A temperature-sensitive hydrogel based on natural melanin and application thereof

Abstract

The present application belongs to the technical field of medical products, and particularly relates to a temperature-sensitive hydrogel based on natural melanin and application thereof. The present application aims to solve the technical problem that clinical treatment drugs for psoriasis include topical drugs, immunosuppressants and various targeted biological agents, and there are problems such as drug resistance, local adverse reactions, recurrence after drug withdrawal, invalidity for some patients, high price, infection, high risk of cancer and great systemic toxic side effects at the present stage. The technical scheme of the present application is a temperature-sensitive hydrogel based on natural melanin, which is prepared by loading natural melanin on amphiphilic high molecular polymer. In the present application, natural melanin nanoparticles and 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 administration of psoriasis and safe and effective anti-inflammatory treatment.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical product technology, specifically relating to a thermosensitive hydrogel based on natural melanin and its applications. Background Technology

[0002] Psoriasis is a chronic, inflammatory skin disease mediated by an immune response, characterized by erythema and scaling. Its incidence rate is approximately 0.14-1.99% of the world's population. It can affect systemic diseases and seriously endanger human physical and mental health. Psoriasis is prone to relapse and difficult to cure. Currently, clinically effective treatments for psoriasis mainly consist of systemic immunosuppressants and biologics. While these are highly effective, they are expensive and carry risks such as drug resistance, local adverse reactions, relapse after discontinuation, high cost, infection, high risk of cancer, and serious toxic side effects. At a societal level, the fact that psoriasis is incurable and requires lifelong treatment with a long course not only places a huge medical burden on society but also increases the economic burden on patients. There is an urgent clinical need for safe and effective new drugs to treat psoriasis. Safe, non-toxic, and inexpensive natural products offer more possibilities for the development of new psoriasis drugs. On the technical level, the stratum corneum of psoriatic lesions is significantly thickened, resulting in poor transdermal drug delivery, low concentration, and unsatisfactory effects. Currently, there is no ideal and efficient drug formulation among topical psoriasis preparations that can simultaneously achieve effective adhesion and transdermal penetration to the thickened stratum corneum.

[0003] Thermosensitive hydrogels, as a promising novel delivery system, have been widely used in precision medicine for tumor treatment, skin therapy, and anti-infection, thanks to their unique physicochemical properties, namely, the ability to achieve a sol-gel phase transition under temperature stimulation. In a flowing state, thermosensitive hydrogels can uniformly cover irregular skin lesions, and then undergo a phase transition within a very short time, continuously adhering to and remaining on the skin surface in a gel state. This characteristic provides conditions for the adhesion of internally loaded drug molecules and their sustained action on the skin. The *Journal of Nanobiotechnology* (2022, https: / / doi.org / 10.1186 / s12951-022-01368-y) reported a composite hydrogel using mesoporous zinc oxide / silver nanoparticles (ZnO / Ag) with anti-inflammatory activity as a sustained-release carrier for systemically administered immunosuppressants. This hydrogel was used for the local transdermal delivery and sustained release of systemically administered immunosuppressants, jointly enhancing the anti-inflammatory and anti-proliferative effects of systemically administered immunosuppressants, achieving superior transdermal absorption performance and therapeutic efficacy for psoriasis. Although the above methods have significantly improved transdermal drug delivery for psoriasis, they still have great potential for further improvement compared to the safety considerations of natural drug treatments.

[0004] Natural melanin is widely found in nature and possesses excellent biocompatibility, superior photothermal properties, free radical scavenging ability, and strong chelating capacity. Natural melanin exhibits diverse biomedical applications, including UV radiation protection, cataract symptom relief, cancer treatment, and anti-infective therapy. Studies have reported that natural melanin possesses a unique polyphenolic structure with excellent antioxidant and anti-inflammatory properties. Natural melanin-like nanoparticles with numerous reducing functional groups have shown great potential in tissue repair. They have been successfully applied to treat ROS-related diseases such as ischemic stroke, periodontal disease, acute peritonitis, and acute kidney / lung injury. Hydrogels containing artificial melanin-like nanoparticles have also demonstrated excellent tissue adhesion and antioxidant properties in tissue repair. However, the application of thermosensitive hydrogels containing natural melanin in the anti-inflammatory treatment of psoriasis has not yet been reported. Summary of the Invention

[0005] This invention provides an application of a thermosensitive hydrogel based on natural melanin in the treatment of psoriasis. The technical problem solved is the application of a thermosensitive hydrogel based on natural active pharmaceutical ingredients in the treatment of psoriasis. As a safe, effective, and inexpensive topical treatment for psoriasis, the natural melanin thermosensitive hydrogel's sol-gel transformation can promote the transdermal penetration of natural melanin into psoriatic lesions and prolong its local retention time, thereby enhancing its local anti-inflammatory therapeutic effect.

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

[0007] Furthermore, the amphiphilic polymer is selected from at least one of D,L-lactide-polyethylene glycol-D,L-lactide copolymer, polyvinylpyrrolidone, or polyethylene.

[0008] The natural melanin is extracted from at least one of squid ink, black rice, or black sesame.

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

[0010] S1. Preparation of melanin nanoparticles: Crude natural melanin is dissolved in solvent A, then mixed with solvent B, reacted, purified, and melanin nanoparticles are obtained.

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

[0012] Specifically, in step S1, 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, 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] Specifically, in step S1, the reaction time is 12 hours.

[0018] In step S1, the purification process involves at least one of stirring, dialysis, or centrifugation.

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

[0020] Furthermore, the dialysis membrane has a molecular weight cutoff of 1 kDa.

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

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

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

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

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

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

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

[0028] Specifically, polyethylene glycol is heated at 100°C for 1 hour in a vacuum drying oven to remove moisture until constant weight is achieved.

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

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

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

[0032] The present invention also provides the method for preparing a thermosensitive hydrogel.

[0033] The present invention also provides the application of the thermosensitive hydrogel in the preparation of a treatment for psoriasis.

[0034] The beneficial effects of this invention are as follows: This invention mixes natural melanin nanoparticles with an amphiphilic polymer in a sol-gel state, providing a novel carrier for effective transdermal delivery of natural melanin and increasing the local drug concentration of natural melanin. Furthermore, the sol-gel transition of the thermosensitive hydrogel under body temperature stimulation prolongs the retention time of natural melanin in the lesion and enhances its anti-inflammatory effect, significantly downregulating the expression of pro-inflammatory factors. In a mouse psoriasis model, it also significantly improved the erythema and scaling symptoms of mouse skin lesions, achieving effective transdermal drug delivery of natural melanin and anti-inflammatory treatment of psoriasis. In addition, the thermosensitive hydrogel based on natural melanin described in this invention has a simple preparation method, mild process conditions, and is not demanding on reaction conditions, possessing the potential for rapid industrial production. This invention lays the foundation for the clinical translation of natural melanin for the topical treatment of psoriasis and provides a new strategy for the prevention and treatment of psoriasis. Attached Figure Description

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

[0036] Figure 2 The images show the phase transitions 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 thermosensitive hydrogel based on natural melanin prepared in Example 1 in keratinocytes (HaCaT).

[0038] Figure 4 The image shows the ROS fluorescence of HaCaT cells prepared in Example 1, which were incubated with H2O2 and TNF-α using melanin nanoparticles of different concentrations in a thermosensitive hydrogel based on natural melanin.

[0039] Figure 5The images are photographs of the skin on the backs of mice taken on the 5th day after treatment in each treatment group of Example 1.

[0040] Figure 6 The images shown are H&E slices of back skin tissue obtained from each experimental group in Example 1. Detailed Implementation

[0041] Natural melanin is widely found in nature and possesses excellent biocompatibility, photothermal properties, free radical scavenging ability, and strong chelating capacity. Its applications in biomedicine are diverse, including ultraviolet radiation protection, cataract symptom relief, anti-infection, and cancer treatment. Research reports that natural melanin possesses a unique polyphenolic structure with excellent antioxidant and anti-inflammatory properties. Natural melanin-like nanoparticles with numerous reducing functional groups have shown great potential in tissue repair and anti-inflammation.

[0042] Amphiphilic polymer hydrogels are thermosensitive and exist in a gel state at 34–40°C, thus making them suitable as carriers.

[0043] Thermosensitive hydrogels, as a promising new delivery system, have wide applications in oncology, skin diseases, and anti-infection, thanks to their unique physicochemical properties, namely, the ability to achieve a sol-gel phase transition under temperature stimulation. In a flowing state, thermosensitive hydrogels can uniformly cover irregular skin lesions and undergo a phase transition within a very short time, continuously adhering to and remaining on the skin surface in a gel state. This characteristic provides conditions for increasing the adhesion and sustained action of natural melanin on the skin.

[0044] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0045] Example 1: Preparation of a 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 1 hour in a vacuum drying oven to remove moisture until constant weight. After cooling to room temperature, 20 g of the product was weighed and 40 g of D,L-lactide and 0.18 g of stannous isozincate were added as a linker. The mixture was reacted at 140°C for 12 hours under nitrogen protection. After the reaction was completed and cooled, the product was dissolved in ethanol. The filtrate was extracted using an excess of cooled n-pentane (600 mL). The precipitate was dried under vacuum at 45°C until constant weight to obtain D,L-lactide-polyethylene glycol-D,L-lactide copolymer.

[0048] A hydrogel with a mass concentration of 25% was prepared by mixing D,L-lactide-polyethylene glycol-D,L-lactide copolymer with deionized water and stored in a refrigerator at 4°C.

[0049] 2. Preparation of natural melanin nanoparticles

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

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

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

[0053] The morphology of the melanin nanoparticles and hydrogel prepared in this embodiment was characterized as follows:

[0054] like Figure 1 As shown in the scanning electron microscope (SEM) images, the obtained natural melanin nanoparticles have uniform size, with a particle size in the range of 10–17 nm.

[0055] Take a blank thermosensitive hydrogel and a thermosensitive hydrogel based on natural melanin, and place them separately into 10mL wide-mouth bottles. Incubate them at 4℃ for 2 minutes, photographing the results. Then transfer them to a 37℃ incubator for 2 minutes, photographing the results again. Figure 2 As can be seen, both the blank hydrogel and the thermosensitive hydrogel based on natural melanin exhibit a flowing sol state at 4℃, while the flowing sol state transforms into a gel state in a constant temperature chamber at 37℃.

[0056] The sol-gel transition of hydrogels depends on changes in their loss modulus (G”) and storage modulus (G’). To verify its thermosensitive properties, temperature-rheological correlation data of a thermosensitive hydrogel based on natural melanin were collected using a rotational rheometer. The results show that when the temperature is above 34.2℃, the G’ of the photoresponsive thermosensitive hydrogel is greater than G”, forming a gel state, indicating that the transition from a sol to a gel state can be induced under human body temperature.

[0057] Example 2: Therapeutic effect test on in vitro psoriasis cell model

[0058] To verify the safety of in vitro cell therapy using thermosensitive hydrogels based on natural melanin, HaCaT cells in normal condition were cultured in logarithmic phase and co-incubated for 24 h in 96-well plates with different concentrations (0, 5, 10, 25, 50, 100, 150, 200 μg / mL) of natural melanin nanoparticles. Figure 3 The results show that even at a natural melanin concentration as high as 200 μg / mL, there was no significant difference in cell viability after co-incubation. This indicates that natural melanin nanoparticles possess good biocompatibility.

[0059] To verify the anti-inflammatory effect of melanin nanoparticles in a thermosensitive hydrogel based on natural melanin, a HaCaT cell model stimulated by H2O2 and TNF-α was established. The changes in ROS in different treatment groups were observed using a confocal laser scanning microscope (CLSM) to detect reactive oxygen species (ROS) using a fluorescent probe (DCHF-DA: 2',7'-Dichlorodihydrofluorescein diacetate). The groups are as follows:

[0060] Control group: HaCaT cells were not treated in any way;

[0061] Model group: HaCaT cells were co-stimulated with 100 ng / mL TNF-α and 200 μM H2O2.

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

[0063] from Figure 4 As can be seen, the DCHF-DA model group showed significant green fluorescence, indicating that the inflammation model was successfully established. With the addition of natural melanin nanoparticles, the green fluorescence signal decreased significantly, which indicates that natural melanin has excellent ROS scavenging ability.

[0064] Example 3: Testing the therapeutic effect on an animal model of psoriasis.

[0065] The therapeutic effect of the thermosensitive hydrogel based on natural melanin prepared in this embodiment on transdermal spray administration of psoriasis was tested according to the following steps:

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

[0067] Normal group: Normal mice received no treatment;

[0068] Model group: Mice that successfully developed the model were not treated;

[0069] Positive control group: Calcipotriol was applied to the backs of model mice;

[0070] Experimental group: The backs of model mice were coated with a temperature-sensitive hydrogel based on natural melanin.

[0071] Photographs were taken of the skin lesions in each group after each day's treatment. Mice were euthanized after the photography was completed on the tenth day, and their skin tissue was collected and fixed with 4% paraformaldehyde for subsequent H&E staining in histopathology.

[0072] Figure 5 These are representative photographs of the mouse back skin taken on day 10 after treatment in each treatment group of this embodiment. As can be observed from the images, the skin in the experimental group showed a whiter and smoother appearance, more closely resembling normal skin, comparable to the positive control group. This verifies that the hydrogel prepared in this embodiment can achieve a good therapeutic effect on psoriasis.

[0073] Figure 6 These are H&E section images of back skin tissue obtained from each treatment group in this embodiment. Histopathological analysis showed that, compared with the control group, the experimental group had a significantly reduced epidermal thickness, decreased inflammatory cell infiltration in the dermis, and significantly improved hyperkeratosis and parakeratosis, comparable to the positive control group. This result verifies that the natural melanin thermosensitive hydrogel has a significant therapeutic effect on psoriasis inflammation.

Claims

1. A thermosensitive hydrogel based on natural melanin, characterized in that: It is prepared by loading natural melanin onto an amphiphilic polymer; the amphiphilic polymer is a D,L-lactide-polyethylene glycol-D,L-lactide copolymer; the natural melanin is derived from squid ink; the preparation method includes the following steps: S1. Preparation of natural melanin nanoparticles: Crude natural melanin is dissolved in solvent A, then mixed with solvent B, reacted, and purified to obtain melanin nanoparticles; solvent A is at least one of hydrochloric acid, nitric acid, or acetic acid; solvent B is at least one of methanol, ethanol, or ammonium hydroxide; purification is at least one of stirring, dialysis, or centrifugation. S2. Preparation of thermosensitive hydrogel: At room temperature, natural melanin nanoparticles are added to an amphiphilic polymer, stirred evenly, and allowed to stand at 4°C to obtain a thermosensitive hydrogel based on natural melanin; the mass ratio of the natural melanin nanoparticles to the amphiphilic polymer is 1:50~1:100; the stirring time is 20 min~60 min; and the standing time is 12 h~36 h.

2. The thermosensitive hydrogel according to claim 1, characterized in that: In step S2, the mass ratio of natural melanin nanoparticles to amphiphilic polymer is 1:

100.

3. The thermosensitive hydrogel according to claim 1, characterized in that: In step S1, the molecular weight cutoff of the dialysis membrane is 1 kDa.

4. The thermosensitive hydrogel according to claim 1, characterized in that: In step S1, the hydrochloric acid is concentrated hydrochloric acid diluted 10,000 times.

5. The thermosensitive hydrogel according to claim 1, characterized in that: In step S1, the preparation process of the polymer D,L-lactide-polyethylene glycol-D,L-lactide copolymer is as follows: remove moisture from polyethylene glycol to constant weight; add D,L-lactide and stannous isozonate as linkers, and react at 140°C under nitrogen protection; after the reaction is complete, cool, dissolve the obtained product in ethanol, add excess cold n-pentane, filter, and dry the precipitate to constant weight to obtain the product.

6. The thermosensitive hydrogel according to claim 5, characterized in that: It contains at least one of the following features: h. Heat polyethylene glycol in a vacuum drying oven at 100°C for 1 hour to remove moisture until constant weight is achieved; i. The mass ratio of polyethylene glycol, D,L-lactide and stannous isozincate is 20:40:0.18; j. React at 140℃ for 12 h under nitrogen protection; k. Dry the precipitate under vacuum at 45°C until constant weight.

7. The thermosensitive hydrogel according to claim 6, characterized in that: The polyethylene glycol is polyethylene glycol 1000 (PEG1000), polyethylene glycol 1500 (PEG1500), or polyethylene glycol 2000 (PEG2000).

8. The use of the thermosensitive hydrogel according to any one of claims 1 to 7 in the preparation of a medicament for treating psoriasis.

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