High-throughput screening-based design of multifunctional natural polyphenol nano-vesicles to accelerate diabetic wound healing

High-throughput screening-based design of multifunctional natural polyphenol nano-vesicles to accelerate diabetic wound healing | 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 High-throughput screening-based design of multifunctional natural polyphenol nano-vesicles to accelerate diabetic wound healing Xiaoying Zhao, Shenkai Su, Chenyu Wu, Yuxin Deng, Yu Chen, Tanxin Yu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4948405/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Nov, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted 11 You are reading this latest preprint version Abstract Oxidative stress is a major pathological factor that impedes the diabetic wound healing process. Procyanidins (PC) form nanoparticle-vesicles (PPNs) through hydrogen bonding and exhibit good drug delivery capability; however, its application in diabetic wound is not satisfied. To meet the antioxidant needs for treating, high-throughput screening in natural product library (NPL) under in vitro oxidative stress condition was conducted to enhance the antioxidant activity of PPNs. HUVECs treated with TBHP was established as screening model in vitro. Baicalein (BAI) was identified out of 600 + products in the library as the most effective one to combat oxidative stress. Further study showed that PC and BAI may react in equal proportions to synthesize new vesicles, named BPPNs; while BPPNs have ROS responsive and antioxidant effects. Network pharmacology showed that in diabetic wounds, the target genes of PC are mainly enriched in the VEGF-related pathways, while BAI primarily regulates tyrosine phosphorylation. The complementarity between the two has been validated in in vitro and in vivo experiments. In summary, the antioxidant drug BAI, identified through high-throughput screening of NPL, could optimize the biological function of PPNs; the newly-synthesized BPPNs may accelerate diabetic wound healing through dual mechanisms of promoting angiogenesis and combating oxidative stress. Natural product library polyphenol nanovesicles diabetic wound healing antioxidative stress peptidyl-tyrosine phosphorylation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Diabetic wounds are prevalent chronic, unhealing wounds. The wound healing is one of the most intricate processes within the human body, it is broadly categorized into four phases: hemostasis, inflammation, proliferation, and remodeling. It involves the orchestrated regulation of various cell types over time and space [ 1 ]. Reactive oxygen species (ROS), which encompasses superoxide anions (O 2 - •), peroxides, hydroxyl radicals (OH•), and singlet oxygen ( 1 O 2 ) [ 2 ], serve as pivotal regulatory factors in various stages of wound healing. However, an excessive abundance of ROS can lead to oxidative damage in wound-edge cells, hastening cellular senescence and apoptosis, thereby impeding the overall wound healing process [ 3 – 5 ]. Researches indicate that elevated blood glucose levels lead to an upregulation of pro-inflammatory factors, thereby inducing heightened oxidative stress [ 6 ]. Excessive oxidative stress in tissues and a diminished antioxidative capacity result in an oxidative-reductive imbalance, which stands as a primary determinant of the delayed wound healing process in diabetes [ 3 , 7 ]. Therefore, antioxidant stress is an important strategy for treating diabetes wound healing. General clinical treatment for wounds includes restoring skin perfusion, treating infections, controlling metabolism, treating comorbidities, and caring for local trauma. Although these standard treatments may achieve the goal of controlling symptoms, effective treatment for wound healing of diabetes is still limited[ 8 ]. In recent years, research on multifunctional biomaterials for wound healing has been burgeoning. Biomaterials with controllable release of signal molecules can be combined with other treatment methods, which is a promising treatment method for diabetes wound healing[ 9 , 10 ]. Nano-biomaterials have shown great potential in wound healing due to their multifunctionality and superior physicochemical properties. Natural polyphenols are a class of compounds widely present in living organisms, which have antioxidant, anti-inflammatory, anticancer, antibacterial, and antiviral effects, and are widely used in the treatment of various diseases[ 11 ]. In addition to having multiple biological functions, natural polyphenols also possess unique physicochemical properties, which can form nanoparticles by combining with various substances such as drugs, proteins, nucleic acids, metal ions, etc. through hydrogen bonding, hydrophobic interactions, and electrostatic interactions[ 12 , 13 ]. Studies have shown that natural polyphenols on the surface can effectively enhance the endocytosis of nanoparticles[ 14 , 15 ]. Based on these characteristics, natural polyphenols are widely used in the manufacturing of biomaterials, such as nanoparticles[ 16 ], nanocapsules[ 17 ], encapsulation coatings[ 18 ], and so on. Procyanidins (PC), as one of the natural polyphenols, is considered a highly promising biomaterial with good bioactivity and biocompatibility[ 19 , 20 ]. In the structure of PC, a substantial presence of aromatic rings and phenolic hydroxyl groups confers both amphiphilic and amphiphobic characteristics. Furthermore, they readily engage in hydrogen bonding, governing non-covalent interactions and giving rise to intricate networks [ 12 , 21 ]. Using these properties of the PC, a polyphenol bubble was synthesized, named “Procyanidins polyphenol nanovesicles (PPNs)”. In pursuit of enhancing the antioxidative therapeutic potential of these polyphenol nanovesicles, we conducted a high-throughput drug screening utilizing the Natural Product Library (NPL), an assembly of natural compounds known for their exceptional safety profiles. From a selection of 622 natural compounds, we identified the most efficacious antioxidants and optimized the PPNs to achieve superior therapeutic outcomes of diabetic wound healing. 2. Results 2.1. Sustained high levels of oxidative stress in diabetic wound tissue Existing research indicates that in normal wounds, oxidative stress primarily occurs within the initial three days following injury and gradually diminishes as the inflammatory phase subsides [ 7 ]. In contrast, diabetic wounds exhibit sustained, high levels of ROS [ 3 ]. To further substantiate the oxidative stress levels within diabetic wounds, we have established a rat model of diabetes mellitus (DM) following the protocol delineated in Fig. 1 A. The monitoring of blood glucose in the control group (Ctrl) and the DM group confirmed the successful establishment of the diabetes model (Fig. 1 B). Subsequently, a full-thickness incision with a diameter of 20mm was made on the dorsal surface of both the Ctrl group and the DM group rats. As illustrated in Fig. 1 C, the postoperative wound healing process of both rat groups was documented at days 0, 1, 3, 5, 7, 14, and 21. Compared to Ctrl, the DM group exhibited a noticeably delayed wound healing process with a tendency towards non-closure (Fig. 1 D). Considering the pivotal role of oxidative stress in wound healing, we examined the tissue oxidative stress levels at various stages of wound healing (Day 3 – the inflammatory phase, Day 7 – the proliferation phase, and Day 14 – the remodeling phase [ 1 ]) in both the Ctrl and DM groups. The results indicate that, whether on the 3rd day, 7th day, or 14th day of wound healing, the DM group exhibited significantly lower activities of superoxide dismutase (SOD) and protein expression of heme oxygenase-1 (HO-1) compared to the Ctrl group. Moreover, the levels of lipid peroxidation (malondialdehyde, MDA) and expression of cyclooxygenase-2 (COX2) were notably higher in the DM group than in the Ctrl group (Fig. 1 E-G). The aforementioned experimental results indicate that, in comparison to normal wounds, diabetic wound tissues consistently maintain high levels of oxidative stress throughout the entire wound healing process, corroborating previous research findings. 2.2. High-Throughput Screening of Natural Product Libraries and Network Pharmacology Analysis To target antioxidant stress more effectively, to achieve a better therapeutic effect on diabetic wounds. this study employed TBHP (100µM) to induce oxidative stress in HUVECs in vitro. This study used TBHP to induce oxidative stress in HUVECs in vitro, and a high-throughput screening was conducted on the natural product library (NPL) consisting of 622 compounds (Figure S1 A). The specific procedure is illustrated in Fig. 2 A, wherein cell viability is employed as an indicator of a drug's capacity for antioxidant stress. Toxicity tests were conducted on the top five ranking drugs based on cell viability to ascertain the optimal dosages for these five drugs (Figure S1 B). At the optimal concentration, the fluorescence intensity of DCFH-DA was utilized to assess the ROS scavenging capabilities of these five compounds (Fig. 2 B). Subsequently, baicalein (BAI, 5µM), which exhibited the most remarkable antioxidative effect in HUVECs, was selected for further investigations. Using network pharmacology, a Venn diagram was constructed to elucidate the predicted targets of PC and BAI, as well as the disease-related targets involved in diabetic wound healing. Additionally, common targets shared by these compounds were identified (Fig. 2 C and S1C). In order to investigate the respective effects of biological materials and pharmaceuticals on disease treatment, we conducted separate GO enrichment analyses for the common targets of BAI and PC that intersect with the disease but do not overlap. The results of the enrichment analysis indicate that BAI may exert an influence on the peptidyl-tyrosine phosphorylation and cellular response to hydrogen peroxide (Fig. 2 D), whereas PC primarily affects the vascular endothelial growth factor receptor signaling pathway (Fig. 2 E). This suggests that BAI and PC exhibit a potential complementarity in terms of therapeutic efficacy, and their combined action may influence wound healing through distinct processes, namely antioxidant stress and angiogenesis. This opens up the possibility of multifunctional optimization for PPNs. 2.3. Synthesis and Characterization of Baicalein-Procyanidins polyphenol nanovesicles (BPPNs) PC can utilize the physical adsorption of CaCO 3 on polyphenols to form vesicles [ 22 , 23 ], which can de-nucleate into PPNs under acidic conditions. Under the same conditions, new polyphenol vesicles BPPNs were synthesized using equal amounts of PC and BAI. PPNs and BPPNs are collectively referred to as “Polyphenol nanovesicles (PNs)”. As shown in Fig. 3 A, the scanning electron microscopy (SEM) image of PPNs and BPPNs shows that BPPNs are spherical vesicles with a diameter smaller than PPNs. The diameter and Zeta potential of both were measured, and it was found that the diameter of BPPNs was about 500nm, which is half of PPNs, and the Zeta potential of BPPNs was similar to PPNs, indicating that the addition of BAI reduced the size of vesicles and did not affect their stability (Fig. 3 B). The FTIR spectra of BAI, PNNs, and BPPNs are depicted in Fig. 3 C. Within the PPNs, the presence of intermolecular hydrogen bonding is evident, as indicated by a broad absorption peak at 3300 cm -1 , this phenomenon leads to the absence of exposed phenolic hydroxyl groups, consistent with the expected theory [ 12 ]. The BPPNs inherit the phenolic hydroxyl absorption peak exposed by BAI (3400 cm -1 ), signifying that, at a chemical level, the newly synthesized nanoparticles exhibit enhanced antioxidative properties. Furthermore, in comparison to BAI, BPNNs exhibit no absorption peak around 1655 cm -1 , implying that ketone carbonyl disappeared during the synthesis process of the nanovesicles, a possible addition reaction occurred between the carbonyl group in the BAI structure and the phenolic hydroxyl group in PC. This potentially led to the formation of hemiketals that effectively preserve the chemical structure of BAI. Based on this, create a synthesized illustrative diagram as depicted in Fig. 3 D. Analyzing from a chemical perspective, the hemiketals can protect the carbonyl structure in BAI, decompose after pH change, and release complete BAI and PC molecules under the mildly acidic environment found in diabetic wounds [ 24 ], respectively playing their respective therapeutic roles. According to the molecular formula (PC: C 30 H 26 O 13 , BAI: C 15 H 10 O 5 ) and XPS analysis of PNs involving carbon (C) and oxygen (O) atoms, the results indicate that the content of PC and BAI in BPPNs is approximately in a 1:1 ratio (Fig. 3 E). The peak fitting results also confirmed the successful synthesis of BPPNs (Fig. 3 F). In summary, it can be inferred that in BPPNs, PC, and BAI can undergo proportional addition reaction to generate hemiketals, which are then connected by hydrogen bonds to synthesize smaller-diameter polyphenol nanovesicles. In order to further investigate the biological activity of PNs, toxicity tests were conducted on PNs (Figure S2 A and B). The maximum safe concentration within which PNs were chosen for subsequent experiments was determined to be 4µg/ml (After conversion, the content of BAI and PC in 4 µg/ml BPPNs is approximately 5 µM each). To facilitate the in vitro monitoring of the extracellular uptake of PNs, we employed the fluorescent dye FITC to label PNs. Under both conditions with and without TBHP stimulation, cellular uptake of PNs was recorded at 1h and 2h (Fig. 3 G). Quantitative results, as shown in Fig. 3 H, reveal that both PPNs and BPPNs exhibit time-dependent and ROS-corresponding uptake. Notably, BPPNs demonstrate a greater cellular uptake efficiency in comparison to PNNs, which may be attributed to the smaller size and increased ease of cellular internalization associated with BPPNs. After co-treatment with PNs and TBHP for two hours, the localization relationship between PNs and cellular organelles was assessed using LysoTracker and MitoTracker (Fig. 3 I and J). The quantitative results of Pearson’s correlation coefficient indicate that both types of vesicles can achieve lysosomal escape and co-localize with mitochondria under oxidative stress (Fig. 3 K). In summary, compared with PPNs, the newly synthesized BPPNs have been optimized in characterization. BPPNs are a novel type of polyphenolic nanovesicles with smaller particle size, higher cellular uptake efficiency, lysosomal escape, and mitochondrial ROS response properties. 2.4. In vitro angiogenic activity of BPPNs Based on the network pharmacology and enrichment analysis results from Figs. 2 C and 2 E, it is speculated that PC may participate in or affect the VEFG signal pathway in the course of diabetes wound healing. In response to VEGF stimulation, endothelial cells sprout from the capillaries and then complete the entire process of angiogenesis with the proliferation, migration, and tubular formation of endothelial cells, ultimately providing nutrients for wound tissue, which is a key step in wound healing [ 1 , 25 ]. To further clarify the impact of PNs on angiogenesis, we conducted relevant tests on HUVECs. Scratch assays were conducted on attached HUVECs. Western blot results indicated that both BPPNs and PPNs effectively upregulated the expression of vascular angiogenesis-related proteins, VEGF, and its upstream regulator HIF-1α (Fig. 4 A, B). The cells were treated with BAI (5µM), PPNs (4µg/ml), or BPPNs (4µg/ml) for 24 hours, with HUVEC migration recorded at 0h, 12h, and 24 h (Fig. 4 C). The results reveal that, when compared to the control group, BAI exhibited no significant impact on HUVECs’ migration. However, PPNs synthesized via PC significantly enhanced scratch healing. Notably, BPPNs containing BAI also have similar cell migration-promoting effects to PPNs (Fig. 4 D). Following this, we subsequently performed tube formation assays on HUVECs from different treatment groups on the Matrigel ® matrix (Fig. 4 E), and quantified the number of nodes, meshes, branches, and total tube length (Fig. 4 F). The results demonstrated that BAI had almost no effect on the tube formation of HUVECs, while BPPNs exhibited a promotion of tube formation activity similar to that of PPNs. Overall, although BAI does not affect tube formation, BPPNs can effectively upregulate angiogenic-related proteins, promote the migration and tube formation of HUVECs, and obtain similar promoting tube activity as PPNs. 2.5. In vitro antioxidant activity of BPPNs To further explore the in vitro antioxidant activity of BPPNs, HUVECs were divided into five groups as shown in Fig. 5 , with a treatment duration of 24 hours, and a series of tests were conducted. The CCK-8 results indicated that BAI, PPNs, and BPPNs could all mitigate the reduction in cell viability caused by TBHP, with the rescue effect increasing in the order of PNNs < BAI < BPPNs (Fig. 5 A). Compared to the Ctrl group, the oxidative stress level in HUVECs of the TBHP group was significantly elevated (marked decrease in SOD activity and significant increase in MDA content), while the antioxidative stress capabilities of PPNs, BAI, and BPPNs were enhanced (Fig. 5 B). ROS probes and JC-1 staining were performed on HUVECs under different treatments (Fig. 5 C and E). Oxidative stress induced by TBHP led to an increase in intracellular ROS and mitochondrial damage, with BPPNs showing the best capability in counteracting ROS and repairing mitochondrial damage, followed by BAI (Figs. 5 D and F). Therefore, it is speculated that the prominent antioxidant activity of BPPNs is largely attributed to BAI, which was identified through high-throughput screening in NPL. Based on the outcomes of network pharmacology and enrichment analyses (Figs. 2 C and D), it is hypothesized that the exceptional antioxidative efficacy of BAI may be attributable to its regulatory function on tyrosine phosphorylation. Protein phosphorylation modification, particularly prevalent and functionally paramount, occurs in over 30% of cellular proteins. Despite tyrosine phosphorylation modifications (P-Tyr) constituting less than 1% of all protein phosphorylation modifications, they play a pivotal role in nearly every physiological process within the cell [ 26 , 27 ]. Studies have indicated that a high-fat diet in mice can affect the P-Tyr of liver proteins [ 28 ]. Proteins within pathways such as JAK1/STAT3[ 29 , 30 ], Erk1/2[ 31 ], and Akt [ 32 ], all containing tyrosine residues, are subject to increased levels of tyrosine phosphorylation due to oxidative stress, thereby activating their respective signaling pathways. To validate the impact of BPPNs on P-Tyr and their antioxidative effects at the protein level, Western blot experiments were conducted (Fig. 5 G). The results revealed that BAI significantly inhibits the tyrosine phosphorylation of pathway proteins JAK1, STAT3, Erk1/2, and Akt, markedly modulates oxidative stress-related proteins (upregulating HO-1 and downregulating COX-2), and suppresses the expression of the mitochondria apoptosis-related protein Cyto C. These modulatory effects are notably superior to those of PPNs but inferior to BPPNs (Fig. 5 H). Drawing Fig. 5 I based on the above experimental results and literature search[ 33 , 34 ]. In summary, BPPNs can effectively counteract in vitro oxidative stress caused by TBHP by inhibiting tyrosine phosphorylation of pathway proteins, while also playing a role in mitochondrial protection (Fig. 5 I). 2.6. BPPNs accelerate diabetic wound healing In consideration of the application of PNs in diabetic wounds, we employed a photosensitive hydrogel with tissue adhesiveness, HA-NB, to anchor the PNs at the wound locations [ 35 , 36 ]. The establishment of a diabetic wound model in SD rats was followed by the random allocation of these subjects into four groups, as depicted in Fig. 6 . Initially, we evaluated the impact of treatments with HA-NB, PPNs@HA-NB, and BPPNs@HA-NB on the principal organs—heart, liver, spleen, lungs, and kidneys—through H&E staining of organ tissue sections. The absence of pathological alterations in these stained sections suggests that HA-NB, PPNs@HA-NB, and BPPNs@HA-NB do not exhibit organ toxicity, as illustrated in Supplementary Fig. 3. Figures 6 A and B depict the wound healing progression within fourteen days across four treatment groups, accompanied by a quantification of the wound areas (Fig. 6 C). Compared to the DM group, the application of HA-NB significantly enhances the healing of diabetic wounds, likely attributable to the reduced risk of infection at the diabetic wound sites facilitated by the coverage provided by HA-NB[ 36 ]. It is particularly noteworthy that the BPPNs@HA-NB group exhibited a higher rate of diabetic wound healing compared to the PPNs@HA-NB group. Furthermore, we employed laser Doppler scan images to characterize the functional vasculature with blood flow (Fig. 6 D). It was observed that BPPNs@HA-NB could effectively upregulate angiogenesis around the wound during the proliferative phase of healing, thereby abbreviating the protracted proliferative phase induced by diabetes (Fig. 6 E). Subsequent histopathological evaluation of the neotissue on day 14 post-injury using H&E and Masson's trichrome staining is presented in Fig. 6 F. Quantitative analyses, as shown in Fig. 6 G, revealed that diabetic wound tissues treated with BPPNs@HA-NB had thicker granulation tissue, shorter wound lengths, and higher collagen deposition compared to the PPNs@HA-NB group. These findings collectively suggest that BPPNs significantly accelerate diabetic wound healing and promote the progression of wound healing processes in diabetes, in comparison to PNNs. To investigate the intrinsic mechanisms underlying the therapeutic efficacy of BPPNs in the treatment of diabetic wounds, we further assessed the levels of oxidative stress, angiogenesis, and collagen within the tissue 14 days post-injury. The detection results for Superoxide Dismutase (SOD) and Malondialdehyde (MDA) indicated that, compared to the other three groups, the BPPNs@HA-NB group significantly reduced the level of oxidative stress in diabetic wound tissues (Fig. 7 A). Immunofluorescence co-staining for CD31 and α-SMA, markers of neovascularization (Fig. 7 B), with quantification of fluorescence expression shown in Fig. 7 C, demonstrated that the vascular density in the BPPNs@HA-NB group was comparatively higher. As depicted in Fig. 7 D, immunohistochemical analysis of oxidative stress-related markers (COX2 and HO-1), mitochondrial apoptosis markers (Cyto C), angiogenesis-related markers (VEGF), and Collagen III (COL3), with quantitative results, revealed that BPPNs@HA-NB could downregulate COX2 and Cyto C protein expression and upregulate the relative expression of HO-1, VEGF, and COL3 compared to the other three groups. This suggests that BPPNs may accelerate diabetic wound healing by counteracting oxidative stress, protecting mitochondria, promoting angiogenesis, and enhancing collagen deposition. In summary, optimized with BAI, the newly synthesized polyphenol vesicles, BPPNs, demonstrate enhanced efficacy in counteracting the excessive oxidative stress encountered during the progression of diabetic wound healing, compared to the original anthocyanin polyphenol vesicles, PPNs. Moreover, BPPNs inherit the angiogenic-promoting effects of PPNs and, due to their superior antioxidant activity, provide a more "fertile ground" for angiogenesis. Consequently, the results indicate that BPPNs exhibit superior in vivo angiogenesis promotion compared to PPNs. Overall, BPPNs accelerate diabetic wound healing through a dual approach: combating oxidative stress and promoting angiogenesis, showcasing their potential as a multifaceted therapeutic intervention in diabetic wound management (Fig. 8 ). 3. Discussion In this study, we initially identified the level of oxidative stress during the progression of diabetic wound healing. The experimental results are consistent with existing reports [ 7 ], indicating that diabetes leads to a sustained high level of oxidative stress in the peri-wound tissue. Consequently, we induced oxidative stress in HUVECs in vitro and conducted high-throughput screening of over six hundred drugs from the Natural Product Library, identifying BAI as the most effective antioxidant drug in endothelial cells. By mixing BAI with the commonly used polyphenol vesicle framework material PC, in a 1:1 ratio, and utilizing the physical adsorption properties of CaCO3, we synthesized a new nanovesicle, BPPNs. Further exploration of its chemical structure revealed that BAI can undergo an addition reaction with PC in a 1:1 ratio, forming a hemiketal, which then connects into a network through intermolecular hydrogen bonds, ultimately observing a vesicular structure under an electron microscope. The incorporation of BAI into BPPNs results in smaller particle sizes compared to PPNs, thereby enhancing their cellular uptake efficiency. Additionally, BPPNs possess ROS responsiveness and lysosomal escape capabilities, and they can achieve co-localization with mitochondria under oxidative stress conditions. Network pharmacology and GO enrichment analysis suggest that PC may contribute to diabetic wound healing by affecting the VEGF signaling pathway, while BAI could potentially influence protein tyrosine phosphorylation and induce cellular responses to hydrogen peroxide, thereby participating in the progression of diabetic wound healing. Consequently, we further explored the bioactivity of BPPNs synthesized from PC and BAI. The results indicate that under physiological conditions, BAI does not participate in the angiogenesis of HUVECs, but both PPNs and BPPNs significantly promote in vitro tube formation and upregulate VEGF. Under TBHP stimulation, BPPNs exhibit superior effects in inhibiting pathway protein tyrosine phosphorylation, counteracting oxidative stress, and mitochondrial repair, almost equating to the combined bioactivity of BAI and PNNs. In vivo studies further demonstrate that BPPNs are polyphenol vesicles with both angiogenic and antioxidative stress activities, capable of accelerating diabetic wound healing. Diabetes is a chronic disease that poses a severe threat to human health, with over 500 million individuals affected globally by 2021, and an incidence rate that is increasing annually[ 37 ]. The hallmark of diabetes is elevated blood glucose levels, which, over time, can induce oxidative stress, exacerbating hypoxia, inflammation, and cellular apoptosis[ 38 ]. These conditions are at the core of most diabetic complications. Diabetic wounds, a common complication of diabetes, are particularly troubling due to their non-healing nature, which can lead to ulcers and even amputation [ 39 ]. Studies have shown that the non-healing wounds in diabetic patients are primarily due to excessive oxidative stress in the peri-wound tissue and a decrease in the body's antioxidant capacity following diabetes onset [ 3 ]. Thus, this article aims to design an effective antioxidant treatment strategy targeting oxidative stress during the diabetic wound healing process. The occurrence and progression of oxidative stress primarily stem from the generation of ROS and aberrations in signaling pathways [ 40 , 41 ]. ROS induce signaling pathway aberrations, which in turn exacerbate ROS production. Consequently, antioxidant therapy can be broadly categorized into two approaches: chemical removal of peroxides via direct chemical reactions and modulation of signaling pathways through biological means [ 7 ]. A dual-pronged approach is imperative for mitigating oxidative stress effectively. From a chemical standpoint, the material scaffold employed in this study, Procyanidins, harbors abundant phenolic hydroxyl groups, theoretically enabling it to directly counteract oxidative stress by reacting with ROS. Correspondingly, literature also suggests that PC acts as a barrier against oxidative stress by alleviating the burden of free radicals, thereby preventing molecular and cellular damage [ 42 ]. In this research, PPNs formed via physical adsorption and intermolecular hydrogen bonding of PC exhibit ROS responsiveness and demonstrate antioxidative activity both in vitro and in vivo (Figs. 5 and 7 ). However, their impact on pathway proteins and overall antioxidative efficacy is relatively modest, suggesting that the antioxidative activity of PC primarily derives from its chemical structure, enabling direct reaction with ROS. Optimization of PPNs is imperative for achieving enhanced antioxidative activity. Natural products, cherished for their high biological activity, low toxicity, and considerable developmental potential, have garnered significant attention from clinicians and researchers alike. Hence, our focus gravitates toward this realm. Through high-throughput screening of the natural product library under oxidative stress stimulation, BAI emerged as a standout candidate owing to its remarkable antioxidative activity. Interestingly, BAI also possesses a polyphenolic structure within its chemical composition. However, its phenolic hydroxyl content falls considerably short of that found in PC. Consequently, from a chemical perspective, baicalein's ability to directly react with ROS is presumed to be inferior to that of cyanidin-3-glucoside. Thus, we boldly speculate that baicalein is more inclined towards combating oxidative stress through the modulation of signaling pathways. Subsequent network pharmacological analysis validated this conjecture (Figs. 2 C and D), wherein multiple GO biological processes suggested BAI's capacity to influence protein tyrosine phosphorylation. To corroborate this finding, we selectively screened several pathway proteins closely associated with oxidative stress and harboring tyrosine residues, including JAK1, STAT3, Erk1/2, and Akt. Western blot results (Figures G and H) revealed that BAI effectively inhibits the phosphorylation of these pathway proteins, ultimately yielding antioxidative stress and mitochondrial damage repair effects. BPPNs, BAI-optimized polyphenol nanovesicles, unsurprisingly, exhibited superior antioxidative activity both in vitro and in vivo (Figs. 5 and 7 ), underscoring the feasibility of a dual antioxidative strategy via chemical and biological pathways. It's noteworthy that in network pharmacology analysis, PC and BAI have non-overlapping targets with the common targets of diabetic wounds, which separately lead to two key aspects of diabetic wound treatment: angiogenesis and oxidative stress. The angiogenesis-promoting effect of PC has also been verified in both in vivo and in vitro experiments, which endows BPPNs with angiogenic activity that BAI lacks. Therefore, the synthesis of BPPNs not only combines the antioxidative activities of PC and BAI but also achieves a complementary effect of their functions. Nanoparticles (NPs) are commonly used as part of drug delivery systems because they can enhance the pharmacokinetics, bioavailability, and half-life of drug formulations, and reduce the frequency of dosing for gene or drug molecules [ 43 ]. Given the amphiphilic and network structure of BPPNs, it's reasonable to speculate that the polyphenol vesicles we designed and optimized also possess the potential to carry drugs or to transport exogenous proteins, amino acids, or even nucleic acid sequences into cells. Coupled with their inherent biological activity, this could potentially achieve therapeutic effects that advance disease progression. Of course, these potentials still require further development and optimization, and this is merely a presentation of a possibility. In summary, this study, through high-throughput screening of a natural product library, identified BAI as the drug with the best antioxidant stress effect in HUVECs. Utilizing the biological activity and chemical structure of baicalin, the study characterized and functionally optimized proanthocyanidin polyphenol vesicles, resulting in BPPNs with smaller particle size, faster cellular uptake, and stronger ROS response. These vesicles accelerate diabetic wound healing by simultaneously combating oxidative stress and promoting angiogenesis. 4. Conculsion In this study, we synthesized and successfully validated a multifunctional polyphenol nanoparticle vesicle, BPPNs, optimized through natural products, targeting excessive oxidative stress during the healing process of diabetic wounds. Through screening the antioxidant activity of over six hundred natural products from the natural product library, baicalin was selected for its optimal antioxidant stress effect in HVECs. Baicalin undergoes an additive reaction with the polyphenol biomaterial Procyanidins, utilizing intermolecular hydrogen bonding to form BPPNs, which accelerate cellular uptake under oxidative stress conditions and exhibit the biological activities of baicalin and proanthocyanidins intracellularly. BPPNs not only exert antioxidant activity by inhibiting the phosphorylation of pathway proteins such as tyrosine but also accelerate diabetic wound healing by further promoting angiogenesis and collagen deposition. Overall, our research results offer a new therapeutic strategy for the healing of diabetic wounds. 5. Experimental Section/Methods 5.1. Reagents and kits Natural Product Library (#HY-L021) and FluoroTag™ FITC Conjugation Kit (#HY-66019) from MedChemexpress (NJ, USA). Fetal bovine serum (FBS, #10099141C) was obtained from Gibco-Invitrogen (NY, USA). Dulbecco’s Modified Eagle Medium (DMEM, #MA0212), Penicillin/Streptomycin, sterile(100X) (#MA0110), phosphate buffered solution (PBS, #MA0015), 0.25%Trypsin-EDTA, PhenolRed(modified) (#PWL060), Streptozotocin (STZ, #MB1227), Procyanidins (#MB2168-1), Baicalein (#MB7311) and other reagents required for cell culture were obtained from Meilunbio (Dalian, China). High-fat feed (#MD12033) purchased from Medicience (Jiangsu, China). Tert-butyl hydroperoxide solution (TBHP, #418064) and DMSO (#D2650) were purchased from Sigma-Aldrich (St Louis, MO, USA). Cell Counting Kit-8 (#C0040), Protease inhibitor cocktail for general use (#P1005), SDS-PAGE Sample Loading Buffer (5X) (#P0015), Enhanced BCA Protein Assay Kit (#P0010), Hoechst 33258 (#C1011) and BeyoECL Star (#P0018AM) were purchased from Beyotime (Shanghai, China). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA, #D6470), Superoxide Dismutase (SOD) Activity Assay Kit (#BC0175), Malondialdehyde (MDA) Content Assay Kit (#BC0025), Mitochondrial Membrane Potential Assay Kit with JC-1 (#M8650), Hematoxylin-Eosin (HE) Stain Kit (#G1120), Masson's Trichrome Stain Kit (#G1340), DAB Substrate kit (20×) (#DA1010), Mounting Medium (antifading, with DAPI) (#S2110) and were purchased from Solarbio Science & Technology (Beijing, China). MitoTracker™ Red CMXRos (#M7512) and LysoTracker™ Deep Red (#L12492) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (#ab150113), and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) (#ab150080) were purchased from Abcam (Cambridge, UK). Table 1 Primary antibodies were used in the study. Antibody Purpose Product ID and manufacturer p-JAK1 WB #741295, Cell Signaling Technology, USA JAK1 WB #66466, Proteintech, China p-STAT3 WB #9145, Cell Signaling Technology, USA STAT3 WB #12640, Cell Signaling Technology, USA COX2 WB/ IHC #12282, Cell Signaling Technology, USA HO-1 WB/ IHC #10701-1-AP, Proteintech, China Cyto C WB/ IHC #10993-1-AP, Proteintech, China VEGF WB/ IHC #ET1604-28, Huabio, China HIF-1α WB #66730, Proteintech, China CD31 IF #ab281583, Abcam, UK α-SMA IF #BM0002, Boster, China COL3 IHC #ER1906-50, Huabio, China GAPDH WB #2118, Cell Signaling Technology, USA 5.2. Animal model of diabetic full-thickness wounds All animal experiments were approved by the Institutional Ethical Committee of Wenzhou Medical University (wydw2023-0243). The Experimental Animal Center of Wenzhou Medical University (Zhejiang Province, China) provided a total of 40 male 6-week-old SD rats (180-220g). After adaptive feeding for 1 week, high-fat diets were given for 4 weeks. Intraperitoneal injections were administered for 3 days each time: 60mg/kg STZ (T2D, n = 30) until blood glucose stabilizes > 16.67mmol/L; 0.25ml/kg 0.9% NaCl (Ctrl, n = 10). Continue high-fat feeding for 4 weeks. For the wounds model, animals were anesthetized with 2.5% pentobarbital sodium (30 mg/kg) and randomly selected for grouping. After shaving and sterilization, two full-thickness wounds (20 mm in diameter) were made by scissor cutting along the mark on each side of the rat’s back. All the animals were high-fat fed in individual cages and given ad libitum feeding access to food and water, and they were observed every day during the total period of the experiment. At days 0, 1, 3, 5, 7, 14, and 21 posttreatment, the wound area was calculated by tracing the wound margins from rats and was evaluated as a percent area of the original wound using Image J software. 5.3. High-throughput screening of natural product library Using human umbilical vein endothelial cells (HUVECs) to screen 622 drugs from the natural product library. HUVECs were seeded into a 96-well plate (8000 cells/well) and cultured for 24 hours until they adhered to the walls. A blank control group, stimulation group (TBHP 100µM), and drug treatment group (TBHP 100µM + drug 10µM) were set up in the plate, and after 24 hours of treatment, the detection was performed using CCK8, measuring absorbance at a wavelength of 450nm with an enzyme reader, designated as Ac, Ab, and As, respectively. Cell viability was calculated for each drug treatment using the formula and subjected to statistical analysis (n = 3). Cell viability (%) = \(\:\frac{As-Ab\:}{Ac-Ab}\) × 100% \(\:\:\) 5.4. Network pharmacology and GO biological processes enrichment analysis The two-dimensional conformations of Procyanidins and baicalein were downloaded from PubChem’s official website ( https://pubchem.ncbi.nlm.nih.gov/ ). Isomeric Smiles format files were imported into the Swiss Target Prediction ( http://swisstargetprediction.ch/ ) platform, set the attribute to Homo sapiens, and start to predict the potential target of Procyanidins and baicalein. By searching the keyword of diabetic wound healing in the GeneCards ( https://www.genecards.org/ ) database to obtain the disease targets. Compare the three datasets mentioned above by creating a Venn diagram ( https://jvenn.toulouse.inrae.fr/app/index.html ), and performing an enrichment analysis of GO biological processes on the genes at the intersection ( https://metascape.org/gp/index.html ). 5.5. Preparation and characterization of PPNs and BPPNs After adding 1 mL of 1M Na 2 CO 3 solution to a 15 mL conical flask and stirring at 1200 rpm, 1 mL of ddH 2 O is added. Then, 0.5 mL 10mM Procyanidins and baicalein are added to the conical flask. Stirring continues, and 1 mL of 1M CaCl 2 solution is added. Stirring is continued. After allowing it to stand for 15 minutes, the mixture is centrifuged at 1000 rpm and washed with water three times. The washed samples are then mixed with 4 mL of 1M HCl and centrifuged at 3000 rpm until the pH is between 6 and 6.5. Afterward, freeze-drying is performed to obtain BPPNs. As for PNNs, repeat the above process by replacing the baicalein solution with an equimolar solution of Procyanidins. The morphology of nanoparticles was observed by scanning electron microscopy (SEM) (Quanta 250 FEG, FEI). The particle size and Zeta potential analyses of nanoparticles were carried out on the Malvern Zetasizer Nano ZS (Malvern Instruments, UK). Chemical interaction between NA was recorded on a Nicolet IS10 FT-IR instrument (ThermoFisher Scientific, USA) scanning from 4000 to 500 cm − 1 . XPS was measured by ESCALAB 250Xi K-alpha (ThermoFisher Scientific, USA). For BPPBs: C1s/O1s = (71.54%)/(28.46%) =(30PC + 15BAI)/(13PC + 5BAI) → PC/BAI ≈ 1 5.6. Western blot HUVECs were subjected to a 1:4 dilution with a 5X loading buffer and subsequently exposed to a heat treatment at 100°C for 15 minutes. The resultant protein extracts were then resolved through a 12.5% SDS-PAGE gel and transferred onto PVDF membranes. Following this, the membranes were subjected to a blocking procedure involving 5% nonfat milk for 2 hours. After the milk blocking, the membranes underwent a series of washes using TBST and were subsequently incubated with primary antibodies for 8–12 hours at 4°C. Following this primary antibody incubation, the membranes were once again subjected to TBST washes and subsequently incubated with secondary antibodies conjugated with HRP for 2 hours at RT. The visualization of immunoreactive bands was achieved through the utilization of an ECL kit and the bands were detected using a ChemiDoc Imaging System (Bio-Rad, USA). 5.7. In vitro tube formation assay A tube formation assay using Matrigel® matrix (#356234, Corning, USA) was conducted to assess the morphogenesis and tube formation capability of HUVECs under different treatments. In brief, the Matrigel solution was allowed to thaw at 4°C overnight and subsequently dispensed into µ-Slide chambers (10 µL per well, IBIDI, Germany), followed by incubation in a cell incubator for 1 hour to facilitate solidification. A total of 5000 cells, which had been pre-treated with BAI (10µM), PPNs, and BPPNs, were seeded onto the Matrigel-precoated µ-Slide. Tube formation was meticulously observed and quantified, with an average count derived from the assessment of three independent fields, all conducted under an inverted light microscope (Olympus, Japan). 5.8. Scratch-wound healing assay Cultivate HUVECs in a 6-well culture plate until they adhere to the walls. Scratch the cell surface gently using the tip of a sterile 200-µL pipette. Remove floating cells with PBS and continue with a complete culture medium containing 5% FBS. Capture images of the scratches at 0 hours, 12 hours, and 24 hours, respectively, under an inverted microscope. 5.9. Assessment of blood flow in the wound area The blood flow in the wound area was assessed by a laser Doppler imager (MoorLDI-2; Moor Instruments Limited, Devon, UK). Briefly, the rats were anesthetized with 2.5% pentobarbital sodium (30 mg/kg), shaved, and then gently fixed onto a black platform. MoorLDI Review V6.1 software was used to quantify the results. 5.10. Histology analysis and immunohistochemistry On the 14th day, the rats were perfused with physiological saline. The wound was fixed with 4% paraformaldehyde at 4°C, followed by paraffin embedding. The embedded tissues were sectioned and stained with hematoxylin and eosin (H&E) as well as Masson's trichrome stain to assess inflammation, epidermal regeneration, granulation tissue, and collagen deposition. Immunohistochemistry and immunofluorescence were performed to evaluate the expression of various markers. 5.11. Statistical analysis Numerical data, presented as mean values accompanied by their respective standard deviations (mean ± SD), are depicted based on the results of a minimum of three distinct experiments unless stated otherwise. Statistical analysis of the data was conducted through a one-way analysis of variance (ANOVA), followed by Tukey's post-hoc analysis, utilizing GraphPad Prism 7.0 (La Jolla, CA, USA). Statistical significance was established at a threshold of P < 0.05, and comparisons were made at two significance levels, *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 against the specified reference group. Declarations Data Availability The data used to support the findings of this study are available from the corresponding authors upon request. Ethics approval and consent to participate All animal experiments were approved by the Institutional Ethical Committee of Wenzhou Medical University (wydw2023-0243). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interest. Authors’ contributions XY. Zhao: Writing – original draft, Methodology, Formal analysis, Conceptualization. SK Su: Methodology. CY. Wu: Methodology, Formal analysis. YX. Deng: Methodology. Y. Chen: Methodology. TX. Yu: Methodology. CC. Li: Methodology. YK. Zhang: Methodology. XY. Wang: Project administration. YF. Zhou: Funding acquisition. XL. Zhang: Writing – review & editing, Methodology, Formal analysis. All authors have read and approved the content of the manuscript. Acknowledgements The authors thank Professor Linyong Zhu from the school of Biomedical engineering, Shanghai Jiao Tong University, for providing us with HA-NB hydrogel. This study was supported by Zhejiang Provincial Natural Science Foundation of China (LY22H060008). References Rodrigues M, et al. Wound Healing: A Cellular Perspective. Physiol Rev. 2019;99(1):665–706. http://doi.org/10.1152/physrev.00067.2017 . Chen L, et al. The role of antioxidants in photoprotection: a critical review. J Am Acad Dermatol. 2012;67(5):1013–24. http://doi.org/10.1016/j.jaad.2012.02.009 . Cano Sanchez M, et al. Targeting Oxidative Stress and Mitochondrial Dysfunction in the Treatment of Impaired Wound Healing: A Systematic Review. Antioxid (Basel). 2018;7(8). http://doi.org/10.3390/antiox7080098 . Dunnill C, et al. 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03:55:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4948405/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4948405/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-024-02950-2","type":"published","date":"2024-11-21T15:58:17+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65203291,"identity":"c058bec9-dfa4-46c9-83de-18f91e72cdac","added_by":"auto","created_at":"2024-09-24 17:23:44","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":644963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment of the Diabetic Wound Model and detection of oxidative stress levels in tissues.\u003c/strong\u003e A. Schematic representation of the establishment of the diabetic wound model. B. Blood glucose levels in the control group (Ctrl) and the diabetic group (DM). C. Representative photographs of diabetic wounds for Ctrl and DM groups on the 0, 1st, 3rd, 5th, 7th, 14th, and 21st day. D. Quantitative analysis of wound area change in comparison with the original wound. E. SOD and MDA level of Ctrl and DM on day 3, 7 and 14. F. Corresponding immunohistochemistry staining of COX2 and HO-1 on day 3, 7 and 14. G. Immunohistochemical quantification of COX2 and HO-1 in image F (n=5). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.005.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4948405/v1/fd02c1d05f4e8c020a2eb1e1.jpeg"},{"id":65202351,"identity":"eeea3d88-53ab-4495-90aa-644941b8c8fa","added_by":"auto","created_at":"2024-09-24 17:07:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":317888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh-throughput Drug Screening, Network Pharmacology, and GO Enrichment Analysis. \u003c/strong\u003eA. The process diagram and analytical outcomes of high-throughput drug screening in NPL. B. Effects of different drugs with TBHP on ROS levels in HUVECs (n=3). *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.005, ****P \u0026lt; 0.001, vs. that of Baicalein. C. Venn diagram for the potential target of procyanidins and baicalein and the disease targets of diabetic wound healing. D. Bubble chart of the biological process category terms from GO enrichment analysis for the 77 common targets. E. Bubble chart of the biological process category terms from GO enrichment analysis for the 11 common targets.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4948405/v1/acc2d078ea350f6159bdd2c0.png"},{"id":65202352,"identity":"f0a5cc58-a43e-4ddb-ad42-a52d1bb5280b","added_by":"auto","created_at":"2024-09-24 17:07:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":854639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Synthesis and Characterization of PPNs and BPPNs. A. SEM image of PPNs and BPPNs.\u003c/strong\u003e Scale bar: 500 nm. B. Particle size and Zeta potential of PPNs and BPPNs. C. FTIR spectra of BAI, PPNs and BPPNs. D. Schematic of PPNs and BPPNs synthesis. E. XPS full spectrum of PPNs and BPPNs. F. The high-resolution XPS spectrum for O1s of BPPNs. G. Images of HUVECs taking up PPNs and BPPNs. Green: FITC-labeled PNs; Blue: Hoechst (nuclei). Scale bar: 100 μm. H. Quantitative measurement of FITC fluorescence intensity in G. I. Confocal microscope images locating FITC-labeled PPNs and BPPNs relative to lysosomes in HUVECs cells with TBHP. Green: FITC-labeled PNs; Red: LysoTracker (lysosome); Blue: Hoechst (nuclei). Scale bar: 10 μm. J. Confocal microscope images locating FITC-labeled PPNs and BPPNs relative to mitochondrions in HUVECs cells with TBHP. Green: FITC-labeled PNs; Red: MitoTracker (mitochondrion); Blue: Hoechst (nuclei). Scale bar: 10 μm. K. Pearson’s correlation coefficients indicating the degree of PNs/lysosome and PNs/Mitochondrion colocalization in I and J : R=1 (perfect colocalization), R=0 (no colocalization). n=3, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.005, ****P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4948405/v1/0d57abbb75c2959ae0ff1677.png"},{"id":65202353,"identity":"695272a8-f179-4a22-974a-a996b7e07639","added_by":"auto","created_at":"2024-09-24 17:07:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":363706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe angiogenic properties of BPPNs in vitro. \u003c/strong\u003eA-B. Western blots of VEGF and HIF-1α by HUVECs incubated in different preparations for 24 h. C. Representative images of HUVEC migration after treating for 0, 6, 12 h, scale bar: 500 μm. D. Quantify the migration of HUVEC. E. Representative images of HUVEC tubular formation after treating for 4 h, scale bar: 200 μm. F. Quantify the tubular formation of HUVEC. n=3, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.005, ****P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4948405/v1/cf49092bb820b23bc9fa32ea.png"},{"id":65202760,"identity":"e4930ec6-9180-4e98-b24a-4ddabcc5f200","added_by":"auto","created_at":"2024-09-24 17:15:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2423160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro antioxidant activity of BPPNs. \u003c/strong\u003eA. Effects of different preparations with TBHP on cell viability in HUVECs. B. Effects of different preparations on SOD and MDA level in HUVECs. C. DCFH-DA staining of HUVECs with different preparations. Scale bar: 100 μm. D. Effects of different preparations on ROS levels in HUVECs. E-F. JC-1 staining images of HUVECs with different treatments. JC-1 aggregates: normal mitochondria (red); JC-1 monomers: unhealthy mitochondrial (green). Scale bar: 100 μm. G-H. Western blots of p-JAK1, JAK1, p-STAT3, STAT3, p-Erk1/2. Erk1/2, p-Akt, Akt, HO-1, COX2 and Cyto C by HUVECs incubated in different treatments. I. The antioxidant mechanism diagram of BPPNs. n=3, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.005, ****P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4948405/v1/dc212a468c9ea8f5fd2fcdb4.png"},{"id":65202358,"identity":"6f611a91-a3cc-40ec-8060-e84ad2c80f84","added_by":"auto","created_at":"2024-09-24 17:07:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":992022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo evaluation of BPPNs in the diabetic rat full-thickness wounds model.\u003c/strong\u003e A. Representative photographic images of the wounds healing process with different treatments on day 0, 1, 3, 5, 7 and 14. B. Traces of wound-bed closure during 14 days for each treatment. C. percentage of wound area after treatment with the different preparations on day 1, 3, 5, 7 and 14. D. Representative Laser Doppler scan images on the diabetic wound after treatment on day 0, 1, 3, 5, 7 and 14. E. Quantification of blood flow volume at day 1, 3, 5, 7 and 14 using moorLDI Review V6.1 software. F. H\u0026amp;E and masson staining evaluation of wound regeneration after treatment with the different preparations on the 14th day. The black dashed line show the border between the wound tissue and surrounding healthy skin tissue. G. Quantification of granulation tissue thicknesses, \u0026nbsp;the length of wound and collagen volume fraction for different treatments on the 14th day. n=5, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.005.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4948405/v1/914338f650a749f6a48d33f8.png"},{"id":65202359,"identity":"2cf785bf-a892-4464-a8db-6f333869be9b","added_by":"auto","created_at":"2024-09-24 17:07:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":700270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the therapeutic efficacy after BPPNs treatment. \u003c/strong\u003eA. Effects of different preparations on SOD and MDA level on day 14. B. Immunofluorescence staining of neovascularizationat the wound tissue with different treatments on day 14. α-SMA (green), CD31 (red), and DAPI (blue). Scale bar: 100 μm. C. Quantitative analysis of the blood vessel density on day 14. D. Corresponding immunohistochemistry staining of COX2, HO-1, Cyto C, VEGF and COL3 on day 14. Scale bar: 200 μm. E-I. Immunohistochemical quantification of COX2, HO-1, HIF-1α, VEGF and COL3 , respectively. n=5, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.005.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4948405/v1/390eb984a6058ad0e632adeb.png"},{"id":65202355,"identity":"885e78ba-2cda-41c8-8c30-059fe318115e","added_by":"auto","created_at":"2024-09-24 17:07:44","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":879896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic overview of the preparation and application of BPPNs for diabetic wound healing. \u003c/strong\u003eA. The synthesis diagram of BPPNs. B. BPPNs was applied on the diabetic rat, wherein it promoted wound healing by reducing the level of ROS and triggering angiogenesis.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4948405/v1/deca50af450d9c92dd1c4410.jpeg"},{"id":69835734,"identity":"fe767b9f-5bb9-47b4-881b-2c56b273f606","added_by":"auto","created_at":"2024-11-25 16:14:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8061104,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4948405/v1/6c4581e3-1b34-41b9-b7f9-03b8f28ce9fa.pdf"},{"id":65202356,"identity":"81c10674-7657-43e0-a89f-fb1d0dc45c4f","added_by":"auto","created_at":"2024-09-24 17:07:44","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1554141,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4948405/v1/bf9ab513de4e600a7b688f94.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"High-throughput screening-based design of multifunctional natural polyphenol nano-vesicles to accelerate diabetic wound healing","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDiabetic wounds are prevalent chronic, unhealing wounds. The wound healing is one of the most intricate processes within the human body, it is broadly categorized into four phases: hemostasis, inflammation, proliferation, and remodeling. It involves the orchestrated regulation of various cell types over time and space [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Reactive oxygen species (ROS), which encompasses superoxide anions (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e\u0026bull;), peroxides, hydroxyl radicals (OH\u0026bull;), and singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], serve as pivotal regulatory factors in various stages of wound healing. However, an excessive abundance of ROS can lead to oxidative damage in wound-edge cells, hastening cellular senescence and apoptosis, thereby impeding the overall wound healing process [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Researches indicate that elevated blood glucose levels lead to an upregulation of pro-inflammatory factors, thereby inducing heightened oxidative stress [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Excessive oxidative stress in tissues and a diminished antioxidative capacity result in an oxidative-reductive imbalance, which stands as a primary determinant of the delayed wound healing process in diabetes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, antioxidant stress is an important strategy for treating diabetes wound healing.\u003c/p\u003e \u003cp\u003eGeneral clinical treatment for wounds includes restoring skin perfusion, treating infections, controlling metabolism, treating comorbidities, and caring for local trauma. Although these standard treatments may achieve the goal of controlling symptoms, effective treatment for wound healing of diabetes is still limited[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In recent years, research on multifunctional biomaterials for wound healing has been burgeoning. Biomaterials with controllable release of signal molecules can be combined with other treatment methods, which is a promising treatment method for diabetes wound healing[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Nano-biomaterials have shown great potential in wound healing due to their multifunctionality and superior physicochemical properties.\u003c/p\u003e \u003cp\u003eNatural polyphenols are a class of compounds widely present in living organisms, which have antioxidant, anti-inflammatory, anticancer, antibacterial, and antiviral effects, and are widely used in the treatment of various diseases[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition to having multiple biological functions, natural polyphenols also possess unique physicochemical properties, which can form nanoparticles by combining with various substances such as drugs, proteins, nucleic acids, metal ions, etc. through hydrogen bonding, hydrophobic interactions, and electrostatic interactions[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Studies have shown that natural polyphenols on the surface can effectively enhance the endocytosis of nanoparticles[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Based on these characteristics, natural polyphenols are widely used in the manufacturing of biomaterials, such as nanoparticles[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], nanocapsules[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], encapsulation coatings[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and so on.\u003c/p\u003e \u003cp\u003eProcyanidins (PC), as one of the natural polyphenols, is considered a highly promising biomaterial with good bioactivity and biocompatibility[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the structure of PC, a substantial presence of aromatic rings and phenolic hydroxyl groups confers both amphiphilic and amphiphobic characteristics. Furthermore, they readily engage in hydrogen bonding, governing non-covalent interactions and giving rise to intricate networks [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Using these properties of the PC, a polyphenol bubble was synthesized, named \u0026ldquo;Procyanidins polyphenol nanovesicles (PPNs)\u0026rdquo;. In pursuit of enhancing the antioxidative therapeutic potential of these polyphenol nanovesicles, we conducted a high-throughput drug screening utilizing the Natural Product Library (NPL), an assembly of natural compounds known for their exceptional safety profiles. From a selection of 622 natural compounds, we identified the most efficacious antioxidants and optimized the PPNs to achieve superior therapeutic outcomes of diabetic wound healing.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Sustained high levels of oxidative stress in diabetic wound tissue\u003c/h2\u003e \u003cp\u003eExisting research indicates that in normal wounds, oxidative stress primarily occurs within the initial three days following injury and gradually diminishes as the inflammatory phase subsides [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In contrast, diabetic wounds exhibit sustained, high levels of ROS [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To further substantiate the oxidative stress levels within diabetic wounds, we have established a rat model of diabetes mellitus (DM) following the protocol delineated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. The monitoring of blood glucose in the control group (Ctrl) and the DM group confirmed the successful establishment of the diabetes model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Subsequently, a full-thickness incision with a diameter of 20mm was made on the dorsal surface of both the Ctrl group and the DM group rats. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, the postoperative wound healing process of both rat groups was documented at days 0, 1, 3, 5, 7, 14, and 21. Compared to Ctrl, the DM group exhibited a noticeably delayed wound healing process with a tendency towards non-closure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Considering the pivotal role of oxidative stress in wound healing, we examined the tissue oxidative stress levels at various stages of wound healing (Day 3 \u0026ndash; the inflammatory phase, Day 7 \u0026ndash; the proliferation phase, and Day 14 \u0026ndash; the remodeling phase [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]) in both the Ctrl and DM groups. The results indicate that, whether on the 3rd day, 7th day, or 14th day of wound healing, the DM group exhibited significantly lower activities of superoxide dismutase (SOD) and protein expression of heme oxygenase-1 (HO-1) compared to the Ctrl group. Moreover, the levels of lipid peroxidation (malondialdehyde, MDA) and expression of cyclooxygenase-2 (COX2) were notably higher in the DM group than in the Ctrl group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G). The aforementioned experimental results indicate that, in comparison to normal wounds, diabetic wound tissues consistently maintain high levels of oxidative stress throughout the entire wound healing process, corroborating previous research findings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. High-Throughput Screening of Natural Product Libraries and Network Pharmacology Analysis\u003c/h2\u003e \u003cp\u003eTo target antioxidant stress more effectively, to achieve a better therapeutic effect on diabetic wounds. this study employed TBHP (100\u0026micro;M) to induce oxidative stress in HUVECs in vitro. This study used TBHP to induce oxidative stress in HUVECs in vitro, and a high-throughput screening was conducted on the natural product library (NPL) consisting of 622 compounds (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). The specific procedure is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, wherein cell viability is employed as an indicator of a drug's capacity for antioxidant stress. Toxicity tests were conducted on the top five ranking drugs based on cell viability to ascertain the optimal dosages for these five drugs (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). At the optimal concentration, the fluorescence intensity of DCFH-DA was utilized to assess the ROS scavenging capabilities of these five compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Subsequently, baicalein (BAI, 5\u0026micro;M), which exhibited the most remarkable antioxidative effect in HUVECs, was selected for further investigations.\u003c/p\u003e \u003cp\u003eUsing network pharmacology, a Venn diagram was constructed to elucidate the predicted targets of PC and BAI, as well as the disease-related targets involved in diabetic wound healing. Additionally, common targets shared by these compounds were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and S1C). In order to investigate the respective effects of biological materials and pharmaceuticals on disease treatment, we conducted separate GO enrichment analyses for the common targets of BAI and PC that intersect with the disease but do not overlap. The results of the enrichment analysis indicate that BAI may exert an influence on the peptidyl-tyrosine phosphorylation and cellular response to hydrogen peroxide (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), whereas PC primarily affects the vascular endothelial growth factor receptor signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This suggests that BAI and PC exhibit a potential complementarity in terms of therapeutic efficacy, and their combined action may influence wound healing through distinct processes, namely antioxidant stress and angiogenesis. This opens up the possibility of multifunctional optimization for PPNs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.3. Synthesis and Characterization of Baicalein-Procyanidins polyphenol nanovesicles (BPPNs)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003ePC can utilize the physical adsorption of CaCO\u003csub\u003e3\u003c/sub\u003e on polyphenols to form vesicles [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], which can de-nucleate into PPNs under acidic conditions. Under the same conditions, new polyphenol vesicles BPPNs were synthesized using equal amounts of PC and BAI. PPNs and BPPNs are collectively referred to as \u0026ldquo;Polyphenol nanovesicles (PNs)\u0026rdquo;. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the scanning electron microscopy (SEM) image of PPNs and BPPNs shows that BPPNs are spherical vesicles with a diameter smaller than PPNs. The diameter and Zeta potential of both were measured, and it was found that the diameter of BPPNs was about 500nm, which is half of PPNs, and the Zeta potential of BPPNs was similar to PPNs, indicating that the addition of BAI reduced the size of vesicles and did not affect their stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The FTIR spectra of BAI, PNNs, and BPPNs are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. Within the PPNs, the presence of intermolecular hydrogen bonding is evident, as indicated by a broad absorption peak at 3300 cm\u003csup\u003e-1\u003c/sup\u003e, this phenomenon leads to the absence of exposed phenolic hydroxyl groups, consistent with the expected theory [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The BPPNs inherit the phenolic hydroxyl absorption peak exposed by BAI (3400 cm\u003csup\u003e-1\u003c/sup\u003e), signifying that, at a chemical level, the newly synthesized nanoparticles exhibit enhanced antioxidative properties. Furthermore, in comparison to BAI, BPNNs exhibit no absorption peak around 1655 cm\u003csup\u003e-1\u003c/sup\u003e, implying that ketone carbonyl disappeared during the synthesis process of the nanovesicles, a possible addition reaction occurred between the carbonyl group in the BAI structure and the phenolic hydroxyl group in PC. This potentially led to the formation of hemiketals that effectively preserve the chemical structure of BAI. Based on this, create a synthesized illustrative diagram as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. Analyzing from a chemical perspective, the hemiketals can protect the carbonyl structure in BAI, decompose after pH change, and release complete BAI and PC molecules under the mildly acidic environment found in diabetic wounds [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], respectively playing their respective therapeutic roles. According to the molecular formula (PC: C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eO\u003csub\u003e13\u003c/sub\u003e, BAI: C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e) and XPS analysis of PNs involving carbon (C) and oxygen (O) atoms, the results indicate that the content of PC and BAI in BPPNs is approximately in a 1:1 ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The peak fitting results also confirmed the successful synthesis of BPPNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). In summary, it can be inferred that in BPPNs, PC, and BAI can undergo proportional addition reaction to generate hemiketals, which are then connected by hydrogen bonds to synthesize smaller-diameter polyphenol nanovesicles.\u003c/p\u003e \u003cp\u003eIn order to further investigate the biological activity of PNs, toxicity tests were conducted on PNs (Figure S2 A and B). The maximum safe concentration within which PNs were chosen for subsequent experiments was determined to be 4\u0026micro;g/ml (After conversion, the content of BAI and PC in 4 \u0026micro;g/ml BPPNs is approximately 5 \u0026micro;M each). To facilitate the in vitro monitoring of the extracellular uptake of PNs, we employed the fluorescent dye FITC to label PNs. Under both conditions with and without TBHP stimulation, cellular uptake of PNs was recorded at 1h and 2h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Quantitative results, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, reveal that both PPNs and BPPNs exhibit time-dependent and ROS-corresponding uptake. Notably, BPPNs demonstrate a greater cellular uptake efficiency in comparison to PNNs, which may be attributed to the smaller size and increased ease of cellular internalization associated with BPPNs. After co-treatment with PNs and TBHP for two hours, the localization relationship between PNs and cellular organelles was assessed using LysoTracker and MitoTracker (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI and J). The quantitative results of Pearson\u0026rsquo;s correlation coefficient indicate that both types of vesicles can achieve lysosomal escape and co-localize with mitochondria under oxidative stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK).\u003c/p\u003e \u003cp\u003eIn summary, compared with PPNs, the newly synthesized BPPNs have been optimized in characterization. BPPNs are a novel type of polyphenolic nanovesicles with smaller particle size, higher cellular uptake efficiency, lysosomal escape, and mitochondrial ROS response properties.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.4.\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eangiogenic activity of BPPNs\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eBased on the network pharmacology and enrichment analysis results from Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, it is speculated that PC may participate in or affect the VEFG signal pathway in the course of diabetes wound healing. In response to VEGF stimulation, endothelial cells sprout from the capillaries and then complete the entire process of angiogenesis with the proliferation, migration, and tubular formation of endothelial cells, ultimately providing nutrients for wound tissue, which is a key step in wound healing [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo further clarify the impact of PNs on angiogenesis, we conducted relevant tests on HUVECs. Scratch assays were conducted on attached HUVECs. Western blot results indicated that both BPPNs and PPNs effectively upregulated the expression of vascular angiogenesis-related proteins, VEGF, and its upstream regulator HIF-1α (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). The cells were treated with BAI (5\u0026micro;M), PPNs (4\u0026micro;g/ml), or BPPNs (4\u0026micro;g/ml) for 24 hours, with HUVEC migration recorded at 0h, 12h, and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The results reveal that, when compared to the control group, BAI exhibited no significant impact on HUVECs\u0026rsquo; migration. However, PPNs synthesized via PC significantly enhanced scratch healing. Notably, BPPNs containing BAI also have similar cell migration-promoting effects to PPNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Following this, we subsequently performed tube formation assays on HUVECs from different treatment groups on the Matrigel\u003csup\u003e\u0026reg;\u003c/sup\u003e matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), and quantified the number of nodes, meshes, branches, and total tube length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). The results demonstrated that BAI had almost no effect on the tube formation of HUVECs, while BPPNs exhibited a promotion of tube formation activity similar to that of PPNs. Overall, although BAI does not affect tube formation, BPPNs can effectively upregulate angiogenic-related proteins, promote the migration and tube formation of HUVECs, and obtain similar promoting tube activity as PPNs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.5.\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eantioxidant activity of BPPNs\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo further explore the in vitro antioxidant activity of BPPNs, HUVECs were divided into five groups as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, with a treatment duration of 24 hours, and a series of tests were conducted. The CCK-8 results indicated that BAI, PPNs, and BPPNs could all mitigate the reduction in cell viability caused by TBHP, with the rescue effect increasing in the order of PNNs\u0026thinsp;\u0026lt;\u0026thinsp;BAI\u0026thinsp;\u0026lt;\u0026thinsp;BPPNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Compared to the Ctrl group, the oxidative stress level in HUVECs of the TBHP group was significantly elevated (marked decrease in SOD activity and significant increase in MDA content), while the antioxidative stress capabilities of PPNs, BAI, and BPPNs were enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). ROS probes and JC-1 staining were performed on HUVECs under different treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and E). Oxidative stress induced by TBHP led to an increase in intracellular ROS and mitochondrial damage, with BPPNs showing the best capability in counteracting ROS and repairing mitochondrial damage, followed by BAI (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and F). Therefore, it is speculated that the prominent antioxidant activity of BPPNs is largely attributed to BAI, which was identified through high-throughput screening in NPL.\u003c/p\u003e \u003cp\u003eBased on the outcomes of network pharmacology and enrichment analyses (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D), it is hypothesized that the exceptional antioxidative efficacy of BAI may be attributable to its regulatory function on tyrosine phosphorylation. Protein phosphorylation modification, particularly prevalent and functionally paramount, occurs in over 30% of cellular proteins. Despite tyrosine phosphorylation modifications (P-Tyr) constituting less than 1% of all protein phosphorylation modifications, they play a pivotal role in nearly every physiological process within the cell [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Studies have indicated that a high-fat diet in mice can affect the P-Tyr of liver proteins [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Proteins within pathways such as JAK1/STAT3[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], Erk1/2[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and Akt [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], all containing tyrosine residues, are subject to increased levels of tyrosine phosphorylation due to oxidative stress, thereby activating their respective signaling pathways. To validate the impact of BPPNs on P-Tyr and their antioxidative effects at the protein level, Western blot experiments were conducted (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). The results revealed that BAI significantly inhibits the tyrosine phosphorylation of pathway proteins JAK1, STAT3, Erk1/2, and Akt, markedly modulates oxidative stress-related proteins (upregulating HO-1 and downregulating COX-2), and suppresses the expression of the mitochondria apoptosis-related protein Cyto C. These modulatory effects are notably superior to those of PPNs but inferior to BPPNs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Drawing Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI based on the above experimental results and literature search[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, BPPNs can effectively counteract in vitro oxidative stress caused by TBHP by inhibiting tyrosine phosphorylation of pathway proteins, while also playing a role in mitochondrial protection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. BPPNs accelerate diabetic wound healing\u003c/h2\u003e \u003cp\u003eIn consideration of the application of PNs in diabetic wounds, we employed a photosensitive hydrogel with tissue adhesiveness, HA-NB, to anchor the PNs at the wound locations [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The establishment of a diabetic wound model in SD rats was followed by the random allocation of these subjects into four groups, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Initially, we evaluated the impact of treatments with HA-NB, PPNs@HA-NB, and BPPNs@HA-NB on the principal organs\u0026mdash;heart, liver, spleen, lungs, and kidneys\u0026mdash;through H\u0026amp;E staining of organ tissue sections. The absence of pathological alterations in these stained sections suggests that HA-NB, PPNs@HA-NB, and BPPNs@HA-NB do not exhibit organ toxicity, as illustrated in Supplementary Fig.\u0026nbsp;3.\u003c/p\u003e \u003cp\u003eFigures \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B depict the wound healing progression within fourteen days across four treatment groups, accompanied by a quantification of the wound areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Compared to the DM group, the application of HA-NB significantly enhances the healing of diabetic wounds, likely attributable to the reduced risk of infection at the diabetic wound sites facilitated by the coverage provided by HA-NB[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. It is particularly noteworthy that the BPPNs@HA-NB group exhibited a higher rate of diabetic wound healing compared to the PPNs@HA-NB group. Furthermore, we employed laser Doppler scan images to characterize the functional vasculature with blood flow (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). It was observed that BPPNs@HA-NB could effectively upregulate angiogenesis around the wound during the proliferative phase of healing, thereby abbreviating the protracted proliferative phase induced by diabetes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Subsequent histopathological evaluation of the neotissue on day 14 post-injury using H\u0026amp;E and Masson's trichrome staining is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF. Quantitative analyses, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, revealed that diabetic wound tissues treated with BPPNs@HA-NB had thicker granulation tissue, shorter wound lengths, and higher collagen deposition compared to the PPNs@HA-NB group. These findings collectively suggest that BPPNs significantly accelerate diabetic wound healing and promote the progression of wound healing processes in diabetes, in comparison to PNNs.\u003c/p\u003e \u003cp\u003eTo investigate the intrinsic mechanisms underlying the therapeutic efficacy of BPPNs in the treatment of diabetic wounds, we further assessed the levels of oxidative stress, angiogenesis, and collagen within the tissue 14 days post-injury. The detection results for Superoxide Dismutase (SOD) and Malondialdehyde (MDA) indicated that, compared to the other three groups, the BPPNs@HA-NB group significantly reduced the level of oxidative stress in diabetic wound tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Immunofluorescence co-staining for CD31 and α-SMA, markers of neovascularization (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), with quantification of fluorescence expression shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, demonstrated that the vascular density in the BPPNs@HA-NB group was comparatively higher. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, immunohistochemical analysis of oxidative stress-related markers (COX2 and HO-1), mitochondrial apoptosis markers (Cyto C), angiogenesis-related markers (VEGF), and Collagen III (COL3), with quantitative results, revealed that BPPNs@HA-NB could downregulate COX2 and Cyto C protein expression and upregulate the relative expression of HO-1, VEGF, and COL3 compared to the other three groups. This suggests that BPPNs may accelerate diabetic wound healing by counteracting oxidative stress, protecting mitochondria, promoting angiogenesis, and enhancing collagen deposition.\u003c/p\u003e \u003cp\u003eIn summary, optimized with BAI, the newly synthesized polyphenol vesicles, BPPNs, demonstrate enhanced efficacy in counteracting the excessive oxidative stress encountered during the progression of diabetic wound healing, compared to the original anthocyanin polyphenol vesicles, PPNs. Moreover, BPPNs inherit the angiogenic-promoting effects of PPNs and, due to their superior antioxidant activity, provide a more \"fertile ground\" for angiogenesis. Consequently, the results indicate that BPPNs exhibit superior in vivo angiogenesis promotion compared to PPNs. Overall, BPPNs accelerate diabetic wound healing through a dual approach: combating oxidative stress and promoting angiogenesis, showcasing their potential as a multifaceted therapeutic intervention in diabetic wound management (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIn this study, we initially identified the level of oxidative stress during the progression of diabetic wound healing. The experimental results are consistent with existing reports [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], indicating that diabetes leads to a sustained high level of oxidative stress in the peri-wound tissue. Consequently, we induced oxidative stress in HUVECs in vitro and conducted high-throughput screening of over six hundred drugs from the Natural Product Library, identifying BAI as the most effective antioxidant drug in endothelial cells. By mixing BAI with the commonly used polyphenol vesicle framework material PC, in a 1:1 ratio, and utilizing the physical adsorption properties of CaCO3, we synthesized a new nanovesicle, BPPNs. Further exploration of its chemical structure revealed that BAI can undergo an addition reaction with PC in a 1:1 ratio, forming a hemiketal, which then connects into a network through intermolecular hydrogen bonds, ultimately observing a vesicular structure under an electron microscope. The incorporation of BAI into BPPNs results in smaller particle sizes compared to PPNs, thereby enhancing their cellular uptake efficiency. Additionally, BPPNs possess ROS responsiveness and lysosomal escape capabilities, and they can achieve co-localization with mitochondria under oxidative stress conditions. Network pharmacology and GO enrichment analysis suggest that PC may contribute to diabetic wound healing by affecting the VEGF signaling pathway, while BAI could potentially influence protein tyrosine phosphorylation and induce cellular responses to hydrogen peroxide, thereby participating in the progression of diabetic wound healing. Consequently, we further explored the bioactivity of BPPNs synthesized from PC and BAI. The results indicate that under physiological conditions, BAI does not participate in the angiogenesis of HUVECs, but both PPNs and BPPNs significantly promote in vitro tube formation and upregulate VEGF. Under TBHP stimulation, BPPNs exhibit superior effects in inhibiting pathway protein tyrosine phosphorylation, counteracting oxidative stress, and mitochondrial repair, almost equating to the combined bioactivity of BAI and PNNs. In vivo studies further demonstrate that BPPNs are polyphenol vesicles with both angiogenic and antioxidative stress activities, capable of accelerating diabetic wound healing.\u003c/p\u003e \u003cp\u003eDiabetes is a chronic disease that poses a severe threat to human health, with over 500\u0026nbsp;million individuals affected globally by 2021, and an incidence rate that is increasing annually[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The hallmark of diabetes is elevated blood glucose levels, which, over time, can induce oxidative stress, exacerbating hypoxia, inflammation, and cellular apoptosis[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These conditions are at the core of most diabetic complications. Diabetic wounds, a common complication of diabetes, are particularly troubling due to their non-healing nature, which can lead to ulcers and even amputation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Studies have shown that the non-healing wounds in diabetic patients are primarily due to excessive oxidative stress in the peri-wound tissue and a decrease in the body's antioxidant capacity following diabetes onset [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus, this article aims to design an effective antioxidant treatment strategy targeting oxidative stress during the diabetic wound healing process.\u003c/p\u003e \u003cp\u003eThe occurrence and progression of oxidative stress primarily stem from the generation of ROS and aberrations in signaling pathways [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. ROS induce signaling pathway aberrations, which in turn exacerbate ROS production. Consequently, antioxidant therapy can be broadly categorized into two approaches: chemical removal of peroxides via direct chemical reactions and modulation of signaling pathways through biological means [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A dual-pronged approach is imperative for mitigating oxidative stress effectively. From a chemical standpoint, the material scaffold employed in this study, Procyanidins, harbors abundant phenolic hydroxyl groups, theoretically enabling it to directly counteract oxidative stress by reacting with ROS. Correspondingly, literature also suggests that PC acts as a barrier against oxidative stress by alleviating the burden of free radicals, thereby preventing molecular and cellular damage [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In this research, PPNs formed via physical adsorption and intermolecular hydrogen bonding of PC exhibit ROS responsiveness and demonstrate antioxidative activity both in vitro and in vivo (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, their impact on pathway proteins and overall antioxidative efficacy is relatively modest, suggesting that the antioxidative activity of PC primarily derives from its chemical structure, enabling direct reaction with ROS. Optimization of PPNs is imperative for achieving enhanced antioxidative activity.\u003c/p\u003e \u003cp\u003eNatural products, cherished for their high biological activity, low toxicity, and considerable developmental potential, have garnered significant attention from clinicians and researchers alike. Hence, our focus gravitates toward this realm. Through high-throughput screening of the natural product library under oxidative stress stimulation, BAI emerged as a standout candidate owing to its remarkable antioxidative activity. Interestingly, BAI also possesses a polyphenolic structure within its chemical composition. However, its phenolic hydroxyl content falls considerably short of that found in PC. Consequently, from a chemical perspective, baicalein's ability to directly react with ROS is presumed to be inferior to that of cyanidin-3-glucoside. Thus, we boldly speculate that baicalein is more inclined towards combating oxidative stress through the modulation of signaling pathways. Subsequent network pharmacological analysis validated this conjecture (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D), wherein multiple GO biological processes suggested BAI's capacity to influence protein tyrosine phosphorylation. To corroborate this finding, we selectively screened several pathway proteins closely associated with oxidative stress and harboring tyrosine residues, including JAK1, STAT3, Erk1/2, and Akt. Western blot results (Figures G and H) revealed that BAI effectively inhibits the phosphorylation of these pathway proteins, ultimately yielding antioxidative stress and mitochondrial damage repair effects. BPPNs, BAI-optimized polyphenol nanovesicles, unsurprisingly, exhibited superior antioxidative activity both in vitro and in vivo (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), underscoring the feasibility of a dual antioxidative strategy via chemical and biological pathways.\u003c/p\u003e \u003cp\u003eIt's noteworthy that in network pharmacology analysis, PC and BAI have non-overlapping targets with the common targets of diabetic wounds, which separately lead to two key aspects of diabetic wound treatment: angiogenesis and oxidative stress. The angiogenesis-promoting effect of PC has also been verified in both in vivo and in vitro experiments, which endows BPPNs with angiogenic activity that BAI lacks. Therefore, the synthesis of BPPNs not only combines the antioxidative activities of PC and BAI but also achieves a complementary effect of their functions.\u003c/p\u003e \u003cp\u003eNanoparticles (NPs) are commonly used as part of drug delivery systems because they can enhance the pharmacokinetics, bioavailability, and half-life of drug formulations, and reduce the frequency of dosing for gene or drug molecules [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Given the amphiphilic and network structure of BPPNs, it's reasonable to speculate that the polyphenol vesicles we designed and optimized also possess the potential to carry drugs or to transport exogenous proteins, amino acids, or even nucleic acid sequences into cells. Coupled with their inherent biological activity, this could potentially achieve therapeutic effects that advance disease progression. Of course, these potentials still require further development and optimization, and this is merely a presentation of a possibility.\u003c/p\u003e \u003cp\u003eIn summary, this study, through high-throughput screening of a natural product library, identified BAI as the drug with the best antioxidant stress effect in HUVECs. Utilizing the biological activity and chemical structure of baicalin, the study characterized and functionally optimized proanthocyanidin polyphenol vesicles, resulting in BPPNs with smaller particle size, faster cellular uptake, and stronger ROS response. These vesicles accelerate diabetic wound healing by simultaneously combating oxidative stress and promoting angiogenesis.\u003c/p\u003e"},{"header":"4. Conculsion","content":"\u003cp\u003eIn this study, we synthesized and successfully validated a multifunctional polyphenol nanoparticle vesicle, BPPNs, optimized through natural products, targeting excessive oxidative stress during the healing process of diabetic wounds. Through screening the antioxidant activity of over six hundred natural products from the natural product library, baicalin was selected for its optimal antioxidant stress effect in HVECs. Baicalin undergoes an additive reaction with the polyphenol biomaterial Procyanidins, utilizing intermolecular hydrogen bonding to form BPPNs, which accelerate cellular uptake under oxidative stress conditions and exhibit the biological activities of baicalin and proanthocyanidins intracellularly. BPPNs not only exert antioxidant activity by inhibiting the phosphorylation of pathway proteins such as tyrosine but also accelerate diabetic wound healing by further promoting angiogenesis and collagen deposition. Overall, our research results offer a new therapeutic strategy for the healing of diabetic wounds.\u003c/p\u003e"},{"header":"5. Experimental Section/Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e5.1. Reagents and kits\u003c/h2\u003e \u003cp\u003eNatural Product Library (#HY-L021) and FluoroTag\u0026trade; FITC Conjugation Kit (#HY-66019) from MedChemexpress (NJ, USA). Fetal bovine serum (FBS, #10099141C) was obtained from Gibco-Invitrogen (NY, USA). Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM, #MA0212), Penicillin/Streptomycin, sterile(100X) (#MA0110), phosphate buffered solution (PBS, #MA0015), 0.25%Trypsin-EDTA, PhenolRed(modified) (#PWL060), Streptozotocin (STZ, #MB1227), Procyanidins (#MB2168-1), Baicalein (#MB7311) and other reagents required for cell culture were obtained from Meilunbio (Dalian, China). High-fat feed (#MD12033) purchased from Medicience (Jiangsu, China). Tert-butyl hydroperoxide solution (TBHP, #418064) and DMSO (#D2650) were purchased from Sigma-Aldrich (St Louis, MO, USA). Cell Counting Kit-8 (#C0040), Protease inhibitor cocktail for general use (#P1005), SDS-PAGE Sample Loading Buffer (5X) (#P0015), Enhanced BCA Protein Assay Kit (#P0010), Hoechst 33258 (#C1011) and BeyoECL Star (#P0018AM) were purchased from Beyotime (Shanghai, China). 2\u0026prime;,7\u0026prime;-Dichlorofluorescin diacetate (DCFH-DA, #D6470), Superoxide Dismutase (SOD) Activity Assay Kit (#BC0175), Malondialdehyde (MDA) Content Assay Kit (#BC0025), Mitochondrial Membrane Potential Assay Kit with JC-1 (#M8650), Hematoxylin-Eosin (HE) Stain Kit (#G1120), Masson's Trichrome Stain Kit (#G1340), DAB Substrate kit (20\u0026times;) (#DA1010), Mounting Medium (antifading, with DAPI) (#S2110) and were purchased from Solarbio Science \u0026amp; Technology (Beijing, China). MitoTracker\u0026trade; Red CMXRos (#M7512) and LysoTracker\u0026trade; Deep Red (#L12492) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Goat Anti-Mouse IgG H\u0026amp;L (Alexa Fluor\u0026reg; 488) (#ab150113), and Goat Anti-Rabbit IgG H\u0026amp;L (Alexa Fluor\u0026reg; 594) (#ab150080) were purchased from Abcam (Cambridge, UK).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimary antibodies were used in the study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePurpose\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProduct ID and manufacturer\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-JAK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#741295, Cell Signaling Technology, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJAK1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#66466, Proteintech, China\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep-STAT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#9145, Cell Signaling Technology, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSTAT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#12640, Cell Signaling Technology, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOX2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWB/ IHC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#12282, Cell Signaling Technology, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHO-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWB/ IHC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#10701-1-AP, Proteintech, China\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCyto C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWB/ IHC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#10993-1-AP, Proteintech, China\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVEGF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWB/ IHC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#ET1604-28, Huabio, China\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHIF-1α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#66730, Proteintech, China\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCD31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#ab281583, Abcam, UK\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-SMA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#BM0002, Boster, China\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCOL3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIHC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#ER1906-50, Huabio, China\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGAPDH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e#2118, Cell Signaling Technology, USA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e5.2. Animal model of diabetic full-thickness wounds\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Institutional Ethical Committee of Wenzhou Medical University (wydw2023-0243). The Experimental Animal Center of Wenzhou Medical University (Zhejiang Province, China) provided a total of 40 male 6-week-old SD rats (180-220g). After adaptive feeding for 1 week, high-fat diets were given for 4 weeks. Intraperitoneal injections were administered for 3 days each time: 60mg/kg STZ (T2D, n\u0026thinsp;=\u0026thinsp;30) until blood glucose stabilizes\u0026thinsp;\u0026gt;\u0026thinsp;16.67mmol/L; 0.25ml/kg 0.9% NaCl (Ctrl, n\u0026thinsp;=\u0026thinsp;10). Continue high-fat feeding for 4 weeks.\u003c/p\u003e \u003cp\u003eFor the wounds model, animals were anesthetized with 2.5% pentobarbital sodium (30 mg/kg) and randomly selected for grouping. After shaving and sterilization, two full-thickness wounds (20 mm in diameter) were made by scissor cutting along the mark on each side of the rat\u0026rsquo;s back. All the animals were high-fat fed in individual cages and given ad libitum feeding access to food and water, and they were observed every day during the total period of the experiment. At days 0, 1, 3, 5, 7, 14, and 21 posttreatment, the wound area was calculated by tracing the wound margins from rats and was evaluated as a percent area of the original wound using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e5.3. High-throughput screening of natural product library\u003c/h2\u003e \u003cp\u003eUsing human umbilical vein endothelial cells (HUVECs) to screen 622 drugs from the natural product library. HUVECs were seeded into a 96-well plate (8000 cells/well) and cultured for 24 hours until they adhered to the walls. A blank control group, stimulation group (TBHP 100\u0026micro;M), and drug treatment group (TBHP 100\u0026micro;M\u0026thinsp;+\u0026thinsp;drug 10\u0026micro;M) were set up in the plate, and after 24 hours of treatment, the detection was performed using CCK8, measuring absorbance at a wavelength of 450nm with an enzyme reader, designated as Ac, Ab, and As, respectively. Cell viability was calculated for each drug treatment using the formula and subjected to statistical analysis (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003eCell viability (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{As-Ab\\:}{Ac-Ab}\\)\u003c/span\u003e\u003c/span\u003e \u0026times; 100%\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e5.4. Network pharmacology and GO biological processes enrichment analysis\u003c/h2\u003e \u003cp\u003eThe two-dimensional conformations of Procyanidins and baicalein were downloaded from PubChem\u0026rsquo;s official website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Isomeric Smiles format files were imported into the Swiss Target Prediction (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://swisstargetprediction.ch/\u003c/span\u003e\u003cspan address=\"http://swisstargetprediction.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) platform, set the attribute to Homo sapiens, and start to predict the potential target of Procyanidins and baicalein. By searching the keyword of diabetic wound healing in the GeneCards (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database to obtain the disease targets. Compare the three datasets mentioned above by creating a Venn diagram (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jvenn.toulouse.inrae.fr/app/index.html\u003c/span\u003e\u003cspan address=\"https://jvenn.toulouse.inrae.fr/app/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and performing an enrichment analysis of GO biological processes on the genes at the intersection (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://metascape.org/gp/index.html\u003c/span\u003e\u003cspan address=\"https://metascape.org/gp/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5.5. Preparation and characterization of PPNs and BPPNs\u003c/h2\u003e \u003cp\u003eAfter adding 1 mL of 1M Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e solution to a 15 mL conical flask and stirring at 1200 rpm, 1 mL of ddH\u003csub\u003e2\u003c/sub\u003eO is added. Then, 0.5 mL 10mM Procyanidins and baicalein are added to the conical flask. Stirring continues, and 1 mL of 1M CaCl\u003csub\u003e2\u003c/sub\u003e solution is added. Stirring is continued. After allowing it to stand for 15 minutes, the mixture is centrifuged at 1000 rpm and washed with water three times. The washed samples are then mixed with 4 mL of 1M HCl and centrifuged at 3000 rpm until the pH is between 6 and 6.5. Afterward, freeze-drying is performed to obtain BPPNs. As for PNNs, repeat the above process by replacing the baicalein solution with an equimolar solution of Procyanidins.\u003c/p\u003e \u003cp\u003eThe morphology of nanoparticles was observed by scanning electron microscopy (SEM) (Quanta 250 FEG, FEI). The particle size and Zeta potential analyses of nanoparticles were carried out on the Malvern Zetasizer Nano ZS (Malvern Instruments, UK). Chemical interaction between NA was recorded on a Nicolet IS10 FT-IR instrument (ThermoFisher Scientific, USA) scanning from 4000 to 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. XPS was measured by ESCALAB 250Xi K-alpha (ThermoFisher Scientific, USA).\u003c/p\u003e \u003cp\u003eFor BPPBs:\u003c/p\u003e \u003cp\u003eC1s/O1s = (71.54%)/(28.46%) =(30PC\u0026thinsp;+\u0026thinsp;15BAI)/(13PC\u0026thinsp;+\u0026thinsp;5BAI) \u0026rarr; PC/BAI\u0026thinsp;\u0026asymp;\u0026thinsp;1\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e5.6. Western blot\u003c/h2\u003e \u003cp\u003eHUVECs were subjected to a 1:4 dilution with a 5X loading buffer and subsequently exposed to a heat treatment at 100\u0026deg;C for 15 minutes. The resultant protein extracts were then resolved through a 12.5% SDS-PAGE gel and transferred onto PVDF membranes. Following this, the membranes were subjected to a blocking procedure involving 5% nonfat milk for 2 hours. After the milk blocking, the membranes underwent a series of washes using TBST and were subsequently incubated with primary antibodies for 8\u0026ndash;12 hours at 4\u0026deg;C. Following this primary antibody incubation, the membranes were once again subjected to TBST washes and subsequently incubated with secondary antibodies conjugated with HRP for 2 hours at RT. The visualization of immunoreactive bands was achieved through the utilization of an ECL kit and the bands were detected using a ChemiDoc Imaging System (Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e5.7. \u003cem\u003eIn vitro\u003c/em\u003e tube formation assay\u003c/h2\u003e \u003cp\u003eA tube formation assay using Matrigel\u0026reg; matrix (#356234, Corning, USA) was conducted to assess the morphogenesis and tube formation capability of HUVECs under different treatments. In brief, the Matrigel solution was allowed to thaw at 4\u0026deg;C overnight and subsequently dispensed into \u0026micro;-Slide chambers (10 \u0026micro;L per well, IBIDI, Germany), followed by incubation in a cell incubator for 1 hour to facilitate solidification. A total of 5000 cells, which had been pre-treated with BAI (10\u0026micro;M), PPNs, and BPPNs, were seeded onto the Matrigel-precoated \u0026micro;-Slide. Tube formation was meticulously observed and quantified, with an average count derived from the assessment of three independent fields, all conducted under an inverted light microscope (Olympus, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e5.8. Scratch-wound healing assay\u003c/h2\u003e \u003cp\u003eCultivate HUVECs in a 6-well culture plate until they adhere to the walls. Scratch the cell surface gently using the tip of a sterile 200-\u0026micro;L pipette. Remove floating cells with PBS and continue with a complete culture medium containing 5% FBS. Capture images of the scratches at 0 hours, 12 hours, and 24 hours, respectively, under an inverted microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e5.9. Assessment of blood flow in the wound area\u003c/h2\u003e \u003cp\u003eThe blood flow in the wound area was assessed by a laser Doppler imager (MoorLDI-2; Moor Instruments Limited, Devon, UK). Briefly, the rats were anesthetized with 2.5% pentobarbital sodium (30 mg/kg), shaved, and then gently fixed onto a black platform. MoorLDI Review V6.1 software was used to quantify the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e5.10. Histology analysis and immunohistochemistry\u003c/h2\u003e \u003cp\u003eOn the 14th day, the rats were perfused with physiological saline. The wound was fixed with 4% paraformaldehyde at 4\u0026deg;C, followed by paraffin embedding. The embedded tissues were sectioned and stained with hematoxylin and eosin (H\u0026amp;E) as well as Masson's trichrome stain to assess inflammation, epidermal regeneration, granulation tissue, and collagen deposition. Immunohistochemistry and immunofluorescence were performed to evaluate the expression of various markers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e5.11. Statistical analysis\u003c/h2\u003e \u003cp\u003eNumerical data, presented as mean values accompanied by their respective standard deviations (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), are depicted based on the results of a minimum of three distinct experiments unless stated otherwise. Statistical analysis of the data was conducted through a one-way analysis of variance (ANOVA), followed by Tukey's post-hoc analysis, utilizing GraphPad Prism 7.0 (La Jolla, CA, USA). Statistical significance was established at a threshold of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and comparisons were made at two significance levels, *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.005, ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 against the specified reference group.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the findings of this study are available from the corresponding authors upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Ethical Committee of Wenzhou Medical University (wydw2023-0243).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXY. Zhao:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; original draft, Methodology, Formal analysis, Conceptualization. \u003cstrong\u003eSK Su:\u0026nbsp;\u003c/strong\u003eMethodology.\u003cstrong\u003e\u0026nbsp;CY. Wu:\u0026nbsp;\u003c/strong\u003eMethodology, Formal analysis. \u003cstrong\u003eYX. Deng:\u003c/strong\u003e Methodology.\u003cstrong\u003e\u0026nbsp;Y. Chen:\u0026nbsp;\u003c/strong\u003eMethodology. \u003cstrong\u003eTX. Yu:\u0026nbsp;\u003c/strong\u003eMethodology. \u003cstrong\u003eCC. Li:\u0026nbsp;\u003c/strong\u003eMethodology. \u003cstrong\u003eYK. Zhang:\u0026nbsp;\u003c/strong\u003eMethodology. \u003cstrong\u003eXY. Wang:\u0026nbsp;\u003c/strong\u003eProject administration. \u003cstrong\u003eYF. Zhou:\u0026nbsp;\u003c/strong\u003eFunding acquisition. \u003cstrong\u003eXL. Zhang:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; review \u0026amp; editing, Methodology, Formal analysis. All authors have read and approved the content of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Professor Linyong Zhu from the school of Biomedical engineering, Shanghai Jiao Tong University, for providing us with HA-NB hydrogel. This study was supported by Zhejiang Provincial Natural Science Foundation of China (LY22H060008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRodrigues M, et al. Wound Healing: A Cellular Perspective. 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Int J Nanomed. 2022;17:3125\u0026ndash;45. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.2147/IJN.S372211\u003c/span\u003e\u003cspan address=\"10.2147/IJN.S372211\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\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":"Natural product library, polyphenol nanovesicles, diabetic wound healing, antioxidative stress, peptidyl-tyrosine phosphorylation","lastPublishedDoi":"10.21203/rs.3.rs-4948405/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4948405/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxidative stress is a major pathological factor that impedes the diabetic wound healing process. Procyanidins (PC) form nanoparticle-vesicles (PPNs) through hydrogen bonding and exhibit good drug delivery capability; however, its application in diabetic wound is not satisfied. To meet the antioxidant needs for treating, high-throughput screening in natural product library (NPL) under in vitro oxidative stress condition was conducted to enhance the antioxidant activity of PPNs. HUVECs treated with TBHP was established as screening model in vitro. Baicalein (BAI) was identified out of 600\u0026thinsp;+\u0026thinsp;products in the library as the most effective one to combat oxidative stress. Further study showed that PC and BAI may react in equal proportions to synthesize new vesicles, named BPPNs; while BPPNs have ROS responsive and antioxidant effects. Network pharmacology showed that in diabetic wounds, the target genes of PC are mainly enriched in the VEGF-related pathways, while BAI primarily regulates tyrosine phosphorylation. The complementarity between the two has been validated in in vitro and in vivo experiments. In summary, the antioxidant drug BAI, identified through high-throughput screening of NPL, could optimize the biological function of PPNs; the newly-synthesized BPPNs may accelerate diabetic wound healing through dual mechanisms of promoting angiogenesis and combating oxidative stress.\u003c/p\u003e","manuscriptTitle":"High-throughput screening-based design of multifunctional natural polyphenol nano-vesicles to accelerate diabetic wound healing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-24 17:07:39","doi":"10.21203/rs.3.rs-4948405/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-22T05:43:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-17T14:58:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-15T15:26:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-15T04:47:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137820633627456216563304039541887702996","date":"2024-09-02T13:45:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"125501440624053510686460838060577114212","date":"2024-09-02T07:13:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282277101831006387444975815211775213663","date":"2024-09-01T14:26:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-01T12:43:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-24T17:58:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-24T17:57:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-08-21T03:54:01+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":"541b15b3-0095-4d7c-8ca1-e60db28bb8c4","owner":[],"postedDate":"September 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T16:08:46+00:00","versionOfRecord":{"articleIdentity":"rs-4948405","link":"https://doi.org/10.1186/s12951-024-02950-2","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2024-11-21 15:58:17","publishedOnDateReadable":"November 21st, 2024"},"versionCreatedAt":"2024-09-24 17:07:39","video":"","vorDoi":"10.1186/s12951-024-02950-2","vorDoiUrl":"https://doi.org/10.1186/s12951-024-02950-2","workflowStages":[]},"version":"v1","identity":"rs-4948405","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4948405","identity":"rs-4948405","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}