Preparation method of PNIPAm hydrogel system and application of PNIPAm hydrogel system in treatment of psoriasis skin lesion

Abstract

The invention provides a preparation method of a PNIPAm hydrogel system and application of the PNIPAm hydrogel system in treatment of psoriasis skin lesion, and relates to the technical field of biomedicine, the PNIPAm hydrogel system, an M2 exosome and cerium dioxide are included, and the PNIPAm hydrogel system carries cerium dioxide and wraps the M2 exosome. The invention successfully prepares a PNIPAm hydrogel system, and also discloses an application of the PNIPAm hydrogel system in treatment of psoriasis. The M2 type macrophage exosome possibly affects the psoriasis microenvironment by affecting the polarization state of macrophages so as to achieve the treatment effect, meanwhile, the M2 type macrophage exosome can regulate expression of inflammatory factors, reduce expression of pro-inflammatory factors such as TNF-alpha and IL-1beta, and up-regulate expression of anti-inflammatory factors such as IL-10, so that the psoriasis treatment effect is improved, and the psoriasis treatment effect is improved. Therefore, the inflammatory reaction is relieved. According to the application, the application of the PNIPAm hydrogel in treatment of psoriasis is explored, and research shows that the PNIPAm hydrogel as an efficient and safe drug delivery carrier has a relatively good application prospect in treatment of psoriasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a preparation method of a PNIPAm hydrogel system and application of the system in treating psoriasis lesions. Background Art

[0002] Psoriasis is a chronic, recurrent, systemic autoimmune disease characterized by red papules or plaques on the skin covered with multiple layers of silvery white scales, which can be localized or widespread on the human body. Psoriasis is a multi-gene inheritance that affects 2-4% of the world's population, and 5% to 20% of patients develop psoriatic arthritis, which causes joint pain and stiffness in the affected area, movement disorders, and even disability.

[0003] In the existing technology, the treatment of psoriasis and its arthritis faces multiple challenges. Although there are many treatment methods on the market, the efficacy varies, and they are often accompanied by side effects such as dry skin, redness, swelling, and stinging, and may even cause more serious systemic reactions, greatly affecting the daily quality of life and mental health of patients.

[0004] In addition, the chronic recurrence of psoriasis is also a major problem in current treatment. How to effectively control the disease, prolong the remission period, and reduce the frequency of recurrence is the focus of common concern for doctors and patients. Summary of the invention

[0005] The purpose of the present invention is to solve the technical problem of lack of drugs for treating psoriasis in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A PNIPAm hydrogel system comprises a PNIPAm hydrogel system, M2 exosomes and cerium dioxide, wherein the PNIPAm hydrogel system carries cerium dioxide and is wrapped outside the M2 exosomes.

[0008] Preferably, the preparation method of the PNIPAm hydrogel system comprises the following steps:

[0009] S1: Preparation of M2 exosomes

[0010] S2: PNIPAM composite gel preparation

[0011] S3: Preparation of hydrogel system:

[0012] The M2 exosomes prepared in S1 were treated with Tris(2-carboxyethyl)phosphine;

[0013] Excess exosomes, CeO 2，The composite gel was stirred with PNIPAm at 4°C overnight to obtain a PNIPAm composite gel material encapsulating M2 exosomes and carrying CeO2.

[0014] Preferably, the method for preparing M2 exosomes in S1 is as follows:

[0015] Cultivate macrophages and induce them to become M2 cells.

[0016] When the cell confluence density was greater than 70%, the cells were washed 2-3 times with PBS.

[0017] After washing, serum-free medium was added and cultured for 24 hours.

[0018] The supernatant was taken and centrifuged using density gradient ultracentrifugation to prepare exosomes.

[0019] Preferably, the density gradient ultracentrifugation method in S1 is carried out in an environment of 4° C., and the specific steps are as follows:

[0020] Centrifuge at 300 × g for 15 min and collect the supernatant;

[0021] Centrifuge at 2000 × g for 15 min and collect the supernatant;

[0022] Centrifuge at 10,000 × g for 30 minutes and collect the supernatant;

[0023] The mixture was centrifuged at 150,000 × g for 90 min, the supernatant was removed, and the remaining precipitate was resuspended in PBS and filtered with a 0.22 μm filter to obtain M2 exosomes.

[0024] Preferably, the specific steps of S2 are as follows:

[0025] NIPAm and APS were added into water and heated at 60°C for 5-7h to form a white PNIPAm composite gel.

[0026] Preferably, the ratio of NIPAm to APS in S2 is 1:10.

[0027] The present application also provides the use of a PNIPAm hydrogel system in the preparation of a drug for treating psoriasis, wherein the PNIPAm hydrogel system is the PNIPAm hydrogel system described above.

[0028] The PNIPAm hydrogel system of this application encapsulates M2 exosomes and carries cerium dioxide. The cerium dioxide nanosystem has good enzyme activity and biosafety, and can successfully remove the DNA skeleton of neutrophil extracellular traps (NETs), reduce psoriasis skin inflammation, and at the same time, M2 macrophage exosomes can regulate the expression of inflammatory factors, reduce pro-inflammatory factors, and achieve therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow cytometry identification diagram of M2 exosomes in one embodiment of the present invention;

[0030] Figure 2 This is a transmission electron microscopy photograph of M2 exosomes in one embodiment of the present invention;

[0031] Figure 3 This is a graph showing the particle size and potential of M2 exosomes in one embodiment of the present invention;

[0032] Figure 4 This is a diagram showing CeO2 enzyme activity in one embodiment of the present invention, used to show CeO2

[0033] SOD activity and hydroxyl free radical scavenging rate;

[0034] Figure 5 This is a diagram showing the ROS scavenging ability of cerium dioxide nanoparticles in one embodiment of the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with specific embodiments.

[0036] A method for preparing a PNIPAm hydrogel system comprises the following steps:

[0037] S1: M2 exosome preparation:

[0038] Cultivate macrophages and induce them to become M2 cells.

[0039] When the cell confluence density was greater than 70%, the cells were washed 2-3 times with PBS.

[0040] After washing, serum-free medium was added and cultured for 24 hours.

[0041] The supernatant was taken and centrifuged using density gradient ultracentrifugation to prepare exosomes.

[0042] In one embodiment, the density gradient ultracentrifugation is performed at 4°C, and the specific steps are as follows:

[0043] Centrifuge at 300 × g for 15 min and collect the supernatant;

[0044] Centrifuge at 2000 × g for 15 min and collect the supernatant;

[0045] Centrifuge at 10,000 × g for 30 minutes and collect the supernatant;

[0046] The mixture was centrifuged at 150,000 × g for 90 min, the supernatant was removed, and the remaining precipitate was resuspended in PBS and filtered with a 0.22 μm filter to obtain M2 exosomes.

[0047] S2: Preparation of PNIPAM composite gel:

[0048] Add 3.5 g NIPAm and 35 mg APS to 3.5 mL water;

[0049] Heat at 60℃ for 5-7h to form a white PNIPAm composite gel

[0050] S3: Preparation of hydrogel system:

[0051] The exosomes obtained in S1 were treated with 0.5, 1, and 2 mM Tris (2-carboxyethyl) phosphine (tris (2-carboxyethyl) phosphine hydrochloride) at 37°C for 30 min.

[0052] Then excess exosomes, 100 mg CeO 2， The mixture was stirred with 20 mL of PNIPAm composite gel at 4°C overnight to obtain a PNIPAm composite gel material encapsulating M2 exosomes and carrying CeO2.

[0053] The present application also provides a PNIPAm hydrogel system, using the above-mentioned preparation method, wherein the PNIPAm hydrogel system includes M2 macrophage exosomes and PNIPAm hydrogel, and the PNIPAm hydrogel encapsulates the M2 exosomes and carries cerium dioxide.

[0054] Based on the PNIPAm hydrogel system described above, the present application also provides the use of the PNIPAm hydrogel system in the preparation of drugs for treating psoriasis.

[0055] The above contents are described below in conjunction with specific embodiments.

[0056] Example 1: Identification of exosomes:

[0057] S1: Add 20 μL of fluorescently labeled antibodies, F480 and CD206 antibodies, to the M2 cell samples prepared in step 1 of the above preparation method, respectively, mix well, and incubate at 37° C. for 30 min in the dark;

[0058] S2: Add 1 mL of pre-cooled PBS, centrifuge at 4°C and remove the supernatant;

[0059] S3: Resuspend in 50 μL pre-cooled PBS

[0060] S4: On the flow cytometer, pay attention to the gradient dilution to avoid the sample clogging the injection needle

[0061] The experimental results are as follows Figure 1As shown, the M2 cell sample prepared in step 1 can be identified as M2 cells based on the flow cytometry results, and thus the subsequently collected samples can be identified as M2 exosomes.

[0062] Example 2: M2 exosome morphology detection:

[0063] After ultrasonic dispersion of the exosomes, a suspension was prepared. A few drops were dropped onto the electron microscope copper grid using a pipette. After drying (or blotting with filter paper), it became a sample for electron microscope observation. The test results were as follows: Figure 2 shown.

[0064] Example 3: M2 exosome potential and particle size detection:

[0065] Take 20 mg of the sample, add it to pure water for dispersion, ultrasonicate for 1 min, and drop it into the test chamber.

[0066] Experimental results: Wait for the intensity to be moderate, repeat the above three times, and get the average result and curve spectrum of the sample. Figure 3 As shown, the particle size of M2 exosomes is about 100 nm and exhibits a slightly negative potential.

[0067] Example 4: Verification of the anti-inflammatory properties of the PNIPAm hydrogel system:

[0068] 1. Prepare instruments: any specification of microplate reader (450±10nm wavelength), ordinary disposable 96-well microplate, micropipette (single-channel pipette, multi-channel pipette), 37℃ constant temperature incubator (or water bath), ordinary low-speed centrifuge

[0069] 2. According to the Nanjing Jiancheng kit operation table, add the sample to be tested, distilled water, enzyme working solution, enzyme diluent, and substrate application solution, gently shake the well plate to mix, incubate at 37℃ for 20 minutes, take out the microplate reader reading at 450nm, and calculate according to the formula described in the manual

[0070] The results are as follows Figure 4 As shown, the prepared PNIPAm hydrogel system has enzyme activity, including hydroxyl radical scavenging rate, SOD activity, and anti-inflammatory properties.

[0071] Example 5: PNIPAm hydrogel system neutralizes ROS verification:

[0072] 1. Prepare cells: Evenly spread the target cells in a six-well plate, perform corresponding treatments on the experimental group and the control group, and set up a blank tube (i.e., no probe loaded) until the cell density reaches 80%-95%.

[0073] 2. Washing: Wash the cells in the six-well plate 1-2 times with PBS and discard the PBS.

[0074] 3. Digestion: Use trypsin to digest the cells in the six-well plate, transfer them to a 1.5 mL EP tube, and mark it.

[0075] 4. Centrifugation: 1200 rpm, room temperature, centrifugation for 5 minutes.

[0076] 5. Probe preparation: Prepare 2 μL DCFH-DA stock solution: 3 mL serum-free culture medium, mix by inverting (keep away from light).

[0077] 6. Add probe: discard 4 culture media, add 1mL serum-free culture medium to the blank tube, add 1mL prepared probe solution to the rest, and resuspend the cells (be careful to avoid light).

[0078] 7. Incubation: Incubate at 37°C in dark for 20 min.

[0079] 8. Centrifugation: 1200 rpm, room temperature, centrifuge for 5 minutes, discard the supernatant.

[0080] 9. Washing: Wash with 1 mL of cold PBS, centrifuge at 1200 rpm, room temperature for 5 min, discard the supernatant, and repeat twice (be careful to avoid light).

[0081] 10. Loading: Resuspend the cells in 500 μL cold PBS, transfer to flow cytometry tube, and load onto the machine.

[0082] The results are as follows Figure 5 As shown, the PNIPAm hydrogel system prepared in the present application has antioxidant properties, and the PNIPAm hydrogel system helps to neutralize ROS, which is a key factor in promoting the development of psoriasis. The flow cytometry results prove the ability to reduce ROS at different concentrations, thereby reflecting the efficacy of psoriasis.

[0083] Experimental materials and sources in the above examples:

[0084] Total superoxide dismutase (SOD) assay kit, Nanjing completed Hydroxyl Radical Test Kit Nanjing completed Reactive oxygen species detection kit Solebao NIPAm、APS Sigma Tris(2-carboxyethyl)phosphine Aladdin

[0085] Cerium dioxide nanoparticles (CeO2), as an emerging rare earth nanomaterial, have shown great potential for application in the field of antioxidant and anti-inflammatory. These nanoparticles have attracted attention for their excellent antioxidant capacity, especially in simulating the activity of antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD). Because its ROS scavenging mechanism is similar to the biological process used by endogenous enzymes in our body (such as superoxide dismutase [SOD] and catalase). As a potential therapeutic candidate, CeO2's endogenous antioxidant properties help neutralize ROS while inhibiting the activity of inflammatory cytokines, which are key factors driving the development of psoriasis.

[0086] M2 macrophage exosomes may affect the psoriasis microenvironment by affecting the polarization state of macrophages, thereby playing a therapeutic role. At the same time, M2 macrophage exosomes can regulate the expression of inflammatory factors, reduce the expression of proinflammatory factors such as TNF-α and IL-1β, and upregulate the expression of anti-inflammatory factors such as IL-10, thereby alleviating the inflammatory response.

[0087] In view of the anti-inflammatory properties of CeO2 nanoparticles and M2 macrophage exosomes, a PNIPAm composite hydrogel system encapsulating M2 exosomes and carrying cerium dioxide was developed to reduce ROS and alleviate inflammatory responses. This strategy not only targets the clearance of skin inflammation, but also achieves local delivery of CeO2 to the lesion site through the sustained release properties of PNIPAm hydrogel, thereby improving the therapeutic effect and reducing systemic side effects. Through this innovative treatment method, researchers have provided a new perspective for the treatment of psoriasis, which is expected to achieve more effective inflammation control and skin lesion repair.

[0088] In summary, the present invention verifies the successful synthesis of the material through characterization experiments, and draws conclusions through in vitro experiments that the PNIPAm composite hydrogel system has good enzyme activity and biosafety, and can successfully reduce ROS levels. This application explores the application of the multifunctional hydrogel system in the treatment of psoriasis, and through research, it is shown that the PNIPAm composite hydrogel, as an efficient and safe drug delivery carrier, has good application prospects for the treatment of psoriasis.

Claims

1. A PNIPAm hydrogel system, characterized in that: It includes a PNIPAm hydrogel system, M2 exosomes and cerium dioxide. The PNIPAm hydrogel system carries cerium dioxide and is wrapped outside the M2 exosomes.

2. A PNIPAm hydrogel system according to claim 1, characterized in that: The preparation method of the PNIPAm hydrogel system comprises the following steps: S1: Preparation of M2 exosomes S2: PNIPAM composite gel preparation S3: Preparation of hydrogel system: The M2 exosomes prepared in S1 were treated with Tris(2-carboxyethyl)phosphine; Excess exosomes, CeO2 and PNIPAm composite gel were stirred at 4°C overnight to obtain a PNIPAm composite gel material encapsulating M2 exosomes and carrying CeO2.

3. A PNIPAm hydrogel system according to claim 2, characterized in that: The method for preparing M2 exosomes in S1 is as follows: Cultivate macrophages and induce them to become M2 cells. When the cell confluence density was greater than 70%, the cells were washed 2-3 times with PBS. After washing, serum-free medium was added and cultured for 24 hours. The supernatant was taken and centrifuged using density gradient ultracentrifugation to prepare exosomes.

4. A PNIPAm hydrogel system according to claim 3, characterized in that: The density gradient ultracentrifugation method in S1 is carried out in a 4°C environment, and the specific steps are as follows: Centrifuge at 300 × g for 15 min and collect the supernatant; Centrifuge at 2000 × g for 15 min and collect the supernatant; Centrifuge at 10,000 × g for 30 minutes and collect the supernatant; The mixture was centrifuged at 150,000 × g for 90 min, the supernatant was removed, and the remaining precipitate was resuspended in PBS and filtered with a 0.22 μm filter to obtain M2 exosomes.

5. A PNIPAm hydrogel system according to claim 4, characterized in that: The specific steps of S2 are as follows: NIPAm and APS were added into water and heated at 60°C for 5-7h to form a white PNIPAm composite gel.

6. A PNIPAm hydrogel system according to claim 5, characterized in that: The ratio of NIPAm to APS in the S2 is 1:

10.

7. Application of PNIPAm hydrogel system in the preparation of drugs for treating psoriasis, characterized in that: The PNIPAm hydrogel system is the PNIPAm hydrogel system described in claims 1-6.

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