A gelatin / polyacrylamide drug-loaded hydrogel dressing, construction method and application thereof

Abstract

The application provides a gelatin / polyacrylamide drug-loaded hydrogel dressing, a construction method and application thereof, and belongs to the fields of tissue engineering, material science and biology. A novel double-network interpenetrating hydrogel GEMC is prepared based on gelatin grafting dopamine GEDA, acrylamide and nanoclay NC as raw materials, and combined with curcumin nanomedicine CCNPs. The prepared GEMC hydrogel has good oxidation resistance, excellent mechanical properties (tensile, compression and viscoelasticity), excellent tissue adhesion (human, pig skin and glass) and good biocompatibility. In addition, the GEMC can be used as a drug release platform and can visually track the drug. The skin ulcer model caused by radiotherapy of mice proves that the GEMC hydrogel dressing can promote the skin regeneration of the radiotherapy wound, promote angiogenesis and reduce the inflammatory response. The current research results have great application potential in the treatment of skin ulcer caused by local radiotherapy of breast cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of tissue engineering, materials science and biology, and particularly relates to a gelatin / polyacrylamide hydrogel dressing, a construction method and applications thereof. BACKGROUND

[0002] Breast cancer is the highest incidence of cancer in women worldwide in recent years. At present, the main intervention method for the treatment of early breast cancer is local treatment, including surgical resection, local radiotherapy or adjuvant surgical treatment. In radiotherapy, although a large dose of X-ray irradiation can effectively kill cancer cells, it usually produces serious side effects. A large number of studies have shown that about 85%-95% of breast cancer patients have different degrees of skin damage due to radiotherapy. Unlike ordinary burns and ulcers, radiation directly damages the skin and its deep tissue cells, and the wound is more difficult to heal and more complex, with the most obvious manifestations of skin necrosis, inflammation, ulceration, etc. In addition, the excessive production of reactive oxygen species (ROS) will cause more serious inflammation, leading to a long time of non-healing of the wound of the patient, thus severely limiting the widespread application of radiotherapy. In view of the above, it is a key factor for the successful treatment of skin damage after radiotherapy to eliminate inflammation, promote angiogenesis, and improve the antioxidant capacity of the skin to avoid skin infection.

[0003] Nowadays, researchers have developed many clinical wound dressings for skin damage after radiotherapy. For example, necrotic skin debridement, traditional corticosteroids and anti-inflammatory drug dressings such as triethanolamine cream. However, due to the lack of uniform evaluation criteria, the conclusions of various studies often contradict each other and lack universality. In addition, traditional dry dressings, such as the use of absorbent cotton, absorbent gauze and sterile dressings, have limited therapeutic effect, and frequent dressing changes will also deepen the pain of patients. Therefore, it is still a great challenge to prepare an effective and high-quality solution for treating skin damage after breast cancer radiotherapy. Studies have shown that a moist environment under physiological conditions is conducive to the growth of granulation and the division of skin cells, thereby promoting complete healing of the wound. In view of the above considerations, a high-quality wound dressing should have good biocompatibility, maintain a moist environment, protect the wound from microbial invasion, and absorb wound exudates, etc. Hydrogels, as classic polymers with three-dimensional porous network structures, have shown great potential in wound dressings. However, traditional hydrogels still have many shortcomings, such as only providing a moist environment, weak mechanical strength, poor tensile properties and lack of adhesion, etc., which seriously limit their application in biological wound dressings. Therefore, it is necessary to develop a composite hydrogel through innovative strategies to improve its mechanical properties.

[0004] Polyacrylamide (PAM), as a cationic polymer, on the one hand has a higher water retention performance, which can keep the wound moist; secondly, PAM also has a higher water absorption performance, which can absorb the excess exudate of the wound, and help to maintain the normal physiological environment. In addition, PAM also has good biocompatibility and biosafety. Based on the above considerations, PAM has been favored in wound dressings in recent years. However, pure PAM has some deficiencies in the application of clinical wound dressings, for example, PAM can only form a loose cross-linked network structure and provide elasticity, but it is brittle and easy to break, which seriously affects its application in wound dressings. In order to solve this mechanical problem, a rigid dense cross-linked first network is introduced to protect the acrylamide network and maintain pressure, and finally a double cross-linked composite network hydrogel with toughness and elasticity coexisting is formed, which has become a more favored topic for researchers. Based on the above considerations, researchers have also prepared a series of double network cross-linked hydrogels, such as gelatin / polyacrylamide (Gelatin / PAM) DN hydrogel, alginate-calcium ion / polyacrylamide DN hydrogel, xanthan gum / polyacrylamide DN hydrogel and κ-carrageenan / polyacrylamide double physical cross-linked hydrogel. In addition, due to the excellent mechanical properties, biocompatibility and drug loading of double network hydrogel, it is widely used as wound dressings. For example, Zeng et al. prepared a pH and temperature dual-responsive microgel-embedded hydrogel by introducing poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAAm-co-AAc) microgel particles into a polyacrylamide (PAAM) / chitosan (CS) semi-IPN. The prepared hydrogel has excellent mechanical properties such as stretchability, compressibility and elasticity, which can significantly promote the wound healing process.

[0005] It is worth noting that compared with normal skin damage, radiotherapy can also cause more ROS synthesis, resulting in more serious oxidative damage and cytotoxicity. Therefore, it is worth studying to use antioxidant materials to reduce skin damage caused by radiotherapy. Inspired by mussels, catechol-mediated reversible non-covalent bonds have been shown to impart super-tissue adhesion and antioxidant capacity to hydrogels, thereby improving the mechanical properties of hydrogels. Therefore, grafting dopamine onto gelatin will impart good antioxidant properties to gelatin. Liu et al. prepared multifunctional hydrogels with adhesion, conductivity, antioxidant and antibacterial activity by grafting dopamine onto gelatin (GelDA) and coating graphene oxide with polydopamine (PGO), and also provided an application direction for multifunctional wound dressings to promote wound healing. However, it is worth noting that for adhesion hydrogels to achieve adhesion properties, catechol groups must be oxidized by oxygen or oxidizing agents. Therefore, the choice of oxidizing agent during the reaction is also a key factor. Traditional oxidizing agents often rapidly oxidize or cross-link catechol groups, resulting in problems such as short adhesion time and limited reuse. Most reported mussel-inspired adhesive hydrogels exhibit low mechanical strength and poor deformation ability, thus hindering the toughness and stretchability of hydrogel dressings.

[0006] Nanoclay has excellent biocompatibility and biodegradability, and has weak alkalinity, which can partially oxidize catechol structures such as dopamine, solve the drawbacks of dopamine peroxidation, and provide a weak alkaline environment that can facilitate the polymerization effect of acrylamide. Most importantly, the layered structure of nanoclay provides a large interfacial contact area, which can interact with polymers or biomolecules, and also improve drug loading capacity for better local drug release platforms. In summary, it is still a great challenge to prepare a wound dressing that has excellent adhesion, mechanical properties, antioxidant properties, and can perform local drug release while addressing the problems of wound repair and long-term local drug therapy after radiotherapy. SUMMARY

[0007] Therefore, the purpose of the present application is to provide a new drug-loaded wound hydrogel dressing (GEMC) for the treatment of skin ulcers, inflammation and other serious skin damage caused by local radiotherapy of breast cancer. The therapeutic effect of the drug-loaded wound hydrogel dressing (GEMC) on skin ulcers caused by radiotherapy of breast cancer is studied.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is:

[0009] A preparation method of a gelatin / polyacrylamide drug-loaded hydrogel dressing, comprising the following steps:

[0010] Step one, at room temperature, gelatin grafting dopamine (GEDA) and nanoclay (NC) are dissolved in deionized water and stirred thoroughly to obtain a mixed solution, and the mixed solution is in contact with oxygen molecules in the air during the stirring process. The exposure time of the air is 5 hours, and the purpose of the contact between the mixed solution and the oxygen molecules in the air is to oxidize the catechol groups in GEDA, thereby improving the adhesion of the material.

[0011] Further, 0.5-0.7 grams of GEDA and 0.1-0.2 grams of NC are added to every 10 ml of deionized water, and preferably 0.6 grams of GEDA and 0.15 grams of NC are added to every 10 ml of deionized water.

[0012] Further, in step one, the stirring speed is 15-30 rpm, and preferably 20 rpm.

[0013] Step two, preparation of GEMC composite hydrogel

[0014] 2.1) At room temperature, the monomers acrylamide (AM), N,N'-methylenebisacrylamide (MBAA) and curcumin nanoparticles (CCNPs) are mixed and then added to the mixed solution obtained in step one and stirred thoroughly to make them uniformly mixed. During this mixing process, the curcumin nanoparticles (CCNPs) act as a nano-drug and do not participate in chemical reactions in the GEMC composite hydrogel, but only undergo physical doping; MBAA acts as a crosslinking agent and polymerizes with AM, and can produce crosslinking between AM chains, thereby forming a polyacrylamide (PAM) three-dimensional network instead of unconnected polyacrylamide linear chains.

[0015] 2.2) Then, ammonium persulfate (APS) is added to the solution obtained in step 2.1) and allowed to react for 10 minutes, and then tetramethyl ethylene diamine (TEMED) is added dropwise to the mixed solution. During this mixing process, APS is used to generate free radicals, and the free radicals initiate AM polymerization; TEMED catalyzes the generation of free radicals by APS and initiates the polymerization of AM monomers.

[0016] 2.3) After the completion of the addition of TEMED, the mixed solution is ultrasonically vibrated for a few minutes at room temperature to remove air bubbles, and then it is quickly transferred to a reaction mold using a rubber bulb dropper, and polymerized at 50-70℃ for 1-3 hours to obtain a GEMC composite hydrogel with the same shape as the mold. During the polymerization reaction, if the temperature is too high, the decomposition rate of the initiator will be accelerated, the activity of the free radicals will be larger, and the reaction will be difficult to control; if the temperature is too low, the reaction will be too slow and it will be difficult to generate macromolecular chain polymers. Therefore, increasing the temperature to 50-70℃, which is the optimal temperature for the reaction, helps to generate macromolecular chain polymers.

[0017] 2.4) Finally, the GEMC composite hydrogel was sealed in a sterilized glass bottle and stored in a 4℃ refrigerator to prevent water evaporation.

[0018] Further, the adding amount of each substance in step two is: 3 grams of monomer AM, 0.01 grams of MBAA, 0.4 grams of CCNPs, 0.24 grams of APS and 20 μL of TEMED are added in every 10 ml of deionized water in step one.

[0019] Further, in step 2.1), the stirring speed is 15-30 rpm, preferably 20 rpm.

[0020] Further, in step 2.3), the polymerization reaction temperature is preferably 60℃, and the time is preferably 2h.

[0021] Further, in step 2.3), the size of the mold is 35 mm in diameter.

[0022] A gelatin / polyacrylamide drug-loaded hydrogel dressing based on gelatin / dopamine (GEDA), polyacrylamide (PAM) and nanoclay (NC) combined with curcumin nanomedicine (CCNPs) is prepared into a double-network interpenetrating hydrogel, which is a brown transparent hydrogel material, has rich functional groups (C-O, C=O, C-N and N-H), and has obvious pore structure and is doped with CCNPs curcumin nanomedicine, wherein the curcumin nanomedicine is prepared according to the patent 2022110502973 granted by the applicant in 2022.

[0023] The application of a gelatin / polyacrylamide drug-loaded hydrogel dressing is applied to the treatment of skin ulcers caused by tumor radiotherapy, that is, the repair of ulcerated skin caused by radiotherapy, and the type of tumor cells is breast cancer cells, which can promote skin regeneration at the ulcer site, has good biological safety, can inhibit the growth of breast cancer cells, and achieves the purpose of wound healing.

[0024] Analysis of the principles and innovations of the present application:

[0025] (1) The GEMC hydrogel prepared by the present application has good antioxidant property, excellent mechanical property (tensile, compression and viscoelasticity), excellent tissue adhesion (human, pig skin and glass) and good biocompatibility.

[0026] (2) The GEMC hydrogel prepared by the application contains three materials, gelatin dopamine grafting (GEDA), acrylamide (AM) and nanoclay (NC), all of which have high biocompatibility, no immunogenicity and no toxicity. GEDA contains rich o-diphenol groups, has excellent antioxidant properties, and is conducive to cell adhesion, growth and reproduction. Acrylamide forms polyacrylamide through polymerization reaction, has high water retention performance, can keep the wound moist, has high water absorption performance, can absorb excess exudate of the wound, helps to maintain normal physiological environment, and most importantly, has good biocompatibility and low biological toxicity, so it is widely studied in hydrogel dressings. NC not only has important regenerative medicine properties, such as cell adhesion and advantages conducive to cell diffusion, but also has the ability to regulate drug release, can increase the viscosity of polymer ink and stabilize the polymer network.

[0027] (3) The GEMC hydrogel prepared by the application can be used as a drug release platform and can visually track the released drugs.

[0028] (4) The GEMC hydrogel prepared by the application shows good biological safety and has obvious cytotoxicity to breast cancer cells, which can inhibit the growth of breast cancer cells.

[0029] (5) The GEMC hydrogel prepared by the application can promote skin regeneration at the ulceration site of the mouse after radiotherapy, promote angiogenesis while reducing inflammatory response, and thus achieve the purpose of wound healing.

[0030] Compared with the prior art, the application has the following beneficial effects:

[0031] (1) The GEMC hydrogel of the application has good biological safety and good tumor treatment effect.

[0032] (2) The GEMC hydrogel of the application has good antioxidant properties, excellent mechanical properties (tensile, compression and viscoelasticity), and excellent tissue adhesion (human, pig skin and glass).

[0033] (3) The GEMC hydrogel of the application can be used as a drug release platform and can visually track the released drugs.

[0034] (4) The GEMC hydrogel of the application can promote skin regeneration at the ulceration site of the mouse after radiotherapy, promote angiogenesis while reducing inflammatory response, through the dual effect of hydrogel and drug release.

[0035] In summary, the research of the application shows that the gelatin / polyacrylamide drug-loaded hydrogel dressing has great application potential in treating skin ulceration caused by local radiotherapy of breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 Scanning Electron Microscope (SEM) image of PAM (polymer of AM alone, control) hydrogel, scale: 100 μιη;

[0037] Figure 2 Scanning Electron Microscope (SEM) image of PAM (polymer of AM alone, control) hydrogel, scale: 100 μιη; Figure 1 Magnified SEM image of PAM hydrogel, scale 10 μιη;

[0038] Figure 3 Scanning Electron Microscope (SEM) image of AMNC (polymer of AM and NC, control) hydrogel, scale: 100 μιη;

[0039] Figure 4 Scanning Electron Microscope (SEM) image of AMNC (polymer of AM and NC, control) hydrogel, scale: 100 μιη; Figure 3 Magnified SEM image of AMNC hydrogel, scale 10 μιη;

[0040] Figure 5 Scanning Electron Microscope (SEM) image of GEM (polymer of GEDA, AM and NC, control) hydrogel, scale: 100 μιη;

[0041] Figure 6 Scanning Electron Microscope (SEM) image of GEM (polymer of GEDA, AM and NC, control) hydrogel, scale: 100 μιη; Figure 5 Magnified SEM image of GEM hydrogel, scale 10 μιη;

[0042] Figure 7 Scanning Electron Microscope (SEM) image of GEM (polymer of GEDA, AM and NC, control) hydrogel, scale: 100 μιη;

[0043] Figure 8 Scanning Electron Microscope (SEM) image of GEM (polymer of GEDA, AM and NC, control) hydrogel, scale: 100 μιη; Figure 7 Magnified SEM image of GEMC hydrogel, scale 10 μιη;

[0044] Figure 9 Full scan XPS spectra of PAM, AMNC, GEM and GEMC;

[0045] Figure 10 High resolution XPS of oxygen element of PAM;

[0046] Figure 11 High resolution XPS of nitrogen element of PAM;

[0047] Figure 12 High resolution XPS of carbon element of PAM;

[0048] Figure 13 High resolution XPS of oxygen element of AMNC;

[0049] Figure 14 High resolution XPS of nitrogen element of AMNC;

[0050] Figure 15 High resolution XPS of oxygen element for GEMC;

[0051] Figure 16 High resolution XPS of nitrogen element for GEMC;

[0052] Figure 17 High resolution XPS of carbon element for GEMC;

[0053] Figure 18 High resolution XPS of oxygen element for GEMC;

[0054] Figure 19 High resolution XPS of nitrogen element for GEMC;

[0055] Figure 20 High resolution XPS of carbon element for GEMC;

[0056] Figure 21 High resolution XPS of oxygen element for GEMC;

[0057] Figure 22 Fourier transform infrared (FTIR) spectra of PAM, AMNC, GEM and GEMC;

[0058] Figure 23 Absorption spectra of PAM, AMNC, GEM and GEMC;

[0059] Figure 24 Fluorescence spectra of PAM at different excitation wavelengths;

[0060] Figure 25 Fluorescence spectra of AMNC at different excitation wavelengths;

[0061] Figure 26 Fluorescence spectra of GEM at different excitation wavelengths;

[0062] Figure 27 Fluorescence spectra of GEMC at different excitation wavelengths;

[0063] Figure 28 Degradation rate of PAM at different days;

[0064] Figure 29 Degradation rate of AMNC at different days;

[0065] Figure 30 Degradation rate of GEM at different days;

[0066] Figure 31 Degradation rate of GEMC at different days;

[0067] Figure 32 Swelling rates of PAM, AMNC, GEM, and GEMC at different times;

[0068] Figure 33 Drug release curves of GEMC at different days and different pH values;

[0069] Figure 34 Images showing drug release from GEMC hydrogel in a buffer solution at pH 5.0 under bright field and 365 nm irradiation conditions (at different time points);

[0070] Figure 35 Images showing drug release from GEMC hydrogel in a buffer solution at pH 6.8 under bright field and 365 nm irradiation conditions (at different time points);

[0071] Figure 36 Images showing drug release from GEMC hydrogel in a buffer solution at pH 7.4 under bright field and 365 nm irradiation conditions (at different time points);

[0072] Figure 37 Images showing drug release from GEMC hydrogel in a buffer solution at pH 8.0 under bright field and 365 nm irradiation conditions (at different time points); Figure 38 For live / dead cell staining to detect drug release of GEM, GEMC and CCNPs at 1 day (NIH-3T3 cells), scale bar, 200 μm;

[0073] Figure 39 For live / dead cell staining to detect drug release from GEM, GEMC and CCNPs at 3 days (NIH-3T3 cells), scale bar, 200 μm;

[0074] Figure 40 For live / dead cell staining to detect drug release from GEM, GEMC and CCNPs at 7 days (NIH-3T3 cells), scale bar, 200 μm;

[0075] Figure 41 For live / dead cell staining to detect drug release from GEM, GEMC and CCNPs at 14 days (NIH-3T3 cells), scale bar, 200 μm;

[0076] Figure 42 For live / dead cell staining to detect drug release of GEM, GEMC and CCNPs at 1 day (MDA-MB-231 cells), scale bar, 200 μm;

[0077] Figure 43Live / dead cell staining to detect GEM, GEMC and CCNPs drug release at 3 days (MDA-MB-231 cells), scale bar, 200 pm;

[0078] Figure 44 Live / dead cell staining to detect GEM, GEMC and CCNPs drug release at 7 days (MDA-MB-231 cells), scale bar, 200 pm;

[0079] Figure 45 Live / dead cell staining to detect GEM, GEMC and CCNPs drug release at 14 days (MDA-MB-231 cells), scale bar, 200 pm;

[0080] Figure 46 Storage modulus G' and loss modulus G" as a function of angular frequency for PAM, AMNC, GEM and GEMC;

[0081] Figure 47 Stress-strain curves in compression test for PAM, AMNC, GEM and GEMC;

[0082] Figure 48 Stress-strain curves in tensile test for PAM, AMNC, GEM and GEMC;

[0083] Figure 49 Qualitative determination of the adhesion of PAM, AMNC GEM and GEMC hydrogels to human skin;

[0084] Figure 50 Quantitative analysis of the adhesion energy of PAM, AMNC, GEM and GEMC hydrogels on glass surfaces;

[0085] Figure 51 Quantitative analysis of the adhesion strength of PAM, AMNC, GEM and GEMC hydrogels on glass surfaces;

[0086] Figure 52 Quantitative analysis of the adhesion energy of PAM, AMNC, GEM and GEMC hydrogels on pig skin surfaces;

[0087] Figure 53 Quantitative analysis of the adhesion strength of PAM, AMNC, GEM and GEMC hydrogels on pig skin surfaces;

[0088] Figure 54 Efficiency of PAM, AMNC, GEM and GEMC hydrogels in scavenging DPPH radicals;

[0089] Figure 55 Efficiency of PAM, AMNC, GEM and GEMC hydrogels in scavenging ABTS radicals;

[0090] Figure 56 To detect ROS in NIH-3T3 cells incubated with PAM, AMNC, GEM, and GEMC using the DCFHDA probe;

[0091] Figure 57 To detect ROS in MDA-MB-231 cells incubated with PAM, AMNC, GEM, and GEMC using the DCFHDA probe;

[0092] Figure 58 Images of live / dead cells stained in GEMC hydrogel for NIH-3T3 cells;

[0093] Figure 59 Images of live / dead cells stained in GEMC hydrogel for MDA-MB-231 cells;

[0094] Figure 60 SEM images of NIH-3T3 cells on GEMC hydrogel at different culture times;

[0095] Figure 61 SEM images of MDA-MB-231 cells on GEMC hydrogel at different culture times;

[0096] Figure 62 Blood compatibility studies of negative controls, PAM, AMNC, GEM, GEMC hydrogels, and positive controls;

[0097] Figure 63 The hemolysis rate was assessed for negative controls, PAM, AMNC, GEM, GEMC hydrogels, and positive controls.

[0098] Figure 64 A timeline for the use of GEMC hydrogel in the treatment of post-radiotherapy wound ulceration;

[0099] Figure 65 Photographs showing the healing process of radiation-induced skin damage treated with hydrogel dressings (GEM, GEMC);

[0100] Figure 66 In vivo fluorescence imaging of mice with radiation-induced skin damage at 1, 3, 7, and 14 days after GEMC hydrogel treatment;

[0101] Figure 67 For quantitative analysis Figure 68 Average fluorescence signal at the wound site over time.

[0102] Figure 69 Quantitative detection of tumor necrosis factor-α (TNF-α) in wounds at different treatment times (3, 7, and 14 days);

[0103] Figure 70 Quantitative detection of interleukin-10 (IL-10) factor in the wound surface for different treatment times (3, 7, 14 days);

[0104] Figure 71 Immunohistochemical CD68 antibody staining of the wound surface for different treatment times (3, 7, 14 days);

[0105] Figure 72 H&E staining of the wound surface for different treatment times (3, 7, 14 days);

[0106] Figure 73 Masson staining of the wound surface for different treatment times (3, 7, 14 days);

[0107] Figure 74 Immunohistochemical CD31 antibody staining of the wound surface for different treatment times (3, 7, 14 days);

[0108] Figure 1 Schematic diagram of the present application. DETAILED DESCRIPTION

[0109] In order to further understand the present application, the preferred embodiments of the present application are described below in conjunction with specific embodiments, but it should be understood that these descriptions are only to further illustrate the features and advantages of the present application and are not a limitation on the patent claims of the present application.

[0110] The present application provides a new type of gelatin / polyacrylamide drug-loaded hydrogel dressing based on skin repair for ulceration caused by breast cancer radiotherapy. Gelatin dopamine grafting (GEDA), polyacrylamide (PAM) and nanoclay (NC) are combined with curcumin nanomedicine (CCNPs) to prepare. Among them, the curcumin nanomedicine is prepared by the patent 2022110502973 of the applicant in 2022.

[0111] 1. The present application also provides a preparation method of the above-mentioned gelatin / polyacrylamide drug-loaded hydrogel dressing:

[0112] Step one, dissolve gelatin dopamine grafting (GEDA) and nanoclay (NC) in deionized water, stir thoroughly and expose the ingredients to air; 0.6 grams of GEDA and 0.15 grams of NC are added to every 10 ml of deionized water, and the exposure time in air is 5 hours. Among them, the stirring speed is 20 rpm.

[0113] Step two, mix monomer acrylamide (AM), N,N'-methylene bisacrylamide (MBAA) and curcumin nanoparticles (CCNPs), wherein the curcumin nanoparticles (CCNPs) are carbon dots; dissolve them in the deionized water solution of step one and mix and stir until uniform. Then add the initiator ammonium persulfate (APS), and after 10 min of reaction, drop tetramethyl ethylenediamine (TEMED) into the mixed solution. Finally, ultrasonically vibrate the mixed solution at room temperature for a few minutes to remove air bubbles, and then quickly transfer it to a reaction mold with a rubber bulb dropper. After polymerization at 60°C for 2h, GEMC composite hydrogel with the same shape as the mold is obtained. Finally, these hydrogels are sealed in sterilized glass bottles and stored in a refrigerator at a speed of 4°C to prevent water evaporation. The corresponding 3 grams of monomer acrylamide, 0.01 grams of N,N'-methylene bisacrylamide, 0.4 grams of carbon dot-curcumin nanoparticles, 0.24 grams of ammonium persulfate and 20 μL of tetramethyl ethylenediamine are added to every 10 ml of deionized water. The size of the mold is 35 mm in diameter. The stirring speed is 20 rpm.

[0114] 2. The present application also provides the use of the above-mentioned gelatin / polyacrylamide drug-loaded hydrogel dressing in the treatment of skin ulcers caused by breast cancer radiotherapy.

[0115] Step one, subcutaneously inoculate breast cancer MDA-MB-231 cell lines (8 x 10 6 / 200 μL) into the left limb of nude mice. Seven days after inoculation, a mouse breast cancer model is established, and the tumor-bearing mice are fixed on the operating bed for tumor resection.

[0116] Step two, on the third day after tumor resection, the mice are anesthetized and fixed on the operating platform, and the left upper limb skin of the mice is irradiated with a biological X-ray instrument at 25Gy (1.19Gy / min). After irradiation, all mice are observed daily and fed to the tenth day. When the skin at the irradiated site begins to ulcerate, it is considered that the radiotherapy damage model has been successfully established.

[0117] Step three, after the establishment of the mouse radiotherapy damage model, GEMC hydrogel is pasted on the radiotherapy damage site, and mice without dressing are used as blank controls, and mice treated with GEM hydrogel are used as treatment controls. Since the hydrogel does not need to be fixed with other materials, and continuous exposure to air will cause a certain amount of water loss, the GEM and GEMC hydrogel dressings are replaced every three days. On the 3rd, 7th and 14th days after treatment, the wounds are observed and photographed, and skin specimens are collected at the above-mentioned time points for tumor necrosis factor-α (TNF-α), interleukin 10 (IL-10) detection and section staining analysis to detect the treatment effect of GEMC on the mouse breast cancer radiotherapy damage model.

[0118] Example 1

[0119] First step, synthesis of gelatin / polyacrylamide-based drug-loaded hydrogel

[0120] Step one, GEDA 6%, w:v) and NC (1.5%, w:v) were dissolved in 20 mL of super-solution, charged with stirring, exposed to air for 5 hours, the purpose was to partially oxidize GEDA, increase the adhesion conditions.

[0121] Step two, after the reaction was completed, the monomer acrylamide (AM, 40%, w:v), MBAA (0.3%, w:v) and CCNPs (0.4g) were mixed and dissolved in the above GEDA super-solution for 10 minutes.

[0122] Step three, 0.3% (w:v) initiator APS was added to the above mixture, and it was left for 10 min, and 5 μL TEMED was added dropwise. Finally, the mixed solution was ultrasonically vibrated for a few minutes at room temperature to eliminate air bubbles, and then quickly transferred to a reaction mold (the size of the mold was 35 mm in diameter) with a rubber dropper.

[0123] Step four, polymerization at 60°C for 2 hours to obtain GEMC hydrogel with the same shape as the mold. Finally, these hydrogels were sealed in sterile glass bottles and stored in a refrigerator at 4°C to prevent water precipitation and evaporation.

[0124] Step five, in addition, PAM AMNC and GEM hydrogels were prepared as control groups by the same method as above. Only AM was used as raw material for polymerization to form PAM hydrogel. AMNC hydrogel was formed by using AM and NC as raw materials. The preparation process of GEM hydrogel is the same as GEMC, except that CCNPs are not added.

[0125] Preparation and characterization of the synthesized GEMC hydrogel.

[0126] 1.1 Morphology and structure analysis of GEMC

[0127] First, the morphology of PAM, AMNC, GEM and GEMC was analyzed by scanning electron microscopy (SEM). Figure 2 The scanning electron microscope image of PAM is shown in Figure 1 The local enlargement of Figure 3 From the image, it can be seen that PAM only presents a dense surface morphology by the polymerization of acrylamide, and is smooth and flat. It is well known that NC has a typical layered structure, which can promote chemical bonding with the polymer during the polymerization process, thereby increasing the cross-linking degree of the polymer. Therefore, compared with pure PAM, the surface density of AMNC is increased after adding NC, and the pore structure is obviously Figure 4 , Figure 3 The scanning electron microscope image of GEMC is shown in Figure 5(Local magnification). With the addition of GEDA ( Figure 6 , Figure 5 for Figure 7 (A local magnification) shows that the GEM hydrogel exhibits an interconnected porous structure with a significantly increased pore size, which is the result of the copolymerization of GEDA and PAM. Figure 8 The microstructure of GEMC was revealed. The results show that GEMC maintains the porous network structure of GEM due to CCNP doping, while adding more particles, indicating that GEMC successfully underwent PAM polymerization and non-covalent crosslinking with GEDA, loading the nanomaterial CCNPs (… Figure 7 for Figure 9 (A magnified view of a part).

[0128] Secondly, this application underwent XPS testing, and the results are as follows: Figures 10-12 As shown in the full-scan XPS spectrum, three peaks were observed for PAM, AMNC, GEM, and GEMC. These peaks belong to C1s (286.72 eV), N1s (398.28 eV), and O1s (528.67 eV), respectively. The functional group structure of the hydrogel was then further analyzed. Figures 13-15 The O1s, N1s, and C1s functional groups of PAM were displayed, indicating that PAM contains C=O and CN. Furthermore, high-resolution XPS analysis using AMNC revealed the C=O and CN functional groups (…). Figure 16 Next, the GEM hydrogel was analyzed, and the results showed that the high-resolution spectrum of O1s ( Figure 17 The spectrum can be divided into two peaks, C=O (284.5 eV) and CO (285.8 eV), mainly due to the addition of the GEDA chemical bond. The high-resolution spectrum of N1s (…) Figure 18 The spectrum shows two peaks, located at 399.06 eC (CN) and 400.02 eV (NH), respectively. The C1s band ( Figure 19 The values ​​correspond to 284.46 eV (CC / C=C), 285.70 eV (CN / CO), and 287.62 eV (C=O). These results indicate that GEDA has been successfully loaded into the GEM polymer. Figure 20 The high-resolution XPS spectrum of the O1s of GEMC, containing CO and C=O, is shown. Notably, the CO content increases significantly due to the addition of CCNPs. Furthermore, the high-resolution spectrum of the N1s is also shown. Figure 21 The results showed two fitted peaks, corresponding to CN (399.17 eV) and NH (400.81 eV), which are identical to the GEM components, indicating successful mixing of GEDA. C1s( Figure 22) can be divided into C-C / C=C (283.97 eV), C-N / C-O (285.10 eV), C=O (287.56 eV), in addition, the content of C-N / C-O functional groups is high, mainly due to the doping of CCNPs and GEDA. In summary, GEMC successfully polymerizes PAM and loads GEDA and CCNPs.

[0129] Next, the present application further analyzes the functional groups of PAM, AMNC, GEM and GEMC by Fourier transform infrared spectroscopy (FTIR). The results are shown in Figure 23 PAM, AMNC, GEM and GEMC exhibit C=O vibration absorption band at 1647 cm -1 -1. The stretching vibration spectrum of N-H bond and O-H bond is observed at 3056 cm -1 -1, and the display wavelength of GEMC and GEM corresponds. In summary, the functional groups in the above FTIR are consistent with the XPS results.

[0130] 1.2 Optical properties of GEMC

[0131] The luminescent properties of GEMC are analyzed by luminescent properties. Figure 24 The absorption spectra of PAM, AMNC, GEM and GEMC are shown respectively. The results show that PAM, AMNC and GEM have no light absorption ability, while GEMC has obvious absorption peak, and the position of the absorption peak is consistent with that of CCNPs. The results show that the doping of CCNPs endows GEMC with light absorption ability. Next, the fluorescence spectra of PAM( Figure 25 ), AMNC( Figure 26 ), GEM( Figure 27 ) and GEMC( Figure 31 ) are detected respectively. The results show that PAM, AMNC and GEM have no fluorescence phenomenon, while GEMC shows good luminescence behavior after excitation at different wavelengths. The luminescence intensity is consistent with the change trend of CCNPs, which further shows that GEMC hydrogel has good luminescence performance.

[0132] 1.3 Degradation performance and water absorption performance test of GEMC

[0133] Next, the present application studies the degradation of GEMC in different PH buffer solutions( Figure 28 ). At the same time, PAM( Figure 29 ), AMNC( Figure 30 ) and GEM( Figure 32) hydrogel as a control. The results show that the four hydrogels have good degradation effect, and the degradation rate is the largest when pH = 6.8. That is, when pH = 6.8, the degradation rate of PAM is 33.45%, the degradation rate of AMNC is 37.94%, and the degradation rate of GEM is 45.37%, indicating that the addition of GEDA increases the degradation rate of the hydrogel. The degradation rate of GEMC hydrogel at pH = 6.5 (47.13%) is basically the same as that of GEM hydrogel. The results show that the addition of CCNPs does not affect the degradation performance of GEM hydrogel. In summary, GEMC hydrogel has obvious pH-responsive degradation performance and is more sensitive to weak acidic environment, so GEMC hydrogel has application potential in tumor treatment applications.

[0134] Then, the water absorption rate of the hydrogel within 48 hours was studied, as shown in Figure 33 Within 24h of water absorption, the water absorption rate of the hydrogel increased rapidly. The 24h water absorption rate of PAM (714.81%), AMNC (680.53%), GEM (518.36%) and GEMC (469.08%) was more than 400%. With the extension of water absorption time, the water absorption rate of each hydrogel tended to be stable after 24h, and reached the maximum water absorption rate at 48h. It is worth noting that the double network cross-linked GEM (529.36%) and GEMC (499.46%) are slightly lower than PAM (720.81%) and AMNC (763.52%) hydrogels, but the water absorption performance is still very good. The water absorption effect of GEM and GEMC is basically the same, indicating that the addition of CCNPS does not affect the water absorption performance of GEMC.

[0135] 1.3 Drug release detection of GEMC

[0136] Drug release research is an important content of drug release system research. Figures 34-37The drug release behavior of GEMC in different pH buffer solutions (8.0, 7.4, 6.5 and 5.0) was shown. The different pH values of the buffer solution were used to simulate different environments, corresponding to the wound site (pH = 8.0), plasma (pH = 7.4), extracellular environment of tumor cells (pH = 6.5) and inside tumor cells (pH = 5.0), respectively. The results showed that the cumulative drug release amount of GEMC hydrogel in the above different buffer solutions increased with time, and reached a drug release equilibrium state at 42 d, and the release efficiency was greatly affected by pH. When the release time was 42 days, GEMC hydrogel reached the maximum drug release rate (90.06%) at pH = 6.5, indicating that GEMC hydrogel was more likely to release in the weak acidic environment. At 42 days, the final drug release rate of pH = 5.0 (78.40%), pH = 7.4 (70.48%) and pH = 8.0 (75.02%) was not higher than that of GEMC hydrogel, but all reached more than 70%. The above results showed that the degradation of GEMC hydrogel also accelerated its drug release behavior. In addition, it also showed that GEMC hydrogel could achieve good drug release effect in the slightly acidic tumor environment and the wound environment.

[0137] Next, the present application visually analyzed the drug release by images under bright field and 365 nm conditions. As shown in Figures 38-41 , the color of GEMC hydrogel in different pH buffer solutions gradually deepened with the extension of time. When the drug release time reached 42 days, the images under bright field and 365 nm light under different pH conditions were compared, and the results showed that the color of the solution under different pH conditions reached the maximum color of the same pH group. Notably, the color was the deepest at pH = 6.5. In addition, the solutions with different pH values (8.0, 7.4, 6.5 and 5.0) had different fluorescence emissions at 365 nm, which was mainly due to the solvent effect (different pH). These results showed that GEMC hydrogel had good drug release effect, which was consistent with the quantitative analysis results of drug release. In summary, GEMC hydrogel was beneficial to the release of drugs in the acidic environment of tumor, and still had good sustained release effect in the wound injury environment, and had certain application prospect in tumor treatment and wound healing.

[0138] Next, the present application further investigated the toxicity of GEM, GEMC and CCNPs and the drug release of GEMC from the cell level, and the blank cells (NIH-3T3 and MDA-MB-231 cells) without material treatment were used as the control group. The present application carried out live / dead cell staining experiment, and it was worth noting that Hoechst staining showed that the cell nucleus emitted blue fluorescence. The live cells stained by calcein emitted green fluorescence, and the dead cells stained by iodine pyridine emitted red fluorescence Figures 42-45The cell staining images of GEM, GEMC and CCNPs treated NIH-3T3 cells at 1, 3, 7 and 14 days are shown respectively. Figure 46 The cell staining of MDA-MB-231 cells treated with GEM, GEMC 19 and CCNPs for 1, 3, 7 and 14 days is shown respectively. The results show that the green fluorescence intensity of GEM, GEMC and CCNPS treated NIH-3T3 cells gradually increases with the extension of the action time. At 14 days, although the red fluorescence is enhanced, there is still a large amount of green fluorescence, and cell death is caused by too many cells and insufficient oxygen in the cell space, which does not affect the overall growth of the cells. The results show that GEM has good biocompatibility with CCNPs, and the drug release of GEMC does not cause damage to normal cells. For MDA-MB-231 cells treated with different materials (GEM, GEMC and CCNPs), the red fluorescence of MDA-MB-231 cells treated with GEMC gradually increases with time, and almost no green fluorescence can be seen at 14 days, indicating that the cells have basically died. Experiments have proved that GEMC has good drug release effect and good tumor killing ability. As for CCNPs, it is difficult to see green fluorescence after CCNPs treat MDA-MB-231 for one day, and the number of cells can be seen from the cell nucleus staining. With the increase of treatment time, MDA-MB-231 cells almost lose activity, and the cell nucleus has been dissolved at 14 days, indicating that CCNPs can directly kill tumor cells and significantly inhibit the growth of tumor cells. In summary, GEMC hydrogel has a long-term drug release effect and can inhibit the growth of MDA-MB-231 cells for up to 14 days, and is harmless to normal cells, and is expected to become a new drug delivery platform for local drug release and inhibition of tumor growth.

[0139] 1.4Mechanical property test of GEMC hydrogel

[0140] Good mechanical properties are the key and challenge of hydrogel wound dressings. First, the mechanical properties of GEMC were characterized by dynamic rheological test. The changes of storage modulus G' and loss modulus G" of different hydrogels (PAM, AMNC, GEM and GEMC) at different angular frequencies were tested by frequency measurement method respectively. Figure 47). The results show that the G" of PAM hydrogel is always significantly greater than G' at different angular frequencies, which can be attributed to the collapse of its hydrogel network. For AMNC hydrogel, the G' of the sample begins to decline after the angular frequency is greater than 46 rad / s, which is mainly due to the destruction of its hydrogel network. The above results show that the mechanical properties of PAM and AMNC hydrogels are not stable. For GEM and GEMC, within the angular frequency range of 0.1-100 rad / s, the G' of the sample is always greater than G", which can be attributed to the addition of GEDA, which increases the crosslinking degree of the sample, increases G', indicating that GEM and GEMC hydrogels both have good elastic properties and stable mechanical structure.

[0141] Next, the present application carries out compression test on the hydrogel. Figure 48 The stress-strain curves of each hydrogel are shown. Since none of the test pieces broke when the compression strain reached 60%, the stress here is defined as the maximum compressive stress. The data shows that the compressive stress of GEM and GEMC is smaller than that of PAM and AMNC. The results show that GEM and GEMC are easier to compress, have better compression performance, and are softer than PAM and AMNC, which lays a foundation for the subsequent bonding of materials.

[0142] The mechanical properties of the prepared hydrogels (PAM, AMNC, GEM and GEMC) are characterized by uniaxial tensile test. Figure 49 The stress-strain curves of the above hydrogel samples are shown, and the results show that the fracture strain of GEM and GEMC is 382.43±4.34% and 313.63±2.45%, respectively. In summary, CCNPs do not affect the tensile properties of GEMC on the one hand, and on the other hand, the result of mixed double network indeed helps to obtain the tensile properties and high toughness of GEMC hydrogel.

[0143] 1.5 Adhesion performance test of GEMC hydrogel

[0144] The adhesion of hydrogel samples to skin tissue is an important factor in wound dressing applications. In order to facilitate observation, the present application first shows the deformation, stretching and adhesion of PAM, AMNC, GEM and GEMC on human skin Figure 50 ). In fact, PAM is easily peeled off from the skin under the action of external force, and a little force will make it break, so it can only be pulled a little. In addition, although the elasticity and viscosity of AMNC are slightly improved compared with PAM, it still cannot fully adhere to human skin. While GEM and GEMC can both exhibit good adhesion on the skin, it can be seen that GEMC hydrogel has higher skin adhesion and is more closely adhered to human skin, which may be due to the rich polyphenol structure in GEDA and CCNPs.

[0145] Next, the adhesion behavior of each hydrogel on glass and pig skin tissue was further analyzed by the universal testing machine. The results showed that the adhesive energy of GEMC on glass was 2.93 J m -2 ( Figure 51 ), the adhesive strength was 4.42 KPa Figure 52 ), which was higher than PAM (adhesive energy was 1.47 J m -2 , adhesive strength was 1.78 KPa), AMNC (adhesive energy was 2.00 J m -2 , adhesive strength was 3.24 KPa) and GEM (adhesive energy was 3.43 J m -2 , adhesive strength was 3.97 KPa). In addition, Figure 53 and Figure 54 showed the adhesive energy and adhesive strength of different hydrogels on pig skin tissue. The results also showed that GEMC had better adhesive properties. In summary, GEMC had good adhesive properties, which laid the foundation for its application in wound dressings.

[0146] 1.6 Antioxidant test of GEMC hydrogel

[0147] It is well known that breast cancer is prone to serious wound ulceration during radiotherapy, and the wound is prone to produce reactive oxygen species (ROS), which hinders wound healing. Therefore, effectively removing ROS from the wound and improving the antioxidant properties of wound dressings is the key to treating post-radiotherapy wound damage. In this paper, the DPPH and ABTS radical scavenging method was used to test the antioxidant capacity of GEMC hydrogel. First, Figure 55 shows the DPPH radical scavenging effect of different hydrogel samples. Among the hydrogels, PAM (0.96 ± 0.31%) and AMNC (3.49 ± 0.47%) had almost no antioxidant capacity, and the antioxidant activity of GEMC hydrogel (65.41 ± 3.26%) was significantly higher than that of GEM hydrogel (43.09 ± 2.67%). Next, the oxidation performance of each material was further verified by the ABTS radical scavenging method Figure 56 . The results showed that GEDA, CCNPS and GEMC hydrogels indeed had good antioxidant capacity, which was consistent with the DPPH radical scavenging effect.

[0148] Next, the antioxidant effect of GEMC hydrogel on cells was studied by using the oxidation-sensitive fluorescent probe DCFHDA as a cell-permeable ROS-sensitive fluorescent marker. Figure 57 and Figure 58The different materials' ability to eliminate NIH-3T3 cells and MDA-MB-231 cells is shown respectively. It is worth mentioning that all groups of cells were treated with hydrogen peroxide before the cells were incubated with the samples. The results show that when the cells are treated with hydrogen peroxide (control group), strong green fluorescence can be observed, indicating that the average reactive oxygen species content of NIH-3T3 and MDA-MB-231 cells is high. For NIH-3T3 and MDA-MB-231 cells, only weak green fluorescence is observed after incubation with GEM and GEMC, indicating that the above-mentioned samples have good ROS scavenging ability. In addition, it is also observed that the green fluorescence of the GEMC group is weaker than that of the GEM group, which is consistent with the results of the in vitro DPPH and ABTS free radical scavenging experiments. In summary, GEMC hydrogel has good ROS scavenging ability and good antioxidant performance.

[0149] 1.7 Biocompatibility test of GEMC hydrogel

[0150] The biocompatibility of the sample is an important factor related to the in vivo animal experiment. Therefore, the present application analyzes the influence of GEM and GEMC hydrogels on the survival of different cells (NIH-3T3 and MDA-MB-231). NIH-3T3 and MDA-MB-231 cells were inoculated on GEM and GEMC hydrogels, respectively, and the influence of the above-mentioned materials on the proliferative activity of normal cells (NIH-3T3 cells) and tumor cells (MDA-MB-231) was evaluated by live / dead cell staining. Figure 59 The staining of GEMC loaded with NIH-3T3 cells is shown. Figure 60 The staining of GEMC loaded with MDA-MB-231 cells is shown. Here, the present application chooses different dyes to stain the cells, using Hoechst 33342 as a nuclear stain to emit blue fluorescence (excitation wavelength of 405 nm). Using calcein AM as a cytoplasmic dye, green fluorescence is obtained under 488 nm excitation. Dead cells are stained with iodine pyridine (PI), which emits red fluorescence (excitation wavelength of 559 nm). The results show that after 1 day and 3 days of treatment, the green fluorescence of the cells in each group is enhanced, although the green fluorescence is less, but there is a trend of increase, indicating that the cells in each group begin to adhere and proliferate on the hydrogel. With the passage of time, MDA-MB-231 cells incubated with GEMC begin to show obvious red fluorescence on the 7th day, and the green fluorescence is weakened. No green fluorescence is observed on the 14th day, indicating that MDA-MB-231 cells have a significant activity inhibition effect on GEMC hydrogel.

[0151] In order to further verify the influence of GEMC on the proliferative activity of MDA-MB231 cells, the present application observes the morphology of the cells after treatment by scanning electron microscope. Figure 61The GEMC-loaded NIH-3T3 cells showed normal cell morphology, and the cell aggregation phenomenon increased with the extension of incubation time, indicating that the GEMC hydrogel indeed has good biocompatibility and does not harm normal cells. For the MDA-MB-231 cells treated with GEMC, as shown in Figure 62 , on the third day of cell inoculation, the cell growth slowed down significantly, and the cell morphology began to be abnormal. With the extension of culture time, the number of cells decreased significantly, and the cells had no morphology, indicating that the GEMC hydrogel has a certain cytotoxicity to MDA-MB-231 cells and can effectively inhibit the normal growth of the cells, also indicating that GEMC has great potential in the treatment of local breast cancer.

[0152] 1.8 Application of GEMC Hydrogel in Repairing Radiation Damage of Breast Cancer

[0153] Before GEMC is used to treat radiation damage, the biosafety of each group of hydrogels is first evaluated. The mouse blood compatibility test is as shown in Figure 63 , in which 2% PBS mouse blood suspension is used as the negative control group, and 2% ultrapure water mouse blood suspension is used as the positive control group. PAM, AMNC, GEM and GEMC hydrogels are mixed with 2% PBS mouse blood suspension, respectively, and the results show that no hemolysis occurs in the hydrogel samples. Further, the hemolysis rate Figure 64 ) further proves that the GEMC hydrogel sample has good blood safety.

[0154] Then, local radiotherapy is performed at a dose of 25 Gy, and on the 10th day after radiotherapy (when the skin starts to ulcerate), GEM and GEMC are applied to the local ulcerated skin (treatment process as shown in Figure 66 ). Then, the treatment effect of GEMC hydrogel on radiation damage is studied. As shown in 65, it can be observed that before the hydrogel patch treatment (0 days), the tissues of all groups show obvious skin ulceration, and with the extension of treatment time, the control group shows more severe skin ulceration, redness and exudate. The GEM group has a certain alleviating effect compared with the control group. However, for the GEMC group, no more severe ulceration occurs during the whole treatment process, and the wound gradually heals. For the occurrence of the above phenomenon, it is guessed by the present application that in addition to the unique moist environment and antioxidant effect of the hydrogel, the important factor for promoting wound healing is also the sustained release of nano-drugs (CCNPs). Therefore, the in vivo drug release behavior of GEMC hydrogel is analyzed by using a small animal imaging instrument. As shown in Figure 67As shown, there was no fluorescence phenomenon in the control group and the GEM group, while for the GEMC group, the fluorescence at the wound site gradually increased with the extension of the treatment time, which was due to the accumulation of nanomedicines (CCNPs) at the damaged site caused by the release of drugs due to the regular replacement of hydrogel dressings. Figure 68 The average fluorescence signal of the wound damage site at different treatment times is shown. It is further proved that the GEMC hydrogel can continuously release at the wound site, has excellent imaging ability, and has potential application value in the field of drug imaging tracking.

[0155] The side effect treatment of skin ulcers after radiotherapy will also be accompanied by the occurrence of inflammatory reactions, thereby delaying the wound healing process, so the analysis and detection of chronic inflammation are also essential work. First, the present application collected the damaged sites of mice at 3, 7, and 14 days after treatment, and detected the expression of mouse tumor necrosis factor a (TNF-ɑ, pro-inflammatory factor) and mouse interleukin 10 (IL-10, anti-inflammatory factor) by enzyme-linked immunosorbent assay (ELISA) method. The results are as follows Figure 69 As shown, the expression of TNF-ɑ in the GEM and GEMC groups was lower than that in the control group at 3, 7, and 14 days after treatment, and the expression of TNF-ɑ in the GEMC group continued to decrease compared with the GEM and control groups, and at 14 days, the expression of TNF-ɑ in the GEMC group was basically close to normal skin. It is proved that the GEMC hydrogel significantly inhibits the expression of pro-inflammatory factors. As for IL-10 Figure 70 ), the expression of IL-10 in the GEMC group was significantly higher than that in the other groups at the third day of treatment, indicating that the anti-inflammatory effect is the largest at the third day. With the extension of time, at 14 days after treatment, the expression of IL-10 in the GEMC group was lower than that in the control group and the GEM group, and similar to that in normal tissue, indicating that GEMC can inhibit the occurrence of inflammatory reactions and promote wound healing. Next, the present application further evaluates the macrophage infiltration in the damaged area by immunohistochemical CD68 staining, and the results are as follows Figure 71 As shown, at the third day after treatment, each group showed the presence of a large number of macrophages. With the extension of treatment time, the macrophages in the GEMC group were significantly lower than those in the control group and the GEM group at 7 days. At 14 days after treatment, the expression of CD68 in the GEMC group was basically close to normal tissue. In summary, the GEMC hydrogel can effectively inhibit the spread of inflammation caused by radiotherapy damage and promote wound healing.

[0156] In order to further evaluate the regeneration effect of epidermal cells, the present application collected the wound tissue of mice at 3, 7, and 14 days after treatment for H&E staining. The results are as follows Figure 72As shown, the untreated mice (control group) were always in a state of skin damage within 14 days, indicating that the wound ulceration caused by radiotherapy was difficult to achieve self-healing effect. Compared with the GEM group, the mice treated by GEMC group had faster wound healing effect, and the granulation tissue at the wound site began to appear on the seventh day, the skin began to enter the healing process, and the inflammatory cells began to decrease. On the 14th day of treatment, the granulation tissue of the GEMC group was significantly narrowed, the inflammatory cells basically disappeared, the hair follicles increased, and the staining phenomenon similar to normal skin tissue was shown.

[0157] Next, the present application uses Masson staining Figure 73 to evaluate the amount of collagen deposition in the wound tissue. Among them, the collagen fibers are marked in blue, and the staining intensity represents the collagen content. The wound site of the control group has less and less collagen deposition over time, indicating that under the condition of no treatment, the skin ulceration is getting worse and worse. Compared with the GEM group, the GEMC hydrogel group has a more significant deepening of blue with the increase of treatment time, indicating that the degree of collagen generation is more excellent, and at 14 days, obvious hair follicles and Langerhans cells are seen, and complete epidermis is generated. In addition, the present application determines the effect of GEMC hydrogel on neovascularization of the damaged site by immunohistochemical staining of CD31 antibody at 3, 7, 14 days after treatment. As ​ shown, at 14 days of treatment, it can be clearly observed that the GEMC hydrogel group has more neovascularization and hair follicles than the control group and the GEM group.

[0158] Obviously, the above embodiments are only examples for the purpose of clarity, and are not limited to the embodiments. Based on the above description, those skilled in the art can make other different forms of changes or variations. Here, it is not necessary and impossible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for constructing a gelatin / polyacrylamide-based drug-loaded hydrogel dressing, characterized in that, Includes the following steps: Step 1: At room temperature, gelatin-grafted dopamine (GEDA) and nano-clay (NC) are dissolved in deionized water and stirred thoroughly to obtain a mixed solution. During the stirring process, the mixed solution comes into contact with oxygen molecules in the air, causing the catechol groups in GEDA to oxidize. Step 2: Preparation of GEMC composite hydrogel 2.1) At room temperature, the monomers acrylamide AM, N,N'-methylenebisacrylamide MBAA and curcumin nanoparticles CCNPs were mixed and added to the mixed solution obtained in step one, and stirred thoroughly to make it uniformly mixed; 2.2) Next, add the initiator ammonium persulfate (APS) to the solution obtained in step 2.1), react for 10 min, and then add tetramethylethylenediamine (TEMED) dropwise into the mixed solution; 2.3) After the TEMED is added, the mixed solution is ultrasonically vibrated at room temperature to remove air bubbles, and polymerized at 50-70℃ for 1-3 h to obtain GEMC composite hydrogel; the gelatin / polyacrylamide drug-loaded hydrogel dressing is a double-network interpenetrating hydrogel prepared by combining gelatin grafted with dopamine GEDA, polyacrylamide PAM, nano clay NC and curcumin nanomedicine CCNPs. It is a brown transparent hydrogel material with abundant functional groups, obvious porous structure and doped with CCNPs curcumin nanomedicine.

2. The method for constructing a gelatin / polyacrylamide drug-loaded hydrogel dressing according to claim 1, characterized in that, In step one, 0.5-0.7 g of GEDA and 0.1-0.2 g of NC are added to every 10 ml of deionized water, and the stirring time is 5 hours.

3. The method for constructing a gelatin / polyacrylamide drug-loaded hydrogel dressing according to claim 1, characterized in that, In step one, 0.6 g of GEDA and 0.15 g of NC are added to every 10 ml of deionized water.

4. The method for constructing a gelatin / polyacrylamide drug-loaded hydrogel dressing according to claim 1, characterized in that, The amounts of each substance added in step two are as follows: 3 g of monomer AM, 0.01 g of MBAA, 0.4 g of CCNPs, 0.24 g of APS and 20 μL of TEMED are added to every 10 ml of deionized water in step one.

5. The method for constructing a gelatin / polyacrylamide drug-loaded hydrogel dressing according to claim 1, characterized in that, In step one, the stirring speed is 15-30 rpm; in step 2.1), the stirring speed is 15-30 rpm.

6. The method for constructing a gelatin / polyacrylamide drug-loaded hydrogel dressing according to claim 1, characterized in that, In step one, the stirring speed is 20 rpm; in step 2.1), the stirring speed is 20 rpm.

7. The method for constructing a gelatin / polyacrylamide drug-loaded hydrogel dressing according to claim 1, characterized in that, In step 2.3), the polymerization reaction temperature is 60℃ and the time is 2h.

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