Temperature-sensitive hydrogel based on natural melanin and application thereof

Abstract

The invention belongs to the technical field of medical products, and particularly relates to temperature-sensitive hydrogel based on natural melanin and application of the temperature-sensitive hydrogel. The invention aims to solve the technical problems of drug resistance, local adverse reaction, relapse after drug withdrawal, ineffectiveness of part of patients, high price, infection, high risk of cancer, large systemic toxic and side effects and the like in clinical treatment drugs for psoriasis, including local drugs, immunosuppressants and various targeted biological agents, at the present stage. According to the technical scheme, the temperature-sensitive hydrogel based on the natural melanin is prepared by loading the natural melanin on an amphiphilic high-molecular polymer. According to the invention, the natural melanin nanoparticles and the amphiphilic high-molecular polymer are mixed in a sol state, so that the natural nano active drug melanin with excellent biocompatibility is used for transdermal drug delivery of psoriasis and safe and effective anti-inflammatory treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical products, and particularly relates to a thermosensitive hydrogel based on natural melanin and application thereof. Background Art

[0002] Psoriasis is a chronic, inflammatory skin disease mediated by immune response, with erythema and scaling as the main clinical manifestations. The incidence rate is about 0.14-1.99% of the world's population. It can involve systemic lesions and seriously endanger human physical and mental health. Psoriasis is prone to relapse and difficult to cure. Currently, the clinically effective psoriasis treatment drugs are mainly systemic immunosuppressants and biological agents. Although they have significant efficacy, they are expensive. At the same time, there are drug resistance, local adverse reactions, recurrence after discontinuation of medication, high prices, infection, high risk of cancer and serious toxic side effects. At the social level, psoriasis cannot be cured and requires lifelong treatment with a long course of treatment. This not only creates a huge medical burden on society, but also increases the economic pressure on patients. There is an urgent need for safe and effective new drugs for the treatment of psoriasis in clinical practice. Safe, non-toxic and low-cost natural products provide more possibilities for the development of new drugs for psoriasis. On the technical level, the stratum corneum of psoriasis lesions is significantly thickened, resulting in poor transdermal delivery of local drugs, low concentrations, and unsatisfactory effects. Currently, there is no ideal and efficient drug preparation among psoriasis topical preparations that can simultaneously achieve effective adhesion and transdermal penetration in areas of thickened stratum corneum.

[0003] As a promising new delivery system, thermosensitive hydrogel has been widely used in precision treatment of tumor treatment, skin treatment and anti-infection, thanks to its unique physicochemical properties, that is, it can achieve sol-gel phase transition under temperature stimulation. Thermosensitive hydrogel can evenly cover irregular skin lesions in a flowing state, and then phase transition in a very short time, continuously adhering to and staying on the skin surface in a gel state. This property provides conditions for the adhesion of drug molecules carried inside and their continuous action on the skin. Journal of Nanobiotechnology (2022, https: / / doi.org / 10.1186 / s12951-022-01368-y) reported a composite hydrogel with mesoporous zinc oxide silver nanoparticles (ZnO / Ag) with anti-inflammatory activity as a sustained-release carrier of systemically administered immunosuppressants, which is used for local transdermal delivery and sustained release of systemically administered immunosuppressants, and jointly enhances the anti-inflammatory and anti-proliferative effects of systemically administered immunosuppressants, achieving superior transdermal absorption performance and therapeutic effects of psoriasis. Although the above-mentioned approach has significantly improved the transdermal treatment of psoriasis, it still has great potential for further improvement due to safety considerations compared to natural drug treatments.

[0004] Natural melanin is widely present in nature, has excellent biocompatibility, excellent photothermal properties, free radical scavenging ability and strong chelating ability. Natural melanin exhibits a variety of biomedical applications, including UV radiation protection, cataract symptom relief, cancer treatment, and anti-infection treatment. Studies have reported that natural melanin has a unique polyphenol structure and excellent antioxidant and anti-inflammatory properties. Natural melanin-like nanoparticles with a large number of reducing functional groups have shown great potential in tissue repair. They have been successfully used to treat ROS-related diseases such as ischemic stroke, periodontal disease, acute peritonitis and acute kidney / lung injury. Hydrogels containing artificial melanin-like nanoparticles also exhibit excellent tissue adhesion and antioxidant properties in tissue repair. However, there has been no research report on the anti-inflammatory treatment of psoriasis using thermosensitive hydrogels containing natural melanin. Summary of the invention

[0005] The present invention provides an application of a thermosensitive hydrogel developed based on natural melanin in the treatment of psoriasis. The technical problem to be solved is to construct an application of a thermosensitive hydrogel developed based on natural active drugs in the treatment of psoriasis. As a safe, effective and inexpensive topical treatment drug for psoriasis, the sol-gel transition of the natural melanin thermosensitive hydrogel can promote the percutaneous penetration of natural melanin in psoriasis lesions and prolong the local retention time, thereby enhancing its local anti-inflammatory treatment effect.

[0006] The technical solution of the present invention is a thermosensitive hydrogel based on natural melanin, which is prepared by loading natural melanin on an amphiphilic high molecular polymer.

[0007] Furthermore, the amphiphilic high molecular polymer is at least one of D,L-lactide-polyethylene glycol-D,L-lactide copolymer, polyvinyl pyrrolidone or polyethylene.

[0008] Wherein, the natural melanin is extracted from at least one of cuttlefish ink, black rice or black sesame.

[0009] The present invention also provides a method for preparing the thermosensitive hydrogel, comprising the following steps:

[0010] S1. Preparation of melanin nanoparticles: dissolving crude natural melanin in solvent A, then mixing with solvent B, reacting, and purifying to obtain melanin nanoparticles;

[0011] S2. Preparation of thermosensitive hydrogel: At room temperature, melanin nanoparticles are added to an amphiphilic high molecular polymer, stirred evenly, and allowed to stand at 4°C to obtain a thermosensitive hydrogel based on natural melanin.

[0012] Specifically, in step S1, the solvent A is at least one of hydrochloric acid, nitric acid or acetic acid.

[0013] Furthermore, the hydrochloric acid is concentrated hydrochloric acid diluted 10,000 times.

[0014] Specifically, in step S1, the solvent B is at least one of methanol, ethanol or ammonium hydroxide.

[0015] Specifically, the concentration of ethanol is 99.5%.

[0016] Specifically, the mass fraction of ammonium hydroxide is 28-30%.

[0017] Particularly, in step S1, the reaction time is 12 h.

[0018] Wherein, in step S1, the purification is at least one of stirring, dialysis or centrifugation.

[0019] Specifically, the purification uses a dialysis membrane.

[0020] Furthermore, the molecular weight cut-off of the dialysis membrane is 1 kDa.

[0021] Preferably, in step S2, the stirring time is 20 min to 60 min.

[0022] Preferably, in step S2, the standing time is 12 h to 36 h.

[0023] Wherein, in step S2, the mass ratio of the natural melanin nanoparticles to the amphiphilic high molecular polymer is 1:50 to 1:100.

[0024] Preferably, the mass ratio of natural melanin nanoparticles to amphiphilic high molecular polymer is 1:100.

[0025] Furthermore, in step S2, the amphiphilic high molecular polymer is D,L-lactide-polyethylene glycol-D,L-lactide copolymer, polyvinyl pyrrolidone and / or polyethylene.

[0026] Specifically, the preparation process of the polymer D,L-lactide-polyethylene glycol-D,L-lactide copolymer is as follows: eliminate the water content of polyethylene glycol to constant weight; add D,L-lactide and stannous isozincate as linkers, and react at 140°C under nitrogen protection; cool after the reaction is completed, dissolve the obtained product in ethanol, add excess cold n-pentane, filter, and dry the precipitate to constant weight.

[0027] Specifically, the polyethylene glycol is polyethylene glycol 1000 (PEG1000), polyethylene glycol 1500 (PEG1500) or polyethylene glycol 2000 (PEG2000).

[0028] Specifically, polyethylene glycol was heated in a vacuum oven at 100°C for 1 h to eliminate moisture to a constant weight.

[0029] Furthermore, the mass ratio of polyethylene glycol, D,L-lactide and stannous isozincate is 20:40:0.18.

[0030] Preferably, the reaction is carried out at 140° C. for 12 h under nitrogen protection.

[0031] The precipitate was dried under vacuum at 45°C to a constant weight.

[0032] The invention also provides a thermosensitive hydrogel obtained by the preparation method.

[0033] The present invention also provides application of the thermosensitive hydrogel in preparing a method for treating psoriasis.

[0034] Beneficial effects of the present invention: The present invention mixes natural melanin nanoparticles with amphiphilic high molecular polymers in a sol state, which not only provides a new carrier for local effective transdermal delivery of natural melanin, but also improves the local drug concentration of natural melanin; and the sol-gel transition of the thermosensitive hydrogel under body temperature stimulation also prolongs the local retention time of natural melanin in the skin lesions, and enhances its anti-inflammatory effect, significantly downregulating the expression of pro-inflammatory factors; in the mouse psoriasis model, it also significantly improves the erythema and scaling symptoms of mouse skin lesions, achieving effective transdermal administration of natural melanin and anti-inflammatory treatment of psoriasis. In addition, the thermosensitive hydrogel based on natural melanin described in the present invention has a simple preparation method, mild process conditions, and is not harsh on reaction conditions, and has the potential for rapid industrial production. The present invention lays a foundation for the clinical transformation of natural melanin for local treatment of psoriasis and provides a new strategy for the prevention and treatment of psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a scanning electron microscope (SEM) photograph of melanin nanoparticles of the thermosensitive hydrogel based on natural melanin prepared in Example 1.

[0036] Figure 2 These are phase change images of the blank control hydrogel and the thermosensitive hydrogel based on natural melanin prepared in Example 1 at different temperatures.

[0037] Figure 3 This is a toxicity test of melanin nanoparticles of different concentrations in the natural melanin-based thermosensitive hydrogel prepared in Example 1 in keratinocytes (HaCaT).

[0038] Figure 4 The prepared Example 1 is based on the different concentrations of melanin nanoparticles and H in the natural melanin thermosensitive hydrogel 2 O 2 ROS fluorescence image of HaCaT cells co-incubated with TNF-α.

[0039] Figure 5 These are photos of the back skin of mice taken during the treatment of each treatment group in Example 1 on the 5th day after the treatment was completed.

[0040] Figure 6 These are H&E slice images of the back skin tissue obtained from each experimental group in Example 1. DETAILED DESCRIPTION

[0041] Natural melanin is widely present in nature and has excellent biocompatibility, photothermal properties, free radical scavenging ability and strong chelating ability. The application of natural melanin in biomedicine is diverse, including ultraviolet radiation protection, cataract symptom relief, anti-infection and cancer treatment. Studies have reported that natural melanin has a unique polyphenol structure and has excellent antioxidant and anti-inflammatory properties. Natural melanin-like nanoparticles with a large number of reducing functional groups have shown great potential in tissue repair and anti-inflammatory aspects.

[0042] Amphiphilic polymer hydrogels are thermosensitive and are in a gel state at 34-40°C, so they are used as carriers.

[0043] As a promising new delivery system, thermosensitive hydrogel has a wide range of applications in tumors, skin diseases and anti-infection, thanks to its unique physicochemical properties, namely, it can achieve sol-gel phase transition under temperature stimulation. Thermosensitive hydrogel can evenly cover irregular skin lesions in a flowing state and undergo phase transition in a very short time, continuously adhering to and staying on the skin surface in a gel state. This property provides conditions for increasing the adhesion and continuous action of natural melanin on the skin.

[0044] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0045] Example 1 Preparation of thermosensitive hydrogel based on natural melanin

[0046] 1. Synthesis of D,L-lactide-polyethylene glycol-D,L-lactide copolymer

[0047] Polyethylene glycol 1500 (PEG1500) was heated at 100°C for 1h in a vacuum drying oven to eliminate moisture to constant weight; after cooling to room temperature, 20g was weighed, 40g of D,L-lactide and 0.18g of stannous isozincate were added as a linker, and the mixture was reacted at 140°C for 12h under nitrogen protection; after the reaction was completed and cooled, the obtained product was dissolved in ethanol, and an excess of cold n-pentane (600mL) was used to extract it from the filtrate, and the precipitate was vacuum dried at 45°C to constant weight to obtain D,L-lactide-polyethylene glycol-D,L-lactide copolymer.

[0048] D,L-lactide-polyethylene glycol-D,L-lactide copolymer was mixed with deionized water to prepare a hydrogel with a mass concentration of 25%, and the hydrogel was stored in a refrigerator at 4°C.

[0049] 2. Preparation of natural melanin nanoparticles

[0050] Under magnetic stirring, 2.4 mL of ethanol (Vokay, CAS No. 64-17-5, 99.5%) and 5.4 mL of ammonium hydroxide (sigma, CAS Nol: 1336-21-6; mass fraction of 28% to 30%) were mixed evenly; concentrated hydrochloric acid was diluted 1:10000 times, and cuttlefish melanin (purchased from Sigma, CAS Nol: 8049-97-6) was added; the above two solutions were mixed and reacted for 12 hours, and purified using a dialysis membrane (molecular weight: ~1kDa) to remove excess reactants, and finally uniform natural melanin nanoparticles were obtained.

[0051] 3. Preparation of thermosensitive hydrogel based on natural melanin

[0052] The natural melanin nanoparticle solution obtained in step 2 is dissolved in ethanol, and D,L-lactide-polyethylene glycol-D,L-lactide copolymer in a sol state is added at room temperature (the mass ratio of natural melanin nanoparticles to D,L-lactide-polyethylene glycol-D,L-lactide copolymer is 1:100), stirred at room temperature for 0.5 h, and allowed to stand in a 4°C refrigerator for 24 h to obtain a thermosensitive hydrogel based on natural melanin.

[0053] The melanin nanoparticles and hydrogel prepared in this example were characterized in terms of morphology:

[0054] like Figure 1 As shown, it can be seen from the scanning electron microscope (SEM) photos that the obtained natural melanin nanoparticle target product has a uniform size, and the particle size of the nanoparticles is in the range of 10 to 17 nm.

[0055] Take the blank thermosensitive hydrogel and the thermosensitive hydrogel based on natural melanin, put them into 10 mL wide-mouth bottles, place them in a 4°C refrigerator for 2 minutes, take photos and record, and then transfer them to a 37°C constant temperature box for 2 minutes and take photos and record. Figure 2 It can be seen that at 4°C, both the blank hydrogel and the thermosensitive hydrogel based on natural melanin present a flowing sol state. In a constant temperature box at 37°C, the flowing sol state transforms into a gel state.

[0056] The sol-gel transition of the hydrogel depends on the changes in its loss modulus (G”) and storage modulus (G’). In order to verify its thermosensitive properties, the temperature-rheological related data of the thermosensitive hydrogel based on natural melanin were collected by a rotational rheometer. From the results, it can be seen that when the temperature is higher than 34.2°C, the G’ of the photoresponsive thermosensitive hydrogel is greater than G”, forming a gel state, indicating that it can be induced to transition from the sol state to the gel state under the human body temperature environment.

[0057] Example 2 Testing of therapeutic effects on in vitro psoriasis cell models

[0058] To verify the safety of in vitro cell therapy based on natural melanin thermosensitive hydrogel, HaCaT cells in normal state were cultured in logarithmic phase and incubated with natural melanin nanoparticles at different concentrations (0, 5, 10, 25, 50, 100, 150, 200 μg / mL) in 96-well plates for 24 h. Figure 3 It can be seen that when the concentration of natural melanin is as high as 200 μg / mL, there is no significant difference in the cell survival rate after co-incubation. This result shows that natural melanin nanoparticles have good biocompatibility.

[0059] Next, to verify the anti-inflammatory effect of melanin nanoparticles in thermosensitive hydrogel based on natural melanin, a H 2 O 2 The HaCaT cell model stimulated by TNF-α and TNF-α was used to observe the changes of ROS in different treatment groups using a laser scanning confocal microscope (CLSM: Confocal laser scanning microscope) using a reactive oxygen species (ROS) fluorescent probe (DCHF-DA: 2',7'-Dichlorodihydrofluorescein diacetate). The groups are as follows:

[0060] Blank group: HaCaT cells were not treated;

[0061] Model group: 100 ng / mL TNF-α and 200 μM H 2 O 2 The medium was used to co-stimulate the culture of HaCaT cells;

[0062] Experimental group: The model group was co-incubated with 50 μg / mL melanin nanoparticles.

[0063] from Figure 4 It can be seen that the green fluorescence of DCHF-DA in the model group is significant, indicating that the inflammatory model was successfully established. With the addition of natural melanin nanoparticles, the green fluorescence signal decreased significantly. This result shows that natural melanin has excellent ROS scavenging ability.

[0064] Example 3 Testing the therapeutic effect on psoriasis animal model

[0065] The therapeutic effect of the natural melanin-based thermosensitive hydrogel prepared in this example on psoriasis via transdermal spraying was tested according to the following steps:

[0066] Female C57BL / 6J mice aged 6 to 8 weeks were randomly divided into 3 groups (n=5). The hair in the central area of ​​the back of the mice was carefully shaved with an electric hair remover to form an exposed area of ​​2 cm×3 cm. A mild depilatory cream was then used to remove the surface hair. Imiquimod cream (Imiquimod, IMQ) 62.5 mg / d was applied to the back of mice in each group for 5 consecutive days. IMQ was discontinued from the 6th day and treatment was started with daily topical application of a thermosensitive hydrogel based on natural melanin at a therapeutic dose of 0.5 mL / d.

[0067] Normal group: normal mice were not treated;

[0068] Model group: mice with successful modeling were not treated;

[0069] Positive control group: Calcipotriol was applied on the back of model mice;

[0070] Experimental group: The back of model mice was coated with thermosensitive hydrogel based on natural melanin.

[0071] After each treatment, the skin lesions of each group were photographed. After the tenth day of photography, the mice were euthanized, and the mouse skin tissues were collected and fixed with 4% paraformaldehyde for subsequent H&E staining of histopathology.

[0072] Figure 5 The following are representative photos of the back skin of mice taken after the treatment of each treatment group in this example on the 10th day after the treatment. It can be observed from the figure that the skin of the experimental group showed white and smooth changes, which was closer to normal skin and comparable to the positive control group, which verified that the hydrogel prepared in this example can achieve a good therapeutic effect on psoriasis.

[0073] Figure 6The H&E slice images of the back skin tissue obtained for each treatment group in this example. Histopathological analysis showed that compared with the control group, the epidermal thickness of the experimental group was significantly reduced, the inflammatory cell infiltration in the dermis was reduced, and the hyperkeratosis and parakeratosis were significantly improved, which was comparable to the positive control group. This result verified that the natural melanin thermosensitive hydrogel was effective in treating psoriasis inflammation.

Claims

1. A thermosensitive hydrogel based on natural melanin, characterized in that: It is prepared by loading natural melanin with amphiphilic polymer; Preferably, the amphiphilic high molecular polymer is at least one of D,L-lactide-polyethylene glycol-D,L-lactide copolymer, polyvinyl pyrrolidone or polyethylene; Preferably, the natural melanin comes from cuttlefish ink, black rice and / or black sesame.

2. The method for preparing the thermosensitive hydrogel according to claim 1, characterized in that: The steps include: S1. Preparation of natural melanin nanoparticles: dissolving crude natural melanin in solvent A, then mixing with solvent B, reacting, and purifying to obtain melanin nanoparticles; S2. Preparation of thermosensitive hydrogel: At room temperature, natural melanin nanoparticles are added to an amphiphilic high molecular polymer, stirred evenly, and allowed to stand at 4°C to obtain a thermosensitive hydrogel based on natural melanin.

3. The preparation method according to claim 2, characterized in that: Contains at least one of the following features: a. In step S1, the solvent A is at least one of hydrochloric acid, nitric acid or acetic acid; b. In step S1, the solvent B is at least one of methanol, ethanol or ammonium hydroxide; c. In step S1, the purification is at least one of stirring, dialysis or centrifugation; d. In step S2, the stirring time is 20 min to 60 min; e. In step S2, the standing time is 12 h to 36 h; f. In step S2, the mass ratio of the natural melanin nanoparticles to the amphiphilic polymer is 1:50-1:100; g. In step S2, the amphiphilic high molecular polymer is D,L-lactide-polyethylene glycol-D,L-lactide copolymer, polyvinyl pyrrolidone and / or polyethylene.

4. The preparation method according to claim 3, characterized in that: In step S2, the mass ratio of natural melanin nanoparticles to amphiphilic high molecular polymer is 1:

100.

5. The preparation method according to claim 3, characterized in that: The molecular weight cut-off of the dialysis membrane is 1 kDa.

6. The preparation method according to claim 3, characterized in that: The hydrochloric acid is concentrated hydrochloric acid diluted 10000 times.

7. The preparation method according to claim 3, characterized in that: The preparation process of the polymer D,L-lactide-polyethylene glycol-D,L-lactide copolymer is as follows: eliminate the water content of polyethylene glycol to constant weight; add D,L-lactide and stannous isozincate as linkers, and react at 140°C under nitrogen protection; cool after the reaction is completed, dissolve the obtained product in ethanol, add excess cold n-pentane, filter, and dry the precipitate to constant weight.

8. The preparation method according to claim 7, characterized in that: Contains at least one of the following features: h. Heat the polyethylene glycol in a vacuum drying oven at 100°C for 1 h to eliminate moisture until constant weight is reached; i. The mass ratio of polyethylene glycol, D,L-lactide and stannous isozincate is 20:40:0.18; j. React at 140°C for 12 h under nitrogen protection; k. Dry the precipitate in vacuum at 45°C to constant weight.

9. The preparation method according to claim 7, characterized in that: The polyethylene glycol is polyethylene glycol 1000 (PEG1000), polyethylene glycol 1500 (PEG1500) or polyethylene glycol 2000 (PEG2000).

10. Use of the thermosensitive hydrogel prepared by the preparation method according to claim 1 or any one of claims 2 to 9 in the preparation of treatment for psoriasis.

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