ZnO colludes with C. acnes in healing delay and scar hyperplasia by barrier destruction

ZnO colludes with C. acnes in healing delay and scar hyperplasia by barrier destruction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ZnO colludes with C. acnes in healing delay and scar hyperplasia by barrier destruction Fenglan Zhang, Tianyi Wang, Wenqiao Wang, Yaqian Lv, Yingshan Qu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5823650/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 May, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted 6 You are reading this latest preprint version Abstract As an important component of sunscreen products for sensitive skin, the potential damage mechanism of ZnO nanoparticles on skin surface with barrier structure or function defect caused by Cutibacterium acnes (C. acnes) has not been elucidated, which poses a serious challenge for reasonable selection of sunscreen products for acne-infected skin. In this work, we demonstrated for the first time that C. acnes induced significant changes in the membrane permeability and intracellular pH of fibroblasts through lipase up-regulation and lipid peroxidation, promoting endocytosis and ionization of ZnO NPs. High amounts of Zn2 + further delayed acne wound healing and aggravated scar hyperplasia by intervening matrix metalloproteinase-9 (MMP-9) and TGF-β1/Smad pathway. MMP9 was confirmed to be the key target of ZnO in delaying acne wound healing by the wound regulatory effects of MMP9 agonist and MMP9 inhibitor. In summary, this work clarified the interaction mechanism between ZnO NPs and acne skins, providing guideline for the application of physical sunscreens for special skins. ZnO NPs membrane permeability MMP-9 wound healing scar hyperplasia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Skin, which is the largest organ and outermost layer of the human body, easily suffers from various diseases (such as acne, atopic dermatitis and psoriasis) [ 1 – 4 ]. Any dysfunction of the skin barrier may lead to a vicious cycle that aggravates barrier damage and thus leads to various skin diseases [ 5 , 6 ]. The microbiological barrier plays an important role in regulating the colonization of pathogens and the release of inflammatory cytokines by skin resident pathogens [ 7 ]. However, when over proliferation of Cutibacterium acnes ( C. acnes ) in the skin microbiological barrier occurred, the disorder of the microbiological barrier will further lead to the damage of chemical, physical, and immune barriers [ 8 , 9 ]. Specifically, disturbance of the microbial barrier can lead to abnormal lipid metabolism and acidic environmental damage, which weakens the antibacterial ability of the chemical barrier [ 10 ]. At the same time, the proliferation of harmful bacteria will degrade the corneum tightener protein and destroy the integrity of the physical barrier [ 11 ]. These changes further trigger the imbalance of immune regulation and the overactivation of innate immunity, which aggravate the inflammatory response [ 12 ]. The failure of chemical barrier, the destruction of physical barrier and the imbalance of immune barrier jointly aggravate the pathological process of skin diseases. On the skin surface, the microbial community is mainly composed of bacteria belonging to the genera Corynebacterium, Cutibacterium acnes , and Staphylococcus, the Interaction of which is essential for the maintenance of skin health [ 13 , 14 ]. The commensal bacterium C. acnes , which is important for regulating skin homeostasis and preventing the colonization of other harmful pathogens, can also serve as an opportunistic pathogen of acne vulgaris [ 15 ]. C. acnes produces propionic acid, the release of which induces a low pH in the skin micro-environment where C. acnes over colonize, posing a severe challenge to the physiological function of surrounding cells [ 16 ]. In addition, existing studies have shown that C. acnes promotes acne inflammation by activating inflammatory cells, keratinocytes, monocytes, and sebaceous gland cells. Induced the secretion of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α [ 17 , 18 ]. C. acnes can also promote the secretion of pro-inflammatory cytokines by activating monocytes or via the innate immune receptors toll-like receptors (TLR), TLR2, and TLR4 [ 19 ]. Therefore, the severe micro-environment imbalance in acne wounds caused by C. acnes over colonization poses a serious challenge to the integrity of skin barrier. To prevent UV damage, sunscreen is recommended to be adopted in order to protect the skin barrier. Compared with chemical sunscreen, physical sunscreen is more difficult to be absorbed by the skin or produce allergic phenomena, showing more satisfactory safety and stability, so it is more suitable for sensitive skin or children's skin [ 20 ]. Zinc oxide nanoparticles (ZnO NPs), as an most common inorganic sunscreen component, are widely used as a physical sunscreen due to its excellent shortwave UV insulation [ 21 – 23 ]. Many studies have confirmed that ZnO NPs barely penetrate the skin barrier and cause cell damage when the skin barrier remains intact [ 24 ]. Zvyagin et al. [ 25 ] and Roberts al. [ 26 ] were the first to confirm that ZnO NPs do not penetrate the stratum corneum of the skin with the help of non-invasive multiphoton imaging, and this finding was also verified by later studies [ 27 – 29 ]. However, when the skin barrier is impaired in certain inflammatory skin diseases, such as psoriasis, atopic dermatitis, and seborrheic dermatitis, ZnO NPs deposition in cells and significant cell damage have been observed [ 30 ]. Zn 2+ is a key auxiliary component of key metabolic regions of many enzyme molecules involved in protein synthesis, such as ribonucleic acid (ribonucleic acid, RNA) polymerase, and therefore, an appropriate amount of Zn 2+ is necessary for cells to maintain normal physiological function [ 31 ]. However, excessive Zn 2+ can block calcium signaling, thereby selectively inhibiting cell growth [ 32 ]. In addition, excessive Zn 2+ promotes the production of endogenous reactive oxygen species (ROS) and disrupts mitochondrial membrane potential [ 33 – 35 ]. In addition, previous studies have shown that long-term application of ZnO NPs has significant effects on the survival rate, oxidative stress, cell proliferation and apoptosis of Enzootic Bovine Leukosis (EBL) cells [ 36 ].For the above reasons, an abnormal increase in intracellular Zn 2+ can lead to severe cell damage, which is often used as a novel anti-tumor and antibacterial strategy [ 37 – 39 ]. In the face of the severe changes in the micro-environment of acne wounds, especially the acidification of the skin micro-environment induced by over-colonized C. acnes , the safety of ZnO NPs and the potential interaction mechanism between ZnO NPs and skin barrier have not received enough attention and systematic analysis [ 40 ]. In this study, we demonstrated for the first time that C. acnes , as the “accomplice” of ZnO NPs, induced significant changes in the cell membrane permeability and intracellular pH environment of fibroblasts through lipase up-regulation and lipid peroxidation, providing the necessary conditions for the opportunistic entry and ionization of ZnO NPs. After uptake by fibroblasts, ZnO NPs decomposed into Zn 2+ , which down-regulated matrix metalloproteinase-9 (MMP-9) and TGF-β1 expression and the downstream TGF-β1/Smad signaling pathway. This further results in a decrease in α-smooth muscle actin (α-SMA) and collagen synthesis and a delay in acne wound healing (Scheme 1 ). In summary, focusing on the changes of cell membrane permeability, this study confirmed the specific mechanism and action pathway of C. acnes acting as an accomplice to cause irreversible damage to the acne skin barrier by ZnO NPs, which provided meaningful theoretical guidance for the application of ZnO NPs-containing sunscreens. Results and Discussion C. acnes promotes acidification of neighboring cells by disrupting the membrane permeability pH in the traumatic environment plays a key role in the process of wound healing. As one of the important parameters of the wound microenvironment, intracellular and extracellular pH influences intracellular metabolism (enzyme activity, macromolecule synthesis, metabolite transport) and cell cycle-related biological behaviors (inflammation, collagen formation, and angiogenesis) [ 41 ]. In normal skin, the skin surface presents an acidic environment (pH 4–6), and the pH value gradually decreases from the basal layer (pH ≈ 7) to the stratum corneum [ 42 ]. This pH gradient not only guarantees the function of the epidermal barrier (including the inhibition of microbial colonization and the regulation of keratinase activity), but is also essential for the maintenance of keratinocyte differentiation homeostasis. In chronic wound skin, the central area of the wound is alkaline (~ 7.5) due to persistent inflammation and hypoxia, while the marginal area is maintained with low pHe (~ 6.5) due to active metabolism. This gradient difference in chronic skin wounds inhibits the proliferation and centripetal migration of marginal cells, resulting in delayed wound healing [ 43 ]. C. acnes reduces local skin pH through synergistic action in multiple pathways. C. acnes secretes lipase to decompose triacylglycerol in sebum and release short-chain fatty acids dominated by propionic acid [ 44 ]. Studies have shown that pH of the skin surface could be decreased by increased free fatty acid and propionic acid content by the fermentation of glucose in C. acnes [ 45 ]. At the same time, lactic acid and sulfur-containing acidic products are generated through glycolysis and protein metabolism [ 46 ]. In addition, the host immune response further exacerbates the acidification. For example, neutrophil respiratory bursts rely on glycolysis, which results in significant production of lactic acid [ 47 ]. Moreover, the imbalance of microenvironment induced by C. acnes led to the failure of buffer system such as bicarbonate, and the blockage of hair follicles caused by abnormal keratosis promoted the stroke of anoxic environment, which further promoted the proliferation and continuous acid production of C. acnes [ 48 ]. The pH of the supernatant of the medium after co-incubation of C. acnes with L929 cells at different concentrations (Control, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 and 10 9 CFU/mL) all decreased gradually with the increase of time (Fig S1 ), indicating the propionic acid accumulation of C. acnes and the decrease of pHe, which has been demonstrated to be closely related with the up-regulated expression of lipase [ 49 ]. Lipase can hydrolyze triglycerides into glycerol and fatty acids, further leading to lipid peroxidation and the destruction of cell membrane structure and function [ 50 , 51 ]. In this work, lactate dehydrogenase content (Fig. S2 ), trypan blue staining (Fig. S3, Fig. S4A) and intracellular pH (Fig. S4B) were respectively analyzed and proved that C. acnes could destroy C. acnes through the combined action of propionic acid and lipase. Membrane homeostasis of fibroblasts in C. acnes infected tissue. Previous studies have confirmed that low pHe significantly inhibits cell proliferation, migration and angiogenesis by regulating acid-sensitive ion channels (ASICs) and matrix metalloproteinases (MMPs) [ 43 ]. The result of differential gene cluster heat map (Fig. S5) showed that the down-regulation of MMP-9 in C. acnes group was higher than that in control group. CCK-8 analysis confirmed that with the increase of co-incubation time and bacterial concentration, C. acnes infection induced a significant decline in the viability of L929 cells. In addition, L929 cells could achieve self-repair under low bacterial concentration (10 3 -10 4 CFU/mL), but significantly lost self-repair ability and showed irreversible damage and apoptosis under high bacterial concentration (> 10 4 CFU/mL) (Fig. S6). Therefore, the above results confirmed that the damage of C. acnes to L929 cells was closely related to the acidification of extracellular microenvironment by bacterial metabolites (such as propionic acid), inhibition of MMP-9 activity and activation of lipid peroxidation. Alternatively, the lipase inhibitor orlistat was added to the bacterial suspension to further evaluate the damage of the skin cell membrane by the lipase produced by C. acnes [ 52 ]. The lipase content and extracellular free fatty acid content secreted by C. acnes increased significantly with time, which was inhibited by the addition of orlistat (Fig. 1 A-B). In addition, the conclusion of intracellular lipid peroxidation analysis (Fig. 1 C-E) was similar to the above results. C. acnes treatment significantly up-regulated ROS and MDA levels and down-regulated GSH levels, which were all reversed by treatment of orlistat. Co-culture of C. acnes resulted in the leakage of LDH in L929 cells and the increase in the number of Trypan blue-stained cells, as well as serious damage to membrane permeability, which could be inhibited by orlistat (Fig. 1 F-G). These results fully demonstrated that C. acnes induced lipid peroxidation of adjacent cells and cell membrane damage through lipase secretion, which played an important role in the process of C. acnes disrupting skin cell homeostasis in acne tissues. Moreover, C. acnes transported secreted propionic acid on the basis of destroying the cell membrane structure, resulting in significant down-regulation of intracellular pH and serious dysregulation of skin homeostasis, which could not be achieved in pH 6.4 group (Fig. 1 H-I). Characterization of ZnO NPs Recently, the biosafety of ZnO NPs of skin cells attracted increasing attention due to the wider application in the field of sun protection, medicine and chemical engineering [ 53 , 54 ]. Prior to analyzing the effects of ZnO NPs on acne tissue, ZnO NPs adopted in this work were characterized in detail. SEM and TEM images showed the morphology and average size of ZnO NPs (20.30 ± 6.7 nm, Fig. S7-10). This diameter met the size requirements for sun protection products between 20 and 100 nm, which facilitated reflection and absorption of ultraviolet rays between 280 and 400 nm, including UVB and some UVC. In addition, the X-Ray Diffraction (XRD) of ZnO NPs were consistent with the standard X-ray diffraction spectra of ZnO in the JCPDs data card (PDF#79–0205, Fig. S8). The hydrodynamic size and zeta potential of ZnO NPs in distilled water were 20.30 ± 6.7 nm and − 8.38 mV, respectively. These above results were consistent with data in other ZnO NPs related studies [ 55 ]. C. acnes act as an "accomplice" to assist ZnO NPs enter the acidified cells When C. acnes was colonized in subcutaneous tissue sites and abnormally proliferated in skin tissues, the significant changes in adjacent cell membrane permeability and cell acidification induced by C. acnes raised a "red light" on the safety of ZnO NPs in acne sites. ZnO NPs might lead to a series of unsafe biological behaviors after entering the skin cells with damaged cell membranes. According to BioTEM images (Fig. 2 A), L929 cells with normal membrane structure (Fig. 2 A-I) showed serious cell membrane damage after co-incubation with C. acnes (Fig. 2 A-II), which was consistent with the results in Fig. 1 . Almost no ZnO NPs entered L929 cells and no serious organelle damage was observed when ZnO NPs were treated with L929 cells alone (Fig. 2 A-III). However, a large number of ZnO NPs crossed the damaged cell membrane into the cell by the assistance of C. acnes , and mitochondrial damage and endoplasmic reticulum vacuoles and damaged nuclei were observed (Fig. 2 A-IV). This confirmed that C. acnes play a key role in the entry of ZnO NPs into skin cells. Furthermore, ICP analysis proved significantly higher intracellular zinc levels in L929 with the help of C. acnes (Fig. 2 B) and the conversion of ZnO to Zn 2+ in the acidified intracellular environment. Confocal laser scanning microscope images and relative quantitative results of Zn 2+ (Fig. 2 C-D) also confirmed the above conclusions. These results indicated that C. acnes , as an "accomplice", assisted ZnO NPs to enter the acidified cells, in which ZnO NPs are further converted to Zn 2+ in an acidic environment. Ionization of ZnO NPs can cause severe cytotoxicity, which is confirmed by CCK-8 assay (Fig. 2 E) and Calcein-AM/PI staining (Fig. 2 F). These results both confirmed that cytotoxicity of L929 cells was not observed after direct contact with ZnO NPs for 9 h but occurred after 12 h, which provided guidance for the use time of sunscreen products containing ZnO NPs. However, L929 cells showed more serious injury and apoptosis at any treatment time under the co-treatment of C. acnes and ZnO NPs. In summary, those above results provided sufficient evidence that C. acnes not only directly damaged L929 cells, but also acted as an "accomplice" to assist ZnO NPs to enter the cell interior and induce cell ion homeostasis interference and cell apoptosis through further ionization of ZnO (Fig. 2 G). Mechanism analysis of skin cell damage induced by the combination of C. acnes and ZnO NPs Since C. acnes can assist ZnO NPs to enter cells by changing membrane permeability. In order to further explore the specific damage mechanism of ZnO NPs on skin cells, intracellular H 2 O 2 , MDA, GSH contents and intracellular ROS were detected. After treating acne tissue with 50 µg/mL ZnO NPs for 9 h, the intracellular H 2 O 2 level was significantly higher than that in normal tissue (P < 0.001, ***). The expression of intracellular MDA and GSH were also consistent with intracellular H 2 O 2 (Fig. 3 A-C), proving the destruction of membrane permeability and oxidative damage of skin cells under the combination of C. acnes and ZnO NPs. In addition, C. acnes + ZnO treatment resulted in a gradual increase in intracellular ROS levels with the increase of time, indicating that C. acnes helped ZnO NPs induce more intense oxidative stress in skin cells (Fig. 3 D, Fig. S11). And C. acnes promoted the decrease of the mitochondrial membrane potential of L929 cells induced by ZnO NPs treatment with the extension of time (Fig. 3 E, Fig S12). Therefore, the above experimental results confirmed the specific signaling pathways and mechanisms by which ZnO NPs damage skin cells with the assistance of C. acnes . Transcriptome analysis Although the mechanism by which C. acnes helps ZnO NPs to enter skin cells has been elucidated, the cytological behavior of ZnO NPs after ionization into the cytoplasm has not been fully understood, in view of which, transcriptome analysis was used to explore the signaling pathways and key targets Zn 2+ is involved in acne skin tissue. L929 cells were treated with ZnO NPs, C. , and C. +ZnO NPs respectively to compare the influence of ZnO NPs on skin cells in normal environment and acne environment. The intergroup correlation behavior analysis (Fig. 4 A) showed significant difference between ZnO group and Control group, and between C. +ZnO group and C. group, indicating remarkable influence of ZnO NPs both on normal and acne skin cells. Specifically, volcano map analysis calculated out 2451 down-regulated and 2808 up-regulated genes in normal skin cells after ZnO NPs treatment (Fig. 4 B), and 40 down-regulated and 79 up-regulated genes in acne cells after ZnO NPs treatment (Fig. 4 C). According to GO functional enrichment analysis and KEGG enrichment analysis, the significantly ZnO-regulating genes in normal skin cells were mainly SMAD protein signal transduction, epithelial- mesenchymal transformation (EMT) and negative regulation of Transforming growth factor β (TGF-β) signaling pathway (Fig. 4 D-E). In classical TGF-β signaling, TGF-β1 first activates TGF-β receptor I (TβRI) and TβRII. TβRII first autophosphorylates and then phosphorylates TβRI, resulting in TβRI activation and C-terminal phosphorylation of SMAD. The phosphorylated SMADs then form complexes with the co-mediators SMAD and SMAD4, which translocate to the nucleus, where they bind to the gene promoter. In collaboration with different transcription factors and cofactors, these complexes control the transcription of hundreds of genes. Although ZnO NPs treatment resulted in similar changes of gene expression of TGF-β, EMT and SMAD in normal skin tissues and acne tissues (Fig. 4 F and G), the regulation of above gene expression by ZnO NPs in acne skin was more remarkable, indicating more serious cell damage in acne tissue caused by ZnO NPs. TGF-β signaling pathway is closely related to the occurrence of EMT. The SMAD-dependent pathway is the most typical pathway for TGF-β signaling. When TGF-β binds to the receptor complex on the cell surface, activated type I receptor TGF-β1 phosphorylates the signaling protein Smad2/3, forms a complex with Smad4, enters the nucleus, binds to other transcription factors SNAI1, SNAI2, ZEB, TWIST1, etc., and interacts with each other. Transcriptome analysis in this study confirmed the interference of ZnO NPs in the down-regulation of TGF-β/SMAD/EMT signaling axis in acne tissue repair process. Next, KEGG enrichment network diagram was constructed and analyzed to further explore the specific mechanism and target of ZnO NPs damage to acne skin cells (Fig. 5 A), and the differential genes concentrated on the TGF-β signaling pathway related to wound healing were determined to be TGF-β1, SMAD2 and SMAD4, which were all down-regulated. To explore the mutual regulatory effects of the proteins corresponding to the differential genes, 119 differential genes were uploaded to the STRING database and the protein-protein interaction (PPI) network was constructed (Fig. 5 B). The top five target proteins with the highest correlation in the network were MMP9, TGF-β1, Egr1, SMAD2 and Atm, and MMP9 was observed to be highly correlated with TGF-β1 and SMAD4 in the TGF-β signaling pathway. Zn 2+ inhibits the activity and maturation of MMP-9 and disrupts TGF-β1/Smad signaling through both direct and indirect pathways. On the one hand, Zn 2+ stabilizes the propeptide-catalytic domain interaction by competitively binding cysteine thiol group (Cys 69 ) in the PRCGVPD motif of the MMP-9 propeptide domain, delaying the activation of zymogen to maintain its latent state [ 56 – 59 ]. On the other hand, high concentration of Zn 2+ inhibits its activity by binding to histidine residues at the catalytic site of serine proteases (such as plasminase), hinding the maturation of pro-MMP-9 [ 60 – 62 ]. Activated MMP-9 can cooperate with the Tolloid proteinase family to cut potential TGF-β, resulting in the release of mature TGF-β1 polypeptide [ 63 ]. Activated TGF-β1 regulates transcription of genes associated with wound healing behavior, including α-smooth muscle actin, type I collagen, and type III collagen, by participating in TGF-β1/Smad signaling. Therefore, combined with previous reports and new findings in this study, MMP9 is considered to be the target of ZnO NPs in downregulating TGF-β signaling pathway and interfering with acne skin tissue repair. Mechanism validation of ZnO NPs participating in TGF-β signaling pathway in an acne microenvironment To further verify the role of Zn 2+ released by ZnO NPs in the regulatory mechanisms and targets of TGF-β signaling pathway in acne skin tissues, L929 cells treated with ZnO NPs, C. , C. +ZnO NPs were detected by Westen-blotting for MMP9, TGF-β and SMAD4, respectively. The results in Fig. 6 A-D showed that the expression of MMP9, TGF-β and SMAD4 related proteins in acne skin cells treated with ZnO NPs were significantly down-regulated. Further, the effect of ZnO NPs on the expression of type I collagen and type III collagen located downstream of the TGF-β signaling pathway was investigated. Immunofluorescence (Fig. 6 E) and relative quantitative results (Fig. 6 F-G) showed the down-regulation of type I collagen and type III collagen in acne skin cells treated with ZnO NPs. Therefore, these results proved the credibility of GO functional analysis and KEGG pathway signaling analysis and confirmed that ZnO NPs can significantly hinder skin healing in acne tissue by interfering with the activity of TGF-β signaling pathway and the reconstruction of collagen network. In vivo validation of ZnO NPs delaying acne skin healing Zn 2+ released by ZnO NPs in acne environment interferes with tissue healing and repair, which was further demonstrated in Sprague-Dawley rat skin (Fig. 7 A). The rats were randomly divided into four groups (five in each group), and the skin of the four groups was treated with PBS, PBS + carbomer + ZnO NPs (PCZ), C. acnes ( C. ), C. + carbomer + ZnO NPs ( C. CZ), respectively, in which the infection of C. acnes resulted in acne wounds on the skin of rats in the C. and C. CZ groups. The skin parts that had been treated at different time points were photographed. Skin pH test strips were used to measure the pHe of normal skin and acne skin (Fig. S13). The results confirmed that the pHe of the skin surface without C. acnes was 5.8, and that of the skin surface infected with C. acnes was 5.5, which confirmed the effect of C. acnes on the skin pHe and its support for the ionization of ZnO NPs in acidic environment. Although the effect of C. acnes on skin surface pH is well established, there is no clear pH boundary between "healthy" and "acne" skin. The decomposition of ZnO NPs into Zn 2+ is ph-dependent. The concentration of Zn 2+ released by ZnO NPs gradually increases with the decrease of pH [ 64 ]. In healthy skin with a pH of about 5.8, a small amount of ZnO NPs is decomposed into Zn 2+ , which can be effectively blocked by a dense cuticle barrier or cleared by epidermal metallothione chelation [ 65 ]. In contrast, due to the release of propionic acid and other fatty acids, more Zn 2+ release of ZnO NPs appeared in acne lesions with a pH of around 5.5. In addition, 20–50 µm micropores in hair follicle corners in acne skin induced further destruction of barrier integrity [ 66 ]. which can help dissolved Zn 2+ penetrate into the dermis along damaged hair follicle channels and interfere with fibroblast function [ 67 ]. The analysis of Zn 2+ content in skin by ICP showed the significantly higher Zn 2+ content in acne skin than that in normal skin (Fig S14), suggesting the promotion of ZnO NPs ionization in acne environment at lower pH. Further, the content of Zn 2+ in the epidermis, dermis and subcutaneous tissues of the skin (Fig S15) confirmed that in normal skin, Zn 2+ mainly distributed in the epidermis, but its concentration in the dermis and subcutaneous tissues could be almost ignored, indicating that Zn 2+ hardly penetrated the skin barrier under normal conditions. However, the concentration of ZnO NPs in epidermal layer, dermis layer and subcutaneous tissue in acne skin was significantly higher than that in normal tissue, suggesting more significant penetration and damage to skin tissues of different depths under acne environment. ZnO NPs treatment caused no significant change in the skin of normal mice (Fig. 7 B) but significantly healing delay in acne skin (Fig. 7 C-D). However, further hematoxylin and eosin (H&E) staining (Fig. 7 E) confirmed that ZnO NPs treatment resulted in the thickening of normal skin stratum corneum, and that the section diameter of acne tissue after ZnO NPs treatment (2.055 mm) was significantly larger than C. group (1.075 mm) at day 7. In addition, ZnO NPs treatment did not significantly affect the growth of bacteria in acne sites (Fig. S16-17), thus ruling out the possibility of the effect of different microbial infections on wound healing. Skin samples of each group were histochemically stained to further clarify the specific mechanism of ZnO NPs on acne tissue wounds. Masson staining (Fig. 8 A-B) showed that ZnO NPs treatment could reduce the content of new collagen fibers in normal skin, and resulted in a more significant decrease in the generation of new collagen fibers in acne sites. Sirius red staining (Fig. 8 C) showed that ZnO NPs treatment significantly reduced the birefringence of type III collagen (green) in acne tissues, indicating a significant decline in the generation of new type III collagen fibers and potential scar risk. Therefore, the Sirius red staining not only verified the results of Masson tricolor staining analysis, but also distinguished the types of collagen in normal skin and acne skin, which was of great significance for the functional evaluation of the new collagen network. In addition, immunohistochemical staining of type III collagen (Fig. 8 D-E) and type I collagen (Fig. 8 F-G) proved that ZnO NPs treatment decreased the content of type I and III collagen fibers in acne skin. Meanwhile, ZnO NPs treatment resulted in a more significant decrease in type III collagen than type I collagen, indicating that ZnO NPs not only delayed the healing of acne skin, but also put the acne skin at risk of scar hyperplasia. At the same time, the expression of mesenchymal marker α-SMA in the tissue of acne skin treated with ZnO NPs was significantly weakened (Fig. 8 H-I), suggesting that ZnO NPs significantly inhibited skin EMT behavior in acne environment, which was consistent with the results of transcriptome analysis. ICP analysis of Zn 2+ at all skin levels (Fig.S15) shows that the content of Zn 2+ in the dermis at the acne site is significantly higher than in the normal dermis and that Zn 2+ down-regulated the expression and activity of MMP-9 by direct combination with the MMP-9 catalytic domain (Zn 2+ dependent active site). This leads to the continuous deposition of collagen fibers and the formation of scars. In addition, L929 cells could achieve self-repair under low bacterial concentration (10 3 -10 4 CFU/mL), but significantly lost self-repair ability and showed irreversible damage and apoptosis under high bacterial concentration (> 10 4 CFU/mL) (Fig. S6). These results suggest that the early abnormal collagen deposition is highly correlated with the repair of L929 cell function, that is, the abnormal collagen deposition caused by infection of low concentration of C. acnes is reversible, while the abnormal collagen deposition caused by infection of high concentration of C. acnes may be irreversible. The target of MMP-9 of Zn 2+ interfering with the healing and repair of acne tissue was confirmed by treatment of MMP-9 activator (MMP-9-IN-1) and MMP-9 inhibitor (β-Neo-Endorphin) (Fig. S14). After the following treatments: C. acnes (C.), C. acnes + Carbomer + ZnO NPs ( C. CZ), C. acnes + Carbomer + ZnO NPs + MMP-9-IN-1 ( C. CZ + M), C. acnes + Carbomer + ZnO + Beta-neo-endorphin ( C. CZ + N), Skin sites were photographed at different time points (Fig. 9 A). Consistent with previous results, ZnO NPs treatment caused a significant delay in acne skin healing. And the combination of ZnO NPs and MMP-9-IN-1 further delayed acne skin healing, but the combined treatment of ZnO NPs with β-Neo-Endorphin accelerated acne skin healing (Fig. 9 B-D). The above results not only further verified the biological mechanism by which Zn 2+ of ZnO NPs blocks wound healing by targeting MMP-9 in the acne environment, but also suggested the efficient reversal of the delay of acne skin healing by combination of ZnO NPs and MMP-9 activator, which provides effective clinical treatment options for acne skin healing interfered by sunscreen products (Fig. 9 E). Conclusion In this work, the effective dose of ZnO NPs was determined based on the commonly used concentration of ZnO NPs in sunscreen products. To evaluate the true state of fibroblasts in the acne environment, cell and animal models of acne were established, respectively. First, the biological behavior of C. acnes to disrupt acne skin homeostasis by inducing acidification and membrane damage in normal cells was revealed. On this basis, the specific molecular mechanism of ZnO NPs in delaying skin tissue healing and inducing scar hyperplasia was confirmed by combined analysis of transcriptomics and antibody neutralization technology. In summary, focusing on the specificity of acne tissue, this work proved the specific mechanism and action pathway of C. acnes acting as an accomplice of ZnO NPs to cause irreversible damage to the acne skin from the animal-cell-molecular level. This study confirmed that ZnO NPs in the acne environment interferes with collagen metabolic homeostasis by inhibiting the MMP-9/TGF-β axis, leading to delayed healing of acne skin. The use of molecular targeting agents enables precise regulation of MMP-9 activity, which helps to reverse the healing delay. In addition, in order to avoid the potential risk of ZnO NPs on acne skin, the preferred choice of cosmetics that do not contain ZnO NPs, modified ZnO formulations (such as pH responsive coatings), or contain MMP-9 activators in acne activities is recommended. According to the conclusion of this work, the biosafety risk of ZnO NPs in sunscreen products is mainly attributed to the release of Zn 2+ and its effects on subsequent cell metabolism and collagen secretion. The surface modification of ZnO NPs is a potential option to reduce the solubility toxicity of ZnO NPs. For example, surface coatings or element doping can effectively regulate the dissolution of ZnO NPs, thereby reducing their potential toxicity and improving product safety. For example, lipid coating significantly improves the stability of ZnO NPs and inhibits the aggregation and dissolution of ZnO NPs, resulting in a reduction of over 60% in the release of Zn² and a threefold reduction in cytotoxicity [ 68 ]. Mannose modification, by coating the ZnO NPs surface via a hydrogen bond network, reduces the Zn 2+ release rate by 50% [ 69 ]. Al 3+ or Mn 2+ doping alters the ZnO electronic structure by lattice substitution, enhancing chemical inertness. For example, the dissolution rate of Al-doped ZnO in the physiological environment is 70% lower than in the undoped system and the antioxidant stability is significantly increased [ 70 ], whereas Mn doping further inhibits ROS production and reduces Zn 2+ mediated cellular damage [ 71 ]. These modification strategies delay the dissolution of ZnO NPs in the acidic environment of the skin and reduce the release of Zn 2+ , providing a guarantee for the safe application of ZnO in sunscreen. This study provides important clinical product development guidance for the care of acne skin: surfacing modified ZnO NPs (such as SiO 2 -coated or lipid-coated ZnO NPs) are recommended for the development of sunscreen products for acne-prone populations. In addition, the concentration of ZnO in sunscreen products is recommended to be limited to less than 10 µg/mL. In addition, a "stage-differentiated" treatment strategy is considered necessary during the clinical repair of acne. Acne patients in the acute stage are recommended to use sunscreens without ZnO NPs and barrier repair drugs, while acne patients in the repair stage are recommended to use MMP-9 activators or Zn 2+ chelating dressings to promote wound healing and reverse scarring. These findings not only provide guidance for the optimization of acne-specific sunscreen products, but also supports the formulation of personalized care programs for acne patients. Furthermore, the cellular intervention targets of ZnO NPs in the unique acne environment was clarified, which shows important clinical guiding significance in the application of sunscreen products in different pathological skin environments as well as clinical protocol for acne skin repair. Declarations Author Contributions F.Z.: Conceptualization, methodology, data curation; T.W.: Data curation, validation; W.W.: Formal Analysis; Y.L.: Resources; Y.Q.: Investigation; D.L.: Software; X.S.: Reagents; X.K.: writing - original draft, Conceptualization, funding acquisition; C. W.: Evaluation of Animal Experiments; J.S.: Conceptualization, supervision, funding acquisition. Acknowledgement F.Z. and T.W. contributed to the work equally and should be regarded as co-first authors. This work was supported by the Natural Science Foundation of Shandong Province (Grant No. ZR2022MH187), National Natural Science Foundation of China (Grant No. 32101137). Materials And Methods Materials Zinc Oxide nanoparticles (ZnO NPs) and Oleic Acid (OA) were provided by Sigma (St Louis, MO). Orlistat, Roswell Park Memorial Institute (RPMI) medium 1640, phosphate buffered saline (PBS), Cell Counting Kit-8 (CCK8), Fluo-3 AM, Calcein AM/PI Double Staining Kit, Lipase (LPS) Activity Assay Kit, Amplex Red Free Fatty Acid(FFA) Assay Kit, Reactive Oxygen Species (ROS) Assay Kit, GSH Assay Kit and Hydrogen Peroxide (H 2 O 2 ) Assay Kit were provided by Beijing Solarbio Science & Technology Co., Ltd. Mito Tracker Red CMXRos, Hoechst 33342, Trypan Blue Staining Cell Viability Assay Kit and BCECF AM were provided by Beyotime, China. Cell Malondialdehyde (MDA) assay kit and Lactate dehydrogenase (LDH) assay kit were provided by Nanjing Jiancheng Biotechnology Research Institute Co., Ltd. Anti-MMP-9, anti-TGF-β1, anti-SMAD-4 and goat anti-rabbit secondary antibody were provided by Wuhan Service-bio Technology Co., Ltd. MMP-9-IN-1 and β-Neo-Endorphin were provided by Shanghai Aladdin Biochemical Technology Co., Ltd. Microbial/cell culture and animals Cutibacterium acnes ( C. acnes ) was provided by Shanghai Microbiological Culture Collection Co., Ltd. Activated C. acnes were inoculated into the Brucella blood AGAR culture plate. The Bradner blood AGAR culture plates inoculated with C. acnes were immediately placed into anaerobic gas-producing bags, which were incubated in a constant temperature incubator (37℃, 5% CO 2 ). Preparation of the bacterial suspension: Cultured C. acnes were diluted in gradient (10 5 CFU/mL, 10 6 CFU/mL, 10 7 CFU/mL, 10 8 CFU/mL, and 10 9 CFU/mL) with FT. The bacterial suspension obtained was used as the bacterial solution for cell experiments. In addition, the bacterial solution was diluted to 10 9 CFU/mL with PBS buffer for animal experiments. NCTC clone 929 cells (L929) were provided by BeyoClickTM, China. At 37℃ and 5% CO 2 , L929 cells were cultured using RPMI 1640 medium, which contained 1% double antibodies (100 μL·mL -1 penicillin and 100 mg·mL -1 streptomycin) and 10% fetal bovine serum. Sprague-Dawley rats (3 w, 160-180 g) were provided by Qingdao Darenfucheng Animal Technology Co., Ltd. Each rat was randomly placed in a cage with free access to food and water. After one week of adaptive feeding, rats were used for the preparation of an acne animal model and other treatments at later stages. Characterization A transmission electron microscope (TEM, HT7700, Hitachi, Japan) was used to observe the microstructure and morphology of the nanoparticles at 80 kV. Scanning electron microscopy (SEM, JSM-7500F, JEOL, Japan) was used to analyze the surface morphology of the nanoparticles at 2 kV. A Zeta Simeter (ZEN3700, Malvern, Germany) was used to measure the Zeta potential and dynamic light scattering (DLS) of the nanoparticles. X-ray powder diffractometer (XRPD; Crystal phase analysis was performed using a Bruker D8 Advance X-ray diffractometer. A TCS SP5 laser scanning microscope (CLSM, LEICA, Germany) was used to acquire fluorescence images of cells or tissues. The CCK-8 method was used to detect cell viability after treatment of nanoparticles at 450 nm on Microplate Reader (Thermo Scientific Multiskan™ SkyHigh). Fluorescence microscopy (Olympus, Japan) was used to obtain immunofluorescence images of the cells. Determination of pH and lipase content of bacterial supernatants Activated C. acnes were seeded on FT at 4℃ at concentrations of 10 5 CFU/mL, 10 6 CFU/mL, 10 7 CFU/mL, 10 8 CFU/mL and 10 9 CFU/mL respectively. C. acnes supernatant was obtained by centrifugation after being cultured for 6, 12, and 24 h in a cell incubator at 37℃ and 5% CO 2 . The pH of the supernatant at different time periods was measured by a pH meter (PB-10). In addition, FT inoculated with C. acnes (10 9 CFU/mL) was simultaneously supplemented with 10 μM Orlistat, which was labeled as C. + Orlistat group. And C. + Orlistat group was cultured in a cell incubator at 37℃ for 6, 12, and 24 h, respectively. The supernatant was obtained by centrifugation, and the lipase content secreted by C. acnes at each time point was detected by lipase test kit. Construction of an in vitro model of acne-like L929 cells 2 mL of L929 cell suspension (1×10 5 cells/well) was seeded into 12-well plates for culture. At 70% to 80% confluence of the cells, the old medium was replaced with 2 mL of a new medium pH 6.4, which was labeled as the pH 6.4 group. Then, 1 mL C. acnes suspension (1×10 9 CFU/mL) was inoculated in transwell chambers, which were immersed in 12-well plates of L929 cells cultured with normal medium for 6 h. therefore, an in vitro model of acne-like L929 cells was established. which was labeled as C. acnes ( C. ) group. At the same time as C. acnes suspension was inoculated in a transwell chamber, 10 μL of 1 mM Orlistat was added and then immersed in 12-well plates of L929 cells cultured with normal medium for 6 h and labeled as C. + Orlistat group. Analysis of the effect of C. acnes treatment on free fatty acid content L929 cells were treated and grouped according to the above method: control group, pH 6.4 group, C. Group and C. + Orlistat group. L929 cells in all groups were treated for 6 h and then were treated with amplex red free fatty acid assay kit respectively. Analysis of the effect of C. acnes and ZnO NPs treatment on intracellular ROS production L929 cells were given different treatments: control group, pH 6.4 group, C. group, C. + Orlistat group, control + ZnO NPs (50 μg/mL) group and C. + ZnO NPs (50 μg/mL) group. L929 cells in the first four groups were treated for 6 h. L929 cells in the last two groups were treated for 0, 3, 6, 9, 12 h and then were treated with Reactive Oxygen Species Assay Kit respectively. Analysis of the influence of C. acnes and ZnO NPs treatment on intracellular MDA and GSH L929 cells were given different treatments: control group, pH 6.4 group, C. group, C. + Orlistat group, control + ZnO NPs (50 μg/mL) group and C. + ZnO NPs (50 μg/mL) group. L929 cells in the first four groups were treated for 6 h. L929 cells in the last two groups were treated for 0, 3, 6, 9, 12 h and then were treated with malondialdehyde kit and glutathione kit respectively. Analysis of the influence of C. acnes treatment on LDH content in cell supernatant L929 cells were given different treatments: control group, pH 6.4 group, C. Group with different concentrations (10 5 CFU/mL, 10 6 CFU/mL, 10 7 CFU/mL, 10 8 CFU/mL, 10 9 CFU/mL) and C. + Orlistat group (10 9 CFU/mL C. acnes + 10 μL/1 mM Orlistat). L929 cells in each group were treated for 6, 12, 24 h and then were treated with Lactate dehydrogenase assay kit respectively. Analysis of the influence of C. acnes treatment on membrane permeability L929 cells were given different treatments: control group, pH 6.4 group, C. Group with different concentrations (10 5 CFU/mL, 10 6 CFU/mL, 10 7 CFU/mL, 10 8 CFU/mL, 10 9 CFU/mL) and C. + Orlistat group (109 CFU/mL C. acnes + 10 μL/1 mM Orlistat). L929 cells in each group were treated for 6, 12, 24 h and then were treated with Trypan Blue Staining Cell Viability Assay Kit respectively. Analysis of the influence of C. acnes treatment on intracellular pH L929 cells were given different treatments: control group, pH 6.4 group, C. Group and C. + Orlistat group. L929 cells in all groups were treated for 6 h and then were treated with BCECF AM respectively. BioTEM for ZnO NPs endocytosis L929 cells were given different treatments: control group, pH 6.4 group, ZnO NPs Group and ZnO NPs + Orlistat group (ZnO NPs: 50 μg/mL). L929 cells in all groups were treated for 6 h. The cells were washed by PBS and collected in a centrifuge tube, and then were fixed with 2.5% (v/v) glutaraldehyde. Finally cells in each group were subjected to bioTEM and ZnO NPs endocytosis was observed. Cytotoxicity evaluation 200 μL L929 cells (1×10 4 cells/well) were cultured in 96-well plates for 24 h. The cells are divided into the following five groups: Control group, C. group, Orlistat group, ZnO NPs group, C. + ZnO NPs group. For the control group, L929 cells were cultured under standard conditions. For C. group, 200 μL of C. acnes suspensions with different concentrations (10 5 CFU/mL, 10 6 CFU/mL, 10 7 CFU/mL, 10 8 CFU/mL, 10 9 CFU/mL) were inoculated into 96-well plates which had been cultured with L929 cells, and the C. acnes /cells mixed solutions were incubated for 3, 6, 9, 12 h, 24 h. For the Orlistat group, 100 μL C. acnes suspension (2×10 9 CFU/mL) and 100 μL of Orlistat (20 μM) solution were added to 96-well plates containing L929 cells, and all the above components were incubated for 6, 12, and 24 h. for ZnO NPs group, 12.5 μg·mL -1 , 25 μg·mL -1 and 50 μg·mL -1 ZnO NPs were added to L929 cell culture medium and incubated for 3, 6, 9 and 12 h, respectively. For the C. +ZnO NPs group, 200 μL C. acnes suspension (1×10 9 CFU/mL) was inoculated on a 96-well plate in which L929 cells had been cultured, and the mixed solutions were incubated for 6 h. After that, the old mediums were removed and all the cells were cleaned twice with PBS. L929 cells were then treated with 12.5 μg·mL -1 , 25 μg·mL -1 and 50 μg·mL -1 ZnO NPs for 3, 6, 9 and 12 h, respectively. All groups were treated with Cell Counting Kit-8 respectively. Inductively coupled plasma (ICP) for Zn 2+ content analysis L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 μg·mL -1 ) group, C. +ZnO NPs (50 μg·mL -1 ) group. L929 cells in all groups were treated for 0, 3, 6, 12 h. The cells were broken under an ice water bath with an ultrasonic cell breaker. The intracellular Zn 2+ content was detected by inductively coupled plasma emission spectrometer. There were three parallels in each group. Analysis of intracellular Zn 2+ content L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 μg·mL -1 ) group, C. +ZnO NPs (50 μg·mL -1 ) group. L929 cells in all groups were treated for 0, 3, 6, 12 h and then were treated with Zinquin ethyl ester respectively. Evaluation of mitochondrial membrane potential and apoptosis of L929 cells L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 μg·mL -1 ) group, C. + ZnO NPs (50 μg·mL -1 ) group. L929 cells in all groups were treated for 0, 3, 6, 9, 12 h and then were treated with Mito Tracker Red CMXRos and Calcein AM/PI Double Staining Kit respectively. Detection of type I collagen and type III collagen secretion L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 μg·mL -1 ) group, C. +ZnO NPs (50 μg·mL -1 ) group. L929 cells in all groups were treated for 6 h. All cells were then incubated overnight with COL-I and COL-III antibodies at 4℃, then were incubated with Cyanine 3-labeled goat anti-rabbit immunoglobulin G (IgG) at room temperature away from light. The cells were then added to 4', 6-diaminidine 2-phenylindole dye and incubated at room temperature away from light. The cells were encapsulated and photographed by fluorescence microscopy. Western-blotting analysis of MMP9 、 TGF-β1 和 SDAD4 L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 μg·mL -1 ) group, C. + ZnO NPs (50 μg·mL -1 ) group. L929 cells in all groups were treated for 6 h. Nucleoprotein and cytoplasmic protein extraction kits containing 1 mM phosphatase inhibitors were used to treat cells after PBS washing. The cell lysate was heated in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer. The proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane, where the separation process was then blocked by skim milk. The polyvinylidene fluoride film was incubated with primary antibodies including anti-MMP9, anti-TGF-β1 and anti-SDAD4 at 4℃ overnight. After washing with TBS containing Tween-20 (TBST) buffer, polyvinylidene fluoride film coupled with horseradish peroxidase (HRP) was incubated at room temperature. Enhanced chemiluminescent reagent (ECL) kits and automated chemiluminescence image analysis systems were used to detect the expression of MMP9, TGF-β1 and SDAD4 in the cells of above groups. Transcriptome analysis L929 cells were divided into the following four groups: control group, C. group, ZnO NPs (50 μg·mL -1 ) group, C. +ZnO NPs (50 μg·mL -1 ) group. L929 cells in all groups were treated for 6 h. The cells in each group were added with total RNA extraction reagent (TRIzol) reagent. The subsequent RNA sequencing was performed by eukaryotic mRNA sequencing based on Illumina sequencing platform by Shanghai NOhe Zhiyuan Bioinformation Technology Co., LTD. DESeq2 software was used for ZnO NPs group VS control group and C. +ZnO NPs group VS C. group transcriptional behavior differential analysis (DEGs) and further functional analysis of DEGs. After the differential genes were obtained through gene expression analysis, the functions of differential genes were enriched and analyzed to find the signaling pathways and molecular regulatory mechanisms that play a key role in the differential biological behavior. ClusterProfiler software was used to complete GO functional enrichment analysis and KEGG path enrichment analysis of DEGs sets. Among them, GO analysis, which is based on the GO database (Gene Ontology), was responsible for the classification of genes according to Biological process (BP), Cellular component (Cellular component), biological process (BP). CC) and Molecular function (MF). KEGG pathway enrichment, which is based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database, was responsible for statistical analysis of the differential signal pathways. After the target genes were identified, Pearson or Spearman algorithm was used to obtain the correlation coefficient between genes, which provided support for the drawing of the visual network map. In this work, STRING database was used for interaction network analysis of target proteins. In addition, Cytoscape software was adopted for Protein-protein interaction (PPI) analysis. Preparation of ZnO NPs supported gels 0.1g of carbomer powder was added to 10 mL of PBS buffer. The solution was stirred evenly, and 1% (m/v) carbomer gel was prepared. In addition, 50 μg/mL PBS solution of ZnO NPs was prepared and ultrasounded for 30 min. After 10 mL of the above ZnO NPs suspension was added with 0.1g carbomer powder and the solution was stirred evenly, ZnO NPs (50 μg/mL) carbomer gel was prepared. Evaluation of in vivo effects of ZnO NPs on acne skin tissue In this study, all animal procedures were conformed to the Guide for the Care and Use of Laboratory Animals and performed following the guidelines and the protocol approved by the application of the experimental animal ethical project of Qingdao Agricultural University (Approval No.20220072). After the rats were fed adaptively for one week and the rats' back hair was shaved, 0.5 mL oleic acid was applied once a day to the back skin for 7 days. From day 8, 0.2 mL C. acnes (1×10 9 CFU/mL) was injected into the dermis for 14 days, and acne-like inflammatory lesion model was established. The rats were randomly divided into six groups: Control group (PBS, n=5), Carbomer + ZnO NPs group (CZ, n=5), C. acnes group ( C. , n=5), C. acnes + Carbomer + ZnO NPs group ( C. CZ, n=5), C. acnes + Carbomer + ZnO NPs + MMP-9-IN -1 group (CCZ+M, n=5), C. acnes + Carbomer + ZnO NPs + β-Neo-Endorphin group ( C. CZ+N, n=5). From the 15th day to the 21st day of modeling, rats in different groups were treated as follows: for control group and C. acnes group, 1 mL PBS was applied on the back of rats every day; for the CZ group and the C. Z group, the back of rats was coated with 1 mL ZnO NPs carbomer gel every day. for C. CZM group, the back of rats was coated with 1 mL ZnO NPs and MMP-9-IN-1 carbomer gel every day. for the C. CZβ group, rats were coated with 1 mL ZnO NPs and β-Neo-Endorphin carbomer gel daily on their backs. The acne wounds were photographed on day 15 and day 21 respectively, and the area of acne wounds was recorded. On day 22, all anesthetized rats were killed by neck amputation and then microsurgical scissors were used to separate the acne wounds on the back of the rats. Skin pH value, Bacterial colony count, Zn level, HE staining, Masson staining, Sirius red staining, and immunohistochemical analysis were respectively performed on the back acne wounds. Specifically, the wound tissues of the Control group, CZ group, C. group and C. CS group on the last day of treatment were placed in 4% paraformaldehyde, dehydrated by alcohol gradients of different concentrations, embedded in paraffin wax and were prepared into tissue sections with thickness of 5 mm. When measuring the skin's pH, the test strip was first moistened in distilled water, then applied to both normal skin and acne areas. It was removed after five seconds, and the color was compared with the reference chart provided on the test strip container. When the number of bacteria in the wound tissue was measured, the wound tissue of the C. and C. C groups was placed in sterile PBS on the last day. After homogenizing and continuously diluting with sterile PBS, the diluent was inoculated on Columbia blood AGAR plate and incubated at 37℃ for 36 h. Finally, C. acnes was selectively isolated and the total number of colonies was counted. In order to determine the pathological condition of the acne tissue, the wound tissue was analyzed by HE staining. After dewaxing and H&E staining, the tissue sections were viewed microscopically and photographed in full scan. In order to evaluate the effects of ZnO NPs on collagen fibers and muscle fibers in acne tissues, acne tissues were subjected to Masson's trichrome staining analysis. After undergoing dewaxing and Masson staining, sections of acne tissue were observed and photographed. To evaluate the effects of ZnO NPs on type I collagen and type III collagen in acne tissues, the acne tissues were stained with Sirius red. After dewaxing, Sirius red staining, the tissue sections were placed under polariscope for observation and photography of collagen fibers. In addition, the distribution of type I collagen, type III collagen and smooth actin in acne tissues was also confirmed by immunohistochemical analysis. The acne tissue sections of each group were labeled with Collagen I, Collagen III and α-SMA monoclonal antibodies, respectively, and then incubated with second anti-antibody. Finally, the section samples were viewed under a fluorescence microscope and photographed. To determine Zn levels in acne tissues, acne tissues of each group were placed on sterile PBS on the last day of treatment to remove residual ZnO NPs. After that, inductively coupled plasma analyzer was used to determine the content of Zn in the tissues. References Szabó K, Bolla BS, Erdei L, Balogh F, Kemény L. Are the Cutaneous Microbiota a Guardian of the Skin’s Physical Barrier? The Intricate Relationship between Skin Microbes and Barrier Integrity. Int J Mol Sci. 2023;24:15962. Eyerich S, Eyerich K, Traidl-Hoffmann C, Biedermann T. Cutaneous Barriers and Skin Immunity: Differentiating A Connected Network. Trends Immunol. 2018;39:315–27. Rajkumar J, Chandan N, Lio P, Shi V. The Skin Barrier and Moisturization: Function, Disruption, and Mechanisms of Repair. Skin Pharmacol Physiol. 2023;36:174–85. Naik S, Bouladoux N, Linehan JL, Han SJ, Harrison OJ, Wilhelm C, et al. Commensal-dendritic-cell interaction specifies a unique protective skin immune signature. Nature. 2015;520:104–8. Harris-Tryon TA, Grice EA. Microbiota and maintenance of skin barrier function. Science. 2022;376:940–5. Alwan W, Di Meglio P. Guardians of the barrier: Microbiota engage AHR in keratinocytes to mantain skin homeostasis. Cell Host Microbe. 2021;29:1213–6. Lee H-J, Kim M. Skin Barrier Function and the Microbiome. Int J Mol Sci. 2022;23:13071. Boyanova L. Cutibacterium Acnes (Formerly Propionibacterium Acnes): Friend or Foe? Future Microbiology. 2023; 18:235 – 44. Scharschmidt TC. Antibiotics for Acne—A Pilot Study of Collateral Damage to the Skin Microbiome. JAMA Dermatology. 2019;155:419–21. Mempel M, Schmidt T, Weidinger S, Schnopp C, Ring J, Abeck D, et al. Role of Staphylococcus Aureus Surface-Associated Proteins in the Attachment to Cultured HaCaT Keratinocytes in a New Adhesion Assay. J Invest Dermatology. 1998;111:452–6. Namjoshi S, Caccetta R, Benson HAE. Skin Peptides: Biological Activity and Therapeutic Opportunities. J Pharm Sci. 2008;97:2524–42. Goodarzi H, Trowbridge J, Gallo RL. Innate Immunity: A Cutaneous Perspective. Clin Rev Allerg Immunol. 2007;33:15–26. Grice EA, Kong HH, Conlan S, Deming CB, Davis J, Young AC, et al. Topographical and Temporal Diversity of the Human Skin Microbiome. Science. 2009;324:1190–2. Harris-Tryon TA, Grice EA. Microbiota and maintenance of skin barrier function. Science. 2022;376:940–5. Dréno B, Pécastaings S, Corvec S, Veraldi S, Khammari A, Roques C. Cutibacterium acnes (Propionibacterium acnes) and acne vulgaris: a brief look at the latest updates. J Eur Acad Dermatol Venereol. 2018;32:5–14. Youn SH, Choi CW, Choi JW, Youn SW. The skin surface pH and its different influence on the development of acne lesion according to gender and age. Skin Res Technol. 2013;19:131–6. Contassot E, French LE. New Insights into Acne Pathogenesis: Propionibacterium Acnes Activates the Inflammasome. J Invest Dermatol. 2014;134:310–3. Agak GW, Qin M, Nobe J, Kim M-H, Krutzik SR, Tristan GR, et al. Propionibacterium acnes Induces an IL-17 Response in Acne Vulgaris that Is Regulated by Vitamin A and Vitamin D. J Invest Dermatology. 2014;134:366–73. Lim H-J, Kang S-H, Song Y-J, Jeon Y-D, Jin J-S. Inhibitory Effect of Quercetin on Propionibacterium acnes-induced Skin Inflammation. Int Immunopharmacol. 2021;96:107557. Serpone N, Dondi D, Albini A. Inorganic and organic UV filters: Their role and efficacy in sunscreens and suncare products. Inorg Chim Acta. 2007;360:794–802. Scharffetter-Kochanek K, Wlaschek M, Brenneisen P, Schauen M, Blaudschun R, Wenk J. UV-Induced Reactive Oxygen Species in Photocarcinogenesis and Photoaging Biol. Biol Chem. 1997;378:1247–57. Katsambas A, Nicolaidou E. Cutaneous malignant melanoma and sun exposure. Recent developments in epidemiology. Arch Dermatol. 1996;132:444–50. Singh TA, Das J, Sil PC. Zinc oxide nanoparticles: A comprehensive review on its synthesis, anticancer and drug delivery applications as well as health risks. Adv Colloid Interface Sci. 2020;286:102317. Leite-Silva VR, Sanchez WY, Studier H, Liu DC, Mohammed YH, Holmes AM, et al. Human skin penetration and local effects of topical nano zinc oxide after occlusion and barrier impairment. Eur J Pharm Biopharm. 2016;104:140–7. Zvyagin AV, Zhao X, Gierden A, Sanchez W, Ross JA, Roberts MS. Imaging of zinc oxide nanoparticle penetration in human skin in vitro and in vivo. J Biomed Opt. 2008;13:064031. Roberts MS, Roberts MJ, Robertson TA, Sanchez W, Thörling C, Zou Y, et al. In vitro and in vivo imaging of xenobiotic transport in human skin and in the rat liver. J Biophotonics. 2008;1:478–93. Holmes AM, Song Z, Moghimi HR, Roberts MS. Relative Penetration of Zinc Oxide and Zinc Ions into Human Skin after Application of Different Zinc Oxide Formulations. ACS Nano. 2016;10:1810–9. Watkinson AC, Bunge AL, Hadgraft J, Lane ME. Nanoparticles do not penetrate human skin–a theoretical perspective. Pharm Res. 2013;30:1943–6. Filipe P, Silva JN, Silva R, Cirne de Castro JL, Marques Gomes M, Alves LC, et al. Stratum corneum is an effective barrier to TiO2 and ZnO nanoparticle percutaneous absorption. Skin Pharmacol Physiol. 2009;22:266–75. Chen R-J, Lee Y-H, Yeh Y-L, Wang Y-J, Wang B-J. The Roles of Autophagy and the Inflammasome during Environmental Stress-Triggered Skin Inflammation. Int J Mol Sci. 2016;17:2063. Faa G, Nurchi VM, Ravarino A, Fanni D, Nemolato S, Gerosa C, et al. Zinc in gastrointestinal and liver disease. Coord Chem Rev. 2008;252:1257–69. Choi S, Cui C, Luo Y, Kim S-H, Ko J-K, Huo X, et al. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orail. FASEB J. 2018;32:404–16. Cen D, Ge Q, Xie C, Zheng Q, Guo J, Zhang Y et al. ZnS@BSA Nanoclusters Potentiate Efficacy Cancer Immunotherapy Adv Mater. 2021; 49: n/a-n/a Zhao H, Li T, Yao C, Gu Z, Liu C, Li J, et al. Dual Roles of Metal-Organic Frameworks as Nanocarriers for miRNA Delivery and Adjuvants for Chemodynamic Therapy. ACS Appl Mater Interfaces. 2021;13:6034–42. Su Y, Cockerill I, Wang Y, Qin Y-X, Chang L, Zheng Y, et al. Zinc-Based Biomaterials for Regeneration and Therapy. Trends Biotechnol. 2019;37:428–41. Li M, Ma Y, Lian X, Lu Y, Li Y, Xi Y, et al. Study on the biological effects of ZnO nanosheets on EBL cells. Front Bioeng Biotechnol. 2022;10:915749. Yan J, Liu C, Wu Q, Zhou J, Xu X, Zhang L, et al. Mineralization of pH-Sensitive Doxorubicin Prodrug in ZIF-8 to Enable Targeted Delivery to Solid Tumors. Anal Chem. 2020;92:11453–61. Zheng P, Ding B, Zhu G, Wang M, Li C, Lin J. Biodegradable Hydrogen Peroxide Nanogenerator for Controllable Cancer Immunotherapy Via Modulating Cell Death Pathway from Apoptosis to Pyroptosis. Chem Eng J. 2022;450:137967. Du M, Chen ZJ. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science. 2018;361:704–9. Moradi Tuchayi S, Makrantonaki E, Ganceviciene R, Dessinioti C, Feldman SR, Zouboulis CC. Acne vulgaris. Nat Rev Dis Primers. 2015;1:15029. Schreml S, Szeimies R, Karrer S, Heinlin J, Landthaler M, Babilas P. The impact of the pH value on skin integrity and cutaneous wound healing. Acad Dermatol Venereol. 2010;24:373–8. Ohman H, Vahlquist A. In vivo studies concerning a pH gradient in human stratum corneum and upper epidermis. Acta Derm Venereol. 1994;74:375–9. Schreml S, Meier RJ, Kirschbaum M, Kong SC, Gehmert S, Felthaus O, et al. Luminescent Dual Sensors Reveal Extracellular pH-Gradients and Hypoxia on Chronic Wounds That Disrupt Epidermal Repair. Theranostics. 2014;4:721–35. Yu T, Xu X, Liu Y, Wang X, Wu S, Qiu Z, et al. Multi-omics signatures reveal genomic and functional heterogeneity of Cutibacterium acnes in normal and diseased skin. Cell Host Microbe. 2024;32:1129–e11468. Fluhr JW, Kao J, Jain M, Ahn SK, Feingold KR, Elias PM. Generation of Free Fatty Acids from Phospholipids Regulates Stratum Corneum Acidification and Integrity. J Invest Dermatol. 2001;117:44–51. Carney SA, Hall M, Ricketts CR. The adenosine triphosphate content and lactic acid production of guinea-pig skin after mild heat damage. Br J Dermatol. 1976;94:291–4. Zhong G, Guo Y, Gong X, Xu M, Wang Q, Wu M et al. Enhanced glycolysis by ATPIF1 gene inactivation increased the anti-bacterial activities of neutrophils through induction of ROS and lactic acid. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2023; 1869:166820. Almoughrabie S, Cau L, Cavagnero K, O’Neill AM, Li F, Roso-Mares A, et al. Commensal Cutibacterium acnes induce epidermal lipid synthesis important for skin barrier function. Sci Adv. 2023;9:eadg6262. Jeong S, Lee J, Im BN, Park H, Na K. Combined photodynamic and antibiotic therapy for skin disorder via lipase-sensitive liposomes with enhanced antimicrobial performance. Biomaterials. 2017;141:243–50. Strauss JS, Stranieri AM. Acne treatment with topical erythromycin and zinc: Effect on Propionibacterium acnes and free fatty acid composition. J Am Acad Dermatol. 1984;11:86–9. Kang R, Zeng L, Zhu S, Xie Y, Liu J, Wen Q, et al. Lipid Peroxidation Drives Gasdermin D-Mediated Pyroptosis in Lethal Polymicrobial Sepsis. Cell Host Microbe. 2018;24:97–e1084. Borovicka J, Schwizer W, Guttmann G, Hartmann D, Kosinski M, Wastiel C, et al. Role of lipase in the regulation of postprandial gastric acid secretion and emptying of fat in humans: a study with orlistat, a highly specific lipase inhibitor. Gut. 2000;46:774–81. Mohammed YH, Holmes A, Haridass IN, Sanchez WY, Studier H, Grice JE, et al. Support for the Safe Use of Zinc Oxide Nanoparticle Sunscreens: Lack of Skin Penetration or Cellular Toxicity after Repeated Application in Volunteers. J Invest Dermatol. 2019;139:308–15. Smijs TG, Pavel S. Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus on their safety and effectiveness. NSA. 2011;4:95–112. Alenezi NA, Al-Qurainy F, Tarroum M, Nadeem M, Khan S, Salih AM, et al. Zinc Oxide Nanoparticles (ZnO NPs), Biosynthesis, Characterization and Evaluation of Their Impact to Improve Shoot Growth and to Reduce Salt Toxicity on Salvia officinalis In Vitro Cultivated. Processes. 2022;10:1273. Han H, Du B, Pan X, Liu J, Zhao Q, Lian X, et al. CADPE inhibits PMA-stimulated gastric carcinoma cell invasion and matrix metalloproteinase-9 expression by FAK/MEK/ERK-mediated AP-1 activation. Mol Cancer Res. 2010;8:1477–88. Kjeldsen L, Johnsen AH, Sengeløv H, Borregaard N. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem. 1993;268:10425–32. Jeong Y-J, Cho H-J, Chung F-L, Wang X, Hoe H-S, Park K-K, et al. Isothiocyanates suppress the invasion and metastasis of tumors by targeting FAK/MMP-9 activity. Oncotarget. 2017;8:63949–62. Augoff K, Hryniewicz-Jankowska A, Tabola R, Stach K. MMP9: A Tough Target for Targeted Therapy for Cancer. Cancers (Basel). 2022;14:1847. Schmidt AE, Shah P, Gauthier EM, Bajaj SP. Protease Domain Sodium, Calcium, and Zinc Sites in Factor VIIa: Crystal Structures and Kinetic Studies. Blood. 2004;104:122–122. Kanuru M, Raman R, Aradhyam GK. Serine Protease Activity of Calnuc. J Biol Chem. 2013;288:1762–73. Yeung K-S, Meanwell NA, Qiu Z, Hernandez D, Zhang S, McPhee F, et al. Structure–activity relationship studies of a bisbenzimidazole-based, Zn2+-dependent inhibitor of HCV NS3 serine protease. Bioorg Med Chem Lett. 2001;11:2355–9. Kobayashi T, Kim HJ, Liu X, Sugiura H, Kohyama T, Fang Q, et al. Matrix metalloproteinase-9 activates TGF-β and stimulates fibroblast contraction of collagen gels. Am J Physiol Lung Cell Mol Physiol. 2014;306:L1006–15. Bian S-W, Mudunkotuwa IA, Rupasinghe T, Grassian VH. Aggregation and Dissolution of 4 nm ZnO Nanoparticles in Aqueous Environments: Influence of pH, Ionic Strength, Size, and Adsorption of Humic Acid. Langmuir. 2011;27:6059–68. Sun F, Li R, Jin F, Zhang H, Zhang J, Wang T, et al. Dopamine/zinc oxide doped poly( N -hydroxyethyl acrylamide)/agar dual network hydrogel with super self-healing, antibacterial and tissue adhesion functions designed for transdermal patch. J Mater Chem B. 2021;9:5492–502. Tegenaw AB, Yimer AA, Beyene TT. Boosting the photocatalytic activity of ZnO-NPs through the incorporation of C-dot and preparation of nanocomposite materials. Heliyon. 2023;9:e20717. Liu X, Li J, Zhu L, Huang J, Zhang Q, Wang J, et al. Mechanistic insights into zinc oxide nanoparticles induced embryotoxicity via H3K9me3 modulation. Biomaterials. 2024;311:122679. Cao D, Shu X, Zhu D, Liang S, Hasan M, Gong S. Lipid-coated ZnO nanoparticles synthesis, characterization and cytotoxicity studies in cancer cell. Nano Convergence. 2020;7:14. Piasek A, Kominko H, Zielina M, Banach M, Pulit-Prociak J. Mannose-modified ZnO particles for controlled Zn 2+ release as potential drug carriers. Chem Pap. 2025;32:287–6. Tseng Z-L, Chiang C-H, Chang S-H, Wu C-G. Surface engineering of ZnO electron transporting layer via Al doping for high efficiency planar perovskite solar cells. Nano Energy. 2016;28:311–8. Xie Q, Liu P, Zeng D, Xu W, Wang L, Zhu Z, et al. Dual Electrostatic Assembly of Graphene Encapsulated Nanosheet-Assembled ZnO‐Mn‐C Hollow Microspheres as a Lithium Ion Battery Anode. Adv Funct Mater. 2018;28:1707433. Additional Declarations No competing interests reported. Supplementary Files supplementary.docx Graphicalabstract.docx Revisedsupplementary.docx Cite Share Download PDF Status: Published Journal Publication published 31 May, 2025 Read the published version in Journal of Nanobiotechnology → Version 1 posted Editorial decision: Accepted 22 Apr, 2025 Reviews received at journal 08 Apr, 2025 Reviewers agreed at journal 08 Apr, 2025 Reviewers invited by journal 08 Apr, 2025 Submission checks completed at journal 04 Apr, 2025 First submitted to journal 01 Apr, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5823650","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":440772502,"identity":"9353158d-5d9a-4dec-b5a9-9c571f8a616e","order_by":0,"name":"Fenglan Zhang","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fenglan","middleName":"","lastName":"Zhang","suffix":""},{"id":440772503,"identity":"638dbe5c-889a-4f06-be05-70551286e7cb","order_by":1,"name":"Tianyi Wang","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Tianyi","middleName":"","lastName":"Wang","suffix":""},{"id":440772504,"identity":"5277df4f-7aa0-462c-a3ae-9c0cf436d7c9","order_by":2,"name":"Wenqiao Wang","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Wenqiao","middleName":"","lastName":"Wang","suffix":""},{"id":440772505,"identity":"7888a259-cbc0-4262-9fc2-a78772bac356","order_by":3,"name":"Yaqian Lv","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yaqian","middleName":"","lastName":"Lv","suffix":""},{"id":440772506,"identity":"880c3f4d-c0c0-44b8-a1f6-237302d86619","order_by":4,"name":"Yingshan Qu","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yingshan","middleName":"","lastName":"Qu","suffix":""},{"id":440772507,"identity":"99c6fad4-a1e0-4f3c-9e2c-23d9037d3d98","order_by":5,"name":"Danping Liu","email":"","orcid":"","institution":"Qingdao Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Danping","middleName":"","lastName":"Liu","suffix":""},{"id":440772508,"identity":"bc972618-a89a-4baa-8be4-57f8a1839cc8","order_by":6,"name":"Xiaoyue Sun","email":"","orcid":"","institution":"Talent Beauty Biotech (Qingdao) Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyue","middleName":"","lastName":"Sun","suffix":""},{"id":440772509,"identity":"ee9588b9-a9e5-44d6-ae23-2a37ddda8fe4","order_by":7,"name":"Xiaoying Kong","email":"","orcid":"","institution":"Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoying","middleName":"","lastName":"Kong","suffix":""},{"id":440772510,"identity":"7e35aa61-2b08-462e-9c4b-846fd4ab42a4","order_by":8,"name":"Changyuan Wang","email":"","orcid":"","institution":"University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital)","correspondingAuthor":false,"prefix":"","firstName":"Changyuan","middleName":"","lastName":"Wang","suffix":""},{"id":440772511,"identity":"79391d00-da74-418d-95f3-a51d58ee84df","order_by":9,"name":"Jinsheng Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACCcYGgwQGBiBiYHwAEUogXguzAZFaEMrYkNh4gPzs5oaChzvq8vil269V81TcY+BnzzFg+LkDtxaDOwcbDBLPHC6WnHOm7DbPmWIGyZ43Boy9Z/BokUgEamk7kLjhRk7abd62BAaDGzkGzIxteBw2A6ylLnE/UEsxSIs9IS0MN8BamBM3SKQfYwbbIkFAiwFEy+HEGTdymIH+SeCROPOs4GAvXoelPzP8CXRY/4z0hx/eVCTI8bcnb3zwE5/DgNEBjUEeMM0DIg7g1QCM9AcQmv0BAYWjYBSMglEwUgEAeSFULxsrI9QAAAAASUVORK5CYII=","orcid":"","institution":"University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital)","correspondingAuthor":true,"prefix":"","firstName":"Jinsheng","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-01-14 03:23:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5823650/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5823650/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-025-03414-x","type":"published","date":"2025-05-31T15:57:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80345119,"identity":"dc18b8b5-f7ec-4dfa-960a-bbd0ee40d64d","added_by":"auto","created_at":"2025-04-10 19:34:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":230592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. acnes\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e on skin cell membrane and pH and mechanism analysis. \u003c/strong\u003e(A) Lipase content of \u003cem\u003eC. acnes\u003c/em\u003esupernatant. (B) Free fatty acid content of L929 cell supernatant. (C) Laser confocal images of L929 cells stained with DCFH-DA. (D-F) ELISA results of MDA (D), GSH (E) and LDH (F) in L929 cells. (G) Trypan blue staining images of L929 cells (scale bar: 50 μm). (H) L929 cell BCECF/AM dye laser confocal images (scale bar: 100 μm). (I) Fluorescence relative quantification results from laser confocal images of intracellular BCECF/AM staining.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/c537fd3c87c9b9e91abf5ee1.jpg"},{"id":80345120,"identity":"3b45805d-6dd6-4b26-a162-ff4ec94926cd","added_by":"auto","created_at":"2025-04-10 19:34:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":540915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the damage mechanism of ZnO NPs on L929 cells in the acne environment. \u003c/strong\u003e(A) Biological transmission electron microscopy of L929 cells after different treatments (I: Control; II: 50 μg/mL ZnO NPs; III: \u003cem\u003eC.\u003c/em\u003e; IV: \u003cem\u003eC.\u003c/em\u003e + 50 μg/mL ZnO NPs). (B) Results of ICP analysis of intracellular Zn. (C) L929 cell by Zinquin ethyl ester CLSM image after dyeing (scale: 20 microns). (D) Fluorescence relative quantitative results of CLSM images after Zinquin ethyl ester staining. (E) ZnO NPs L929 cell toxicity to normal and acne environment analysis (E1: Control; E2: 12.5 μg/mL ZnO NPs; E3: 25 μg/mL ZnO NPs; E4: 50 μg/mL ZnO NPs; E5: C.; E6: C. + 12.5 μg/mL ZnO NPs; E7: C.+25 μg/mL ZnO NPs; E8: C.+50 μg/mL ZnO NPs). (F) CLSM images of L929 cells after AM and PI staining (scale bar: 200 μm). (G) Diagram of the cell membrane permeability mechanism of C. acne disrupting L929 cells.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/66ad0da50f49defc7dd159d5.jpg"},{"id":80346177,"identity":"9fcc31f3-8ccc-40b4-984f-729d8b50036f","added_by":"auto","created_at":"2025-04-10 19:58:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":241413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in oxidative stress levels in L929 cells by the combined treatment of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC. acnes\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and ZnO NPs.\u003c/strong\u003e (A-C) Contents of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (A), MDA (B) and GSH (C) in L929 cells. (D) CLSM image of L929 cells stained with DCFH-DA (scale bar: 100 μm). (E) CLSM image of L929 cells stained with Mito Tracker Red CMXRos (scale bar: 20 μm).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/220074aba2e4735decd6dc83.jpg"},{"id":80345771,"identity":"f3424748-8bab-46a5-8ea7-a493c0109288","added_by":"auto","created_at":"2025-04-10 19:50:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":276917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of normal and acne skin cells treated with ZnO NPs.\u003c/strong\u003e (A) Correlation heat map between groups. (B) Volcano plot of differentially expressed genes of normal skin cells before and after ZnO NPs treatment. Red dots represent up-regulated genes, and green dots represent down-regulated genes, and blue dots represent genes with no difference. (C) Volcano plot of differentially expressed genes of acne skin cells before and after ZnO NPs treatment. (D, E) GO enrichment analysis (D) and KEGG enrichment analysis (E) of differentially expressed genes of normal skin cells before and after ZnO NPs treatment. (F, G) GO enrichment analysis (F) and KEGG enrichment analysis (G) of differentially expressed genes of acne skin cells before and after ZnO NPs treatment.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/51014f119d8711d673474c0a.jpg"},{"id":80345566,"identity":"d3519b63-97be-4ca5-a443-750585dfe1c4","added_by":"auto","created_at":"2025-04-10 19:42:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":125804,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG enrichment network diagram (A) and protein-protein interaction (PPI) network analysis (B) of differentially expressed genes of acne skin cells before and after ZnO NPs treatment. The density of connecting lines is positively correlated with the degree of interaction association.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/486a6d64f846e503da91b90d.jpg"},{"id":80345569,"identity":"c1240d23-6ec7-41a6-8f4a-61976a849245","added_by":"auto","created_at":"2025-04-10 19:42:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":266364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro analysis of the mechanism by which ZnO NPs delayed wound healing in the acne environment.\u003c/strong\u003e (A) Western-blotting of MMP9, TGF-β1 and SMAD4 (I: Control; II: \u003cem\u003eC.\u003c/em\u003e; III: Control +ZnO NPs; V: \u003cem\u003eC.\u003c/em\u003e+ ZnO NPs). (B-D) Relative quantitative results of Western-blotting for TGF-β1 (B), MMP9 (C), and SMAD4 (D) Immunofluorescence images of collagen type I and III. (F, G) Immunofluorescence relative quantification results of type I collagen (F) and type III collagen (G).\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/fefc1dc98e3152b39b5cbd04.jpg"},{"id":80345126,"identity":"110a4764-f04a-44c8-90ba-457283470874","added_by":"auto","created_at":"2025-04-10 19:34:58","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":232154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo evaluation of the effect of ZnO NPs on acne wounds.\u003c/strong\u003e (A) Schematic of the animal model and in vivo treatment design. (B) Picture of acne wound (scale: 1 cm). (C, D) Wound healing trace and wound healing rate analysis. (E) H\u0026amp;E plot of normal skins and acne skins before and after ZnO NPs treatment (P: PBS; PCZ: PBS+ Carbomer + ZnO NPs; \u003cem\u003eC.\u003c/em\u003e: \u003cem\u003eC. acnes\u003c/em\u003e; C: \u003cem\u003eC. acnes\u003c/em\u003e+ Carbomer + ZnO NPs).\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/8d0ef1e8d69a26e64ec3417f.jpg"},{"id":80345773,"identity":"ab8e3e5b-2f1a-4642-9ebc-6b31485594ce","added_by":"auto","created_at":"2025-04-10 19:50:58","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":343414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo evaluation of histological staining.\u003c/strong\u003e (A) Mason Trichromatic dyeing. (B) Relative quantitative analysis of collagen fibers. (C) Sirius red staining. (D) Immunohistochemical staining of collagen type III. (E) Relative quantitative analysis of collagen type III immunohistochemistry. (F) Immunohistochemical staining of collagen type I. (G) Relative quantitative analysis of type I collagen immunohistochemistry. (H) a-SMA immunohistochemical staining. (I) Relative quantitative analysis of a-SMA immunohistochemistry.\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/52f32375e0ba10144e1120d7.jpg"},{"id":80345778,"identity":"4a0f27f9-2e75-45ee-bbf6-7c049996bb46","added_by":"auto","created_at":"2025-04-10 19:50:59","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":179548,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of MMP9 inhibitor and MMP9 activator on the healing of acne wounds treated with ZnO NPs.\u003c/strong\u003e (A) Schematic diagram of the in vivo experimental procedure. (B) Images of rat acne after different treatments (scale: 1 cm). (C, D) Analysis of acne wound healing traces and wound healing rates after different treatments (\u003cem\u003eC.\u003c/em\u003e: \u003cem\u003eC. acnes\u003c/em\u003e;\u003cem\u003e C.\u003c/em\u003eCZ: \u003cem\u003eC. acnes\u003c/em\u003e+ Carbomer + ZnO NPs; \u003cem\u003eC.\u003c/em\u003eCZ+M: \u003cem\u003eC. acnes\u003c/em\u003e+ Carbomer + ZnO NPs + MMP-9-IN-1; \u003cem\u003eC.\u003c/em\u003eCZ+N: \u003cem\u003eC. acnes\u003c/em\u003e + Carbomer +ZnO NPs + β-Neo-Endorphin). (E) Analysis of the mechanism of MMP9 inhibitor and MMP9 activator on acne healing.\u003c/p\u003e","description":"","filename":"Picture10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/8e86f512e0c2f9b0bc4aedbf.jpg"},{"id":83782864,"identity":"aa76bd9c-c765-48c3-8c4b-7b534765fb43","added_by":"auto","created_at":"2025-06-02 16:07:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3982461,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/6e16f839-dcc4-4cb7-bee0-8b2dcd9d4ab3.pdf"},{"id":80345125,"identity":"23e13bd9-a1bd-408f-9f17-a1c536a96e17","added_by":"auto","created_at":"2025-04-10 19:34:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2224244,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/472408609ede4c3c1cafedd3.docx"},{"id":80345135,"identity":"4daf42fc-74ff-409d-a383-06a037c76444","added_by":"auto","created_at":"2025-04-10 19:34:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5169791,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/9c0a91098a48439298d7151d.docx"},{"id":80345576,"identity":"0ffdb970-f7ce-4945-ab34-b037b7b61658","added_by":"auto","created_at":"2025-04-10 19:42:59","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":6751675,"visible":true,"origin":"","legend":"","description":"","filename":"Revisedsupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-5823650/v1/cd18ca06f050da9cd533f283.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"ZnO colludes with C. acnes in healing delay and scar hyperplasia by barrier destruction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSkin, which is the largest organ and outermost layer of the human body, easily suffers from various diseases (such as acne, atopic dermatitis and psoriasis) [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Any dysfunction of the skin barrier may lead to a vicious cycle that aggravates barrier damage and thus leads to various skin diseases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The microbiological barrier plays an important role in regulating the colonization of pathogens and the release of inflammatory cytokines by skin resident pathogens [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, when over proliferation of \u003cem\u003eCutibacterium acnes\u003c/em\u003e (\u003cem\u003eC. acnes\u003c/em\u003e) in the skin microbiological barrier occurred, the disorder of the microbiological barrier will further lead to the damage of chemical, physical, and immune barriers [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Specifically, disturbance of the microbial barrier can lead to abnormal lipid metabolism and acidic environmental damage, which weakens the antibacterial ability of the chemical barrier [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. At the same time, the proliferation of harmful bacteria will degrade the corneum tightener protein and destroy the integrity of the physical barrier [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These changes further trigger the imbalance of immune regulation and the overactivation of innate immunity, which aggravate the inflammatory response [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The failure of chemical barrier, the destruction of physical barrier and the imbalance of immune barrier jointly aggravate the pathological process of skin diseases.\u003c/p\u003e \u003cp\u003eOn the skin surface, the microbial community is mainly composed of bacteria belonging to the genera Corynebacterium, \u003cem\u003eCutibacterium acnes\u003c/em\u003e, and Staphylococcus, the Interaction of which is essential for the maintenance of skin health [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The commensal bacterium \u003cem\u003eC. acnes\u003c/em\u003e, which is important for regulating skin homeostasis and preventing the colonization of other harmful pathogens, can also serve as an opportunistic pathogen of acne vulgaris [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eC. acnes\u003c/em\u003e produces propionic acid, the release of which induces a low pH in the skin micro-environment where \u003cem\u003eC. acnes\u003c/em\u003e over colonize, posing a severe challenge to the physiological function of surrounding cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In addition, existing studies have shown that \u003cem\u003eC. acnes\u003c/em\u003e promotes acne inflammation by activating inflammatory cells, keratinocytes, monocytes, and sebaceous gland cells. Induced the secretion of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. \u003cem\u003eC. acnes\u003c/em\u003e can also promote the secretion of pro-inflammatory cytokines by activating monocytes or via the innate immune receptors toll-like receptors (TLR), TLR2, and TLR4 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, the severe micro-environment imbalance in acne wounds caused by \u003cem\u003eC. acnes\u003c/em\u003e over colonization poses a serious challenge to the integrity of skin barrier.\u003c/p\u003e \u003cp\u003eTo prevent UV damage, sunscreen is recommended to be adopted in order to protect the skin barrier. Compared with chemical sunscreen, physical sunscreen is more difficult to be absorbed by the skin or produce allergic phenomena, showing more satisfactory safety and stability, so it is more suitable for sensitive skin or children's skin [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Zinc oxide nanoparticles (ZnO NPs), as an most common inorganic sunscreen component, are widely used as a physical sunscreen due to its excellent shortwave UV insulation [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Many studies have confirmed that ZnO NPs barely penetrate the skin barrier and cause cell damage when the skin barrier remains intact [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Zvyagin et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and Roberts al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] were the first to confirm that ZnO NPs do not penetrate the stratum corneum of the skin with the help of non-invasive multiphoton imaging, and this finding was also verified by later studies [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, when the skin barrier is impaired in certain inflammatory skin diseases, such as psoriasis, atopic dermatitis, and seborrheic dermatitis, ZnO NPs deposition in cells and significant cell damage have been observed [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Zn\u003csup\u003e2+\u003c/sup\u003e is a key auxiliary component of key metabolic regions of many enzyme molecules involved in protein synthesis, such as ribonucleic acid (ribonucleic acid, RNA) polymerase, and therefore, an appropriate amount of Zn\u003csup\u003e2+\u003c/sup\u003e is necessary for cells to maintain normal physiological function [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, excessive Zn\u003csup\u003e2+\u003c/sup\u003e can block calcium signaling, thereby selectively inhibiting cell growth [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In addition, excessive Zn\u003csup\u003e2+\u003c/sup\u003e promotes the production of endogenous reactive oxygen species (ROS) and disrupts mitochondrial membrane potential [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, previous studies have shown that long-term application of ZnO NPs has significant effects on the survival rate, oxidative stress, cell proliferation and apoptosis of Enzootic Bovine Leukosis (EBL) cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].For the above reasons, an abnormal increase in intracellular Zn\u003csup\u003e2+\u003c/sup\u003e can lead to severe cell damage, which is often used as a novel anti-tumor and antibacterial strategy [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the face of the severe changes in the micro-environment of acne wounds, especially the acidification of the skin micro-environment induced by over-colonized \u003cem\u003eC. acnes\u003c/em\u003e, the safety of ZnO NPs and the potential interaction mechanism between ZnO NPs and skin barrier have not received enough attention and systematic analysis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated for the first time that \u003cem\u003eC. acnes\u003c/em\u003e, as the \u0026ldquo;accomplice\u0026rdquo; of ZnO NPs, induced significant changes in the cell membrane permeability and intracellular pH environment of fibroblasts through lipase up-regulation and lipid peroxidation, providing the necessary conditions for the opportunistic entry and ionization of ZnO NPs. After uptake by fibroblasts, ZnO NPs decomposed into Zn\u003csup\u003e2+\u003c/sup\u003e, which down-regulated matrix metalloproteinase-9 (MMP-9) and TGF-β1 expression and the downstream TGF-β1/Smad signaling pathway. This further results in a decrease in α-smooth muscle actin (α-SMA) and collagen synthesis and a delay in acne wound healing (Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In summary, focusing on the changes of cell membrane permeability, this study confirmed the specific mechanism and action pathway of \u003cem\u003eC. acnes\u003c/em\u003e acting as an accomplice to cause irreversible damage to the acne skin barrier by ZnO NPs, which provided meaningful theoretical guidance for the application of ZnO NPs-containing sunscreens.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eC. acnes\u003c/b\u003e \u003cb\u003epromotes acidification of neighboring cells by disrupting the membrane permeability\u003c/b\u003e\u003c/p\u003e \u003cp\u003epH in the traumatic environment plays a key role in the process of wound healing. As one of the important parameters of the wound microenvironment, intracellular and extracellular pH influences intracellular metabolism (enzyme activity, macromolecule synthesis, metabolite transport) and cell cycle-related biological behaviors (inflammation, collagen formation, and angiogenesis) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In normal skin, the skin surface presents an acidic environment (pH 4\u0026ndash;6), and the pH value gradually decreases from the basal layer (pH\u0026thinsp;\u0026asymp;\u0026thinsp;7) to the stratum corneum [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. This pH gradient not only guarantees the function of the epidermal barrier (including the inhibition of microbial colonization and the regulation of keratinase activity), but is also essential for the maintenance of keratinocyte differentiation homeostasis. In chronic wound skin, the central area of the wound is alkaline (~\u0026thinsp;7.5) due to persistent inflammation and hypoxia, while the marginal area is maintained with low pHe (~\u0026thinsp;6.5) due to active metabolism. This gradient difference in chronic skin wounds inhibits the proliferation and centripetal migration of marginal cells, resulting in delayed wound healing [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. \u003cem\u003eC. acnes\u003c/em\u003e reduces local skin pH through synergistic action in multiple pathways. \u003cem\u003eC. acnes\u003c/em\u003e secretes lipase to decompose triacylglycerol in sebum and release short-chain fatty acids dominated by propionic acid [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Studies have shown that pH of the skin surface could be decreased by increased free fatty acid and propionic acid content by the fermentation of glucose in \u003cem\u003eC. acnes\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. At the same time, lactic acid and sulfur-containing acidic products are generated through glycolysis and protein metabolism [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In addition, the host immune response further exacerbates the acidification. For example, neutrophil respiratory bursts rely on glycolysis, which results in significant production of lactic acid [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Moreover, the imbalance of microenvironment induced by \u003cem\u003eC. acnes\u003c/em\u003e led to the failure of buffer system such as bicarbonate, and the blockage of hair follicles caused by abnormal keratosis promoted the stroke of anoxic environment, which further promoted the proliferation and continuous acid production of \u003cem\u003eC. acnes\u003c/em\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The pH of the supernatant of the medium after co-incubation of \u003cem\u003eC. acnes\u003c/em\u003e with L929 cells at different concentrations (Control, 10\u003csup\u003e3\u003c/sup\u003e, 10\u003csup\u003e4\u003c/sup\u003e, 10\u003csup\u003e5\u003c/sup\u003e, 10\u003csup\u003e6\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/mL) all decreased gradually with the increase of time (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating the propionic acid accumulation of \u003cem\u003eC. acnes\u003c/em\u003e and the decrease of pHe, which has been demonstrated to be closely related with the up-regulated expression of lipase [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Lipase can hydrolyze triglycerides into glycerol and fatty acids, further leading to lipid peroxidation and the destruction of cell membrane structure and function [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In this work, lactate dehydrogenase content (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), trypan blue staining (Fig. S3, Fig. S4A) and intracellular pH (Fig. S4B) were respectively analyzed and proved that \u003cem\u003eC. acnes\u003c/em\u003e could destroy \u003cem\u003eC. acnes\u003c/em\u003e through the combined action of propionic acid and lipase. Membrane homeostasis of fibroblasts in \u003cem\u003eC. acnes\u003c/em\u003e infected tissue. Previous studies have confirmed that low pHe significantly inhibits cell proliferation, migration and angiogenesis by regulating acid-sensitive ion channels (ASICs) and matrix metalloproteinases (MMPs) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The result of differential gene cluster heat map (Fig. S5) showed that the down-regulation of MMP-9 in \u003cem\u003eC. acnes\u003c/em\u003e group was higher than that in control group. CCK-8 analysis confirmed that with the increase of co-incubation time and bacterial concentration, \u003cem\u003eC. acnes\u003c/em\u003e infection induced a significant decline in the viability of L929 cells. In addition, L929 cells could achieve self-repair under low bacterial concentration (10\u003csup\u003e3\u003c/sup\u003e-10\u003csup\u003e4\u003c/sup\u003e CFU/mL), but significantly lost self-repair ability and showed irreversible damage and apoptosis under high bacterial concentration (\u0026gt;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e CFU/mL) (Fig. S6). Therefore, the above results confirmed that the damage of \u003cem\u003eC. acnes\u003c/em\u003e to L929 cells was closely related to the acidification of extracellular microenvironment by bacterial metabolites (such as propionic acid), inhibition of MMP-9 activity and activation of lipid peroxidation. Alternatively, the lipase inhibitor orlistat was added to the bacterial suspension to further evaluate the damage of the skin cell membrane by the lipase produced by \u003cem\u003eC. acnes\u003c/em\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The lipase content and extracellular free fatty acid content secreted by \u003cem\u003eC. acnes\u003c/em\u003e increased significantly with time, which was inhibited by the addition of orlistat (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). In addition, the conclusion of intracellular lipid peroxidation analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E) was similar to the above results. \u003cem\u003eC. acnes\u003c/em\u003e treatment significantly up-regulated ROS and MDA levels and down-regulated GSH levels, which were all reversed by treatment of orlistat. Co-culture of \u003cem\u003eC. acnes\u003c/em\u003e resulted in the leakage of LDH in L929 cells and the increase in the number of Trypan blue-stained cells, as well as serious damage to membrane permeability, which could be inhibited by orlistat (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-G). These results fully demonstrated that \u003cem\u003eC. acnes\u003c/em\u003e induced lipid peroxidation of adjacent cells and cell membrane damage through lipase secretion, which played an important role in the process of \u003cem\u003eC. acnes\u003c/em\u003e disrupting skin cell homeostasis in acne tissues. Moreover, \u003cem\u003eC. acnes\u003c/em\u003e transported secreted propionic acid on the basis of destroying the cell membrane structure, resulting in significant down-regulation of intracellular pH and serious dysregulation of skin homeostasis, which could not be achieved in pH 6.4 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of ZnO NPs\u003c/h2\u003e \u003cp\u003eRecently, the biosafety of ZnO NPs of skin cells attracted increasing attention due to the wider application in the field of sun protection, medicine and chemical engineering [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Prior to analyzing the effects of ZnO NPs on acne tissue, ZnO NPs adopted in this work were characterized in detail. SEM and TEM images showed the morphology and average size of ZnO NPs (20.30\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7 nm, Fig. S7-10). This diameter met the size requirements for sun protection products between 20 and 100 nm, which facilitated reflection and absorption of ultraviolet rays between 280 and 400 nm, including UVB and some UVC. In addition, the X-Ray Diffraction (XRD) of ZnO NPs were consistent with the standard X-ray diffraction spectra of ZnO in the JCPDs data card (PDF#79\u0026ndash;0205, Fig. S8). The hydrodynamic size and zeta potential of ZnO NPs in distilled water were 20.30\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7 nm and \u0026minus;\u0026thinsp;8.38 mV, respectively. These above results were consistent with data in other ZnO NPs related studies [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eC. acnes\u003c/b\u003e \u003cb\u003eact as an \"accomplice\" to assist ZnO NPs enter the acidified cells\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhen \u003cem\u003eC. acnes\u003c/em\u003e was colonized in subcutaneous tissue sites and abnormally proliferated in skin tissues, the significant changes in adjacent cell membrane permeability and cell acidification induced by \u003cem\u003eC. acnes\u003c/em\u003e raised a \"red light\" on the safety of ZnO NPs in acne sites. ZnO NPs might lead to a series of unsafe biological behaviors after entering the skin cells with damaged cell membranes. According to BioTEM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), L929 cells with normal membrane structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-I) showed serious cell membrane damage after co-incubation with \u003cem\u003eC. acnes\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-II), which was consistent with the results in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Almost no ZnO NPs entered L929 cells and no serious organelle damage was observed when ZnO NPs were treated with L929 cells alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-III). However, a large number of ZnO NPs crossed the damaged cell membrane into the cell by the assistance of \u003cem\u003eC. acnes\u003c/em\u003e, and mitochondrial damage and endoplasmic reticulum vacuoles and damaged nuclei were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-IV). This confirmed that \u003cem\u003eC. acnes\u003c/em\u003e play a key role in the entry of ZnO NPs into skin cells. Furthermore, ICP analysis proved significantly higher intracellular zinc levels in L929 with the help of \u003cem\u003eC. acnes\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and the conversion of ZnO to Zn\u003csup\u003e2+\u003c/sup\u003e in the acidified intracellular environment. Confocal laser scanning microscope images and relative quantitative results of Zn\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D) also confirmed the above conclusions. These results indicated that \u003cem\u003eC. acnes\u003c/em\u003e, as an \"accomplice\", assisted ZnO NPs to enter the acidified cells, in which ZnO NPs are further converted to Zn\u003csup\u003e2+\u003c/sup\u003e in an acidic environment. Ionization of ZnO NPs can cause severe cytotoxicity, which is confirmed by CCK-8 assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eE) and Calcein-AM/PI staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results both confirmed that cytotoxicity of L929 cells was not observed after direct contact with ZnO NPs for 9 h but occurred after 12 h, which provided guidance for the use time of sunscreen products containing ZnO NPs. However, L929 cells showed more serious injury and apoptosis at any treatment time under the co-treatment of \u003cem\u003eC. acnes\u003c/em\u003e and ZnO NPs. In summary, those above results provided sufficient evidence that \u003cem\u003eC. acnes\u003c/em\u003e not only directly damaged L929 cells, but also acted as an \"accomplice\" to assist ZnO NPs to enter the cell interior and induce cell ion homeostasis interference and cell apoptosis through further ionization of ZnO (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMechanism analysis of skin cell damage induced by the combination of\u003c/b\u003e \u003cb\u003eC. acnes\u003c/b\u003e \u003cb\u003eand ZnO NPs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSince \u003cem\u003eC. acnes\u003c/em\u003e can assist ZnO NPs to enter cells by changing membrane permeability. In order to further explore the specific damage mechanism of ZnO NPs on skin cells, intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, MDA, GSH contents and intracellular ROS were detected. After treating acne tissue with 50 \u0026micro;g/mL ZnO NPs for 9 h, the intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e level was significantly higher than that in normal tissue (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ***). The expression of intracellular MDA and GSH were also consistent with intracellular H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C), proving the destruction of membrane permeability and oxidative damage of skin cells under the combination of \u003cem\u003eC. acnes\u003c/em\u003e and ZnO NPs. In addition, \u003cem\u003eC. acnes\u003c/em\u003e\u0026thinsp;+\u0026thinsp;ZnO treatment resulted in a gradual increase in intracellular ROS levels with the increase of time, indicating that \u003cem\u003eC. acnes\u003c/em\u003e helped ZnO NPs induce more intense oxidative stress in skin cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, Fig. S11). And \u003cem\u003eC. acnes\u003c/em\u003e promoted the decrease of the mitochondrial membrane potential of L929 cells induced by ZnO NPs treatment with the extension of time (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, Fig S12). Therefore, the above experimental results confirmed the specific signaling pathways and mechanisms by which ZnO NPs damage skin cells with the assistance of \u003cem\u003eC. acnes\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTranscriptome analysis\u003c/h3\u003e\n\u003cp\u003eAlthough the mechanism by which \u003cem\u003eC. acnes\u003c/em\u003e helps ZnO NPs to enter skin cells has been elucidated, the cytological behavior of ZnO NPs after ionization into the cytoplasm has not been fully understood, in view of which, transcriptome analysis was used to explore the signaling pathways and key targets Zn\u003csup\u003e2+\u003c/sup\u003e is involved in acne skin tissue. L929 cells were treated with ZnO NPs, \u003cem\u003eC.\u003c/em\u003e, and \u003cem\u003eC.\u003c/em\u003e+ZnO NPs respectively to compare the influence of ZnO NPs on skin cells in normal environment and acne environment. The intergroup correlation behavior analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) showed significant difference between ZnO group and Control group, and between \u003cem\u003eC.\u003c/em\u003e+ZnO group and \u003cem\u003eC.\u003c/em\u003e group, indicating remarkable influence of ZnO NPs both on normal and acne skin cells. Specifically, volcano map analysis calculated out 2451 down-regulated and 2808 up-regulated genes in normal skin cells after ZnO NPs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), and 40 down-regulated and 79 up-regulated genes in acne cells after ZnO NPs treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). According to GO functional enrichment analysis and KEGG enrichment analysis, the significantly ZnO-regulating genes in normal skin cells were mainly SMAD protein signal transduction, epithelial- mesenchymal transformation (EMT) and negative regulation of Transforming growth factor β (TGF-β) signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn classical TGF-β signaling, TGF-β1 first activates TGF-β receptor I (TβRI) and TβRII. TβRII first autophosphorylates and then phosphorylates TβRI, resulting in TβRI activation and C-terminal phosphorylation of SMAD. The phosphorylated SMADs then form complexes with the co-mediators SMAD and SMAD4, which translocate to the nucleus, where they bind to the gene promoter. In collaboration with different transcription factors and cofactors, these complexes control the transcription of hundreds of genes. Although ZnO NPs treatment resulted in similar changes of gene expression of TGF-β, EMT and SMAD in normal skin tissues and acne tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and G), the regulation of above gene expression by ZnO NPs in acne skin was more remarkable, indicating more serious cell damage in acne tissue caused by ZnO NPs. TGF-β signaling pathway is closely related to the occurrence of EMT. The SMAD-dependent pathway is the most typical pathway for TGF-β signaling. When TGF-β binds to the receptor complex on the cell surface, activated type I receptor TGF-β1 phosphorylates the signaling protein Smad2/3, forms a complex with Smad4, enters the nucleus, binds to other transcription factors SNAI1, SNAI2, ZEB, TWIST1, etc., and interacts with each other. Transcriptome analysis in this study confirmed the interference of ZnO NPs in the down-regulation of TGF-β/SMAD/EMT signaling axis in acne tissue repair process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, KEGG enrichment network diagram was constructed and analyzed to further explore the specific mechanism and target of ZnO NPs damage to acne skin cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and the differential genes concentrated on the TGF-β signaling pathway related to wound healing were determined to be TGF-β1, SMAD2 and SMAD4, which were all down-regulated. To explore the mutual regulatory effects of the proteins corresponding to the differential genes, 119 differential genes were uploaded to the STRING database and the protein-protein interaction (PPI) network was constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The top five target proteins with the highest correlation in the network were MMP9, TGF-β1, Egr1, SMAD2 and Atm, and MMP9 was observed to be highly correlated with TGF-β1 and SMAD4 in the TGF-β signaling pathway. Zn\u003csup\u003e2+\u003c/sup\u003e inhibits the activity and maturation of MMP-9 and disrupts TGF-β1/Smad signaling through both direct and indirect pathways. On the one hand, Zn\u003csup\u003e2+\u003c/sup\u003e stabilizes the propeptide-catalytic domain interaction by competitively binding cysteine thiol group (Cys\u003csup\u003e69\u003c/sup\u003e) in the PRCGVPD motif of the MMP-9 propeptide domain, delaying the activation of zymogen to maintain its latent state [\u003cspan additionalcitationids=\"CR57 CR58\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. On the other hand, high concentration of Zn\u003csup\u003e2+\u003c/sup\u003e inhibits its activity by binding to histidine residues at the catalytic site of serine proteases (such as plasminase), hinding the maturation of pro-MMP-9 [\u003cspan additionalcitationids=\"CR61\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Activated MMP-9 can cooperate with the Tolloid proteinase family to cut potential TGF-β, resulting in the release of mature TGF-β1 polypeptide [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Activated TGF-β1 regulates transcription of genes associated with wound healing behavior, including α-smooth muscle actin, type I collagen, and type III collagen, by participating in TGF-β1/Smad signaling. Therefore, combined with previous reports and new findings in this study, MMP9 is considered to be the target of ZnO NPs in downregulating TGF-β signaling pathway and interfering with acne skin tissue repair.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMechanism validation of ZnO NPs participating in TGF-β signaling pathway in an acne microenvironment\u003c/h3\u003e\n\u003cp\u003eTo further verify the role of Zn\u003csup\u003e2+\u003c/sup\u003e released by ZnO NPs in the regulatory mechanisms and targets of TGF-β signaling pathway in acne skin tissues, L929 cells treated with ZnO NPs, \u003cem\u003eC.\u003c/em\u003e, \u003cem\u003eC.\u003c/em\u003e+ZnO NPs were detected by Westen-blotting for MMP9, TGF-β and SMAD4, respectively. The results in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-D showed that the expression of MMP9, TGF-β and SMAD4 related proteins in acne skin cells treated with ZnO NPs were significantly down-regulated. Further, the effect of ZnO NPs on the expression of type I collagen and type III collagen located downstream of the TGF-β signaling pathway was investigated. Immunofluorescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and relative quantitative results (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-G) showed the down-regulation of type I collagen and type III collagen in acne skin cells treated with ZnO NPs. Therefore, these results proved the credibility of GO functional analysis and KEGG pathway signaling analysis and confirmed that ZnO NPs can significantly hinder skin healing in acne tissue by interfering with the activity of TGF-β signaling pathway and the reconstruction of collagen network.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIn vivo validation of ZnO NPs delaying acne skin healing\u003c/h3\u003e\n\u003cp\u003eZn\u003csup\u003e2+\u003c/sup\u003e released by ZnO NPs in acne environment interferes with tissue healing and repair, which was further demonstrated in Sprague-Dawley rat skin (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The rats were randomly divided into four groups (five in each group), and the skin of the four groups was treated with PBS, PBS\u0026thinsp;+\u0026thinsp;carbomer\u0026thinsp;+\u0026thinsp;ZnO NPs (PCZ), \u003cem\u003eC. acnes\u003c/em\u003e (\u003cem\u003eC.\u003c/em\u003e), \u003cem\u003eC.\u003c/em\u003e+ carbomer\u0026thinsp;+\u0026thinsp;ZnO NPs (\u003cem\u003eC.\u003c/em\u003eCZ), respectively, in which the infection of \u003cem\u003eC. acnes\u003c/em\u003e resulted in acne wounds on the skin of rats in the \u003cem\u003eC.\u003c/em\u003e and \u003cem\u003eC.\u003c/em\u003eCZ groups. The skin parts that had been treated at different time points were photographed. Skin pH test strips were used to measure the pHe of normal skin and acne skin (Fig. S13). The results confirmed that the pHe of the skin surface without \u003cem\u003eC. acnes\u003c/em\u003e was 5.8, and that of the skin surface infected with \u003cem\u003eC. acnes\u003c/em\u003e was 5.5, which confirmed the effect of \u003cem\u003eC. acnes\u003c/em\u003e on the skin pHe and its support for the ionization of ZnO NPs in acidic environment. Although the effect of \u003cem\u003eC. acnes\u003c/em\u003e on skin surface pH is well established, there is no clear pH boundary between \"healthy\" and \"acne\" skin. The decomposition of ZnO NPs into Zn\u003csup\u003e2+\u003c/sup\u003e is ph-dependent. The concentration of Zn\u003csup\u003e2+\u003c/sup\u003e released by ZnO NPs gradually increases with the decrease of pH [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. In healthy skin with a pH of about 5.8, a small amount of ZnO NPs is decomposed into Zn\u003csup\u003e2+\u003c/sup\u003e, which can be effectively blocked by a dense cuticle barrier or cleared by epidermal metallothione chelation [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In contrast, due to the release of propionic acid and other fatty acids, more Zn\u003csup\u003e2+\u003c/sup\u003e release of ZnO NPs appeared in acne lesions with a pH of around 5.5. In addition, 20\u0026ndash;50 \u0026micro;m micropores in hair follicle corners in acne skin induced further destruction of barrier integrity [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. which can help dissolved Zn\u003csup\u003e2+\u003c/sup\u003e penetrate into the dermis along damaged hair follicle channels and interfere with fibroblast function [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. The analysis of Zn\u003csup\u003e2+\u003c/sup\u003e content in skin by ICP showed the significantly higher Zn\u003csup\u003e2+\u003c/sup\u003e content in acne skin than that in normal skin (Fig S14), suggesting the promotion of ZnO NPs ionization in acne environment at lower pH. Further, the content of Zn\u003csup\u003e2+\u003c/sup\u003e in the epidermis, dermis and subcutaneous tissues of the skin (Fig S15) confirmed that in normal skin, Zn\u003csup\u003e2+\u003c/sup\u003e mainly distributed in the epidermis, but its concentration in the dermis and subcutaneous tissues could be almost ignored, indicating that Zn\u003csup\u003e2+\u003c/sup\u003e hardly penetrated the skin barrier under normal conditions. However, the concentration of ZnO NPs in epidermal layer, dermis layer and subcutaneous tissue in acne skin was significantly higher than that in normal tissue, suggesting more significant penetration and damage to skin tissues of different depths under acne environment. ZnO NPs treatment caused no significant change in the skin of normal mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) but significantly healing delay in acne skin (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D). However, further hematoxylin and eosin (H\u0026amp;E) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e7\u003c/span\u003eE) confirmed that ZnO NPs treatment resulted in the thickening of normal skin stratum corneum, and that the section diameter of acne tissue after ZnO NPs treatment (2.055 mm) was significantly larger than \u003cem\u003eC.\u003c/em\u003e group (1.075 mm) at day 7. In addition, ZnO NPs treatment did not significantly affect the growth of bacteria in acne sites (Fig. S16-17), thus ruling out the possibility of the effect of different microbial infections on wound healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSkin samples of each group were histochemically stained to further clarify the specific mechanism of ZnO NPs on acne tissue wounds. Masson staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B) showed that ZnO NPs treatment could reduce the content of new collagen fibers in normal skin, and resulted in a more significant decrease in the generation of new collagen fibers in acne sites. Sirius red staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003eC) showed that ZnO NPs treatment significantly reduced the birefringence of type III collagen (green) in acne tissues, indicating a significant decline in the generation of new type III collagen fibers and potential scar risk. Therefore, the Sirius red staining not only verified the results of Masson tricolor staining analysis, but also distinguished the types of collagen in normal skin and acne skin, which was of great significance for the functional evaluation of the new collagen network. In addition, immunohistochemical staining of type III collagen (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-E) and type I collagen (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003eF-G) proved that ZnO NPs treatment decreased the content of type I and III collagen fibers in acne skin. Meanwhile, ZnO NPs treatment resulted in a more significant decrease in type III collagen than type I collagen, indicating that ZnO NPs not only delayed the healing of acne skin, but also put the acne skin at risk of scar hyperplasia. At the same time, the expression of mesenchymal marker α-SMA in the tissue of acne skin treated with ZnO NPs was significantly weakened (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003eH-I), suggesting that ZnO NPs significantly inhibited skin EMT behavior in acne environment, which was consistent with the results of transcriptome analysis.\u003c/p\u003e \u003cp\u003eICP analysis of Zn\u003csup\u003e2+\u003c/sup\u003e at all skin levels (Fig.S15) shows that the content of Zn\u003csup\u003e2+\u003c/sup\u003e in the dermis at the acne site is significantly higher than in the normal dermis and that Zn\u003csup\u003e2+\u003c/sup\u003e down-regulated the expression and activity of MMP-9 by direct combination with the MMP-9 catalytic domain (Zn\u003csup\u003e2+\u003c/sup\u003e dependent active site). This leads to the continuous deposition of collagen fibers and the formation of scars. In addition, L929 cells could achieve self-repair under low bacterial concentration (10\u003csup\u003e3\u003c/sup\u003e-10\u003csup\u003e4\u003c/sup\u003e CFU/mL), but significantly lost self-repair ability and showed irreversible damage and apoptosis under high bacterial concentration (\u0026gt;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e CFU/mL) (Fig. S6). These results suggest that the early abnormal collagen deposition is highly correlated with the repair of L929 cell function, that is, the abnormal collagen deposition caused by infection of low concentration of \u003cem\u003eC. acnes\u003c/em\u003e is reversible, while the abnormal collagen deposition caused by infection of high concentration of \u003cem\u003eC. acnes\u003c/em\u003e may be irreversible.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe target of MMP-9 of Zn\u003csup\u003e2+\u003c/sup\u003e interfering with the healing and repair of acne tissue was confirmed by treatment of MMP-9 activator (MMP-9-IN-1) and MMP-9 inhibitor (β-Neo-Endorphin) (Fig. S14). After the following treatments: \u003cem\u003eC. acnes\u003c/em\u003e (C.), \u003cem\u003eC. acnes\u003c/em\u003e\u0026thinsp;+\u0026thinsp;Carbomer\u0026thinsp;+\u0026thinsp;ZnO NPs (\u003cem\u003eC.\u003c/em\u003eCZ), \u003cem\u003eC. acnes\u003c/em\u003e\u0026thinsp;+\u0026thinsp;Carbomer\u0026thinsp;+\u0026thinsp;ZnO NPs\u0026thinsp;+\u0026thinsp;MMP-9-IN-1 (\u003cem\u003eC.\u003c/em\u003eCZ\u0026thinsp;+\u0026thinsp;M), \u003cem\u003eC. acnes\u003c/em\u003e\u0026thinsp;+\u0026thinsp;Carbomer\u0026thinsp;+\u0026thinsp;ZnO\u0026thinsp;+\u0026thinsp;Beta-neo-endorphin (\u003cem\u003eC.\u003c/em\u003eCZ\u0026thinsp;+\u0026thinsp;N), Skin sites were photographed at different time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Consistent with previous results, ZnO NPs treatment caused a significant delay in acne skin healing. And the combination of ZnO NPs and MMP-9-IN-1 further delayed acne skin healing, but the combined treatment of ZnO NPs with β-Neo-Endorphin accelerated acne skin healing (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-D). The above results not only further verified the biological mechanism by which Zn\u003csup\u003e2+\u003c/sup\u003e of ZnO NPs blocks wound healing by targeting MMP-9 in the acne environment, but also suggested the efficient reversal of the delay of acne skin healing by combination of ZnO NPs and MMP-9 activator, which provides effective clinical treatment options for acne skin healing interfered by sunscreen products (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e9\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this work, the effective dose of ZnO NPs was determined based on the commonly used concentration of ZnO NPs in sunscreen products. To evaluate the true state of fibroblasts in the acne environment, cell and animal models of acne were established, respectively. First, the biological behavior of \u003cem\u003eC. acnes\u003c/em\u003e to disrupt acne skin homeostasis by inducing acidification and membrane damage in normal cells was revealed. On this basis, the specific molecular mechanism of ZnO NPs in delaying skin tissue healing and inducing scar hyperplasia was confirmed by combined analysis of transcriptomics and antibody neutralization technology. In summary, focusing on the specificity of acne tissue, this work proved the specific mechanism and action pathway of \u003cem\u003eC. acnes\u003c/em\u003e acting as an accomplice of ZnO NPs to cause irreversible damage to the acne skin from the animal-cell-molecular level. This study confirmed that ZnO NPs in the acne environment interferes with collagen metabolic homeostasis by inhibiting the MMP-9/TGF-β axis, leading to delayed healing of acne skin. The use of molecular targeting agents enables precise regulation of MMP-9 activity, which helps to reverse the healing delay. In addition, in order to avoid the potential risk of ZnO NPs on acne skin, the preferred choice of cosmetics that do not contain ZnO NPs, modified ZnO formulations (such as pH responsive coatings), or contain MMP-9 activators in acne activities is recommended. According to the conclusion of this work, the biosafety risk of ZnO NPs in sunscreen products is mainly attributed to the release of Zn\u003csup\u003e2+\u003c/sup\u003e and its effects on subsequent cell metabolism and collagen secretion. The surface modification of ZnO NPs is a potential option to reduce the solubility toxicity of ZnO NPs. For example, surface coatings or element doping can effectively regulate the dissolution of ZnO NPs, thereby reducing their potential toxicity and improving product safety. For example, lipid coating significantly improves the stability of ZnO NPs and inhibits the aggregation and dissolution of ZnO NPs, resulting in a reduction of over 60% in the release of Zn\u0026sup2; and a threefold reduction in cytotoxicity [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Mannose modification, by coating the ZnO NPs surface via a hydrogen bond network, reduces the Zn\u003csup\u003e2+\u003c/sup\u003e release rate by 50% [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Al\u003csup\u003e3+\u003c/sup\u003e or Mn\u003csup\u003e2+\u003c/sup\u003e doping alters the ZnO electronic structure by lattice substitution, enhancing chemical inertness. For example, the dissolution rate of Al-doped ZnO in the physiological environment is 70% lower than in the undoped system and the antioxidant stability is significantly increased [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], whereas Mn doping further inhibits ROS production and reduces Zn\u003csup\u003e2+\u003c/sup\u003e mediated cellular damage [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. These modification strategies delay the dissolution of ZnO NPs in the acidic environment of the skin and reduce the release of Zn\u003csup\u003e2+\u003c/sup\u003e, providing a guarantee for the safe application of ZnO in sunscreen. This study provides important clinical product development guidance for the care of acne skin: surfacing modified ZnO NPs (such as SiO\u003csub\u003e2\u003c/sub\u003e-coated or lipid-coated ZnO NPs) are recommended for the development of sunscreen products for acne-prone populations. In addition, the concentration of ZnO in sunscreen products is recommended to be limited to less than 10 \u0026micro;g/mL. In addition, a \"stage-differentiated\" treatment strategy is considered necessary during the clinical repair of acne. Acne patients in the acute stage are recommended to use sunscreens without ZnO NPs and barrier repair drugs, while acne patients in the repair stage are recommended to use MMP-9 activators or Zn\u003csup\u003e2+\u003c/sup\u003e chelating dressings to promote wound healing and reverse scarring. These findings not only provide guidance for the optimization of acne-specific sunscreen products, but also supports the formulation of personalized care programs for acne patients. Furthermore, the cellular intervention targets of ZnO NPs in the unique acne environment was clarified, which shows important clinical guiding significance in the application of sunscreen products in different pathological skin environments as well as clinical protocol for acne skin repair.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eF.Z.: Conceptualization, methodology, data curation; T.W.: Data curation, validation; W.W.: Formal Analysis; Y.L.: Resources; Y.Q.: Investigation; D.L.: Software; X.S.: Reagents; X.K.: writing - original draft, Conceptualization, funding acquisition; C. W.: Evaluation of Animal Experiments; J.S.: Conceptualization, supervision, funding acquisition.\u003c/p\u003e\n\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eF.Z. and T.W. contributed to the work equally and should be regarded as co-first authors. This work was supported by the Natural Science Foundation of Shandong Province (Grant No. ZR2022MH187), National Natural Science Foundation of China (Grant No. 32101137).\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZinc Oxide nanoparticles (ZnO NPs) and Oleic Acid (OA) were provided by Sigma (St Louis, MO). Orlistat, Roswell Park Memorial Institute (RPMI) medium 1640, phosphate buffered saline (PBS), Cell Counting Kit-8 (CCK8), Fluo-3 AM, Calcein AM/PI Double Staining Kit, Lipase (LPS) Activity Assay Kit, Amplex Red Free Fatty Acid(FFA) Assay Kit, Reactive Oxygen Species (ROS) Assay Kit, GSH Assay Kit and Hydrogen Peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) Assay Kit were provided by Beijing Solarbio Science \u0026amp; Technology Co., Ltd. Mito Tracker Red CMXRos, Hoechst 33342, Trypan Blue Staining Cell Viability Assay Kit and BCECF AM were provided by Beyotime, China. Cell Malondialdehyde (MDA) assay kit and Lactate dehydrogenase (LDH) assay kit were provided by Nanjing Jiancheng Biotechnology Research Institute Co., Ltd. Anti-MMP-9, anti-TGF-β1, anti-SMAD-4 and goat anti-rabbit secondary antibody were provided by Wuhan Service-bio Technology Co., Ltd. MMP-9-IN-1 and β-Neo-Endorphin were provided by Shanghai Aladdin Biochemical Technology Co., Ltd.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobial/cell culture and animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCutibacterium acnes\u003c/em\u003e (\u003cem\u003eC. acnes\u003c/em\u003e) was provided by Shanghai Microbiological Culture Collection Co., Ltd. Activated \u003cem\u003eC. acnes\u003c/em\u003e were inoculated into the Brucella blood AGAR culture plate. The Bradner blood AGAR culture plates inoculated with \u003cem\u003eC. acnes\u003c/em\u003e were immediately placed into anaerobic gas-producing bags, which were incubated in a constant temperature incubator (37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e).\u003c/p\u003e\n\u003cp\u003ePreparation of the bacterial suspension: Cultured \u003cem\u003eC. acnes\u003c/em\u003e were diluted in gradient (10\u003csup\u003e5\u003c/sup\u003e CFU/mL, 10\u003csup\u003e6\u003c/sup\u003e CFU/mL, 10\u003csup\u003e7\u003c/sup\u003e CFU/mL, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, and 10\u003csup\u003e9\u003c/sup\u003e CFU/mL) with FT. The bacterial suspension obtained was used as the bacterial solution for cell experiments. In addition, the bacterial solution was diluted to 10\u003csup\u003e9\u003c/sup\u003e CFU/mL with PBS buffer for animal experiments.\u003c/p\u003e\n\u003cp\u003eNCTC clone 929 cells (L929) were provided by BeyoClickTM, China. At 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e, L929 cells were cultured using RPMI 1640 medium, which contained 1% double antibodies (100 μL·mL\u003csup\u003e-1\u003c/sup\u003e penicillin and 100 mg·mL\u003csup\u003e-1\u003c/sup\u003e streptomycin) and 10% fetal bovine serum.\u003c/p\u003e\n\u003cp\u003eSprague-Dawley rats (3 w, 160-180 g) were provided by Qingdao Darenfucheng Animal Technology Co., Ltd. Each rat was randomly placed in a cage with free access to food and water. After one week of adaptive feeding, rats were used for the preparation of an acne animal model and other treatments at later stages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA transmission electron microscope (TEM, HT7700, Hitachi, Japan) was used to observe the microstructure and morphology of the nanoparticles at 80 kV. Scanning electron microscopy (SEM, JSM-7500F, JEOL, Japan) was used to analyze the surface morphology of the nanoparticles at 2 kV. A Zeta Simeter (ZEN3700, Malvern, Germany) was used to measure the Zeta potential and dynamic light scattering (DLS) of the nanoparticles. X-ray powder diffractometer (XRPD; Crystal phase analysis was performed using a Bruker D8 Advance X-ray diffractometer. A TCS SP5 laser scanning microscope (CLSM, LEICA, Germany) was used to acquire fluorescence images of cells or tissues. The CCK-8 method was used to detect cell viability after treatment of nanoparticles at 450 nm on Microplate Reader (Thermo Scientific Multiskan™ SkyHigh). Fluorescence microscopy (Olympus, Japan) was used to obtain immunofluorescence images of the cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of pH and lipase content of bacterial supernatants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActivated \u003cem\u003eC. acnes\u003c/em\u003e were seeded on FT at 4℃ at concentrations of 10\u003csup\u003e5\u003c/sup\u003e CFU/mL, 10\u003csup\u003e6\u003c/sup\u003e CFU/mL, 10\u003csup\u003e7\u003c/sup\u003e CFU/mL, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL and 10\u003csup\u003e9\u003c/sup\u003e CFU/mL respectively. \u003cem\u003eC. acnes\u003c/em\u003e supernatant was obtained by centrifugation after being cultured for 6, 12, and 24 h in a cell incubator at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e. The pH of the supernatant at different time periods was measured by a pH meter (PB-10). In addition, FT inoculated with \u003cem\u003eC. acnes\u003c/em\u003e (10\u003csup\u003e9\u003c/sup\u003e CFU/mL) was simultaneously supplemented with 10 μM Orlistat, which was labeled as \u003cem\u003eC.\u003c/em\u003e+ Orlistat group. And \u003cem\u003eC.\u003c/em\u003e+ Orlistat group was cultured in a cell incubator at 37℃ for 6, 12, and 24 h, respectively. The supernatant was obtained by centrifugation, and the lipase content secreted by \u003cem\u003eC. acnes\u003c/em\u003e at each time point was detected by lipase test kit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of an in vitro model of acne-like L929 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2 mL of L929 cell suspension (1×10\u003csup\u003e5\u003c/sup\u003e cells/well) was seeded into 12-well plates for culture. At 70% to 80% confluence of the cells, the old medium was replaced with 2 mL of a new medium pH 6.4, which was labeled as the pH 6.4 group. Then, 1 mL \u003cem\u003eC. acnes\u003c/em\u003e suspension (1×10\u003csup\u003e9\u003c/sup\u003e CFU/mL) was inoculated in transwell chambers, which were immersed in 12-well plates of L929 cells cultured with normal medium for 6 h. therefore, an in vitro model of acne-like L929 cells was established. which was labeled as \u003cem\u003eC. acnes\u003c/em\u003e (\u003cem\u003eC.\u003c/em\u003e) group. At the same time as \u003cem\u003eC. acnes\u003c/em\u003e suspension was inoculated in a transwell chamber, 10 μL of 1 mM Orlistat was added and then immersed in 12-well plates of L929 cells cultured with normal medium for 6 h and labeled as \u003cem\u003eC.\u003c/em\u003e+ Orlistat group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the effect of \u003cem\u003eC. acnes\u003c/em\u003e treatment on free fatty acid content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were treated and grouped according to the above method: control group, pH 6.4 group, \u003cem\u003eC.\u003c/em\u003e Group and \u003cem\u003eC.\u003c/em\u003e + Orlistat group. L929 cells in all groups were treated for 6 h and then were treated with amplex red free fatty acid assay kit respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the effect of \u003cem\u003eC. acnes\u003c/em\u003e and ZnO NPs treatment on intracellular ROS production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were given different treatments: control group, pH 6.4 group, \u003cem\u003eC.\u003c/em\u003e group, \u003cem\u003eC.\u003c/em\u003e+ Orlistat group, control + ZnO NPs (50 μg/mL) group and \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ ZnO NPs (50 μg/mL) group. L929 cells in the first four groups were treated for 6 h. L929 cells in the last two groups were treated for 0, 3, 6, 9, 12 h and then were treated with Reactive Oxygen Species Assay Kit respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the influence of \u003cem\u003eC. acnes\u003c/em\u003e and ZnO NPs treatment on intracellular MDA and GSH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were given different treatments: control group, pH 6.4 group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ Orlistat group, control + ZnO NPs (50 μg/mL) group and \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ ZnO NPs (50 μg/mL) group. L929 cells in the first four groups were treated for 6 h. L929 cells in the last two groups were treated for 0, 3, 6, 9, 12 h and then were treated with malondialdehyde kit and glutathione kit respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the influence of \u003cem\u003eC. acnes\u003c/em\u003e treatment on LDH content in cell supernatant\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were given different treatments: control group, pH 6.4 group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003eGroup with different concentrations (10\u003csup\u003e5\u003c/sup\u003e CFU/mL, 10\u003csup\u003e6\u003c/sup\u003e CFU/mL, 10\u003csup\u003e7\u003c/sup\u003e CFU/mL, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, 10\u003csup\u003e9\u003c/sup\u003e CFU/mL) and \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ Orlistat group (10\u003csup\u003e9\u003c/sup\u003e CFU/mL \u003cem\u003eC. acnes\u003c/em\u003e+ 10 μL/1 mM Orlistat). L929 cells in each group were treated for 6, 12, 24 h and then were treated with Lactate dehydrogenase assay kit respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the influence of \u003cem\u003eC. acnes\u003c/em\u003e treatment on membrane permeability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were given different treatments: control group, pH 6.4 group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003eGroup with different concentrations (10\u003csup\u003e5\u003c/sup\u003e CFU/mL, 10\u003csup\u003e6\u003c/sup\u003e CFU/mL, 10\u003csup\u003e7\u003c/sup\u003e CFU/mL, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, 10\u003csup\u003e9\u003c/sup\u003e CFU/mL) and \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ Orlistat group (109 CFU/mL \u003cem\u003eC. acnes\u003c/em\u003e+ 10 μL/1 mM Orlistat). L929 cells in each group were treated for 6, 12, 24 h and then were treated with Trypan Blue Staining Cell Viability Assay Kit respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the influence of \u003cem\u003eC. acnes\u003c/em\u003e treatment on intracellular pH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were given different treatments: control group, pH 6.4 group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003eGroup and \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ Orlistat group. L929 cells in all groups were treated for 6 h and then were treated with BCECF AM respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioTEM for ZnO NPs endocytosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were given different treatments: control group, pH 6.4 group, ZnO NPs Group and ZnO NPs + Orlistat group (ZnO NPs: 50 μg/mL). L929 cells in all groups were treated for 6 h. The cells were washed by PBS and collected in a centrifuge tube, and then were fixed with 2.5% (v/v) glutaraldehyde. Finally cells in each group were subjected to bioTEM and ZnO NPs endocytosis was observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCytotoxicity evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e200 μL L929 cells (1×10\u003csup\u003e4\u003c/sup\u003e cells/well) were cultured in 96-well plates for 24 h. The cells are divided into the following five groups: Control group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup, Orlistat group, ZnO NPs group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ ZnO NPs group. For the control group, L929 cells were cultured under standard conditions. For \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup, 200 μL of \u003cem\u003eC. acnes\u003c/em\u003e suspensions with different concentrations (10\u003csup\u003e5\u003c/sup\u003eCFU/mL, 10\u003csup\u003e6\u003c/sup\u003e CFU/mL, 10\u003csup\u003e7\u003c/sup\u003e CFU/mL, 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, 10\u003csup\u003e9\u003c/sup\u003e CFU/mL) were inoculated into 96-well plates which had been cultured with L929 cells, and the \u003cem\u003eC. acnes\u003c/em\u003e/cells mixed solutions were incubated for 3, 6, 9, 12 h, 24 h. For the Orlistat group, 100 μL \u003cem\u003eC. acnes\u003c/em\u003e suspension (2×10\u003csup\u003e9\u003c/sup\u003e CFU/mL) and 100 μL of Orlistat (20 μM) solution were added to 96-well plates containing L929 cells, and all the above components were incubated for 6, 12, and 24 h. for ZnO NPs group, 12.5 μg·mL\u003csup\u003e-1\u003c/sup\u003e, 25 μg·mL\u003csup\u003e-1\u003c/sup\u003e and 50 μg·mL\u003csup\u003e-1\u003c/sup\u003e ZnO NPs were added to L929 cell culture medium and incubated for 3, 6, 9 and 12 h, respectively. For the \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ZnO NPs group, 200 μL \u003cem\u003eC. acnes\u003c/em\u003e suspension (1×10\u003csup\u003e9\u0026nbsp;\u003c/sup\u003eCFU/mL) was inoculated on a 96-well plate in which L929 cells had been cultured, and the mixed solutions were incubated for 6 h. After that, the old mediums were removed and all the cells were cleaned twice with PBS. L929 cells were then treated with 12.5 μg·mL\u003csup\u003e-1\u003c/sup\u003e, 25 μg·mL\u003csup\u003e-1\u003c/sup\u003e and 50 μg·mL\u003csup\u003e-1\u003c/sup\u003e ZnO NPs for 3, 6, 9 and 12 h, respectively. All groups were treated with Cell Counting Kit-8 respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInductively coupled plasma (ICP) for Zn\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e2+\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;content analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were divided into the following four groups: control group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup, ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group. L929 cells in all groups were treated for 0, 3, 6, 12 h. The cells were broken under an ice water bath with an ultrasonic cell breaker. The intracellular Zn\u003csup\u003e2+\u003c/sup\u003e content was detected by inductively coupled plasma emission spectrometer. There were three parallels in each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of intracellular Zn\u003csup\u003e2+\u003c/sup\u003e content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were divided into the following four groups: control group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup, ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group. L929 cells in all groups were treated for 0, 3, 6, 12 h and then were treated with Zinquin ethyl ester respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of mitochondrial membrane potential and apoptosis of L929 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were divided into the following four groups: control group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup, ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group. L929 cells in all groups were treated for 0, 3, 6, 9, 12 h and then were treated with Mito Tracker Red CMXRos and Calcein AM/PI Double Staining Kit respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of type I collagen and type III collagen secretion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were divided into the following four groups: control group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup, ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group. L929 cells in all groups were treated for 6 h. All cells were then incubated overnight with COL-I and COL-III antibodies at 4℃, then were incubated with Cyanine 3-labeled goat anti-rabbit immunoglobulin G (IgG) at room temperature away from light. The cells were then added to 4', 6-diaminidine 2-phenylindole dye and incubated at room temperature away from light. The cells were encapsulated and photographed by fluorescence microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern-blotting analysis of MMP9\u003c/strong\u003e\u003cstrong\u003e、\u003c/strong\u003e\u003cstrong\u003eTGF-β1\u003c/strong\u003e\u003cstrong\u003e和\u003c/strong\u003e\u003cstrong\u003eSDAD4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were divided into the following four groups: control group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup, ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group. L929 cells in all groups were treated for 6 h. Nucleoprotein and cytoplasmic protein extraction kits containing 1 mM phosphatase inhibitors were used to treat cells after PBS washing. The cell lysate was heated in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer. The proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane, where the separation process was then blocked by skim milk. The polyvinylidene fluoride film was incubated with primary antibodies including anti-MMP9, anti-TGF-β1 and anti-SDAD4 at 4℃ overnight. After washing with TBS containing Tween-20 (TBST) buffer, polyvinylidene fluoride film coupled with horseradish peroxidase (HRP) was incubated at room temperature. Enhanced chemiluminescent reagent (ECL) kits and automated chemiluminescence image analysis systems were used to detect the expression of MMP9, TGF-β1 and SDAD4 in the cells of above groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL929 cells were divided into the following four groups: control group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup, ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ZnO NPs (50 μg·mL\u003csup\u003e-1\u003c/sup\u003e) group. L929 cells in all groups were treated for 6 h. The cells in each group were added with total RNA extraction reagent (TRIzol) reagent. The subsequent RNA sequencing was performed by eukaryotic mRNA sequencing based on Illumina sequencing platform by Shanghai NOhe Zhiyuan Bioinformation Technology Co., LTD. DESeq2 software was used for ZnO NPs group VS control group and \u003cem\u003eC.\u0026nbsp;\u003c/em\u003e+ZnO NPs group VS \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup transcriptional behavior differential analysis (DEGs) and further functional analysis of DEGs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter the differential genes were obtained through gene expression analysis, the functions of differential genes were enriched and analyzed to find the signaling pathways and molecular regulatory mechanisms that play a key role in the differential biological behavior. ClusterProfiler software was used to complete GO functional enrichment analysis and KEGG path enrichment analysis of DEGs sets. Among them, GO analysis, which is based on the GO database (Gene Ontology), was responsible for the classification of genes according to Biological process (BP), Cellular component (Cellular component), biological process (BP). CC) and Molecular function (MF). KEGG pathway enrichment, which is based on the KEGG (Kyoto Encyclopedia of Genes and Genomes) database, was responsible for statistical analysis of the differential signal pathways.\u003c/p\u003e\n\u003cp\u003eAfter the target genes were identified, Pearson or Spearman algorithm was used to obtain the correlation coefficient between genes, which provided support for the drawing of the visual network map. In this work, STRING database was used for interaction network analysis of target proteins. In addition, Cytoscape software was adopted for Protein-protein interaction (PPI) analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of ZnO NPs supported gels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e0.1g of carbomer powder was added to 10 mL of PBS buffer. The solution was stirred evenly, and 1% (m/v) carbomer gel was prepared. In addition, 50 μg/mL PBS solution of ZnO NPs was prepared and ultrasounded for 30 min. After 10 mL of the above ZnO NPs suspension was added with 0.1g carbomer powder and the solution was stirred evenly, ZnO NPs (50 μg/mL) carbomer gel was prepared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of in vivo effects of ZnO NPs on acne skin tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, all animal procedures were conformed to the Guide for the Care and Use of Laboratory Animals and performed following the guidelines and the protocol approved by the application of the experimental animal ethical project of Qingdao Agricultural University (Approval No.20220072). After the rats were fed adaptively for one week and the rats' back hair was shaved, 0.5 mL oleic acid was applied once a day to the back skin for 7 days. From day 8, 0.2 mL \u003cem\u003eC. acnes\u003c/em\u003e (1×10\u003csup\u003e9\u003c/sup\u003e CFU/mL) was injected into the dermis for 14 days, and acne-like inflammatory lesion model was established. The rats were randomly divided into six groups: Control group (PBS, n=5), Carbomer + ZnO NPs group (CZ, n=5), \u003cem\u003eC. acnes\u003c/em\u003e group (\u003cem\u003eC.\u003c/em\u003e, n=5), \u003cem\u003eC. acnes\u003c/em\u003e + Carbomer + ZnO NPs group (\u003cem\u003eC.\u0026nbsp;\u003c/em\u003eCZ, n=5), \u003cem\u003eC. acnes\u003c/em\u003e + Carbomer + ZnO NPs + MMP-9-IN\u003csup\u003e-1\u003c/sup\u003e group (CCZ+M, n=5), \u003cem\u003eC. acnes\u003c/em\u003e + Carbomer + ZnO NPs + β-Neo-Endorphin group (\u003cem\u003eC.\u0026nbsp;\u003c/em\u003eCZ+N, n=5). From the 15th day to the 21st day of modeling, rats in different groups were treated as follows: for control group and \u003cem\u003eC. acnes\u003c/em\u003e group, 1 mL PBS was applied on the back of rats every day; for the CZ group and the \u003cem\u003eC.\u0026nbsp;\u003c/em\u003eZ group, the back of rats was coated with 1 mL ZnO NPs carbomer gel every day. for \u003cem\u003eC.\u0026nbsp;\u003c/em\u003eCZM group, the back of rats was coated with 1 mL ZnO NPs and MMP-9-IN-1 carbomer gel every day. for the \u003cem\u003eC.\u0026nbsp;\u003c/em\u003eCZβ group, rats were coated with 1 mL ZnO NPs and β-Neo-Endorphin carbomer gel daily on their backs. The acne wounds were photographed on day 15 and day 21 respectively, and the area of acne wounds was recorded. On day 22, all anesthetized rats were killed by neck amputation and then microsurgical scissors were used to separate the acne wounds on the back of the rats. Skin pH value, Bacterial colony count, Zn level, HE staining, Masson staining, Sirius red staining, and immunohistochemical analysis were respectively performed on the back acne wounds.\u003c/p\u003e\n\u003cp\u003eSpecifically, the wound tissues of the Control group, CZ group, \u003cem\u003eC.\u0026nbsp;\u003c/em\u003egroup and \u003cem\u003eC.\u0026nbsp;\u003c/em\u003eCS group on the last day of treatment were placed in 4% paraformaldehyde, dehydrated by alcohol gradients of different concentrations, embedded in paraffin wax and were prepared into tissue sections with thickness of 5 mm. When measuring the skin's pH, the test strip was first moistened in distilled water, then applied to both normal skin and acne areas. It was removed after five seconds, and the color was compared with the reference chart provided on the test strip container. When the number of bacteria in the wound tissue was measured, the wound tissue of the \u003cem\u003eC.\u003c/em\u003e and \u003cem\u003eC.\u003c/em\u003eC groups was placed in sterile PBS on the last day. After homogenizing and continuously diluting with sterile PBS, the diluent was inoculated on Columbia blood AGAR plate and incubated at 37℃ for 36 h. Finally, \u003cem\u003eC. acnes\u003c/em\u003e was selectively isolated and the total number of colonies was counted.\u003c/p\u003e\n\u003cp\u003eIn order to determine the pathological condition of the acne tissue, the wound tissue was analyzed by HE staining. After dewaxing and H\u0026amp;E staining, the tissue sections were viewed microscopically and photographed in full scan.\u003c/p\u003e\n\u003cp\u003eIn order to evaluate the effects of ZnO NPs on collagen fibers and muscle fibers in acne tissues, acne tissues were subjected to Masson's trichrome staining analysis. After undergoing dewaxing and Masson staining, sections of acne tissue were observed and photographed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the effects of ZnO NPs on type I collagen and type III collagen in acne tissues, the acne tissues were stained with Sirius red. After dewaxing, Sirius red staining, the tissue sections were placed under polariscope for observation and photography of collagen fibers.\u003c/p\u003e\n\u003cp\u003eIn addition, the distribution of type I collagen, type III collagen and smooth actin in acne tissues was also confirmed by immunohistochemical analysis. The acne tissue sections of each group were labeled with Collagen I, Collagen III and α-SMA monoclonal antibodies, respectively, and then incubated with second anti-antibody. Finally, the section samples were viewed under a fluorescence microscope and photographed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine Zn levels in acne tissues, acne tissues of each group were placed on sterile PBS on the last day of treatment to remove residual ZnO NPs. After that, inductively coupled plasma analyzer was used to determine the content of Zn in the tissues.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSzab\u0026oacute; K, Bolla BS, Erdei L, Balogh F, Kem\u0026eacute;ny L. Are the Cutaneous Microbiota a Guardian of the Skin\u0026rsquo;s Physical Barrier? The Intricate Relationship between Skin Microbes and Barrier Integrity. Int J Mol Sci. 2023;24:15962.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEyerich S, Eyerich K, Traidl-Hoffmann C, Biedermann T. Cutaneous Barriers and Skin Immunity: Differentiating A Connected Network. Trends Immunol. 2018;39:315\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajkumar J, Chandan N, Lio P, Shi V. The Skin Barrier and Moisturization: Function, Disruption, and Mechanisms of Repair. Skin Pharmacol Physiol. 2023;36:174\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaik S, Bouladoux N, Linehan JL, Han SJ, Harrison OJ, Wilhelm C, et al. Commensal-dendritic-cell interaction specifies a unique protective skin immune signature. Nature. 2015;520:104\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris-Tryon TA, Grice EA. Microbiota and maintenance of skin barrier function. Science. 2022;376:940\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlwan W, Di Meglio P. Guardians of the barrier: Microbiota engage AHR in keratinocytes to mantain skin homeostasis. Cell Host Microbe. 2021;29:1213\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee H-J, Kim M. Skin Barrier Function and the Microbiome. Int J Mol Sci. 2022;23:13071.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyanova L. Cutibacterium Acnes (Formerly Propionibacterium Acnes): Friend or Foe? Future Microbiology. 2023; 18:235\u0026thinsp;\u0026ndash;\u0026thinsp;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScharschmidt TC. Antibiotics for Acne\u0026mdash;A Pilot Study of Collateral Damage to the Skin Microbiome. JAMA Dermatology. 2019;155:419\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMempel M, Schmidt T, Weidinger S, Schnopp C, Ring J, Abeck D, et al. Role of Staphylococcus Aureus Surface-Associated Proteins in the Attachment to Cultured HaCaT Keratinocytes in a New Adhesion Assay. J Invest Dermatology. 1998;111:452\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNamjoshi S, Caccetta R, Benson HAE. Skin Peptides: Biological Activity and Therapeutic Opportunities. J Pharm Sci. 2008;97:2524\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoodarzi H, Trowbridge J, Gallo RL. Innate Immunity: A Cutaneous Perspective. Clin Rev Allerg Immunol. 2007;33:15\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrice EA, Kong HH, Conlan S, Deming CB, Davis J, Young AC, et al. Topographical and Temporal Diversity of the Human Skin Microbiome. Science. 2009;324:1190\u0026ndash;2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarris-Tryon TA, Grice EA. Microbiota and maintenance of skin barrier function. Science. 2022;376:940\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDr\u0026eacute;no B, P\u0026eacute;castaings S, Corvec S, Veraldi S, Khammari A, Roques C. Cutibacterium acnes (Propionibacterium acnes) and acne vulgaris: a brief look at the latest updates. J Eur Acad Dermatol Venereol. 2018;32:5\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoun SH, Choi CW, Choi JW, Youn SW. The skin surface pH and its different influence on the development of acne lesion according to gender and age. Skin Res Technol. 2013;19:131\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eContassot E, French LE. New Insights into Acne Pathogenesis: Propionibacterium Acnes Activates the Inflammasome. J Invest Dermatol. 2014;134:310\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgak GW, Qin M, Nobe J, Kim M-H, Krutzik SR, Tristan GR, et al. Propionibacterium acnes Induces an IL-17 Response in Acne Vulgaris that Is Regulated by Vitamin A and Vitamin D. J Invest Dermatology. 2014;134:366\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim H-J, Kang S-H, Song Y-J, Jeon Y-D, Jin J-S. Inhibitory Effect of Quercetin on Propionibacterium acnes-induced Skin Inflammation. Int Immunopharmacol. 2021;96:107557.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerpone N, Dondi D, Albini A. Inorganic and organic UV filters: Their role and efficacy in sunscreens and suncare products. Inorg Chim Acta. 2007;360:794\u0026ndash;802.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScharffetter-Kochanek K, Wlaschek M, Brenneisen P, Schauen M, Blaudschun R, Wenk J. UV-Induced Reactive Oxygen Species in Photocarcinogenesis and Photoaging Biol. Biol Chem. 1997;378:1247\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatsambas A, Nicolaidou E. Cutaneous malignant melanoma and sun exposure. Recent developments in epidemiology. Arch Dermatol. 1996;132:444\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh TA, Das J, Sil PC. Zinc oxide nanoparticles: A comprehensive review on its synthesis, anticancer and drug delivery applications as well as health risks. Adv Colloid Interface Sci. 2020;286:102317.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeite-Silva VR, Sanchez WY, Studier H, Liu DC, Mohammed YH, Holmes AM, et al. Human skin penetration and local effects of topical nano zinc oxide after occlusion and barrier impairment. Eur J Pharm Biopharm. 2016;104:140\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZvyagin AV, Zhao X, Gierden A, Sanchez W, Ross JA, Roberts MS. Imaging of zinc oxide nanoparticle penetration in human skin in vitro and in vivo. J Biomed Opt. 2008;13:064031.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts MS, Roberts MJ, Robertson TA, Sanchez W, Th\u0026ouml;rling C, Zou Y, et al. In vitro and in vivo imaging of xenobiotic transport in human skin and in the rat liver. J Biophotonics. 2008;1:478\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolmes AM, Song Z, Moghimi HR, Roberts MS. Relative Penetration of Zinc Oxide and Zinc Ions into Human Skin after Application of Different Zinc Oxide Formulations. ACS Nano. 2016;10:1810\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatkinson AC, Bunge AL, Hadgraft J, Lane ME. Nanoparticles do not penetrate human skin\u0026ndash;a theoretical perspective. Pharm Res. 2013;30:1943\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFilipe P, Silva JN, Silva R, Cirne de Castro JL, Marques Gomes M, Alves LC, et al. Stratum corneum is an effective barrier to TiO2 and ZnO nanoparticle percutaneous absorption. Skin Pharmacol Physiol. 2009;22:266\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen R-J, Lee Y-H, Yeh Y-L, Wang Y-J, Wang B-J. The Roles of Autophagy and the Inflammasome during Environmental Stress-Triggered Skin Inflammation. Int J Mol Sci. 2016;17:2063.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaa G, Nurchi VM, Ravarino A, Fanni D, Nemolato S, Gerosa C, et al. Zinc in gastrointestinal and liver disease. Coord Chem Rev. 2008;252:1257\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi S, Cui C, Luo Y, Kim S-H, Ko J-K, Huo X, et al. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orail. FASEB J. 2018;32:404\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCen D, Ge Q, Xie C, Zheng Q, Guo J, Zhang Y et al. ZnS@BSA Nanoclusters Potentiate Efficacy Cancer Immunotherapy Adv Mater. 2021; 49:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003en/a-n/a\u003c/span\u003e\u003cspan address=\"http://n/a-n/a\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao H, Li T, Yao C, Gu Z, Liu C, Li J, et al. Dual Roles of Metal-Organic Frameworks as Nanocarriers for miRNA Delivery and Adjuvants for Chemodynamic Therapy. ACS Appl Mater Interfaces. 2021;13:6034\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu Y, Cockerill I, Wang Y, Qin Y-X, Chang L, Zheng Y, et al. Zinc-Based Biomaterials for Regeneration and Therapy. Trends Biotechnol. 2019;37:428\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Ma Y, Lian X, Lu Y, Li Y, Xi Y, et al. Study on the biological effects of ZnO nanosheets on EBL cells. Front Bioeng Biotechnol. 2022;10:915749.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan J, Liu C, Wu Q, Zhou J, Xu X, Zhang L, et al. Mineralization of pH-Sensitive Doxorubicin Prodrug in ZIF-8 to Enable Targeted Delivery to Solid Tumors. Anal Chem. 2020;92:11453\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng P, Ding B, Zhu G, Wang M, Li C, Lin J. Biodegradable Hydrogen Peroxide Nanogenerator for Controllable Cancer Immunotherapy Via Modulating Cell Death Pathway from Apoptosis to Pyroptosis. Chem Eng J. 2022;450:137967.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDu M, Chen ZJ. DNA-induced liquid phase condensation of cGAS activates innate immune signaling. Science. 2018;361:704\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoradi Tuchayi S, Makrantonaki E, Ganceviciene R, Dessinioti C, Feldman SR, Zouboulis CC. Acne vulgaris. Nat Rev Dis Primers. 2015;1:15029.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchreml S, Szeimies R, Karrer S, Heinlin J, Landthaler M, Babilas P. The impact of the pH value on skin integrity and cutaneous wound healing. Acad Dermatol Venereol. 2010;24:373\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhman H, Vahlquist A. In vivo studies concerning a pH gradient in human stratum corneum and upper epidermis. Acta Derm Venereol. 1994;74:375\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchreml S, Meier RJ, Kirschbaum M, Kong SC, Gehmert S, Felthaus O, et al. Luminescent Dual Sensors Reveal Extracellular pH-Gradients and Hypoxia on Chronic Wounds That Disrupt Epidermal Repair. Theranostics. 2014;4:721\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu T, Xu X, Liu Y, Wang X, Wu S, Qiu Z, et al. Multi-omics signatures reveal genomic and functional heterogeneity of Cutibacterium acnes in normal and diseased skin. Cell Host Microbe. 2024;32:1129\u0026ndash;e11468.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFluhr JW, Kao J, Jain M, Ahn SK, Feingold KR, Elias PM. Generation of Free Fatty Acids from Phospholipids Regulates Stratum Corneum Acidification and Integrity. J Invest Dermatol. 2001;117:44\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarney SA, Hall M, Ricketts CR. The adenosine triphosphate content and lactic acid production of guinea-pig skin after mild heat damage. Br J Dermatol. 1976;94:291\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhong G, Guo Y, Gong X, Xu M, Wang Q, Wu M et al. Enhanced glycolysis by ATPIF1 gene inactivation increased the anti-bacterial activities of neutrophils through induction of ROS and lactic acid. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2023; 1869:166820.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlmoughrabie S, Cau L, Cavagnero K, O\u0026rsquo;Neill AM, Li F, Roso-Mares A, et al. Commensal \u003cem\u003eCutibacterium acnes\u003c/em\u003e induce epidermal lipid synthesis important for skin barrier function. Sci Adv. 2023;9:eadg6262.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeong S, Lee J, Im BN, Park H, Na K. Combined photodynamic and antibiotic therapy for skin disorder via lipase-sensitive liposomes with enhanced antimicrobial performance. Biomaterials. 2017;141:243\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrauss JS, Stranieri AM. Acne treatment with topical erythromycin and zinc: Effect on Propionibacterium acnes and free fatty acid composition. J Am Acad Dermatol. 1984;11:86\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang R, Zeng L, Zhu S, Xie Y, Liu J, Wen Q, et al. Lipid Peroxidation Drives Gasdermin D-Mediated Pyroptosis in Lethal Polymicrobial Sepsis. Cell Host Microbe. 2018;24:97\u0026ndash;e1084.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorovicka J, Schwizer W, Guttmann G, Hartmann D, Kosinski M, Wastiel C, et al. Role of lipase in the regulation of postprandial gastric acid secretion and emptying of fat in humans: a study with orlistat, a highly specific lipase inhibitor. Gut. 2000;46:774\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammed YH, Holmes A, Haridass IN, Sanchez WY, Studier H, Grice JE, et al. Support for the Safe Use of Zinc Oxide Nanoparticle Sunscreens: Lack of Skin Penetration or Cellular Toxicity after Repeated Application in Volunteers. J Invest Dermatol. 2019;139:308\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmijs TG, Pavel S. Titanium dioxide and zinc oxide nanoparticles in sunscreens: focus on their safety and effectiveness. NSA. 2011;4:95\u0026ndash;112.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlenezi NA, Al-Qurainy F, Tarroum M, Nadeem M, Khan S, Salih AM, et al. Zinc Oxide Nanoparticles (ZnO NPs), Biosynthesis, Characterization and Evaluation of Their Impact to Improve Shoot Growth and to Reduce Salt Toxicity on Salvia officinalis In Vitro Cultivated. Processes. 2022;10:1273.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan H, Du B, Pan X, Liu J, Zhao Q, Lian X, et al. CADPE inhibits PMA-stimulated gastric carcinoma cell invasion and matrix metalloproteinase-9 expression by FAK/MEK/ERK-mediated AP-1 activation. Mol Cancer Res. 2010;8:1477\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKjeldsen L, Johnsen AH, Sengel\u0026oslash;v H, Borregaard N. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem. 1993;268:10425\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeong Y-J, Cho H-J, Chung F-L, Wang X, Hoe H-S, Park K-K, et al. Isothiocyanates suppress the invasion and metastasis of tumors by targeting FAK/MMP-9 activity. Oncotarget. 2017;8:63949\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAugoff K, Hryniewicz-Jankowska A, Tabola R, Stach K. MMP9: A Tough Target for Targeted Therapy for Cancer. Cancers (Basel). 2022;14:1847.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmidt AE, Shah P, Gauthier EM, Bajaj SP. Protease Domain Sodium, Calcium, and Zinc Sites in Factor VIIa: Crystal Structures and Kinetic Studies. Blood. 2004;104:122\u0026ndash;122.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanuru M, Raman R, Aradhyam GK. Serine Protease Activity of Calnuc. J Biol Chem. 2013;288:1762\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeung K-S, Meanwell NA, Qiu Z, Hernandez D, Zhang S, McPhee F, et al. Structure\u0026ndash;activity relationship studies of a bisbenzimidazole-based, Zn2+-dependent inhibitor of HCV NS3 serine protease. Bioorg Med Chem Lett. 2001;11:2355\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi T, Kim HJ, Liu X, Sugiura H, Kohyama T, Fang Q, et al. Matrix metalloproteinase-9 activates TGF-β and stimulates fibroblast contraction of collagen gels. Am J Physiol Lung Cell Mol Physiol. 2014;306:L1006\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBian S-W, Mudunkotuwa IA, Rupasinghe T, Grassian VH. Aggregation and Dissolution of 4 nm ZnO Nanoparticles in Aqueous Environments: Influence of pH, Ionic Strength, Size, and Adsorption of Humic Acid. Langmuir. 2011;27:6059\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun F, Li R, Jin F, Zhang H, Zhang J, Wang T, et al. Dopamine/zinc oxide doped poly(\u003cem\u003eN\u003c/em\u003e -hydroxyethyl acrylamide)/agar dual network hydrogel with super self-healing, antibacterial and tissue adhesion functions designed for transdermal patch. J Mater Chem B. 2021;9:5492\u0026ndash;502.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTegenaw AB, Yimer AA, Beyene TT. Boosting the photocatalytic activity of ZnO-NPs through the incorporation of C-dot and preparation of nanocomposite materials. Heliyon. 2023;9:e20717.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Li J, Zhu L, Huang J, Zhang Q, Wang J, et al. Mechanistic insights into zinc oxide nanoparticles induced embryotoxicity via H3K9me3 modulation. Biomaterials. 2024;311:122679.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao D, Shu X, Zhu D, Liang S, Hasan M, Gong S. Lipid-coated ZnO nanoparticles synthesis, characterization and cytotoxicity studies in cancer cell. Nano Convergence. 2020;7:14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePiasek A, Kominko H, Zielina M, Banach M, Pulit-Prociak J. Mannose-modified ZnO particles for controlled Zn\u003csup\u003e2+\u003c/sup\u003e release as potential drug carriers. Chem Pap. 2025;32:287\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTseng Z-L, Chiang C-H, Chang S-H, Wu C-G. Surface engineering of ZnO electron transporting layer via Al doping for high efficiency planar perovskite solar cells. Nano Energy. 2016;28:311\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Q, Liu P, Zeng D, Xu W, Wang L, Zhu Z, et al. Dual Electrostatic Assembly of Graphene Encapsulated Nanosheet-Assembled ZnO‐Mn‐C Hollow Microspheres as a Lithium Ion Battery Anode. Adv Funct Mater. 2018;28:1707433.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ZnO NPs, membrane permeability, MMP-9, wound healing, scar hyperplasia","lastPublishedDoi":"10.21203/rs.3.rs-5823650/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5823650/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs an important component of sunscreen products for sensitive skin, the potential damage mechanism of ZnO nanoparticles on skin surface with barrier structure or function defect caused by Cutibacterium acnes (C. acnes) has not been elucidated, which poses a serious challenge for reasonable selection of sunscreen products for acne-infected skin. In this work, we demonstrated for the first time that C. acnes induced significant changes in the membrane permeability and intracellular pH of fibroblasts through lipase up-regulation and lipid peroxidation, promoting endocytosis and ionization of ZnO NPs. High amounts of Zn2\u0026thinsp;+\u0026thinsp;further delayed acne wound healing and aggravated scar hyperplasia by intervening matrix metalloproteinase-9 (MMP-9) and TGF-β1/Smad pathway. MMP9 was confirmed to be the key target of ZnO in delaying acne wound healing by the wound regulatory effects of MMP9 agonist and MMP9 inhibitor. In summary, this work clarified the interaction mechanism between ZnO NPs and acne skins, providing guideline for the application of physical sunscreens for special skins.\u003c/p\u003e","manuscriptTitle":"ZnO colludes with C. acnes in healing delay and scar hyperplasia by barrier destruction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 19:34:53","doi":"10.21203/rs.3.rs-5823650/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accepted","date":"2025-04-23T02:15:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-08T19:39:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"249784861457871702443066450125125654810","date":"2025-04-08T19:34:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-08T15:17:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-04T07:19:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-04-01T10:15:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2fdfd2f-7f85-44e4-aaaf-668fcaa8c75e","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:00:40+00:00","versionOfRecord":{"articleIdentity":"rs-5823650","link":"https://doi.org/10.1186/s12951-025-03414-x","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2025-05-31 15:57:24","publishedOnDateReadable":"May 31st, 2025"},"versionCreatedAt":"2025-04-10 19:34:53","video":"","vorDoi":"10.1186/s12951-025-03414-x","vorDoiUrl":"https://doi.org/10.1186/s12951-025-03414-x","workflowStages":[]},"version":"v1","identity":"rs-5823650","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5823650","identity":"rs-5823650","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}