAB@MN treatment. Conclusions In conclusion, we successfully construct a photodynamic waste-powered zero-waste “ferroptosis amplifier”, which works in synergy with PDT to enhance the treatment efficacy for HS. Similar to the event of a whale fall which injects new life into the deep-sea ecosystem, the designed FAB@MN significantly enhances the catalytic efficiency of HO-1, ultimately converting useless heme into an “auxiliary energy source” for ferroptosis. Notably, FAB@MN achieved significant anti-scarring activity with just a single light exposure, overcoming the drawbacks of traditional PDT which requires multiple drug administrations and light exposures, making it more suitable for clinical application. More importantly, the strategy not only provides an outlet for photodynamic waste, but also offers a new perspective for the combined use of PDT and ferroptosis in the treatment of HS. Methods Cell culture This research project has been approved by the Ethics Committee of Chongqing Medical University. Nine HS tissues and the matched normal skin tissues were provided by patients undergoing surgical treatment. All participants in this study did not have any known systemic diseases and were not receiving any treatments that could potentially affect the study results. In brief, tissues were first cut into 3 mm0-thick slices, and then digested overnight with 0.25% neutral protease II. Subsequently, the epidermal layer was peeled off, and further digestion was carried out for 4–6 hours at 37°C with 0.2% Collagenase II. After digestion, cells were filtered and collected using a cell strainer with a 75µm pore size, then resuspended in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS, and cultured at 37°C in an atmosphere of 5% CO 2 . The method of isolating fibroblasts from normal skin was similar to the aforementioned process for obtaining HSFs with minor modifications. Preparation of FAB@MN The synthesis of FAB@MN followed a detailed protocol. Initially, 5 mg of 5-ALA was dissolved in PBS, while 5 mg of baicalin was dissolved in ethanol. Then, 10 mg of HFn was dissolved in Tris-HCl buffer (20 mM, pH 7.5). The solutions of 5-ALA and baicalin were gradually added to the HFn solution under gentle stirring to prevent ethanol-induced denaturation of the protein. After mixing, the blend was subsequently incubated in a 60°C water bath for 4 hours, followed by centrifugation at 12,000 rpm for 15 mins. The supernatant was collected and dialyzed four times in PBS using a dialysis bag (10 kDa), with the dialysis fluid replaced every 4 hours to remove unencapsulated baicalin and 5-ALA. Subsequently, the obtained FAB NPs were mixed with a 20% PVA solution and poured into a PDMA mold, then placed under a vacuum of 600 mmHg for 1 min to ensure the solution completely filled the MN cavities. Finally, it was dried overnight in the dark before being demolded. Characterizations of FAB NPs and FAB@MN The protein structure changes of HFn were characterized with size-exclusion chromatography (SEC, Agilent 1260 Infinity II, USA) and circular dichroism (CD, JASCO J-1500, Japan). Particle size and zeta potential of the materials were measured using a Zetasizer Nano ZS90 (Malvern, UK). The ultraviolet absorption spectra of the materials were determined using a multimode microplate reader (PerkinElmer EnSpire, USA). Nanoparticle morphology was visualized using a JEM-1400 Plus transmission electron microscope (TEM, Tokyo, Japan), with grid staining performed using 1% (w/v) uranyl acetate for several minss before observation. Molecular docking was performed with Auto Dock Vina1.2.0, and molecular dynamics simulation was performed by the GROMACS (2022) program. The fabricated MN patches were then examined by scanning electron microscopy (SEM, FEI Hillsboro, USA), and their mechanical properties were assessed using a universal material testing machine (INSTRON, USA). To evaluate the skin penetration ability of FAB@MN (with RhB-labeled FAB NPs), we vertically inserted it into rabbit ear HS for 5 minss. The treated skin sample was then embedded in optimal cutting temperature (OCT), and sliced into 10 µm sections using a cryotome (CM1860, Leica). Subsequently, these skin sections were placed on adhesive microscope slides and observed using a multi-function microscope. Targeting ability and cellular uptake of FAB NPs To investigate the targeting ability of FAB NPs towards HSFs, RhB-labeled FAB NPs were added into HSFs for 4 h. Then, cells were fixed in 4% cold formaldehyde, and the nuclei and cytoskeleton were stained with DAPI and Actin-Tracker respectively. The fluorescence emitted from the treated cells was evaluated using a CLSM (Leica TCS SP8, Germany) and flow cytometry (BD Biosciences, USA). To further assess the deep penetration capability of FAB NPs in a 3D cell model, we initially planted 5 × 104 HSFs in ultra-low attachment round-bottom 48-well plates (Corning, USA) to cultivate 3D cell spheroids. After the formation of 3D scarring spheroids, the fluorescent-labeled NPs were co-inculcated for 4 hours, and the fluorescence images were captured and analyzed using a CLSM. In vitro drug release and subcellular colocalization of FAB NPs To examine the release profiles of FAB NPs, 5 mL of FAB NPs solution were put into a dialysis bag (10 kDa) that was directly immersed into 20 mL PBS at 37°C with pH values of 7.4 and 5.0. At predetermined time intervals, the concentrations of the released drugs were tested using HPLC (Agilent, USA). Stability evaluation was conducted by incubating FAB NPs with cell culture medium containing 10% FBS for 48 hours, and assessed their stability using a Zetasizer Nano ZS90 (Malvern, UK). For the subcellular colocalization imaging experiments, HSFs were seeded into 48-well plates at a density of 1 × 10 4 cells/well and incubated with RhB-labeled FAB NPs. After removing the culture medium, the cells were washed twice with cold PBS. Lysosomes and nuclei were stained with Lyso-Tracker and Hoechst for 30 and 10 mins respectively. After staining, the intracellular fluorescence signals were captured with CLSM. In vitro analysis of 5-ALA metabolic products in HSFs To evaluate the metabolic products of 5-ALA generated in HSFs, the cells were co-cultured with different materials in the dark for 4 hours, then irradiated with a 633 nm laser for 10 minss, and subsequently incubated at 37°C for 24 hours. For heme assessment, the cells were gently scraped and subjected to ultrasonication. The cell suspension was then centrifuged at 10,000 rpm for 10 minss to collect the supernatant, and the heme content was analyzed using an ELISA kit. Simultaneously, the cells were stained with COP-1 (Ruixi Biotechnology, China) for CO detection and FerroOrange (Beiren Chemical Technology, Beijing) for Fe 2+ detection. These metabolic products were then examined using a fluorescence microscope, allowing for the measurement of fluorescence signals for PpIX, CO, Fe 2+ , and BV (autofluorescence). Molecular docking of baicalin with Nrf2 was performed using Auto Dock Vina 1.2.0. In vitro cytotoxicity assay In vitro cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8). In brief, HSFs were seeded at a density of 5000 cells/well in a 96-well plate and cultured for 24 hours. Subsequently, these cells were exposed to various experimental materials for 4 hours, followed by laser irradiation at 633 nm (40 mW/cm², LWRPD630, China) for 10 minss. After an additional 24-hour incubation period, a diluted CCK-8 solution was administered to each well and incubated at 37°C for 4 hours. The absorbance at 450 nm was then measured using a microplate reader. Similarly, the treated HSFs underwent Annexin V-FITC/PI staining following the supplied protocol and were then analyzed with flow cytometry. Ferroptosis evaluation of FAB@MN HSFs were seeded at a density of 5 × 10 4 cells/well in 24-well plates and cultured overnight. Following 24 hours of exposure to various treatments, the HSFs were stained with DCFH-DA for ROS and BODIPY 581/591-C11 for LPO analysis, each for 30 mins, and analyzed using a fluorescence microscope and flow cytometry. In addition, the MDA and GSH amount were monitored using an MDA and GSH assay kit according to the instruction. The iron concentration was determined with ICP-OES (iCAP 6300, ThermoFisher, USA). To assess the mitochondrial structure and MMP of HSFs, JC-1, TMRM and Mito-Tracker probe were incubated with cells for 30 min respectively, and the intracellular fluorescence was evaluated with CLSM or flow cytometry. For a detailed examination of the mitochondrial ultrastructure, the treated cells were fixed with 2.5% glutaraldehyde and observed using biological transmission electron microscopy (Bio-TEM, Tokyo, Japan). Western blot analysis was conducted as follows: Protein concentrations in the collected samples were determined using the BCA protein assay. The expression levels of GAPDH, xCT, GPX4, P62, PINK1, and Parkin were then analyzed through polyacrylamide gel electrophoresis. Mitophagy was evaluated using a dual staining method with Lyso-Tracker Green and Mito-Tracker Deep Red. Specifically, the treated HSFs were first stained with Mito-Tracker Deep Red at 37 ℃ for 30 mins, then incubated with Lyso-Tracker Green for another 30 minss, and finally stained with Hoechst for 10 mins to label the cell nuclei. In vivo anti-scarring efficacy of FAB@MN The HS model was established using a previously detailed method. Under anesthesia, a 1 cm diameter full-thickness wound was created on the ventral side near the proximal end of the rabbit's ear, with careful removal of the perichondrium to avoid damaging the cartilage beneath. Four weeks post-surgery, a firm and dense HS had formed at the wound sites, and these scars were randomly divided into four groups: Control group, FA@MN group, FA@MN group and FAB@MN group. Four hours post MN application, the treated regions were exposed to laser irradiation for 10 minss, and this process was repeated weekly for a total of four weeks. The scar morphology within each group was captured and recorded using a digital camera. Upon completion of the treatment, ultrasound imaging of the scars was conducted using the Aixplorer ultrasound system (Super-Sonic Imagine, France), enabling direct measurement of scar thickness. Specifically, ultrasound transmission gel was then uniformly applied over the scarred region, and the scars were analyzed with an ultrasonic probe. Following these procedures, the rabbits were sacrificed and HS tissues were collected for histological analysis through HE, Masson and Sirius Red staining. Additionally, immunohistochemical staining with GPX4 antibody was performed. Statistical Analysis All analyses and comparisons were performed using SPSS 20.0 software (IBM, USA). Results were presented as mean ± standard deviation. The t-test was employed for comparisons between two groups, while one-way ANOVA was used for analyzing the variance among multiple groups. P-value < 0.05 indicated a significant difference between groups. Declarations Acknowledgements Y.C. and S.W. contributed equally to this work. This work was supported by the National Science Fund for Excellent Young Scholars (32322044), National Natural Science Foundation of China (32071362), Key International (Regional) Joint Research Program funded by National Natural Science Foundation of China (NSFC) (82220108019), the Science Fund for Distinguished Young Scholars of Chongqing (CSTB2022NSCQ-JQX0012), the CQMU Program for Youth Innovation in FutureMedicine (W0077). All animal experiments were in accordance with the ARRIVE guidelines, and were approved by the Animal Ethics Committee of Chongqing Medical University. Author Contributions Y.C., S.W., Q.Q.H. and T.C. conceived the idea and designed the experiments; Y.C., S.W., C.X.M., Y.W., D.Q.F. prepared and characterized the FAB@MN; Y.C., S.W., Y.P.L., X.C., J.L.Z., X.M. and Z.X.Y performed in vitro and in vivo experiments. 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Feng, X., Wang, S., Sun, Z., et al. Ferroptosis Enhanced Diabetic Renal Tubular Injury via HIF-1α/HO-1 Pathway in db/db Mice. Front Endocrinol (Lausanne) , 12 , 626390 (2021). Yu, F., Zhang, Q., Liu, H., et al. Dynamic O-GlcNAcylation coordinates ferritinophagy and mitophagy to activate ferroptosis. Cell Discov . 8 , 40 (2022). Li, J., Li, M., Ge, Y., et al. β-amyloid protein induces mitophagy-dependent ferroptosis through the CD36/PINK/PARKIN pathway leading to blood-brain barrier destruction in Alzheimer's disease. Cell Biosci . 12 , 69 (2022). Wang, H., Liu, C., Zhao, Y., et al. Mitochondria regulation in ferroptosis. Eur J Cell Biol . 99, 151058 (2020) Kumar, A. V., Mills, J., Lapierre, L. R. Selective Autophagy Receptor p62/SQSTM1, a Pivotal Player in Stress and Aging. Front Cell Dev Biol . 10 , 793328 (2022). Li, J., Li, M., Ge, Y., et al. β-amyloid protein induces mitophagy-dependent ferroptosis through the CD36/PINK/PARKIN pathway leading to blood-brain barrier destruction in Alzheimer's disease. Cell Biosci . 12 , 69 (2022). Yang, P., Li, J., Zhang, T., et al. Ionizing radiation-induced mitophagy promotes ferroptosis by increasing intracellular free fatty acids. Cell Death Differ . 30 , 2432–2445 (2023). Doric, Z., Nakamura, K. Mice with disrupted mitochondria used to model Parkinson's disease. Nature . 599 , 558–560 (2021) Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files SupportingInformation.pdf Scheme1.png Scheme 1. Schematic illustration of FAB@MN on HS treatment. ASchematic illustration of the fabrication process of a FAB@MN patch. B The targeting of FAB@MN for HSFs. C Scheme illustrating the formation of a ferroptosis amplifier utilizing photodynamic waste. D Schematic illustration of the treatment process of FAB@MN for HS. Cite Share Download PDF Status: Published Journal Publication published 18 Sep, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4498276","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":313641437,"identity":"480eecb1-051f-42ed-bdc9-4d63a1040db6","order_by":0,"name":"Tao 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University","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Ji","suffix":""}],"badges":[],"createdAt":"2024-05-29 16:00:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4498276/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4498276/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-63438-7","type":"published","date":"2025-09-18T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58253855,"identity":"5761004b-b66e-4234-93dc-a717dd692526","added_by":"auto","created_at":"2024-06-13 04:28:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1451025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe synthesis and characterization of FAB@MN. A\u003c/strong\u003e Schematic illustration of the steps for preparation of FAB@MN. \u003cstrong\u003eB \u003c/strong\u003eTEM and \u003cstrong\u003eC \u003c/strong\u003edigital images of HFn and FAB NPs solutions (scale bar: 20 μm). \u003cstrong\u003eD \u003c/strong\u003eDLS and Zeta potential analysis, data represent the mean ± SD (n = 3). \u003cstrong\u003eE\u003c/strong\u003e UV-Vis spectra of 5-ALA, baicalin, HFn and FAB NPs. \u003cstrong\u003eF\u003c/strong\u003e Native-PAGE characterization. \u003cstrong\u003eG\u003c/strong\u003e (i) The SEC and (ii) CD analysis of HFn, FB NPs and FAB NPs. \u003cstrong\u003eH\u003c/strong\u003e The schematic diagram of MNs fabrication and transdermal drug delivery. \u003cstrong\u003eI\u003c/strong\u003e Digital photo and \u003cstrong\u003eJ\u003c/strong\u003e mechanical compression test of PVP MN and FAB@MN patches. \u003cstrong\u003eK\u003c/strong\u003e SEM image of FAB@MN. \u003cstrong\u003eL \u003c/strong\u003eTypical images of rabbit HS skin after applying FAB@MN (scale bars: 2 mm). \u003cstrong\u003eM\u003c/strong\u003e (i) Fluorescent photograph of FAB@MN (FAB NPs were labeled by red RhB) (scale bars: 50 μm) and (ii) distribution of RhB-labeled FAB NPs in the depths of rabbit ear HS skin after FAB@MN insertion for 5min (scale bar: 100 μm).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4498276/v1/cc823ed608777363bbd5d32a.png"},{"id":58254141,"identity":"546efd12-37c8-43b9-b20d-b03667241caf","added_by":"auto","created_at":"2024-06-13 04:36:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1009695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe targeting, penetration and kinetics of drug release profile of FAB NPs. A \u003c/strong\u003eMolecular docking of 5-ALA and baicalin with HFn. \u003cstrong\u003eB \u003c/strong\u003eTargeting ability of FAB NPs towards HSFs. i) The schematic diagram illustrating the difference in uptake of FAB NPs by fibroblasts and HSFs. ii) Protein and iii) relative mRNA expression of TFR1 in fibroblasts and HSFs (n = 3). iv) The fluorescence images of RhB-labeled FAB NPs uptake in fibroblasts and HSFs (green, cytoskeleton; blue, nucleus; red, RhB-labeled FAB NPs; scale bar: 10 μm). \u003cstrong\u003eC \u003c/strong\u003eThe deep penetration of FAB NPs in 3D scarring spheroids. i) The fabrication and treatment process of scarring spheroids. ii) CLSM images of scarring spheroids after different treatments. (Green, FITC-labeled 5-ALA; Red, RhB-labeled baicalin. scale bar: 50 μm). iii) 3D surface plot of the 75μm sections in different groups. \u003cstrong\u003eD \u003c/strong\u003eThe subcellular location and release profile of FAB NPs. i) Representative CLSM images of RhB-labeled FAB NPs colocalized with lysosomes after endocytosis (green, lysosome; blue, nucleus; red, RhB-labeled FAB NPs; scale bar: 5 μm). ii) Cumulative release profiles of 5-ALA and baicalin from FAB NPs in solutions at different pH values \u003cem\u003ein vitro.\u003c/em\u003e iii) The FEL of 5-ALA-HFn (left) and baicalin-HFn (right). \u003cstrong\u003eE\u003c/strong\u003eScheme illustrating the therapeutic strategies and characteristics of FAB NPs for HSFs. Data represent the mean ± SD (n = 3). \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 indicates significant statistical difference.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4498276/v1/c0a5dacd7bb5b785a8fe42d6.png"},{"id":58253858,"identity":"8915219c-c635-4fc8-8720-ba609fc03cac","added_by":"auto","created_at":"2024-06-13 04:28:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":667512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiosynthesis of therapeutic metabolites in HSFs following treatment with FAB@MN.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Quantitative analysis of intracellular heme in different groups by Elisa kits. \u003cstrong\u003eB,C\u003c/strong\u003e Fluorescence images and the corresponding 3D surface plot of \u003cstrong\u003e(B) \u003c/strong\u003eCO and \u003cstrong\u003e(C)\u003c/strong\u003e Fe\u003csup\u003e2+\u003c/sup\u003e in HSFs with various treatments.\u003cstrong\u003e D\u003c/strong\u003e Molecular docking visualization results of baicalin with HO-1 protein. \u003cstrong\u003eE \u003c/strong\u003eWestern blot and \u003cstrong\u003eF \u003c/strong\u003ethe corresponding quantitative analysis of Nrf2 and HO-1 proteins in different groups (n = 3). \u003cstrong\u003eG\u003c/strong\u003e Cell apoptosis after being treated with different materials by flow cytometry. \u003cstrong\u003eH \u003c/strong\u003eStatistical analysis of the cell viability of HSFs (n = 3). \u003cstrong\u003eI \u003c/strong\u003eScheme illustrating FAB NPs enhanced their cytotoxic effect by recycling photodynamic waste heme.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4498276/v1/51c01cce4f0deba1d92d47c8.png"},{"id":58253862,"identity":"dcbf17b0-1ea9-4d70-95c8-48cbdb1bd81c","added_by":"auto","created_at":"2024-06-13 04:28:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1516110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFAB@MN-induced continuous ferroptosis in HSFs.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Schematic illustration to reveal the mechanism of photodynamic waste augmented ferroptosis in HSFs. \u003cstrong\u003eB\u003c/strong\u003e ICP-OES measurement of intracellular iron. \u003cstrong\u003eC\u003c/strong\u003eThe GSH and \u003cstrong\u003eD\u003c/strong\u003e MDA levels in HSFs with different treatments. Error bars are means ±SD (n = 3). \u003cstrong\u003eE\u003c/strong\u003e Fluorescence images of intracellular LPO in different groups by staining with C11-BODIPY581/591 probe (scale bar: 50 μm). \u003cstrong\u003eF \u003c/strong\u003eWestern blot analysis and\u003cstrong\u003e G \u003c/strong\u003edensitometric analysis of the expression of GPX4 and xCT proteins in HSFs treated with different interventions (n = 3). \u003cstrong\u003eH\u003c/strong\u003eHeatmap of expressed mRNA in different groups. \u003cstrong\u003eI \u003c/strong\u003eFluorescent assessment of MMP level in HSFs using JC-1 fluorescent probe (scale bar: 50 μm). \u003cstrong\u003eJ\u003c/strong\u003eRepresentative fluorescent images of mitochondrial morphology in HSFs using Mito-Tracker Deep Red fluorescent probe. Cell nuclei were stained by blue fluorescent Hoechst 33342 (scale bar: 10 μm).\u003cstrong\u003e K\u003c/strong\u003e TEM images of mitochondrial ultrastructure. Structures colored red indicate mitochondria (scale bar:1 μm and 500 nm). \u003cstrong\u003eL \u003c/strong\u003eFlow cytometry analysis of MMP in HSFs stained by TMRM probe. \u003cstrong\u003eM\u003c/strong\u003e Statistical data of mitochondrial individuals in HSFs based on fluorescent images with Mito-Tracker Deep Red probe (n = 3).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4498276/v1/112f044b7a48a864aefe4505.png"},{"id":58254142,"identity":"7a906eb4-a1ea-41c7-ac8a-6aa11309e8fe","added_by":"auto","created_at":"2024-06-13 04:36:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1567294,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe mechanism of FAB@MN-induced ferroptosis in HSFs. A\u003c/strong\u003e Volcano plot of DEGs in HSFs upon FAB@MN treatments. \u003cstrong\u003eB\u003c/strong\u003e GO functional analysis of DEGs in FAB@MN-treated HSFs. \u003cstrong\u003eC\u003c/strong\u003e KEGG pathway analysis of DEGs in HSFs upon FAB@MN treatments. \u003cstrong\u003eD\u003c/strong\u003e Circular visualization of the results of functional enrichment analysis. \u003cstrong\u003eE \u003c/strong\u003ePPI network of DEGs in HSFs upon FAB@MN treatments. \u003cstrong\u003eF\u003c/strong\u003e (i) TEM image of mitophagosomes in FAB@MN-treated HSFs. (ii) Representative fluorescence images of Mito-Tracker and Lyso-Tracker with Hoechst to show the formation of mitophagosomes in HSFs (scar bar: 5 μm and 1 μm). (iii) Scheme diagram of the formation of lysosome and mitochondria. \u003cstrong\u003eG \u003c/strong\u003eWestern blot analysis of the expression of P62, LC3B, PINK1 and Parkin proteins in HSFs treated with different interventions. \u003cstrong\u003eH\u003c/strong\u003e Statistical analysis of the cell viability of HSFs (n = 3). \u003cstrong\u003eI \u003c/strong\u003eWestern blot analysis of the expression of xCT and GPX4. \u003cstrong\u003eJ\u003c/strong\u003e CLSM images of Fe\u003csup\u003e2+\u003c/sup\u003e upon different treatments (scar bar: 25 μm). \u003cstrong\u003eK \u003c/strong\u003eFluorescence images of LPO stained with C11-BODIPY581/591 probe (scale bar: 50 μm). \u003cstrong\u003eL\u003c/strong\u003e Schematic of the ferroptosis mechanism induced by mitophagy.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4498276/v1/87ec83d3269002472f2e54aa.png"},{"id":58254401,"identity":"61f30671-fb2f-4bba-b8cf-e103067da959","added_by":"auto","created_at":"2024-06-13 04:44:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1681230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe therapeutic efficacy of FAB@MN in rabbit ear HS models. A \u003c/strong\u003eTime arrangement for animal experiments. \u003cstrong\u003eB\u003c/strong\u003e Representative photographs of the scars with four rounds of treatments. \u003cstrong\u003eC\u003c/strong\u003e Ultrasound images of scars after predetermined treatments (scale bars: 2 mm). \u003cstrong\u003eD \u003c/strong\u003eHE, \u003cstrong\u003eE\u003c/strong\u003e Masson, Sirus red and GPX4 immunohistochemical staining of scar tissues after various treatments. \u003cstrong\u003eF \u003c/strong\u003eScheme diagram of efficient anti-scarring efficacy of FAB@MN treatment.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4498276/v1/d9c8da529a4871ab6eded95f.png"},{"id":91683253,"identity":"c38a5e6e-086d-498e-8d09-06e8144cf634","added_by":"auto","created_at":"2025-09-19 07:05:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11141000,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4498276/v1/52d35448-d72d-4107-aa09-2034aa1f367f.pdf"},{"id":58253859,"identity":"67bb517b-cc32-4724-8972-63d88e523f39","added_by":"auto","created_at":"2024-06-13 04:28:55","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1046480,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupportingInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4498276/v1/bfb011eec44d38f977913e29.pdf"},{"id":58253857,"identity":"36af2de7-0cb3-4b3c-8abb-2d67389e3e76","added_by":"auto","created_at":"2024-06-13 04:28:54","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":408026,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. Schematic illustration of FAB@MN on HS treatment. A\u003c/strong\u003eSchematic illustration of the fabrication process of a FAB@MN patch. \u003cstrong\u003eB\u003c/strong\u003e The targeting of FAB@MN for HSFs. \u003cstrong\u003eC\u003c/strong\u003e Scheme illustrating the formation of a ferroptosis amplifier utilizing photodynamic waste. \u003cstrong\u003eD\u003c/strong\u003e Schematic illustration of the treatment process of FAB@MN for HS.\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4498276/v1/4567b01d3b6d35663cda0e8c.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Photodynamic metabolite-powered zero-waste “ferroptosis amplifier” for enhanced hypertrophic scar therapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHypertrophic scar (HS), characterized by excessive proliferation of fibroblasts and over-deposition of extracellular matrix, has been regarded as a complex skin disorder, imposing substantial social and financial burdens\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Especially for scars on the face, they not only cause various physical discomforts, such as itching and pain, but also lead to anatomical deformities, severely hindering the patient's mental health and social interactions, affecting the quality of life\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The Food and Drug Administration (FDA) has approved photodynamic therapy (PDT) using 5-aminolevulinic acid (5-ALA) for HS treatment\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As a precursor of photosensitizer, 5-ALA is metabolically converted to protoporphyrin IX (PpIX), which generates abundant reactive oxygen species (ROS) for hypertrophic scar fibroblasts (HSFs) killing upon light exposure. However, PpIX further metabolizes into free heme, which not only leads to the loss of photosensitizer but may also induce toxicity to normal tissues\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. For the past few decades, 5-ALA-generated heme has been considered a useless metabolic waste. Despite attempts to reduce its production, the effects have been minimal\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. From the perspective of natural material cycle, even waste materials can provide energy for new life processes under suitable conditions. This is evidenced by the phenomenon of whale falls in nature: when a giant whale dies and sinks to the seabed, its body becomes a rich food source for deep-sea organisms, thus promoting the flourishing development of life in the ecosystem. Inspired by this natural phenomenon of \"a whale fall brings life to thousands,\" we are exploring the possibility of turning these useless heme into potential \"auxiliary energy source\" to amplify the therapeutic efficacy of PDT.\u003c/p\u003e \u003cp\u003eNumerous studies have demonstrated heme can be degrade into Fe\u003csup\u003e2+\u003c/sup\u003e and CO through the action of heme oxygenase-1 (HO-1), thereby inducing iron overload and ferroptosis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Coincidentally, it has been observed that HSFs were naturally addicted to iron, suggesting ferroptosis as a potential alternative treatment\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Inspired by this, 5-ALA-generated heme may become a potential energy source for ferroptosis, amplifying the therapeutic effect for HS. However, due to the limited HO-1 quantity within cells, catalyzing the excessive accumulation of heme to facilitate the process of ferroptosis presents a significant challenge. Therefore, efforts to improve the expression of HO-1 and optimize the utilization of photodynamic waste are critical for establishing an efficient ferroptosis bioreactor post-PDT.\u003c/p\u003e \u003cp\u003eBaicalin is a flavonoid extracted from the root of scutellaria baicalensis. As one of the oldest medicinal plants first recorded in Shennong Bencaojing, a Chinese ancient pharmacopeia, baicalin has been widely used to treat inflammation, infection and cancer for centuries\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Recent studies have reported that baicalin can significantly promote the accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), which in turn upregulated the expression levels of HO-1\u003csup\u003e17\u0026ndash;18\u003c/sup\u003e. These findings suggest the co-delivery of 5-ALA and baicalin could potentially accelerate the biotransformation of useless heme, thereby promoting ferroptosis in HSFs. Moreover, further investigations have revealed baicalin's inherent capacity to induce ferroptosis in cancer treatments\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This dual-action of baicalin, coupled with the generated heme, may form a continuous ferroptosis bioreactor within HSFs. Compared to other ferroptosis inducers such as erastin, baicalin is more affordable and exhibits lower toxicity to normal tissues. However, its limited water solubility, and non-specific targeting severely restrict its clinical application\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Recently, nanotechnology has been applied to overcome the poor bioavailability of hydrophobic drugs and enhance their therapeutic efficiency\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Of note, human H-ferritin (HFn) has garnered significant interests, due to its excellent drug loading capacity and intrinsic targeting ability to transferrin receptor 1 (TfR1), which is highly expressed in most tumor cell\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Moreover, TFR1 has been found to be overexpressed on HSFs recently, suggesting that HFn-based delivery strategies may be useful for targeted therapies against HS\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHerein, a zero-waste \u0026ldquo;ferroptosis amplifier\u0026rdquo; has been constructed by co-encapsulating 5-ALA and baicalin into a HFn nanocage (FAB NPs). To improve transdermal delivery efficacy \u003cem\u003ein vivo\u003c/em\u003e, these FAB NPs were further integrated with polyvinylpyrrolidone (PVP) microneedles (FAB@MNs), providing a painless and convenient method of application. On one hand, after traversing the epidermal barrier, FAB@MNs can target HSFs through the TFR1-HFn interaction, thereby enhancing drug delivery and retention within cells. On the other hand, a synergistic therapy for HS can be implemented by PDT and ferroptosis based on multistage cascade metabolites. Specifically, the 5-ALA first biotransformed into PpIX to initiate PDT. Subsequently, the baicalin was released to trigger ferroptosis. Meanwhile, the baicalin could also serve as an HO-1 activator to facilitate the breakdown of photodynamic waste heme into Fe\u003csup\u003e2+\u003c/sup\u003e and CO, which contributed to the amplification of the ferroptosis process. Unlike traditional approaches that solely focus on the immediate effects of PDT, this strategy emphasizes transforming photodynamic waste into an \u0026ldquo;auxiliary energy source\u0026rdquo; to trigger the subsequent ferroptosis cascade. Just as every whale fall in the ocean heralds the abundance of new life, each short-term session of PDT powers a continuous ferroptosis bioreaction, thereby allowing FAB@MN to realize significant anti-scarring outcomes following a single laser irradiation. Despite the potential for further optimization, we anticipate more breakthroughs in this field, potentially bringing revolutionary changes to HS treatments. Importantly, such a \u0026ldquo;waste to energy\u0026rdquo; strategy not only reflects the spirit of scientific exploration inspired by nature but also introduces a new perspective on utilizing photodynamic waste to enhance the synergistic efficacy of PDT and ferroptosis, opening up a new path for treating other similar diseases.\u003c/p\u003e \u003cp\u003e \u003cb\u003eScheme 1. Schematic illustration of FAB@MN on HS treatment. A\u003c/b\u003e Schematic illustration of the fabrication process of a FAB@MN patch. \u003cb\u003eB\u003c/b\u003e The targeting of FAB@MN for HSFs. \u003cb\u003eC\u003c/b\u003e Scheme illustrating the formation of a ferroptosis amplifier utilizing photodynamic waste. \u003cb\u003eD\u003c/b\u003e Schematic illustration of the treatment process of FAB@MN for HS.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eFabrication and characteristics of FAB@MN\u003c/h2\u003e\n \u003cp\u003eHerein, we reported a one-pot method to co-encapsulate 5-ALA and baicalin within the hollow core of the HFn nanocage (FAB NP), which was subsequently incorporated into PVP MN patches (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Specifically, 5-ALA and baicalin were mixed with a solution of HFn, and then the mixture was heated to 60 ℃. It has been demonstrated that hyperthermia can expand the channels of HFn, thereby facilitating the encapsulation of small molecules without damaging the protein structures\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Transmission electron microscopy (TEM), dynamic light scattering (DLS), and Zeta potential analysis were performed to characterize HFn and the newly synthesized FAB NPs. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, HFn exhibited a typical ring-like structure with a hollow core in the center, whereas FAB NPs presented as solid spheres, possibly due to the filling of 5-ALA and baicalin. Notably, the dual encapsulation of 5-ALA and baicalin did not alter the high mono-dispersity and good colloidal stability of HFn, which was crucial for its potential in biomedical treatments (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Moreover, the solution of FAB NPs exhibited a clear orange-yellow color, indicating successful encapsulation of baicalin and its transformation from a water-insoluble form into a water-soluble one (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC and Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). According to the DLS data, the average diameters of HFn and FAB NPs were 10.11 nm and 11.68 nm, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). The slight increase in size of FAB NPs compared to HFn may also be attributed to the drug encapsulation within HFn. Additionally, the Zeta potential remained negative both before and after drug loading with no significant differences, suggesting that the drugs were encapsulated within the HFn nanocage rather than being adsorbed onto the shell (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eThe ultraviolet-visible (UV-Vis) absorption spectra confirmed the successful drug encapsulation within HFn, with characteristic absorption of 5-ALA and baicalin respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). And high-performance liquid chromatography (HPLC) analysis determined the encapsulation efficiencies of 54.92% for 5-ALA and 37.6% for baicalin. To confirm that the drug loading process did not affect the structure of HFn, comprehensive analyses including native polyacrylamide gel electrophoresis (Native-PAGE), circular dichroism spectrum (CD) and size-exclusion chromatography (SEC) were performed. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF, the FAB NPs appeared as a\u0026thinsp;\u0026asymp;\u0026thinsp;440 kDa protein in Native-PAGE, similar to that of HFn. And the CD spectra and SEC elution profiles of FAB NPs also closely matched those of HFn, indicating the secondary structure and oligomerization states of HFn remained unchanged after drug encapsulation (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eG (i-ii)). The stability of FAB NPs in cell culture media was also assessed, revealing that no significant change in size occurred over a 48-hour period under physiological conditions (Figure S2).\u003c/p\u003e\n \u003cp\u003eTo facilitate the \u003cem\u003ein-vivo\u003c/em\u003e biomedical applications, we developed a percutaneous delivery system for FAB NPs utilizing PVP MNs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eH). Specifically, a 20% PVP solution containing FAB NPs was cast into a poly-dimethylsiloxane (PDMS) mold to dry overnight. The optical images demonstrated that the fabricated MN patch consisted of an array of 225 needles spread over a 14 \u0026times; 14 mm\u0026sup2; area, and the patch turned slightly yellow after loading with FAB NPs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eI). The scanning electron microscopy (SEM) further revealed that each MN was pyramid-shaped, with 340 \u0026micro;m in diameter at the base and 800 \u0026micro;m in height (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eK). Additionally, the mechanical hardness of the fabricated FAB@MN was evaluated, showing a force of 0.11N/needle which was sufficient for skin penetration (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Intriguingly, the mechanical strength of FAB@MN was found to be higher than that of the blank PVP MN, possibly due to the hydrogen bonding and electrostatic interactions between the proteins and polymers. The penetration ability of FAB@MN was further demonstrated on rabbit ear HS models, which showed regularly spaced perforations in the skin post-application (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eL). And the fluorescence staining images revealed these MNs successfully penetrated the epidermal layer, capable of delivering RhB-labeled FAB NPs to the deeper dermal layer (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eM (i-ii)).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eTargeting ability and programmable release behavior of FAB NPs\u003c/h2\u003e\n \u003cp\u003eAlthough various nanodrugs have been explored to eliminate HSFs, the development of their targeting capabilities has often been neglected. Recently, the HFn protein drug delivery system has been widely used in cancer treatments, becoming a hotspot in the field of nanomedicine\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Researches have shown the HFn not only possessed basic characteristics of protein-based nanoparticles, but also displayed notable features including excellent drug loading capacity, high targeting specificity towards TFR1, and acid-responsive release \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In this study, HFn was used to encapsulate 5-ALA and baicalin, aiming to optimize the therapeutic effects of free drugs while simultaneously reducing damage to normal tissues. To gain deeper insights into the therapeutic potential of FAB NPs for HS, their targeting ability and release behavior were meticulously characterized.\u003c/p\u003e\n \u003cp\u003eThe strong and stable molecular interaction was crucial to ensuring the high targeting specificity of FAB NPs. In this regard, dual molecular blind docking studies were carried out to delve into the complex molecular interactions of FAB NPs, with 5-ALA and baicalin serving as ligands to co-interact with HFn (PDB ID: 7CK9). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, the analysis revealed a significant interaction between 5-ALA, baicalin and HFn, mediated through the formation of fourteen contacts involving five hydrogen bonds (GLN58, LYS143, GLU61, ASN109, GLU116), seven van der waals (ALA144, GLU147, TYR54, GLU140, ASN25, GLN83, SER113) and two pi-alkyl interactions (ARG22, LEU26). And the binding energies for 5-ALA and baicalin with HFn were calculated as -4.4 and \u0026minus;\u0026thinsp;9.4 kcal\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e respectively, indicating that the resulting complex structures were stable, laying a solid foundation for FAB NPs to exhibit targeting ability similar to HFn. Numerous studies have utilized HFn to target the overexpressed TFR1 in tumor cells, yielding favorable therapeutic outcomes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Inspired by this, we examined the expression of TFR1 in HSFs to explore the possibility of HFn-mediated targeted therapy for HS. According to the results from western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR), the TfR1 level in HSFs was significantly higher than that in normal fibroblasts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB(i-iii) and Figure S3). And the immunohistochemical staining of HS samples also demonstrated the increased TFR1 levels in cells, which potentially offered more binding sites for FAB NPs (Figure S4). To verify the targeting ability of FAB NPs towards HSFs, we constructed RhB-labeled FAB NPs and co-incubated them with normal fibroblasts and HSFs. As shown in Fig.\u0026nbsp;3B (iv) and Figure S5, HSFs had an eightfold higher fluorescence intensity than normal fibroblasts, highlighting FAB NPs\u0026apos; superior targeting efficacy to HSFs. Flow cytometry also confirmed these findings, with results consistent with the observed fluorescence patterns (Figure S6).\u003c/p\u003e\n \u003cp\u003eAs many drugs proven effective in 2D cell cultures lose their efficacy in dense scar tissues \u003cem\u003ein vivo\u003c/em\u003e, we further investigated the deep penetration abilities of FAB NPs in 3D scarring spheroid models using confocal laser scanning microscopy (CLSM) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC (i)). As show in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC (ii), the spheroids treated with free 5-ALA and baicalin remained relative dim, with drugs predominantly accumulating at the periphery. In sharp contrast, spheroids incubated with FAB NPs exhibited widespread fluorescence throughout the entire structure. The fluorescence intensity of treated spheroids was further quantified by 3D surface plots, revealing obvious fluorescence signals even reaching the core of spheroids after incubation with the FAB NPs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC(iii)). These results suggested that the strong targeting ability of FAB NPs facilitated drug penetration into dense scars, potentially further enhancing the therapeutic efficacy \u003cem\u003ein vivo\u003c/em\u003e. Considering the excellent targeting and penetration abilities exhibited by FAB NPs in both 2D and 3D scar models, we proceeded to assess the specific cytotoxicity of FAB NPs against HSFs. For that, normal fibroblasts and HSFs were exposed to various concentrations of FAB NPs, and the CCK8 assay indicated that FAB NPs displayed significantly higher cytotoxicity towards HSFs, further affirming their outstanding selective therapeutic effect on HS (Figure S7).\u003c/p\u003e\n \u003cp\u003eThe subcellular location and release behavior of FAB NPs were also explored. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD (i) and Figure S8, a large portion of FAB NPs colocalized with LysoTracker (a typical marker of lysosome) after internalization. Recent studies have demonstrated the acidic-responsive dissociation property of HFn, which suggested a great potential for FAB NPs to release 5-ALA and baicalin in acidic compartments of lysosomes, thus minimizing premature leakage during administration\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Following this, the release behavior of FAB NPs was examined under lysosome-mimicking conditions \u003cem\u003ein vitro\u003c/em\u003e. As expected, a complete release of 5-ALA and baicalin was observed after 24 hours of incubation at pH 5.0, significantly faster than that at pH 7.4. Moreover, notable discrepancies were observed in the release rates of 5-ALA and baicalin, particularly within the initial 4 hours, with approximately 81.41% of 5-ALA and 53.87% of baicalin released (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD (ii)). To gain a deeper insight into the mechanisms behind the differential release behavior of the two drugs, we conducted molecular dynamics simulations and computed the free energy landscape (FEL) and free binding energy for each drug-protein interaction (Fig. 3l). In cases where the protein-ligand interaction was weak or unstable, the FEL exhibited multiple minima clusters. Conversely, strong and stable interactions resulted in nearly single and smooth energy clusters in the potential energy distribution. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD (iii), the 5-ALA-HFn complex had four small energy clusters, whereas the baicalin-HFn complex exhibited only a single concentrated energy cluster, indicating the binding affinity between HFn and baicalin was stronger. Additionally, the binding free energy of 5-ALA-HFn was calculated as -10.68 kcal\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, higher than that of baicalin-HFn at -42.06 kcal\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A lower binding free energy often indicated a stronger binding affinity, which may explain why baicalin was released more slowly than 5-ALA from the FAB NP (Figure S9). The diagram in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE summarized the therapeutic strategies and characteristics of FAB NPs for treating HSFs. Initially, the fabricated FAB NPs can selectively bind to the significantly overexpressed TFR1 on the surface of HSFs, thereby achieving precise drug localization and reducing the side effects on surrounding tissues. Once internalized by the cells, FAB NPs were degraded within the lysosomal acidic environment, initiating a programmable sequential release of encapsulated agents. Specifically, the rapid release of 5-ALA quickly initiated PDT, laying the groundwork for early treatment, whereas the slow release of baicalin provided lasting support for the therapy. This sequential release pattern allowed for a programmable approach to treating HS, significantly improving the precision and efficiency of the therapy.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eEnhanced anti-scarring effects through FAB@MN-mediated photodynamic waste reutilization\u003c/h2\u003e\n \u003cp\u003e5-ALA served as a precursor of photosensitizer PpIX, demonstrating notable photodynamic activity against HSFs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. However, a significant challenge arose as PpIX was quickly metabolized into non-photosensitive heme, which significantly diminished its ability to produce ROS\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Currently, various strategies have focused on promoting immediate PpIX accumulation during PDT, while overlooking the negative metabolic impacts of PpIX conversion to heme and the potential to reutilize photodynamic waste\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Inspired by the energy cycling phenomenon of whale falls, we developed a \u0026ldquo;waste to energy\u0026rdquo; strategy by integrating 5-ALA and baicalin into a single nanotherapeutic platform to fully reutilize photodynamic waste heme for enhanced anti-scarring therapy. In this strategy, FAB@MN initially released 5-ALA to biosynthesize PpIX, which can be transformed into heme. And then, the released baicalin boosted the expression of HO-1, significantly accelerating the decomposition of heme into therapeutically beneficial Fe\u003csup\u003e2+\u003c/sup\u003e, CO and biliverdin. Through this method, we transformed the \u0026quot;end\u0026quot; metabolic waste into a new energy against HSFs, thereby enhancing the anti-scarring effects.\u003c/p\u003e\n \u003cp\u003eTo explore the effects of FAB@MN on PpIX metabolism regulation and subsequent heme utilization, we compared the main upstream and downstream metabolites within the 5-ALA-PpIX-heme-Fe\u003csup\u003e2+\u003c/sup\u003e/CO/ biliverdin metabolic axis among different groups. By employing Enzyme-Linked Immunosorbent Assay (ELISA) and fluorescence imaging techniques, the levels of PpIX and heme were initially quantified. As shown in Figure S10, both FA@MN and FAB@MN groups displayed a similar elevation in PpIX levels, indicating the successful delivery of 5-ALA and equivalent photodynamic activity of FA@MN and FAB@MN. However, the levels of heme, a downstream metabolite of PpIX, revealed significant differences between the two groups. In the FA@MN group, heme was significantly increased compared to the control group, while changes in the FAB@MN group were minimal, suggesting that FAB@MN may reduce the synthesis of heme or promote its degradation (Fig.\u0026nbsp;3A). Following this clue, we further tracked the decomposition products of heme, including Fe\u003csup\u003e2+\u003c/sup\u003e, CO and biliverdin, to gain a deeper understanding of the underlying mechanism by which FAB@MN acted upon heme. As depicted in Fig.\u0026nbsp;3B and 3C, compared to the FA@MN group which displayed almost no fluorescence for Fe\u003csup\u003e2+\u003c/sup\u003e and CO, the FAB@MN group showed pronounced fluorescence signals, confirming the role of baicalin in promoting heme degradation. However, the fluorescence of biliverdin in the FAB@MN group did not differ from other groups, possibly due to the presence of abundant biliverdin reductase enzymes in the human body that facilitated biliverdin\u0026rsquo;s rapid degradation (Figure S11)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Additionally, the FB@MN group also exhibited minor Fe\u003csup\u003e2+\u003c/sup\u003e signals but lacked any CO signals, which may result from the independent ferroptosis effect of baicalin, aligning with the findings of prior research\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Notably, the introduction of HO-1 inhibitors significantly reversed the observed increase in CO and Fe\u003csup\u003e2+\u003c/sup\u003e within the FAB@MN group, further implying the close correlation between the enhanced heme decomposition activity and HO-1 proteins.\u003c/p\u003e\n \u003cp\u003eAs the rate-limiting enzymes catalyzing oxidative degradation of heme, HO-1 has been regarded as an important regulator in the 5-ALA-PpIX-heme- Fe\u003csup\u003e2+\u003c/sup\u003e/CO/ biliverdin metabolic axis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Extensive mechanistic studies have revealed that HO-1 gene responded to positive regulation by Nrf2, we therefore examined the expression of Nrf2 and HO-1 in HSFs to figure out the exact molecular mechanisms of FAB@MN\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. As shown in Fig.\u0026nbsp;3D and 3E, the FAB@MN significantly increased endogenous Nrf2 expression, while this increase up-reregulated the expression of HO-1 at protein levels. Conversely, there was no obvious change in the expression level of Nrf2 and HO-1 in the FA@MN group, underscoring baicalin as the critical element enhancing the activity of the Nrf2/HO-1 pathway. To better understand the interactions between baicalin and Nrf2/HO-1 signaling axis at the atomic level, molecular docking studies were subsequently conducted. As shown in Fig.\u0026nbsp;3F, baicalin formed hydrogen bond interactions with multiple residues of Nrf2 such as ARG-456, ASN-507 and GLN-26, showing a robust binding affinity of -8.8 kcal\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These findings suggested that baicalin can directly target the inner cavity of Nrf2, activating the Nrf2/HO-1 signaling pathway and promoting heme degradation. From the results above, it was confirmed that FAB@MN can decompose photodynamic waste heme into an auxiliary energy source under the action of baicalin. This \u0026ldquo;waste to energy\u0026rdquo; process was similar to how a whale fall bring life to thousands, suggesting the potential to amplify anti-scarring effects after PDT. We, therefore, sought to validate the hypothesis.\u003c/p\u003e\n \u003cp\u003eAs indicated by CCK-8 assay, the relative cell viability in FA@MN and FB@MN groups was reduced to 72.2% and 61.9% respectively, implying poor therapeutic efficacy of PDT and baicalin-mediated killing effects (Fig.\u0026nbsp;3H). In contrast, only 13.8% of HSFs survived in the FAB@MN group after a single irradiation, which was far greater than the cumulative effect of FA@MN and FB@MN. The flow cytometry analysis also showed the highest apoptosis rate in FAB@MN group, further confirming the enhanced therapeutic effects of FAB@MN (Fig.\u0026nbsp;3G). Building on these findings, it can be concluded that FAB@MN not only generated the photosensitizer PpIX to initiate PDT under irradiation, but also upregulated HO-1 to convert heme into a significant amount of Fe\u003csup\u003e2+\u003c/sup\u003e and CO (Fig.\u0026nbsp;3I). This chemical reaction storm has been demonstrated to enhance the anti-scarring effects of PDT, truly achieving the conversion of waste into treasure.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 3. Biosynthesis of therapeutic metabolites in HSFs following treatment with FAB@MN. A\u003c/strong\u003e Quantitative analysis of intracellular heme in different groups by Elisa kits. \u003cstrong\u003eB,C\u003c/strong\u003e Fluorescence images and the corresponding 3D surface plot of \u003cstrong\u003e(B)\u003c/strong\u003e CO and \u003cstrong\u003e(C)\u003c/strong\u003e Fe\u003csup\u003e2+\u003c/sup\u003e in HSFs with various treatments. \u003cstrong\u003eD\u003c/strong\u003e Molecular docking visualization results of baicalin with HO-1 protein. \u003cstrong\u003eE\u003c/strong\u003e Western blot and \u003cstrong\u003eF\u003c/strong\u003e the corresponding quantitative analysis of Nrf2 and HO-1 proteins in different groups (n\u0026thinsp;=\u0026thinsp;3). \u003cstrong\u003eG\u003c/strong\u003e Cell apoptosis after being treated with different materials by flow cytometry. \u003cstrong\u003eH\u003c/strong\u003e Statistical analysis of the cell viability of HSFs (n\u0026thinsp;=\u0026thinsp;3). \u003cstrong\u003eI\u003c/strong\u003e Scheme illustrating FAB NPs enhanced their cytotoxic effect by recycling photodynamic waste heme.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eFAB@MN-induced continuous ferroptosis in HSFs\u003c/h3\u003e\n\u003cp\u003eFerroptosis was widely recognized as a new form of programmed cell death, closely associated with the accumulation of Fe\u003csup\u003e2+\u003c/sup\u003e and lipid peroxidation (LPO)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Recent studies have shown that the heme decomposition products, specifically Fe\u003csup\u003e2+\u003c/sup\u003e and CO, have the potential to promote ferroptosis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Based on this, we speculated that the FAB@MN-mediated reutilization of photodynamic waste heme could amplify the ferroptosis process, thereby enhancing killing effects on HSFs. Furthermore, studies have indicated that baicalin not only acted as a promoter of HO-1 to assist in the decomposition of heme but may also exert an independent ferroptosis effect, and this dual-action effect may help FAB@MN to provide dual assurance for continuous ferroptosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eTo clarify the role of ferroptosis in the action of FAB@MN, we introduced deferoxamine (DFO) as a ferroptosis inhibitor in cell viability assays. As shown, the cell viability in the FAB@MN group recovered from 14.9\u0026ndash;76.2%, which meant ferroptosis played a dominant role in FAB@MN-induced cell death independent of PDT (Figure S12). According to our strategy, FAB@MN transformed photodynamic waste heme into a chemical storm of Fe\u003csup\u003e2+\u003c/sup\u003e and CO. This cascade, in conjunction with baicalin\u0026apos;s inherent ferroptosis potential, was anticipated to initiate robust and sustained ferroptosis in HSFs. To demonstrate it, we investigated several ferroptosis biomarkers across different groups. As shown in Figure S13 and S14, the cells treated with FAB@MN had the highest levels of ROS, which was partly due to the PDT induced by PpIX, and partly a result of continuous ferroptosis. Additionally, FAB@MN-treated HSFs exhibited the highest intracellular iron content, which was 2.44-fold, 2.61-fold and 1.84-fold higher than those observed in Control, FA@MN and FB@MN groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). While the level of antioxidants such as GSH dropped to 39.43% of the control group after treatment with FAB@MN, significantly lower than those in other treatment groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, upon evaluating changes in intracellular LPO with the C11-BODIPY591/581 probe, it was found the green fluorescence intensity in HSFs treated with FAB@MN was higher than that in the FB@MN group, indicating increased LPO production through the biotransformation of heme post-PDT (Fig.\u0026nbsp;5E and Figure S14). And the end product of lethal LPO, intracellular malondialdehyde (MDA), was also increased by 1241.18% in FAB@MN-treated HSFs, far exceeding that in other groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). It was noteworthy that the use of the iron chelator DFO can significantly reverse the aforementioned abnormal ferroptosis biomarkers in the FAB@MN group, further confirming that FAB@MN promoted ferroptosis in HSFs.\u003c/p\u003e\n\u003cp\u003eBesides, the expression levels of two typical negative regulators against ferroptosis, xCT and GPX4, were also evaluated. The dysfunction of xCT led to the inactivation of GPX4, resulting in the legitimate accumulation of LPO and ultimately triggering ferroptosis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF-\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH, the protein and gene expression levels of GPX4 and xCT in FAB@MN group were much lower than those in FB@MN group, evidencing the augmented ferroptosis caused by the reutilization of heme. In addition to the Fe\u003csup\u003e2+\u003c/sup\u003e produced from heme degradation directly promoting ferroptosis, studies have also shown that the generated CO can also induce cellular ferroptosis, which mechanism involved downregulating the activity of GPX4 and increasing the concentration of ROS\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Furthermore, mRNA expression of additional ferroptosis-promoting genes, including ACSL4, FTH1, and TFR1, was significantly higher in the FAB@MN group, compared to the control group and others (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e\n\u003cp\u003eAs previously reported, mitochondrial dysfunction was closely involved in ferroptosis. Therefore, we investigated the mitochondrial structure and mitochondrial membrane potential (MMP) of HSFs treated by different materials. Initially, JC-1 probe was utilized as the fluorescent indictor for MMP detection. As depicted in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eI, FAB@MN-treated HSFs exhibited the brightest green fluorescence and weakest red fluorescence, revealing FAB@MN caused the most severe MMP depolarization within cells. In addition, according to the flow cytometry results, the intensity of TMRM fluorescence signal decreased the most in FAB@MN group, further supporting the significant depolarization of MMP induced by FAB@MN (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eL). Subsequently, Mito-Tracker staining was employed to evaluate the mitochondrial structure. Unlike the elongated and interconnected mitochondria seen in the control group, the mitochondria of HSFs treated with FAB@MN were abnormally rod-like, spherical and fragmented (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Notably, all observed mitochondrial damage observed in the FAB@MN group could be reversed to levels comparable with the control group by co-treatment with DFO, conforming the mitochondrial damage in HSFs was induced by FAB@MN-mediated ferroptosis. Furthermore, alterations in mitochondrial ultrastructure were characterized by TEM. As results shown, the HSFs treated with FAB@MN experienced severe mitochondrial shrinkage with diminished crista, which was consistent with the characteristic mitochondrial structure observed in ferroptosis (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eK)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The mitochondrial number and length were further quantified with ImageJ. As expected, the FAB@MN-treated HSFs exhibited significant changes in mitochondrial structure, including an increase in the number of mitochondria and a reduction in branch length. However, these changes were reversed by the iron chelator DFO (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eM and Figure S15). Collectively, by deeply delving into the potential of 5-ALA-generated heme in the process of ferroptosis, FAB@MN achieved sustained ferroptotic effects on HSFs, ultimately enhancing the anti-scarring efficacy of PDT. The superiority of this strategy lied in its ability to not only combine PDT and ferroptosis, but also to fully utilize the photodynamic waste as beneficial resources for ferroptosis, thereby amplifying therapeutic effects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 4.\u003c/strong\u003e \u003cstrong\u003eFAB@MN-induced continuous ferroptosis in HSFs.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Schematic illustration to reveal the mechanism of photodynamic waste augmented ferroptosis in HSFs. \u003cstrong\u003eB\u003c/strong\u003e ICP-OES measurement of intracellular iron. \u003cstrong\u003eC\u003c/strong\u003e The GSH and \u003cstrong\u003eD\u003c/strong\u003e MDA levels in HSFs with different treatments. Error bars are means \u0026plusmn;SD (n = 3). \u003cstrong\u003eE\u003c/strong\u003e Fluorescence images of intracellular LPO in different groups by staining with C11-BODIPY581/591 probe (scale bar: 50 \u0026mu;m). \u003cstrong\u003eF\u0026nbsp;\u003c/strong\u003eWestern blot analysis and\u003cstrong\u003e\u0026nbsp;G\u0026nbsp;\u003c/strong\u003edensitometric analysis of the expression of GPX4 and xCT proteins in HSFs treated with different interventions (n = 3). \u003cstrong\u003eH\u003c/strong\u003e Heatmap of expressed mRNA in different groups. \u003cstrong\u003eI\u0026nbsp;\u003c/strong\u003eFluorescent assessment of MMP level in HSFs using JC-1 fluorescent probe (scale bar: 50 \u0026mu;m). \u003cstrong\u003eJ\u003c/strong\u003e Representative fluorescent images of mitochondrial morphology in HSFs using Mito-Tracker Deep Red fluorescent probe. Cell nuclei were stained by blue fluorescent Hoechst 33342 (scale bar: 10 \u0026mu;m).\u003cstrong\u003e\u0026nbsp;K\u003c/strong\u003e TEM images of mitochondrial ultrastructure. Structures colored red indicate mitochondria (scale bar:1 \u0026mu;m and 500 nm). \u003cstrong\u003eL\u0026nbsp;\u003c/strong\u003eFlow cytometry analysis of MMP in HSFs stained by TMRM probe.\u0026nbsp;\u003cstrong\u003eM\u003c/strong\u003e Statistical data of mitochondrial individuals in HSFs based on fluorescent images with Mito-Tracker Deep Red probe (n = 3).\u0026nbsp;\u003c/p\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eMechanism of FAB@MN-induced ferroptosis in HSFs\u003c/h2\u003e\n \u003cp\u003eIn HSFs, FAB@MN acted as a persistent inducer of ferroptosis by utilizing photodynamical waste heme and the inherent ferroptosis property of baicalin. To further explore its underlying mechanisms of ferroptosis, mRNA-sequencing analysis was conducted on FAB@MN-treated HSFs. As shown in Fig.\u0026nbsp;5A, the volcano plot revealed a total of 2314 differentially expressed genes (DEGs) following the treatment with FAB@MN, of which 1364 DEGs were up-regulated and 1049 DEGs were down-regulated. According to gene ontology (GO) enrichment analysis, DEGs were significantly enriched in biological processes such as mitochondrial membrane permeability, stress response to metal ion, fatty acid oxidation and oxidative stress (Fig.\u0026nbsp;5B). Further, we conducted gene set enrichment analysis (GSEA) based on Kyoto Encyclopedia of Genes and Genomes (KEGG) database to reveal the changes in cellular signaling pathways of HSFs induced by FAB@MN. The results showed that HIF1, mitophagy and ferroptosis pathways were significantly activated, with genes such as HO-1, P62, xCT, and LC3B involved in these processes (Fig.\u0026nbsp;5C, D). Numerous studies have demonstrated that the activation of the HIF-1 pathway was also closely linked to ferroptosis and mitophagy, emphasizing their critical role in the damage to HSFs caused by FAB@MN\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Recently, as research into the mechanisms of ferroptosis and mitophagy has deepened, people have become increasingly aware of the potential close relationship between the two phenomena. In this study, we conducted protein-protein interaction network analysis (PPI) on DEGs enriched in both ferroptosis and mitophagy. As shown in Fig.\u0026nbsp;5E, the proteins encoded by genes mainly involved in ferroptosis and mitophagy exhibited extensive interactions, indicating a possible association between FAB@MN-induced ferroptosis and mitophagy. Existing researches have suggested that excessive activation of mitophagy may significantly increase the release of iron ions from the mitochondria\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. These iron ions then participated in the LPO reaction chain, further exacerbating cell membrane damage and leading to ferroptosis. Based on this, we hypothesized that FAB@MN not only induced ferroptosis but also triggered mitophagy, and there might be a direct or indirect relationship between them.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig.\u0026nbsp;5F (i), TEM analysis indicated FAB@MN significantly enhanced the formation of mitophagosomes in HSFs. And the fluorescence imaging also revealed a marked increase in colocalization between mitochondria (stained with Mito-Tracker) and lysosomes (stained with Lyso-Tracker), further supporting the enhancement of mitophagy activity (Fig.\u0026nbsp;5F (ii-iii)). To elucidate the molecular mechanism of FAB@MN-induced mitophagy, the expression level of several mitophagy-related proteins including LC3B, P62, PINK1 and Parkin were subsequently measured by immunoblotting assays (Fig.\u0026nbsp;5G). As expected, FAB@MN markedly upregulated the protein ratio of LC3B-II and LC3B-I, while decreasing P62 levels, indicating the formation of autophagosomes and occurrence of autophagy. Furthermore, the abundance of PINK1 and Parkin proteins also significantly increased, suggesting that FAB@MN activated mitophagy via classical PINK1/ Parkin pathway. As the key proteins that regulated mitophagy, PINK1 and Parkin interacted with each other to promote the recognition and clearance of damaged mitochondria\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Under normal conditions, PINK1 was rapidly degraded. However, when mitochondria were damaged, it accumulated and acted as a beacon to recruit Parkin from the cytosol to the surface of mitochondria. Subsequently, Parkin, endowed with E3 ubiquitin ligase activity, can tag these damaged mitochondria, thereby attracting a large amount of P62 to the mitochondrial outer membrane and promoting the ubiquitination of LC3B I. This process has been shown to ultimately lead to autophagosomes engulfing the damaged mitochondria, thus triggering mitophagy.\u003c/p\u003e\n \u003cp\u003eNumerous studies have confirmed that excessive activation of mitophagy can promote the occurrence of ferroptosis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. To explore the effect of mitophagy on FAB@MN-induced ferroptosis, we employed the autophagy inhibitor chloroquine (CQ) to co-treat with HSFs. Surprisingly, it was found the CQ could significantly restore the cell viability of FAB@MN from 16.7\u0026ndash;67.9% (Fig.\u0026nbsp;5H). Furthermore, CQ treatment notably counteracted the reduction of GPX4 and xCT proteins, and decreased the fluorescence signals of Fe\u003csup\u003e2+\u003c/sup\u003e and LPO in FAB@MN-treated HSFs (Fig.\u0026nbsp;5I-K). These observations suggested that ferroptosis induced by FAB@MN occurred in a mitophagy-dependent manner, which enhanced our comprehension of the anti-scarring effects of FAB@MN. Currently, the function of mitophagy remained controversial. For example, in neuronal cells, mitophagy cleared damaged mitochondria to maintain normal physiological conditions; however, mitophagy induced by ionizing radiation may promote ferroptosis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. In this study, FAB@MN excessively activated mitophagy through the PINK1/Parkin pathway, leading to the accumulation of Fe\u003csup\u003e2+\u003c/sup\u003e and LPO within cells, which ultimately triggered ferroptosis (Fig.\u0026nbsp;5L). This finding suggested that further research into the regulation of mitophagy could improve the therapeutic efficacy of FAB@MN for HS, and provided important theoretical support for the future application of FAB@MN in other related therapeutic areas.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 5. The mechanism of FAB@MN-induced ferroptosis in HSFs. A\u003c/strong\u003e Volcano plot of DEGs in HSFs upon FAB@MN treatments. \u003cstrong\u003eB\u003c/strong\u003e GO functional analysis of DEGs in FAB@MN-treated HSFs. \u003cstrong\u003eC\u003c/strong\u003e KEGG pathway analysis of DEGs in HSFs upon FAB@MN treatments. \u003cstrong\u003eD\u003c/strong\u003e Circular visualization of the results of functional enrichment analysis. \u003cstrong\u003eE\u003c/strong\u003e PPI network of DEGs in HSFs upon FAB@MN treatments. \u003cstrong\u003eF\u003c/strong\u003e (i) TEM image of mitophagosomes in FAB@MN-treated HSFs. (ii) Representative fluorescence images of Mito-Tracker and Lyso-Tracker with Hoechst to show the formation of mitophagosomes in HSFs (scar bar: 5 \u0026micro;m and 1 \u0026micro;m). (iii) Scheme diagram of the formation of lysosome and mitochondria. \u003cstrong\u003eG\u003c/strong\u003e Western blot analysis of the expression of P62, LC3B, PINK1 and Parkin proteins in HSFs treated with different interventions. \u003cstrong\u003eH\u003c/strong\u003e Statistical analysis of the cell viability of HSFs (n\u0026thinsp;=\u0026thinsp;3). \u003cstrong\u003eI\u003c/strong\u003e Western blot analysis of the expression of xCT and GPX4. \u003cstrong\u003eJ\u003c/strong\u003e CLSM images of Fe\u003csup\u003e2+\u003c/sup\u003e upon different treatments (scar bar: 25 \u0026micro;m). \u003cstrong\u003eK\u003c/strong\u003e Fluorescence images of LPO stained with C11-BODIPY581/591 probe (scale bar: 50 \u0026micro;m). \u003cstrong\u003eL\u003c/strong\u003e Schematic of the ferroptosis mechanism induced by mitophagy.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e \u003cstrong\u003eanti-scarring effects of FAB@MN\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eEncouraged by the superior therapeutic efficacy of FAB@MN observed \u003cem\u003ein vitro\u003c/em\u003e, we proceeded to monitor its anti-scarring activity \u003cem\u003ein vivo\u003c/em\u003e. Specifically, we established rabbit ear HS models, and applied treatments once a week for a month (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, the rabbits receiving PBS, FA@MN and FB@MN still exhibited typical HS features with dark-red surfaces and thick uplift after four-week treatments. However, in FAB@MN group, the HS appeared smooth and closely resembled surrounding normal skin. Ultrasound images further demonstrated that FAB@MN led to the most significant reduction in scar thickness, decreasing from 2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 to 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC and Figure S16). As predicted, FA@MN and FB@MN had limited therapeutic effects \u003cem\u003ein vivo\u003c/em\u003e, indicating that short-term PDT or baicalin-induced ferroptosis alone was ineffective. In contrast, FAB@MN leveraged a series of photodynamic waste to establish a sustained bioreactor integrating PDT and ferroptosis, opening up new avenues to effectively treat and prevent scar recurrence.\u003c/p\u003e\n \u003cp\u003eHistological analyses were further performed to assess the therapeutic outcomes of various treatments. Hematoxylin\u0026thinsp;\u0026minus;\u0026thinsp;eosin (HE) staining showed the HS treated with FAB@MN has returned to the smooth appearance of normal skin, whereas the other three groups still exhibited varying degrees of protuberance, accompanied by densely packed and disorganized muscle fibers (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD). And the results of Masson staining also demonstrated a noticeable reduction in collagen deposition at the HS site following treatment with FAB@MN (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE and Figure S16). Further examination of collagen fiber was conducted via Sirius red staining. Notably, the control, FA@MN and FB@MN groups exhibited abundant red-orange type I collagen densely distributed at the HS sites, while in contrast, the FAB@MN group displayed increased deposition of green-stained type III collagen fibers, suggesting the abnormal collagen was significantly remodeled by FAB@MN (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). Besides, the GPX4 level in HS tissues was also evaluated to assess the ferroptosis effects via immunohistochemical staining. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE and Figure S16, FAB@MN notably reduced the area of GPX4-positive cells and exhibited a more effective therapeutic outcome than FB@MN, likely due to the ferroptosis cascade initiated by photodynamic waste.\u003c/p\u003e\n \u003cp\u003eThe biosafety of FAB@MN \u003cem\u003ein vivo\u003c/em\u003e was verified by the HE staining of vital organs (major organs (heart, liver, spleen, lung, and kidney), as well as hematological and biochemical analyses. It was observed no significant organ damage occurred in FAB@MN group, and all blood parameters were within the normal limits after one-month treatment, demonstrating its negligible systemic toxicity \u003cem\u003ein vivo\u003c/em\u003e (Figure S17-S18). Overall, these findings underscored the remarkable anti-scarring ability of FAB@MN with favorable biocompatibility \u003cem\u003ein vivo\u003c/em\u003e, and the process was depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF. On one hand, owing to its excellent targeting delivery and programmable release behavior, FAB@MN not only accurately targeted pathological HSFs without damaging normal tissues, but also optimized treatment timing, thereby enhancing treatment specificity and effectiveness. On the other hand, the utilization of photodynamic waste provided auxiliary energy for ferroptosis, further enhancing the synergistic therapeutic effect of PDT and ferroptosis. Based on the significant anti-scarring effects demonstrated by FAB@MN in \u003cem\u003ein vivo\u003c/em\u003e experiments, this strategy was expected to provide patients with more efficient treatment options.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eFigure 6.\u003c/strong\u003e The therapeutic efficacy of FAB@MN in rabbit ear HS models. A\u0026nbsp;\u003c/strong\u003eTime arrangement for animal experiments. \u003cstrong\u003eB\u003c/strong\u003e Representative photographs of the scars with four rounds of treatments. \u003cstrong\u003eC\u003c/strong\u003e Ultrasound images of scars after predetermined treatments (scale bars: 2 mm). \u003cstrong\u003eD\u0026nbsp;\u003c/strong\u003eHE, \u003cstrong\u003eE\u003c/strong\u003e Masson, Sirus red and GPX4 immunohistochemical staining of scar tissues after various treatments.\u0026nbsp;\u003cstrong\u003eF\u0026nbsp;\u003c/strong\u003eScheme diagram of efficient anti-scarring efficacy of FAB@MN treatment.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, we successfully construct a photodynamic waste-powered zero-waste \u0026ldquo;ferroptosis amplifier\u0026rdquo;, which works in synergy with PDT to enhance the treatment efficacy for HS. Similar to the event of a whale fall which injects new life into the deep-sea ecosystem, the designed FAB@MN significantly enhances the catalytic efficiency of HO-1, ultimately converting useless heme into an \u0026ldquo;auxiliary energy source\u0026rdquo; for ferroptosis. Notably, FAB@MN achieved significant anti-scarring activity with just a single light exposure, overcoming the drawbacks of traditional PDT which requires multiple drug administrations and light exposures, making it more suitable for clinical application. More importantly, the strategy not only provides an outlet for photodynamic waste, but also offers a new perspective for the combined use of PDT and ferroptosis in the treatment of HS.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThis research project has been approved by the Ethics Committee of Chongqing Medical University. Nine HS tissues and the matched normal skin tissues were provided by patients undergoing surgical treatment. All participants in this study did not have any known systemic diseases and were not receiving any treatments that could potentially affect the study results. In brief, tissues were first cut into 3 mm0-thick slices, and then digested overnight with 0.25% neutral protease II. Subsequently, the epidermal layer was peeled off, and further digestion was carried out for 4\u0026ndash;6 hours at 37\u0026deg;C with 0.2% Collagenase II. After digestion, cells were filtered and collected using a cell strainer with a 75\u0026micro;m pore size, then resuspended in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS, and cultured at 37\u0026deg;C in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e. The method of isolating fibroblasts from normal skin was similar to the aforementioned process for obtaining HSFs with minor modifications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of FAB@MN\u003c/h2\u003e \u003cp\u003eThe synthesis of FAB@MN followed a detailed protocol. Initially, 5 mg of 5-ALA was dissolved in PBS, while 5 mg of baicalin was dissolved in ethanol. Then, 10 mg of HFn was dissolved in Tris-HCl buffer (20 mM, pH 7.5). The solutions of 5-ALA and baicalin were gradually added to the HFn solution under gentle stirring to prevent ethanol-induced denaturation of the protein. After mixing, the blend was subsequently incubated in a 60\u0026deg;C water bath for 4 hours, followed by centrifugation at 12,000 rpm for 15 mins. The supernatant was collected and dialyzed four times in PBS using a dialysis bag (10 kDa), with the dialysis fluid replaced every 4 hours to remove unencapsulated baicalin and 5-ALA. Subsequently, the obtained FAB NPs were mixed with a 20% PVA solution and poured into a PDMA mold, then placed under a vacuum of 600 mmHg for 1 min to ensure the solution completely filled the MN cavities. Finally, it was dried overnight in the dark before being demolded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCharacterizations of FAB NPs and FAB@MN\u003c/h2\u003e \u003cp\u003eThe protein structure changes of HFn were characterized with size-exclusion chromatography (SEC, Agilent 1260 Infinity II, USA) and circular dichroism (CD, JASCO J-1500, Japan). Particle size and zeta potential of the materials were measured using a Zetasizer Nano ZS90 (Malvern, UK). The ultraviolet absorption spectra of the materials were determined using a multimode microplate reader (PerkinElmer EnSpire, USA). Nanoparticle morphology was visualized using a JEM-1400 Plus transmission electron microscope (TEM, Tokyo, Japan), with grid staining performed using 1% (w/v) uranyl acetate for several minss before observation. Molecular docking was performed with Auto Dock Vina1.2.0, and molecular dynamics simulation was performed by the GROMACS (2022) program. The fabricated MN patches were then examined by scanning electron microscopy (SEM, FEI Hillsboro, USA), and their mechanical properties were assessed using a universal material testing machine (INSTRON, USA). To evaluate the skin penetration ability of FAB@MN (with RhB-labeled FAB NPs), we vertically inserted it into rabbit ear HS for 5 minss. The treated skin sample was then embedded in optimal cutting temperature (OCT), and sliced into 10 \u0026micro;m sections using a cryotome (CM1860, Leica). Subsequently, these skin sections were placed on adhesive microscope slides and observed using a multi-function microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTargeting ability and cellular uptake of FAB NPs\u003c/h2\u003e \u003cp\u003eTo investigate the targeting ability of FAB NPs towards HSFs, RhB-labeled FAB NPs were added into HSFs for 4 h. Then, cells were fixed in 4% cold formaldehyde, and the nuclei and cytoskeleton were stained with DAPI and Actin-Tracker respectively. The fluorescence emitted from the treated cells was evaluated using a CLSM (Leica TCS SP8, Germany) and flow cytometry (BD Biosciences, USA). To further assess the deep penetration capability of FAB NPs in a 3D cell model, we initially planted 5 \u0026times; 104 HSFs in ultra-low attachment round-bottom 48-well plates (Corning, USA) to cultivate 3D cell spheroids. After the formation of 3D scarring spheroids, the fluorescent-labeled NPs were co-inculcated for 4 hours, and the fluorescence images were captured and analyzed using a CLSM.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003edrug release and subcellular colocalization of FAB NPs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo examine the release profiles of FAB NPs, 5 mL of FAB NPs solution were put into a dialysis bag (10 kDa) that was directly immersed into 20 mL PBS at 37\u0026deg;C with pH values of 7.4 and 5.0. At predetermined time intervals, the concentrations of the released drugs were tested using HPLC (Agilent, USA). Stability evaluation was conducted by incubating FAB NPs with cell culture medium containing 10% FBS for 48 hours, and assessed their stability using a Zetasizer Nano ZS90 (Malvern, UK). For the subcellular colocalization imaging experiments, HSFs were seeded into 48-well plates at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well and incubated with RhB-labeled FAB NPs. After removing the culture medium, the cells were washed twice with cold PBS. Lysosomes and nuclei were stained with Lyso-Tracker and Hoechst for 30 and 10 mins respectively. After staining, the intracellular fluorescence signals were captured with CLSM.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eanalysis of 5-ALA metabolic products in HSFs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo evaluate the metabolic products of 5-ALA generated in HSFs, the cells were co-cultured with different materials in the dark for 4 hours, then irradiated with a 633 nm laser for 10 minss, and subsequently incubated at 37\u0026deg;C for 24 hours. For heme assessment, the cells were gently scraped and subjected to ultrasonication. The cell suspension was then centrifuged at 10,000 rpm for 10 minss to collect the supernatant, and the heme content was analyzed using an ELISA kit. Simultaneously, the cells were stained with COP-1 (Ruixi Biotechnology, China) for CO detection and FerroOrange (Beiren Chemical Technology, Beijing) for Fe\u003csup\u003e2+\u003c/sup\u003e detection. These metabolic products were then examined using a fluorescence microscope, allowing for the measurement of fluorescence signals for PpIX, CO, Fe\u003csup\u003e2+\u003c/sup\u003e, and BV (autofluorescence). Molecular docking of baicalin with Nrf2 was performed using Auto Dock Vina 1.2.0.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecytotoxicity assay\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e cytotoxicity was assessed using the Cell Counting Kit-8 (CCK-8). In brief, HSFs were seeded at a density of 5000 cells/well in a 96-well plate and cultured for 24 hours. Subsequently, these cells were exposed to various experimental materials for 4 hours, followed by laser irradiation at 633 nm (40 mW/cm\u0026sup2;, LWRPD630, China) for 10 minss. After an additional 24-hour incubation period, a diluted CCK-8 solution was administered to each well and incubated at 37\u0026deg;C for 4 hours. The absorbance at 450 nm was then measured using a microplate reader. Similarly, the treated HSFs underwent Annexin V-FITC/PI staining following the supplied protocol and were then analyzed with flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFerroptosis evaluation of FAB@MN\u003c/h2\u003e \u003cp\u003eHSFs were seeded at a density of 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in 24-well plates and cultured overnight. Following 24 hours of exposure to various treatments, the HSFs were stained with DCFH-DA for ROS and BODIPY 581/591-C11 for LPO analysis, each for 30 mins, and analyzed using a fluorescence microscope and flow cytometry. In addition, the MDA and GSH amount were monitored using an MDA and GSH assay kit according to the instruction. The iron concentration was determined with ICP-OES (iCAP 6300, ThermoFisher, USA). To assess the mitochondrial structure and MMP of HSFs, JC-1, TMRM and Mito-Tracker probe were incubated with cells for 30 min respectively, and the intracellular fluorescence was evaluated with CLSM or flow cytometry. For a detailed examination of the mitochondrial ultrastructure, the treated cells were fixed with 2.5% glutaraldehyde and observed using biological transmission electron microscopy (Bio-TEM, Tokyo, Japan). Western blot analysis was conducted as follows: Protein concentrations in the collected samples were determined using the BCA protein assay. The expression levels of GAPDH, xCT, GPX4, P62, PINK1, and Parkin were then analyzed through polyacrylamide gel electrophoresis. Mitophagy was evaluated using a dual staining method with Lyso-Tracker Green and Mito-Tracker Deep Red. Specifically, the treated HSFs were first stained with Mito-Tracker Deep Red at 37 ℃ for 30 mins, then incubated with Lyso-Tracker Green for another 30 minss, and finally stained with Hoechst for 10 mins to label the cell nuclei.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eanti-scarring efficacy of FAB@MN\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe HS model was established using a previously detailed method. Under anesthesia, a 1 cm diameter full-thickness wound was created on the ventral side near the proximal end of the rabbit's ear, with careful removal of the perichondrium to avoid damaging the cartilage beneath. Four weeks post-surgery, a firm and dense HS had formed at the wound sites, and these scars were randomly divided into four groups: Control group, FA@MN group, FA@MN group and FAB@MN group. Four hours post MN application, the treated regions were exposed to laser irradiation for 10 minss, and this process was repeated weekly for a total of four weeks. The scar morphology within each group was captured and recorded using a digital camera. Upon completion of the treatment, ultrasound imaging of the scars was conducted using the Aixplorer ultrasound system (Super-Sonic Imagine, France), enabling direct measurement of scar thickness. Specifically, ultrasound transmission gel was then uniformly applied over the scarred region, and the scars were analyzed with an ultrasonic probe. Following these procedures, the rabbits were sacrificed and HS tissues were collected for histological analysis through HE, Masson and Sirius Red staining. Additionally, immunohistochemical staining with GPX4 antibody was performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll analyses and comparisons were performed using SPSS 20.0 software (IBM, USA). Results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The t-test was employed for comparisons between two groups, while one-way ANOVA was used for analyzing the variance among multiple groups. P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated a significant difference between groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.C. and S.W. contributed equally to this work. This work was supported by the National Science Fund for Excellent Young Scholars (32322044), National Natural Science Foundation of China (32071362), Key International (Regional) Joint Research Program funded by National Natural Science Foundation of China (NSFC) (82220108019), the Science Fund for Distinguished Young Scholars of Chongqing (CSTB2022NSCQ-JQX0012), the CQMU Program for Youth Innovation in FutureMedicine (W0077). All animal experiments were in accordance with the ARRIVE guidelines, and were approved by the Animal Ethics Committee of Chongqing Medical University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.C., S.W., Q.Q.H. and T.C. conceived the idea and designed the experiments; Y.C., S.W., C.X.M., Y.W., D.Q.F. prepared and characterized the FAB@MN; Y.C., S.W., Y.P.L., X.C., J.L.Z., X.M. and Z.X.Y performed in vitro and in vivo experiments. X.Y.Z., Y.C. and S.W. contributed to analysis and discussion of the results; Y.C. and S.W. wrote the paper; Q.Q.H., P.J. and T.C. revised the manuscript; T.C. supervised the overall study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence\u0026nbsp;\u003c/strong\u003eand requests for materials should be addressed to Tao Chen.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are provided in the main text and the Supplementary Information. Additional information can be obtained from the corresponding author upon request. Source data are also provided with this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eYang, Z. R., Suo, H., Fan, J. W., et al. 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Mice with disrupted mitochondria used to model Parkinson\u0026apos;s disease. \u003cem\u003eNature\u003c/em\u003e. \u003cstrong\u003e599\u003c/strong\u003e, 558\u0026ndash;560 (2021)\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":false,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ferroptosis, hypertrophic scars, mitophagy, photodynamic therapy, photodynamic waste, recycle","lastPublishedDoi":"10.21203/rs.3.rs-4498276/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4498276/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypertrophic scar (HS) is a somatopsychic disease that significantly affects quality of life. 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) shows promise for HS treatment, while challenges like poor transdermal delivery and the accumulation of photodynamic by-products restrict its effectiveness. Inspired by the natural phenomenon that a whale fall brings life to thousands, this study proposes a zero-waste strategy by leveraging the photodynamic metabolite heme to establish a \u0026ldquo;ferroptosis amplifier\u0026rdquo;, which allows these metabolic wastes to be transformed into new sources of energy, thereby amplifying ferroptosis response following PDT. This is achieved by encapsulating 5-ALA and baicalin within human H-ferritin (HFn), subsequently incorporated into polyvinylpyrrolidone (PVP) microneedles (FAB@MN). The FAB@MN exhibits excellent targeting towards hypertrophic scar fibroblasts (HSFs) and pH-responsive programmed drug release. The treatment begins with the release of 5-ALA, which is converted into PpIX to activate PDT. Baicalin is then released, which directly triggers ferroptosis while also facilitating the breakdown of photodynamic waste heme into Fe\u003csup\u003e2+\u003c/sup\u003e and CO, thereby amplifying ferroptosis. Unlike conventional PDT only focuses on immediate effects, this approach uses photodynamic waste to fuel a sustained ferroptosis response after PDT, offering a new path for treatment.\u003c/p\u003e","manuscriptTitle":"Photodynamic metabolite-powered zero-waste “ferroptosis amplifier” for enhanced hypertrophic scar therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-13 04:28:50","doi":"10.21203/rs.3.rs-4498276/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b040f78b-39ef-4296-917c-f5537c464cca","owner":[],"postedDate":"June 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33163357,"name":"Biological sciences/Biotechnology/Biomaterials/Bioinspired materials"},{"id":33163358,"name":"Biological sciences/Biological techniques/Nanobiotechnology/Nanoparticles"}],"tags":[],"updatedAt":"2025-09-19T07:05:45+00:00","versionOfRecord":{"articleIdentity":"rs-4498276","link":"https://doi.org/10.1038/s41467-025-63438-7","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-09-18 04:00:00","publishedOnDateReadable":"September 18th, 2025"},"versionCreatedAt":"2024-06-13 04:28:50","video":"","vorDoi":"10.1038/s41467-025-63438-7","vorDoiUrl":"https://doi.org/10.1038/s41467-025-63438-7","workflowStages":[]},"version":"v1","identity":"rs-4498276","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4498276","identity":"rs-4498276","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}