A double-layer hydrogel microneedle combined with photothermal therapy and chemotherapy and a preparation method and use thereof

Abstract

The application provides a kind of double-layer hydrogel microneedle combined with photothermal therapy and chemotherapy and its preparation method and use, belong to the field of biomedical materials.The microneedle in the hydrogel of the application is composed of a needle tip layer prepared from FG MN needle tip premixing solution and a backing layer prepared from CS / PEG-CHO / SA / CuSO4 double network hydrogel.The hydrogel microneedle of the application has good cell compatibility and biocompatibility, and is safe;At the same time, the hydrogel microneedle has good mechanical strength and drug loading capacity, can be dissolved after penetrating into the skin, release drugs to play effect.The hydrogel microneedle also has good photothermal performance, good effect on oral squamous carcinoma, can avoid tissue defect caused by tumor resection, and avoid inconvenience to patients' life.In addition, the hydrogel microneedle of the application has antibacterial effect, can promote the repair of infected soft tissue defect, and has good wound repair effect.The hydrogel microneedle of the application has good application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and particularly relates to a double-layer hydrogel microneedle combined with photothermal therapy and chemotherapy and a preparation method and application thereof. BACKGROUND

[0002] Oral squamous cell carcinoma (OSCC) is a common malignant tumor of human head and neck caused by the development of mucosal epithelial cells in the tongue, floor of the mouth, buccal mucosa and other parts, mainly caused by related factors such as smoking, alcoholism and HPV16 virus infection. Because its early lesions are not obvious, it is generally manifested as leukoplakia and erythema, and has high local invasiveness, high metastasis and high recurrence rate, making its treatment more difficult, and ultimately resulting in a low 5-year survival rate of patients, generally only 50% to 60%. Due to the high invasiveness of OSCC, it will continuously infiltrate the soft tissues such as the muscle and skin around the tumor to cause soft tissue defects, which seriously affects the patient's physiology and psychology. In addition, there are a large number of microorganisms in the oral environment, which are easy to cause infection at the tissue breakdown site where the tumor grows, and even some flora will promote the development process of oral squamous carcinoma. According to the global cancer statistics data in 2020, oral cancer patients in China account for 8% of the total number of oral cancer patients in the world, of which oral squamous carcinoma accounts for more than 90%, and there are a large number of patients in China whose quality of life and life quality are affected due to oral squamous carcinoma.

[0003] At present, oral squamous carcinoma is often treated by surgical resection, chemotherapy and radiotherapy in clinic, but surgical resection will lead to significant loss and damage of facial function, such as difficulty in swallowing, eating and speaking, and psychological trauma caused by facial defects, and part of the elderly patients cannot carry out surgical treatment. Even if extensive surgical resection is performed, the recurrence rate of oral cancer is also high. And the whole body toxic side effects of chemotherapy and radiotherapy are large, which will reduce the quality of life of patients, and the treatment effect is also difficult to meet the clinical demand. Therefore, adopting a new treatment strategy is a problem that needs to be solved in clinic.

[0004] At present, many scholars have applied targeted therapy to the treatment of cancer, such as nanoparticles, nanocapsules, magnetically responsive microbubbles and the like. But most of them are intravenous administration, which has the problems of unstable accumulation concentration at the action site, whole body toxicity, and pain accompanying administration. And part of the local administration needs the drug to penetrate through the stratum corneum for administration and other shortcomings.

[0005] Therefore, the controllable, effective, local and low side effect treatment method is the ideal treatment method that many oral squamous carcinoma patients are eager to achieve. Photothermal therapy is a control response treatment method that has received more and more attention in recent years. It can not only achieve the control purpose through an external light exciter, but also can directly kill tumor cells through the heat energy converted by a photothermal agent during tumor treatment, so as to achieve the purpose of ablation of the tumor. However, the single photothermal treatment method of the tumor needs a large dose of the photothermal agent or a high-power external light exciter to be excited, which is not conducive to the safety treatment of the patient. In order to safely apply the photothermal treatment and ensure the treatment effectiveness, the photothermal treatment combined with chemotherapy is a better combined treatment strategy. The photothermal treatment can directly kill tumor cells and reduce the dosage of the chemotherapy drug, so as to achieve the combined anti-tumor effect.

[0006] The photothermal therapy refers to that a photothermal conversion agent absorbs external light energy and converts the light energy into heat energy for tumor ablation. At present, the photothermal therapy has more application researches in the aspects of tumor treatment, promotion of bone healing, anti-microbial infection and skin repair. The photothermal agent mainly includes melanin analog polymers, coordination polyphenol polymers and heavy metal nanoparticles and the like. Different photothermal agents have different properties and can be synthesized by different methods or carry different drugs, so as to have higher application value. For example, the gold nanorod (GNR) is a kind of gold nanomaterial. Due to the transverse and longitudinal surface plasmon resonance peaks of the morphological structure, the gold nanorod has unique and adjustable physical and chemical properties and optical properties. The gold nanorod can adjust the aspect ratio (AR) according to the synthesis method, so as to adjust the absorption wavelength to meet the requirements of different use conditions and is widely used in the biological and medical fields. At present, the GNR is mainly synthesized by a seed growth method. The GNR prepared by the method has cetyltrimethylammonium bromide (CTAB) cationic surfactant on the surface. The CTAB can damage the cell membrane and cause serious damage to the cells.

[0007] The chemotherapy is a commonly used anti-tumor treatment method in the clinic at present. However, the chemotherapy is usually performed by a systemic administration mode such as oral administration or intravenous drip. In addition to the treatment effect on the tumor site, the chemotherapy also produces toxic side effects on the remaining parts, which reduces the life quality of the patient. In addition, the chemotherapy drug should be used within a certain concentration range. The higher the concentration is, the stronger the side effect is. The lower the concentration is, the worse the treatment effect is. These reasons become the limitations of the use of the chemotherapy drug. For example, 5-FU is a chemotherapy drug commonly used for treating head and neck tumors in the clinic. After the 5-FU is injected into the vein and distributed in the whole body, it reaches the tumor site. Not only is the amount of the drug acting on the tumor site unstable, but also the systemic toxicity is caused. For example, the immune system, liver, kidney, small intestine and even myocardial toxicity, the specific manifestations are bone marrow suppression, neurotoxicity, nausea and vomiting, leukopenia and the like. Therefore, a local and low side effect administration mode is a good way to safely and effectively use the chemotherapy drug.

[0008] Microneedle is a local targeted drug delivery transdermal drug delivery system that has attracted more and more attention in recent years. It is generally prepared from various metals or high molecular materials, and contains a micrometer or nanometer needle-shaped array of needle body and backing layer. Microneedles are diverse, and can be divided into five categories according to different drug delivery principles: solid, hollow, coated, dissolvable and hydrogel microneedles. In solid, hollow and coated microneedles, drugs enter the body through the holes generated by the action of microneedles, the hollow tubes of microneedles and the surface of microneedle bodies, respectively; dissolvable microneedles are prepared from dissolvable polymer materials carrying drugs, and have the characteristics of high drug loading capacity; hydrogel microneedles are prepared from cross-linked hydrogels, and most current researches use them for dermal fluid extraction and detection. Microneedles have the following characteristics and advantages: (1) minimally invasive, painless, and direct drug delivery to the epidermis or dermis through the stratum corneum; (2) local action can reduce the dosage of chemotherapeutic drugs and systemic toxic side effects; (3) easy self-management and high efficiency of drug delivery. Through the delivery of photothermal conversion agents and chemotherapeutic drugs by microneedle system, the drugs can directly reach the action site and reduce drug side effects.

[0009] In the inventors' previous study "Hyaluronic acid microneedles carrying curcumin nanoparticles and new indocyanine green can inhibit the proliferation of human tongue squamous cell carcinoma cells" (Xie et al., Zhejiang University: Medical Edition, 2022, 51(5): 585-593.), the photothermal agent IR820 and the chemotherapeutic drug curcumin nanoparticles were jointly carried in HA and prepared into an integrated dissolvable microneedle. In its in vitro treatment of oral squamous cell carcinoma, photothermal therapy and chemotherapy showed good synergy. However, it was also found that there were problems to be solved. After the release of the integrated microneedle system, the tumor can be ablated by combined treatment, but the soft tissues such as muscles and skin infiltrated by the tumor have not been repaired, which still affects the facial tissue integrity and quality of life of the patient. At the same time, the microorganisms in the tumor micro-environment (TME, tumor micro-environment) of oral squamous cell carcinoma make it difficult for the soft tissues affected by the tumor to repair and heal. In order to solve the above problems, the present application aims to construct a double-layer microneedle system that can quickly dissolve the needle tip to resist tumors and the backing layer can promote the repair of infected soft tissues. SUMMARY

[0010] The purpose of the present application is to provide a double-layer hydrogel microneedle for combined photothermal therapy and chemotherapy, as well as a preparation method and use thereof.

[0011] The present application provides a double-layer hydrogel microneedle for combined photothermal therapy and chemotherapy, which is composed of a needle tip layer prepared from an FG MN needle tip premix liquid and a backing layer prepared from a CS / PEG-CHO / SA / CuSO4 double network hydrogel;

[0012] The FG MN needle tip premix liquid is obtained by uniformly mixing sodium hyaluronate, 5-fluorouracil, gelatin and gold nanorods in water.

[0013] The CS / PEG-CHO / SA / CuSO4 double network hydrogel is obtained by mixing a Schiff base hydrogel and a coordination hydrogel, the Schiff base hydrogel is obtained by mixing a chitosan aqueous solution and an aldehyde group polyethylene glycol aqueous solution, and the coordination hydrogel is obtained by mixing a CuSO4 aqueous solution and a sodium alginate aqueous solution.

[0014] Further, the FG MN needle tip premix liquid is obtained by uniformly mixing a sodium hyaluronate aqueous solution, a 5-fluorouracil aqueous solution, a gelatin aqueous solution, water and a gold nanorod aqueous dispersion;

[0015] Preferably,

[0016] The preparation method of the FG MN needle tip premix liquid comprises the following steps: uniformly mixing a 5-fluorouracil aqueous solution and water; then uniformly mixing a gold nanorod aqueous dispersion and a gelatin aqueous solution, and finally uniformly mixing a sodium hyaluronate aqueous solution.

[0017] Further, the volume ratio of the sodium hyaluronate aqueous solution, the 5-fluorouracil aqueous solution, the gelatin aqueous solution, the water and the gold nanorod aqueous dispersion is 5:(1-2):(0.1-0.5):(3-4):(0.1-1);

[0018] Preferably,

[0019] The volume ratio of the sodium hyaluronate aqueous solution, the 5-fluorouracil aqueous solution, the gelatin aqueous solution, the water and the gold nanorod aqueous dispersion is 5:1.5:0.2:3.2:0.5.

[0020] Further, the concentration of the sodium hyaluronate aqueous solution is 1-5wt%;

[0021] And / or, the concentration of the 5-fluorouracil aqueous solution is 1-5wt%;

[0022] And / or, the concentration of the gelatin aqueous solution is 10-20wt%;

[0023] Preferably,

[0024] The concentration of the sodium hyaluronate aqueous solution is 3wt%;

[0025] And / or, the concentration of the 5-fluorouracil aqueous solution is 1wt%;

[0026] And / or, the concentration of the gelatin aqueous solution is 20wt%.

[0027] Further, the preparation method of the gold nanorod water dispersion liquid comprises the following steps:

[0028] (1) uniformly mixing an aqueous solution of HAuCl4·3H2O, an aqueous solution of cetyltrimethylammonium bromide and an aqueous solution of NaBH4, and then activating to obtain a seed solution;

[0029] (2) adding an aqueous solution of HAuCl4·3H2O, an aqueous solution of H2SO4, an aqueous solution of AgNO3, an aqueous solution of vitamin C and the seed solution obtained in step (1) into the aqueous solution of cetyltrimethylammonium bromide, uniformly mixing, and then standing overnight;

[0030] (3) centrifuging the growth solution after standing overnight in step (2), removing the supernatant, and then resuspending with water to obtain a gold nanorod water dispersion liquid; and centrifuging 100 mL of the growth solution and finally dispersing into 10 mL of the gold nanorod water dispersion liquid;

[0031] Preferably,

[0032] In step (1), the volume ratio of the aqueous solution of HAuCl4·3H2O, the aqueous solution of cetyltrimethylammonium bromide and the aqueous solution of NaBH4 is (0.1-1):(7-8):(0.1-1);

[0033] And / or, in step (1), the temperature of the activation is 25-35℃, and the time of the activation is 1-5h;

[0034] And / or, in step (2), the volume ratio of the aqueous solution of cetyltrimethylammonium bromide, the aqueous solution of HAuCl4·3H2O, the aqueous solution of H2SO4, the aqueous solution of AgNO3, the aqueous solution of vitamin C and the seed solution is 100:(1-5):(1-5):(0.1-1):(0.1-1):(0.1-1);

[0035] And / or, in step (2), when the aqueous solution of HAuCl4·3H2O, the aqueous solution of H2SO4, the aqueous solution of AgNO3, the aqueous solution of vitamin C and the seed solution obtained in step (1) are added into the aqueous solution of cetyltrimethylammonium bromide, the stirring speed is kept at 1000-2000 rpm / min;

[0036] And / or, in step (2), after the uniform mixing, the solution is stirred until it turns dark burgundy, and then it is left to stand overnight;

[0037] And / or, in step (2), the temperature of the standing overnight is 25-35℃;

[0038] And / or, in step (3), the speed of the centrifugation is 10000-20000 rpm / min, and the time of the centrifugation is 10-30 min;

[0039] More preferably,

[0040] In step (1), the volume ratio of the HAuCl4·3H2O aqueous solution, the cetyltrimethylammonium bromide aqueous solution and the NaBH4 aqueous solution is 0.1:7.5:0.6;

[0041] And / or, in step (2), the volume ratio of the cetyltrimethylammonium bromide aqueous solution, the HAuCl4·3H2O aqueous solution, the H2SO4 aqueous solution, the AgNO3 aqueous solution, the vitamin C aqueous solution and the seed solution is 100:2.04:2:0.9:0.8:0.24.

[0042] Further,

[0043] In step (1), the concentration of the HAuCl4·3H2O aqueous solution is 20-30 mM;

[0044] And / or, in step (1), the concentration of the cetyltrimethylammonium bromide aqueous solution is 0.1-1 M;

[0045] And / or, in step (1), the concentration of the NaBH4 aqueous solution is 10-20 mM;

[0046] And / or, in step (2), the concentration of the cetyltrimethylammonium bromide aqueous solution is 0.1-1 M;

[0047] And / or, in step (2), the concentration of the HAuCl4·3H2O aqueous solution is 20-30 mM;

[0048] And / or, in step (2), the concentration of the H2SO4 aqueous solution is 0.1-1 M;

[0049] And / or, in step (2), the concentration of the AgNO3 aqueous solution is 0.01-0.05 M;

[0050] And / or, in step (2), the concentration of the vitamin C aqueous solution is 0.1-1 M;

[0051] Preferably,

[0052] In step (1), the concentration of the HAuCl4·3H2O aqueous solution is 24 mM;

[0053] And / or, in step (1), the concentration of the cetyltrimethylammonium bromide aqueous solution is 0.1 M;

[0054] And / or, in step (1), the concentration of the NaBH4 aqueous solution is 10 mM;

[0055] And / or, in step (2), the concentration of the aqueous solution of cetyltrimethylammonium bromide is 0.1 M;

[0056] And / or, in step (2), the concentration of the aqueous solution of HAuCl4·3H2O is 24 mM;

[0057] And / or, in step (2), the concentration of the aqueous solution of H2SO4 is 0.5 M;

[0058] And / or, in step (2), the concentration of the aqueous solution of AgNO3 is 0.01 M;

[0059] And / or, in step (2), the concentration of the aqueous solution of vitamin C is 0.1 M.

[0060] Further, the volume ratio of the Schiff base hydrogel to the coordination hydrogel is 130: (30-100);

[0061] And / or, in the Schiff base hydrogel, the volume ratio of the aqueous solution of chitosan to the aqueous solution of aldehyde-based polyethylene glycol is (7-10): 4;

[0062] And / or, in the coordination hydrogel, the volume ratio of the aqueous solution of CuSO4 to the aqueous solution of sodium alginate is (10-80): 160;

[0063] Preferably,

[0064] The volume ratio of the Schiff base hydrogel to the coordination hydrogel is 130: 100;

[0065] And / or, in the Schiff base hydrogel, the volume ratio of the aqueous solution of chitosan to the aqueous solution of aldehyde-based polyethylene glycol is 9:4;

[0066] And / or, in the coordination hydrogel, the volume ratio of the aqueous solution of CuSO4 to the aqueous solution of sodium alginate is 40:160.

[0067] Further, the preparation method of the CS / PEG-CHO / SA / CuSO4 double network hydrogel comprises the following steps:

[0068] 1) Mix the aqueous solution of CuSO4 and the aqueous solution of aldehyde-based polyethylene glycol uniformly to obtain a mixed solution;

[0069] 2) Mix the aqueous solution of sodium alginate and the aqueous solution of chitosan uniformly to obtain a mixed solution;

[0070] 3) Mix the mixed solution obtained in step 1) and the mixed solution obtained in step 2) uniformly according to the volume ratio of the Schiff base hydrogel to the coordination hydrogel to obtain the CS / PEG-CHO / SA / CuSO4 double network hydrogel;

[0071] Preferably,

[0072] The concentration of the CuSO4 aqueous solution is 0.01-0.1M;

[0073] And / or, the concentration of the aldehyde-based polyethylene glycol aqueous solution is 20-30wt%;

[0074] And / or, the concentration of the sodium alginate aqueous solution is 1-5wt%;

[0075] And / or, the concentration of the chitosan aqueous solution is 5-10wt%;

[0076] More preferably,

[0077] The concentration of the CuSO4 aqueous solution is 0.05M;

[0078] And / or, the concentration of the aldehyde-based polyethylene glycol aqueous solution is 30wt%;

[0079] And / or, the concentration of the sodium alginate aqueous solution is 2wt%;

[0080] And / or, the concentration of the chitosan aqueous solution is 6wt%.

[0081] The application also provides a preparation method of the aforementioned double-layer hydrogel microneedle, which comprises the following steps:

[0082] (A) adding the FG MN needle tip premix solution into the microneedle template, removing bubbles and drying;

[0083] (B) adding the CS / PEG-CHO / SA / CuSO4 double-network hydrogel into the microneedle template of step (A), removing bubbles and drying, to obtain the double-layer hydrogel microneedle.

[0084] The application also provides the use of the aforementioned double-layer hydrogel microneedle in the preparation of a medical material for resisting oral squamous carcinoma and / or promoting soft tissue defect repair;

[0085] Preferably,

[0086] The oral squamous carcinoma is oral squamous cell carcinoma;

[0087] And / or, the soft tissue defect repair is infectious soft tissue defect repair.

[0088] Oral squamous carcinoma is a malignant tumor that threatens the life safety of patients at all times, and the most important treatment method at present is surgical resection, which can cause significant tissue defects to the patients and thus affect their quality of life. The systemic toxicity of radiotherapy and chemotherapy is also a great burden to the patients. The present application can better meet the needs of patients by combining photothermal therapy and chemotherapy and applying them locally to the tumor site. The 5-FU in the microneedle system is applied locally to the tumor site to reduce systemic toxicity and achieve the purpose of anti-tumor; GNR is ablated by heat production induced by 808nm near infrared light; and the CS / PEG-CHO / SA / CuSO4 double network hydrogel backing layer can promote the repair of infectious soft tissue.

[0089] Compared with the prior art, the present application has the following beneficial effects:

[0090] The present application successfully prepares a double-layer hydrogel microneedle combined with photothermal therapy and chemotherapy, which has good cell compatibility and biocompatibility and good safety; at the same time, the hydrogel microneedle has good mechanical strength and drug loading capacity, can be dissolved after penetrating the skin, and release drugs to play an effect. The hydrogel microneedle also has good photothermal performance, good anti-oral squamous carcinoma effect, can avoid tissue defects caused by tumor resection, and avoid inconvenience to the patient's life. In addition, the hydrogel microneedle of the present application has antibacterial effect, can promote the repair of infectious soft tissue defects, and has good wound repair effect. The hydrogel microneedle of the present application has good application prospect.

[0091] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modification, replacement or change can be made without departing from the above technical idea of the present application.

[0092] The above content of the present application will be further described in detail through the specific embodiments in the form of examples. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 It is the ultraviolet absorption spectrum of gold nanorods (GNR) at 400-900nm wavelength.

[0094] Figure 2 It is the TEM picture of gold nanorods (GNR), and the scale in the picture is 20nm.

[0095] Figure 3 It is the determination result of photothermal conversion efficiency of gold nanorods (GNR): A is the temperature rise-fall curve of GNR; B is the relationship diagram of-lntheta and t.

[0096] Figure 4 Zeta potential values of GNRs before and after surface modification.

[0097] Figure 5 Cell survival rates of L929 cells and Cal-27 cells after co-culturing with different concentrations of GNRs and GNR / HA: A, L929 cells; B, Cal-27 cells; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0098] Figure 6 Oscillatory shear modulus detection results of CS solution and PEG-CHO solution with volume ratios of 7:4, 8:4, 9:4 and 10:4, respectively.

[0099] Figure 7 Cell survival rates of L929 cells after co-culturing with coordination hydrogels prepared from CuSO4 solution and SA solution with different volume ratios.

[0100] Figure 8 Rheological properties and swelling rates of double network hydrogels prepared from Schiff base hydrogels and coordination hydrogels with different volume ratios: A, oscillatory shear modulus detection results; B, swelling rate results.

[0101] Figure 9 FT-IR spectra and SEM images of CS / PEG-CHO / SA / CuSO4 double network hydrogel (volume ratio of Schiff base hydrogel to coordination hydrogel is 130:100): A, FT-IR spectra; B, SEM images, scale bar = 300 μm.

[0102] Figure 10 Macroscopic images of different microneedles: A, one-piece HA microneedle; B, hydrogel HA MN microneedle; C, hydrogel FG MN microneedle; scale bar = 2 mm.

[0103] Figure 11 Stereo microscope observation images and scanning electron microscope images of different microneedles: A, stereo microscope observation image of one-piece HA microneedle; B, stereo microscope observation image of hydrogel HA MN microneedle; C, stereo microscope observation image of hydrogel FG MN microneedle; scale bar of stereo microscope observation image = 200 μm; D, scanning electron microscope image and local magnified image of FG MN microneedle; scale bars = 1 mm and 300 μm, respectively.

[0104] Figure 12 Stereo microscope observation images of FG MN microneedles after action of different weight loads; scale bar = 200 μm.

[0105] Figure 13The FG MN micro-needle penetration performance results in the skin: A is the macroscopic view of the mouse skin after the micro-needle treatment, scale = 2 mm; B is the H&E section of the mouse skin after the micro-needle treatment, scale = 200 μm.

[0106] Figure 14 The body microscope observation chart of the FG MN micro-needle after penetrating the skin for 0, 15, 30 and 45 min, scale = 200 μm.

[0107] Figure 15 The 5-FU content determination results of the FU MN micro-needle tip: A is the 5-FU ultraviolet absorption spectrum chart; B is the 5-FU standard curve; C is the average content of 5-FU in the FU MN micro-needle.

[0108] Figure 16 The GNR content in the GNR MN micro-needle.

[0109] Figure 17 The photothermal performance determination results of different micro-needles: A is the first cycle photothermal chart of different micro-needles; B is the temperature rise curve of different micro-needles in 3 photothermal cycles.

[0110] Figure 18 The cell survival rate after different cells were co-cultured with different concentrations of CS / PEG-CHO / SA / CuSO4 double network hydrogel extracts: A is the L929 cell; B is the Cal-27 cell.

[0111] Figure 19 The cell survival rate after the Cal-27 cell was co-cultured with different concentrations of GNR MN micro-needle extracts.

[0112] Figure 20 The micro-needle in vitro anti-oral squamous cell carcinoma cell experiment results: A is the cell live and dead staining chart of the Cal-27 cell co-cultured with the blank group, the FU group, the GNR+NIR group and the FG+NIR group for 48 h, scale = 200 μm, green fluorescence is the live cell, and red fluorescence is the dead cell; B is the cell survival rate of each group cultured for 2 h and 48 h, *** is P < 0.001, **** is P < 0.0001.

[0113] Figure 21 The photothermal treatment results chart of the GNR MN+NIR group (GNR+NIR) and the FG MN+NIR group (FG+NIR): A is the photothermal chart; B is the photothermal curve chart; C is the temperature 3D chart of the mouse irradiated for 5 min in the FG MN+NIR group.

[0114] Figure 22The body weight change graph of tumor mice after starting treatment by blank group (CON), 5-FU MN group (5-FU MN), GNR MN+NIR group (GNR+NIR) and FG MN+NIR group (FG+NIR) respectively.

[0115] Figure 23 The tumor change results of tumor mice after 0, 2, 4, 6 and 14 days of starting treatment by blank group (CON), 5-FU MN group (5-FU or 5-FU MN), GNR MN+NIR group (GNR+NIR) and FG MN+NIR group (FG+NIR) respectively: A is the macrograph of mice in each group; B is the relative tumor volume change histogram, *** is P<0.001.

[0116] Figure 24 The tumor change results of tumor mice after 14 days of starting treatment by blank group (CON), 5-FU MN group (5-FU), GNR MN+NIR group (GNR+NIR) and FG MN+NIR group (FG+NIR) respectively: A is the tumor macrograph; B is the relative tumor volume histogram, * is P<0.05, ** is P<0.01, *** is P<0.001.

[0117] Figure 25 The macrograph and H&E section staining of heart, liver, spleen, lung and kidney of tumor mice after 14 days of starting treatment by blank group (CON), 5-FU MN group (5-FU), GNR MN+NIR group (GNR+NIR) and FG MN+NIR group (FG+NIR) respectively. The scale of heart, liver, spleen and lung is 400 μm, and the scale of kidney is 200 μm.

[0118] Figure 26 The cell proliferation results of L929 cells co-cultured with CS / PEG-CHO / SA / CuSO4 double network hydrogel extract for 2d and 4d: A is the light microscope graph of cells; B is the cell survival rate, * is P<0.05, *** is P<0.001, scale = 500 μm.

[0119] Figure 27 The antibacterial performance results of CS / PEG-CHO / SA / CuSO4 double network hydrogel: A is the blank group; B is 200 μL of hydrogel; C is 800 μL of hydrogel.

[0120] Figure 28 The macrograph of infected full-thickness skin defect, the template size is 1.5 cm x 1.5 cm.

[0121] Figure 29The results of treating 0, 3, 7 and 14 days of infectious full-thickness skin defects by no treatment (blank group), hydrogel microneedle (hydrogel group), FG solution microneedle (FG solution group) and FG hydrogel microneedle (FG hydrogel group) are as follows: A is a wound macrograph; B is a wound area change graph, 0d (black), 3d (yellow), 7d (orange), 14d (green).

[0122] Figure 30 The wound area statistics graph of treating 0, 3, 7 and 14 days of infectious full-thickness skin defects by no treatment (blank group), hydrogel microneedle (hydrogel group), FG solution microneedle (FG solution group) and FG hydrogel microneedle (FG hydrogel group) is as follows: ** is P<0.01.

[0123] Figure 31 The wound skin H&E section staining of treating 7 and 14 days of infectious full-thickness skin defects by no treatment (blank group), hydrogel microneedle (hydrogel group), FG solution microneedle (FG solution group) and FG hydrogel microneedle (FG hydrogel group) is as follows: the thumbnail scale = 2mm, the enlarged view scale = 400μm (yellow arrow: S.aureus flora; red arrow: capillary / microvessel; green arrow: hair follicle; blue arrow: gland; blue circle: granuloma).

[0124] Figure 32 The wound section (A) Masson staining section of treating 7 and 14 days of infectious full-thickness skin defects by no treatment (blank group), hydrogel microneedle (hydrogel group), FG solution microneedle (FG solution group) and FG hydrogel microneedle (FG hydrogel group) is as follows: the thumbnail scale = 2mm, the orange enlarged view scale = 400μm, the red enlarged view scale = 100μm; (B) the relative collagen area statistics graph after 14 days of treatment; (C) the collagen color concentration distribution curve after 14 days of treatment, ns is P>0.05, * is P<0.05, **** is P<0.0001. DETAILED DESCRIPTION

[0125] The raw materials and equipment used in the specific embodiments of the application are known products, which are obtained by purchasing commercially available products.

[0126] If the solvent is not specified in the solution or dispersion in the specific embodiments, the solvent is water.

[0127] Example 1, preparation of double-layer hydrogel microneedle of the application

[0128] 1, synthesis and characterization of gold nanorods (GNRs)

[0129] 1.1 synthesis of gold nanorods (GNRs)

[0130] GNRs were synthesized by seed-mediated growth method, the synthesis steps are as follows:

[0131] (1) Preparation of vessels: all vessels were washed and then soaked in 0.01 M dilute hydrochloric acid solution for more than 8 h, then washed with ultrapure water and dried.

[0132] (2) Preparation of solution: CTAB solution (0.1 M, 3.6 g of CTAB powder was weighed and added to 120.0 mL of ultrapure water, and dissolved into a colorless transparent clear solution in a 60°C water bath), NaBH4 solution (10.0 mM, 37.0 mg of NaBH4 powder was dissolved in 10.0 mL of ultrapure water, and after complete dissolution, 1.0 mL was diluted in 9.0 mL of ultrapure water and placed in a 4°C refrigerator), HAuCl4·3H2O solution (24.0 mM, 1.0 g of HAuCl4·3H2O powder was dissolved in 105.7 mL of ultrapure water and stored in a 4°C refrigerator), H2SO4 solution (0.5 M, 133.0 μL of 98% concentrated sulfuric acid was added to 4.8 mL of ultrapure water while stirring), AgNO3 solution (0.01 M, 85.0 mg of AgNO3 powder was weighed and added to 5.0 mL of ultrapure water, and after complete dissolution, 1.0 mL was added to 9.0 mL of ultrapure water), Vitamin C solution (Vc, 0.1 M, 52.9 mg of Vc powder was weighed and added to 3.0 mL of ultrapure water).

[0133] (3) Synthesis of seed solution: 0.1 mL of HAuCl4·3H2O solution was added to 7.5 mL of CTAB solution, magnetically stirred for 2 min, then 0.6 mL of NaBH4 solution was added, and the solution was stirred until it turned brown yellow, and activated at room temperature for 2 h.

[0134] (4) Synthesis of growth solution: 100 mL of CTAB solution was kept under rapid magnetic stirring at 1000 rpm / min, and HAuCl4·3H2O solution 2.04 mL, H2SO4 solution 2.0 mL, AgNO3 solution 0.9 mL, Vc solution 0.8 mL were added in turn. Within 30 s after adding the Vc solution, 0.24 mL of seed solution was added, and the stirring was continued until the solution turned deep wine red, and then it was left to stand at room temperature overnight at 25°C.

[0135] (5) The prepared growth solution was centrifuged at 10000 rpm / min for 10 min, and after removing the supernatant, ultrapure water was added to resuspend the GNR, which was repeated twice to remove excess CTAB. Finally, the prepared GNR was stored in the form of a water dispersion, and each 100 mL of growth solution was centrifuged and finally dispersed into 10 mL.

[0136] 1.2 Characterization of gold nanorods (GNRs)

[0137] The gold nanorods (GNRs) prepared above were subjected to relevant characterization.

[0138] 1.2.1 Property and morphology determination of GNRs

[0139] Property determination of gold nanorods (GNRs): The gold nanorod dispersion liquid (the dispersion liquid resuspended with ultrapure water above) was taken to determine its absorbance spectrum at 400 nm to 900 nm wavelength, and the GNR spectrum was observed to determine whether the GNR synthesis was successful. The aspect ratio (AR) was calculated according to the following formula:

[0140] AR = (λ max -418) / 96, λ max is the wavelength corresponding to the highest absorbance value in the ultraviolet absorption spectrum.

[0141] Morphology determination of gold nanorods (GNRs): The morphology of GNRs was observed by transmission electron microscopy (TEM), and the length was measured using Image J software, and the aspect ratio was calculated.

[0142] The ultraviolet absorption spectrum of gold nanorods (GNRs) at 400-900 nm wavelength is shown in Figure 1 : The short axis wavelength of the synthesized gold nanorods is at 540 nm, and the long axis wavelength is at 808 nm. It indicates that the gold nanorods are successfully synthesized. The AR is calculated to be 4.06 according to the two wavelengths and absorbance of the ultraviolet absorption spectrum.

[0143] Figure 2 is a TEM picture of gold nanorods (GNRs). As shown in Figure 2 : The gold nanorods present a short rod structure, and the length distribution is determined by Image J to be: the long axis is 57.6 ± 3.9 nm, and the short axis is 13.4 ± 1.1 nm. The AR is calculated by transmission electron microscopy to be: 4.3 ± 0.3, which is basically consistent with the aspect ratio calculated according to the ultraviolet absorption spectrum.

[0144] 1.2.2, Determination of GNR photo-thermal conversion efficiency

[0145] Take 0.2 mL of GNR dispersion liquid in a 96-well plate, determine its absorbance value at 808 nm, and then place it under an 808 nm near-infrared light exciter. Record the temperature every 30 seconds until the temperature reaches equilibrium, then turn off the exciter and continue to measure the temperature every 30 seconds until the solution returns to room temperature. Draw the temperature-time change curve, and calculate the photo-thermal conversion efficiency according to the following formula:

[0146] (1) η = [hS (T m -T r ) - Q0] / I (1-10 -A808 );

[0147] (2) τs = m d C d / hS;

[0148] (3) t = -τ s ln[(T c -T r ) / (T m -T r )];

[0149] (4) Q0= hS(T m.w -T r ),

[0150] where hS is the overall substitution calculation (h is the heat transfer coefficient; S is the support surface area); T m represents the highest point temperature of the material temperature-time curve, T c is the real-time measured temperature, T r represents the room temperature, Q0 is the pure water correction calculation value, T m.w represents the highest point temperature in the pure water temperature-time curve, I is the near-infrared exciter power, A 808 represents the absorbance value of the gold rod at 808 nm in the ultraviolet full spectrum, m d , C d respectively represent the mass and heat capacity of the dispersion liquid.

[0151] As Figure 3 shown: according to the formula calculation, the -lnθ and time curve [θ = (T c -T r ) / (T m -T r )] is drawn, τ s = 176.4 is calculated, and the photo-thermal conversion efficiency of the synthesized gold nanorod is calculated to be 29.3%.

[0152] 2. Surface modification and characterization of gold nanorods (GNRs)

[0153] 2.1 Surface modification of gold nanorods (GNRs)

[0154] (1) The hyaluronic acid (HA) solution (15.0 mg / mL, 1.0 mL) is added to the GNR dispersion liquid (1.0 mL), and the HA and GNR are fully contacted and uniformly mixed by magnetic stirring for 30 min, and combined by mutual attraction through opposite charges, to obtain a GNR / HA mixed dispersion liquid.

[0155] (2) Add 10.0 mL of ultrapure water to dilute the GNR / HA mixed dispersion liquid, centrifuge at 10000 rpm / min for 10 min, remove the supernatant, and then add 10.0 mL of ultrapure water to resuspend the GNR / HA. Centrifuge again at 10000 rpm / min for 10 min, and remove the supernatant, and add 1.0 mL of ultrapure water to resuspend the GNR / HA.

[0156] 2.2 Characterization of GNR surface modification

[0157] The gold nanorods (GNRs) prepared above and the GNRs after surface modification (GNR / HA) were subjected to relevant characterization.

[0158] 2.2.1 Zeta potential detection

[0159] GNR / HA dispersion liquid and GNR dispersion liquid each 1.0 mL were dispersed in an ultrasonic oscillator for 30 min. The Zeta potential changes of GNR before and after modification (GNR and GNR / HA) were determined by Malvern particle size analyzer.

[0160] As shown in Figure 4 : The Zeta potential value of GNR dispersion liquid before modification is positive, +21.4 mV, and the Zeta potential value of GNR / HA dispersion liquid after surface modification of GNR is negative, -51.7 mV. By coating the gold nanorods with HA material, the positive potential of GNR is successfully modified to negative potential. It can reduce the damage of CTAB to cells and improve the biocompatibility of GNR.

[0161] 2.2.2 Cell compatibility experiment before and after GNR modification

[0162] The survival rate of cells after co-culturing with GNR before and after modification was determined by CCK-8 method, and the improvement of HA modified GNR on the cell compatibility of GNR was analyzed.

[0163] (1) Material preparation: After ultrasonic vibration of GNR dispersion liquid and GNR / HA dispersion liquid for 30 min, both were gradiently diluted with 10% fetal bovine serum-containing α-MEM medium and DMEM medium to 0, 1, 2, 3, 4, 5 μg / mL.

[0164] (2) Passaging / Seedling: Remove the culture medium from L929 and Cal-27 cells, rinse with sterile PBS buffer and remove the medium. Digest with trypsin for 1 min and 3 min respectively, then terminate digestion with α-MEM and DMEM medium containing 10% fetal bovine serum, respectively, and resuspend the cells. Centrifuge at 1200 rpm / min for 3 min, remove the supernatant, and resuspend the cells in culture medium. Add 1 / 3 of the resuspended cells to a T25 culture dish, and add 2 times the volume of α-MEM and DMEM medium containing serum, respectively. Mix well and place in a cell culture incubator. Count the remaining resuspended cells using a hemocytometer and then count them at 5.0 × 10⁻⁶ cells / mL. 3 One hole per hole and 2.0 × 10 4 L929 and Cal-27 cells were seeded into 96-well plates at a density of 0.1 mL per well and incubated in a cell culture incubator (5% CO2, 37°C) for 24 h.

[0165] (3) Co-culture with materials: After the cells adhered to the wall, the culture medium was replaced with GNR dispersion medium containing GNR concentrations of 1, 2, 3, 4, and 5 μg / mL and GNR / HA dispersion medium, respectively. The cells were then placed in a cell culture incubator (5% CO2, 37℃) for co-culture for 24 h.

[0166] (4) Cell viability determination: After removing the GNR dispersion and GNR / HA dispersion, each well was rinsed with 0.1 mL of sterile PBS buffer and removed. Then, medium containing 10% CCK-8 reagent was added (0.1 mL of CCK-8 solution was added to 0.9 mL of α-MEM medium). After incubation for 60 min, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula:

[0167] Cell viability (%) = (Absorbance C - Absorbance A) / (Absorbance B - Absorbance A), where A is the 10% CCK-8 reagent control group, B is the blank experimental group, and C is the cell viability of each experimental group. After calculating the cell viability, a bar chart of cell viability is plotted and the significance of differences is calculated. The cell viability of each subsequent experiment is calculated using the same formula.

[0168] like Figure 5As shown: the cell survival rate of L929 cells and Cal-27 cells gradually decreased with the increase of GNR concentration, whether it was the GNR dispersion solution before modification or the GNR / HA dispersion solution after modification. However, the cell survival rate of L929 cells and Cal-27 cells co-cultured with the GNR / HA dispersion solution was significantly higher than that of the corresponding concentration of GNR dispersion solution before modification. When the GNR concentration was 1 μg / mL, the survival rate of L929 cells co-cultured with the GNR / HA dispersion solution reached 99.9%, and the survival rate of Cal-27 cells reached 94.6%, while the survival rates of L929 cells and Cal-27 cells of the corresponding concentration of GNR before modification were 86.7% and 47.2%, respectively. Therefore, the cell compatibility of gold nanorods can be significantly improved by modifying GNR with HA.

[0169] 3. Preparation of CS / PEG-CHO / SA / CuSO4 double network hydrogel

[0170] 3.1 Explore the ratio of CS solution to PEG-CHO solution in hydrogel

[0171] Take the concentration of 6wt% chitosan (CS) solution and the concentration of 30wt% aldehyde polyethylene glycol (PEG-CHO) solution, mix and stir according to the volume ratio of CS solution to PEG-CHO solution of 7:4, 8:4, 9:4, 10:4, respectively, to prepare Schiff base hydrogel, and determine the rheological curve, select the volume ratio with higher storage modulus and carry out subsequent experiments.

[0172] The rheological curve is shown in Figure 6 The storage modulus is ranked as 9:4>8:4>10:4>7:4, and the Schiff base hydrogel with a volume ratio of 9:4 has the highest storage modulus and the best mechanical properties. Therefore, the volume ratio of CS solution (concentration of 6wt%) to PEG-CHO solution (concentration of 30wt%) is determined as 9:4 and subsequent experiments are carried out.

[0173] 3.2 Explore the ratio of CuSO4 solution (0.05M) to SA solution (2wt%) in hydrogel

[0174] (1) Material preparation: CuSO4 solution (0.05M, 125mg CuSO4·5H2O powder dissolved in 10.0mL pure water) and sodium alginate (SA) solution (2.0wt%, 0.2g powder SA dissolved in 10.0mL pure water) were mixed and stirred according to the volume ratio of 80:160, 60:160, 40:160, 30:160, 20:160, 10:160, respectively, to prepare 0.2mL of coordination hydrogel.

[0175] After the prepared hydrogel was extracted with 5.0 mL of α-MEM medium without serum for 24 h, it was filtered through a sterile filter of 220 nm, fetal bovine serum was added, and a hydrogel extraction medium containing 10% fetal bovine serum was prepared.

[0176] (2) Plate: the steps are the same as the passage of subculture / plate in "2.2.2" above.

[0177] (3) Co-culture with materials: after the cells adhere, the medium is replaced with the hydrogel extraction medium in each volume ratio, and the L929 cells in the 96-well plate are co-cultured for 24 h.

[0178] (4) Determination of cell survival rate: the steps are the same as the determination of cell survival rate in "2.2.2" above. The volume ratio of CuSO4 solution (0.05 M) to SA solution (2.0%) is determined and subsequent experiments are carried out.

[0179] As shown in Figure 7 : when the volume ratio of CuSO4 solution to SA solution is 40:160, it has good cell compatibility. Therefore, the volume ratio of CuSO4 solution (0.05 M) to SA solution (2.0 wt%) is determined to be 40:160 and subsequent experiments are carried out.

[0180] 3.3 Exploration of the ratio of Schiff base hydrogel to coordination hydrogel

[0181] Schiff base hydrogel and coordination hydrogel are prepared into injectable hydrogel with double network cross-linking in the ratio of 130:30, 130:60, and 130:100. Their mechanical properties (rheological curves) are determined by a rheometer, and the ratio with appropriate mechanical strength and swelling rate is selected for subsequent research.

[0182] The swelling rate determination method is as follows: the prepared hydrogel is placed in a microneedle mold, dried at room temperature for 24 h, peeled off and soaked in water, and the excess water is absorbed with filter paper at 0, 5, 10, 20, 30, 45, 60, 75, 90, 120, 180, 240, 300, 360, 480, 1200, and 1440 min, respectively, and weighed, and a swelling curve graph is drawn.

[0183] The preparation process of Schiff base hydrogel is as follows: a 6 wt% CS solution is added to a 30 wt% PEG-CHO solution, the volume ratio of the CS solution to the PEG-CHO solution is 9:4, and stirring is performed until gelation.

[0184] The preparation process of coordination hydrogel is as follows: a 0.05 M CuSO4 solution is added to a 2.0 wt% SA solution, the volume ratio of the CuSO4 solution to the SA solution is 40:160, and stirring is performed until gelation.

[0185] The preparation process of the double network cross-linked hydrogel (CS / PEG-CHO / SA / CuSO4 double network hydrogel) is as follows: CuSO4 solution (concentration of 0.05 M) is added to PEG-CHO solution (concentration of 30 wt%), and SA solution (concentration of 2.0 wt%) is added to CS solution (concentration of 6 wt%) at the same time, and the mixture is stirred uniformly, and the CS / SA mixture is added to the CuSO4 / PEG-CHO mixed solution according to the total volume ratio (the volume ratio of the Schiff base hydrogel and the coordination hydrogel is 130:30, 130:60, 130:100), and stirring is performed until gelation. The volume ratio of CuSO4 solution (0.05 M) to SA solution (2.0 wt%) is 40:160; the volume ratio of CS solution (concentration of 6 wt%) to PEG-CHO solution (concentration of 30 wt%) is 9:4, and subsequent experiments are performed.

[0186] The surface structure of the double network cross-linked hydrogel is determined as follows: 1.0 mL of the prepared CS / PEG-CHO / SA / CuSO4 double network hydrogel is placed in a 5.0 mL centrifuge tube, frozen in a-20℃ refrigerator, dried by a freeze dryer, and finally the network structure of the hydrogel is observed by a scanning electron microscope.

[0187] The rheological properties and swelling rate of the double network hydrogel prepared by different volume ratios of Schiff base hydrogel and coordination hydrogel are shown in Figure 8 , and Figure 8 As shown in A and 8B: the hydrogel with a volume ratio of 130:100 has suitable mechanical properties and a lower swelling rate, which is more suitable for the design requirements of microneedles, so the ratio of 130:100 is selected for subsequent experiments. The hydrogel with a volume ratio of 130:30 has a very high swelling rate and a too high modulus, which cannot completely fill the microneedle template and is not suitable for preparing microneedles. Therefore, the CS / PEG-CHO / SA / CuSO4 double network hydrogel prepared by the Schiff base hydrogel and the coordination hydrogel with a volume ratio of 130:100 is used in subsequent experiments.

[0188] The FT-IR spectrum of the CS / PEG-CHO / SA / CuSO4 double network hydrogel (the volume ratio of the Schiff base hydrogel and the coordination hydrogel is 130:100) is shown in Figure 9 A, and the scanning electron microscope (SEM) image is shown in Figure 9 B: the double network hydrogel has a characteristic absorption peak of C=N at 1643 cm -1 , which indicates the presence of the Schiff base bond in the double network hydrogel. The SEM image of the double network hydrogel shows that it has a uniform and consistent porous structure with a pore size of 99.8±3.5 μm.

[0189] 4、Preparation and characterization of microneedles

[0190] 4.1 Microneedle tip premix solution preparation

[0191] Weigh 1.2 g of sodium hyaluronate (HA) powder and dissolve it in 40.0 mL of pure water to prepare a 3.0% (30.0 mg / mL) HA solution. Weigh 0.1 g of 5-FU powder and add it to 10.0 mL of pure water, and dissolve it in a 60°C water bath to prepare a 10.0 mg / mL 5-FU solution. Weigh 2.0 g of gelatin and dissolve it in 10.0 mL of pure water to prepare a 0.2 g / mL gelatin solution.

[0192] After mixing 1.5 mL of 5-FU solution and 3.2 mL of pure water, add 0.5 mL of GNR dispersion prepared above and 0.2 mL of gelatin solution, mix well, and finally add 5.0 mL of HA solution, and stir for 1 h with a magnetic stirrer to prepare the FGMN tip premix solution. After mixing GNR and HA, the surface of GNR can be modified by HA.

[0193] When preparing the HA MN tip premix solution, replace the GNR dispersion and 5-FU solution in the FGMN tip premix solution with the same volume of pure water and mix well; when preparing the FU MN tip premix solution, replace the GNR dispersion in the FGMN tip premix solution with the same volume of pure water and mix well; when preparing the GNR MN tip premix solution, replace the 5-FU solution in the FGMN tip premix solution with the same volume of pure water and mix well.

[0194] 4.2 Integrated microneedle and hydrogel microneedle preparation

[0195] (1) Take 1 mL of the above-mentioned FG MN tip premix solution and add it to the PDMS microneedle template, and remove the bubbles by vacuumizing for 3 min in a vacuum drying box. To prevent the presence of small bubbles, after vacuumizing to remove the bubbles, place the PDMS template containing the tip premix solution in a 4°C refrigerator to dissolve the bubbles overnight.

[0196] (2) Take it out of the refrigerator and dry it in a 37°C oven for 4 h, remove the excess solution in the PDMS template by a cell scraper (keep the tip cavity full), and then add 0.8 mL of the CS / PEG-CHO / SA / CuSO4 double network hydrogel prepared in advance to the cavity of the PDMS microneedle template, and make the hydrogel fill the PDMS template (if the hydrogel contains bubbles after preparation, remove them by centrifuging for 10 s). The CS / PEG-CHO / SA / CuSO4 double network hydrogel is prepared according to the above-mentioned volume ratio of Schiff base hydrogel to coordination hydrogel of 130:100.

[0197] (3) Dry at room temperature of 25℃ for 12h, then carefully peel the microneedle from the PDMS template to get FG MN microneedle (double-layer hydrogel microneedle of the present application).

[0198] FU MN microneedle, GNR MN microneedle and HA MN microneedle are prepared in the same way, except that the FG MN tip premix solution is replaced by FU MN tip premix solution, GNR MN tip premix solution and HA MN tip premix solution. FU MN microneedle, GNR MN microneedle and HA MN microneedle all belong to hydrogel microneedle.

[0199] HA MN (without Cu) microneedle is prepared in the same way as HA MN microneedle, the only change is that the CuSO4 solution used for hydrogel in the preparation process of HA MN is replaced by pure water.

[0200] Integrated HA microneedle and integrated FG microneedle (FG solution microneedle) completely fill the PDMS microneedle mold with HA MN tip premix solution and FG MN tip premix solution respectively, perform bubble extraction and dissolve microbubbles in the refrigerator, then directly dry at room temperature for 12h, then peel off to get the corresponding microneedle. No CS / PEG-CHO / SA / CuSO4 double network hydrogel is used in the preparation process of microneedle.

[0201] As shown in Figure 10 : The integrated HA microneedle, HA MN microneedle and FG MN microneedle prepared are observed by stereomicroscope. The microneedle patch size is 1.5cm x 1.5cm, which is composed of 20 x 20 needle body array. It can be found that there is a clear color difference between hydrogel microneedle and integrated HA microneedle. The hydrogel microneedle presents green color due to the presence of Cu 2+ , and the needle body color in FG MN patch is darker, presenting deep burgundy color due to the addition of GNR in the tip. The needle body part of HA MN is colorless and transparent.

[0202] 4.3 Characterization of microneedle

[0203] 4.3.1 Morphological characterization of microneedle

[0204] The tip and backing layer of integrated HA microneedle, hydrogel HA MN microneedle and hydrogel FG MN microneedle are observed by stereomicroscope respectively. The tip of FG MN microneedle is observed by scanning electron microscope.

[0205] As shown in Figure 11As shown, both the integrated HA microneedle backing layer and the needle tip part were colorless and transparent under a stereomicroscope; the HAMN needle tip was colorless and transparent, and the backing layer was green and transparent; the FG MN needle tip was purple red, and the backing layer was blue-green and transparent. Meanwhile, SEM observation of the FG MN needle tip and the connection part of the backing layer showed a clear boundary, indicating that the double-layer hydrogel microneedle was successfully prepared, and the needle tip was a four-sided pyramid, with a bottom width of 194 ± 1.9 μm and a distance between the two needle tips of 643 ± 2.0 μm.

[0206] 4.3.2 Mechanical properties of microneedles

[0207] To evaluate whether the microneedles can maintain the structural integrity during pressing towards the skin. The FG MN microneedle tip was placed upward on a hard desktop, and 0, 10, 50, 100, 250, and 500 g weights were placed on the top of the microneedle, respectively. After 5 min, the deformation of the microneedle tip was observed under a stereomicroscope.

[0208] As shown in Figure 12 It can be seen that under the action of 10 g and 50 g weights, the FG MN microneedle tip can maintain good morphology and structural integrity without deformation. Under the action of 100 g and 250 g weights, part of the needle tip is bent, but the microneedle structure remains intact without breaking. Even under the action of 500 g weight, the microneedle tip is bent, but the needle body still maintains the complete structure, indicating that the microneedle tip has good mechanical properties.

[0209] 4.3.3 In vitro penetration performance of microneedles

[0210] To observe the penetration ability and depth of the microneedles into the skin of nude mice, the FG MN microneedle was inserted into the skin of nude mice in vitro, pressed and kept for 5 min, and then removed. The skin of nude mice was fixed with paraformaldehyde, dehydrated, embedded, sectioned, HE stained, and observed and photographed under an optical microscope.

[0211] By inserting the microneedle into the skin of nude mice in vitro, and staining the skin after removing the microneedle and observing the penetration by sectioning, it was found that the microneedle tip could penetrate the skin of nude mice, and the hole after penetration could be seen in the stained skin, as shown in Figure 13 A, with obvious penetration marks. Through Figure 13 B skin penetration sectioning, it was found that the microneedle could pierce the stratum corneum, but did not penetrate the dermis, indicating that the microneedle had the ability to penetrate the skin and did not produce pain.

[0212] 4.3.4 In vitro dissolution performance of microneedles

[0213] The surface of the pig skin was removed from the extracorporeal pig skin and placed on the electric blanket at 37°C. Five FGMN microneedles were inserted into the extracorporeal pig skin, and were maintained for 0, 15, 30, and 45 min, respectively. The microneedle tip was observed by a body microscope and photographed at different time points.

[0214] As shown in Figure 14 , the tip of the microneedle was found to be obviously passivated after 15 min of insertion, and a tetrahedral structure was also observed. The tip of the microneedle still had a large amount of purple-red material. After 30 min of insertion, the tip of the microneedle was further dissolved and passivated, and the tip of the microneedle had a small amount of purple-red material. After 45 min of insertion, the tip of the microneedle had substantially no material, indicating that the tip of the microneedle was dissolved, and indicating that the tip of the microneedle had a drug-dissolving performance.

[0215] 4.3.5 Determination of the content of 5-FU in the microneedle

[0216] (1) Standard curve preparation: 5-FU powder was dissolved in 0.1 M dilute HCl solution, 2 mL of the solution was added to a sample cell, and the ultraviolet absorption spectrum of 5-FU at 200 nm to 800 nm was determined by an ultraviolet spectrophotometer to determine the absorption peak wavelength of 5-FU. Then, 1 mg / mL of 5-FU solution was prepared (20 mg of 5-FU powder was dissolved in 20 mL of 0.1 M dilute HCl solution), and 0.1 M dilute HCl solution was used to gradually dilute the solution to 1, 2.5, 3.75, 5, 6.25, 7.5, 10, 12.5, 15, 17.5, 20, and 25 μg / mL. The absorbance of the 5-FU solution at 265 nm was determined, and a standard curve was drawn.

[0217] (2) Determination of the drug loading amount of the microneedle: three prepared FU MN microneedles and one HAMN microneedle were dissolved in 10 mL of 0.1 M dilute HCl solution. 2 mL of the dissolution solution of the HAMN microneedle was added to a sample cell and the absorbance at 265 nm was determined to correct the background value. Then, 2 mL of the dissolution solution of the FU MN microneedle was added to another sample cell, and the absorbance at 265 nm was determined. The concentration of 5-FU and the drug loading amount were calculated.

[0218] The ultraviolet absorption spectrum of the 5-FU solution was determined, as shown in Figure 15 A, and the 5-FU solution had a maximum absorption peak at an ultraviolet wavelength of 265 nm. The absorbance values of 5-FU solutions at different concentrations at 265 nm were determined, and a standard curve was drawn, as shown in Figure 15 B. The regression equation was Y=0.05841X-0.002384, and R 2= 0.9996. The average content of 5-FU in the FU MN microneedle was calculated to be 200.5 ± 19.6 μg by measuring the UV absorbance of the FU MN microneedle leachate.

[0219] 4.3.6 Determination of GNR content in microneedles

[0220] Aqua regia preparation: In a fume hood, 9.0 mL of concentrated nitric acid was added to 3.0 mL of H2SO4 to prepare 12.0 mL of aqua regia, which was placed in 3 10.0 mL volumetric flasks, and 4.0 mL of aqua regia was placed in each flask.

[0221] After dissolving 3 GNR MN microneedles in 10.0 mL of pure water, 1.0 mL of the leachate was added to 4.0 mL of aqua regia, which was digested at room temperature for 1 h, and then digested at 74°C for 2 h and acid was removed. The solution was diluted to 10.0 mL with pure water, and the content of Au in the solution was determined by ICP-OES to calculate the content of GNR in the microneedle.

[0222] As shown in Figure 16 , the GNR content in the GNR MN microneedle was determined to be 2.9 ± 0.4 μg.

[0223] 4.3.7 Determination of photothermal performance of microneedles

[0224] To explore the photothermal stability of GNR, the photothermal stability of microneedles (repeated photothermal performance) was determined by 3 cycles of photothermal irradiation. The HAMN microneedle, HA MN (without Cu) microneedle, FU MN microneedle, GNR MN microneedle and FG MN microneedle were irradiated with an 808 nm near-infrared laser at 0.4 W / cm 2 for 5 min, the exciter was turned off for 5 min, then the 808 nm near-infrared laser was turned on for 5 min, and then turned off for 5 min, for a total of 2 cycles. The temperature was recorded every 60 s and photographed, and the cycle photothermal temperature change curve was drawn.

[0225] By irradiating different microneedles with a near-infrared exciter for 3 cycles, the results are shown in Figure 17 . The HAMN microneedle without Cu was heated by about 2.9-3.2°C, the HAMN microneedle and FU MN microneedle were heated by 8.6-8.7°C after 5 min of near-infrared light irradiation, and the GNR MN microneedle and FG MN microneedle were both heated by 14.8°C after 5 min of near-infrared light irradiation. And in the cycle photothermal, the microneedles showed good repeated photothermal performance. In 3 cycles of photothermal, the HA MN microneedle and FU MN microneedle could be heated to 32.5-33.0°C, and the GNR MN microneedle and FG MN microneedle could be heated to 38.0-39.0°C. The results show that the FGN MN microneedle has good photothermal performance and has the potential to achieve anti-tumor effect through photothermal therapy.

[0226] The beneficial effects of the present application are demonstrated below through specific test examples.

[0227] Test Example 1, CS / PEG-CHO / SA / CuSO4 double network hydrogel cell compatibility experiment

[0228] The L929 cell and Cal-27 cell survival rates co-cultured with the CS / PEG-CHO / SA / CuSO4 hydrogel were detected by the CCK-8 method to analyze the in vitro biocompatibility of the hydrogel.

[0229] (1) Hydrogel material extract preparation: 0.8 mL of hydrogel was prepared by the CS / PEG-CHO / SA / CuSO4 hydrogel preparation method described in Example 1 (Schiff base hydrogel to coordination hydrogel volume ratio 130:100), and the prepared hydrogel was extracted with 10.0 mL of serum-free a-MEM culture medium and DMEM culture medium for 24 h, then filtered with a 220 nm sterile filter, and FBS was added to prepare 100% hydrogel extraction medium containing 10% FBS. The 100% hydrogel extraction medium was diluted with 10% FBS medium to 50%, 25%, 10%, 5%, and 0% by gradient dilution.

[0230] (2) Passage / seed plate: the passage / seed plate operation in Example 1 “2.2.2” was followed.

[0231] (3) Co-culture with the material: after the cells adhered, the culture medium was replaced with the hydrogel extraction medium, and the cells were placed in a cell culture incubator (5% CO2, 37°C) for co-culture for 24 h.

[0232] (4) Determination of cell survival rate: the hydrogel extraction medium was removed, and the remaining steps were the same as the operation for determining the cell survival rate in Example 1 “2.2.2”.

[0233] As shown in Figure 18 A, the L929 cell survival rates co-cultured with 5% to 100% hydrogel extraction medium for 24 h and 48 h were all greater than 90.0%. At the same time, the L929 cells co-cultured with 100% concentration hydrogel extraction liquid for 24 h and 48 h showed obvious cell proliferation effect compared with the blank control group (0% group), and the cell survival rates reached 112.4% and 120.7%, respectively, showing good cell compatibility to L929 cells. As shown in Figure 18 B, the Cal-27 cell survival rates co-cultured with 5% to 100% hydrogel extraction medium for 24 h and 48 h were also higher than 90.0%. The CS / PEG-CHO / SA / CuSO4 double network hydrogel showed good cell compatibility.

[0234] Test Example 2, GNR MN microneedle cell compatibility

[0235] The cell survival rate of Cal-27 cells co-cultured with GNR MN microneedles was detected by CCK-8 method to analyze the in vitro cell compatibility of GNR MN microneedles.

[0236] (1) Microneedle extract preparation: The GNR MN microneedles prepared according to the method of Example 1 were immersed in 10.0 mL of serum-free DMEM medium for 24 h, and the remaining steps were the same as the preparation of hydrogel material extract in Test Example 1.

[0237] (2) Seeding: The steps were the same as the passaging / seeded operation in Example 1 "2.2.2".

[0238] (3) Co-culture with materials: After the cells adhered, the culture medium was replaced with GNR MN immersion medium, and the cells were placed in a cell incubator (5% CO2, 37°C) for co-culture for 24 h.

[0239] (4) Determination of cell survival rate: The steps were the same as the operation of determining the cell survival rate in Example 1 "2.2.2".

[0240] The results are shown in Figure 19 Cal-27 cells cultured in immersion medium showed good cell compatibility, and the cell survival rate of all groups of Cal-27 cells at 24 h and 48 h was higher than 90.0%, showing good cell compatibility.

[0241] Test Example 3, in vitro anti-oral squamous cell carcinoma cell experiment of microneedle

[0242] In order to study the anti-oral squamous cell carcinoma cell performance of the tip part of the double-layer hydrogel microneedle, this test example used CCK-8 method and cell live and dead fluorescent staining method to detect the in vitro anti-tumor effect of one-piece FG microneedle (prepared by using the tip part of the tip pre-mixed solution material of the double-layer hydrogel microneedle).

[0243] (1) Grouping and material preparation: The experiment was divided into 4 groups, in turn (1) blank group, (2) FU group, (3) GNR+NIR group and (4) FG+NIR group. The FU group used the FU MN tip pre-mixed solution in Example 1 to prepare the one-piece FU microneedle according to the one-piece microneedle method in Example 1; the GNR+NIR group used the GNR MN tip pre-mixed solution in Example 1 to prepare the one-piece GNR microneedle according to the one-piece microneedle method in Example 1; the FG+NIR group used the FG MN tip pre-mixed solution in Example 1 to prepare the one-piece FG microneedle according to the one-piece microneedle method in Example 1. The amount of each microneedle pre-mixed solution was 133 μL, which was dissolved in DMEM medium containing 10% fetal bovine serum after 30 min ultraviolet sterilization.

[0244] (2) Plate: Cal-27 cells were seeded in 24-well plates at a density of 8.0 x 10 4 cells / well (0.5 mL) and the remaining steps were the same as the passaging / plate operation in Example 1 "2.2.2".

[0245] (3) Photothermal therapy and co-culture with materials: After the cells adhered, the culture medium of each group was replaced according to the group as follows: (1) DMEM medium, (2) DMEM medium containing 5-FU, (3) DMEM medium containing GNR, and (4) DMEM medium containing 5-FU and GNR. The Cal-27 cells in groups (3) and (4) were irradiated with a near-infrared exciter at 0.4 W / cm 2 for 5 min, and then the cells were placed in a cell incubator (5% CO2, 37°C) for co-culture for 2 h and 48 h.

[0246] (4) Determination of cell survival rate: The culture medium of each group was removed, and 0.4 mL of sterile PBS buffer was added to each well for rinsing and removal. Medium containing 10% CCK-8 reagent was added, and after incubation for 60 min, the absorbance at 450 nm was determined by a microplate reader, and the cell survival rate was calculated and plotted.

[0247] (5) Cell live / dead staining: Cal-27 cells for observing cell live / dead by fluorescence staining were prepared by adding 0.4 mL of live / dead staining solution (5.0 μL of PI reagent was added to 10.0 mL of PBS buffer, vortexed, and then 5.0 μL of Calcein reagent was added and vortexed) to each well after removing the culture medium of each group and rinsing with sterile PBS buffer. After incubation at room temperature for 35 min, the cell live / dead was observed by inverted laser confocal microscopy, in which live cells showed green fluorescence and dead cells showed red fluorescence.

[0248] The experimental results are shown in Figure 20 B. The survival rate of Cal-27 cells co-cultured for 2 h was the highest in the blank group, and the survival rate of cells in the experimental group treated with pure chemotherapy drugs (FU group) was reduced to 96.3%, the survival rate of cells in the experimental group treated with pure photothermal therapy (GNR+NIR group) was further reduced to 90.8%, and the survival rate of cells in the experimental group treated with chemotherapy drugs combined with photothermal therapy (FG+NIR group) was the lowest, which was 79.0%. The survival of Cal-27 cells co-cultured for 48 h is shown in Figure 20As shown in A and 20B, the cell survival rate of the rest of the experimental groups is further reduced, except for the blank group. The cell survival rate of the FU group is 16.3%, the cell survival rate of the GNR+NIR group is 5.4%, and the cell survival rate of the FG+NIR group is only 1.0%. The FG+NIR group shows a synergistic effect of the combined treatment of chemotherapy drugs and photothermal therapy on the inhibition and killing of Cal-27 cells. It shows that the double-layer hydrogel microneedle has a synergistic inhibitory effect on the proliferation of oral squamous cell carcinoma cells, and can effectively resist oral squamous cell carcinoma cells.

[0249] Test Example 4, Study on the Anti-Oral Squamous Cell Carcinoma Subcutaneous Tumor of Double-Layer Hydrogel Microneedle in Vivo

[0250] 1. Establishing a subcutaneous tumor model of oral squamous cell carcinoma

[0251] (1) After the cultured Cal-27 cells were digested, suspended, collected and centrifuged, the supernatant was removed, resuspended with PBS buffer, counted, and then diluted to a cell concentration of 5×10 6 cells / 0.1 mL of cell suspension.

[0252] (2) The skin at the injection site of the nude mice was disinfected with an alcohol cotton ball, and 0.1 mL of the mixed Cal-27 cell suspension was injected subcutaneously on the back of the nude mice through a 1 mL sterile syringe to form a circular ball.

[0253] (3) Continue to feed the nude mice for 7 days. Check the tumor formation of the nude mice. The tumor at the injection site grows normally (the diameter reaches about 4 mm), is not absorbed, and is considered to be successful modeling.

[0254] 2. Double-Layer Hydrogel Microneedle Anti-Oral Squamous Cell Carcinoma Subcutaneous Tumor Experiment

[0255] (1) The successfully modeled nude mice were randomly divided into 4 groups (n=5), which were blank group, 5-FU MN group, GNR MN+NIR group, and FG MN+NIR group, respectively.

[0256] (2) After the nude mice were anesthetized, the body weight and tumor volume size (recorded as V0) of the nude mice in each group were measured.

[0257] (3) The blank group was not treated; the 5-FU MN group was treated with the FU MN microneedle prepared in Example 1; the GNR MN+NIR group and the FG MN+NIR group were respectively inserted into the GNR MN microneedle and the FG MN microneedle prepared in Example 1, and then irradiated with a near-infrared light exciter for 5 min (0.4 W / cm 2 ), and the temperature change was recorded and plotted with an infrared imager during the irradiation.

[0258] (4) The start of treatment of nude mice is recorded as D0, and the microneedle patch is replaced every 2 days thereafter and the operation is repeated as before, for a total of 4 times. Continue to raise to D14, and record the body weight and tumor volume of the nude mice every 2 days (recorded as V t ).

[0259] (5) The nude mice are sacrificed at D14, the tumor volume is measured, and the internal organs of the nude mice in each group are sampled, and the relative tumor volume is calculated and plotted. The tumor volume calculation and relative tumor volume calculation formula are as follows:

[0260] Tumor volume = (length x width 2 ) / 2; Relative tumor volume = V t / V0

[0261] The light-thermal treatment temperature of the microneedle is shown in Figure 21 , and the light-thermal temperature of the GNR MN microneedle and the FG MN microneedle is maintained at 51-52°C, which can achieve the effective temperature for treating tumors, while the temperature is not higher than 52°C, ensuring the safety of the light-thermal treatment of the microneedle. At the same time, as shown in the 3D temperature display diagram of Figure 21 C, the light-thermal microneedle treatment of the tumor can cause local heating of the tumor and reduce damage to the peritumoral tissue.

[0262] As shown in Figure 22 , the body weight changes of the nude mice in each group were basically the same in the 14 days of treatment. This shows that the treatment does not affect the normal life activities of the mice.

[0263] The anti-tumor situation is shown in Figure 23 , and the 4 groups of subcutaneous tumors gradually increased with time in 14 days, among which the relative tumor volume of the nude mice in the blank group increased most significantly, and the relative tumor volume was always higher than that of the other 3 groups after the start of treatment, and the relative tumor volume size at the 14th day was: blank group > 5-FU MN group > GNR MN+NIR group > FG MN+NIR group.

[0264] As shown in Figure 24As shown, the relative tumor volume of the blank group reached 5.6 times of the V0 of the blank group at 14 days. The treatment effects of the 5-FUMN group and the GNR MN+NIR group were similar, and the relative tumor volumes were 3.6 and 3.0, respectively. This indicates that the chemotherapy alone and the photothermal therapy alone have partial inhibitory effects on the tumor, but the treatment effects are limited. The FG MN+NIR group, i.e., the chemotherapy combined with the photothermal therapy microneedle group, can basically maintain the tumor size before treatment (the relative tumor volume D6=1.1) in the first 6 days of treatment. After the treatment is stopped, the tumor gradually increases, and the relative tumor volume reaches 1.8 at 14 days, which is still significantly lower than those of the other three groups, indicating that the chemotherapy combined with the photothermal microneedle treatment used by the FG MN+NIR has a synergistic inhibitory effect on the growth of the oral squamous cell carcinoma.

[0265] 3. Histological analysis

[0266] After the treatment according to "2", the visceral tissues of the sample nude mice were fixed with 4% paraformaldehyde solution for 24 h, and after dehydration, embedding and sectioning, H&E staining was performed, and whether lesions were produced in the viscera was observed by upright fluorescence microscopy and photographed.

[0267] The five organs of the heart, liver, spleen, lung and kidney of the nude mice in each group were dyed to evaluate the systemic toxicity of the microneedle treatment of each group. The results are shown in Figure 25 As shown, the heart, liver, spleen and lung of each group of nude mice had no obvious lesions. Except for the FG MN+NIR group, the kidneys of the other groups all had different numbers of bleeding spots. This indicates that the FG MN microneedle not only has a good inhibitory effect on the tumor, but also does not cause significant impact on the kidney, and is safer to use.

[0268] Test Example 5: CS / PEG-CHO / SA / CuSO4 double network hydrogel promotes cell proliferation experiment

[0269] The cell survival rates of the CS / PEG-CHO / SA / CuSO4 double network hydrogel and L929 cells co-cultured for 2 days and 4 days were determined by the CCK-8 method and photographed to verify the effect of the double network hydrogel on promoting the proliferation of L929 cells. The preparation method of the CS / PEG-CHO / SA / CuSO4 double network hydrogel is the same as that in Example 1 (Schiff base hydrogel and coordination hydrogel volume ratio 130:100).

[0270] (1) Preparation of hydrogel material extract: the steps are the same as those in Test Example 1 for the preparation of the hydrogel material extract, but no gradient dilution is performed.

[0271] (2) Plate seeding: L929 cells were inoculated in two 24-well plates at a density of 1.0x10 4 cells / well (500 μL), and the remaining steps were the same as those in Example 1 "2.2.2" for the subculture / plate seeding operation.

[0272] (3) Co-culture with materials: After the cells adhere, the culture medium is replaced with the hydrogel extraction medium, and the cells are placed in a cell incubator (5% CO2, 37°C) for co-culture for 2d and 4d, respectively.

[0273] (4) Determination of cell survival rate: The hydrogel extraction medium is removed, each well is rinsed with 400 μL of sterile PBS buffer and removed. Then, the medium containing 10% CCK-8 reagent is added, and after incubation for 60 min, the reagent in each well is added to the 96-well plate, and the absorbance at 450 nm wavelength is determined by an enzyme marker, and the cell survival rate is calculated and plotted. To observe the relationship between the number of cells at 2d and 4d, the 2d blank experiment group is used instead of the 4d blank experiment group when calculating the cell survival rate at 4d.

[0274] Through the co-culture experiment of L929 cells and hydrogel extraction medium, it is found that the hydrogel extraction medium has good promoting effect on the proliferation of L929 cells, so further verification of the promoting effect of CCK-8 method on the proliferation of L929 cells is carried out. As shown in Figure 26 , L929 cells co-cultured with hydrogel extraction medium for 2d and 4d respectively all show higher cell survival rate than the blank control group. Among them, the cell survival rate of L929 cells co-cultured with hydrogel extraction medium for 2d reaches 113.6%, and the cell survival rate of L929 cells co-cultured for 4d reaches 211.4%. The cell survival rate of the blank experiment group at 4d is 148.0%. It shows that the CS / PEG-CHO / SA / CuSO4 double network hydrogel has good promoting effect on the proliferation of L929 cells, which further shows that the double-layer hydrogel microneedle of the application is beneficial to promote the repair of soft tissue defects.

[0275] Test Example 6, antibacterial performance of CS / PEG-CHO / SA / CuSO4 double network hydrogel

[0276] The prepared CS / PEG-CHO / SA / CuSO4 double network hydrogel is placed in the center of the LB solid medium plate coated with S. aureus, and placed in an incubator (5% CO2, 37°C) for co-culture for 24h, and the antibacterial performance of the hydrogel is observed and photographed. The preparation method of CS / PEG-CHO / SA / CuSO4 double network hydrogel is the same as that of Example 1 (volume ratio of Schiff base hydrogel to coordination hydrogel 130:100).

[0277] As shown in Figure 27 , the hydrogel has good inhibitory performance on S. aureus in direct contact, and different volumes of hydrogel show similar antibacterial effect, that is, the double network hydrogel has contact antibacterial effect.

[0278] Test Example 7, in vivo skin repair effect of double-layer hydrogel microneedle

[0279] 1. Construction of an infectious full-thickness skin defect model

[0280] The in vivo skin repair effect of the double-layer hydrogel microneedle was verified by applying the FG MN microneedle to the infectious full-thickness skin defect model of the rat.

[0281] The method of constructing the infectious full-thickness skin defect model is as follows:

[0282] (1) After the rat was anesthetized with isoflurane, the hair on its back was removed.

[0283] (2) After iodine solution disinfection, a full-thickness skin defect wound of 1.5 cm x 1.5 cm was formed with surgical scissors.

[0284] (3) 0.1 mL (1.0 x 10 8 cfu / mL) of S. aureus bacterial solution was added dropwise. After the bacterial solution was slightly dried, the wound was bandaged with air-permeable gauze.

[0285] (4) After 24 h, the gauze was cut open, and the wound appeared to be infected, indicating that the infectious full-thickness skin defect model was successfully constructed.

[0286] As shown in Figure 28 , there was obvious purulent exudate on the surface of the muscle of the infectious full-thickness skin defect model, which was slightly whitish and showed a purulent wet appearance. This indicates that the infectious full-thickness skin defect model was successfully constructed.

[0287] 2. In vivo skin repair experiment

[0288] (1) The successfully modeled rats were randomly divided into 4 groups, namely the blank group, the hydrogel group, the FG solution group, and the FG hydrogel microneedle group (n = 9).

[0289] (2) The blank group was not treated; the hydrogel group used hydrogel microneedles in the skin defect (microneedles prepared according to the one-piece microneedle preparation method of Example 1 using CS / PEG-CHO / SA / CuSO4 double-network hydrogel); the FG solution group used FG solution microneedles in the skin defect (microneedles prepared according to the one-piece microneedle preparation method of Example 1 using the FG MN needle tip premix prepared in Example 1); and the FG hydrogel microneedle group used the FG MN microneedles prepared in Example 1 in the skin defect. Subsequently, the wound was re-bandaged with air-permeable gauze.

[0290] (3) Each group was sampled on days 3, 7, and 14, respectively, and the wound was photographed. The wound area was calculated by Image J software, converted to relative wound area, and plotted. The calculation formula is as follows:

[0291] Relative wound area = Sd / S d＝0 ×100%;S d is the wound area at d days, S d＝0 is the wound area at 0 days.

[0292] After the modeling of the infectious full-thickness skin defects was successful, different microneedle treatments were added, and the results are shown in Figure 29 A, the 0d wound surface of all the rats had obvious purulent exudate. On the 3rd day after treatment, the wounds of the four groups of rats were all covered with obvious scab layer. By the 7th day, the FG hydrogel group began to have a more obvious wound contraction trend than the other three groups. By the 14th day, the skin defects treated by the FG hydrogel microneedle group had almost no defective wounds, followed by the hydrogel microneedle group, while the blank group and the FG solution group still had a larger wound area.

[0293] In order to visually compare the changes in wound healing, the wound areas of the four groups of microneedle treatments were arranged in the order of 0d (black), 3d (yellow), 7d (orange), and 14d (green), as shown in Figure 29 B, different colors were used to represent and superimpose, and finally each group of wound changes was plotted in a square black frame with a side length of 1.5 cm. As shown in Figure 30 , on the 3rd day of treatment, the repair area of the damaged skin of each group was less than 30.0%. On the 7th day after treatment, the skin defect area of the blank group of rats was still 64.2%, while the FG hydrogel group had a defect skin area of only 38.3%, and the defect areas of the hydrogel group and the FG solution group were both lower than that of the blank group, being 55.9% and 59.4%, respectively. By the 14th day after treatment, the FG hydrogel group of rats had the smallest skin defect area, only 0.5%. The repair trend of the other three groups was similar to that on the 7th day, with the hydrogel group being second only to the FG hydrogel group, with a defect area of only 4.2%, while the FG solution group had a defect area of 6.4%. The skin defect area of the blank group of rats was 10.4%, and the repair effect was the worst. The experimental results show that the double-layer hydrogel microneedle prepared by the present application has the effect of synergistically promoting the healing of infectious wounds.

[0294] 3. Histological analysis

[0295] After being treated according to “2”, the skin tissues sampled at 7 and 14 days were fixed with 4% paraformaldehyde solution for 24 h, dehydrated, embedded, sectioned, and then subjected to H&E and Masson staining. The skin regeneration was observed and photographed by upright fluorescence microscopy, and the collagen area and collagen staining intensity were quantitatively analyzed by Image J.

[0296] By observing the HE staining of the sections, the repair degree and inflammation of the rat skin at 7d and 14d were evaluated, respectively. As shown in Figure 31As shown, after 7 days of different treatments for each group, the HE staining section of the blank group can see only a small amount of new epithelial tissue produced. At the same time, there are still a large number of S. aureus colonies, and the skin tissue has formed a large mass of fibrous tissue with granulation tissue fibrosis, but there are still a large number of new granulation tissue, indicating that the blank group of rats is still in the inflammatory response process. In addition, the blank group can also observe the formation of granuloma due to S. aureus infection.

[0297] And the hydrogel group and the FG solution group also have a large number of new granulation tissue, but the hydrogel group is less S. aureus colonies, and the FG solution group can still see obvious S. aureus colonies. This may be due to the antibacterial effect of chitosan and Cu 2+ At the same time, the backing layer hydrogel plays a water absorption role during the treatment, transferring part of the bacteria to the inside of the hydrogel, reducing the stimulation to the rat body, while keeping the wound moist and promoting the epithelialization of the granulation tissue, but the degree of epithelialization is still limited. The skin section of the FG solution group shows that only a small amount of new epithelial tissue is produced, and it is still in the inflammatory response.

[0298] Unlike the first three groups, the FG hydrogel group, although the section also shows the formation of granuloma, but it has basically no residual S. aureus colonies, which may be due to the antibacterial effect of chitosan and Cu 2+ in the hydrogel components. At the same time, the granulation tissue of the rat wound has basically completed fibrosis, and most of the capillaries have completed closure or evolved into small blood vessels. It shows that the FG hydrogel group of rats has experienced the inflammatory response and has begun to transform from the fibrotic granulation tissue to the collagen fiber repair process.

[0299] After 14 days of treatment of infected skin defects, the fibroblasts of the blank group, the hydrogel group and the FG solution group have basically transformed into fibrocytes, but the blank group still has some capillaries and basically no hair follicle growth, while the hydrogel group and the FG solution group only have a small amount of hair follicle growth. The repair effect of the FG hydrogel group is the best, and the fibroblasts produce a large amount of collagen fibers, in addition to a large number of hair follicles and gland growth, and have basically completed the repair of the skin structure.

[0300] Because collagen is an important product of skin repair, it can more directly reflect the skin repair situation, and the collagen staining is observed to observe the collagen production. For example Figure 32As shown in the figure, at 7 days, a small amount of collagen fibers were produced in each group. At 14 days, the relative collagen area of the blank group, hydrogel group and FG solution group reached 58.5%, 74.9% and 72.2%, respectively, while the FG hydrogel group produced more collagen, reaching 81.3%. At the same time, it can be observed from the slice graph that the newly generated collagen in the first three groups is reticular fiber, which is the type III collagen with a relatively high content in the wound repair process. The newly generated collagen in the FG hydrogel group is type I collagen, which is thick in shape and has a relatively high content in the normal state. Through quantitative detection of the color of the collagen in the four groups, it was found that Figure 32 As shown in the figure, the blank group has the lightest blue collagen, the hydrogel group and the FG solution group have similar collagen color, and the FG hydrogel group has the darkest collagen. The above shows that the skin repair state of the FG hydrogel group is faster, and can better play the role of skin soft tissue repair.

[0301] In summary, the double-layer hydrogel microneedle for combined photothermal therapy and chemotherapy is successfully prepared, which has good cell compatibility and biocompatibility, and good safety; at the same time, the hydrogel microneedle has good mechanical strength and drug loading capacity, can be dissolved after penetrating the skin, and release drugs to play the effect. The hydrogel microneedle also has good photothermal performance, good effect on oral squamous carcinoma, can avoid tissue defects caused by tumor resection, and avoid inconvenience to patients' life. In addition, the hydrogel microneedle has antibacterial effect, can promote the repair of infected soft tissue defects, and has good wound repair effect. The hydrogel microneedle has good application prospect.

Claims

1. A double-layer hydrogel microneedle combining photothermal therapy and chemotherapy, characterized in that: The microneedle is composed of a tip layer prepared from the FG MN tip premix liquid and a backing layer prepared from the CS / PEG-CHO / SA / CuSO4 double network hydrogel; The FG MN tip premix liquid is obtained by uniformly mixing sodium hyaluronate, 5-fluorouracil, gelatin and gold nanorods in water; The CS / PEG-CHO / SA / CuSO4 double network hydrogel is obtained by mixing a Schiff base hydrogel and a coordination hydrogel, wherein the Schiff base hydrogel is obtained by mixing a chitosan aqueous solution and an aldehyde polyethylene glycol aqueous solution, and the coordination hydrogel is obtained by mixing a CuSO4 aqueous solution and a sodium alginate aqueous solution.

2. The double-layer hydrogel microneedle of claim 1, wherein: The FG MN tip premix liquid is obtained by mixing sodium hyaluronate, 5-fluorouracil, gelatin, water and gold nanorod aqueous dispersion.

3. The double-layer hydrogel microneedle according to claim 2, wherein: The preparation method of the FG MN tip premix liquid comprises the following steps: uniformly mixing 5-fluorouracil aqueous solution and water; then uniformly mixing gold nanorod aqueous dispersion and gelatin aqueous solution, and finally uniformly mixing sodium hyaluronate aqueous solution.

4. The double-layer hydrogel microneedle of claim 3, wherein: The volume ratio of the sodium hyaluronate aqueous solution, 5-fluorouracil aqueous solution, gelatin aqueous solution, water and gold nanorod aqueous dispersion is 5: (1-2): (0.1-0.5): (3-4): (0.1-1).

5. The double-layer hydrogel microneedle according to claim 4, wherein: The volume ratio of the sodium hyaluronate aqueous solution, 5-fluorouracil aqueous solution, gelatin aqueous solution, water and gold nanorod aqueous dispersion is 5:1.5:0.2:3.2:0.

5.

6. The double-layer hydrogel microneedle of claim 5, wherein: The concentration of the sodium hyaluronate aqueous solution is 1-5 wt%; And / or, the concentration of the 5-fluorouracil aqueous solution is 1-5 wt%; And / or, the concentration of the gelatin aqueous solution is 10-20 wt%.

7. The double-layer hydrogel microneedle according to claim 6, wherein: The concentration of the sodium hyaluronate aqueous solution is 3 wt%; And / or, the concentration of the 5-fluorouracil aqueous solution is 1 wt%; And / or, the concentration of the gelatin aqueous solution is 20 wt%.

8. The double-layer hydrogel microneedle of claim 5, wherein: The preparation method of the gold nanorod aqueous dispersion comprises the following steps: (1) uniformly mixing HAuCl4·3H2O aqueous solution, cetyltrimethylammonium bromide aqueous solution and NaBH4 aqueous solution to obtain a synthesis seed solution; (2) adding HAuCl4·3H2O aqueous solution, H2SO4 aqueous solution, AgNO3 aqueous solution, vitamin C aqueous solution and the seed solution obtained in step (1) into the cetyltrimethylammonium bromide aqueous solution, uniformly mixing and then standing overnight; (3) centrifuging the growth solution after standing overnight in step (2), resuspending with water after removing the supernatant to obtain a gold nanorod aqueous dispersion; and 100 mL of the growth solution is finally dispersed into 10 mL of the gold nanorod aqueous dispersion.

9. The double-layer hydrogel microneedle according to claim 8, wherein: In step (1), the volume ratio of the HAuCl4·3H2O aqueous solution, the cetyltrimethylammonium bromide aqueous solution and the NaBH4 aqueous solution is (0.1-1): (7-8): (0.1-1); And / or, in step (1), the activation temperature is 25-35℃, and the activation time is 1-5h; And / or, in step (2), the volume ratio of the cetyltrimethylammonium bromide aqueous solution, the HAuCl4·3H2O aqueous solution, the H2SO4 aqueous solution, the AgNO3 aqueous solution, the vitamin C aqueous solution and the seed solution is 100: (1-5): (1-5): (0.1-1): (0.1-1): (0.1-1); And / or, in step (2), when the cetyltrimethylammonium bromide aqueous solution is added with the HAuCl4·3H2O aqueous solution, the H2SO4 aqueous solution, the AgNO3 aqueous solution, the vitamin C aqueous solution and the seed solution obtained in step (1), the stirring speed is kept at 1000-2000 rpm / min; And / or, in step (2), after the mixing is uniform, the solution is stirred until it turns dark wine red and then is left to stand overnight; And / or, in step (2), the temperature for the overnight standing is 25-35℃; And / or, in step (3), the centrifugation speed is 10000-20000 rpm / min, and the centrifugation time is 10-30 min.

10. The double-layer hydrogel microneedle according to claim 9, wherein: In step (1), the volume ratio of the HAuCl4·3H2O aqueous solution, the cetyltrimethylammonium bromide aqueous solution and the NaBH4 aqueous solution is 0.1:7.5:0.6; And / or, in step (2), the volume ratio of the cetyltrimethylammonium bromide aqueous solution, the HAuCl4·3H2O aqueous solution, the H2SO4 aqueous solution, the AgNO3 aqueous solution, the vitamin C aqueous solution and the seed solution is 100:2.04:2:0.9:0.8:0.

24.

11. The double-layer hydrogel microneedle according to claim 10, wherein: In step (1), the concentration of the HAuCl4·3H2O aqueous solution is 20-30 mM; And / or, in step (1), the concentration of the cetyltrimethylammonium bromide aqueous solution is 0.1-1 M; And / or, in step (1), the concentration of the NaBH4 aqueous solution is 10-20 mM; And / or, in step (2), the concentration of the cetyltrimethylammonium bromide aqueous solution is 0.1-1 M; And / or, in step (2), the concentration of the HAuCl4·3H2O aqueous solution is 20-30 mM; And / or, in step (2), the concentration of the H2SO4 aqueous solution is 0.1-1 M; And / or, in step (2), the concentration of the AgNO3 aqueous solution is 0.01-0.05 M; And / or, in step (2), the concentration of the vitamin C aqueous solution is 0.1-1 M.

12. The double-layer hydrogel microneedle according to claim 11, wherein: In step (1), the concentration of the HAuCl4·3H2O aqueous solution is 24 mM; In step (1), the concentration of the cetyltrimethylammonium bromide aqueous solution is 0.1 M; In step (1), the concentration of the NaBH4 aqueous solution is 10 mM; In step (2), the concentration of the cetyltrimethylammonium bromide aqueous solution is 0.1 M; In step (2), the concentration of the HAuCl4·3H2O aqueous solution is 24 mM; In step (2), the concentration of the H2SO4 aqueous solution is 0.5 M; In step (2), the concentration of the AgNO3 aqueous solution is 0.01 M; In step (2), the concentration of the vitamin C aqueous solution is 0.1 M.

13. The double-layer hydrogel microneedle of claim 1, wherein: The volume ratio of the Schiff base hydrogel to the coordination hydrogel is 130: (30-100); In the Schiff base hydrogel, the volume ratio of the chitosan aqueous solution to the aldehyde-functionalized polyethylene glycol aqueous solution is (7-10): 4; In the coordination hydrogel, the volume ratio of the CuSO4 aqueous solution to the sodium alginate aqueous solution is (10-80):

160.

14. The double-layer hydrogel microneedle according to claim 13, wherein: The volume ratio of the Schiff base hydrogel to the coordination hydrogel is 130: 100; In the Schiff base hydrogel, the volume ratio of the chitosan aqueous solution to the aldehyde-functionalized polyethylene glycol aqueous solution is 9: 4; In the coordination hydrogel, the volume ratio of the CuSO4 aqueous solution to the sodium alginate aqueous solution is 40:

160.

15. The double-layer hydrogel microneedle of claim 14, wherein: The preparation method of the CS / PEG-CHO / SA / CuSO4 double-network hydrogel comprises the following steps: 1) uniformly mixing the CuSO4 aqueous solution and the aldehyde-functionalized polyethylene glycol aqueous solution to obtain a mixed solution; 2) uniformly mixing the sodium alginate aqueous solution and the chitosan aqueous solution to obtain a mixed solution; 3) uniformly mixing the mixed solution obtained in step 1) and the mixed solution obtained in step 2) according to the volume ratio of the Schiff base hydrogel to the coordination hydrogel to obtain the CS / PEG-CHO / SA / CuSO4 double-network hydrogel.

16. The double-layer hydrogel microneedle according to claim 15, wherein: The concentration of the CuSO4 aqueous solution is 0.01-0.1 M; In step (2), the concentration of the H2SO4 aqueous solution is 0.5 M; In step (2), the concentration of the AgNO3 aqueous solution is 0.01 M; In step (2), the concentration of the vitamin C aqueous solution is 0.1 M.

17. The double-layer hydrogel microneedle according to claim 16, wherein: The concentration of the CuSO4 aqueous solution is 0.05 M; In step (2), the concentration of the H2SO4 aqueous solution is 0.5 M; In step (2), the concentration of the AgNO3 aqueous solution is 0.01 M; In step (2), the concentration of the vitamin C aqueous solution is 0.1 M.

18. A method of making the double-layer hydrogel microneedle of any one of claims 1-17, characterized by: It comprises the following steps: (A) adding the FG MN tip premix solution into the microneedle template, removing the bubbles and drying; (B) The CS / PEG-CHO / SA / CuSO4 double network hydrogel is added into the microneedle template of step (A), and after removing the bubbles, it is dried to obtain the microneedle.

19. Use of the double-layer hydrogel microneedle according to any one of claims 1-17 in the preparation of a medical material for resisting oral squamous cell carcinoma and / or promoting repair of soft tissue defects.

20. The use according to claim 19, characterized in that: the oral squamous cell carcinoma is oral squamous cell carcinoma; and / or, the repair of soft tissue defects is repair of infectious soft tissue defects.

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