Functionalized hydrogel for treating atopic dermatitis and preparation method and application thereof

Abstract

The application relates to the technical field of medical materials, and discloses a functionalized hydrogel for treating atopic dermatitis and a preparation method and application thereof, the preparation method comprising the following steps: taking g-C3N4 powder as raw material to prepare C3N4 gel with a three-dimensional network structure; taking g-C3N4 powder and CDs powder as raw material, adding a matrix solution, and uniformly ultrasonically dispersing; adding a crosslinking agent solution to make the solution fully undergo crosslinking reaction to prepare a CDs / g-C3N4 crosslinking solution; pouring the above-mentioned CDs / g-C3N4 crosslinking solution into the above-mentioned C3N4 gel, standing for gelation, forming a functionalized modified carbon nitride gel three-dimensional network structure, and freeze-drying to obtain a functionalized three-dimensional crosslinking hydrogel. The functionalized three-dimensional crosslinking hydrogel prepared by the application has the advantages of degradability and the ability to penetrate through an intact skin barrier; and can be used for treating atopic dermatitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical materials, in particular to a functionalized hydrogel for treating atopic dermatitis and a preparation method and application thereof. BACKGROUND

[0002] Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by skin barrier dysfunction, Th2 / Th17 type immune imbalance and itch-inflammation vicious cycle. Its global incidence continues to rise, with a prevalence rate of 15-30% in children and 2-10% in adults. Its pathological mechanism involves the disorder of physical, immune, neural and other multi-dimensional barrier systems.

[0003] Existing treatment methods such as topical glucocorticoids, calcineurin inhibitors and JAK inhibitors can only provide short-term relief from symptoms, and long-term use can cause side effects such as skin atrophy, systemic immune suppression and drug resistance. In addition, the pathogenesis of AD is closely related to the disorder of skin microbiome, and traditional antibiotic or probiotic therapy has problems such as broad-spectrum killing and low survival rate of flora transplantation. The complexity of the pathological mechanism of AD and the limitations of existing therapies point to the necessity of multi-modal collaborative therapy, and there is an urgent need to develop an efficient treatment strategy that can simultaneously repair the skin barrier, regulate immunity and restore microbial homeostasis.

[0004] Photothermal therapy (PTT) is a new treatment mode that generates heat effects through light-induced local temperature rise, simultaneously generates reactive oxygen species (ROS), and establishes direct or indirect electron transfer pathways, forming a "heat-ROS-electric field" multi-physical field synergistic network, which theoretically has the potential to simultaneously intervene in the three pathological dimensions of AD. However, traditional PTT systems face the following technical bottlenecks: first, metal-based nano-photothermal agents (such as gold nanorods, molybdenum sulfide) are not biodegradable, and long-term retention can cause oxidative stress and organ toxicity; second, rigid nanomaterials are difficult to penetrate the intact skin barrier, often requiring the use of invasive methods such as nanoneedles or electroporation, which increases the risk of skin damage; third, the photothermal dose has poor adaptability to individual skin heterogeneity, and excessive temperature can cause epidermal burns or insufficient temperature rise, resulting in poor treatment effect; fourth, the correlation mechanism between photothermal energy transfer efficiency and flora metabolic regulation is not clear. Although the multi-physical field synergistic mechanism of photothermal therapy opens up a new path for AD treatment, breakthroughs in its technical bottlenecks require dual innovation in materials science and precise regulation technology. SUMMARY

[0005] The present application relates to the technical field of medical materials, in particular to a functionalized hydrogel for treating atopic dermatitis and a preparation method and application thereof.

[0006] To achieve the above object, the technical scheme of the present application is as follows:

[0007] The present application provides a functionalized hydrogel for treating atopic dermatitis and a preparation method and application thereof, comprising the following steps:

[0008] S1, preparing a C3N4 gel with a three-dimensional network structure by using g-C3N4 powder as raw material

[0009] S2, preparing a CDs / g-C3N4 cross-linking solution by using g-C3N4 powder and CDs powder as raw material

[0010] The g-C3N4 powder and the CDs powder are added into the matrix solution and uniformly dispersed by ultrasonic treatment; the cross-linking agent solution is slowly added dropwise, and continuous stirring is performed to ensure that the cross-linking agent solution is uniformly dispersed, so that the solution is fully cross-linked.

[0011] S3, preparing a functionalized three-dimensional cross-linked hydrogel

[0012] The CDs / g-C3N4 cross-linking solution of step S2 is poured into the C3N4 gel of step S1, and after standing and gelation, a functionalized modified carbon nitride gel three-dimensional network structure is formed, and the functionalized three-dimensional cross-linked hydrogel is obtained by freeze-drying.

[0013] The volume / mass ratio of the CDs / g-C3N4 cross-linking solution to the C3N4 gel is 8-12 ml:0.1 g.

[0014] Preferably, the cross-linking agent solution is a CaCl2 solution, and the volume molar concentration of the CaCl2 solution is 0.1-0.5 M.

[0015] Preferably, the mass concentration of the CaCl2 solution relative to the CDs / g-C3N4 cross-linking solution is 1wt%-8wt%.

[0016] Preferably, the matrix is one or more of sodium alginate, chitosan, cellulose derivatives and agar.

[0017] Preferably, the mass / volume fraction of the matrix solution is 1-5%.

[0018] Preferably, the mass ratio of the g-C3N4 powder to the CDs powder is 8-12:1, and the cross-linking reaction time is not less than 30 min.

[0019] Preferably, the C3N4 gel is obtained by dispersing g-C3N4 powder into an alkaline solution, ultrasonic crushing, centrifugation, freeze-drying to obtain an intermediate; then dispersing the intermediate into an ethanol aqueous solution with a volume fraction of 45-55%, ultrasonic treatment, transferring to an autoclave for reaction for more than 10 h; cooling to room temperature, and freeze-drying.

[0020] Preferably, the alkaline solution is CO3 2- The salt solution is NaOH solution with a volume molar concentration of 4-8 M; the mass-volume ratio of the g-C3N4 powder body and the alkaline solution is 9-11 mg:1 ml.

[0021] Preferably, the intermediate is a mixture of two-dimensional porous C3N4 nanosheets and one-dimensional nanowires; the mass-volume ratio of the intermediate and the aqueous ethanol solution is not more than 200 mg:30 ml.

[0022] The application also provides a functionalized three-dimensional cross-linked hydrogel prepared by the preparation method of the functionalized hydrogel.

[0023] The application also provides application of the above functionalized three-dimensional cross-linked hydrogel in treating atopic dermatitis.

[0024] In summary, the application has the following beneficial effects:

[0025] The functionalized three-dimensional cross-linked hydrogel prepared by the application has good therapeutic effect on atopic dermatitis; has the advantages of degradability, and does not cause oxidative stress reaction and organ toxicity in long-term use; can penetrate the complete skin barrier without the aid of invasive means such as nanoneedle or electroporation, thereby reducing the risk of skin damage; and has high adaptability of photothermal dose to individual skin heterogeneity. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a flowchart of the preparation method of the functionalized hydrogel;

[0027] Figure 2 is a material morphology characterization diagram of 3D CDs / CN;

[0028] Figure 3 is an X-ray diffraction diagram of CN before and after loading CDs, and B is an ultraviolet absorption diffuse reflection diagram of CN before and after loading CDs;

[0029] Figure 4 is a skin tissue staining section and skin wound recovery of mice in different groups;

[0030] Figure 5 is the serum IgE concentration level of mice in different groups;

[0031] Figure 6 is the growth of Staphylococcus aureus in different groups. DETAILED DESCRIPTION

[0032] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0033] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0034] It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this invention can be combined with each other. The invention will now be described in detail with reference to the embodiments.

[0035] like Figure 1 As shown, the present invention provides a method for preparing a functionalized hydrogel, comprising the following steps:

[0036] S1. Using g-C3N4 powder as raw material, prepare C3N4 gel with a three-dimensional network structure.

[0037] The C3N4 gel of this invention is prepared by dispersing 500 mg of g-C3N4 powder in 50 ml of 1 M K2CO3 aqueous solution and 50 ml of 6 M NaOH solution, respectively. After ultrasonic disruption and centrifugation, a mixture of two-dimensional porous C3N4 nanosheets and one-dimensional nanowires (intermediate) is collected, washed multiple times with water until neutral, and then freeze-dried. 200 mg of the intermediate is dispersed in 30 ml of 50% (v / v) ethanol aqueous solution, ultrasonicated for 30 min, transferred to a hydrothermal reactor, and maintained at 120 °C for 10 h. After cooling to room temperature, the C3N4 gel with a complete three-dimensional structure is obtained by freeze-drying.

[0038] S2. Using g-C3N4 powder and CDs powder as raw materials, prepare CDs / g-C3N4 crosslinking solution.

[0039] Add g-C3N4 powder and CDs powder to a matrix solution with a mass ratio of 8–12:1, and ultrasonically disperse until uniform. Slowly add a 0.1–0.5 M CaCl2 solution to achieve a CaCl2 concentration of 1 wt%–8 wt% in the CDs / g-C3N4 crosslinking solution, while continuously stirring to ensure the CaCl2 concentration remains constant. 2+ Disperse evenly to allow the solution to undergo a full cross-linking reaction; the reaction time should be no less than 30 minutes.

[0040] The above matrix solution (surfactant solution) can be one or more of sodium alginate, chitosan, cellulose derivatives, and agar.

[0041] S3, preparing a functionalized three-dimensional crosslinked hydrogel

[0042] The CDs / g-C3N4 crosslinked solution of step S2 is poured into the C3N4 gel of step S1, and left to stand for 2 h, and a functionalized modified carbon nitride gel three-dimensional network structure is formed after gelation, and freeze-drying obtains the functionalized three-dimensional crosslinked hydrogel (3D CDs / CN).

[0043] The volume-mass ratio of the CDs / g-C3N4 crosslinked solution to the C3N4 gel is 8-12 ml:0.1 g.

[0044] It should be noted that: the g-C3N4 powder of the application can be obtained by using melamine or urea as a precursor, placing it in a crucible, and calcining at 550 DEG C for 4 hours in a high-temperature muffle furnace to obtain yellow g-C3N4 powder.

[0045] The CDs powder of the application is prepared by using citric acid and urea as raw materials, dissolving in deionized water to form a mixed solution with a volume molar concentration of citric acid of 0.1-1 M and a volume molar concentration of urea of 0.1-0.5 M, transferring to a hydrothermal reactor and reacting at 180 DEG C for 12 hours; after the reaction, the obtained CDs solution is cooled to room temperature, centrifuged, washed several times with deionized water, and freeze-dried to obtain the CDs powder.

[0046] Example 1

[0047] The g-C3N4 powder and the CDs powder are added to a 3% mass fraction sodium alginate solution in a mass ratio of 10:1, and ultrasonically dispersed uniformly; a 0.3 M volume molar concentration CaCl2 solution is slowly added, so that the mass concentration of the CaCl2 solution in the CDs / g-C3N4 crosslinked solution is 5 wt%, and continuous stirring is ensured to ensure uniform dispersion of the Ca 2 The 50 mg CDs / g-C3N4 crosslinked solution is poured into 0.5 g C3N4 gel, left to stand for 2 h, and a functionalized modified carbon nitride gel three-dimensional network structure is formed after gelation, and freeze-drying obtains the functionalized three-dimensional crosslinked hydrogel (3D CDs / CN).

[0048] Example 2

[0049] The g-C3N4 powder and the CDs powder are added into a 1% mass fraction chitosan solution in a mass ratio of 8:1 and ultrasonically dispersed uniformly; a 0.1M CaCl2 solution is slowly added dropwise, so that the mass concentration of the CaCl2 solution in the CDs / g-C3N4 crosslinking solution is 8wt%, and stirring is continued to ensure that Ca 2+ is uniformly dispersed, so that the solution fully undergoes a crosslinking reaction, and the reaction time is not less than 30min, to obtain a CDs / g-C3N4 crosslinking solution. 40ml of the CDs / g-C3N4 crosslinking solution is poured into 0.5g of the C3N4 gel, and left to stand for 2h, so that a functionalized carbon nitride gel three-dimensional network structure is formed after gelation, and functionalized three-dimensional crosslinked hydrogel (3D CDs / CN) is obtained by freeze-drying.

[0050] Example 3

[0051] The g-C3N4 powder and the CDs powder are added into a 5% mass fraction mixed solution of cellulose derivatives and agar in a mass ratio of 12:1 and ultrasonically dispersed uniformly; a 0.5M CaCl2 solution is slowly added dropwise, so that the mass concentration of the CaCl2 solution in the CDs / g-C3N4 crosslinking solution is 1wt%, and stirring is continued to ensure that Ca 2+ is uniformly dispersed, so that the solution fully undergoes a crosslinking reaction, and the reaction time is not less than 30min, to obtain a CDs / g-C3N4 crosslinking solution. 60ml of the CDs / g-C3N4 crosslinking solution is poured into 0.5g of the C3N4 gel, and left to stand for 2h, so that a functionalized carbon nitride gel three-dimensional network structure is formed after gelation, and functionalized three-dimensional crosslinked hydrogel (3D CDs / CN) is obtained by freeze-drying.

[0052] The C3N4 gel (CN), the CDs powder and the CDs / g-C3N4 crosslinking solution (CDs / CN) in Example 1 are characterized by transmission electron microscopy, and the results are shown in Figures 2-3 .

[0053] The TEM results show that the CN powder is in a sheet layer shape, the particle size of the CDs is about 2-3nm, and the CDs are uniformly loaded on the CN layer. The CDs loading does not change the CN phase, but the characteristic peak intensity increases, and the CN crystallinity is improved (see Figure 3 A). After the CDs are loaded, the absorption band edge is red-shifted, and the range is expanded to nearly 1000nm (see Figure 3 B).

[0054] Application Examples

[0055] 1. Experimental Methods

[0056] 1.1 Construction of an atopic dermatitis mouse model:

[0057] On the first day of the experiment, 5% concentration of p-hydroxybenzoate (Mep) and DMSO solvent dissolved in DMSO were applied to the skin of the shaved back of the mice for sensitization, 100 μl / d, for 7 days, negative control group: only DMSO solvent, model group: 5% concentration of p-hydroxybenzoate

[0058] The application time and application dose of the control group were exactly the same as those of the model group.

[0059] 1.2 Experimental groups of the model group:

[0060] 5 model mice were placed in a dark environment, which was the dark control group; 5 model mice were placed in a dark environment and applied with the functionalized three-dimensional cross-linked hydrogel material prepared in Example 1, which was the material dark degradation group; 5 model mice were placed in a light environment, which was the light control group; 5 model mice were placed in a light environment and applied with the functionalized three-dimensional cross-linked hydrogel material prepared in this example, which was the material light degradation group.

[0061] 1.3 Animal experiment method

[0062] The mice (C57BL / 6) of the control group and the model group were anesthetized with anesthetic reagent injection liquid, and the hair on the back of each mouse was shaved off with an electric shaver. Then the limbs were fixed, and the skin was disinfected with 75% alcohol. A skin wound with an area of about 80 mm 2 was caused by using a sterile surgical scissors. The wounds of the mice in the material light degradation group and the 3D CDs / CN hydrogel covered light degradation group and the material dark degradation group were covered with the material, and the wounds of the mice in the material light degradation group were irradiated with visible light for 2 hours. The wounds of the mice in the material dark degradation group were not treated with light, which could be used as a control.

[0063] The lighting parameters are as follows: 300W xenon lamp, equipped with ultraviolet cutoff filter; illumination intensity: ≈100 mW cm -2 . The mice were examined every day to ensure that the hydrogel material was applied correctly. On the 1st, 3rd, 5th and 7th days, the wound surface was photographed with a digital camera. At the same time, the wound area was measured with Image J software. The mice were recorded and scored for scratching, and the total IgE in the serum was detected with an Elisa kit, HE staining was taken, and PCR was tested to determine the amount of S. aureus in the infected skin tissue at the specified time point, the specimen was collected and homogenized in 1 ml of PBS. Colony number determination and laser confocal testing were performed.

[0064] The mice were sacrificed on the 7th day after infection, and the infected skin of the wound was taken, homogenized, diluted appropriately, and then coated on mannitol sodium chloride agar medium (G+ bacteria) or blood plate (G+ bacteria), cultured for 18-24h, and the colonies were counted and converted into the bacterial content (cfu / g) per gram of tissue. For the Streptococcus pyogenes experiment, the colonies growing on the blood plate were picked and subjected to antibacterial circle sensitivity test with bacitracin paper, to verify that the colonies growing on the blood plate were the inoculated infection bacteria.

[0065] 2. Experimental results

[0066] 2.1 Functionalized three-dimensional cross-linked hydrogel is beneficial to the recovery of skin wound lesions in mice

[0067] From the above, Figure 4 It can be seen that after 1 week of treatment, the H&E staining results of the skin tissue show that the epidermal proliferation of the mice in the material photodegradation group (3D CDs / CN+light group) is significantly reduced, and it is beneficial to the recovery of skin wound lesions in mice.

[0068] 2.2 Functionalized three-dimensional cross-linked hydrogel has anti-inflammatory effect

[0069] From the above, Figure 5 It can be seen that the IgE of the untreated mice (dark control group and light control group) increases after Mep stimulation, and significantly decreases after the application of 3D CDs / CN light treatment.

[0070] 2.3 Functionalized three-dimensional cross-linked hydrogel has the effect of inhibiting Staphylococcus aureus

[0071] From the above, Figure 6 It can be seen that the Staphylococcus aureus infection of the untreated mice (dark control group and light control group) is severe after Mep stimulation, and the Staphylococcus aureus significantly decreases after the application of 3D CDs / CN light treatment. It shows that the photothermal property of the 3D CDs / CN light group can obviously inhibit the growth of Staphylococcus aureus.

[0072] In summary, the functionalized three-dimensional cross-linked hydrogel (3D CDs / CN) prepared by the application has good efficacy for treating atopic dermatitis.

[0073] The above-described embodiments only express the relatively optimal implementation of the present application, and the description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A method for preparing a functionalized hydrogel, characterized in that, Includes the following steps: S1. Using g-C3N4 powder as raw material, prepare C3N4 gel with a three-dimensional network structure. The C3N4 gel was prepared by dispersing g-C3N4 powder in an alkaline solution at a mass-to-volume ratio of 9-11 mg:1 ml, followed by ultrasonic disruption, centrifugation, and freeze-drying to obtain an intermediate. The intermediate was then dispersed in a 45-55% (v / v) ethanol aqueous solution, ultrasonically treated, and transferred to a hydrothermal reactor for reaction for at least 10 hours. After cooling to room temperature, it was freeze-dried. The alkaline solution was a CO3 solution with a volume molar concentration of 0.5-1.5 M. 2- Salt water solution and NaOH solution with a volume molar concentration of 4~8M; The intermediate is a mixture of two-dimensional porous C3N4 nanosheets and one-dimensional nanowires; the mass-to-volume ratio of the intermediate to the aqueous ethanol solution is no more than 200 mg: 30 ml. S2. Preparation of CDs / g-C3N4 crosslinking solution Add g-C3N4 powder and CDs powder in a mass ratio of 8~12:1 to the matrix solution and disperse them evenly by ultrasonication; slowly add the crosslinking agent dropwise while continuously stirring to ensure that the crosslinking agent is evenly dispersed and that the solution undergoes a full crosslinking reaction; The matrix is ​​one or more of sodium alginate, chitosan and agar, and the crosslinking agent is a CaCl2 solution with a volume molar concentration of 0.1~0.5 M. The CDs powder is prepared by dissolving citric acid and urea in deionized water to form a mixture with a citric acid volume molar concentration of 0.1-1 M and a urea volume molar concentration of 0.1-0.5 M. The mixture is then transferred to a hydrothermal reactor and reacted at 180°C for 12 hours. After the reaction, the mixture is cooled to room temperature, centrifuged to obtain the CDs solution, washed several times with deionized water, and then freeze-dried. S3. Preparation of functionalized three-dimensional cross-linked hydrogels According to a volume-to-mass ratio of 8~12ml:0.1g, the CDs / g-C3N4 crosslinking solution from step S2 was poured into the C3N4 gel from step S1. After standing and gelling, a functionalized modified carbon nitride gel three-dimensional network structure was formed. The gel was then freeze-dried to obtain a functionalized three-dimensional crosslinked hydrogel capable of preparing drugs for treating atopic dermatitis.

2. The method for preparing a functionalized hydrogel according to claim 1, characterized in that, The mass concentration of the CaCl2 solution relative to the CDs / g-C3N4 crosslinking solution is 1wt%~8wt%.

3. The method for preparing a functionalized hydrogel according to claim 1, characterized in that, The matrix solution has a mass-volume fraction of 1-5%.

4. The method for preparing a functionalized hydrogel according to claim 1, characterized in that, The crosslinking reaction time is not less than 30 minutes.

5. The method for preparing a functionalized hydrogel according to claim 1, characterized in that, The reaction temperature in the hydrothermal reactor is 120°C.

6. The functionalized three-dimensional cross-linked hydrogel prepared by the preparation method according to any one of claims 1 to 5.

7. The use of the functionalized three-dimensional cross-linked hydrogel as described in claim 6 in the preparation of drugs for treating atopic dermatitis.

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