Bionic nano melanin protective agent with photo-thermal responsiveness and ultraviolet-visible light dual-band protection function as well as preparation method and application of bionic nano melanin protective agent

Abstract

The invention belongs to the technical field of fine chemicals, and provides a bionic nano melanin protective agent with photo-thermal responsiveness and an ultraviolet-visible light dual-band protection function as well as a preparation method and application of the bionic nano melanin protective agent. The preparation method comprises the following steps: mixing dopamine hydrochloride with a Tris buffer solution for reaction, and then carrying out light-shielding reaction with a tris (hydroxymethyl) aminomethane hydrochloride solution and sulfydryl polyethylene glycol to obtain PDA-coated PEG nanoparticles; the preparation method comprises the following steps: mixing Pluronic F-127 with phosphate buffered normal saline, and reacting to obtain PF-127 hydrogel; and mixing the PF-127 hydrogel with the PDA-coated PEG nanoparticles, and carrying out a light-shielding reaction, so as to obtain the bionic nano melanin protective agent with photothermal responsiveness and an ultraviolet-visible light dual-band protection function. The method is simple, green and environment-friendly, and the obtained protective agent has photo-thermal response performance and a lasting and safe sunscreen effect and has good application potential in the aspect of cold skin disease protection.

Description

Technical Field

[0001] This invention relates to the field of fine chemicals technology, and in particular to a biomimetic nano-melanin protective agent with photothermal responsiveness and UV-Vis dual-band protection, its preparation method and application. Background Technology

[0002] Prolonged exposure to sunlight exposes human skin to various photodamage risks. Ultraviolet (UV) radiation is the primary spectral component causing photodamage. UVB (290-320 nm) can penetrate the basal layer of the epidermis, inducing excessive proliferation of keratinocytes and acanthosis. UVA (320-400 nm) can penetrate deep into the superficial dermis, causing collagen fiber breakage, vasodilation, and Langerhans cell loss. It also accelerates photoaging by activating the NF-κB pathway, promoting the release of inflammatory factors such as IL-6 and TNF-α. Furthermore, high-energy visible light (HEV, 400-450 nm, especially the 400-420 nm blue light band) is also a concern. It can induce mitochondrial DNA damage and inhibit fibroblast proliferation by generating reactive oxygen species (ROS), leading to a decrease in dermal thickness. Its penetration depth can reach subcutaneous tissue, causing new photodamage problems such as pigmentation, photosensitive dermatitis, and circadian rhythm disruption.

[0003] Currently, sun protection methods mainly focus on UV protection, but there are three major technical bottlenecks: physical sunscreens (such as ZnO and TiO2) rely on reflection or scattering mechanisms to work, but nano-sized particles have photocatalytic activity, and long-term use can easily induce ROS generation and clog hair follicles; chemical sunscreens (such as para-aminobenzoic acid derivatives and cinnamic acid esters) are easily absorbed by the skin, posing a potential risk of endocrine disruption, and have poor photostability, requiring frequent reapplication; natural plant extracts (such as flavonoids and polyphenols) have antioxidant capabilities, but they are prone to photodegradation in high-radiation environments such as high altitudes, and their protection against HEV blue light is limited.

[0004] Polydopamine (PDA), as a biomimetic melanin material, has a light absorption range covering a wide wavelength band of 200-800 nm. Theoretically, it can simultaneously protect against ultraviolet and near-infrared (NIR) light and can convert light energy into heat energy through photothermal conversion (with a conversion efficiency of over 65% according to literature), showing potential in the prevention of skin diseases in cold environments. However, PDA currently still has two main drawbacks: (1) Nano-aggregation: PDA particles are prone to irreversible aggregation, resulting in a decrease in light absorption coefficient of more than 30%; (2) Insufficient extensibility: PDA film is significantly brittle in dry state, making it difficult to form a uniform protective layer on the skin surface.

[0005] With the increasing time spent using electronic screens, skin damage caused by HEV blue light has become a new type of occupational health problem. Existing sunscreen products generally have a protection efficiency of less than 40% in the 400-450 nm wavelength range, and there is a lack of material systems that can simultaneously protect against UV and HEV blue light and also have photothermal regulation functions.

[0006] Therefore, developing a novel photoprotectant that can synergistically block ultraviolet light and high-energy blue light, while also possessing self-healing properties, good spreadability, and environmental adaptability, is key to addressing skin health challenges in extreme environments such as plateaus and polar regions, as well as against the backdrop of urban light pollution. Summary of the Invention

[0007] In view of this, the present invention provides a biomimetic nano melanin protective agent with photothermal responsiveness and UV-Vis dual-band protection function, as well as its preparation method and application, to solve the problems that existing sunscreen products cannot achieve UV-Vis dual-band protection and that polydopamine is prone to aggregation, which is not conducive to long-term storage and has poor spreadability on the skin surface.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for preparing a biomimetic nano-melanin protective agent with photothermal responsiveness and UV-Vis dual-band protection, comprising the following steps: 1) Mix dopamine hydrochloride with Tris buffer and react to obtain polydopamine particles; 2) PDA@PEG nanoparticles were obtained by mixing polydopamine particles with tris(hydroxymethyl)aminomethane hydrochloride solution and mercapto polyethylene glycol and carrying out a light-protected reaction. 3) Pluronic F-127 was mixed with phosphate-buffered saline and reacted to obtain PF-127 hydrogel; 4) PF-127 hydrogel was mixed with PDA@PEG nanoparticles and subjected to a light-shielding reaction to obtain a biomimetic nano melanin protectant with photothermal responsiveness and UV-Vis dual-band protection function. There is no specific order requirement for steps 2) and 3).

[0009] Preferably, the mass-to-volume ratio of dopamine hydrochloride to Tris buffer in step 1) is 0.1~0.2 g: 10~20 mL; the molar concentration of Tris buffer is 9~11 mmol / L and the pH is 8~9; the reaction time in step 1) is 4~24 h.

[0010] Preferably, the mass-to-volume ratio of Pluronic F-127 to phosphate-buffered saline in step 3) is 20-25 g: 75-80 mL.

[0011] Preferably, in step 2), the mass-to-volume ratio of the polydopamine particles to the tris(hydroxymethyl)aminomethane hydrochloride solution and mercapto polyethylene glycol is 0.06~0.1 g: 50 mL: 0.08~0.12 g; and the pH of the tris(hydroxymethyl)aminomethane hydrochloride solution is 8.0~8.1.

[0012] Preferably, the temperature for the light-protected reaction in step 2) is 20~25℃ and the time is 12~24 h.

[0013] Preferably, the diameter of the PDA@PEG nanoparticles in step 2) is 50~150 nm.

[0014] Preferably, the reaction temperature in step 3) is 0~4℃ and the time is 12~24 h.

[0015] Preferably, the mass ratio of PF-127 hydrogel to PDA@PEG nanoparticles in step 4) is 85~95:5~15.

[0016] Preferably, the temperature for the light-protected reaction in step 4) is 0~4℃ and the time is 12~24 h.

[0017] The present invention also provides a biomimetic nano-melanin protectant with photothermal responsiveness and UV-Vis dual-band protection function prepared by the above preparation method.

[0018] The present invention also provides an application of the above-mentioned biomimetic nano-melanin protective agent with photothermal responsiveness and ultraviolet-visible dual-band protection function in the preparation of products for preventing cold skin diseases.

[0019] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: 1. The polydopamine (PDA) particles used in this invention possess a broad absorption spectrum covering the entire visible light region, with a maximum absorption peak in the ultraviolet region, effectively protecting against blue light and broadly absorbing ultraviolet and near-infrared (NIR) light. These particles can convert absorbed light radiation into heat energy in a non-radiative form. Based on the photothermal effect of PDA@PEG nanoparticles, the protective agent described in this invention shows good application potential in protecting against cold-induced skin diseases and in cold environments. Furthermore, polydopamine (PDA), as a natural aromatic polymer, possesses excellent ultraviolet protection properties. After modification with polyethylene glycol (PEG), the dispersibility of PDA is significantly improved, effectively inhibiting the tendency of nanoparticles to aggregate, while enhancing its stability and further reducing cytotoxicity, thereby achieving a long-lasting and safe sun protection effect. Moreover, the nano-sized PDA@PEG particles, in a fully dispersed state, can also enhance the light scattering effect, further improving the overall protection performance against ultraviolet rays.

[0020] 2. This invention uses Pluronic F-127 as a matrix, with a mass-to-volume ratio of 20-25 g to 75-80 mL of phosphate-buffered saline. This not only provides moisturizing and a hydrophilic environment, promoting stratum corneum hydration, but also forms a drug reservoir, enabling controlled drug release (equivalent to PDA@PEG being continuously released and absorbed by the skin surface within the hydrogel), thereby improving the stability of the formulation (PDA@PEG nanoparticles) and enhancing its compatibility with the skin. Furthermore, at the dosage of Pluronic F-127 described in this invention, PDA@PEG nanoparticles can partially penetrate into the skin, exerting a protective effect in vivo. In addition, at the dosage of Pluronic F-127 described in this invention, the physical limitations imposed by the gel mesh on nanoparticle movement and release can be minimized, while also avoiding the problem of high viscosity inhibiting nanoparticle diffusion to the skin surface. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 The images show the PF-127 hydrogel from Example 1 and the physical samples of the mixture of PDA@PEG nanoparticles and PF-127 hydrogel. Figure 1 The image on the left is a picture of the actual PF-127 hydrogel. Figure 1The image on the right shows the actual product after PDA@PEG nanoparticles were mixed with PF-127 hydrogel; Figure 2 The images shown are transmission electron microscopy (TEM) images and particle size analysis diagrams of the PDA@PEG nanoparticles in Example 1. Figure 2 In the image, 'a' represents a transmission electron microscope (TEM) image of the PDA@PEG nanoparticles. Figure 2 In the figure, b represents the particle size analysis diagram of PDA@PEG nanoparticles; Figure 3 This is a schematic diagram illustrating the dispersion of PDA and PDA@PEG nanoparticles in Example 1, wherein... Figure 3 In the diagram, 'a' represents the dispersion of the PDA at 0 h. Figure 3 In the diagram, b represents the dispersion of PDA@PEG nanoparticles at 0 h. Figure 3 In the diagram, 'c' represents the dispersion of the PDA after 72 hours. Figure 3 In the figure, d represents the dispersion of PDA@PEG nanoparticles after 72 h (observed under static conditions of room temperature 25℃ and relative humidity 50%). Figure 4 The UV-Vis absorption spectra of PDA and PDA@PEG nanoparticles in Example 1 are shown in the range of 200–800 nm. Figure 5 This is a graph showing the free radical scavenging rates of PDA and PDA@PEG nanoparticles in Example 1; Figure 6 The image shows the protective effect of PDA and PDA@PEG nanoparticles against UVB and blue light (BL) in Example 1. Figure 6 In the figure, 'a' represents the UVB protection effect of the protective agent composed of PDA@PEG nanoparticles and PF-127 hydrogel. Figure 6 In the diagram, b represents the UVB protection provided by a mixture of PDA and PF-127 hydrogel. Figure 6 In the diagram, 'c' represents the UVB protection provided by the PF-127 hydrogel. Figure 6 In the diagram, 'd' represents the UVB protection without any protective agent applied. Figure 6 In the figure, 'e' represents the protection of BL by a protective agent composed of PDA@PEG nanoparticles and PF-127 hydrogel. Figure 6 In the figure, f represents the protection of BL by a protective agent composed of PDA and PF-127 hydrogel. Figure 6 In the figure, g represents the protection of BL by PF-127 hydrogel. Figure 6 In the diagram, 'h' represents the protection of BL without any protective agent applied. Figure 7 The image shows the photothermal conversion infrared spectrum of the PDA@PEG nanoparticles in Example 1. Figure 7In this context, 'a' represents the infrared thermal image. Figure 7 In the graph, b represents the temperature-time variation curve; Figure 8 The image shows the immunofluorescence of ROS in Hacat cells induced by UVB irradiation with different amounts of PDA@PEG nanoparticles in Example 1. Figure 8 In the image, 'a' represents the immunofluorescence image of reactive oxygen species (ROS). Figure 8 In the figure, b represents the bar chart of quantitative analysis of average fluorescence intensity; Figure 9 This is a diagram showing the effect of PDA@PEG nanoparticles from Example 1 on the skin of a mouse back under UVB irradiation. Figure 9 In the image, 'a' represents the skin on the back of a mouse that has not been exposed to UVB. Figure 9 In the image, b represents the back skin of a mouse that has not been coated with a protective agent and has been exposed to UVB for two weeks. Figure 9 Image c in the figure shows the back skin of a mouse coated with PF-127 hydrogel and irradiated with UVB for 2 weeks. Figure 9 In the image, d represents the back skin of a mouse coated with a protective agent consisting of 99.9 wt% PF-127 hydrogel and 0.1 wt% PDA@PEG nanoparticles, and then irradiated with UVB for 2 weeks. Figure 9 In the figure, 'e' represents the back skin of a mouse coated with a protective agent consisting of 99.8 wt% PF-127 hydrogel and 0.2 wt% PDA@PEG nanoparticles and irradiated with UVB for 2 weeks. Figure 10 This is a laser confocal image showing the effect of PDA@PEG nanoparticles on filaggrin (FLG), a key protein in the skin barrier of mice, under UVB irradiation, as described in Example 1. Figure 10 In the image, 'a' represents a laser confocal image of the back of a mouse (Control group) that has not been exposed to UVA+UVB radiation. Figure 10 In the image, b is a laser confocal image of the back of a mouse exposed to UVA+UVB radiation without any protective coating. Figure 10 In the image, c represents a laser confocal image of the back of a mouse (UVA+UVB+Sunscreen group) treated only with commercial sunscreen and exposed to UVA+UVB radiation. Figure 10 In the image, d represents a laser confocal image of the back of a mouse (UVA+UVB+Sunscreen+2.5wt%PDA group) treated with a protective agent containing 2.5wt% PDA@PEG nanoparticles in combination with a commercial sunscreen and subjected to UVA+UVB irradiation. Figure 10 In the image, 'e' represents a laser confocal image of the back of a mouse (UVA+UVB+Sunscreen+5wt%PDA group) treated with a protective agent containing 5wt% PDA@PEG nanoparticles in combination with a commercial sunscreen and subjected to UVA+UVB irradiation. Figure 10f in the figure is a laser confocal image of the back of a mouse (UVA+UVB+Sunscreen+10wt%PDA group) treated with a protective agent containing 10wt% PDA@PEG nanoparticles in combination with commercial sunscreen and exposed to UVA+UVB irradiation. Detailed Implementation

[0023] This invention provides a method for preparing a biomimetic nano-melanin protective agent with photothermal responsiveness and UV-Vis dual-band protection, comprising the following steps: 1) Mix dopamine hydrochloride with Tris buffer and react to obtain polydopamine particles; 2) PDA@PEG nanoparticles were obtained by mixing polydopamine particles with tris(hydroxymethyl)aminomethane hydrochloride solution and mercapto polyethylene glycol and carrying out a light-protected reaction. 3) Pluronic F-127 was mixed with phosphate-buffered saline and reacted to obtain PF-127 hydrogel; 4) PF-127 hydrogel was mixed with PDA@PEG nanoparticles and subjected to a light-shielding reaction to obtain a biomimetic nano melanin protectant with photothermal responsiveness and UV-Vis dual-band protection function. There is no specific order requirement for steps 2) and 3).

[0024] In this invention, the mass-to-volume ratio of dopamine hydrochloride to Tris buffer in step 1) is 0.1-0.2 g: 10-20 mL, preferably 0.12-0.18 g: 12-18 mL, more preferably 0.15-0.16 g: 15-16 mL; the molar concentration of the Tris buffer is 9-11 mmol / L, preferably 9.2-10.8 mmol / L, more preferably 9.5-10.5 mmol / L, more preferably 10 mmol / L; the pH of the Tris buffer is 8-9, preferably 8.3-8.7, more preferably 8.4-8.6, more preferably 8.5; the reaction time in step 1) is 4-24 h, preferably 5-22 h, more preferably 8-20 h, more preferably 10-15 h.

[0025] In this invention, the mass-to-volume ratio of Pluronic F-127 to phosphate-buffered saline in step 3) is 20-25 g: 75-80 mL, preferably 21-24 g: 76-79 mL, and more preferably 22-23 g: 77-78 mL.

[0026] In this invention, the higher the mass-to-volume ratio of dopamine hydrochloride to Tris buffer in step 1), the larger the particle size of the polydopamine particles.

[0027] In this invention, the preferred termination operation of the reaction in step 1) is to add citric acid to adjust the pH of the reaction system to 5-6 when the solution color of the reaction system changes from colorless and transparent to light pink, then to brownish-yellow, and finally to dark brown or brownish-black. This adjustment is more preferably 5.2-5.8 and even more preferably 5.5.

[0028] In this invention, after the reaction in step 1) is completed, it is preferable to further include sequential centrifugation and centrifugal washing operations; the centrifugation speed is preferably 10000~13000 rpm, more preferably 11000~12500 rpm, and even more preferably 11500~12000 rpm; the centrifugation time is preferably 12~18 min, more preferably 13~16 min, and even more preferably 15 min; the solvent for centrifugal washing is preferably deionized water; the centrifugal washing speed is preferably 10000~13000 rpm, more preferably 11000~12500 rpm, and even more preferably 11500~12000 rpm.

[0029] In this invention, the mass-to-volume ratio of the polydopamine particles to the tris(hydroxymethyl)aminomethane hydrochloride solution and mercapto polyethylene glycol in step 2) is 0.06~0.1 g: 50 mL: 0.08~0.12 g, preferably 0.07~0.09 g: 50 mL: 0.09~0.11 g, and more preferably 0.08 g: 50 mL: 0.10 g; the pH of the tris(hydroxymethyl)aminomethane hydrochloride solution is 8.0~8.1, preferably 8.02~8.08, and more preferably 8.05.

[0030] In this invention, the temperature of the light-protected reaction in step 2) is 20~25℃, preferably one of 20℃, 21℃, 22℃, 23℃, 24℃, and 25℃; the time of the light-protected reaction is 12~24 h, preferably 14~22 h, more preferably 15~20 h, and more preferably 16~18 h.

[0031] In this invention, the formation of protein crowns on the surface of nanoparticles (NPs) participates in the interaction between nanoparticles and biological systems. Studies have shown that surface proteins depend on the surface properties of NPs. The adhesiveness of PDA, as described in step 2), makes PDA nanoparticles very easy to aggregate in water and biological media. Unmodified PDA nanoparticles adsorb proteins on their surface, disrupting the colloidal stability of NPs and easily accumulating in the presence of secreted proteins in the skin layer. Simultaneously, PDA nanoparticles can interact with other ligands through hydrogen bonding, π-π stacking, metal coordination, and electrostatic interactions. Surface modification of PDA is extremely easy and does not require complex coupling reactions or purification steps. Polyethylene glycol (PEG) modification can minimize the adsorption of protein-like skin secretions on the NP surface and maintain colloidal stability by reducing charge-based interactions between proteins and small molecules. Furthermore, PEG modification can also give NPs good steric stability; the repeated application of hydrophilic PEG increases its solubility in buffers and serum, reduces uptake by the reticuloendothelial system, and prolongs its circulation time in vivo, thereby achieving tissue targeting. At the same time, PEG is inexpensive and widely used, and its modification of PDA nanoparticles further reduces the cytotoxicity of PDA.

[0032] In this invention, the diameter of the PDA@PEG nanoparticles in step 2) is 50~150 nm, preferably 60~140 nm, more preferably 70~120 nm, and even more preferably 80~100 nm.

[0033] In this invention, the reaction temperature in step 3) is 0~4℃, preferably one of 0℃, 1℃, 2℃, 3℃, and 4℃; the reaction time is 12~24 h, preferably 14~22 h, more preferably 15~20 h, and more preferably 16~18 h.

[0034] In this invention, Pluronic F-127 mentioned in step 3) is abbreviated as PF-127, which is composed of polyoxyethylene and polyoxypropylene units and has the linear molecular formula (C3H6O·C2H4O). x It is a white powdery solid that can be dissolved in phosphate-buffered saline (PBS) at 4°C.

[0035] In this invention, the mass ratio of PF-127 hydrogel to PDA@PEG nanoparticles in step 4) is 85~95:5~15, preferably 86~92:8~14, and more preferably 88~90:10~12.

[0036] In this invention, the temperature of the light-protected reaction in step 4) is 0~4℃, preferably one of 0℃, 1℃, 2℃, 3℃, and 4℃; the time of the light-protected reaction is 12~24 h, preferably 14~22 h, more preferably 15~20 h, and more preferably 16~18 h.

[0037] In this invention, the reactions in steps 1), 2), 3), and 4) are all carried out under magnetic stirring; the stirring speed is preferably 400-800 rpm, more preferably 500-700 rpm, and even more preferably 550-600 rpm.

[0038] The present invention also provides a biomimetic nano-melanin protectant with photothermal responsiveness and UV-Vis dual-band protection function prepared by the above preparation method.

[0039] The present invention also provides an application of the above-mentioned biomimetic nano-melanin protective agent with photothermal responsiveness and ultraviolet-visible dual-band protection function in the preparation of products for preventing cold skin diseases.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] 1) Disperse 0.15 g of dopamine hydrochloride in 15 mL of Tris buffer (molar concentration of 10 mmol / L, pH of 8.5), and then magnetically stir at 5500 rpm for 24 h at room temperature. Observe that the color of the reaction solution turns dark brown / brownish-black. Add citric acid to adjust the pH of the reaction system to 5.5 to terminate the reaction. Then centrifuge at 12000 rpm for 15 min, and wash with deionized water at 12000 rpm to obtain polydopamine particles (PDA), which are then lyophilized for later use. 2) Dissolve 0.1 g of the polydopamine particles obtained in step 1) in 50 mL of tris(hydroxymethyl)aminomethane hydrochloride solution (pH 8.0), and add 0.10 g of mercaptopolyethylene glycol (PEG). Then, stir magnetically in the dark at 25 °C and 400 rpm for 24 h, and then purify by dialysis to obtain PDA@PEG nanoparticles with a particle size of 150±20 nm. 3) Add 20 g of Pluronic F-127 particles to 80 mL of phosphate-buffered saline at 4 °C, and then magnetically stir for 24 h at 4 °C and 300 rpm to obtain PF-127 hydrogel. 4) Mix 85 g of PF-127 hydrogel at 4℃ with 15 g of PDA@PEG nanoparticles, and then react with magnetic stirring at 4℃ and 200 rpm for 24 h in the dark to obtain a biomimetic nano melanin protectant with photothermal properties and UV-Vis dual-band protection.

[0043] Figure 1 The images show the PF-127 hydrogel from Example 1 and the physical samples of the mixture of PDA@PEG nanoparticles and PF-127 hydrogel. Figure 1 The image on the left is a picture of the actual PF-127 hydrogel. Figure 1 The image on the right shows a physical picture of the mixture of PDA@PEG nanoparticles and PF-127 hydrogel. Figure 1 As can be seen, the pure PF-127 hydrogel (left) is a colorless and transparent semi-solid state, while the composite PDA@PEG / PF-127 sample (right) shows a uniform dark brown or brownish-black color, and the overall texture is uniform with no obvious phase separation or particle aggregation.

[0044] Figure 2 The images shown are transmission electron microscopy (TEM) images and particle size analysis diagrams of the PDA@PEG nanoparticles in Example 1. Figure 2 In the image, 'a' represents a transmission electron microscope (TEM) image of the PDA@PEG nanoparticles. Figure 2 In the diagram, b represents the particle size analysis of PDA@PEG nanoparticles; from Figure 2 As can be seen from a~b in the figure, PDA@PEG nanoparticles can be uniformly dispersed in PF-127 hydrogel. Electron microscopy shows a uniform spherical structure with good dispersion and uniform particle size, with NPs (average particle size of PDA@PEG nanoparticles) ≈ 119.4 nm.

[0045] Figure 3 This is a schematic diagram illustrating the dispersion of PDA and PDA@PEG nanoparticles in Example 1, wherein... Figure 3 In the diagram, 'a' represents the dispersion of the PDA at 0 h. Figure 3 In the diagram, b represents the dispersion of PDA@PEG nanoparticles at 0 h. Figure 3 In the diagram, 'c' represents the dispersion of the PDA after 72 hours. Figure 3 In the diagram, 'd' represents the dispersion of PDA@PEG nanoparticles after 72 h. The specific testing method was as follows: the colloidal stability of the nanoparticles was evaluated using the static sedimentation observation method. The specific steps are as follows: 1. Sample preparation: Accurately weigh 4.0 mg each of lyophilized polydopamine particles (PDA NPs) and PDA@PEG nanoparticles (PDA@PEG NPs) powder, and place them in 2 mL transparent glass sample bottles respectively.

[0046] 2. Solution preparation: Accurately add 2.0 mL of phosphate-buffered saline to each sample vial to prepare PDA NPs dispersion and PDA@PEG NPs dispersion with a concentration of 2 mg / mL.

[0047] 3. Initial State Recording: After sealing both sample vials, vigorously vortex for 2 minutes to ensure complete and uniform dispersion of the nanoparticles in the PBS. Then, allow to stand at room temperature and immediately photograph the initial state at 0 hours (corresponding to...). Figure 3 (a and b in the text).

[0048] 4. Long-term static observation: Place the sample vials at room temperature (25℃) for 72 hours, keeping the environment stable and free from vibration.

[0049] 5. Endpoint Status Recording: After standing for 72 hours, photograph the two sample vials again and record their dispersion status (corresponding to...). Figure 3 (c and d in the text).

[0050] Results analysis: By comparing the macroscopic state of the dispersion, the height of the supernatant, and the sedimentation at the bottom after 0 hours and 72 hours, the long-term dispersion stability of the nanoparticles was qualitatively evaluated.

[0051] from Figure 3 As shown in figures a-d, after standing for 72 h, PDA in both 2 mg / mL PDA and PDA@PEG solutions exhibited significant sedimentation and aggregation, while PDA@PEG nanoparticles showed significantly better dispersibility in solution compared to PDA. This result clearly demonstrates that the colloidal stability of PDA@PEG nanoparticles was significantly improved after modification with thiol-PEG. Unmodified PDA nanoparticles in PBS rapidly aggregated and precipitated due to their high surface energy and the charge shielding effect caused by salt ions, resulting in a clear upper layer and a large amount of sediment at the bottom after 72 hours. In contrast, PDA@PEG nanoparticles maintained a uniform dispersion after 72 hours without significant precipitation. This proves that the PEG chains effectively prevented the nanoparticles from approaching and aggregating through steric hindrance.

[0052] Figure 4 The UV-Vis absorption spectra of PDA and PDA@PEG nanoparticles in Example 1 are shown in the 200-800 nm range; the specific detection process is as follows: 1. Preparation of test samples: Sample composition: The test samples were pure PDA nanoparticle aqueous dispersion and pure PDA@PEG nanoparticle aqueous dispersion. The solvent was ultrapure water.

[0053] Sample concentration and dosage: To simulate low doping levels in the final product and demonstrate its high efficiency, two nanoparticle dispersions, each with a concentration of 0.1 mg / mL, were precisely prepared. Ultrapure water was used as both the diluent and the reference solution. For testing, 100 μL of each dispersion was placed in a standard high-transparency 96-well plate for measurement.

[0054] 2. Test methods and instruments: Testing instrument: Ultraviolet-visible spectrophotometer was used for testing.

[0055] Test parameters: The scanning wavelength range was set to 200 nm to 800 nm, with ultrapure water as the reference blank, and background subtraction was performed. The scanning speed was set to medium speed, and the data interval was 1 nm.

[0056] Test environment: room temperature.

[0057] from Figure 4 As can be seen, at extremely low doses (0.1 wt%) of PDA and PDA@PEG doping, the ultraviolet absorption rate is significantly improved, and the absorption efficiency of PDA@PEG is significantly better than that of PDA.

[0058] Figure 5 This is a graph showing the DPPH radical scavenging rate of PDA and PDA@PEG nanoparticles in Example 1; from Figure 5 As can be seen, the DPPH scavenging efficiency of PDA@PEG nanoparticles is positively correlated with that of PDA@PEG. At the same concentration (60 μg / mL), after PEG optimization, the DPPH free radical scavenging rate of PDA@PEG was increased by 69.2% compared with that of PDA.

[0059] Figure 6 The image shows the protective effect of PDA and PDA@PEG nanoparticles against UVB and blue light (BL) in Example 1. Figure 6 In the diagram, 'a' represents the UVB protection provided by a mixture of PDA@PEG nanoparticles and PF-127 hydrogel (UVB+PDA@PEG+PF127 group). Figure 6 In the diagram, b represents the UVB protection provided by a mixture of PDA and PF-127 hydrogel (UVB+PDA+PF127 group). Figure 6 In the diagram, 'c' represents the UVB protection provided by the PF-127 hydrogel (UVB+PF127 group). Figure 6 In the diagram, 'd' represents the UVB protection without any protective agent (UVB group). Figure 6 In the figure, 'e' represents the protection of BL by a protective agent composed of PDA@PEG nanoparticles and PF-127 hydrogel (BL+PDA@PEG+PF127 group). Figure 6In the figure, f represents the protection of BL by a protective agent composed of PDA and PF-127 hydrogel (BL+PDA+PF127 group). Figure 6 In the figure, g represents the protection of BL by PF-127 hydrogel (BL+PF127 group). Figure 6 The 'h' in the diagram represents the protection against BL without the protective agent applied (BL group). The specific test method is as follows: Sample preparation and application: Composite protective agent groups (a, b, e, f): PDA@PF-127 composite hydrogel or PDA@PEG / PF-127 composite hydrogel prepared in Example 1 were used at 20 mg / cm³. 2 Apply the product evenly to the prepared skinned area (2 cm × 2 cm) on the guinea pig's back.

[0060] Hydrogel control group (c, g): Pure PF-127 hydrogel (20%, w / v) prepared in Example 1 was used at 20 mg / cm³. 2 Apply evenly.

[0061] Blank control group (d, h): This area was not smeared with any sample.

[0062] UVB and Blue Light Irradiation: After application, wait for the hydrogel to form a film on the skin surface, then use a UVB light source with constant intensity (dose of 1~2 MED) and a high-intensity blue light source (intensity of 50~100 J / cm²) respectively. 2 The dorsal skin of guinea pigs in the corresponding groups was irradiated once daily for two consecutive weeks. Pigmentation on the dorsal skin of each group was observed and recorded at the end of the experiment (day 14). Results are as follows: Figure 6 As shown, from Figure 6 a~d and Figure 6 As can be seen from e~h, both the uncoated group and the group coated with PF-127 hydrogel alone showed obvious pigmentation, while the pigmentation of the group coated with the protective agent mixture of PDA@PEG nanoparticles and PF-127 hydrogel in this embodiment was significantly lighter.

[0063] Figure 7 The image shows the photothermal conversion infrared spectrum of the PDA@PEG nanoparticles in Example 1. Figure 7 In this context, 'a' represents the infrared thermal image. Figure 7 In the figure, b represents the temperature-time change curve. Figure 7The test method for 'a' in the figure is as follows: PF-127 hydrogel, a protective agent consisting of 5 wt% PDA@PEG nanoparticles and 95 wt% PF-127 hydrogel, and a protective agent consisting of 10 wt% PDA@PEG nanoparticles and 90 wt% PF-127 hydrogel are applied to the inner side of the skin of the human forearm, respectively, and exposed to simulated sunlight. Infrared thermal images of temperature changes at 0 min and 5 min are recorded by an infrared camera; among them, Figure 7 In the diagram, 1, 2, and 3 refer to PF-127 hydrogel, a protective agent consisting of a mixture of 5 wt% PDA@PEG nanoparticles and 95 wt% PF-127 hydrogel, and a protective agent consisting of a mixture of 10 wt% PDA@PEG nanoparticles and 90 wt% PF-127 hydrogel, respectively. From... Figure 7 As can be seen from 'a', after 5 minutes, the surface temperature of the coating containing PDA@PEG nanoparticles steadily increased and remained at around 37°C, indicating that the protective agent provided by this invention has a good protective effect; from Figure 7 As shown in b, under simulated continuous sunlight irradiation, the surface temperature of the protective agent group containing PDA@PEG nanoparticles rapidly increased with irradiation time and eventually reached a stable plateau, with the temperature rise positively correlated with the PDA@PEG doping level; while the temperature of the pure PF-127 hydrogel group remained essentially unchanged. Specifically, the stable temperature of the sample with a doping level of 10 wt% was significantly higher than that of the sample with a doping level of 5 wt%.

[0064] from Figure 4 and Figure 7 As can be seen, PDA@PEG nanoparticles have high light absorption and photothermal conversion rates, enabling the protective agent to enhance skin moisturizing performance and prevent cold-induced skin diseases even in harsh environments with strong ultraviolet radiation, such as high altitudes and frigid regions.

[0065] Figure 8 The image shows the immunofluorescence of ROS in Hacat cells induced by UVB irradiation with different amounts of PDA@PEG nanoparticles in Example 1. Figure 8 In the image, 'a' represents the immunofluorescence image of reactive oxygen species (ROS). Figure 8 In the figure, b represents the bar chart for quantitative analysis of average fluorescence intensity. The specific test method is as follows: 1. Cells are seeded at a specific density in confocal culture dishes and allowed to adhere and grow to the appropriate density.

[0066] 2. Following the above grouping, replace with fresh culture medium containing different concentrations of P@E (0, 20, 40, 60 μg / mL) and continue culturing for 24 h for pretreatment.

[0067] 3. After pretreatment, discard the culture medium containing nanoparticles and gently wash the cells three times with PBS.

[0068] 4. Except for the Control group, all other cell groups were exposed to 50 mJ / cm². 2 Irradiation was performed under a UVB light source with a specific dose.

[0069] 5. After irradiation, replace with fresh complete culture medium and continue culturing for 6 hours.

[0070] 6. Immediately afterwards, serum-free culture medium containing 10 μM DCFH-DA probe was added to cells in all groups (including the Control group), and the cells were incubated in a cell culture incubator at 37°C in the dark for 25 min.

[0071] 7. After incubation, wash the cells thoroughly three times with serum-free culture medium to remove any probes that have not entered the cells.

[0072] 8. Observe and photograph the green fluorescence under an inverted fluorescence microscope or a confocal microscope (corresponding to...). Figure 8 (a) Simultaneously, quantitative analysis of fluorescence intensity was performed using image analysis software (corresponding to...) Figure 8 (b) in the middle.

[0073] from Figure 8 As can be seen from a~b in the figures, PDA@PEG nanoparticles can effectively reduce ROS generated by UVB irradiation and counteract its induced excessive production of cellular ROS and oxidative damage.

[0074] Figure 9 This is a diagram showing the effect of PDA@PEG nanoparticles from Example 1 on the skin of a mouse back under UVB irradiation. Figure 9 In the image, 'a' represents the skin on the back of a mouse that has not been exposed to UVB. Figure 9 In the image, b represents the back skin of a mouse that has not been coated with a protective agent and has been exposed to UVB for two weeks. Figure 9 Image c in the figure shows the back skin of a mouse coated with PF-127 hydrogel and irradiated with UVB for 2 weeks. Figure 9 In the image, d represents the back skin of a mouse coated with a protective agent consisting of 99.9 wt% PF-127 hydrogel and 0.1 wt% PDA@PEG nanoparticles, and then irradiated with UVB for 2 weeks. Figure 9Image 'e' in the figure represents the back skin of a mouse coated with a protective agent consisting of 99.8 wt% PF-127 hydrogel and 0.2 wt% PDA@PEG nanoparticles, and then irradiated with UVB for 2 weeks. (From...) Figure 9 As can be seen from a to e, the photodamage on the back skin of mice coated with PDA@PEG nanoparticles was significantly reduced, effectively protecting the skin from ultraviolet damage.

[0075] Figure 10 This is a laser confocal image showing the effect of PDA@PEG nanoparticles on filaggrin (FLG), a key protein in the skin barrier of mice, under UVB irradiation, as described in Example 1. Figure 10 In the image, 'a' represents a laser confocal image of the back of a mouse (Control group) that has not been exposed to UVA+UVB radiation. Figure 10 In the image, b is a laser confocal image of the back of a mouse exposed to UVA+UVB radiation without any protective coating. Figure 10 In the image, c represents a laser confocal image of the back of a mouse (UVA+UVB+Sunscreen group) treated only with commercial sunscreen and exposed to UVA+UVB radiation. Figure 10 In the image, d represents a laser confocal image of the back of a mouse (UVA+UVB+Sunscreen+2.5wt%PDA group) treated with a protective agent containing 2.5wt% PDA@PEG nanoparticles in combination with a commercial sunscreen and subjected to UVA+UVB irradiation. Figure 10 In the image, 'e' represents a laser confocal image of the back of a mouse (UVA+UVB+Sunscreen+5wt%PDA group) treated with a protective agent containing 5wt% PDA@PEG nanoparticles in combination with a commercial sunscreen and subjected to UVA+UVB irradiation. Figure 10 f in the figure represents a laser confocal image of the back of a mouse (UVA+UVB+Sunscreen+10wt%PDA group) treated with a protective agent containing 10wt% PDA@PEG nanoparticles in combination with a commercial sunscreen and then exposed to UVA+UVB radiation. Figure 10 As can be seen from a to f in the figure, UVB irradiation significantly damaged the skin barrier of mice. Compared with the UVB irradiation group, the skin barrier damage on the back of mice in the PDA@PEG nanoparticle group was significantly reduced, effectively protecting the skin from UV barrier damage.

[0076] Example 2

[0077] 1) Disperse 0.12 g of dopamine hydrochloride in 16 mL of Tris buffer (molar concentration of 10 mmol / L, pH of 8.5), and then magnetically stir at 500 rpm for 18 h at room temperature. Observe that the color of the reaction solution turns dark brown. Add citric acid to adjust the pH of the reaction system to 5.5 to terminate the reaction. Then centrifuge at 12000 rpm for 15 min, and wash three times with deionized water at 12000 rpm to obtain polydopamine particles (PDA), which are then lyophilized and stored for later use. 2) Dissolve 0.08 g of the polydopamine particles obtained in step 1) in 50 mL of tris(hydroxymethyl)aminomethane hydrochloride solution (pH 8.0), and add 0.08 g of mercaptopolyethylene glycol (PEG, molecular weight 5000 Da). Then, stir magnetically in the dark at 25 °C and 400 rpm for 24 h, and then purify by dialysis with a filter membrane with a molecular weight cutoff of 100 kDa for 24 h to obtain PDA@PEG nanoparticles with a particle size of 120±15 nm. 3) Add 20 g of Pluronic F-127 particles to 80 mL of phosphate-buffered saline (PBS, pH 7.4) at 4 °C, and then magnetically stir for 24 h at 4 °C and 300 rpm to obtain a clear PF-127 hydrogel precursor solution with a concentration of 20% (w / v). 4) Mix 95 g of PF-127 hydrogel at 4℃ with 5 g of PDA@PEG nanoparticles, and then react with magnetic stirring at 4℃ and 200 rpm for 24 h in the dark to obtain a biomimetic nano melanin protectant with photothermal properties and UV-Vis dual-band protection.

[0078] Example 3

[0079] 1) Disperse 0.18 g of dopamine hydrochloride in 12 mL of Tris buffer (molar concentration of 10 mmol / L, pH of 8.5), and then magnetically stir at 600 rpm for 12 h at room temperature. Observe that the color of the reaction solution turns brownish-black. Add citric acid to adjust the pH of the reaction system to 5.5 to terminate the reaction. Then centrifuge at 13000 rpm for 15 min, and wash three times with deionized water at 13000 rpm to obtain polydopamine particles (PDA), which are then lyophilized and stored for later use. 2) Dissolve 0.09 g of the polydopamine particles obtained in step 1) in 50 mL of tris(hydroxymethyl)aminomethane hydrochloride solution (pH 8.0), and add 0.12 g of mercaptopolyethylene glycol (PEG, molecular weight 5000 Da). Then, stir magnetically in the dark at 25 °C and 500 rpm for 24 h, and then purify by dialysis with a filter membrane with a molecular weight cutoff of 100 kDa for 24 h to obtain PDA@PEG nanoparticles with a particle size of 100±20 nm. 3) 23 g of Pluronic F-127 particles were added to 77 mL of phosphate-buffered saline (PBS, pH 7.4) at 4 °C, and then the mixture was magnetically stirred in an open container at 350 rpm for 24 h to obtain a clear PF-127 hydrogel precursor solution with a concentration of 23% (w / v). 4) Mix 85 g of PF-127 hydrogel at 4℃ with 15 g of PDA@PEG nanoparticles, and then react with magnetic stirring at 4℃ and 250 rpm for 18 h in the dark to obtain a biomimetic nano melanin protectant with photothermal properties and UV-Vis dual-band protection.

[0080] Analysis of the above examples and experimental results shows that PF-127, being a biocompatible, non-toxic, and thermally reversible material, can be widely used as a wound dressing. PF-127 exhibits unique reversible thermogel properties in higher concentrations of aqueous solution, allowing it to safely remain on the wound surface. This property is that it is liquid at 4°C and semi-solid gel at room temperature or body temperature, possessing both hydrophilic and lipophilic properties, as well as anti-inflammatory and antibacterial effects. This provides a basis for the adhesion of PDA@PEG nanoparticles to the skin surface and allows for uniform dispersion of PDA@PEG nanoparticles within it, serving as a matrix for the preparation of protective agents. Polydopamine is a major component of human anti-UV melanin. The thermal reversibility of PF-127 hydrogel and the modification of PDA@PEG with polyethylene glycol ensure thorough mixing of polydopamine and PF-127, avoiding denaturation damage caused by ordinary mixing methods such as heating and stirring. The full dispersion of PDA@PEG nanoparticles will further enhance the UV protection capability, which is related to the enhanced UV scattering effect after full dispersion, while avoiding uneven color and pore blockage after application.

[0081] Furthermore, the amphiphilic nature of PF-127 hydrogel allows the protective agent described in this invention to bind tightly to the skin surface, reducing the skin permeability of PDA@PEG nanoparticles while improving usage efficiency; at the same time, the amount of PF-127 used in this invention allows PDA@PEG to partially enter the skin and exert its protective effect in vivo.

[0082] Moreover, the preparation process of the protective agent described in this invention is green and environmentally friendly. It uses biomimetic nano-melanin as the core ingredient, and the product is safe and efficient. It solves a series of problems of traditional sunscreens, such as poor water resistance, easy penetration, free radical damage to the skin, and clogging of pores, and has broad application prospects.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a biomimetic nano-melanin protective agent with photothermal responsiveness and ultraviolet-visible light dual-band protection function, characterized in that, The method comprises the following steps: 1) mixing dopamine hydrochloride with Tris buffer solution to react to obtain polydopamine particles; 2) mixing the polydopamine particles with a tris(hydroxymethyl)aminomethane hydrochloride solution and mercapto polyethylene glycol to perform a light-proof reaction to obtain PDA@PEG nanoparticles; 3) mixing Pluronic F-127 with a phosphate buffered saline to react to obtain a PF-127 hydrogel; 4) mixing the PF-127 hydrogel with the PDA@PEG nanoparticles to perform a light-proof reaction to obtain a biomimetic nano-melanin protective agent with photothermal responsiveness and ultraviolet-visible light dual-band protection function; wherein steps 2) and 3) are not limited in sequence; in step 1), the mass-volume ratio of dopamine hydrochloride to Tris buffer solution is 0.1-0.2 g:10-20 mL; the molar concentration of the Tris buffer solution is 9-11 mmol / L, and the pH is 8-9; in step 1), the reaction time is 4-24 h; in step 3), the mass-volume ratio of Pluronic F-127 to phosphate buffered saline is 20-25 g:75-80 mL.

2. The preparation method of the biomimetic nano-melanin protective agent with photothermal response and UV-Vis dual-band protection function according to claim 1, characterized in that, in step 2), the mass-volume ratio of polydopamine particles to tris(hydroxymethyl)aminomethane hydrochloride solution and mercapto polyethylene glycol is 0.06-0.1 g:50 mL:0.08-0.12 g; the pH of the tris(hydroxymethyl)aminomethane hydrochloride solution is 8.0-8.

1.

3. The method for preparing the biomimetic nano-melanin protective agent with photothermal response and UV-Vis dual-band protection function according to claim 2, characterized in that, in step 2), the light-proof reaction temperature is 20-25℃, and the time is 12-24 h.

4. The preparation method of the biomimetic nano-melanin protective agent with photothermal response and UV-Vis dual-band protection function according to any one of claims 1-3, characterized in that, in step 2), the diameter of the PDA@PEG nanoparticles is 50-150 nm.

5. The method of claim 4, wherein the method of preparing the biomimetic nano-melanin sunscreen agent with photothermal response and dual-band protection function of ultraviolet-visible light is characterized in that, in step 3), the reaction temperature is 0-4℃, and the time is 12-24 h.

6. The method for preparing the biomimetic nano-melanin sunscreen agent with photothermal response and UV-Vis dual-band protection function according to claim 5, characterized in that, in step 4), the mass ratio of the PF-127 hydrogel to PDA@PEG nanoparticles is 85-95:5-15.

7. The method of claim 6, wherein the method of preparing the biomimetic nano-melanin sunscreen agent with photothermal response and dual-band protection function of ultraviolet-visible light is characterized in that, in step 4), the light-proof reaction temperature is 0-4℃, and the time is 12-24 h.

8. The biomimetic nano-melanin protective agent with photothermal responsiveness and ultraviolet-visible light dual-band protection function prepared by the preparation method of any one of claims 1-7.

9. The use of the biomimetic nano-melanin protective agent with photothermal responsiveness and ultraviolet-visible light dual-band protection function of claim 8 in the preparation of a product for preventing cold dermatosis.

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