A Core-Shell-Type Nanosystem Promotes Diabetic Wound Healing Through Photothermal-Responsive Release of Transforming Growth Factor β

A Core-Shell-Type Nanosystem Promotes Diabetic Wound Healing Through Photothermal-Responsive Release of Transforming Growth Factor β | 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 A Core-Shell-Type Nanosystem Promotes Diabetic Wound Healing Through Photothermal-Responsive Release of Transforming Growth Factor β Jinfei Hou, Jiejun Jie, Xinwei Wei, Xiangqian Shen, Qingfang Zhao, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4226321/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Jul, 2024 Read the published version in Journal of Nanobiotechnology → Version 1 posted 11 You are reading this latest preprint version Abstract The treatment of diabetic wounds remains a major clinical challenge owing to bacterial infection, defects in angiogenesis, and the corresponding inhibition of cell activity and extracellular matrix deposition. In this study, a core-shell-type nanosystem was developed using graphdiyne (GDY) nanoparticles covered with gelatin to investigate its effects on diabetic wound healing. The nanoparticles were loaded with transforming growth factor β (TGF-β) via electrostatic self-assembly to promote angiogenesis and cell migration. The photothermal effects of GDY nanoparticles were applied to achieve controllable drug release and antibacterial properties. This nanosystem could rapidly release TGF-β after irradiation by near-infrared rays (NIR) without damaging its biological activities. The associated photothermal antibacterial activity was observed after 30 seconds irradiation of nanoparticles, and the temperature was set at a safe range (<49.6 °C). Besides, the gels possessed good biocompatibility and promoted cell migration in vitro. After implantation, the hydrogels group showed a higher wound healing rate than the control group in diabetic wound mouse models after 14 days and exhibited evident tissue regeneration, including angiogenesis and extracellular matrix deposition. This study presents a method for fabricating antibacterial wound dressings and an effective NIR-response strategy for designing drug-delivery nanosystems loaded with cellular factors. graphdiyne photothermal-responsive transforming growth factor β diabetic wounds Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Diabetic wounds are, the most common complication of diabetes, contributing to severe health costs and economic losses [ 1 ]. Over 1 million patients with diabetes present with diabetic wounds [ 2 , 3 ]. Therefore, developing new wound dressings is essential for treating diabetic wounds [ 4 ]. However, treating diabetic wounds remain challenging because a high-glucose microenvironment can further induce bacterial breeding, resulting in infection-related complications [ 2 , 5 ]. Microangiopathy and revascularization defects can also negatively influence healing [ 6 , 7 ]. The infections and defects of vessels might further trigger a chain of adverse reactions, such as inhibition of cell proliferation and migration [ 8 , 9 ]. Transforming growth factor β (TGF-β) is a cell factor that potentially helps wound healing [ 10 ]. It promotes the growth and migration of epidermal and vascular endothelial cells [ 11 ]. It also promotes angiogenesis and consequent blood flow to wounds by stimulating the local release of other growth factors [ 12 ]. Furthermore, topical use of medicated wound dressings helps avoid potential damage to other tissues in the body. Therefore, TGF-β is an excellent drug candidate for the local treatment of diabetic wounds [ 13 ]. However, there are limitations; cellular factors are likely to degrade in vivo and cannot be sustained for a long time, while diabetic wound healing is chronic and continuous [ 14 ]. Additionally, TGF-β lacks the antibacterial properties that are essential for diabetic wound healing [ 15 ]. To overcome these difficulties, drug-delivery nanosystems may be used to construct appropriate carriers for TGF-β to cure diabetic wounds. The stimulus-response nanosystems (especially the light-response nanosystem) have been widely used to treat wounds [ 16 ]. Drug release could be controllable with an “ON/OFF” switch for light, and the associated temperature rise could result in bacterial death owing to the photothermal effects of these nanosystems. Nevertheless, the high temperature could also deconstruct the molecular structures containing loaded cell factors and harm the surrounding tissues [ 17 , 18 ]. It is essential to develop thermosensitive nanosystems to achieve antibacterial effects at a safe temperature range of < 50℃. Consequently, the core-shell-type is a common method to construct scaffolds in regenerative medicine [ 19 ] and was applied in this study. Graphdiyne (GDY) was chosen as the core part owing to its stability and biocompatibility reported in our previous study [ 20 ]. Most importantly, GDY nanoparticles (NPs) may possess the ability to damage the cell wall of bacteria and produce tiny amounts of reactive oxygen species (ROS), similar to other carbon-based two-dimensional material. It is reasonable to consider that it might be a proper antibacterial and photothermal system functional at relatively safe temperatures. However, the properties to load the drug and kill bacteria in GDY NPs have not been further surveyed. Besides, gelatin (a natural biocompatible material with thermosensitivity) covered the surface of the GDY nanoparticles [ 21 ]. The coverage of gelatin could modify the GDY nanoparticles from anion to cation, which may make TGF-β loading feasible via charge self-assembly [ 22 ]. Moreover, it is possible for GDY/G NPs (GDY NPs covered with gelatin) to release TGF-β responsive to photothermal stimulus without affecting its bioactivity owing to the state change of gelatin from a gel to a liquid between 30–40°C [ 23 ]. In this study, we propose a near-infrared (NIR)-responsive nanosystem based on GDY and gelatin. The GDY/G@TGFβ NPs were further encapsulated in biocompatible polyethylene glycol diacrylate (PEGDA) hydrogels with good mechanical properties. The drug release curve and capacity of the materials to promote cell migration and proliferation, antibacterial effects, and revascularization were determined. Finally, full-thickness wounds in diabetic mouse models were constructed to investigate the ability of the PEGDA-GDY/G@TGFβ hydrogels to promote diabetic wound healing and tissue regeneration. Materials and methods Synthesis of PEGDA-GDY/G@TGFβ hydrogels PEGDA-GDY/G@TGFβ hydrogels were synthesized by the method of electrostatic self-assembly. GDY nanoparticles at concentration of 0.05%, 0.1% and 0.5% were prepared as described before [ 24 ]. After centrifugation at 15000 rpm for at least 10 min, the supernatant is discarded. The deionized water was used to wash the redundant gelatin and then TGF-β1 at the concentration of 100 µg/L was dissolved into the dispersion liquid. After centrifugation as above, the GDY/G@TGFβ solution was prepared. The 10 mg PEGDA and 2 ~ 3 mg lithium pheny l-2,4,6-trimethylbenzoyl phosphinate (LAP) was added to 1 mL solution above and completely dissolved. To construct the hydrogels, blue light irradiation was performed for at least 20 s. For sterilization, the hydrogels were immersed into 75% ethyl alcohol for 12h and then washed with deionized water. Characterization and morphology of PEGDA-GDY/G@TGFβ hydrogels To test the dispersity and morphology, PEGDA-GDY/G@TGFβ solution before crosslinking was characterized with dynamic light scattering and transmission electron microscopy. The 100 µL sample solution were diluted into 1 mL deionized water and then the solution was transferred to the corresponding measuring tube for measurement of particle size and zeta potential in NanoSight (Malvern Panalytical, British). After ultrasonic dispersion for at least 60 min, the sample solution was dripped onto copper grid. Then the redundant liquid was dried and samples were observed by HT7700 microscope (Hitachi, Japan). Besides, Cryoelectron microscopy (Cryo-EM) (S-4800, HITACHI, Japan) was utilized to view the normal surface morphology of hydrogels. Briefly, hydrogels were loaded on a low-temperature sample carrier (Quaroum PP3000T) and then fixed in a cryo-specimen holder The temperature was placed in cold nitrogen (-120°C), and then immediately transferred to the cryo-stage. To remove the ice crystals, the temperature was raised to 90°C and after that the samples were cooled to -180°C for stabilization. The pore sizes were analyzed by the software Image J (America) on the surface images and the frequency distribution histograms were plotted according to the data. The hydrogels were cut into slices at diameter of 10mm and immersed into simulated body fluids (BZ310, Biochemazone, America). After several hours, the immersed hydrogels were weighed. The hydrogels after immersed for 24 hours were performed with tensile experiments described below. Mechanical properties of PEGDA-GDY/G@TGFβ hydrogels Tensile, compressive and adhesion tests Gels were prepared as strips with dimensions (30 mm×10 mm×2 mm) for tensile measurement. The direction of stretching was along the long axis of the hydrogels and the stretching speed was set as 10 mm/min. The constant stretching was kept until the hydrogels ruptured. The tensile modulus (the tangent slope of the stress-strain curve), ultimate strength (strain level at failure) and rupture strain (stress at failure) were calculated according the results. Besides, for compressive tests, hydrogels were cut into slices at the diameter of 10 mm and thickness of 2 mm the compression speed was set as 5 mm/min. Likewise, the compressive modulus was calculated. Besides, hydrogels were prepared as slices with diameter of 20mm and fixed to the one side of mechanical arm. The strength to pull off samples from platform was measured. The experiments above were performed via all-electric dynamic test instrument (Instron, British). All of the above data and plots were obtained using OriginPro software (OriginLab, America). Rheological tests The hydrogels were cut into uniform disks with a diameter of 10 mm and thickness of 1 mm and the rheological tests were performed by using a modular compact rheometer (MCR102, Anto Paar, Germany). The hydrogels were cut into uniform slices at the diameter of 10 mm and 2 mm and the surface of the hydrogels kept smooth. The amplitude sweep (AS) of hydrogels was tested from 0.01–1000% at a constant frequency of 1 Hz and the frequency sweep (FS) of hydrogels was tested from 0.1 Hz to 10 H [ 24 ]. Photothermal effects of PEGDA-GDY/G@TGFβ hydrogels The hydrogels were placed in the culture dish at room temperature and irradiated with 808 nm laser for 2 minutes. The infrared thermal images were captured using an IR camera, and the temperature was recorded at 0 s, 5 s, 10 s, 20 s, 30 s, 45 s, 60 s and 120 s, respectively. Drug release of PEGDA-GDY/G@TGFβ hydrogels The hydrogels were immersed into the 2 mL PBS in tubes, and then 1mL solution were extracted at different time point and 1 mL PBS were supplemented into the tubes. For PGGT-NIR (+) group, the hydrogels were irradiated for 30s before extraction. After sample collection, the concentration of TGFβ in the solution was tested by the enzyme-linked immuno sorbent assay (Elisa) kit. For further experiments, the TGF-β solution at the concentration of 100 µg/L was processed with different temperature for different time and irradiated with NIR for different time. The macro views of solution after procession with Elisa kit were captured and relative amount of TGF-β in solution were measured at absorbance of 450 nm. In vitro biocompatibility tests of PEGDA-GDY/G@TGFβ hydrogels Gel disks with diameters of 10 mm and height of 5 mm were sterilized and immersed into culture medium. HDFs were applied in cytological experiments in vitro . Cell suspension at the concentration of 10 6 /mL was added into the hydrogels. After incubation for 2 h, the hydrogels loaded with HDFs were cultured in low glucose medium (#C11885500BT, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, #10099141, Gibco, USA) and 1% penicillin/streptomycin (P/S, #15070063, Gibco, USA) at 37°C under 5% CO 2 . The medium was exchanged every 2 days, and HDFs were passaged prior to reaching 80% confluence. The FDA/PI staining and CCK8 assay were performed as mentioned previously. The viability of HDFs in hydrogels was calculated according to fluorescence staining images via software Image J. The cytoskeleton was stained with phalloidin-FTIC conjugates at concentration of 0.5 mg/mL. Cell migration experiments affected by hydrogels For cell migration experiments, the extract liquor was prepared by medium immersed with hydrogels for 24 h. HDFs were cultured in a 6-well plate and reached 80% confluence. The sterilized tips were applied to make the scratch through the cell colony. Then the scratch images were captured at 0 h, 8 h, 16 h and 24 h, respectively. The wound width-time curve was plot and the linear regression analysis was utilized to calculate the rate of closure. Assessment of angiogenesis in vitro of hydrogels HUVECs were cultured in RP1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C under 5% CO2. Then HUVECs were collected and resuspended at the concentration of 106/mL. After implantation in gel disks as described before, the cytoskeleton of HUVECs was stained by the solution of phalloidin (p5282, Aldrich-Sigma) at the concentration of 0.1 mg/mL at the different time points of 24 h and 48 h. Then the number of the tube was analyzed and the mean OD values of CD31 (/pixel) were calculated in Image J software. Antibacterial effects in vitro of hydrogels E. coli (Gram-negative, ATCC25922), P. aeruginosa (Gram-negative, ATCC27853), S. aureus (Gram-positive, ATCC43300), and methicillin-resistant S. aureus ( MRSA , ATCC43000) were used to perform antibacterial tests. Single colonies grown on Luria-Bertani (LB) plates were inoculated into LB broth medium at 37°C with shaking at 200 rpm overnight until logarithmic growth (OD590 = 0.8). Bacterial liquid was centrifuged, after which the supernatant was discarded. The bacteria were collected suspended in LB broth medium, and the final bacterial concentration was 1.0 × 10 6 CFU/mL. The bacterial numbers were calculated from the measurement of absorbance at 590 nm using a UV-Vis spectrophotometer. Then experimental conditions were then set up with a group containing 900 µL of 10 4 CFU/mL of bacterial suspension, prepared as above, together with (1) 100 µL PBS, (2) 100 µL of 4 µg/mL ampicillin (AMP), (3) PEGDA hydrogels, (4) PEGDA-GDY hydrogels, (5) PEGDA-GDY/G, and (6) PEGDA-GDY/G@TGFβ, and the groups after irradiated by an 808 nm laser (500 mW/cm 2 ) for 30s. These tubes were then incubated for 2 h at 37°C, after which the bacterial suspensions were coated on LB agar plates for incubation overnight and colonies were counted. Besides, the experiments of the inhibitory zone were performed as follows. The hydrogels were put on the LB agar plates inoculated with various bacteria, and the images were captured at the time point of 24 h after the hydrogels were irradiated with NIR. For antibacterial mechanism experiments, the ROS fluorescence were stained with antibodies described in Table S3 and the intensity of ROS fluorescence was measured via software ZEISS (German). The bacteria of S. aureus at the concentration of 1 × 10 8 CFU/mL were incubated in hydrogels. After NIR-irradiation for 30s, the bacterial suspensions were centrifuged and filtered with a 0.22 µm membrane. The remained supernatant was tested as the leakage of intracellular DNA and RNA by measuring the OD 260 value of each sample using UV-vis measurement. Besides, the HDFs implanted in hydrogels after 48h were collected and the IL-6 were measured via IL-6 ELISA kit (RAB0306, Sigma, America). ROS level in bacteria were measured using the ROS probe (DCFH-DA, HARVEYBIO, China). In vivo implantation evaluation on diabetic wound mouse model All procedures on animals were processed under the National Institute of Health’s Guidelines for the Care and Use of Laboratory Animals and in accordance with the Consent Form for Ethical Review of Animal Experiments (No. N2019066). The C57/8BL6 male mice were intraperitoneally injected with streptozocin (STZ, 50 mg/kg) solution once a day for five days. Diabetic wound model animals were selected from mice with fast glucose levels >11.1 mmol/L for at least two detections. Thirty mice with diabetes were randomly divided into five groups of PEGDA, PEGDA-GDY, PEGDA-GDY/G, PEGDA-GDY/G@TGFβ and PGGT-NIR(+). All operations were performed by the same surgeon under general anesthesia with 2% isoflurane inhalation. After the mice were completely unconscious, the dorsal hair was shaved and applied with a depilatory cream. A full-thickness skin in 8 mm diameter was excised on the dorsa of mice mentioned above to make a diabetic wound mouse model. Then sterilized hydrogels were prepared as slices with an inner diameter of 8 mm and height of 2 mm and fixed on the wounds. The operation region was captured at the time point of 0, 1, 3, 7 and 14 days and the wounds were harvested for 14 days after carbon dioxide asphyxia. Besides, the wound of PGGT-NIR(+) group was irradiated with NIR laser for 30s every day. For histological staining and immunofluorescence staining, the wound skin was fixed and embedded in paraffin and samples were stained according to antibodies shown in Table S3. Besides, the amount of collagen and elastin was measured using the Total Collagen Assay kit (BioVision, USA) and the Fastin Elastin Assay kit (Biocolor, UK). Blood chemistry analysis was performed using Vetscan VS2 (Abaxis) and kits (Bioassay Systems, CA, USA). Statistical analysis All results were shown as mean ± SD. One-way analysis of variance (ANOVA) or two-way ANOVA were performed to analyze differences among three or more groups, followed by Tukey, Sidak or Bonferroni correction and the two-tailed Student’s test was performed to analyze the statistical significance between two group. Statistical significance was set at P < 0.05. Results Fabrication and Characterization of PEGDA-GDY/G@TGFβ Hydrogels The particle size of a 0.1% GDY solution was close to 100 nm and the polymer dispersity index (PDI) was much lower than that of the 0.5% GDY solution ( Fig. S1 ), which indicated that a concentration below 0.5% was more stable and the nanoparticles were less prone to aggregation. Therefore, the 0.1% GDY concentration was selected for this study. Sample solutions from all groups were observed using a transmission electron microscope and analyzed using dynamic light scattering. A gelatin coating was distinctly observed around the GDY NPs ( Fig. S2 ). The gelatin coating caused the GDY NPs to shift from anionic to cationic and made it possible to load negatively charged TGF-β. The process of loading TGF-β and electrostatic self-assembly did not significantly affect the particle sizes, and the zeta potentials of the NPs were slightly decreased after TGF-β loading. Figure 1 A shows the dynamic procedure of the NIR irradiation of the PEGDA-GDY/G@TGFβ solution before cross-linking. Due to the photothermal effects of GDY and thermosensitivity of gelatin, the outer layer of PEGDA-GDY/G@TGFβ was degraded, and the loaded TGF-β was released (Fig. 1 B). The particle sizes of PEGDA-GDY/G@TGFβ significantly decreased and their stability declined after NIR irradiation (Fig. 1 C and Fig. S3 ). The zeta potential of PEGDA-GDY/G@TGFβ significantly decreased after irradiation (Fig. 1 D), further confirming its “covered-uncovered” transition. Cryoelectron microscopy was used to study the surface structures of the hydrogels after cross-linking. The porous morphologies of hydrogels are shown in Fig. 2 A. According to the analysis of pore size distribution in Fig. 2 B-F, the pore sizes of PEGDA-GDY, PEGDA-GDY/G, and PEGDA-GDY/G@TGFβ were close to 30 µm, which has been reported to be beneficial for tissue regeneration [ 7 ]. Interestingly, the addition of GDY had an evident impact on the pore sizes of the hydrogels, and different concentrations of GDY NPs affected the porosities of the hydrogels in different ways ( Fig. S4 ). The GDY NPs also could be observed to uniformly distribute in the hydrogels. Most hydrogel wound dressings have brittle mechanical properties when used on dynamic wounds, especially for joints and axilla, leading to wound infection caused by unnecessary tearing [ 25 ]. Thus, it is important for hydrogel wound dressings to have high mechanical strength and sustained stability [ 26 ]. For most NPs-incorporated hydrogels, their toughness and strengths change distinctly. The GDY NPs in the PEGDA hydrogels enhanced the ultimate strengths, rupture stains, and tensile moduli (Fig. 2 G and Fig. S5A-C ). The PEGDA-GDY/G@TGFβ could withstand tensile stress of 220.69 ± 20.82 kPa and tensile strain of 146.69 ± 21.68%. The compressive modulus of PEGDA-GDY/G@TGFβ also increased after being mixed with the GDY NPs (Fig. 2 H and Fig. S5D ). To examine the hydrodynamic characteristics of the materials, frequency and amplitude sweeps were performed (Fig. 2 I and Fig. S6 ), and it was confirmed that the storage moduli of the GDY-incorporated hydrogels possessed better shear resistance [ 20 ]. We inferred that the enhanced mechanical properties were correlated to the mixture of GDY nanoparticles. Previous studies have reported that the introduction of nanoparticles such as graphene oxide and GDY enhances the hydrogel network and enriches the entanglement of macromolecular chains, effectively dissipating the energy when stretching, compressing, and shearing [ 26 ]. Adhesion is an important property for wound dressings because tight adhesion and close coverages decrease the risk of wound infections, which is one advantage of hydrogel dressing compared to traditional dressings [ 28 , 29 ]. PEGDA-GDY/G@TGFβ hydrogels showed good adhesion strength, approximate to commercial fibrin sealants (Fig. 2 J). Additionally, the PEGDA-GDY/G@TGFβ hydrogel maintained excellent adhesion stability after six strip-adhesion tests and the adhesion only slightly decreased, indicating that it possessed repeated and long-lasting adhesion stability ( Fig. S7 ). Furthermore, the capacity of hydrogel dressing to absorb the excessive blood and tissue exudate was beneficial to wound healing [ 27 ]. In this research, the swelling ratio of hydrogels indicated that the PEGDA-GDY/G@TGFβ hydrogel showed high water absorbability (388.7 ± 23.09%) after incubation in simulated body fluid, which was much higher than that of the PEGDA hydrogel (Fig. 2 K and L ). The swelling significantly affected the tensile modulus of PEGDA-GDY/G@TGFβ hydrogel rather than the PEGDA hydrogel ( Fig. S8 ). This might be correlated with the increase in molecular distance and the associated weakening of the hydrogel network [ 26 ]. Despite the decline in mechanical properties, hydrogels can still meet the requirements of wound dressing under a favorable physiological environment [ 28 ]. The photothermal-responsive properties and stimulus-response drug release characteristics of PEGDA-GDY/G@TGFβ are important for the effectiveness of this nanosystem. Hydrogels containing GDY NPs exhibited a temperature increase after NIR irradiation for a few seconds (Fig. 3 A). The temperature–time curve showed that the temperatures of PEGDA-GDY/G and PEGDA-GDY/G@TGFβ increased slower than that of PEGDA-GDY in the early stage. The curves were similar after 45 s, possibly owing to gelatin degradation (Fig. 3 B). The irradiation time was controlled to within 30 s to exert the antibacterial effects and ensure that the temperature would not be too high to affect TGF-β activity. Additionally, it can be inferred that PEGDA-GDY/G@TGFβ exhibited time-dependent sustained drug release (Fig. 3 C). PGGT-NIR(+) (PEGDA-GDY/G@TGFβ after NIR irradiation) exhibited better drug release than that of PEGDA-GDY/G@TGFβ. The cumulative drug release of PGGT-NIR(+) (77.86 ± 4.92%) was also greater than that of PEGDA-GDY/G@TGFβ (51.20 ± 3.42%). Thus, the rate of change of temperature of the GDY NPs was faster than that of graphene oxide NPs and the targeted temperature was reached in seconds [ 29 ]. This property is beneficial for the stability of protein drugs such as cell factors in drug delivery [ 30 ]. The change of absorbance showed that a change in conditions results in TGF-β denaturation (Fig. 3 D). The temperature increase had more negative effects on TGF-β than NIR-irradiation. To mimic the variation of TGF-β in NIR-responsive drug release, the concentration of TGF-β solution with GDY nanoparticles was measured at different time points after NIR-irradiation (Fig. 3 E). The loss of the amount of TGF-β influenced by the process of NIR-responsive photothermal effects in this study (irradiation for ~ 30 s) was below 3%, which is negligible. It indicated the eminent properties of this nanosystem as a drug carrier for bioactive drugs that were sensitive to temperature and prone to degrade, such as nucleic acid drugs and protein drugs. The key point is that the NIR-controlled drug release could be achieved in 30 seconds of irradiation and at a relatively safe temperature (< 50℃). This is a significant improvement compared to previously described photothermal nanosystems [ 31 ]. In Vitro Antibacterial Effects of PEGDA-GDY/G@TGFβ Hydrogels Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative Escherichia coli and Pseudomonas aeruginosa were used to study the antibacterial activities of the hydrogels [ 32 ]. The GDY-containing groups exhibited outstanding antibacterial properties against S. aureus and E. coli , which are the most common bacteria in diabetic wound infections. Surprisingly, the killing rate of PEGDA-GDY/G@TGFβ hydrogel against P. aeruginosa (74.99 ± 6.52%) was greater than that of ampicillin (AMP) (1.05 ± 0.86%), whereas the killing rate of MRSA by PEGDA-GDY/G@TGFβ hydrogel (74.99 ± 6.52%) showed no significant difference compared to that of AMP (78.52 ± 9.91%). The overall results verified that GDY-incorporated hydrogels could achieve antibacterial effects after NIR-irradiation in 30 s (Fig. 4 A–E and Fig. S9 ). Inhibition zone tests were performed to further assess the antibacterial effects of the hydrogels [ 33 ]. The diameters of the inhibition zones of PEGDA toward the S. aureus group after NIR irradiation were much smaller than those of the other groups (Fig. 4 F and Fig. S10 ). However, the inhibition zones for E. coli, P. aeruginosa , and MRSA did not exhibit significant differences, which might correlate with the differences in the proliferation and membrane structures of the various bacteria [ 34 ]. Moreover, effective photothermal antibacterial therapy usually requires higher temperatures (> 60°C), which are generally higher than physiological temperatures (37°C) and inevitably cause local thermal damage to normal tissues and cells around the wound [ 35 ]. In this research, the temperatures were controlled under 50°C, and the antibacterial effects were similar to those of AMP, which might be associated with potential stress responses of GDY nanoparticles after NIR-irradiation. To confirm that GDY increases the photothermal antibacterial effects, the pure temperature rise via heating was prepared, and the temperature variation was similar to that of NIR-irradiation ( Fig. S11A ). The controlled trial indicated that the antibacterial effects of GDY-dependent photothermal effects was significantly better than that of the pure temperature rise ( Fig. S11B ). The underlying mechanism of antibacterial properties of photothermal effects at safe temperature was further surveyed. It has been reported that graphene oxide nanoparticles could enhance antibacterial efficiency via damage to the bacterial membrane and release of ROS; thus, we inferred that the antibacterial effects at a safe temperature of GDY-incorporated hydrogels might have similar properties [ 36 , 37 ]. The amount of ROS in GDY-incorporated hydrogels was much higher than those of the other groups, and the cell membrane integrity of S. aureus in GDY-incorporated hydrogels decreased after NIR-irradiation, which potentially enhanced the antibacterial effects of PEGDA-GDY/G@TGFβ hydrogel (Fig. 4 G and H ). One reason for the difficulty in healing diabetic wounds is the overactivation of ROS [ 38 , 39 ]. Therefore, we further assessed whether the amount of ROS generated by GDY nanoparticles after NIR-irradiation would cause cell damage. The generated ROS did not interfere with the cell activity of human dermal fibroblasts (HDFs) and it did not activate oxidative stress-related signaling pathways to cause negative effects ( Fig. 4 I and J ). This offered more evidence that the relevant changes in antibacterial properties did not negatively affect the biocompatibility of hydrogels. Cell Biocompatibility and Migration of PEGDA-GDY/G@TGFβ Hydrogels The biocompatibility of hydrogels was assessed using fluorescein diacetate (FDA)/propidium iodide (PI) staining of implanted HDFs. The cell viability in the PEGDA group after cell implantation was lower than that in the other groups, whereas the cell viability in the other groups with GDY NPs was not significantly different (Fig. 5 A–B and Fig. S12 ). Furthermore, the addition of GDY and NIR irradiation did not significantly harm the HDFs according to a cell counting kit-8 (CCK8) assay (Fig. 5 C). This is an important result since the safety of two-dimensional nanomaterials and photothermal effects are important parameters for biomedical applications [ 20 ]. Scratch assays were performed to evaluate the effects of the hydrogels on cell migration during wound healing. The wound widths of PEGDA-GDY/G@TGFβ and PGGT-NIR (+) groups were much higher than those of the other groups after 16 and 24 h (Fig. 5 D and Fig. S13 ). The percentage of wound closure of the PGGT (+) group (88.16 ± 1.40%) at 24 h was much higher than that of the other groups results (Fig. 5 E). Width–time curves were plotted to further test the wound closure rates of the materials (Fig. 5 F). Linear regression was used to calculate the rate of closure. The rate of closure of the PGGT (+) group (36.20 µm/h) was higher than that of the PEGDA-GDY/G@TGFβ group (32.25 µm/h). Endothelial Cell Adhesion and Vascular Formation of PEGDA-GDY/G@TGFβ Hydrogels Blood vessel regeneration is an important factor in diabetic wound healing [ 40 ]. Therefore, angiogenic properties were tested in vitro . Human umbilical vein endothelial cells (HUVECs) were implanted into the hydrogels, and F-actin was stained to measure the adhesion of endothelial cells (ECs). The number of adherent HUVECs containing GDY NPs was greater than that in the PEGDA group (Fig. 6 A). It was previously confirmed that GDY NPs enhance cell adhesion. In addition, the HUVECs in PEGDA-GDY/G@TGFβ and PGGT-NIR(+) groups exhibited cell spreading, and actin filaments were well organized, indicating that the release of TGF-β was beneficial for EC proliferation (Fig. 6 B–C). Tube formation experiments were performed to study the promotion of angiogenesis in the hydrogels. Staining showed that a tubular structure was observed in the PEGDA-GDY/G @TGFβ and PGGT-NIR(+) groups, but not in the PEGDA and PEGDA-GDY groups (Fig. 6 D), which was verified quantitatively (Fig. 6 E). This indicated that the TGF-β in the hydrogels may enhance vascular regeneration. Additionally, the function of ECs was vital for the reconstruction of vessels, as reflected by CD31 staining. The optical density (OD) of the CD31 staining in the groups loaded with TGF-β was much higher than that in the other groups (Fig. 6 F). The plasticity of ECs is mediated by various cytokines, including TGF-β and vessel formulation was involved in TGF-β-regulated SMAD signaling [ 41 , 42 ]. Our research further verified this biological activity and its potential applications in the treatment of chronic wounds. Investigating PEGDA-GDY/G@TGFβ Hydrogels in a Diabetic Wound Mouse Model The hydrogels were implanted into diabetic wound mouse models to study the therapeutic effects of the PEGDA-GDY/G@TGFβ hydrogels. The timeline of the wound-healing experiments is shown in Fig. 7 A. Macroscopic views of the wounds in the different groups were captured on days 0, 1, 3, 7, and 14 (Fig. 7 B). The wounds in the PGGT-NIR (+) group nearly disappeared, whereas defects remained in the PEGDA, PEGDA-GDY, and PEGDA-GDY/G groups. Figure 7 C was generated according to the macro views for clear visualization of the wound changes. The wound-healing patterns of PEGDA-GDY/G@ TGFβ and PGGT-NIR (+) before day 3 were like those of the other groups (Fig. 7 E). In the following days, the wound-healing capacities of PEGDA-GDY/G@TGFβ and PGGT-NIR(+) were better than those of the other groups. The closure rate of the PGGT-NIR(+) group (91.96 ± 3.08%) was higher than that of the PEGDA-GDY/G@TGFβ group (88.44 ± 3.83%) on day 14. Notably, the PEGDA-GDY and PEGDA-GDY/G groups showed better wound healing than the PEGDA group, which may have been related to the antibacterial effects of the GDY NPs. For the histological analysis, the implanted hydrogels were harvested on day 14 and stained with hematoxylin and eosin (H&E), Masson’s trichrome, CD31, and immunofluorescent staining. The H&E and Masson’s trichrome staining revealed different degrees of extracellular matrix deposition (Fig. 7 D). The collagen and fibrins of the unhealed wound areas of the PGGT-NIR (+) group were like those in the normal area, while those of the other groups were arranged in a disorderly fashion. Moreover, the lengths of the wounds were analyzed (Fig. 6 F) and the results were consistent with those of the wound areas. The epithelia of the PEGDA-GDY/G@TGFβ (30.92 ± 6.65 µm) and PGGT-NIR (+) groups (36.77 ± 4.65 µm) were thicker than those of the other groups ( Fig. S14 ). The wound-healing rate was the highest in groups loaded with TGF-β, consistent with previously reported findings that TGF-β could promote cell migration in vivo [ 43 ]. Tissue regeneration (particularly of the extracellular matrix) is important for wound healing [ 44 ]. The deposition of collagen Ⅰ results in scar formation, whereas that of collagen III is beneficial for wound healing [ 4 ]. As shown in Fig. 8 A, collagen I in the PEGDA-GDY/G@TGFβ and PGGT-NIR (+) groups did not show excessive proliferation, whereas the other groups (especially PEGDA-GDY) exhibited collagen I hyperplasia. Additionally, the proliferation of collagen III in the PGGT-NIR (+) group was greater than that in the other groups, and PEGDA barely deposited any collagen Ⅲ. The PGGT-NIR (+) group stimulated the regeneration of the extracellular matrix (collagen and elastin) in the skin wounds and performed better than the other groups (Fig. 8 B–C). Angiogenesis is another important factor that influences diabetic wound healing [ 45 ]. CD31 immunohistochemical staining was performed to assess the ability of the hydrogels to promote vessel generation [ 45 ]. The number of CD31 + vessels per 0.01 mm 2 in the PGGT-NIR (+) group (45.375 ± 5.03) was much higher than that in the PEGDA-GDY/G@TGFβ group (35.25 ± 6.83) (Fig. 7 A and Fig. 7 D). The number of CD31 + vessels in the PEGDA-GDY/G@TGFβ group was significantly higher than that in the other groups, indicating the potential ability of TGF-β to promote revascularization. Nanoparticle biosafety is of vital importance in the clinical applications of nanomedicine and the biosafety of the nanosystem in this study was closely monitored [ 46 ]. There was no significant NP residue or metabolites in the tissues of the heart, liver, lung and kidney (Fig. 8 E). There was no significant difference in the proportion of inflammatory cells in the hydrogels of each group (Fig. 8 F). This demonstrated that the GDY nanosystem in this study either does not cause substantive damage to vital organs or that the nanoparticles rarely enter the blood circulation, having only local effects in the wound. Either possibility indicates good biosafety of the hydrogel for in vivo applications. The blood routine examination and blood biochemistry test shown in Table S1 and S2 further verified this conclusion. Moreover, the response of monocytes/macrophages to nanoparticles during tissue regeneration is important [ 47 ]. CD206 is a marker of anti-inflammatory and proregenerative macrophages and inducible nitric oxide synthase (iNOS) is a marker of proinflammatory macrophages [ 48 ]. The ratio of CD206/iNOS in the PGGT-NIR (+) group was higher than that of the PEGDA group (Fig. 8 G). This indicated that the PEGDA-GDY/G@ TGFβ hydrogels provided a better immune microenvironment for wound healing. Discussion In this study, we developed a PEGDA-GDY/G@TGF-β hydrogel as a wound dressing and investigated its use in diabetic wound healing. The core-shell type is commonly included in smart materials to achieve the “ON/OFF” function of the stimulus-response [ 49 ]. In our study, the smart response was dependent on gelatin thermosensitivity. This prominent feature has been widely used in sacrificial materials and physical and chemical modifications [ 50 ]. On one hand, we can use the charge modifications to shift the charge on GDY NPs from negative to positive to load TGF-β as a drug. On the other hand, heat-induced melting of the gelatin coating during NIR irradiation makes optically controlled drug release possible. Graphdiyne has been explored for potential biomedical applications owing to its excellent properties and stable structure. A mixture of GDY in biomedical scaffolds was investigated to impart the free-radical scavenging ability, strengthen mechanical properties, and enhance cell adhesion [ 51 , 52 ]. However, the potential of GDY NPs as drug carriers and smart materials in nanomedicine was not investigated. In this study, we developed a drug-loaded nanosystem based on GDY NPs. The hydrogels with GDY showed good biocompatibility in vitro and in vivo and were sensitive to photothermal reactions [ 53 ]. Moreover, the rate of change of temperature of the GDY NPs was much quicker than that of graphene oxide NPs and the targeted temperature (approximately 50°C) could be reached in seconds [ 54 ]. This property was beneficial for the stability of protein drugs, such as cell factors [ 55 ]. Most importantly, the antibacterial effects of the NIR irradiation-treated GDY were like those of AMP. Graphdiyne exhibited antibacterial properties for AMP-resistant bacteria (killing rate > 75%). Graphdiyne NPs achieved a high rate of antibacterial effects under 49.5℃ depending on the damage to the membrane of bacteria and potential oxidative stress response and these might strengthen photothermal bactericidal effects. Consequently, the different killing effects on various bacteria correlated with the proliferation model and structure of the bacterial membranes. However, the inhibitory zone experiments cannot prove the diffusion effects of the PEGDA-GDY/G@TGFβ hydrogels on bacteria [ 32 ]. TGF-β is an important cytokine in the induction of endothelial-mesenchymal transition and plays a key role in wound healing [ 56 ]. For example, TGF-β acts as a chemotactic protein of fibroblasts [ 13 ]. The expression of TGF-β receptors by fibroblasts involved in the wound healing has been examined in normal and healed skin, particularly in healing scars [ 10 , 57 ]. In this study, TGF-β was innovatively loaded into GDY/G NPs to be delivered in vivo and in vitro . The groups loaded with TGF-β promoted cell migration in vitro and the wound healing rate of PGGT-NIR(+) was the highest, consistent with previously reported findings [ 58 ]. Reactive oxygen species production (induced by a high-glucose environment) modulates TGF-β signaling through different pathways, including the SMAD pathway [ 59 ]. Activated TGF-β may increase ROS production and suppress antioxidant enzymes, forming a vicious cycle in many fibrotic diseases [ 56 , 60 ]. However, this cycle may be beneficial for the fibrosis of diabetic wounds in the early stages of the disease and angiogenesis in the later stages, which was confirmed in the cell migration tests and tube formation tests. Conclusion We designed and fabricated PEGDA-GDY/G@TGFβ hydrogels with NIR-controlled drug release and antibacterial effects. Moreover, PEGDA-GDY/G@TGFβ hydrogel exhibited the ability to promote cell migration and angiogenesis when applied as a wound dressing for diabetic wounds. This study also suggested the possibility of combining GDY and gelatin as a promising carrier for cell factors and confirmed the sensitivity of GDY NPs in photothermal effects. Most importantly, the excellent properties of PEGDA-GDY/G@TGFβ hydrogels made them suitable for further application in treatment of wounds in clinics. Abbreviations GDY, graphdiyne iNOS, inducible nitric oxide synthase NIR, near-infrared NP, nanoparticle PDI, polymer dispersity index PEGDA, polyethylene glycol diacrylate ROS, reactive oxygen species TGF-β, transforming growth factor β Declarations Acknowledgments J. H., J. J. and X. W. contributed equally to this work. Author contribution Jinfei Hou: Conceptualization, Data curation, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. Junjin Jie: Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft. Xinwei Wei: Data curation, Methodology. Xiangqian Shen: Data curation, Methodology. Qingfang Zhao: Methodology, Software. Xupeng Chai: Data curation, Methodology. Hao Pang: Projection administration, Resource. Zeren Shen: Methodology, Formal analysis. Jinqiang Wang: Data curation, Methodology. Linping Wu: Conceptualization, Methodology, Visualization. Jinghong Xu: Conceptualization, Supervision, Visualization, Writing – review & editing. Funding H. acknowledges financial support fromthe National Natural Science Foundation of China (No. 82302833) and the Young Foundation of the First Affiliated Hospital, Zhejiang University School of Medicine (No. B22127). L-P. Wu. acknowledges financial support from the National Key R&D Program of China (No.2019YFA0110500), the Key Science and Technology Project of Guangzhou City (No.2023B03J1231) and the Guangdong Pearl River Talents Program (No. 2017GC010411). S.Z. acknowledges financial support from Zhejiang Provincial Natural Science Foundation of China (No.LQ22H150005). Availability of data and materials The data are available from the corresponding author upon reasonable request. Ethics approval and consent to participate Relevant studies were carried out with the approval of the National Institute of Health’s Guidelines for the Care and Use of Laboratory Animals and in accordance with the Consent Form for Ethical Review of Animal Experiments. Consent for publication Not applicable. Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4226321","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":289419476,"identity":"f5f918ea-4673-439b-999b-f165f8dcb5e6","order_by":0,"name":"Jinfei Hou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIie3QvQrCMBDA8QMhdQh2jUt9hZNCRejDnAidFDp2cxCSwfouTuLoB9Ql7h0c2sXdsSBiuzm1GQXzgwyB+0NyAJb1g7A5BB4wR50KSkLjxAfG9RwLHZklNR9AUDAs5bk7mTgpxuUBvYGgKCF2BFdtqDWZphpxptFnvMxy4ncQ+rZrf1i+qBP5mkmHopzEA1AsjRJcSaAgJryYJ8T6FACRSaKzuEnGsl6yoGPEu/9yXe/HlcTRSKnTs3qHnqu27UmN4feNd403eoXJlGVZ1h/7APctSmAXj5F2AAAAAElFTkSuQmCC","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jinfei","middleName":"","lastName":"Hou","suffix":""},{"id":289419478,"identity":"244511e2-d914-406b-8293-837ce66ba9ab","order_by":1,"name":"Jiejun Jie","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jiejun","middleName":"","lastName":"Jie","suffix":""},{"id":289419479,"identity":"b7a9adf7-6bd4-488c-b813-190fa9f2c4f5","order_by":2,"name":"Xinwei Wei","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Xinwei","middleName":"","lastName":"Wei","suffix":""},{"id":289419480,"identity":"7c880fd9-38c5-4392-8a3b-0a0af87d8bc3","order_by":3,"name":"Xiangqian Shen","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiangqian","middleName":"","lastName":"Shen","suffix":""},{"id":289419481,"identity":"61985861-435a-48d0-9539-a3385257a50f","order_by":4,"name":"Qingfang Zhao","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Qingfang","middleName":"","lastName":"Zhao","suffix":""},{"id":289419482,"identity":"20b9142d-3dfb-46af-bd39-cf5c3458811d","order_by":5,"name":"Xupeng Chai","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xupeng","middleName":"","lastName":"Chai","suffix":""},{"id":289419483,"identity":"ff47b4f4-4d29-4df4-a4ac-0190fe535083","order_by":6,"name":"Hao Pang","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Pang","suffix":""},{"id":289419484,"identity":"3f1adfea-c50d-44a0-ac3e-52173a458652","order_by":7,"name":"Zeren Shen","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Zeren","middleName":"","lastName":"Shen","suffix":""},{"id":289419485,"identity":"121fc109-3259-4327-b7c3-2fab807b1750","order_by":8,"name":"Jinqiang Wang","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Jinqiang","middleName":"","lastName":"Wang","suffix":""},{"id":289419486,"identity":"d626e5f0-b9f7-4add-927e-7b59e320affd","order_by":9,"name":"Linping Wu","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Linping","middleName":"","lastName":"Wu","suffix":""},{"id":289419487,"identity":"d75c9c06-24f8-40fa-b8ba-4abc72e19cb6","order_by":10,"name":"Jinghong Xu","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jinghong","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-04-06 07:50:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4226321/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4226321/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12951-024-02675-2","type":"published","date":"2024-07-30T15:58:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54590896,"identity":"9ca8eabd-cfd6-49a9-a1bb-d4efafe27a7a","added_by":"auto","created_at":"2024-04-12 17:10:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1223532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe process of drug release of PEGDA-GDY/G@TGFβ. (A)\u003c/strong\u003e Schematic of transformation after NIR-irradiation for PEGDA-GDY/G@TGFβ hydrogels.\u003cstrong\u003e (B) \u003c/strong\u003eTEM images of uncross-linked PEGDA-GDY/G@TGFβ solution in the process of NIR-irradiation. Scale bar, 100 mm.\u003cstrong\u003e \u003c/strong\u003eNIR (-): without NIR-irradiation. NIR (\u003cstrong\u003e|\u003c/strong\u003e): with NIR-irradiation for 30s. NIR (+): with NIR-irradiation for 120s. \u003cstrong\u003e(C) \u003c/strong\u003eThe particle size of uncross-linked PEGDA-GDY/G@TGFβ solution in the process of NIR-irradiation (\u003cem\u003en = 14\u003c/em\u003e). \u003cstrong\u003e(D) \u003c/strong\u003eZeta potential of uncross-linked PEGDA-GDY/G@TGFβ solution in the process of NIR-irradiation (\u003cem\u003en = 5\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226321/v1/f4da6db2f629fa4004ce66e1.jpg"},{"id":54590898,"identity":"90a9d576-c671-4ca7-ba42-8d911efb9db8","added_by":"auto","created_at":"2024-04-12 17:10:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3754535,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological and photothermal characteristics of different hydrogels. (A)\u003c/strong\u003e Macro views and CryoEM images of different hydrogels. The distribution of pore size of \u003cstrong\u003e(B) \u003c/strong\u003ePEGDA, \u003cstrong\u003e(C) \u003c/strong\u003ePEGDA-GDY, \u003cstrong\u003e(D) \u003c/strong\u003ePEGDA-GDY/G and \u003cstrong\u003e(E) \u003c/strong\u003ePEGDA-GDY/G@TGFβ hydrogels. \u003cstrong\u003e(F) \u003c/strong\u003eThe pore size of hydrogels. \u003cem\u003en = 5, ****p \u0026lt; 0.0001, *p \u0026lt; 0.05\u003c/em\u003e, by one-way ANOVA with Tukey correction. \u003cstrong\u003e(G)\u003c/strong\u003e The tensile stress-strain curve of hydrogels. \u003cstrong\u003e(H) \u003c/strong\u003eThe compressive stress-strain of hydrogels.\u003cstrong\u003e (I)\u003c/strong\u003e The shear strain of hydrogels at a constant frequency of 1 rad/s. \u003cstrong\u003e(J) \u003c/strong\u003eThe adhesion strength of different hydrogels after NIR-irradiation. \u003cem\u003en = 5, ***p \u0026lt; 0.001\u003c/em\u003e, by one-way ANOVA with Tukey correction. \u003cstrong\u003e(K)\u003c/strong\u003e The macro view of different hydrogels after immersion in the simulated body fluid.\u003cstrong\u003e (L) \u003c/strong\u003eThe swelling ratio of different hydrogels after immersion in the simulated body fluid. \u003cem\u003en = 5\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226321/v1/1aae579c89726eae64d3434a.jpg"},{"id":54590899,"identity":"73f0bdb2-8be4-416a-a315-d7e9a6c4e3a6","added_by":"auto","created_at":"2024-04-12 17:10:47","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5422466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe photothermal effects of different hydrogels.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Pseudo-color thermal image of different hydrogels with NIR-irradiation at different time points. \u003cstrong\u003e(B) \u003c/strong\u003eThe line chart of temperature changes with time variation for different hydrogels with NIR-irradiation.\u003cstrong\u003e (C) \u003c/strong\u003eThe cumulative release of TGF-β of PEGDA-GDY/G@TGFβ and PGGT-NIR(+). PGGT-NIR(+): PEGDA-GDY/G@TGFβ with NIR-irradiation. \u003cstrong\u003e(D) \u003c/strong\u003eThe staining of TGF-β solution after pure heating and pure NIR-irradiation.\u003cstrong\u003e (E) \u003c/strong\u003eThe relative amount of TGF-β in GDY nanoparticles solution after NIR-irradiation.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226321/v1/41d9e433b685ea236a565c7c.jpg"},{"id":54590901,"identity":"285aaa62-0187-48bf-b1fa-13157e7ff81a","added_by":"auto","created_at":"2024-04-12 17:10:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6115688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe antibacterial effects properties of different hydrogels \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A)\u003c/strong\u003e Macro views of agar plates inoculated with \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cem\u003eMRSA\u003c/em\u003e suspension after immersion with different hydrogels with/without NIR-irradiation. NIR(-): without NIR-irradiation. NIR (+): with NIR-irradiation for 30s. The killing ratio of \u003cstrong\u003e(B) \u003c/strong\u003e\u003cem\u003eS. aureus\u003c/em\u003e, \u003cstrong\u003e(C) \u003c/strong\u003e\u003cem\u003eE. coli\u003c/em\u003e, \u003cstrong\u003e(D)\u003c/strong\u003e \u003cem\u003eP. aeruginosa\u003c/em\u003e and \u003cstrong\u003e(E)\u003c/strong\u003e \u003cem\u003eMRSA\u003c/em\u003e for different hydrogels with/without NIR-irradiation was plotted. \u003cem\u003en = 4\u003c/em\u003e, \u003cem\u003e****p\u003c/em\u003e \u0026lt; 0.0001, byone-way ANOVA with Tukey correction.\u003cstrong\u003e (F) \u003c/strong\u003eThe inhibitory zones of \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e,\u003cem\u003e P. aeruginosa\u003c/em\u003eand \u003cem\u003eMRSA\u003c/em\u003e for different hydrogels with NIR-irradiation at the time point of 24 h. \u003cstrong\u003e(G) \u003c/strong\u003eThe ROS fluorescence intensity of different hydrogels after NIR-irradiation. \u003cem\u003en = 4\u003c/em\u003e, \u003cem\u003e****p\u003c/em\u003e \u0026lt; 0.0001, by one-way ANOVA with Tukey correction. \u003cstrong\u003e(H) \u003c/strong\u003eThe leakages of DNA and RNA of \u003cem\u003eS. aureus\u003c/em\u003e after processing with hydrogels after NIR-irradiation. \u003cem\u003en = 5\u003c/em\u003e, \u003cem\u003e****p\u003c/em\u003e \u0026lt; 0.0001, by one-way ANOVA with Tukey correction. \u003cstrong\u003e(I) \u003c/strong\u003eThe cell viability of HDFs implanted in different hydrogels after NIR-irradiation. \u003cem\u003en = 5\u003c/em\u003e, \u003cem\u003e**p\u003c/em\u003e \u0026lt; 0.01, by one-way ANOVA with Tukey correction. \u003cstrong\u003e(J)\u003c/strong\u003eThe IL-6 level of HDFs implanted in different hydrogels after NIR-irradiation. \u003cem\u003en = 5\u003c/em\u003e, \u003cem\u003e****p\u003c/em\u003e \u0026lt; 0.0001, by one-way ANOVA with Tukey correction.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226321/v1/985100d3398ea1684f5eb959.jpg"},{"id":54590900,"identity":"ddad0a3f-0be8-44af-8a42-931975335534","added_by":"auto","created_at":"2024-04-12 17:10:47","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7555406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e cell biocompatibility of different hydrogels. (A) \u003c/strong\u003eHDFs implantation in hydrogels with FDA (green)/PI (red) staining at 7 days. Scale bar, 200 μm. \u003cstrong\u003e(B) \u003c/strong\u003eViability of HDFs plotted by calculation of the results of biocompatibility experiments. \u003cem\u003en = 3, ****p \u0026lt; 0.0001,\u003c/em\u003e by two-way ANOVA with Tukey correction. \u003cstrong\u003e(C)\u003c/strong\u003e The absorbance at 405 nm for CCK8 assay of different hydrogels. \u003cem\u003en = 5\u003c/em\u003e. \u003cstrong\u003e(D) \u003c/strong\u003eThe images of scratch assays. Scale bar, 200 μm. Black dash line: the edge of scratch. \u003cstrong\u003e(E) \u003c/strong\u003eThe percent of closure area at different time point. \u003cem\u003en = 3. \u003c/em\u003e\u003cstrong\u003e(F)\u003c/strong\u003e The scatter plot with linear regression based on the width of the wound over time for different hydrogels. \u003cem\u003en = 3.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226321/v1/e230923f2bd2ef8e498d36cc.jpg"},{"id":54591541,"identity":"5be3e425-5e1f-4810-bb4b-c2d15816adee","added_by":"auto","created_at":"2024-04-12 17:18:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4922652,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe promotion of angiogenesis properties of different hydrogels \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A) \u003c/strong\u003eF-actin staining images of HUVECs implanted in different hydrogels. \u003cstrong\u003e(B) \u003c/strong\u003eCell area and\u003cstrong\u003e (C) \u003c/strong\u003eFeter’s diameter of different hydrogels was plotted at the time points of 24 h and 48 h.\u003cem\u003e n = 5\u003c/em\u003e, \u003cem\u003e****p \u0026lt; 0.0001, ***p \u0026lt; 0.001, **p \u0026lt; 0.01\u003c/em\u003e, by two-way ANOVA with Tukey correction. \u003cstrong\u003e(D) \u003c/strong\u003eFDA/PI staining of tube formation experiments at 8h.\u003cstrong\u003e \u003c/strong\u003eScale bar, 200 μm.\u003cstrong\u003e \u003c/strong\u003eWhite dash line: tubular structure. \u003cstrong\u003e(E) \u003c/strong\u003eThe number of tubes in tube formation experiments was calculated. \u003cem\u003en = 5, ****p \u0026lt; 0.0001, **p \u0026lt; 0.01,\u003c/em\u003e by one-way ANOVA with Tukey correction. \u003cstrong\u003e(F) \u003c/strong\u003eThe mean OD values of CD31 (/piexl) were analyzed. \u003cem\u003en = 5, ****p \u0026lt; 0.0001, **p \u0026lt; 0.01,\u003c/em\u003e by one-way ANOVA with Tukey correction.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226321/v1/7f75d468f942426fcc4323b5.jpg"},{"id":54591542,"identity":"8e9f1ee1-d82f-4720-b75f-0fcb66f7fe9e","added_by":"auto","created_at":"2024-04-12 17:18:48","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6430139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e implantation of hydrogels on wounds of diabetic mice. (A)\u003c/strong\u003e Schematic demonstrating the timeline of different hydrogels treatment.\u003cstrong\u003e (B) \u003c/strong\u003ePhotographs of the wound after treatment with different hydrogels. Scale bar, 10 mm.\u003cstrong\u003e (C) \u003c/strong\u003eThe projections of the wound at different time points after treatment with different hydrogels. \u003cstrong\u003e(D)\u003c/strong\u003e HE staining images of harvested wound tissues at 14 days. Scale bar, 500 μm, 300μm for magnified view. \u003cstrong\u003e(E)\u003c/strong\u003e The percent of the closure area of wound after treatment with different hydrogels measured by macro views. \u003cem\u003en = 5\u003c/em\u003e. \u003cstrong\u003e(F) \u003c/strong\u003eThe length of the wound after treatment with different hydrogels measured by HE staining images. \u003cem\u003en = 6,****p \u0026lt; \u003c/em\u003e0.0001, \u003cem\u003e***p \u0026lt;\u003c/em\u003e0.001, by one-way ANOVA with Tukey correction.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226321/v1/997097852028101f97ea2211.jpg"},{"id":54590903,"identity":"1a18012a-971b-41fe-b3a1-894ebb6ffb11","added_by":"auto","created_at":"2024-04-12 17:10:48","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":9698861,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e histological assessment of hydrogels on diabetic wound healing. (A) \u003c/strong\u003eImmunofluorescence staining of collagen I (red), collagen III (red) and immunohistochemical staining of CD31 of wounds after treatment with different hydrogels at the time point of 14 days. \u003cstrong\u003e(B)\u003c/strong\u003e Collagen and \u003cstrong\u003e(C)\u003c/strong\u003e elastin content in harvested wounds at the time point of 14 days. \u003cem\u003en = 4\u003c/em\u003e. \u003cem\u003e****p \u0026lt; \u003c/em\u003e0.0001, \u003cem\u003e**p \u0026lt;\u003c/em\u003e 0.01, by one-way ANOVA with Tukey correction. \u003cstrong\u003e(D)\u003c/strong\u003e The number of CD31+ vessels per 0.01 mm\u003csup\u003e2\u003c/sup\u003e in the wound section. \u003cem\u003en = 8, ****p \u0026lt; \u003c/em\u003e0.0001, \u003cem\u003e*p \u0026lt;\u003c/em\u003e 0.05, by one-way ANOVA with Tukey correction. \u003cstrong\u003e(E)\u003c/strong\u003e HE staining of organs after treated with PGGT-NIR (+) group after 14 days. Scale bar, 200 μm.\u003cstrong\u003e (F) \u003c/strong\u003eThe ratio of CD206+ cells and iNOS+ cells in wound treated with different hydrogels after 14 days. \u003cem\u003en = 4, *p \u0026lt;\u003c/em\u003e 0.05, by one-way ANOVA with Tukey correction. \u003cstrong\u003e(G)\u003c/strong\u003e The inflammatory cells in wounds treated with different hydrogels after 14 days. \u003cem\u003en = 5, ****p \u0026lt; \u003c/em\u003e0.0001, \u003cem\u003e*p \u0026lt;\u003c/em\u003e 0.05, by one-way ANOVA with Tukey correction.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4226321/v1/cd48445c389be2560b48df49.jpg"},{"id":61793774,"identity":"67362266-eb99-4d35-9f23-62a5ce1de6e6","added_by":"auto","created_at":"2024-08-05 16:15:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":46141597,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4226321/v1/de058d12-84e6-48f9-95d2-5335d741ac40.pdf"},{"id":54590905,"identity":"f0b3d597-e42a-4d5c-a211-ace389c50d97","added_by":"auto","created_at":"2024-04-12 17:10:49","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":62874863,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4226321/v1/7af93ef6927b86e0d209d753.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Core-Shell-Type Nanosystem Promotes Diabetic Wound Healing Through Photothermal-Responsive Release of Transforming Growth Factor β","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic wounds are, the most common complication of diabetes, contributing to severe health costs and economic losses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Over 1\u0026nbsp;million patients with diabetes present with diabetic wounds [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, developing new wound dressings is essential for treating diabetic wounds [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, treating diabetic wounds remain challenging because a high-glucose microenvironment can further induce bacterial breeding, resulting in infection-related complications [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Microangiopathy and revascularization defects can also negatively influence healing [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The infections and defects of vessels might further trigger a chain of adverse reactions, such as inhibition of cell proliferation and migration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTransforming growth factor β (TGF-β) is a cell factor that potentially helps wound healing [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It promotes the growth and migration of epidermal and vascular endothelial cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It also promotes angiogenesis and consequent blood flow to wounds by stimulating the local release of other growth factors [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, topical use of medicated wound dressings helps avoid potential damage to other tissues in the body. Therefore, TGF-β is an excellent drug candidate for the local treatment of diabetic wounds [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, there are limitations; cellular factors are likely to degrade \u003cem\u003ein vivo\u003c/em\u003e and cannot be sustained for a long time, while diabetic wound healing is chronic and continuous [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, TGF-β lacks the antibacterial properties that are essential for diabetic wound healing [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo overcome these difficulties, drug-delivery nanosystems may be used to construct appropriate carriers for TGF-β to cure diabetic wounds. The stimulus-response nanosystems (especially the light-response nanosystem) have been widely used to treat wounds [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Drug release could be controllable with an \u0026ldquo;ON/OFF\u0026rdquo; switch for light, and the associated temperature rise could result in bacterial death owing to the photothermal effects of these nanosystems. Nevertheless, the high temperature could also deconstruct the molecular structures containing loaded cell factors and harm the surrounding tissues [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It is essential to develop thermosensitive nanosystems to achieve antibacterial effects at a safe temperature range of \u0026lt;\u0026thinsp;50℃.\u003c/p\u003e \u003cp\u003eConsequently, the core-shell-type is a common method to construct scaffolds in regenerative medicine [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and was applied in this study. Graphdiyne (GDY) was chosen as the core part owing to its stability and biocompatibility reported in our previous study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Most importantly, GDY nanoparticles (NPs) may possess the ability to damage the cell wall of bacteria and produce tiny amounts of reactive oxygen species (ROS), similar to other carbon-based two-dimensional material. It is reasonable to consider that it might be a proper antibacterial and photothermal system functional at relatively safe temperatures. However, the properties to load the drug and kill bacteria in GDY NPs have not been further surveyed. Besides, gelatin (a natural biocompatible material with thermosensitivity) covered the surface of the GDY nanoparticles [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The coverage of gelatin could modify the GDY nanoparticles from anion to cation, which may make TGF-β loading feasible via charge self-assembly [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moreover, it is possible for GDY/G NPs (GDY NPs covered with gelatin) to release TGF-β responsive to photothermal stimulus without affecting its bioactivity owing to the state change of gelatin from a gel to a liquid between 30\u0026ndash;40\u0026deg;C [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we propose a near-infrared (NIR)-responsive nanosystem based on GDY and gelatin. The GDY/G@TGFβ NPs were further encapsulated in biocompatible polyethylene glycol diacrylate (PEGDA) hydrogels with good mechanical properties. The drug release curve and capacity of the materials to promote cell migration and proliferation, antibacterial effects, and revascularization were determined. Finally, full-thickness wounds in diabetic mouse models were constructed to investigate the ability of the PEGDA-GDY/G@TGFβ hydrogels to promote diabetic wound healing and tissue regeneration.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of PEGDA-GDY/G@TGFβ hydrogels\u003c/h2\u003e \u003cp\u003ePEGDA-GDY/G@TGFβ hydrogels were synthesized by the method of electrostatic self-assembly. GDY nanoparticles at concentration of 0.05%, 0.1% and 0.5% were prepared as described before [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. After centrifugation at 15000 rpm for at least 10 min, the supernatant is discarded. The deionized water was used to wash the redundant gelatin and then TGF-β1 at the concentration of 100 \u0026micro;g/L was dissolved into the dispersion liquid. After centrifugation as above, the GDY/G@TGFβ solution was prepared. The 10 mg PEGDA and 2\u0026thinsp;~\u0026thinsp;3 mg lithium pheny l-2,4,6-trimethylbenzoyl phosphinate (LAP) was added to 1 mL solution above and completely dissolved. To construct the hydrogels, blue light irradiation was performed for at least 20 s. For sterilization, the hydrogels were immersed into 75% ethyl alcohol for 12h and then washed with deionized water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization and morphology of PEGDA-GDY/G@TGFβ hydrogels\u003c/h2\u003e \u003cp\u003eTo test the dispersity and morphology, PEGDA-GDY/G@TGFβ solution before crosslinking was characterized with dynamic light scattering and transmission electron microscopy. The 100 \u0026micro;L sample solution were diluted into 1 mL deionized water and then the solution was transferred to the corresponding measuring tube for measurement of particle size and zeta potential in NanoSight (Malvern Panalytical, British). After ultrasonic dispersion for at least 60 min, the sample solution was dripped onto copper grid. Then the redundant liquid was dried and samples were observed by HT7700 microscope (Hitachi, Japan). Besides, Cryoelectron microscopy (Cryo-EM) (S-4800, HITACHI, Japan) was utilized to view the normal surface morphology of hydrogels. Briefly, hydrogels were loaded on a low-temperature sample carrier (Quaroum PP3000T) and then fixed in a cryo-specimen holder The temperature was placed in cold nitrogen (-120\u0026deg;C), and then immediately transferred to the cryo-stage. To remove the ice crystals, the temperature was raised to 90\u0026deg;C and after that the samples were cooled to -180\u0026deg;C for stabilization. The pore sizes were analyzed by the software Image J (America) on the surface images and the frequency distribution histograms were plotted according to the data. The hydrogels were cut into slices at diameter of 10mm and immersed into simulated body fluids (BZ310, Biochemazone, America). After several hours, the immersed hydrogels were weighed. The hydrogels after immersed for 24 hours were performed with tensile experiments described below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMechanical properties of PEGDA-GDY/G@TGFβ hydrogels\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eTensile, compressive and adhesion tests\u003c/h2\u003e \u003cp\u003eGels were prepared as strips with dimensions (30 mm\u0026times;10 mm\u0026times;2 mm) for tensile measurement. The direction of stretching was along the long axis of the hydrogels and the stretching speed was set as 10 mm/min. The constant stretching was kept until the hydrogels ruptured. The tensile modulus (the tangent slope of the stress-strain curve), ultimate strength (strain level at failure) and rupture strain (stress at failure) were calculated according the results. Besides, for compressive tests, hydrogels were cut into slices at the diameter of 10 mm and thickness of 2 mm the compression speed was set as 5 mm/min. Likewise, the compressive modulus was calculated. Besides, hydrogels were prepared as slices with diameter of 20mm and fixed to the one side of mechanical arm. The strength to pull off samples from platform was measured. The experiments above were performed via all-electric dynamic test instrument (Instron, British). All of the above data and plots were obtained using OriginPro software (OriginLab, America).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eRheological tests\u003c/h2\u003e \u003cp\u003eThe hydrogels were cut into uniform disks with a diameter of 10 mm and thickness of 1 mm and the rheological tests were performed by using a modular compact rheometer (MCR102, Anto Paar, Germany). The hydrogels were cut into uniform slices at the diameter of 10 mm and 2 mm and the surface of the hydrogels kept smooth. The amplitude sweep (AS) of hydrogels was tested from 0.01\u0026ndash;1000% at a constant frequency of 1 Hz and the frequency sweep (FS) of hydrogels was tested from 0.1 Hz to 10 H [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhotothermal effects of PEGDA-GDY/G@TGFβ hydrogels\u003c/h2\u003e \u003cp\u003eThe hydrogels were placed in the culture dish at room temperature and irradiated with 808 nm laser for 2 minutes. The infrared thermal images were captured using an IR camera, and the temperature was recorded at 0 s, 5 s, 10 s, 20 s, 30 s, 45 s, 60 s and 120 s, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDrug release of PEGDA-GDY/G@TGFβ hydrogels\u003c/h2\u003e \u003cp\u003eThe hydrogels were immersed into the 2 mL PBS in tubes, and then 1mL solution were extracted at different time point and 1 mL PBS were supplemented into the tubes. For PGGT-NIR (+) group, the hydrogels were irradiated for 30s before extraction. After sample collection, the concentration of TGFβ in the solution was tested by the enzyme-linked immuno sorbent assay (Elisa) kit. For further experiments, the TGF-β solution at the concentration of 100 \u0026micro;g/L was processed with different temperature for different time and irradiated with NIR for different time. The macro views of solution after procession with Elisa kit were captured and relative amount of TGF-β in solution were measured at absorbance of 450 nm.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ebiocompatibility tests of PEGDA-GDY/G@TGFβ hydrogels\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGel disks with diameters of 10 mm and height of 5 mm were sterilized and immersed into culture medium. HDFs were applied in cytological experiments \u003cem\u003ein vitro\u003c/em\u003e. Cell suspension at the concentration of 10\u003csup\u003e6\u003c/sup\u003e/mL was added into the hydrogels. After incubation for 2 h, the hydrogels loaded with HDFs were cultured in low glucose medium (#C11885500BT, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, #10099141, Gibco, USA) and 1% penicillin/streptomycin (P/S, #15070063, Gibco, USA) at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. The medium was exchanged every 2 days, and HDFs were passaged prior to reaching 80% confluence. The FDA/PI staining and CCK8 assay were performed as mentioned previously. The viability of HDFs in hydrogels was calculated according to fluorescence staining images via software Image J. The cytoskeleton was stained with phalloidin-FTIC conjugates at concentration of 0.5 mg/mL.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eCell migration experiments affected by hydrogels\u003c/h2\u003e \u003cp\u003eFor cell migration experiments, the extract liquor was prepared by medium immersed with hydrogels for 24 h. HDFs were cultured in a 6-well plate and reached 80% confluence. The sterilized tips were applied to make the scratch through the cell colony. Then the scratch images were captured at 0 h, 8 h, 16 h and 24 h, respectively. The wound width-time curve was plot and the linear regression analysis was utilized to calculate the rate of closure.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssessment of angiogenesis\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eof hydrogels\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHUVECs were cultured in RP1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37\u0026deg;C under 5% CO2. Then HUVECs were collected and resuspended at the concentration of 106/mL. After implantation in gel disks as described before, the cytoskeleton of HUVECs was stained by the solution of phalloidin (p5282, Aldrich-Sigma) at the concentration of 0.1 mg/mL at the different time points of 24 h and 48 h. Then the number of the tube was analyzed and the mean OD values of CD31 (/pixel) were calculated in Image J software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntibacterial effects\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eof hydrogels\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e(Gram-negative, ATCC25922), \u003cem\u003eP. aeruginosa\u003c/em\u003e (Gram-negative, ATCC27853), \u003cem\u003eS. aureus\u003c/em\u003e (Gram-positive, ATCC43300), and \u003cem\u003emethicillin-resistant S. aureus\u003c/em\u003e (\u003cem\u003eMRSA\u003c/em\u003e, ATCC43000) were used to perform antibacterial tests. Single colonies grown on Luria-Bertani (LB) plates were inoculated into LB broth medium at 37\u0026deg;C with shaking at 200 rpm overnight until logarithmic growth (OD590\u0026thinsp;=\u0026thinsp;0.8). Bacterial liquid was centrifuged, after which the supernatant was discarded. The bacteria were collected suspended in LB broth medium, and the final bacterial concentration was 1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e CFU/mL. The bacterial numbers were calculated from the measurement of absorbance at 590 nm using a UV-Vis spectrophotometer. Then experimental conditions were then set up with a group containing 900 \u0026micro;L of 10\u003csup\u003e4\u003c/sup\u003e CFU/mL of bacterial suspension, prepared as above, together with (1) 100 \u0026micro;L PBS, (2) 100 \u0026micro;L of 4 \u0026micro;g/mL ampicillin (AMP), (3) PEGDA hydrogels, (4) PEGDA-GDY hydrogels, (5) PEGDA-GDY/G, and (6) PEGDA-GDY/G@TGFβ, and the groups after irradiated by an 808 nm laser (500 mW/cm\u003csup\u003e2\u003c/sup\u003e) for 30s. These tubes were then incubated for 2 h at 37\u0026deg;C, after which the bacterial suspensions were coated on LB agar plates for incubation overnight and colonies were counted. Besides, the experiments of the inhibitory zone were performed as follows. The hydrogels were put on the LB agar plates inoculated with various bacteria, and the images were captured at the time point of 24 h after the hydrogels were irradiated with NIR.\u003c/p\u003e \u003cp\u003eFor antibacterial mechanism experiments, the ROS fluorescence were stained with antibodies described in Table S3 and the intensity of ROS fluorescence was measured via software ZEISS (German). The bacteria of \u003cem\u003eS. aureus\u003c/em\u003e at the concentration of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL were incubated in hydrogels. After NIR-irradiation for 30s, the bacterial suspensions were centrifuged and filtered with a 0.22 \u0026micro;m membrane. The remained supernatant was tested as the leakage of intracellular DNA and RNA by measuring the OD\u003csub\u003e260\u003c/sub\u003e value of each sample using UV-vis measurement. Besides, the HDFs implanted in hydrogels after 48h were collected and the IL-6 were measured via IL-6 ELISA kit (RAB0306, Sigma, America). ROS level in bacteria were measured using the ROS probe (DCFH-DA, HARVEYBIO, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eimplantation evaluation on diabetic wound mouse model\u003c/b\u003e\u003c/p\u003e \u003cp\u003e All procedures on animals were processed under the National Institute of Health\u0026rsquo;s Guidelines for the Care and Use of Laboratory Animals and in accordance with the Consent Form for Ethical Review of Animal Experiments (No. N2019066). The C57/8BL6 male mice were intraperitoneally injected with streptozocin (STZ, 50 mg/kg) solution once a day for five days. Diabetic wound model animals were selected from mice with fast glucose levels \u0026gt;11.1 mmol/L for at least two detections.\u003c/p\u003e \u003cp\u003eThirty mice with diabetes were randomly divided into five groups of PEGDA, PEGDA-GDY, PEGDA-GDY/G, PEGDA-GDY/G@TGFβ and PGGT-NIR(+). All operations were performed by the same surgeon under general anesthesia with 2% isoflurane inhalation. After the mice were completely unconscious, the dorsal hair was shaved and applied with a depilatory cream. A full-thickness skin in 8 mm diameter was excised on the dorsa of mice mentioned above to make a diabetic wound mouse model. Then sterilized hydrogels were prepared as slices with an inner diameter of 8 mm and height of 2 mm and fixed on the wounds. The operation region was captured at the time point of 0, 1, 3, 7 and 14 days and the wounds were harvested for 14 days after carbon dioxide asphyxia. Besides, the wound of PGGT-NIR(+) group was irradiated with NIR laser for 30s every day. For histological staining and immunofluorescence staining, the wound skin was fixed and embedded in paraffin and samples were stained according to antibodies shown in Table S3. Besides, the amount of collagen and elastin was measured using the Total Collagen Assay kit (BioVision, USA) and the Fastin Elastin Assay kit (Biocolor, UK). Blood chemistry analysis was performed using Vetscan VS2 (Abaxis) and kits (Bioassay Systems, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. One-way analysis of variance (ANOVA) or two-way ANOVA were performed to analyze differences among three or more groups, followed by Tukey, Sidak or Bonferroni correction and the two-tailed Student\u0026rsquo;s test was performed to analyze the statistical significance between two group. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFabrication and Characterization of PEGDA-GDY/G@TGFβ Hydrogels\u003c/h2\u003e \u003cp\u003eThe particle size of a 0.1% GDY solution was close to 100 nm and the polymer dispersity index (PDI) was much lower than that of the 0.5% GDY solution (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), which indicated that a concentration below 0.5% was more stable and the nanoparticles were less prone to aggregation. Therefore, the 0.1% GDY concentration was selected for this study. Sample solutions from all groups were observed using a transmission electron microscope and analyzed using dynamic light scattering. A gelatin coating was distinctly observed around the GDY NPs (\u003cb\u003eFig. S2\u003c/b\u003e). The gelatin coating caused the GDY NPs to shift from anionic to cationic and made it possible to load negatively charged TGF-β. The process of loading TGF-β and electrostatic self-assembly did not significantly affect the particle sizes, and the zeta potentials of the NPs were slightly decreased after TGF-β loading.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows the dynamic procedure of the NIR irradiation of the PEGDA-GDY/G@TGFβ solution before cross-linking. Due to the photothermal effects of GDY and thermosensitivity of gelatin, the outer layer of PEGDA-GDY/G@TGFβ was degraded, and the loaded TGF-β was released (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The particle sizes of PEGDA-GDY/G@TGFβ significantly decreased and their stability declined after NIR irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cb\u003eFig. S3\u003c/b\u003e). The zeta potential of PEGDA-GDY/G@TGFβ significantly decreased after irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), further confirming its \u0026ldquo;covered-uncovered\u0026rdquo; transition.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCryoelectron microscopy was used to study the surface structures of the hydrogels after cross-linking. The porous morphologies of hydrogels are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. According to the analysis of pore size distribution in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-F, the pore sizes of PEGDA-GDY, PEGDA-GDY/G, and PEGDA-GDY/G@TGFβ were close to 30 \u0026micro;m, which has been reported to be beneficial for tissue regeneration [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Interestingly, the addition of GDY had an evident impact on the pore sizes of the hydrogels, and different concentrations of GDY NPs affected the porosities of the hydrogels in different ways (\u003cb\u003eFig. S4\u003c/b\u003e). The GDY NPs also could be observed to uniformly distribute in the hydrogels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMost hydrogel wound dressings have brittle mechanical properties when used on dynamic wounds, especially for joints and axilla, leading to wound infection caused by unnecessary tearing [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Thus, it is important for hydrogel wound dressings to have high mechanical strength and sustained stability [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. For most NPs-incorporated hydrogels, their toughness and strengths change distinctly. The GDY NPs in the PEGDA hydrogels enhanced the ultimate strengths, rupture stains, and tensile moduli (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG \u003cb\u003eand Fig. S5A-C\u003c/b\u003e). The PEGDA-GDY/G@TGFβ could withstand tensile stress of 220.69\u0026thinsp;\u0026plusmn;\u0026thinsp;20.82 kPa and tensile strain of 146.69\u0026thinsp;\u0026plusmn;\u0026thinsp;21.68%. The compressive modulus of PEGDA-GDY/G@TGFβ also increased after being mixed with the GDY NPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH \u003cb\u003eand Fig. S5D\u003c/b\u003e). To examine the hydrodynamic characteristics of the materials, frequency and amplitude sweeps were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI \u003cb\u003eand Fig. S6\u003c/b\u003e), and it was confirmed that the storage moduli of the GDY-incorporated hydrogels possessed better shear resistance [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We inferred that the enhanced mechanical properties were correlated to the mixture of GDY nanoparticles. Previous studies have reported that the introduction of nanoparticles such as graphene oxide and GDY enhances the hydrogel network and enriches the entanglement of macromolecular chains, effectively dissipating the energy when stretching, compressing, and shearing [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdhesion is an important property for wound dressings because tight adhesion and close coverages decrease the risk of wound infections, which is one advantage of hydrogel dressing compared to traditional dressings [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. PEGDA-GDY/G@TGFβ hydrogels showed good adhesion strength, approximate to commercial fibrin sealants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ). Additionally, the PEGDA-GDY/G@TGFβ hydrogel maintained excellent adhesion stability after six strip-adhesion tests and the adhesion only slightly decreased, indicating that it possessed repeated and long-lasting adhesion stability (\u003cb\u003eFig. S7\u003c/b\u003e). Furthermore, the capacity of hydrogel dressing to absorb the excessive blood and tissue exudate was beneficial to wound healing [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this research, the swelling ratio of hydrogels indicated that the PEGDA-GDY/G@TGFβ hydrogel showed high water absorbability (388.7\u0026thinsp;\u0026plusmn;\u0026thinsp;23.09%) after incubation in simulated body fluid, which was much higher than that of the PEGDA hydrogel (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK \u003cb\u003eand L\u003c/b\u003e). The swelling significantly affected the tensile modulus of PEGDA-GDY/G@TGFβ hydrogel rather than the PEGDA hydrogel (\u003cb\u003eFig. S8\u003c/b\u003e). This might be correlated with the increase in molecular distance and the associated weakening of the hydrogel network [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Despite the decline in mechanical properties, hydrogels can still meet the requirements of wound dressing under a favorable physiological environment [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe photothermal-responsive properties and stimulus-response drug release characteristics of PEGDA-GDY/G@TGFβ are important for the effectiveness of this nanosystem. Hydrogels containing GDY NPs exhibited a temperature increase after NIR irradiation for a few seconds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The temperature\u0026ndash;time curve showed that the temperatures of PEGDA-GDY/G and PEGDA-GDY/G@TGFβ increased slower than that of PEGDA-GDY in the early stage. The curves were similar after 45 s, possibly owing to gelatin degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The irradiation time was controlled to within 30 s to exert the antibacterial effects and ensure that the temperature would not be too high to affect TGF-β activity. Additionally, it can be inferred that PEGDA-GDY/G@TGFβ exhibited time-dependent sustained drug release (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). PGGT-NIR(+) (PEGDA-GDY/G@TGFβ after NIR irradiation) exhibited better drug release than that of PEGDA-GDY/G@TGFβ. The cumulative drug release of PGGT-NIR(+) (77.86\u0026thinsp;\u0026plusmn;\u0026thinsp;4.92%) was also greater than that of PEGDA-GDY/G@TGFβ (51.20\u0026thinsp;\u0026plusmn;\u0026thinsp;3.42%). Thus, the rate of change of temperature of the GDY NPs was faster than that of graphene oxide NPs and the targeted temperature was reached in seconds [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This property is beneficial for the stability of protein drugs such as cell factors in drug delivery [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe change of absorbance showed that a change in conditions results in TGF-β denaturation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The temperature increase had more negative effects on TGF-β than NIR-irradiation. To mimic the variation of TGF-β in NIR-responsive drug release, the concentration of TGF-β solution with GDY nanoparticles was measured at different time points after NIR-irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The loss of the amount of TGF-β influenced by the process of NIR-responsive photothermal effects in this study (irradiation for ~\u0026thinsp;30 s) was below 3%, which is negligible. It indicated the eminent properties of this nanosystem as a drug carrier for bioactive drugs that were sensitive to temperature and prone to degrade, such as nucleic acid drugs and protein drugs. The key point is that the NIR-controlled drug release could be achieved in 30 seconds of irradiation and at a relatively safe temperature (\u0026lt;\u0026thinsp;50℃). This is a significant improvement compared to previously described photothermal nanosystems [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eAntibacterial Effects of PEGDA-GDY/G@TGFβ Hydrogels\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGram-positive methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) and Gram-negative \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e were used to study the antibacterial activities of the hydrogels [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The GDY-containing groups exhibited outstanding antibacterial properties against \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e, which are the most common bacteria in diabetic wound infections. Surprisingly, the killing rate of PEGDA-GDY/G@TGFβ hydrogel against \u003cem\u003eP. aeruginosa\u003c/em\u003e (74.99\u0026thinsp;\u0026plusmn;\u0026thinsp;6.52%) was greater than that of ampicillin (AMP) (1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86%), whereas the killing rate of MRSA by PEGDA-GDY/G@TGFβ hydrogel (74.99\u0026thinsp;\u0026plusmn;\u0026thinsp;6.52%) showed no significant difference compared to that of AMP (78.52\u0026thinsp;\u0026plusmn;\u0026thinsp;9.91%). The overall results verified that GDY-incorporated hydrogels could achieve antibacterial effects after NIR-irradiation in 30 s (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;E and \u003cb\u003eFig. S9\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInhibition zone tests were performed to further assess the antibacterial effects of the hydrogels [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The diameters of the inhibition zones of PEGDA toward the \u003cem\u003eS. aureus\u003c/em\u003e group after NIR irradiation were much smaller than those of the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cb\u003eFig. S10\u003c/b\u003e). However, the inhibition zones for \u003cem\u003eE. coli, P. aeruginosa\u003c/em\u003e, and MRSA did not exhibit significant differences, which might correlate with the differences in the proliferation and membrane structures of the various bacteria [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMoreover, effective photothermal antibacterial therapy usually requires higher temperatures (\u0026gt;\u0026thinsp;60\u0026deg;C), which are generally higher than physiological temperatures (37\u0026deg;C) and inevitably cause local thermal damage to normal tissues and cells around the wound [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this research, the temperatures were controlled under 50\u0026deg;C, and the antibacterial effects were similar to those of AMP, which might be associated with potential stress responses of GDY nanoparticles after NIR-irradiation. To confirm that GDY increases the photothermal antibacterial effects, the pure temperature rise via heating was prepared, and the temperature variation was similar to that of NIR-irradiation (\u003cb\u003eFig. S11A\u003c/b\u003e). The controlled trial indicated that the antibacterial effects of GDY-dependent photothermal effects was significantly better than that of the pure temperature rise (\u003cb\u003eFig. S11B\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe underlying mechanism of antibacterial properties of photothermal effects at safe temperature was further surveyed. It has been reported that graphene oxide nanoparticles could enhance antibacterial efficiency via damage to the bacterial membrane and release of ROS; thus, we inferred that the antibacterial effects at a safe temperature of GDY-incorporated hydrogels might have similar properties [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The amount of ROS in GDY-incorporated hydrogels was much higher than those of the other groups, and the cell membrane integrity of \u003cem\u003eS. aureus\u003c/em\u003e in GDY-incorporated hydrogels decreased after NIR-irradiation, which potentially enhanced the antibacterial effects of PEGDA-GDY/G@TGFβ hydrogel (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG \u003cb\u003eand H\u003c/b\u003e). One reason for the difficulty in healing diabetic wounds is the overactivation of ROS [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, we further assessed whether the amount of ROS generated by GDY nanoparticles after NIR-irradiation would cause cell damage. The generated ROS did not interfere with the cell activity of human dermal fibroblasts (HDFs) and it did not activate oxidative stress-related signaling pathways to cause negative effects \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI \u003cb\u003eand J\u003c/b\u003e). This offered more evidence that the relevant changes in antibacterial properties did not negatively affect the biocompatibility of hydrogels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCell Biocompatibility and Migration of PEGDA-GDY/G@TGFβ Hydrogels\u003c/h2\u003e \u003cp\u003eThe biocompatibility of hydrogels was assessed using fluorescein diacetate (FDA)/propidium iodide (PI) staining of implanted HDFs. The cell viability in the PEGDA group after cell implantation was lower than that in the other groups, whereas the cell viability in the other groups with GDY NPs was not significantly different (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;B and \u003cb\u003eFig. S12\u003c/b\u003e). Furthermore, the addition of GDY and NIR irradiation did not significantly harm the HDFs according to a cell counting kit-8 (CCK8) assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This is an important result since the safety of two-dimensional nanomaterials and photothermal effects are important parameters for biomedical applications [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eScratch assays were performed to evaluate the effects of the hydrogels on cell migration during wound healing. The wound widths of PEGDA-GDY/G@TGFβ and PGGT-NIR (+) groups were much higher than those of the other groups after 16 and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and \u003cb\u003eFig. S13\u003c/b\u003e). The percentage of wound closure of the PGGT (+) group (88.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40%) at 24 h was much higher than that of the other groups results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Width\u0026ndash;time curves were plotted to further test the wound closure rates of the materials (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Linear regression was used to calculate the rate of closure. The rate of closure of the PGGT (+) group (36.20 \u0026micro;m/h) was higher than that of the PEGDA-GDY/G@TGFβ group (32.25 \u0026micro;m/h).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEndothelial Cell Adhesion and Vascular Formation of PEGDA-GDY/G@TGFβ Hydrogels\u003c/h2\u003e \u003cp\u003eBlood vessel regeneration is an important factor in diabetic wound healing [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, angiogenic properties were tested \u003cem\u003ein vitro\u003c/em\u003e. Human umbilical vein endothelial cells (HUVECs) were implanted into the hydrogels, and F-actin was stained to measure the adhesion of endothelial cells (ECs). The number of adherent HUVECs containing GDY NPs was greater than that in the PEGDA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). It was previously confirmed that GDY NPs enhance cell adhesion. In addition, the HUVECs in PEGDA-GDY/G@TGFβ and PGGT-NIR(+) groups exhibited cell spreading, and actin filaments were well organized, indicating that the release of TGF-β was beneficial for EC proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB\u0026ndash;C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTube formation experiments were performed to study the promotion of angiogenesis in the hydrogels. Staining showed that a tubular structure was observed in the PEGDA-GDY/G @TGFβ and PGGT-NIR(+) groups, but not in the PEGDA and PEGDA-GDY groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), which was verified quantitatively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). This indicated that the TGF-β in the hydrogels may enhance vascular regeneration. Additionally, the function of ECs was vital for the reconstruction of vessels, as reflected by CD31 staining. The optical density (OD) of the CD31 staining in the groups loaded with TGF-β was much higher than that in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). The plasticity of ECs is mediated by various cytokines, including TGF-β and vessel formulation was involved in TGF-β-regulated SMAD signaling [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Our research further verified this biological activity and its potential applications in the treatment of chronic wounds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eInvestigating PEGDA-GDY/G@TGFβ Hydrogels in a Diabetic Wound Mouse Model\u003c/h2\u003e \u003cp\u003eThe hydrogels were implanted into diabetic wound mouse models to study the therapeutic effects of the PEGDA-GDY/G@TGFβ hydrogels. The timeline of the wound-healing experiments is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA. Macroscopic views of the wounds in the different groups were captured on days 0, 1, 3, 7, and 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The wounds in the PGGT-NIR (+) group nearly disappeared, whereas defects remained in the PEGDA, PEGDA-GDY, and PEGDA-GDY/G groups. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC was generated according to the macro views for clear visualization of the wound changes. The wound-healing patterns of PEGDA-GDY/G@ TGFβ and PGGT-NIR (+) before day 3 were like those of the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). In the following days, the wound-healing capacities of PEGDA-GDY/G@TGFβ and PGGT-NIR(+) were better than those of the other groups. The closure rate of the PGGT-NIR(+) group (91.96\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08%) was higher than that of the PEGDA-GDY/G@TGFβ group (88.44\u0026thinsp;\u0026plusmn;\u0026thinsp;3.83%) on day 14. Notably, the PEGDA-GDY and PEGDA-GDY/G groups showed better wound healing than the PEGDA group, which may have been related to the antibacterial effects of the GDY NPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the histological analysis, the implanted hydrogels were harvested on day 14 and stained with hematoxylin and eosin (H\u0026amp;E), Masson\u0026rsquo;s trichrome, CD31, and immunofluorescent staining. The H\u0026amp;E and Masson\u0026rsquo;s trichrome staining revealed different degrees of extracellular matrix deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The collagen and fibrins of the unhealed wound areas of the PGGT-NIR (+) group were like those in the normal area, while those of the other groups were arranged in a disorderly fashion. Moreover, the lengths of the wounds were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) and the results were consistent with those of the wound areas. The epithelia of the PEGDA-GDY/G@TGFβ (30.92\u0026thinsp;\u0026plusmn;\u0026thinsp;6.65 \u0026micro;m) and PGGT-NIR (+) groups (36.77\u0026thinsp;\u0026plusmn;\u0026thinsp;4.65 \u0026micro;m) were thicker than those of the other groups (\u003cb\u003eFig. S14\u003c/b\u003e). The wound-healing rate was the highest in groups loaded with TGF-β, consistent with previously reported findings that TGF-β could promote cell migration \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTissue regeneration (particularly of the extracellular matrix) is important for wound healing [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The deposition of collagen Ⅰ results in scar formation, whereas that of collagen III is beneficial for wound healing [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, collagen I in the PEGDA-GDY/G@TGFβ and PGGT-NIR (+) groups did not show excessive proliferation, whereas the other groups (especially PEGDA-GDY) exhibited collagen I hyperplasia. Additionally, the proliferation of collagen III in the PGGT-NIR (+) group was greater than that in the other groups, and PEGDA barely deposited any collagen Ⅲ. The PGGT-NIR (+) group stimulated the regeneration of the extracellular matrix (collagen and elastin) in the skin wounds and performed better than the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB\u0026ndash;C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAngiogenesis is another important factor that influences diabetic wound healing [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. CD31 immunohistochemical staining was performed to assess the ability of the hydrogels to promote vessel generation [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The number of CD31\u0026thinsp;+\u0026thinsp;vessels per 0.01 mm\u003csup\u003e2\u003c/sup\u003e in the PGGT-NIR (+) group (45.375\u0026thinsp;\u0026plusmn;\u0026thinsp;5.03) was much higher than that in the PEGDA-GDY/G@TGFβ group (35.25\u0026thinsp;\u0026plusmn;\u0026thinsp;6.83) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The number of CD31\u0026thinsp;+\u0026thinsp;vessels in the PEGDA-GDY/G@TGFβ group was significantly higher than that in the other groups, indicating the potential ability of TGF-β to promote revascularization.\u003c/p\u003e \u003cp\u003eNanoparticle biosafety is of vital importance in the clinical applications of nanomedicine and the biosafety of the nanosystem in this study was closely monitored [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. There was no significant NP residue or metabolites in the tissues of the heart, liver, lung and kidney (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). There was no significant difference in the proportion of inflammatory cells in the hydrogels of each group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). This demonstrated that the GDY nanosystem in this study either does not cause substantive damage to vital organs or that the nanoparticles rarely enter the blood circulation, having only local effects in the wound. Either possibility indicates good biosafety of the hydrogel for \u003cem\u003ein vivo\u003c/em\u003e applications. The blood routine examination and blood biochemistry test shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2\u003c/b\u003e further verified this conclusion. Moreover, the response of monocytes/macrophages to nanoparticles during tissue regeneration is important [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. CD206 is a marker of anti-inflammatory and proregenerative macrophages and inducible nitric oxide synthase (iNOS) is a marker of proinflammatory macrophages [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The ratio of CD206/iNOS in the PGGT-NIR (+) group was higher than that of the PEGDA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). This indicated that the PEGDA-GDY/G@ TGFβ hydrogels provided a better immune microenvironment for wound healing.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we developed a PEGDA-GDY/G@TGF-β hydrogel as a wound dressing and investigated its use in diabetic wound healing. The core-shell type is commonly included in smart materials to achieve the \u0026ldquo;ON/OFF\u0026rdquo; function of the stimulus-response [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In our study, the smart response was dependent on gelatin thermosensitivity. This prominent feature has been widely used in sacrificial materials and physical and chemical modifications [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. On one hand, we can use the charge modifications to shift the charge on GDY NPs from negative to positive to load TGF-β as a drug. On the other hand, heat-induced melting of the gelatin coating during NIR irradiation makes optically controlled drug release possible.\u003c/p\u003e \u003cp\u003eGraphdiyne has been explored for potential biomedical applications owing to its excellent properties and stable structure. A mixture of GDY in biomedical scaffolds was investigated to impart the free-radical scavenging ability, strengthen mechanical properties, and enhance cell adhesion [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. However, the potential of GDY NPs as drug carriers and smart materials in nanomedicine was not investigated. In this study, we developed a drug-loaded nanosystem based on GDY NPs. The hydrogels with GDY showed good biocompatibility \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e and were sensitive to photothermal reactions [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Moreover, the rate of change of temperature of the GDY NPs was much quicker than that of graphene oxide NPs and the targeted temperature (approximately 50\u0026deg;C) could be reached in seconds [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. This property was beneficial for the stability of protein drugs, such as cell factors [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Most importantly, the antibacterial effects of the NIR irradiation-treated GDY were like those of AMP. Graphdiyne exhibited antibacterial properties for AMP-resistant bacteria (killing rate\u0026thinsp;\u0026gt;\u0026thinsp;75%). Graphdiyne NPs achieved a high rate of antibacterial effects under 49.5℃ depending on the damage to the membrane of bacteria and potential oxidative stress response and these might strengthen photothermal bactericidal effects. Consequently, the different killing effects on various bacteria correlated with the proliferation model and structure of the bacterial membranes. However, the inhibitory zone experiments cannot prove the diffusion effects of the PEGDA-GDY/G@TGFβ hydrogels on bacteria [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTGF-β is an important cytokine in the induction of endothelial-mesenchymal transition and plays a key role in wound healing [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. For example, TGF-β acts as a chemotactic protein of fibroblasts [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The expression of TGF-β receptors by fibroblasts involved in the wound healing has been examined in normal and healed skin, particularly in healing scars [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In this study, TGF-β was innovatively loaded into GDY/G NPs to be delivered \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. The groups loaded with TGF-β promoted cell migration \u003cem\u003ein vitro\u003c/em\u003e and the wound healing rate of PGGT-NIR(+) was the highest, consistent with previously reported findings [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Reactive oxygen species production (induced by a high-glucose environment) modulates TGF-β signaling through different pathways, including the SMAD pathway [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Activated TGF-β may increase ROS production and suppress antioxidant enzymes, forming a vicious cycle in many fibrotic diseases [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. However, this cycle may be beneficial for the fibrosis of diabetic wounds in the early stages of the disease and angiogenesis in the later stages, which was confirmed in the cell migration tests and tube formation tests.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe designed and fabricated PEGDA-GDY/G@TGFβ hydrogels with NIR-controlled drug release and antibacterial effects. Moreover, PEGDA-GDY/G@TGFβ hydrogel exhibited the ability to promote cell migration and angiogenesis when applied as a wound dressing for diabetic wounds. This study also suggested the possibility of combining GDY and gelatin as a promising carrier for cell factors and confirmed the sensitivity of GDY NPs in photothermal effects. Most importantly, the excellent properties of PEGDA-GDY/G@TGFβ hydrogels made them suitable for further application in treatment of wounds in clinics.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eGDY, graphdiyne\u003c/p\u003e\n\u003cp\u003eiNOS, inducible nitric oxide synthase\u003c/p\u003e\n\u003cp\u003eNIR, near-infrared\u003c/p\u003e\n\u003cp\u003eNP, nanoparticle\u003c/p\u003e\n\u003cp\u003ePDI,\u0026nbsp;polymer dispersity index\u003c/p\u003e\n\u003cp\u003ePEGDA, polyethylene glycol diacrylate\u003c/p\u003e\n\u003cp\u003eROS, reactive oxygen species\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTGF-\u0026beta;, transforming growth factor \u0026beta;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ. H., J. J. and X. W. contributed equally to this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJinfei Hou: Conceptualization, Data curation, Investigation, Methodology, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Junjin Jie: Conceptualization, Data curation, Formal analysis, Methodology, Writing \u0026ndash; original draft. Xinwei Wei: Data curation, Methodology. Xiangqian Shen: Data curation, Methodology. Qingfang Zhao: Methodology, Software. Xupeng Chai: Data curation, Methodology. Hao Pang: Projection administration, Resource. Zeren Shen: Methodology, Formal analysis. Jinqiang Wang: Data curation, Methodology. Linping Wu: Conceptualization, Methodology, Visualization. Jinghong Xu: Conceptualization, Supervision, Visualization, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH. acknowledges financial support fromthe National Natural Science Foundation of China (No. 82302833) and the Young Foundation of the First Affiliated Hospital, Zhejiang University School of Medicine (No. B22127). L-P. Wu. acknowledges financial support from the National Key R\u0026amp;D Program of China (No.2019YFA0110500), the Key Science and Technology Project of Guangzhou City (No.2023B03J1231) and the Guangdong Pearl River Talents Program (No. 2017GC010411). S.Z. acknowledges financial support from Zhejiang Provincial Natural Science Foundation of China (No.LQ22H150005).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelevant studies were carried out with the approval of the National Institute of Health\u0026rsquo;s Guidelines for the Care and Use of Laboratory Animals and in accordance with the Consent Form for Ethical Review of Animal Experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQiao Y, Ma F, Liu C, Zhou B, Wei Q, Li W, Zhong D, Li Y, Zhou M. 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J Nanobiotechnology. 2022, 20: 213.\u003c/li\u003e\n\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":"graphdiyne, photothermal-responsive, transforming growth factor β, diabetic wounds","lastPublishedDoi":"10.21203/rs.3.rs-4226321/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4226321/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The treatment of diabetic wounds remains a major clinical challenge owing to bacterial infection, defects in angiogenesis, and the corresponding inhibition of cell activity and extracellular matrix deposition. In this study, a core-shell-type nanosystem was developed using graphdiyne (GDY) nanoparticles covered with gelatin to investigate its effects on diabetic wound healing. The nanoparticles were loaded with transforming growth factor β (TGF-β) via electrostatic self-assembly to promote angiogenesis and cell migration. The photothermal effects of GDY nanoparticles were applied to achieve controllable drug release and antibacterial properties. This nanosystem could rapidly release TGF-β after irradiation by near-infrared rays (NIR) without damaging its biological activities. The associated photothermal antibacterial activity was observed after 30 seconds irradiation of nanoparticles, and the temperature was set at a safe range (\u003c49.6 °C). Besides, the gels possessed good biocompatibility and promoted cell migration in vitro. After implantation, the hydrogels group showed a higher wound healing rate than the control group in diabetic wound mouse models after 14 days and exhibited evident tissue regeneration, including angiogenesis and extracellular matrix deposition. This study presents a method for fabricating antibacterial wound dressings and an effective NIR-response strategy for designing drug-delivery nanosystems loaded with cellular factors.","manuscriptTitle":"A Core-Shell-Type Nanosystem Promotes Diabetic Wound Healing Through Photothermal-Responsive Release of Transforming Growth Factor β","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-12 17:10:37","doi":"10.21203/rs.3.rs-4226321/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-04-26T12:31:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-26T07:50:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-16T05:39:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8b836828-829c-450e-9dea-f5121f9daab9","date":"2024-04-12T04:39:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"394777e0-13c2-4fc0-a196-1c2a48bc16e9","date":"2024-04-12T01:04:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c6040d16-b81a-4db7-ad92-a7ca521236c1","date":"2024-04-11T21:04:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58d06135-5793-445f-a374-d0ef74dfdeaf_SNPRID","date":"2024-04-11T18:08:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-11T18:04:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-09T19:52:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-08T17:39:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2024-04-06T07:48:46+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":"7a0fec2e-0257-480e-a751-4e38eed23c7a","owner":[],"postedDate":"April 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-05T16:06:18+00:00","versionOfRecord":{"articleIdentity":"rs-4226321","link":"https://doi.org/10.1186/s12951-024-02675-2","journal":{"identity":"journal-of-nanobiotechnology","isVorOnly":false,"title":"Journal of Nanobiotechnology"},"publishedOn":"2024-07-30 15:58:08","publishedOnDateReadable":"July 30th, 2024"},"versionCreatedAt":"2024-04-12 17:10:37","video":"","vorDoi":"10.1186/s12951-024-02675-2","vorDoiUrl":"https://doi.org/10.1186/s12951-024-02675-2","workflowStages":[]},"version":"v1","identity":"rs-4226321","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4226321","identity":"rs-4226321","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}