Sildenafil hydrogel as well as preparation method and application thereof

Abstract

The invention provides sildenafil hydrogel capable of being used for treating infectious wounds, the hydrogel is mainly composed of egg white and chitosan, sildenafil is added, and egg white and chitosan are used as raw materials for preparing the hydrogel, so that the mechanical property of the hydrogel is enhanced; the adjustable viscosity and biological activity of the materials provide a suitable wet microenvironment for wounds, and the materials have multiple functional advantages. In addition, the egg white and the chitosan become ideal choices for treating infectious wounds due to high economic benefits and safety, and the hydrogel adopting the egg white and the chitosan as the raw materials is easy to prepare and low in cost; a mouse infectious wound model is used for an in-vivo experiment, and it is verified that the hydrogel can promote healing of burn wounds by reducing inflammation, oxidative stress and bacterial load and promoting angiogenesis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to a sildenafil hydrogel and a preparation method and application thereof. Background Art

[0002] Infected wounds are local or systemic infections caused by bacteria, fungi or other microorganisms invading open wounds. They usually occur in surgical incisions, diabetic foot ulcers, pressure sores and burns. Among them, bacterial infections are the most common, mainly caused by pathogens such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). These infections not only delay the healing process, prolong hospitalization, and increase medical costs, but may also lead to serious complications, including sepsis, local tissue necrosis, and even limb loss or death, posing a major threat to the patient's quality of life and safety. The treatment of infected wounds is challenging and usually requires a comprehensive treatment strategy, including debridement, local treatment, the use of antimicrobial dressings, and systemic or local antibiotic therapy. Although these methods can control infection and promote healing to a certain extent, they still have many limitations. For example, the debridement process may cause additional tissue damage, increase pain and discomfort to patients, and the problem of antibiotic resistance has always existed.

[0003] In recent years, the application of hydrogels in the treatment of infected wounds has made significant progress. Hydrogels have many advantages, such as providing a moist healing environment, promoting tissue regeneration, and showing good biocompatibility and drug delivery properties. They can effectively release antimicrobial agents and inhibit microbial growth. However, the clinical application of hydrogels still faces some challenges, including cost, stability, and feasibility of large-scale production. Therefore, it is of great significance to develop a new type of hydrogel dressing with low cost, high efficiency, and significant therapeutic effect to treat infected wounds and promote wound healing.

[0004] In recent years, the application of sildenafil in wound healing and treatment of infected wounds has attracted much attention. The inventors tried to apply sildenafil to hydrogel to prepare a gel that can promote wound healing. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a sildenafil hydrogel and a preparation method and application thereof. The technical scheme adopted by the present invention is as follows: Firstly, a sildenafil hydrogel is provided, which comprises a hydrogel composed of natural egg white and chitosan, and sildenafil added into the hydrogel. The hydrogel containing sildenafil can be used for treating infected wounds and promoting healing.

[0006] Secondly, the preparation method of the above-mentioned sildenafil hydrogel comprises the following steps: S1. Separate egg whites from fresh eggs and stir evenly to obtain egg white liquid; S2. preparing a mixed solution containing 2 wt% sildenafil, 3 wt% chitosan mixture and 0.5 M NaOH; S3. The protein solution prepared in step 1 and the mixed solution prepared in step 2 are mixed in a volume ratio of 1:1 to obtain a gelled product; S4. The gelled product obtained in step 3 is immersed in a DMEM solution to promote gelation and secondary crosslinking, and finally a chitosan-egg white hydrogel CEHS containing sildenafil is obtained.

[0007] Finally, the present invention provides the use of the sildenafil hydrogel in a drug for promoting wound healing, especially for promoting healing of infected wounds.

[0008] The beneficial effects of the present invention are as follows: the present invention provides a novel hydrogel that can be used to treat infected wounds. The hydrogel is mainly composed of egg white and chitosan, and sildenafil is added. The use of egg white and chitosan as raw materials for preparing the hydrogel enhances the mechanical properties of the hydrogel. The adjustable viscosity and biological activity of these materials provide a suitable moist microenvironment for the wound, and have multiple functional advantages. In addition, the high economic benefits and safety of egg white and chitosan make them ideal choices for treating infected wounds. The hydrogel using these as raw materials is easy to prepare and has low cost. In the treatment of infected wounds, chitosan-egg white hydrogel (CEHS) containing sildenafil retains the inherent functions of its two main components and fully utilizes the remarkable efficacy of sildenafil. CEHS has excellent mechanical strength and degradation properties. In vitro cell experiments have shown that it has good biocompatibility, can promote the proliferation and migration of fibroblasts and endothelial cells, and the formation of endothelial cell tubes, while inhibiting the oxidative stress and inflammatory response of macrophages. CEHS also has effective antibacterial activity against Escherichia coli and Staphylococcus aureus; in vivo experiments using mouse infected wound models have shown that CEHS can promote the healing of burn wounds by reducing inflammation, oxidative stress and bacterial load and promoting angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0010] Figure 1 is a schematic diagram of the preparation of chitosan egg white hydrogel (CEHS) containing sildenafil and its application in treating infected wounds in mice. The picture shows: egg white is taken out of the egg, the egg white is mixed with sildenafil and chitosan in an alkaline solution, DMEM is added to promote gelation and secondary cross-linking, and the CEHS provided by the present invention is obtained. Staphylococcus aureus is applied to the wound of mice to establish a mouse wound infection model, and CEHS is applied to the wound of mice to play the role of antibacterial, anti-inflammatory, antioxidant, and promoting angiogenesis, and finally the wound is healed; Figure 2 shows the characteristics of CEHS, including: (A) physical photos of CEH and CEHS; (B) transmission electron microscopy (TEM) photos of CEHS, with a scale bar of 50 μm; (C) FITR detection images of CEH and CEHS; (D) degradation rates of CEH and CEHS in PBS (n = 3); (E) expansion rates of CEH and CEHS in PBS (n = 3); (F) storage (G') and loss modulus (G") of CEHS; (G) shear viscosity of CEHS; (H) absorbance of sildenafil released from CEHS at different time intervals in vitro at a wavelength of 290 nm detected by UV spectroscopy; (I) in vitro release of sildenafil from CEHS in PBS (n = 3); Figure 3 shows the cell compatibility and cell detection of CEHS; (A). CCK-82Jh was used to detect the cell viability of CEHS after co-incubation with 3T3 (n = 4); (B). CCK-8 was used to detect the cell viability of CEHS after co-culture with HUVEC and 3T3 (n = 4); (C). Images of in vitro scratch test of 3T3 after different treatments, the scale bar is 100 μm; (D). Images of in vitro scratch test of HUVEC after different treatments, the scale bar is 100 μm, in the wound healing model; wound closure rate of 3T3 (E) and HUVEC (F) after different treatments (n = 3); (G). HUVEC tube formation test after different treatments in vitro, the scale bar is 50 μm; (H). Quantitative analysis of tube formation in the tube formation test (n = 3); All data are expressed as mean * SD, compared with the control group, ns * 0.05, *p<0.05, **p<0.01, ***p<0.001; Figure 4 shows the in vitro performance of CEHS; (A). Flow cytometric analysis of ROS intensity in RAW264.7 cells after different treatments; (B). Quantitative analysis of ROS intensity corresponding to Figure (A), and the same treatment on inflammatory factors TNF-α (C) and IL-1β (D) in LPS-induced RAW264.7 cells (n = 6); (E). Survival images of Escherichia coli and Staphylococcus aureus on agar plates after different treatments; Inhibition rates of Escherichia coli (F) and Staphylococcus aureus (G) after different treatments (n = 4); All data are expressed as mean ± SD; Figure 5 is the effect evaluation of CEHS treatment on infected wounds in mice; (A). Animal experiment process CEHS was used to treat infected wounds in mice; (B). Healing of infected wounds at different time points; (C). Changes in wound healing rate in different groups (n=3); (D). HE and Masson-stained skin tissue sections of each group on the 5th and 15th days; (E). Quantitative analysis of Masson collagen-positive area; scale bar is 200 μm; Relative content of collagen in different groups on the 5th and 15th days (n=3), all data are expressed as mean * SD, compared with the control group, ns * 0.05, *p<0.05, **p<0.01, ***p<0.001; Figure 6 shows the antioxidant, anti-inflammatory and antibacterial activities of CEHS in vivo; (A). On the 5th and 15th days, the oxidative stress of wound tissues in different groups was evaluated by DHE fluorescence staining; the scale bar is 100 μm; (B). Quantitative analysis of DHE fluorescence intensity in Figure (A), skin index to evaluate the tissue antioxidant capacity of different groups; SOD (C) and MDA (D) levels in different groups changed on the 5th and 15th days (n = 3); the inflammatory indexes of skin tissues in different groups were evaluated; TNF-α (E) and IL-1β levels changed on the 5th and 15th days (n = 3); (F) Changes in different groups on the 5th and 15th days (n = 4); (G). Quantitative analysis of bacteria in wound tissues of different groups on the 5th and 15th days (n = 4); all data are expressed as mean ± SD; compared with the control group, ns * 0.05, *p<0.05, **p<0.05, 0.01, ***p<0.001; Fig. 7 Analysis of in vivo vascularization of vascularized C'EHS Immunohistochemical staining of CD31 (A), α-SMA (C), and VEGF (E) Estrogen levels in different groups on days 5 and 15, scale bar = 100 μm; Quantitative analysis of positive expression of CD31 (B), -SMA (D), and VEGF (F) (n = 3); (F). HYP (G) and TGF-β content; (H) in wound tissues of each group on days 5 and 15 (n = 3). All data are expressed as mean * scale, compared with the control group, ns * 0.05, *P < 0.05, **P < 0.01, ***P < 0.001; Figure 8 In the figure, (A) is the live and dead cell staining after CEH or CEHS was co-incubated with 3T3 cells, (B) is the live and dead cell staining after CEH or CEHS was co-incubated with HUVEC, the scale bar is 50 μm; Fig. 9 CEH or CEHS was co-incubated with 3T3 cells (A) or HUVEC (C). Scale bar is 100 μm. Quantitative analysis of EdU staining in 3T3 cells (B) or HUVEC (D) (n = 3). Fig.10 In the figure, (A) the changes of wound healing rate over time in each group (n = 3), (B) the changes of body weight of mice in each group over time (n = 4); Fig.11 (A) Hemolysis of CEHS, (B) hemolysis rate corresponding to figure (A) (n = 3), (C) HE staining of major organs (kidney, lung, spleen, liver and heart) to evaluate the tissue biocompatibility of CEHS, scale bar is 100 μm. DETAILED DESCRIPTION

[0011] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in combination with experimental test results and accompanying drawings.

[0012] Experimental Materials Fresh eggs were obtained from supermarkets. Chitosan and Dulbecco's Modified Eagle Medium (DMEM) were purchased from Sigma, and sildenafil and NaOH were purchased from Aladdin. Cell counting kit-8 (CCK-8), calcein / PI cell viability / cytotoxicity assay kit, superoxide anion and dihydroethylenediamine (DHE) reactive oxygen species assay kit, and reactive oxygen species assay kit were all purchased from Beyotime. Lipopolysaccharide (LPS) and phosphate buffered saline (PBS) were purchased from Solarbio. Nutrient broth was purchased from Huankai Microbiology. Mouse TNF-α ELISA kit, mouse IL-1β ELISA kit, and mouse TGF-β1 ELISA kit were all purchased from Multi Sciences.

[0013] (Superoxide dismutase (SOD) detection kit, malondialdehyde (MDA) detection kit and hydroxyproline (HYP) detection kit were all purchased from Nanjing Jiancheng Biotechnology Co., Ltd. Superoxide dismutase (SOD) detection kit, malondialdehyde (MDA) detection kit and hydroxyproline (HYP) detection kit were all purchased from Nanjing Jiancheng Bioengineering Institute. Smooth muscle actin (α-SMA) antibody, platelet endothelial cell adhesion molecule-1 (CD31) antibody and vascular endothelial growth factor (VEGF) antibody were all from Abcam.

[0014] HUVEC, NIH 3T3 and RAW 264.7 were obtained from Wuhan Procare Biotechnology Co., Ltd. Escherichia coli and Staphylococcus aureus were purchased from Shanghai Beikangnuo Biotechnology Co., Ltd.; all C57BL / 6 mice (male, 6-8 weeks) were ordered from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.

[0015] Experimental methods 1. Synthesis of chitosan protein hydrogel loaded with sildenafil Carefully separate the egg white from fresh eggs and stir well to obtain a homogeneous liquid. Add the egg white solution to a solution containing 2 wt% (weight / volume) sildenafil, 3 wt% (weight / volume) chitosan, and 0.5 M NaOH, ensuring that the two solutions are mixed in a 1:1 volume ratio. Allow the mixture to gel and then immerse it in Dulbecco's Modified Eagle Medium (DMEM) solution for 12 hours to promote gelation and secondary cross-linking. The final product is chitosan-egg white hydrogel (CEHS) containing sildenafil.

[0016] Chitosan-egg white hydrogel (CEH) without sildenafil was prepared by the same method.

[0017] 2. Characteristics of CEHS To characterize CEHS, scanning electron microscopy (SEM, Hitachi ReguIus8100) was used to observe its morphology and microstructure. The freeze-dried CEH and CEHS samples were analyzed by Fourier transform infrared spectroscopy (FTIR, Nicolet iS 10) in the spectral range of 4000 to 400 cm -1 .

[0018] 3. Degradation Experiment The hydrogel samples were cut into appropriate sizes and their initial weight (M0) was measured with a precision balance. Subsequently, the hydrogel samples were immersed in PBS solution and kept at room temperature. At predetermined time intervals, the hydrogel samples were taken out, gently wiped with filter paper to remove surface moisture, and then weighed (W1) (n = 3). The degradation rate was calculated as follows: Degradation rate = (W0-W1) / W0* 100%.

[0019] 4. Expansion experiment The hydrogel samples were freeze-dried and weighed (M0), and then the samples were immersed in PBS at room temperature. At predetermined time intervals, the samples were taken out and re-weighed (M1) (n = 3). The swelling ratio was calculated as follows: Swelling ratio = (M1– M0) / M0*100%.

[0020] 5. Rheological properties of CEHS The rheological properties of the hydrogels were measured at room temperature using a rheometer (DHR-2, TA) equipped with a 10 mm diameter parallel plate rotor. -1 The oscillatory strain sweep was performed from 0.1% to 100% at an angular frequency of 0.1 to 20 s. -1 The viscosity of the hydrogel was measured by shear rate scanning.

[0021] 6. In vitro drug release studies To evaluate the in vitro drug release ability of CEHS hydrogels, the hydrogel samples were placed in dialysis bags with a molecular weight of 12,000-14,000Da and immersed in PBS at 37°C. The dialysis bags were then placed on a shaker and gently stirred. 200 μL of the supernatant was sampled at predetermined time points and immediately replaced with an equal volume of fresh PBS. The collected supernatant was analyzed for the release concentration of sildenafil at a wavelength of 280 nm using a UV spectrophotometer (Cary 3500, Agilent Technologies) (n = 3).

[0022] 7. Cytocompatibility testing To evaluate the cytotoxicity of CEHS hydrogels on cells, fibroblasts (NIH 3T3) and endothelial cells (human umbilical vein endothelial cells, HUVEC) were selected as the research subjects. After the hydrogels were fully formed, they were immersed in PBS for 8 hours and the corresponding hydrogel eluates were collected. The cytotoxic effects of CEHS hydrogels on 3T3 cells and HUVECs were evaluated using the Cell Counting Kit-8 (CCK-8, Beyotime). First, the cells were seeded into 96-well plates. Then, 20 ml of hydrogel eluate or an equal volume of PBS was added to each well. After incubation, the absorbance of each well at a wavelength of 450 nm was measured using a microplate reader (Varioskan LUX, Thermo Fisher Scientific) (n = 3). Finally, live / dead staining was performed after 72 hours, the cells were fluorescently labeled, and the staining conditions were evaluated using a fluorescence microscope (TE2000-U, Nikon).

[0023] 8. Scratch test HUVEC or 3T3 cells were cultured in six-well plates to 100% confluency and scratched with a 200 μL pipette tip. The cells were then cultured in serum-free DMEM (containing 200 μL of hydrogel eluate or an equal volume of PBS). Wound closure was assessed by comparing the scratch area at 0, 12, and 24 hours (n = 3). Wound closure was quantified using the following formula: Wound healing rate = (0 hour scratch area - 12 hour or 24 hour scratch area) / 0 hour scratch area * 100%.

[0024] 9. Tube Formation Experiment Add 50 μl of Matrigel (ABW 0827245) to each well of a 24-well plate and incubate at 37°C for 30 minutes. 5 HUVECs were seeded at a density of 10 cells per well and treated with medium containing 200 μl of hydrogel eluate or an equal volume of PBS. The tubules formed by HUVECs after 8 h were observed using a microscope (TE2000-U, Nikon), and the number of tubules formed in each group was quantified (n = 3).

[0025] 10. Antioxidant Activity RAW 264.7 cells are the most commonly used cell line to study the antioxidant activity of cells. RAW 264.7 cells were seeded into 6-well plates for culture. Subsequently, 1 mL of Rosup was added to simulate the oxidative stress environment, and 200 microliters of hydrogel extract or an equal volume of PBS was added to co-culture with the cells for 24 hours. The old culture medium was then aspirated and the cells were washed three times with PBS. Then, each well was stained with 1 mL of 10uM DCFH-DA probe to detect the level of intracellular reactive oxygen species (ROS). Finally, the cells were collected and the fluorescence intensity was measured by flow cytometry (n = 3).

[0026] 11. Anti-Inflammatory Activity RAW 264.7 cells respond strongly to inflammatory stimuli and can produce a variety of inflammatory mediators, making them an ideal model for studying cellular anti-inflammatory responses. RAW 264.7 cells were seeded into 24-well plates for culture. 1 μg / mL of LPS was then added to induce an inflammatory environment. The cells were then treated with 200uL of hydrogel extract or an equal amount of PBS for 24 hours. After incubation, the cell supernatant was collected and the levels of TNF-α and IL-1β were determined using commercial ELISA kits (n = 6).

[0027] 12. Antibacterial ability The antibacterial activity of CEHS hydrogel against Escherichia coli and Staphylococcus aureus was evaluated. Escherichia coli and Staphylococcus aureus were cultured in broth medium and incubated in an incubator at 37 °C. 50 mg of CEH hydrogel was added to Staphylococcus aureus (1.0 × 10 5 CFU / mL) suspension and cultured for 24 hours. An equal volume of PBS was added to the control group. Finally, 20 μL of the bacterial suspension was cultured on an agar plate and cultured at 37 °C for 24 hours. The plate was then removed, and the observations were recorded and photographed. In addition, 100 μL of the bacterial suspension was taken and its OD value was measured at a wavelength of 600 nm using a microplate reader (n = 3). The inhibition rate was calculated as follows: Inhibition rate = (OD value of the control group - OD value of the experimental group) / OD value of the control group * 100%.

[0028] 13. Infected Wound Animal Model The full-thickness infected wound mouse model was approved by the Research Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University for animal experiments. Twenty-four healthy male C57BL / 6 mice aged 6 to 8 weeks were randomly divided into three groups: (1) control group; (2) CEH experimental group; (3) CEHS experimental group, with 8 mice in each group. All mice were anesthetized with 3% sodium pentobarbital (50 mg / kg) and the backs were depilated. A 10-mm-diameter skin biopsy punch was used to make a 10-mm circular full-thickness skin wound on the back of each mouse. Then, a Staphylococcus aureus suspension (200 μL, 1 ×10^8 CFU / mL) was applied to the wound.

[0029] If the wound showed obvious signs of infection and the presence of Staphylococcus aureus was confirmed, the model was considered to be successfully established. A circular hydrogel patch with a diameter of 10 mm was attached to the infected wound on the back of each mouse. Wound images were taken with a camera on days 1, 3, 5, 10, and 15, and the wound area was measured using imageJ software (n = 3). The wound healing rate was calculated according to the following formula: Wound healing rate = (A0- A1) / A0×100%, where A0 is the initial wound area and A1 is the actual wound area at the specified time.

[0030] 14. Tissue Staining Skin wound tissue samples were collected from the dorsal side of mice in each group on days 5 and 15. The samples were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections according to standard histological procedures. The sections were routinely stained with hematoxylin and eosin (HE) for histological examination, and collagen deposition in the wound was evaluated by Masson's trichrome staining. Immunohistochemical (IHC) staining of α-SMA (1:200), CD31 (1:200), and VEGF (1:200) in the wound tissue was performed according to standard procedures. Finally, the sections were observed under an optical microscope and quantitatively analyzed. ImageJ software was used for analysis (n = 3).

[0031] 15. ROS Evaluation To evaluate the level of ROS in wound tissue, samples from injured mice were quickly frozen in liquid nitrogen and embedded in optimal cutting temperature (OCT) compound. The embedded tissue blocks were sectioned using a cryostat at a thickness of 8 μm. The sections were then fixed with 4% paraformaldehyde for 1 hour at room temperature. After fixation, the sections were stained with 10 PM dihydroethidium (DHE) for 30 minutes and counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 3 minutes. The sections were observed and photographed using a fluorescence microscope, and quantitative analysis was performed using imageJ software (n = 3).

[0032] 16. ELISA On days 5 and 15, skin wound tissue samples were collected from the dorsal flanks of mice in each group. The samples were rinsed with pre-cooled PBS and blotted on filter paper to remove surface moisture. 20 mg of skin tissue was weighed for subsequent experiments. The skin tissue was homogenized and then centrifuged at 8000 rpm for 5 min at 4°C to remove the supernatant. Subsequently, TNF-α, IL-1β, and TGF-β in the skin tissue samples were quantitatively detected using the corresponding ELISA kits. All procedures were performed according to the manufacturer's instructions (n ​​= 4).

[0033] 17. Concentrations of SOD, MDA and HYP in wound tissue On days 5 and 15, skin wound tissue samples were collected from the dorsal flanks of mice in each group. The samples were rinsed with PBS, minced, homogenized, and centrifuged. The supernatant was then collected. Appropriate assay kits were used to assess the activity of superoxide dismutase (SOD) activity, malondialdehyde (MDA) levels, and hydroxyproline (HYP) content according to the manufacturer's instructions (n ​​= 3).

[0034] 18. In vivo antimicrobial activity To collect wound tissue samples from the dorsal skin of mice at 5 and 15 days after wounding, mice were anesthetized in accordance with approved ethical guidelines. Wound tissue was excised using sterile scissors and forceps, placed in a sterile container, and weighed to ensure consistent handling. Tissue samples were then cut into small pieces and ground to ensure complete release of bacteria. The ground tissue was transferred to a sterile tube and mixed with an appropriate amount of sterile PBS. The tissue homogenate was diluted 50-fold and centrifuged at 3,000 rpm for 10 minutes. The supernatant was discarded and the pellet was resuspended in sterile PBS to form a bacterial suspension. 100 microliters of bacterial suspension was added to each well of a 96-well plate, and the OD value was measured at a wavelength of 600 nm using a microplate reader (n = 4). All operations were performed under sterile conditions to prevent contamination and the steps were kept consistent to ensure reproducibility.

[0035] 19. Statistical analysis Experimental data are presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to evaluate the differences among groups. Statistical significance is as follows: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; "ns" indicates no significant difference. All experiments were performed at least three times (n = 3).

[0036] Experimental test results 1. Preparation and Characterization of CEHS The preparation method of chitosan-protein hydrogel loaded with sildenafil (CEHS) is simple and economical. In short, sildenafil and chitosan are dissolved in NaOH solution, and then the resulting mixture is mixed with protein in a volume ratio of 1:1. The mixture is thoroughly mixed to form a uniform gel, which is then soaked in DMEM solution and subjected to secondary cross-linking after the gel is completely solidified. The final product is CEHS (Figure 2A). First, the microstructure of CEHS was observed using a scanning electron microscope (SEM), and it was found that it had a relatively uniform porous network structure (Figure 2B). Subsequently, CEH and CEHS were freeze-dried, and their Fourier transform infrared spectra (FTIR) were analyzed. The results showed that CEHS had a high molecular weight at 900 cm -1 The characteristic peaks are shown near 3100-3400cm -1 The range shows broad peaks, which are attributed to the carbohydrate structure in the hydrogel. -1 The absorption peak is related to the bending and asymmetric deformation of the CH3 groups in the hydrogel.

[0037] The addition of sildenafil introduced a peak near 1267 cm-1 in the FT-IR spectrum of the hydrogel sample. -1 The new peak of sildenafil was confirmed to be successfully added to the hydrogel (Figure 2C). The degradation ability of the hydrogel was evaluated by degradation test. The degradation rate of CEHS was close to 80% after immersion in PBS for 24 hours. CEHS has a porous network structure, which provides a basic healing environment for absorbing exudate and maintaining moisture. The swelling behavior of freeze-dried CEHS was evaluated by swelling test. The results showed that after immersion in PBS for 24 hours, the swelling ratio reached a peak of 29.86±2.01% (Figure 2E). After 24 hours, the swelling rate gradually decreased at a slower rate, indicating that CEHS has a high initial swelling capacity and relatively stable swelling characteristics in PBS. This absorption capacity enables CEHS to provide a stable moist environment for wound healing.

[0038] The typical viscoelastic properties of CEHS were confirmed by analyzing the storage modulus (G') and loss modulus (G") as a function of frequency (Figure 2F). Beyond a certain range, CEHS collapses rapidly, which is critical for wound dressing applications because it helps the hydrogel provide proper support and recovery when in contact with the wound. As the shear rate increases, the viscosity of CEHS decreases, exhibiting a typical shear-thinning rheological behavior (Figure 2G). This property ensures that the hydrogel is more evenly distributed on the wound tissue in practical applications. The sildenafil release profile of CEHS can be determined by immersing CEHS in PBS and measuring the sildenafil concentration in the supernatant at different time points.

[0039] The release rate of sildenafil was analyzed using ultraviolet spectroscopy (Figure 2H). The results showed that sildenafil was released rapidly within the first 12 hours, and then the release rate gradually decreased until it reached a steady state (Figure 2L). This release characteristic is conducive to quickly reaching a high therapeutic dose of sildenafil in a short period of time, followed by sustained release, thereby prolonging the duration of drug action. This helps to reduce the frequency of administration and maintain efficacy.

[0040] In summary, CEHS has excellent degradation properties and can gradually degrade during the wound healing process, reducing the pain and risks caused by removing the dressing. In addition, the moderate swelling properties enable the hydrogel to better fit the wound and form an effective barrier to prevent external bacterial infection and wound dehydration. In addition, the mechanical strength of CEHS is sufficient to support wound protection and repair. The drug release properties of CEHS not only optimize the drug utilization efficiency, but also reduce the number of dosing times, improving the overall safety and effectiveness of the treatment. In summary, CEHS is an ideal wound healing material with broad application prospects and clinical value.

[0041] Cytocompatibility and interactions of CEHS in vitro Cytocompatibility is a prerequisite for material application. Fibroblasts and endothelial cells are important components of skin tissue; therefore, they are often used to evaluate the biocompatibility of hydrogels in wound applications. 3T3 cells are a mouse fibroblast cell line that are often used to evaluate the cytocompatibility of materials due to their stable growth and sensitivity to environmental changes. HUVEC (human umbilical vein endothelial cells) represent vascular endothelial cells and are an important model for studying angiogenesis and endothelial cell function in wound healing and tissue engineering. The cytotoxicity of CEHS was evaluated using the CCK-8 assay (Figure 3A, 3B). Specifically, the extracts of CEHS were co-cultured with cells, and cell viability was measured for three consecutive days. There was no significant difference in cell viability between the CHE and CEHS experimental groups and the control group.

[0042] In addition, on the third day, live-dead cell staining was performed on the cells in each group (Figure 8A and 8B) to visually display the cell viability status. The staining results showed that most of the cells in the CHE group and the control group were alive. The CEHS experimental group maintained good viability. These experimental results show that CEHS has good cell compatibility and low toxicity and is suitable for use as a biomaterial in tissue engineering applications.

[0043] Scratch assays are often used to simulate the wound healing process and evaluate cell migration and wound closure ability, which is of great significance for studying the effect of CEHS on promoting wound healing at the cellular level. The results of the scratch assay showed that the addition of CEHS significantly increased the cell migration rate and wound coverage ability (Figures 3C, 3D). Specifically, compared with the control group, the wound closure rates of the CHE and CEHS experimental groups were higher, with the wound closure rates of 3T3 cells and HUVEC reaching more than 80% and 90% within 24 hours, respectively (Figures 3E and 3F). The cells in the wound area were more evenly distributed and dense. These research results show that CEHS can effectively promote cell migration and proliferation, and play an important positive role in the wound healing process.

[0044] The formation of endothelial cell tubes is a key step in angiogenesis during wound healing, which can promote blood supply and tissue repair and accelerate wound healing. The results of the tube formation test showed that compared with the control group without hydrogel, HUVECs in the CEH and CEHS experimental groups formed more and longer tubular structures after adding hydrogel. These tubular structures not only increased in number, but also became more regular and stable in morphology (Figure 3G). The number of tubular structures in the CEHS experimental group reached 52.33±5.13 ( Figure 3 H). These results indicate that CEHS can significantly enhance the tube-forming ability of endothelial cells and promote the formation of new blood vessels, thereby accelerating the wound healing process.

[0045] In addition, 5-ethynyl-2'-deoxyuridine (EdU) staining is important for evaluating cell proliferation during wound healing. By performing EdU staining on 3T3 cells and HUVEC, we can visually observe that the fluorescence intensity of the CEHS group was significantly increased compared with the control group (Figure 9A, 9C). Quantitative analysis of fluorescence images showed that the EdU fluorescence intensity of 3T3 cells and HUVEC in the CEHS group was enhanced by 39.32 ± 3.03% and 46.22 ± 5.04%, respectively, compared with the control group (Figure 9B, 9D). These results clearly show that CEHS has a strong ability to promote cell proliferation.

[0046] In conclusion, CEHS not only has good cytocompatibility and low toxicity but also significantly promotes cell migration, proliferation, and endothelial cell tube formation, thereby accelerating the wound healing process.

[0047] 2. In vitro performance of CEHS When treating infected wounds, ROS, inflammation, and bacteria are three major challenges that must be addressed. These factors interact and aggravate each other, profoundly affecting the wound healing process and treatment outcomes. Increased ROS levels not only damage surrounding healthy tissues, but also exacerbate inflammatory responses and disrupt the normal functions of cells and tissues. Although inflammation is a natural response of the body to infection, excessive inflammation in infected wounds may worsen and cause damage to the body, and persistent bacterial infections can complicate wound healing. Therefore, the use of wound dressings with antioxidant, anti-inflammatory, and antibacterial properties can effectively address these multiple challenges, greatly improve the success rate of wound healing, and improve the treatment outcomes of infected wounds.

[0048] RAW 264.7 cells are a classic macrophage model that is widely used in oxidative stress and inflammation research. In order to simulate the oxidative stress environment, RAW264.7 cells were first treated with Rosup and then the hydrogel eluate was added. The cells were then stained with a DCFH-DA probe to detect the level of intracellular ROS. The results showed that the fluorescence intensity of the CEH and CEHS experimental groups was significantly reduced compared with the control group (Figure 4A). Specifically, compared with the control group, the fluorescence intensity of the CEHS experimental group decreased by 50.06±14.77 (Figure 4B), indicating that CEHS can effectively reduce the level of ROS in RAW264.7 cells under oxidative stress conditions, showing its strong antioxidant capacity. To simulate the inflammatory environment, RAW264.7 cells were stimulated with lipopolysaccharide (LPS) to induce the secretion of various inflammatory mediators (such as TNF-α and IL-1β) to simulate the in vivo inflammatory state. The cells were then exposed to hydrogel eluate or PBS solution to evaluate the regulatory effect of CEHS on the cellular inflammatory response.

[0049] ELISA was used to measure TNF-α and IL-1β in the supernatant of each group. The results showed that the concentrations of TNF-α and IL-1β in the CEH and CEHS experimental groups were significantly reduced compared with the control group. Specifically, the concentrations of TNF-α and IL-1β in the CEH group decreased to 223.87±17.17 and 131.26±20.74 pg / mL, respectively, while the concentrations in the CEHS group decreased to 217.33±22.75 and 138.57±16.83 pg / mL, respectively (Figure 4C, 4D). These results indicate that CEHS can effectively inhibit the inflammatory response of RAW264.7 cells under inflammatory conditions, showing its strong anti-inflammatory properties. Escherichia coli and Staphylococcus aureus are the most common bacteria in the skin microenvironment. Therefore, two bacteria were selected to evaluate the in vitro antibacterial activity of CEHS. The results of agar plate assay showed that CEHS had significant antibacterial effects on both Escherichia coli and Staphylococcus aureus (Figure 4E). The antibacterial activities of CEHS against Escherichia coli and Staphylococcus aureus were 70.37 ± 3.42% and 84.82 ± 2.48%, respectively (Figures 4F and 4G). This indicates that CEHS has significant broad-spectrum antimicrobial properties and can effectively inhibit these common pathogens.

[0050] As an innovative hydrogel material, CEHS demonstrates excellent bioactivity in coping with multiple challenges such as oxidative stress, inflammation and bacterial infection, with outstanding antioxidant, anti-inflammatory and antibacterial capabilities. These properties highlight its great clinical application potential in treating infected wounds.

[0051] 3. Effect of CEHS on healing of infected wounds in mice The above in vitro experiments preliminarily verified the potential of CEHS in treating infected wounds. The present invention uses a mouse full-thickness skin infection model to further verify the efficacy of CEHS. Male mice were divided into three groups: a control group (no intervention), a CEH group (treated with CEH), and a CEHS group (treated with CEHS). The entire experimental process is shown in Figure 5A. After the wound was not treated or covered with the corresponding hydrogel, wound photos were taken on the 3rd, 5th, 7th and 10th days to monitor wound healing. On the 5th day, the wound healing rates of the CEH group and the CEHS group were 49.61 ± 4.43% and 62.68 ± 7.01%, respectively. By the 15th day, the wounds in the CEHS group were almost completely healed, and the wound closure was significantly better than that in the CEH group and the control group ( Figure 5 B, 5C), which indicates that CEHS has significant advantages in promoting the healing of infected wounds.

[0052] Analyzing the changes in wound healing rate, the CEHS group was the first to approach 100% healing (Figure 10A), indicating that CEHS is the most effective in promoting the healing of infected wounds. To further verify the therapeutic effect of CEHS, the wound tissues of each group were histopathologically analyzed using HE and Masson staining (Figure 5D).

[0053] HE staining results showed that in the early stage of wound healing (day 5), the control group had obvious inflammatory cell infiltration, while the number of inflammatory cells in the CEH group and the CEHS group was significantly reduced. This reduction shows that CEHS plays a vital role in inhibiting the early inflammatory response. As time goes by, the wound healing enters the late stage (day 15), and the inflammatory cell infiltration in the two experimental groups further decreases, indicating that the wound has successfully transitioned to the proliferation stage. At this stage, both the CEH group and the CEHS group showed signs of epithelial tissue regeneration, which was better than the control group, indicating smooth cell proliferation and tissue repair process. Collagen plays a central role in wound healing, and its deposition, remodeling and metabolism accompany the wound healing process. Therefore, the deposition level of collagen reflects the healing state of the wound.

[0054] Throughout the healing period, the levels of collagen deposition in the CEH and CEHS groups were higher than those in the control group. Quantitative analysis of the Masson collagen-positive area showed that the CEHS group had the highest amount of collagen deposition (Figure 5E). The collagen-positive area in the CEHS group increased significantly on day 5, and by day 15, the collagen staining level stabilized, with the arrangement of collagen fibers becoming more dense and regular. This suggests that CEHS can promote collagen deposition and wound filling in the early stages of repair, and promote the density and regularity of collagen arrangement in the later stages of repair, ultimately helping to enhance the mechanical strength of the tissue, reduce wound space, and enable full maturation and functional recovery of the wound.

[0055] These results suggest that CEHS has great potential to promote the healing of infected wounds. Changes in body weight are not only an indirect indicator of metabolic status and severity of inflammation, but also an important parameter for evaluating efficacy and quality of life (Koh et al., 2005). Therefore, we analyzed the changes in body weight of infected mice. The body weight loss rate of the CEHS group was relatively slow compared with the other two groups (Figure 10B). This difference in body weight loss further demonstrated the therapeutic effect of CEHS on infected wounds.

[0056] 4. Effects of CEHS in vivo According to previous cell experiment results, CEHS has a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, angiogenesis-promoting and cell proliferation-promoting effects. In the mouse infected wound model, the inventors systematically verified the results of the in vitro experiment. First, dihydroethidium (DHE) staining was used to evaluate the degree of oxidative damage in the wound. The stronger the red fluorescence, the higher the level of oxidative stress. The results showed that compared with the 5th day, the fluorescence intensity of all groups decreased on the 15th day, but the fluorescence intensity of the CEH group and the CEHS group was still significantly lower than that of the control group (Figure 6A). This shows that both CEH and CEHS can effectively reduce the level of oxidative stress in the wound. Quantitative analysis of the fluorescence intensity of each group (Figure 6B) showed that the CEHS group had the best antioxidant effect. This result not only confirmed the excellent ability of CEHS in reducing oxidative damage, but also further proved that it can effectively neutralize free radicals and reduce cell damage, laying a solid foundation for completely eliminating oxidative stress in infected wounds. Superoxide dismutase (SOD) is an important antioxidant enzyme that can effectively scavenge superoxide free radicals, thereby reducing the damage to cells caused by oxidative stress. Malondialdehyde (MDA) is the final product of lipid peroxidation, and its elevated levels are often used as a marker of oxidative stress. Reduced SOD activity and elevated MDA levels usually indicate an imbalance in the cellular oxidation and antioxidant systems, reflecting a weakened antioxidant defense system in the body and increased cell and tissue damage.

[0057] Therefore, we evaluated the in vivo antioxidant activity of CEHS by measuring the SOD activity and MDA level in the wound skin tissue of each group. Compared with the control group, the SOD activity of the CEH group and the CEHS group was significantly increased and the MDA level was significantly decreased on the 5th and 15th days (Figure 6C, 6D). Based on these experimental results, we can further infer that CEHS can enhance SOD activity and reduce MDA content, thereby alleviating oxidative stress, protecting skin cells from free radical damage, and ultimately accelerating wound repair and regeneration.

[0058] Excessive or persistent inflammatory response can have a negative impact on wound healing. Chronic inflammation can lead to excessive secretion of cytokines and chemokines, causing tissue damage, fibrosis, and necrosis, which in turn delays the wound healing process. Therefore, balancing the inflammatory response and timely controlling the inflammatory condition are key to the effective healing of infected wounds. To further investigate the anti-inflammatory effect of CEHS in vivo, we used ELISA to detect the levels of two inflammatory cytokines, TNF-α and IL-1β, in the skin tissue of each group of wounds. Compared with the control group, the TNF-α content in the CEH and CEHS groups decreased significantly, especially on the 15th day, when the TNF-α content in the CEHS group was the lowest, with a difference of 49.29%±6.78 pg / mL from the control group (Figure 6E). This suggests that CEHS can more effectively inhibit the overexpression of TNF-α. Similarly, the IL-1β levels in each group showed a similar trend, with a significant decrease in the CEHS group, which was 31.36±7.27 pg / mL compared with the control group (Figure 6F). These findings further validate the anti-inflammatory effect of CEHS, suggesting that it can reduce local inflammatory responses in wounds by inhibiting the expression of TNF-α and IL-1β inflammatory cytokines and potentially promote the healing process.

[0059] In addition, CEHS also showed preliminary antibacterial activity in vitro. To further evaluate the antibacterial ability of CEHS in vivo, we extracted tissue fluid from the infected wound tissue of each group of mice, diluted and centrifuged to prepare bacterial suspension, and then measured the absorbance at a wavelength of 600 nanometers using a microplate reader. The results showed that on the 5th and 15th days, the absorbance values ​​of the CEH group and the CEHS group were significantly lower than those of the control group ( Figure 6 G). This indicates that CEHS has significant in vivo antibacterial activity, while CEH also has similar antibacterial properties. CD31, α-SMA, and vascular endothelial growth factor are important markers of endothelial cells, vascular smooth muscle cells, and angiogenic factors, respectively, and play a key role in evaluating angiogenesis. Increased CD31 expression indicates the formation of new blood vessels and the activity of endothelial cells; monitoring of a-SMA helps to understand the structure and functional status of the vascular wall; increased VEGF levels indicate the activity of angiogenesis and the regulation of related factors. The results showed that the positive expression of CD31, α-SMA, and VEGF in the CEH and CEHS groups was better than that in the control group (Figures 7A, 7C, and 7E). Further quantitative analysis showed that the positive expression of CD31, a-SMA, and VEGF was the highest in the CEHS group (Figures 7B, 7D, and 7F).

[0060] The above results indicate that CEH and CEHS treatment can significantly promote angiogenesis and maturation of vascular smooth muscle cells during wound healing. It is worth noting that CEHS has a more prominent promoting effect on angiogenesis and vascular function. From day 5 to day 15, the expression of α-SMA and VEGF in all groups continued to increase. However, the expression of CD31 decreased slightly. This may be due to the gradual maturation and stabilization of new blood vessels in the late stage of healing, which leads to a decrease in endothelial cell proliferation and migration activity. Therefore, the expression of CD31 also decreased. During this period, the maturation of vascular smooth muscle cells and the expression of angiogenic factors gradually strengthened, promoting the progress of wound healing.

[0061] Hydroxyproline (HYP) is the main component of collagen, and its content changes significantly during angiogenesis, especially during the formation of new blood vessels and the reconstruction of vascular walls. The increase in HYP levels not only shows enhanced collagen synthesis activity, but also reflects the reconstruction and fibrosis of the vascular matrix. In the CEHS group, the HYP level was higher than that in the other two groups. By the 15th day, it reached 7.34±0.92 μg / mg (Figure 7G). These data further confirmed that CEHS promoted the synthesis of collagen and the reconstruction of the vascular matrix by significantly increasing the HYP content, and showed excellent efficacy in promoting the formation and reconstruction of new blood vessels. In addition, transforming growth factor-β (TGF-β) plays an important role in wound healing by promoting the migration and proliferation of vascular endothelial cells and enhancing the expression of matrix metalloproteinases, which greatly promoted the formation and reconstruction of new blood vessels and accelerated the recovery of wounds. The content of TGF-β in wound tissue was determined by enzyme-linked immunosorbent assay, and the results were consistent with the above experiments. The CEHS group had the highest TGF-β content, reaching 136.84 ± 4.96 pg / mL on day 5, and slightly decreased to 133.23 ± 2.98 pg / mL on day 15 ( Figure 7H ).

[0062] In contrast, the TGF-β content was lower in the CEH and control groups. These results further demonstrate the superior performance of the CEHS group in promoting wound healing. The TGF-β content decreased slightly on day 15, which may be due to the fact that the wound healing process entered the remodeling phase and the role of TGF-β shifted to help tissue reorganization and fibrosis maturation. In addition, changes in cell signaling and the local microenvironment may also lead to a decrease in TGF-β concentration.

[0063] In conclusion, compared with the other two groups, the CEHS group not only had significantly higher expression of CD31, α-SMA, and vascular endothelial growth factor, but also had the highest HYP content and peak level of TGF-β in the early stage of wound healing. The CEHS group had a stronger effect on promoting angiogenesis, which was mainly attributed to the addition of sildenafil, which can enhance endothelial cell activity and improve the local microenvironment, thereby greatly promoting the repair and reconstruction of wound tissue and accelerating the healing process.

[0064] Finally, we further verified the safety of CEHS in vivo mainly through hemolysis test and tissue compatibility. The results of hemolysis test showed that CEHS did not cause obvious hemolysis of red blood cells (Figure 11A), and the hemolysis rate was less than 5% ( Fig.11 B), indicating that it is safe for use in vivo. In addition, on the 15th day after applying CEHS to the mouse wound, HE staining of major organs (heart, liver, spleen, lungs, and kidneys) confirmed that the tissue structure of these organs was not significantly different from that of healthy mice (Figure 11C). This finding indicates that CEHS has good tissue biocompatibility. Safety validation further demonstrates the feasibility and effectiveness of CEHS as a treatment strategy for infected wounds.

[0065] In summary, the inventors prepared a hydrogel mainly composed of natural egg white and chitosan, which contained sildenafil, by a simple method and can be used to treat infected wounds. First, this natural hydrogel retains the inherent advantages of its constituent materials, has good degradability, swelling and mechanical strength, meets the basic requirements of wound application, has good biocompatibility, can promote the proliferation and migration of 3T3 cells and HUVEC, and angiogenesis of HUVEC. In addition, it also has antioxidant, anti-inflammatory and antibacterial activities against Escherichia coli and Staphylococcus aureus. In a mouse model of infected wounds, egg white and chitosan hydrogel (CEHS) containing sildenafil effectively promoted wound healing through its excellent antioxidant and anti-inflammatory properties, antibacterial activity and the effect of promoting angiogenesis. Therefore, this multifunctional natural hydrogel with simple preparation has great potential in the field of infected wound treatment.

[0066] Specifically, CEHS can effectively reduce oxidative stress, inhibit the overexpression of inflammatory cytokines such as TNF-α and IL-1β, clear bacterial infection, and promote the formation of new blood vessels, thereby promoting the healing of infected wounds. In short, CEHS provides a promising alternative material for the field of infectious wound healing and has great potential for practical application.

[0067] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A sildenafil hydrogel, characterized in that: The invention comprises a hydrogel and sildenafil added into the hydrogel, and the raw materials of the hydrogel comprise natural egg white and chitosan.

2. The method for preparing a sildenafil hydrogel as described in claim 1, characterized in that: The following steps are involved: S1. Separate egg whites from fresh eggs and stir evenly to obtain egg white liquid; S2. preparing a mixed solution containing 2 wt% sildenafil, 3 wt% chitosan mixture and 0.5 M NaOH; S3. The protein solution prepared in step 1 and the mixed solution prepared in step 2 are mixed in a volume ratio of 1:1 to obtain a gelled product; S4. The gelled product obtained in step 3 is immersed in a DMEM solution to promote gelation and secondary crosslinking, and finally a chitosan-egg white hydrogel CEHS containing sildenafil is obtained.

3. Use of the sildenafil hydrogel as claimed in claim 1 in a medicine for promoting wound healing.

4. Use of the sildenafil hydrogel as claimed in claim 1 in a medicine for treating infected wounds.

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