Responsive hydrogel modulator with self-regulated polyphenol release for accelerating diabetic wound healing via precise immunoregulation

Abstract

Nonhealing chronic wounds are intractable clinical complications of diabetes and are characterized by high protease activity, severe oxidative stress and sustained inflammatory response. In this case, the development of functional hydrogel dressings to modulate the immune microenvironment is a well-known strategy, where the precise stimuli-responsive and spatiotemporally controlled release of bioactive molecules remains a huge challenge. Herein, we developed responsive hydrogels with self-regulated bioactive molecule release based on the protease activity in diabetic wound sites, to serve as a smart immune microenvironment modulator for accelerating wound healing. The hydrogels were fabricated by grafting oxidized hyaluronic acid with epigallocatechin-3-gallate (EGCG) and gelatin methacryloyl (GelMA) under UV irradiation. Resveratrol nanoparticles were further loaded into the hydrogels before gelation to construct a polyphenol delivery system. The prepared hydrogels could achieve the on-demand release of polyphenol upon degradation by protease, as confirmed via degradation and polyphenol release experiments. The released polyphenol was demonstrated to have the capacity to effectively scavenge excessive free radicals, promote macrophage polarization, reduce proinflammatory factor (TNF-α) expression and augment anti-inflammatory factor (IL-10) expression in vitro. Additionally, in vivo rat wound healing model experiment results confirmed that these hydrogels promoted collagen deposition and granulation tissue regeneration, accelerating diabetic wound healing. Based on the protease-responsive degradation characteristic of the hydrogels and high protease activity in the diabetic wound microenvironment, hydrogels with exquisite polyphenol release controllability are promising candidates as dressings for diabetic wound management.

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1. Introduction

Owing to the gradually increasing prevalence of diabetic mellitus worldwide, complications such as diabetic wounds and cardiovascular diseases are unfavorably affecting the quality of life of the diabetic population.¹ Compelling evidence has demonstrated that several million individuals struggle with diabetes, with up to 20% suffering from poor chronic wounds, and even facing the risk of amputation.^2,3 However, there is still an unsatisfied need in terms of diabetic wound management. It has been widely recognized that severe oxidative stress, persistent inflammation and macrophage dysfunction in the high-glucose diabetic microenvironment hinder wound healing.^4–7 Besides, another distinct physiological characteristic in diabetic wounds is the high expression of matrix metalloproteinases (MMPs).^8,9 The activity of multiple MMPs is boosted under the influence of persistent inflammatory response and oxidative stress, which results in a persistently degrading extracellular matrix (ECM), impeding collagen deposition and eventually delaying wound healing.^10,11 These instructive clues indicate that remodeling the diabetic microenvironment is essential for wound management.

Polyphenols composed of abundant phenolic hydroxyl groups and other active chemical groups possess diverse biological activities, such as anti-oxidant and anti-inflammatory capacities.^12,13 Polyphenols were demonstrated to have the capacity to scavenge excess free radicals, alleviate the inflammatory response and modulate macrophage polarization.^14–16 Nevertheless, their drawbacks including inadequate dispersion, unsatisfactory bioavailability and undesirable chemical stability limit their application in the field of diabetic wound management.^17,18 Therefore, based on the premise of improving these shortcomings and the therapeutic effect, polyphenol delivery systems show potential for diabetic wound care.

Hydrogel dressings with a three-dimensional architecture that is similar to the ECM's have attracted increasing attention for application in wound management owing to their characteristic of keeping wounds moist and tissue exudate removal.^19,20 Thus, polyphenol delivery systems based on hydrogels are expected to accelerate diabetic wound healing via immunoregulation.²¹ However, using this strategy, it is difficult to achieve the spatiotemporally precise release of polyphenols to persistently alleviate the inflammatory response and oxidative stress. In comparison, smart hydrogels with stimuli-responsive behavior are capable of responding to intercellular signals such as pH, enzyme and glucose in the complex microenvironment, which is highly desirable in the field of diabetic wound management.^22–24 Gelatin methacryloyl (GelMA) retains the features of gelatin such as good biocompatibility and moisturizing effect and can be crosslinked to form hydrogels with an irregular shape to perfectly fit wounds under external photo-stimulation.^25,26 Besides, a GelMA hydrogel with proteolytic degradability was capable of responding to the MMPs at the diabetic wound site.^27,28 Based on this trait, we propose the hypothesis that introducing polyphenols into GelMA hydrogels for the development of protease-responsive hydrogels will achieve precise polyphenol release to modulate the immune microenvironment in diabetic wounds.

In this study, we constructed protease-responsive hydrogels to achieve the spatiotemporally controlled release of polyphenols, tackling the challenges of high protease activity, severe oxidative stress and sustained inflammatory response at diabetic wound sites. For this purpose, hyaluronan with excellent water retention and immunoregulation properties, as one of the constituents of ECM,²⁹ was grafted with EGCG (Fig. 1A). Secondly, the water-insoluble resveratrol was synthesized as nanoparticles via the Mannich condensation reaction (Fig. 1B), and finally the nanoparticles were added to prepolymers of the obtained EGCG–hyaluronan conjugates and GelMA to prepare functional hydrogels (Fig. 1C). The protease-stimuli degradation behavior of the obtained hydrogels and their corresponding polyphenol release profile were probed (Fig. 1D). Subsequently, the antioxidant and anti-inflammatory properties of the dual polyphenol release system containing EGCG and resveratrol in vitro were investigated (Fig. 1E). Eventually, a streptozotocin-induced rat diabetic wound defect model was created to verify the potential of the hydrogel in wound healing (Fig. 1F).

Fig. 1 Schematic of the fabrication and evaluation of responsive hydrogels. Diagram showing the preparation of (A) OHA–EGCG conjugates via chemical grafting of EGCG on OHA polymers; (B) preparation of resveratrol nanoparticles via nano-assembling water-insoluble resveratrol; (C) development of a functional hydrogel with polyphenol release system composed of EGCG and resveratrol; (D) assessment of hydrogel degradation and corresponding polyphenol release behavior; (E) evaluation of antioxidative and anti-inflammatory properties of the polyphenol release system; (F) investigation on the effect of hydrogels with responsive polyphenol release behavior on diabetic wound healing of rats.

2. Materials and methods

2.1. Chemicals

Hyaluronic acid (MW = 1700–2000 kDa) was acquired from Bloomage BioTechnology Co., Ltd (Jinan, China). Sodium periodate was purchased from Kelong Chemical Reagent (Chengdu, China). Alamar blue cell viability assay reagent, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and phorbol 12-myristate 13-acetate (PMA) were purchased from Solarbio Science & Technology Co., Ltd (Beijing, China). Cell Counting Kit-8 was acquired from Shandong Sparkjade Biotechnology Co., Ltd (Shandong, China). DMEM/high glucose (SP032010500) was purchased from Sperikon Life Science & Biotechnology Co., Ltd. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were provided by Sigma-Aldrich (Missouri, USA). Streptozotocin (STZ) was purchased from Meilun Biotechnology Co., Ltd (Dalian, China). Fluorescein diacetate (FDA) and propidium iodide (PI) were purchased from Shanghai Acmec Biochemical Co., Ltd (Shanghai, China). I2959 photoinitiator was obtained from Aladdin Reagent Co., Ltd (Shanghai, China). MMP-9 protein was acquired from Abbkine Scientific Co., Ltd (Wuhan, China).

2.2. Synthesis of gelatin modified with methacrylate anhydride (GelMA)

Typically, 5.0 g gelatin was dissolved in 50 mL PBS solution (pH = 7.4) at 60 °C under magnetic stirring. Then, 5 mL methacrylate anhydride was pipetted into the solution and the reaction was maintained for 3 h in the dark. The obtained solution was diluted with 100 mL PBS after termination of the reaction, dialyzed in ultrapure (UP) water for 7 days, and finally freeze-dried.

2.3. Synthesis of oxidized hyaluronic acid-polyphenol (OHAP) conjugates

The synthesis of HA endowed with aldehyde groups was described in our previous study.³⁰ Briefly, 2.0 g HA was dissolved in 20 mL UP water with mechanical stirring, and subsequently 2.3 g sodium periodate was added to the solution after HA was thoroughly dissolved. The oxidation process of HA was performed for 24 h in the dark, and then the 5 mL ethylene glycol was pipetted into the solution to end the reaction. The product was dialyzed (MWCO = 14 000 Da) in UP water for 5 days to remove the remaining chemicals, and finally lyophilized to obtain oxidized HA (OHA) sponge.

The method for the preparation of OHAP was based on reported studies.^31,32 Briefly, 2.0 g OHA was dissolved in 20 mL UP water and 0.2 g EGCG was dissolved in a mixed solution (6.8 mL UP water/6.0 mL DMSO/7.2 mL acetic acid), respectively. These two solutions were mixed in a 100 mL flask with mechanical stirring for 48 h. The reaction was processed in the dark and kept at the temperature of 40 °C. The obtained product was sealed in a dialysis bag (MWCO = 14 000 Da) and immersed in UP water containing 15% DMSO for 3 days. Afterwards, the product was transferred to UP water and dialyzed for 5 days. Finally, the polymer of OHAP was lyophilized and stored at 4 °C in the dark.

2.4. Preparation of GelMA-OHAP (GOP) hydrogel

GelMA was dissolved in I2959 photoinitiator solutions (0.5 wt%). The prepared GelMA (6.0 wt%) and OHAP (5 wt%) solutions were mixed in the volume ratio of 20 : 1. The GOP hydrogel was obtained after the mixture was irradiated for 20 s with a 365 nm ultraviolet point light curing device.

2.5. Preparation of resveratrol-loaded GelMA/OHA–EGCG composite hydrogel

2.5.1. Synthesis of resveratrol nanoparticles. Resveratrol (150 mg) and 3-aminophenylboric acid (100 mg) were dissolved in 100 mL ethyl alcohol, and then 200 μL methanal was added into the mixture. The reaction was maintained in the dark at room temperature with continuous stirring for 24 h. The solution was concentrated to 10 mL, and subsequently mixed with ultrapure (UP) water in the volume ratio of 1 : 1. The obtained product was kept in the dark for 24 h to form resveratrol nanoparticles. The resveratrol nanoparticles were collected by centrifugation and rinsed with UP water three times to remove the unreacted feedstock, and finally stored at 4 °C until use.

2.5.2. Preparation of functional hydrogels with polyphenol release system. GelMA (6.0 wt%) was dissolved in I2959 photoinitiator solutions (0.5 wt%). The prepared GelMA and OHAP (5 wt%) solutions were mixed in the volume ratio of 20 : 1. Then, 150 μL hydrogel precursor containing 10 μg or 20 μg resveratrol nanoparticles was UV-irradiated under the same conditions to obtain the PGOP1 and PGOP2 hydrogels, respectively.

2.7. Characterization of resveratrol nanoparticles

The chemical structure of the resveratrol nanoparticles and corresponding polyphenol content in the nanoparticles were analyzed and measured by UV spectroscopy. The size distribution of NPs was determined using a Zetasizer Instrument ((Malvern Instruments). The microstructure of the resveratrol nanoparticles was captured by scanning electron microscopy (Hitachi S-4800, Japan).

2.8. Physicochemical properties characterization of hydrogels

Structure analysis. The alteration in the chemical structure of the feedstocks before and after gelation was assessed by Fourier transform-infrared spectroscopy (Invenio R, Bruker) and ¹H NMR spectroscopy (Avance II-600 MHz, Bruker).

Micromorphology observation. The microstructure of the prepared hydrogels was observed by scanning electron microscopy (Hitachi S-4800, Japan).

Rheological measurement. The rheological behaviors of the hydrogels were appraised using a rheometer (MCR 302, Anton Paar) combined with a 365 nm ultraviolet point light curing device. A time sweep measurement was adopted to probe the gelation kinetics of the hydrogels under the stimulation of UV-irradiation. Frequency sweep and amplitude sweep tests were carried out to investigate the mechanical stability of the hydrogels.

Water absorption evaluation. 150 μL of hydrogel precursor was pipetted into a cylindrical mold and gelled after UV-irradiation. The initial weight of the resultant hydrogels (W₀) was recorded. The weight of the hydrogels immersed in the PBS solution was also measured at different timepoints (W_t) after the water on their surface was removed. The swelling property of the hydrogels was estimated using the ratio of W_t to W₀.

Degradation behavior investigation. The cylindric PGOP2 hydrogel (Φ = 10 mm, h = 5 mm) was prepared after UV-irradiation. The hydrogels were placed in tubes containing 3 mL PBS solution and 20 μL trypsin solution (0.25 wt%). The tubes were subsequently kept on a shaker at 150 rpm and 37 °C. The hydrogels treated with 3 mL PBS solution were set as the control group. The hydrogels were collected at different timepoints and freeze-dried. The weight of the obtained samples was recorded. The remaining mass percent of hydrogel was defined as the ratio of the weight measured at different timepoints (W_t) to the weight measured before the test.

In vitro polyphenol release study. Cylindric hydrogels (Φ = 10 mm, h = 5 mm) were prepared for the polyphenol release experiment. The hydrogels were placed in tubes containing 3 mL PBS solution and 20 μL trypsin solution (0.25 wt%). The hydrogels were immersed in 3 mL MMP-9 solution (300 ng mL⁻¹) in another experiment. These hydrogels were kept on a shaker at 150 rpm and 37 °C. 350 μL of protease solution was added to the tubes after the equal supernatant was collected at different timepoints. The polyphenol content released from the hydrogels was measured based on the protocol of the Folin-phenol reagent method. In brief, 0.3 mL of collected solution was mixed with 1.5 mL freshly prepared Folin-phenol reagent (10 v/v%). After 5 min, 1.2 mL of Na₂CO₃ solution (7.5 w/v%) was added to the obtained mixture, and subsequently the mixed solution was placed in the dark for 30 min. The concentration of polyphenol was measured at 765 nm based on an established calibration curve. At the same time, the polyphenol release from the hydrogels treated with PBS solution was also evaluated.

Free radical scavenging capacity assessment. 300 μL of hydrogel precursor was pipetted into a small bottle, and subsequently gelled by UV-irradiation. 5 mL of ABTS solution (0.35 mM) and DPPH solution (0.3 mM) were added to the small bottles, respectively. At set timepoints, 100 μL of solution was pipetted into a 96-well plate and then 80% methanol solution with an equal volume was supplemented into the small bottle. Besides, photographs were captured to observe the color change to evaluate the free radical scavenging capacity of the hydrogels.

2.9. Cytocompatibility evaluation of hydrogels

The influence of the obtained hydrogels on cell proliferation and spreading was investigated. The cell viability of L929 cells treated with OHAP and resveratrol nanoparticles for 24 h was measured via the CCK-8 assay. On the first and fifth day, the cell proliferation activity of L929 cells cultured with hydrogel extracts at varying concentrations was assessed using the protocol of the Alamar blue assay. Besides, the cells was stained by FDA/PI reagent and their morphology was observed using a laser scanning confocal microscope (LSCM) at set timepoints (LSM 880, Zeiss). The influence of the degradation and products released from the hydrogel treated with MMP-9 on the cell viability of L929 cells was also investigated using the CCK-8 assay.

2.10. Antioxidant and anti-inflammatory activity of hydrogels

2 mL mouse mononuclear macrophages cell (RAW 264.7) suspension at a density of 1.0 × 10⁵ was added into the lower chamber of a Transwell plate. Sterilized cylindric GOP and PGOP hydrogels (Φ = 2.5 cm, h = 3 mm) were placed in the upper chamber after the cells adhered on the surface of the plate. Also, 1 mL of DMEM medium was pipetted into the upper chamber to immerse the hydrogels. The cells were co-cultured with the hydrogels for 24 h. Afterwards, 8 μL PMA solution (1 mg mL⁻¹) was added into the lower chamber to induce an increase in the ROS level in the RAW 264.7 cells. The medium in the lower chamber was replaced by fresh medium containing DCFH-DA (10 μM) after the stimulation of PMA with the cells for up to 2 h. The cells were incubated in the dark for 30 min, rinsed with PBS, and then observed on an LSCM (LSM 880, Zeiss). Besides, the cells treated with the same procedure were collected in tubes, and subsequently their ROS level was detected using a flow cytometer.

The antioxidant property of the hydrogels treated with MMP-9 was also investigated. Firstly, the cylindric hydrogels (Φ = 10 mm and h = 5 mm) were immersed in MMP-containing (300 ng mL⁻¹) DMEM medium for 12 h, while the hydrogels treated with DMEM medium were set as the control group. The hydrogel extracts were used to culture RAW 264.7 cells. The cells were treated using the aforementioned procedure, and then the fluorescence intensity related to ROS was measured with a flow cytometer.

2 mL RAW 264.7 cells at a density of 5.0 × 10⁴ were seeded in the lower chamber of a Transwell plate. Afterwards, 400 ng LPS and 40 ng IFN-γ were combined to stimulate RAW 264.7 cells. The GOP and PGOP hydrogels were added to the upper chamber after the incubation of LPS and IFN-γ with the cells for up to 12 h. The cells were collected in the tubes after culturing for 24 h, and then the macrophage phenotype was detected using a flow cytometer. Besides, q-PCR experiments were also carried out to evaluate the immunoregulation effect of polyphenol on RAW 264.7 cells.

2.11. Diabetic wound healing and histological analyses

The animal experiments were processed according to the institutional guidelines of Sichuan University and approved by the ethics committee of the university. SD rats (ca. 220 g) were provided by the Laboratory Animal Centre of Sichuan University (No. SCU44-250-02). The diabetic rat model was created by two intraperitoneal injections of STZ solution with a dose of 40 mg kg⁻¹. Afterwards, the rats were adaptively fed for a week, and then were randomly divided into four groups. After the treatment of anesthetization, the back hair of the rats was shaven, and their exposed skin tissues were wiped with iodophor and ethyl alcohol. Circular wounds (Φ = 1 cm) were created on the back of the rats, and 150 μL hydrogel prepolymer was added to the wound and gelled under UV-irradiation. The wounds were treated with medical dressings to avoid the movement of the hydrogels. The wound areas were captured and measured via ImageJ. The tissues at the wound site were collected on the 13th day and stained with H&E, Masson trichrome staining and immunofluorescent staining.

2.12. Statistical analysis

The obtained results are presented as mean ± SD and analyzed by One-way ANOVA. P < 0.05 was considered to have a statistical significance.

3. Results and discussion

3.1. Synthesis and characterization of nanoparticles and hydrogels

Hyaluronan was endowed with aldehyde groups via oxidation treatment in the presence of sodium periodate (Fig. 2A). The alteration in the chemical structure of hyaluronan was verified by ¹H NMR measurement. In the ¹H NMR spectra, new peaks appeared in the range of 4.9 to 5.1 ppm, indicating that the aldehyde group on OHA formed hemiacetal proton bonding with the adjacent hydroxyl group, which confirmed the successful synthesis of OHA (Fig. 2B). Subsequently, EGCG was introduced in OHA solution to fabricate OHAP in the presence of acid condition (Fig. 2C). Compared with OHA, the peak intensities in the range 4.9 to 5.1 ppm were decreased in the spectrum of OHAP (Fig. 2B), which resulted from the phenolic reaction between EGCG and OHA. Besides, the peak located at 1730 cm⁻¹ can be attributed to the aldehyde groups of OHA after the oxidation treatment or the carbonyl group of EGCG, as shown in FT-IR spectra of OHAP. The characteristic absorptions of EGCG at 1635 cm⁻¹, 1536 cm⁻¹ and 1453 cm⁻¹ were observed in the FT-IR spectrum of OHAP.³³ Besides, the characteristic absorption peak of EGCG at 280 nm was observed in the ultraviolet spectrum of OHA–EGCG (Fig. S1, ESI†). These results suggest that EGCG was grafted on the OHA polymers. Based on the modular assembly platform developed by our group,^34,35 the water-insoluble resveratrol turned into resveratrol nanoparticles under the effect of aldehyde groups and amino groups (Fig. 2E). The absorption peaks of resveratrol and corresponding nanoparticles were approximately constant in their UV spectra (Fig. S2, ESI†), showing that resveratrol was successfully introduced into the nanoparticles. The average size of the spherical resveratrol nanoparticles was about 334 nm with a PDI of 0.08 (Fig. 2F and G). Furthermore, FT-IR spectra were recorded to analyze the chemical structure of the prepolymers and hydrogels. In the spectrum of GelMA, the bond at 1630 cm⁻¹ was attributed to the stretching vibrations of C=O and C–N, and the absorption of 1542 cm⁻¹ was assigned to the N–H bending vibration and C–H stretching vibration. The ¹H NMR spectra of GelMA and gelatin were also recorded. In comparison with gelatin, new peaks appeared at 5.4 and 5.58 ppm in the GelMA spectrum, which were attributed to the methacrylate groups. Besides, the intensity of the peak for lysine-NH₂ (2.92 ppm) in the spectrum of GelMA obviously decreased due to the graft of MA on the lysine-NH₂ groups (Fig. S3, ESI†), further indicating of successful synthesis of GelMA. The substitution degree in GelMA was about 65.76% based on the analysis of the ¹H NMR spectra. These results indicate that the gelatin was modified with methacrylate. In comparison, the characteristic absorptions of GelMA and OHA were diminished in the spectrum of the hydrogel, demonstrating the occurrence of photo-crosslinking reaction and potential Schiff-base reaction. Besides, the alteration of the liquid prepolymers into a solid hydrogel under UV irradiation via macroscopic observation also indicates the chemical crosslinking. In summary, the nanoparticles and hydrogel prepolymers were successfully synthesized via the analysis of their chemical structure.

Fig. 2 Chemical structure analysis of hydrogel prepolymers and nanoparticles. (A) Schematic of the fabrication of OHA and (B) the corresponding ¹H NMR spectra. (C) Diagram showing the synthesis of OHA–EGCG conjugates; (D) FT-IR spectra of the fabricated prepolymers and hydrogel; (E) diagram showing the development of resveratrol nanoparticles; (F) statistic size distribution with Zetasizer instrument; (G) microstructure observation of resveratrol nanoparticles; (H) observation of sol–gel transition under UV irradiation.

3.2, Rheological properties of hydrogels

The gelation kinetics of the hydrogels were evaluated via time sweep measurements. As shown in Fig. 3A–C, the loss modulus value (G′′) was greater than the corresponding storage modulus value (G′) of the hydrogels in the first two minutes. After exposure of the hydrogel prepolymers to UV irradiation, the polymerization reaction of GelMA and intertwining between the polymers prompted the formation of a three-dimensional network. As a result, the G′ and G′′ values of the hydrogels increased rapidly within several seconds. Given that the increasing rate of G′ was higher than that of G′′, the sol–gel transformation of the hydrogels was completed quickly.³⁶ The G′ value decreased with an increase in the concentration of resveratrol nanoparticles (Fig. S4, ESI†). This is attributed to the fact that the nanoparticles scavenged certain free radicals generated by the photoinitiator, thus reducing the crosslinking of the hydrogels.³⁷ Besides, frequency sweep measurements were performed to investigate the change in modulus with a variation in frequency (Fig. 3D–F). The G′ value was higher than the corresponding G′′ value in the frequency range of 1–100 Hz. The G′ showed a negligible dependence on frequency during the whole test, indicating the mechanical stability of the prepared hydrogels.³⁰ During the strain sweep measurement, the G′ and G′′ values of the hydrogels remained roughly stable with an increase in strain (Fig. 3G–I). An intersection point was not formed in the curves at high strain, suggesting that the network in these hydrogels was not disrupted. Thus, the prepared hydrogels with suitable mechanical properties are capable of fitting the skin as a wound dressing and ensure the peristalsis of wounds.

Fig. 3 Rheological measurements to evaluate the gelation kinetics and mechanical stability of the prepared hydrogels. (A)–(C) Gelation kinetic curves of GOP, PGOP1 and PGOP2 hydrogels under UV stimulation. (D)–(F) Frequency sweep and (G)–(I) strain sweep curves of the fabricated hydrogels.

3.2. Physicochemical properties of hydrogels

The microstructure of the hyaluronan-based hydrogels was observed with a scanning electron microscope. All the hydrogels exhibited a honeycomb-like porous structure (Fig. 4A). The introduction of resveratrol nanoparticles into the GOP hydrogel prepolymers altered their morphology. The pore size of the obtained PGOP hydrogels was greater than that of the GOP hydrogel. It was speculated that the resveratrol nanoparticles eliminated some of free radicals generated by the photoinitiator, resulting in a decrease in the crosslinking density.³⁷ Subsequently, the swelling property of the fabricated hydrogels was investigated. As depicted in Fig. 4B, all the hydrogels showed fast water absorption behavior at the beginning of the measurement. With the extension of time, the swelling rate of the prepared hydrogels became mild. Finally, GOP, PGOP1 and PGOP2 approximately reached the swelling equilibrium state at 10 h. In comparison, the GOP hydrogel exhibited the weakest water absorption behavior. After the resveratrol nanoparticles were introduced in the hydrogels, their swelling ratio improved. Also, the water absorption capacity was positively connected with the content of nanoparticles in the hydrogels. This resulted from the fact that the crosslinking density decreased with an increase in the concentration of the nanoparticles, making it easier for water to infiltrate the pore structure of the hydrogels. According to the above-mentioned experiments, the fabricated polysaccharide composite hydrogels presented good water absorption and water retention capacity, which are conductive to keeping the environment moist and removing the exudate in wounds.³⁸

Fig. 4 Evaluation of the physicochemical properties of hydrogels. (A) SEM images of GOP, PGOP1 and PGOP2 hydrogels at a magnification of 200×; (B) swelling property assessment of hydrogels; (C) degradation behavior and polyphenol release evaluation of hydrogels treated with (D) PBS and (E) protease solution; (F) and (H) ABTS and (G) and (I) DPPH free radicals scavenging capacity investigation of hydrogels.

Besides, due to the high expression of matrix metalloproteinase at diabetic wound sites, the degradation behavior of the hydrogels treated with protease solution was evaluated. Given that the main constituent of the prepared hydrogels was the same, the PGOP2 hydrogel was chosen to investigate the effect of protease on its degradation. During this experiment, the PGOP2 hydrogel treated with PBS solution was set as the control group. The remaining mass percent of hydrogel immersed in PBS solution decreased from 96.84% at 12 h to 82.35% at 48 h (Fig. 4C). In comparison, the PGOP2 hydrogel exhibited fast degradation behavior under the protease treatment conditions. The remaining mass percent reduced from 79.83% at 12 h to 18.06% at 48 h (Fig. 4C). The obvious difference was attributed to the responsiveness of GelMA on protease. Furthermore, the polyphenol release behavior from the prepared hydrogels was probed. The GOP hydrogel presented negligible release behavior in PBS solution (Fig. 4D), while the cumulative polyphenol release was less than 10% in the PGOP hydrogels, indicating the slow release of polyphenol from the hydrogels immersed in PBS solution. By contrast, the hydrogels treated with protease solution exhibited fast polyphenol release, and the amount of released polyphenol was much more than that treated with PBS solution (Fig. 4E and Fig. S5, ESI†). The results can be due to the fact that protease initiated the degradation of hydrogels,³⁹ promoting the release of polyphenol. Furthermore, the effect of OHA–EGCG conjugates on modulating the release of polyphenol was investigated. The PGOP2* hydrogel was fabricated with equal EGCG and resveratrol nanoparticle contents to that in the PGOP2 hydrogel. In the PGOP2* hydrogel formulation, EGCG was simply added to the hydrogel precursors, but not grafted on the OHA chain. As shown in Fig. S6 (ESI†), the cumulative release of polyphenol in the PGOP2* group was higher than that in the PGOP2 group, whether the hydrogels were treated with PBS or protease solution. These results suggested that chemical grafting of EGCG on OHA contributed to modulating the release rate of polyphenol, and thus has potential to achieve persistent immunoregulation in diabetic wound management.

Furthermore, the free radical scavenging activities of the hydrogels on ABTS and DPPH were evaluated. The hydrogels exhibited free radical scavenging capacity in the first 0.5 h (Fig. 4F and H). However, with prolonged time, the capacity of the hydrogel to scavenge the excessive ABTS and DPPH was weakened. After the addition of protease solution at 1.5 h, the ABTS and DPPH free radical levels were significantly reduced at 2.0 h (Fig. 4F–I and Fig. S7, ESI†), indicating that protease could promote polyphenol release and had a negligible effect on the activity of polyphenol to scavenge free radicals. More importantly, the free radical contents decreased at 3.0 h in the GOP and PGOP1 hydrogels. However, in the PGOP2 hydrogel, the free radical level was the lowest at 3.0 h and showed no significant difference in comparison with that at 2.5 h (Fig. 4F–I and Fig. S7, ESI†), demonstrating the fast free radical scavenging capacity of the PGOP2 hydrogel. In summary, the prepared hydrogels showed protease-responsive polyphenol release behavior to scavenge free radicals, promising to continuously modulate the inflammatory microenvironment of diabetic wounds.

3.4. Cytocompatibility of hydrogels

The influence of the hydrogel precursors and hydrogel extracts on cell viability was investigated. The cell viability of the cells decreased with an increase in the concentration of free EGCG, which was ascribed to the damage caused by the high bioactivity of EGCG to cells.⁴⁰ It is worth noting that the cell viability of the L929 cells treated with HA–EGCG conjugates and resveratrol nanoparticles at different concentrations reached up to 90% (Fig. 5A and Fig. S8A, ESI†), indicting the good cytocompatibility of the HA–EGCG conjugates and nanoparticles. Besides, the effect of the hydrogels on cell proliferation and spreading in vitro was investigated using Alamar blue assays and cell live/dead staining. The fluorescence intensity of the cells cultured with the PGOP2 hydrogel extract increased with the incubation time. The concentration of hydrogel extract showed no significant difference in cell fluorescence intensity on the first, third and fifth days (Fig. 5B). Besides, the cell viability of the L929 cells cultured using the PGOP2 hydrogel extract obtained from the MMP-treated hydrogel reached up to 90% (Fig. S8B, ESI†). These results demonstrate that the prepolymers and hydrogel have no toxicity to cells. Furthermore, cell live/dead staining was performed at different timepoints to further verify the cytocompatibility of the hydrogels. The green fluorescence increased with time, and slight red fluorescence was present in all the groups (Fig. 5C), suggesting the favorable cellular affinity of the hydrogels. These collective results confirmed that the prepared hydrogel is capable of supporting cellular spreading and proliferation.

Fig. 5 Cytocompatibility evaluation of hydrogels on L929 cells. (A) Effect of HA–EGCG with varying concentration on cell viability. (B) Cell proliferation measurement through Alamar blue assay to probe the influence of different concentrations of PGOP2 hydrogel extract on cells growth. (C) Cell morphology observation via FDA/PI staining to further assess the cytocompatibility of the hydrogels.

3.5. The antioxidant activity of hydrogels

The excess intracellular ROS levels in cells caused by a high concentration of glucose in diabetic rats would delay the process of wound healing. In this case, the hydrogel with antioxidant activity is beneficial for promoting diabetic wound healing. In this study, the intracellular ROS scavenging property of the prepared hydrogels was investigated via DCFH-DA fluorescent probe measurement. As shown in Fig. 6A, there was little green fluorescence in the negative control group; in comparison, the RAW 264.7 cells in the positive control group exhibited an increase in fluorescence density. The results demonstrate that the intracellular ROS level in the cells was significantly improved with the treatment of PMA. The green fluorescence density was reduced after the PMA-simulated RAW 264.7 cells were cocultured with these polyphenol-containing hydrogels (Φ =2.5 cm and h =3 mm) for 24 h. It is worth noting that the intracellular ROS level in the cells in the PGOP2 hydrogel group was approximately similar to that of the normal cells. This resulted from the fact that the hydrogel with polyphenol release behavior was capable of scavenging the excess intracellular ROS. Furthermore, the flow cytometry results showed that the ROS signals of the RAW 264.7 cells gradually decreased with the hydrogel treatment in comparison with that of the positive control group (Fig. 6B–F). Also, the PGOP2 hydrogel group exhibited the lowest ROS signals (Fig. 6G), which was close to the ROS level of the normal RAW 264.7 cells, indicating that PGOP2 had superb ROS scavenging activity.

Fig. 6 Antioxidant activity of hydrogels. (A) Images of DCFH fluorescence and (B–F) flow cytometry results of RAW 264.7 cells cultured with different hydrogels under PMA stimulation to evaluate their intracellular ROS scavenging property. (G) Average fluorescence intensity of FITC in RAW 264.7 cells treated with different hydrogels.

Besides, the ROS scavenging ability of the hydrogel extracts with different treatments was investigated. The hydrogels (Φ = 10 mm and h = 5 mm) were immersed in MMP/PBS solutions for 12 h, respectively. Subsequently, the obtained hydrogel extracts were used to culture PMA-simulated RAW 264.7 cells. Lastly, the ROS signal of the cells was detected by flow cytometry. The fluorescence intensity of the cells cultured using the hydrogel extracts obtained from PBS treatment was close to that of the positive group, indicating that the excess ROS was not effectively scavenged. In contrast, the fluorescence intensity of the cells cultured using the hydrogel extracts obtained from MMP treatment was significantly reduced (Fig. S9, ESI†). These results suggest that the degradation of the hydrogel and release of polyphenol were necessary for this technology to scavenge ROS. Thus, developing hydrogels with spatiotemporally controlled release behavior is promising to relieve the oxidative stress in diabetic wound sites.

3.6. Anti-inflammatory property of hydrogels

Controlling a sustained inflammatory response in diabetic wounds is of vital significance to the healing of wound defects.⁴¹ In this study, the effect of hydrogels on LPS and IFN-γ combined-treated RAW 264.7 cells was investigated by flow cytometry. Macrophage polarization towards the M1 phenotype increased rapidly, and its proportion reached up to 88.5% after the LPS and IFN-γ treatment in the RAW 264.7 cells (Fig. 7A and B). When the stimulated RAW 264.7 cells were cultured with the hydrogels, the macrophage polarization towards the M1 phenotype decreased, and the proportion was 75.4% for GOP, 68% for PGOP1 and 53.9% for the PGOP2 hydrogel (Fig. 7C–E, respectively). In comparison with that of the positive group, the average fluorescence intensity of APC in the hydrogel groups significantly decreased (Fig. 7F), suggesting that the hydrogels were capable of inhibiting CD86 expression in M1 macrophages. Besides, the anti-inflammatory CD206 expression in the RAW 264.7 cells with different treatments in all groups was also quantitatively analyzed. The proportion of macrophage polarization towards the M2 phenotype was 21.2% for the negative group, 38.9% for the positive group, 78.2% for GOP, 85.8% for PGOP1, and 86.9% for the PGOP2 hydrogel (Fig. 7G–K, respectively). The statistical fluorescence intensity of PE presented a similar trend with the representative flow cytometry of macrophage polarization towards the M2 phenotype. The results showed that the CD206 expression was significantly enhanced in the PGOP hydrogels, indicating that the synergistic effect of HA–EGCG and resveratrol nanoparticles could effectively modulate macrophage polarization. Furthermore, the q-PCR results showed that the introduction of polyphenol was capable of reducing the RNA expression of iNOS and TNF-α, while augmenting the mRNA expression of Arg and IL-10 in comparison with the positive group. More importantly, the mRNA expression of iNOS and TNF-α in the RAW 264.7 cells treated with the PGOP2 hydrogel was several times lower than that in the GOP and PGOP1 hydrogels (Fig. 7M and N, respectively). Compared with the other hydrogels, the RNA expression of Arg and IL-10 significantly improved in the RAW 264.7 cells treated with the PGOP2 hydrogel (Fig. 7O and P, respectively). These results confirm that the synergistic effect of the HA–EGCG conjugates and resveratrol nanoparticles could effectively promote macrophage polarization, which is expected to modulate the inflammatory microenvironment in diabetic wound defects.

Fig. 7 Investigation into the anti-inflammatory property of the hydrogels. Flow cytometry results of CD86 and CD206 expression of RAW 264.7 cells treated with (A) and (G) the PBS solution, (B) and (H) LPS solution, (C) and (I) GOP hydrogel, (D) and (J) PGOP1 hydrogel and (E) and (K) PGOP2 hydrogel, respectively. Average fluorescence intensity of (F) APC and (L) PE in RAW 264.7 cells with different treatments. mRNA expression of (M) iNOS, (N) TNF-α, (O) Arg and (P) IL-10 in RAW 264.7 cells treated with the hydrogels.

3.7. The effect of hydrogels on diabetic wound healing and histology analysis

SD rats were adaptively fed for a week, and then injected with STZ twice to build the diabetic model. After GLU was above 16.7 mmol L⁻¹ persistently for three to five days, the skin defect healing experiments were carried out (Fig. 8A). As shown in Fig. 8B, the wound area decreased with the extension of time in the hydrogel groups. In comparison with the control group, the treatment of the diabetic wounds in the SD rats with the hydrogels improved the wound healing. Specifically, the relative wound area in the control group was 82.73% on the fifth day, which was higher than that of the hydrogel groups. In comparison with the control group, the wound closure rates were 77.78% for GOP, 73.33% for PGOP1, and 65.04% for the PGOP2, respectively. Furthermore, the wound areas were continuously reduced on the seventh and ninth days, and the trend of wound closure was in line with that on the fifth day. Notably, the wound of the control group had a large unhealed area, and the wound closure rate still remained 28.53% after 13 days. In contrast, the diabetic wound in the PGOP2 group was almost completely closed, and the corresponding relative wound area was 4.93%, suggesting the promotion effect of the hydrogels on wound healing. The GOPR hydrogels containing Res NPs exhibited spatiotemporally controlled release behavior, and this release rate was positively connected with the concentration of Res NPs in the hydrogels. It was speculated that the responsive polyphenol release was conductive to reducing the development of inflammation in diabetic wounds and accelerating wound healing.²⁴ As a result, the PGOP2 hydrogel exhibited a significant effect on promoting wound healing.

Fig. 8 Effect of protease-responsive hydrogels with the dual polyphenol system on accelerating diabetic wound healing. (A) Timeline of animal experiments to build the rat diabetic model and investigation of the influence of prepared hydrogels on wound healing. (B) Representative photographs of rat wounds after treatment with hydrogels at different timepoints. (C) Schematic of overlaid wound images for 13 days. (D) Statistical data for the wound closure rate at different timepoints.

The therapeutic effect of the hydrogels on wound healing was further evaluated by H&E and Masson's trichrome staining of the regenerated tissues. As shown in Fig. 9, the wound length decreased with the hydrogel treatment, and the wound length in the PGOP2 group was shorter than that in the other groups, which is consistent with the abovementioned results for the statistical analysis of the relative wound area (Fig. 8B–D). The infiltration of inflammatory cells (brown arrow) at the wounds was still obvious in the control group on the 13th day, indicting the severe inflammatory response. In comparison, this phenomenon was reduced in the other hydrogel groups, demonstrating that the released polyphenol improved the inflammatory microenvironment. Besides, abundant fibroblast cells (green arrow) presented a tightly-packed structure, and blood vessels (blue arrow) containing red cells (purple arrow) were formed at the wound treated with the PGOP2 hydrogel. Notably, the new epidermis was tightly connected to the underlying granulation tissues in the GOPR hydrogel group, whereas a loose connection between the epidermis and granulation tissues was observed in the control and GOP hydrogel groups (Fig. 9A). Besides, the epidermal thickness significantly decreased, accompanied by an increase in the granulation tissue thickness after the wound was treated with the GOPR hydrogel in comparison with that of the control group (Fig. 9B and C, respectively). Compared with the other groups, the PGOP2 group exhibited the most uniformed distribution of tight collagen fibers (red arrow), as demonstrated by Masson's trichrome staining (Fig. 9D). In summary, the regenerated wound tissues treated with the PGOP2 hydrogel were similar to the adjacent normal tissues, suggesting that the PGOP2 hydrogel significantly promoted the diabetic wound healing via the protease-responsive release behavior of polyphenol.

Fig. 9 Effect of polyphenol-containing hydrogels on accelerating diabetic wound healing. (A) H&E and corresponding statistical data for (B) epidermal thickness and (C) granulation tissue thickness of regenerated tissues on the 13th day. (D) Masson's trichrome staining of wound tissue on the 13th day. Brown arrow: inflammatory cell; purple arrow: red cell; green arrow: fibroblast cell; blue arrow: blood vessel; red arrow: collagen fibers.

Besides, immunofluorescent staining was also performed to verify the effect of polyphenol on regulating the inflammatory microenvironment in diabetic wounds. As shown in Fig. 10A, the fluorescent signal of CD86 was reduced in the PGOP hydrogel group compared with the CON and GOP groups. Furthermore, the cells treated with the PGOP hydrogels also exhibited the fluorescent signal of CD206 (Fig. 10B). The ratio of the CD206/CD86 fluorescent signals is shown in Fig. S10 (ESI†). This ratio was significantly higher in the PGOP hydrogel group than that in the CON and GOP groups. In comparison with PGOP1, the ratio of the CD206/CD86 fluorescent signals in the PGOP2 hydrogels also increased with an increase in the concentration of resveratrol nanoparticles. Thus, the results indicate that the released polyphenol was conducive to regulating macrophage polarization, accelerating diabetic wound healing.

Fig. 10 Effect of released polyphenol on the regulation of macrophage polarization. The immunofluorescent staining of wound tissue to mark (A) the macrophage M1 phenotype (CD86) and (B) macrophage M2 phenotype (CD206).

4. Conclusions

In this work, we developed responsive hyaluronan/GelMA composite hydrogels that degrade in the presence of protease to achieve the spatiotemporally controlled release of polyphenol for diabetic wound management. The prepared hydrogels exhibited a porous structure and excellent swelling capacity, which are favorable for keeping wounds moist and removing tissue exudate. Besides, the hydrogels presented soft mechanical strength and their moduli varied in the range of 100 Pa to 300 Pa, which contributed to ensuring the peristalsis of wounds. More importantly, the hydrogels showed responsive polyphenol release behavior under protease solution conditions. The released polyphenol was demonstrated to effectively reduce the ROS level and promote the transition of macrophages towards the M1 phenotype and M2 phenotype in vitro. Furthermore, the animal experiments also confirmed the superb therapeutic functions of the hydrogels on immunoregulation and healing cascades in diabetic wound care. In summary, this study proposed an appealing approach for constructing protease-responsive degradable hydrogels for the precise release of polyphenols to modulate and amplify the inflammatory response and severe oxidative stress in wound defects, promising to serve as hydrogel dressings for diabetic wound management.

Data availability

Data will be made available upon request.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work is supported by the National Natural Science Foundation of China (No. 82102214), the Fundamental Research Funds for the Central University (20826041G4189), the Postdoctor Research Fund of West China Hospital, Sichuan University (2023HXBH013) and the Sichuan Science and Technology Program (2024NSFSC1657 and 2024NSFSC1521).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb02504k

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