Preparation of self-healing hydrogel and application of self-healing hydrogel in promoting wound healing

Abstract

The invention discloses preparation of self-healing hydrogel and application of the self-healing hydrogel in promoting wound healing, and belongs to the technical field of biological medicine. The self-healing hydrogel delivery system is composed of three parts, namely antibacterial metal nanoparticles, mesoporous nanoparticles loaded with the metal nanoparticles, and a hydrogel matrix playing supporting and adhesion roles. Through combination of photothermal therapy, the hydrogel can effectively remove bacteria on wounds, reduce inflammatory response of wound parts, promote migration of fibroblasts and keratinocytes, and play a positive role in rapid healing of the wounds. The self-healing hydrogel has an excellent effect in treatment of infected wounds and diabetic wounds. The hydrogel disclosed by the invention has self-healing effects of antibiosis, anti-inflammation and photo-thermal capacity, can promote wound healing, and provides an important reference for treating wounds by photo-thermal therapy.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to the preparation of a self-healing hydrogel and its application in the treatment of infected and diabetic wounds. Background Technology

[0002] The skin, the largest organ in the human body, plays a vital role in defending against pathogens and infections. However, it is frequently damaged by acute or chronic wounds caused by medical accidents, physiological abnormalities, or mechanical, thermal, or physical factors. Wound healing is a complex process involving intricate mechanisms such as the removal of foreign bodies and inflammation, granulation tissue formation, and scar maturation. Furthermore, wound healing requires the coordinated action of multiple cells and tissues to replace lost or damaged cell and tissue layers. Most skin wounds heal within one to two weeks, but infected wounds, especially full-thickness wounds, can lead to serious complications such as excruciating pain, amputation, and even death. Bacterial infection is one of the biggest obstacles to wound healing. Damage caused by microorganisms can lead to severe tissue damage, prolonged inflammatory responses, and delayed wound healing.

[0003] In recent years, effective treatments such as chemodynamic therapy (CDT), photodynamic therapy (PDT), sonic dynamic therapy (SDT), and photothermal therapy (PTT) have been applied to the treatment of acute and chronic wounds. Among these therapies, PTT promotes wound healing by inhibiting microorganisms around the wound through physical heating, promoting microcirculation, and stimulating cell vitality. It has attracted widespread attention due to its low side effects, good controllability, minimal invasiveness, high selectivity, and low potential to induce multidrug-resistant (MDR) microorganisms. Various inorganic materials (metal oxides, metal sulfides, metal nanoparticles, carbon materials) and organic nanoparticles (porphyrins, dopamine) are being developed as photothermal agents (PTAs) for PTT. Polydopamine (PDA) is an important natural melanin analogue and is considered one of the most promising PTAs due to its ease of preparation, good photostability, good biodegradability, and good biocompatibility. In recent years, researchers have designed PDA nanomaterials in various morphologies (nanotubes, nanospheres, and nanosheets) and different sizes (from 20 to 500 nanometers) for PTT. Among them, mesoporous polydopamine (MPDA) nanoparticles are often used in various drug delivery systems due to their high specific surface area.

[0004] Metal nanoparticles hold great promise for wound healing, enhancing healing efficiency through various mechanisms, such as nano-silver, nano-copper, and nano-gold. Nano-silver possesses potent broad-spectrum antibacterial activity, effectively killing various pathogenic microorganisms and reducing infection risk. It can regulate the production of cytokines and proteins, such as inflammatory factors and matrix metalloproteinases (MMPs), reducing inflammatory infiltration at the wound site. Nano-silver promotes the migration of fibroblasts and keratinocytes, playing a positive role in rapid wound closure. However, despite its excellent healing-promoting effects, high concentrations of nano-silver can inhibit the proliferation of healthy cells. Therefore, designing a formulation that minimizes the dosage and controls the release of nano-silver is crucial.

[0005] Furthermore, hydrogels, with their highly expandable three-dimensional porous structure, have proven to be one of the preferred materials for local wound applications. They can stimulate the body's self-repair mechanisms and promote the development of functional tissues by providing a suitable microenvironment. In addition, hydrogels exhibit excellent biocompatibility, effectively removing excess wound exudate and possessing biodegradable properties. Therefore, the release of silver nanoparticles can be controlled through the dual action of mesoporous materials and hydrogel networks. Combining this with photothermal therapy to enhance its antibacterial and healing-promoting effects is of great significance for clinical wound treatment. Summary of the Invention

[0006] The purpose of this invention is to construct a self-healing hydrogel that enables the sustained release of antibacterial metal nanoparticles, thereby promoting the healing of infected wounds and diabetic wounds through the combined effects of the antibacterial metal nanoparticles, photothermal effect, and hydrogel.

[0007] The technical solution adopted in this invention is as follows: The self-healing hydrogel with antibacterial, anti-inflammatory and photothermal capabilities described in this invention consists of three parts: antibacterial metal nanoparticles, mesoporous nanoparticles loaded with metal nanoparticles, and a hydrogel matrix that provides support and adhesion.

[0008] Preferably, the metal nanoparticles used for antibacterial purposes are silver nanoparticles, gold nanoparticles, copper nanoparticles, or zinc nanoparticles.

[0009] Preferably, the photothermal mesoporous nanoparticles used to load metal nanoparticles are mesoporous polydopamine nanoparticles or mesoporous carbon nanoparticles.

[0010] Preferably, the hydrogel matrix used for supporting and adhering functions is one or more of cationic guar gum, chitosan, guar gum, and xanthan gum.

[0011] The method for preparing the self-healing hydrogel with antibacterial, anti-inflammatory and photothermal properties of the present invention includes the following steps:

[0012] Step 1: Add Pranic F127 and mesitylene (TMB) to an ethanol-water solution and disperse evenly using a probe via ultrasonication. Adjust the pH of the system by adding tris(hydroxymethyl)aminomethane (Tris) solution in a 30–40°C water bath. After adding dopamine hydrochloride, stir in a 30–40°C water bath. After the reaction is complete, centrifuge the sample at 10,000–11,000 rpm for 10–15 min and discard the supernatant. Wash the precipitate twice with deionized water and twice with ethanol, and then wash with a mixture of ethanol and acetone (V... 乙醇 V 丙酮 After rinsing three times with a ratio of 2:1 and vacuum drying, mesoporous polydopamine carrier (MPDA) is obtained.

[0013] Step 2: Add AgNO3 to an ammonia solution and disperse MPDA in an ethanol solution. Add the MPDA solution dropwise to the AgNO3 solution and react in the dark to obtain silver-loaded mesoporous polydopamine nanoparticles Ag@MPDA.

[0014] Step 3: Adjust the pH to dissolve chitosan in the boric acid aqueous solution, dissolve cationic guar gum in PBS buffer, mix the two solutions, stir rapidly and adjust the pH to obtain cationic guar gum-chitosan-boric acid (CGCS) hydrogel.

[0015] Step 4: Disperse the Ag@MPDA nanoparticles into a solution, pour it into the CGCS hydrogel, and stir rapidly to ensure uniform dispersion. Let it stand in a refrigerator to complete the transformation process from sol to gel, obtaining cationic guar gum-chitosan-boric acid / Ag@MPDA(CCPA) hydrogel.

[0016] Preferably, in step 1, the mass ratio of Pluronic F127 to TMB is 1:0.5 to 1:4, the pH value of the alkaline environment is 8 to 10, the volume ratio of water to ethanol in the ethanol aqueous solution is 1:1 to 1:2, and the reaction time is 1 to 6 hours.

[0017] Preferably, in step 2, the mass ratio of AgNO3 to MPDA nanoparticles is 15:1 to 2:1, the amount of ethanol aqueous solution is 20 to 50 mL, the volume ratio of ethanol to water in the ethanol aqueous solution is 1:1 to 1:4, and the reaction time in the dark is 1 to 2 h.

[0018] Preferably, in step 3, the pH is adjusted to 4.5–5.0 to dissolve chitosan in 10–20 mL of boric acid aqueous solution, with a cationic guar gum to chitosan mass ratio of 10:1–2:1. The pH is then adjusted to 7.0–7.4, the stirring speed is 200–1000 rpm, and the stirring time is 10–20 min. The volume of PBS buffer is 10–20 mL.

[0019] Preferably, in step 4, the mass ratio of Ag@MPDA nanoparticles to CGCS hydrogel is 1:500 to 1:100, the stirring speed is 200 to 1000 rpm, the stirring time is 20 to 30 min, the standing temperature is 4 to 10 °C, and the standing time is 12 to 18 h.

[0020] The beneficial effects of this invention are:

[0021] (1) The self-healing CCPA hydrogel prepared in this invention can effectively adhere to the wound surface and provide sustainable treatment.

[0022] (2) The CCPA hydrogel prepared by the present invention exhibits long-lasting antibacterial and anti-inflammatory capabilities at the wound site. The therapeutic effect is further enhanced by the combined effects of Ag@MPDA nanoparticles, photothermal effect and hydrogel.

[0023] (3) By combining photothermal therapy, the hydrogel prepared in this invention can effectively eliminate bacteria on wounds, reduce inflammatory responses at the wound site, promote the migration of fibroblasts and keratinocytes, and play a positive role in rapid wound healing. This hydrogel has excellent effects in the treatment of infected wounds and diabetic wounds. The self-healing hydrogel of this invention, with its antibacterial, anti-inflammatory, and photothermal capabilities, effectively promotes wound healing and provides an important reference for photothermal therapy in wound treatment. Attached Figure Description

[0024] Figure 1 The transmission electron microscopy results of the MPDA nanoparticles prepared in Example 1 show that the prepared MPDA nanoparticles are spherical, uniform in size, and have obvious mesoporous structure with a particle size of about 200 nm.

[0025] Figure 2 The transmission electron microscopy results for the Ag@MPDA nanoparticles prepared in Example 2 show that the surface is loaded with silver nanoparticles with a particle size of approximately 220 nm.

[0026] Figure 3 XPS image of Ag@MPDA nanoparticles prepared in Example 2.

[0027] Figure 4 The image shows the XRD pattern of the Ag@MPDA nanoparticles prepared in Example 2.

[0028] Figure 5 The UV-Vis spectra of the Ag@MPDA nanoparticles prepared in Examples 1 and 2 are shown.

[0029] Figure 6 The image shows the zeta potential of the Ag@MPDA nanoparticles prepared in Examples 1 and 2.

[0030] Figure 7SEM images of the CGCS and CCPA hydrogels prepared in Examples 3 and 4.

[0031] Figure 8 Fourier transform infrared spectra of the CGCS and CCPA hydrogels prepared in Examples 3 and 4.

[0032] Figure 9 The figures show the bonding strength of the CGCS and CCPA hydrogels prepared in Examples 3 and 4, as determined by the bonding test.

[0033] Figure 10 Modulus-strain diagrams of the CGCS and CCPA hydrogels prepared in Examples 3 and 4.

[0034] Figure 11 The figure shows the dynamic step-strain rheological results of the CCPA hydrogel prepared in Example 4.

[0035] Figure 12 The image shows the apparent self-healing properties of the CCPA hydrogel prepared in Example 4.

[0036] Figure 13 The images show DCF / DAPI staining patterns of the CGCS and CCPA hydrogels prepared in Examples 3 and 4.

[0037] Figure 14 The images show bacterial plate coatings of the CGCS and CCPA hydrogels prepared in Examples 3 and 4.

[0038] Figure 15 Digital photographs of the healing process of mouse wound infection models using CGCS and CCPA hydrogels prepared in Examples 3 and 4.

[0039] Figure 16 The graph shows the healing area of ​​mouse wound infection models using CGCS and CCPA hydrogels prepared in Examples 3 and 4.

[0040] Figure 17 Digital photographs of the wound healing process of the CGCS and CCPA hydrogels prepared in Examples 3 and 4.

[0041] Figure 18 The above is a statistical graph showing the healing area of ​​the CGCS and CCPA hydrogels prepared in Examples 3 and 4 in diabetic mouse wound models. Detailed Implementation

[0042] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0043] Example 1

[0044] Dissolve 360 ​​mg of Pronic F127 in 100 mL of ethanol-water solution (V 水 V 乙醇 =1:1), add 400 μL of mesitylene, sonicate to disperse evenly, then add 4 mL of Tris solution (200 mg / mL) dropwise to adjust the system to alkaline pH 8-10. After stirring for 5 min, add 50-100 mg of dopamine hydrochloride, stir at 30℃ for 30 min, and continue the reaction for 2 h. After the reaction is complete, centrifuge at 10000 rpm for 10 min to collect the precipitate, wash 3 times with distilled water and 3 times with ethanol, and then evenly disperse in a mixed solution of ethanol and acetone (V 乙醇 V 丙酮 =2:1), the template agent was removed by sonication for 30 minutes. This operation was repeated 3 times, and the final black solid was MPDA nanoparticles. Transmission electron microscopy showed that the nanoparticles had uniform pores and spherical structures with a diameter of 200 nm. Figure 1 ).

[0045] Example 2

[0046] 40 mg of MPDA nanoparticles were ultrasonically dispersed in 5 mL of an ethanol aqueous solution (V... 乙醇 V 水 =1:1). Add 500 mg AgNO3 to 20 mL of ammonia solution and stir (V 氨水 V 水 =1:10). After AgNO3 was completely dissolved in the ammonia solution, MPDA solution was added dropwise to the AgNO3 solution and stirred in the dark for 1 hour. The mixture was then centrifuged at 10,000 rpm for 10 minutes to collect the precipitate, yielding Ag@MPDA nanoparticles. Transmission electron microscopy (TEM) images showed that silver nanoparticles grew on their surface, with a slightly increased diameter and spherical morphology compared to MPDA. XPS and XRD were used to analyze their elemental composition and the form in which the metals were present. Figure 3-4 ), measuring the Zeta potential of Ag@MPDA ( Figure 6 The results showed that the surface charge changed from -25.6 mV (MPDA) to -21.7 mV (Ag@MPDA). The transmission electron microscopy results of the prepared Ag@MPDA nanoparticles are as follows: Figure 2 As shown, the surface is loaded with silver nanoparticles with a particle size of approximately 220 nm. The UV-Vis spectrum is as follows. Figure 5 As shown.

[0047] Example 3

[0048] Adjust the pH to 4.5, dissolve 50 mg chitosan in 10 mL of boric acid aqueous solution (4%), and dissolve 400 mg cationic guar gum in 10 mL of PBS buffer. Mix the two solutions and stir with a mechanical stirrer for 10 min (500 rpm) and adjust the pH to 7.0-7.4 to obtain CGCS hydrogel.

[0049] Example 4

[0050] 50 mg Ag@MPDA was ultrasonically dispersed in 2 mL of water, and 20 g CGCS hydrogel was added and stirred for 20 min (500 rpm). The mixture was then allowed to stand overnight at 4°C to complete the sol-gel transition, yielding CCP hydrogel. The obtained hydrogels were subjected to scanning electron microscopy, Fourier transform infrared spectroscopy, adhesion testing, and rheological testing. Figure 7-10 ).

[0051] CGCS hydrogel has a porous interconnected structure and a smooth surface when magnified. Figure 7 A). After loading Ag@MPDA nanoparticles, the CCPA hydrogel mainly exhibits a honeycomb structure. Magnification confirmed that the Ag@MPDA nanoparticles are indeed uniformly present in the network structure within the hydrogel in the form of nanoparticles. Figure 7 B). CGCS hydrogel, CCPA hydrogel at approximately 3400, 2930, 1650, 1450 and 810 cm⁻¹. -1 All of these contain OH, CH, sugar ring vibrations, BO and BN coordination bonds. Figure 8 Compared to CGCS and CCP hydrogels, CCPA hydrogel exhibits the best lap shear strength. Figure 9 This is because dopamine nanoparticles provide more polyphenolic structures, which facilitate the formation of more hydrogen bonds between the gel and the tissue surface. Additionally, the quinone structure of oxidized dopamine forms covalent bonds with nucleophilic groups (-NH2, -SH) on the tissue surface. Stress scanning experiments determined that the linear viscoelastic range of CGCS is approximately 0.1%–30%, and that of CCPA is approximately 0.1%–80%. The gel point of both CGCS and CCPA is approximately 230%. Figure 10 Subsequently, the self-healing ability of the designed hydrogel was verified through dynamic step-strain rheological experiments. Figure 11When high strain is applied, the value of G' is lower than that of G″, indicating that the hydrogel has collapsed. Once low strain is restored, the value of G' increases sharply and instantaneously exceeds that of G″, indicating that the sample has recovered to the hydrogel state. This change can be repeated even after three alternating cycles, demonstrating the outstanding self-healing ability of CCPA hydrogel. To further verify the self-healing ability of CCPA hydrogel, sheet-like CCPA hydrogel was cut in half. After being placed in contact at ambient temperature for 1 hour, the two cut parts automatically bonded together to form a complete sheet-like gel. Figure 12 ).

[0052] Example 5

[0053] Take 2 mL of PBS, CGCS hydrogel, CCP hydrogel, and CCPA hydrogel, sterilize with UV light for 4 h, then transfer to a 50 mL centrifuge tube containing 30 mL of complete culture medium and extract until the hydrogel is completely degraded. Take L929 cells in logarithmic growth phase and use 2 × 10⁻⁶ cells / mL of PBS, CGCS hydrogel, CCP hydrogel, and CCPA hydrogel. 4 L929 cells were seeded at a density of 1 cell / well in 24-well plates and cultured for 12 hours. The culture medium was then discarded, and the cells were co-incubated with the extract of CCPA hydrogel for another 24 hours. Cells were stained with a mixture of calcein (AM) and propidium iodide (PI) for 30 minutes, and cell viability was observed under a confocal microscope. None of the CGCS hydrogel, CCP hydrogel, or CCPA hydrogel showed significant toxicity. Figure 13 ).

[0054] Example 6

[0055] Take 6 mL each of Escherichia coli and Staphylococcus aureus in the logarithmic growth phase (concentration 10). 8 CFU / mL, along with 2 mL PBS, CGCS hydrogel, CCP hydrogel, and CCPA hydrogel, were co-cultured in a bacterial incubator for 4 h in 50 mL test tubes. Some CCP and CCPA hydrogel groups were irradiated with 808 nm infrared light for 3 min and then cultured for another 2 h. 100 μL of the bacterial suspension was evenly spread onto solid culture medium and cultured in a bacterial incubator for 12 h, followed by photographing and counting. The CCPA+NIR group showed the best antibacterial effect. Figure 14 ).

[0056] Example 7

[0057] A full-thickness circular wound (diameter = 10 mm) was created on the back of each mouse. 100 μL of Staphylococcus aureus (1.0 × 10⁻⁶) was then injected. 7 A drop of CFU / mL solution was applied to a circular wound surface and spread evenly. Subsequently, 200 μL of PBS, CGCS hydrogel, CCP hydrogel, CCPA hydrogel, and CCP hydrogel + NIR (0.75 W / cm²) were used.2 ,3 min), CCPA hydrogel + NIR (0.75 W / cm 2 (3min), Tegaderm TM Mice were treated with dressings. Wounds were photographed using a digital camera at predetermined time intervals (days 0, 3, 7, and 14). Skin tissue samples were collected on day 14 for H&E and Masson staining. On day 14, the CCPA+NIR group had the smallest open wound area, smooth wound surface, and some newly formed epidermal and dermal tissue. The CCP+NIR group, CCPA group, CCP group, CGCS group, commercially available formulation group, and PBS group had larger open wound areas and uneven scar tissue. Figure 15-16 These results indicate that the CCPA+NIR group achieved the best wound healing outcomes compared to other groups.

[0058] Example 8

[0059] A full-thickness circular wound (diameter = 10 mm) was created on the back of each C57BL / 6 mouse with an established diabetes model. Subsequently, the wound was treated with 200 μL PBS, CGCS hydrogel, CCP hydrogel, CCP hydrogel, and CCP hydrogel + NIR (0.75 W / cm²). 2 ,3 min), CCPA hydrogel + NIR (0.75 W / cm 2 Mice were treated with dressings for 3 minutes. Wounds were photographed using a digital camera at predetermined time intervals (days 0, 3, 7, and 14). Skin tissue samples were collected on day 14 for H&E and Masson staining. On day 14, the CCPA+NIR group had the smallest open wound area, smooth wound surface, and some newly formed epidermal and dermal tissue; while the CCP+NIR, CCPA, CCP, CGCS, and PBS groups had larger open wound areas and significantly uneven scar tissue. Figure 17-18 These results indicate that the CCPA+NIR group achieved the best wound healing outcomes compared to other groups.

Claims

1. A self-healing hydrogel, characterized in that, The self-healing hydrogel described herein has antibacterial, anti-inflammatory, and photothermal capabilities, and is composed of metal nanoparticles for antibacterial purposes, mesoporous nanoparticles for loading the metal nanoparticles, and a hydrogel matrix that provides support and adhesion.

2. The self-healing hydrogel according to claim 1, characterized in that, The metal nanoparticles used for antibacterial purposes are one of Ag nanoparticles, Cu nanoparticles, Au nanoparticles, or Zn nanoparticles.

3. The self-healing hydrogel according to claim 1, characterized in that, The mesoporous nanoparticles used to load metal nanoparticles are a type of mesoporous polydopamine nanoparticles or mesoporous carbon nanoparticles.

4. The self-healing hydrogel according to claim 1, characterized in that, The hydrogel matrix that provides support and adhesion is one or more of cationic guar gum, guar gum, chitosan, and xanthan gum.

5. The method for preparing the self-healing hydrogel according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Dissolve Pronic F127 in an ethanol aqueous solution, add mesitylene and ultrasonically disperse evenly, add tris(hydroxymethyl)aminomethane solution and dopamine hydrochloride, stir and centrifuge to obtain mesoporous polydopamine; (2) The mesoporous polydopamine nanoparticles prepared in step (1) were ultrasonically dispersed in an aqueous ethanol solution and reacted with an aqueous ammonia solution of AgNO3 to obtain silver-loaded mesoporous polydopamine nanoparticles. (3) Adjust the pH to dissolve chitosan in the boric acid aqueous solution, dissolve cationic guar gum in PBS buffer, mix the two solutions, stir rapidly and adjust the pH to obtain cationic guar gum-chitosan-boric acid hydrogel; (4) The dispersion of silver-loaded mesoporous dopamine obtained in step (2) is mixed into the hydrogel in step (3) and stirred rapidly until uniform to obtain cationic guar gum-chitosan-boric acid / silver-loaded mesoporous polydopamine hydrogel.

6. The preparation method according to claim 5, characterized in that, In step (1): the mass ratio of Pranic F127 to mesitylene is 1:0.5 to 1:4, the reaction system is an alkaline environment with a pH of 8 to 10, the volume ratio of water to ethanol in the ethanol aqueous solution is 1:1 to 1:2, and the reaction time is 1 to 6 hours.

7. The preparation method according to claim 5, characterized in that, In step (2): the mass ratio of AgNO3 to mesoporous polydopamine nanoparticles is 10:1 to 2:1, the volume ratio of ethanol to water in the ethanol aqueous solution is 1:1 to 1:4, and the reaction time in the dark is 1 to 2 hours.

8. The preparation method according to claim 5, characterized in that, In step (3): adjust the pH to 4.5-5.0 to dissolve chitosan in 10-20 mL of boric acid aqueous solution, with a cationic guar gum to chitosan mass ratio of 10:1-2:1, then adjust the pH to 7.0-7.4, stir at 200-1000 rpm for 10-20 min; and add 10-20 mL of PBS buffer.

9. The preparation method according to claim 5, characterized in that, In step (4): the mass ratio of silver-loaded mesoporous polydopamine nanoparticles to cationic guar gum-chitosan-boric acid hydrogel is 1:500 to 1:100, the stirring speed is 200 to 1000 rpm, the stirring time is 20 to 30 min, the standing temperature is 4 to 10℃, and the standing time is 12 to 18 h.

10. The application of the self-healing hydrogel according to any one of claims 1-4 in the preparation of treatments for infected wounds, diabetic wounds, and the realization of photothermal wound healing.

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