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

doi:10.21203/rs.3.rs-4948405/v1

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

https://doi.org/10.21203/rs.3.rs-4948405/v1

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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

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 (O₂^-•), peroxides, hydroxyl radicals (OH•), and singlet oxygen (¹O₂) [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.

2.1. 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. 1A. The monitoring of blood glucose in the control group (Ctrl) and the DM group confirmed the successful establishment of the diabetes model (Fig. 1B). 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. 1C, 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. 1D). 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. 1E-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 S1A). The specific procedure is illustrated in Fig. 2A, 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 S1B). At the optimal concentration, the fluorescence intensity of DCFH-DA was utilized to assess the ROS scavenging capabilities of these five compounds (Fig. 2B). 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. 2C 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. 2D), whereas PC primarily affects the vascular endothelial growth factor receptor signaling pathway (Fig. 2E). 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 CaCO₃ 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. 3A, 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. 3B). The FTIR spectra of BAI, PNNs, and BPPNs are depicted in Fig. 3C. 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. 3D. 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: C₃₀H₂₆O₁₃, BAI: C₁₅H₁₀O₅) 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. 3E). The peak fitting results also confirmed the successful synthesis of BPPNs (Fig. 3F). 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. 3G). Quantitative results, as shown in Fig. 3H, 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. 3I 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. 3K).

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. 2C and 2E, 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. 4A, 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. 4C). 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. 4D). Following this, we subsequently performed tube formation assays on HUVECs from different treatment groups on the Matrigel^® matrix (Fig. 4E), and quantified the number of nodes, meshes, branches, and total tube length (Fig. 4F). 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. 5A). 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. 5B). ROS probes and JC-1 staining were performed on HUVECs under different treatments (Fig. 5C 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. 5D 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. 2C 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. 5G). 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. 5H). Drawing Fig. 5I 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. 5I).

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 6A and B depict the wound healing progression within fourteen days across four treatment groups, accompanied by a quantification of the wound areas (Fig. 6C). 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. 6D). 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. 6E). Subsequent histopathological evaluation of the neotissue on day 14 post-injury using H&E and Masson's trichrome staining is presented in Fig. 6F. Quantitative analyses, as shown in Fig. 6G, 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. 7A). Immunofluorescence co-staining for CD31 and α-SMA, markers of neovascularization (Fig. 7B), with quantification of fluorescence expression shown in Fig. 7C, demonstrated that the vascular density in the BPPNs@HA-NB group was comparatively higher. As depicted in Fig. 7D, 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).

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. 2C 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.

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.

5.1. 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 Na₂CO₃ solution to a 15 mL conical flask and stirring at 1200 rpm, 1 mL of ddH₂O is added. Then, 0.5 mL 10mM Procyanidins and baicalein are added to the conical flask. Stirring continues, and 1 mL of 1M CaCl₂ 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.

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).

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You are reading this latest preprint version

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

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Abstract

Figures

1. Introduction

2. Results

2.1. Sustained high levels of oxidative stress in diabetic wound tissue

2.2. High-Throughput Screening of Natural Product Libraries and Network Pharmacology Analysis

2.3. Synthesis and Characterization of Baicalein-Procyanidins polyphenol nanovesicles (BPPNs)

2.4. In vitro angiogenic activity of BPPNs

2.5. In vitro antioxidant activity of BPPNs

2.6. BPPNs accelerate diabetic wound healing

3. Discussion

4. Conculsion

5. Experimental Section/Methods

5.1. Reagents and kits

5.2. Animal model of diabetic full-thickness wounds

5.3. High-throughput screening of natural product library

5.4. Network pharmacology and GO biological processes enrichment analysis

5.5. Preparation and characterization of PPNs and BPPNs

5.6. Western blot

5.7. In vitro tube formation assay

5.8. Scratch-wound healing assay

5.9. Assessment of blood flow in the wound area

5.10. Histology analysis and immunohistochemistry

5.11. Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

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