Multifunctional sunscreen nanoparticles for shielding full-waveband ultraviolet rays, and preparation method and application thereof

Abstract

The application provides a full-waveband ultraviolet shielding multifunctional sunscreen nanoparticle and a preparation method and application thereof. The nanoparticle takes a covalent cross-linking network formed by Mannich reaction of a polyphenol compound and an amino acid and a peptide derivative thereof as a skeleton, and composites an antibacterial nanoparticle in the skeleton. The sunscreen nanoparticle can realize high-efficiency shielding of full-waveband ultraviolet, has antibacterial performance and good antioxidant capacity, and realizes synergistic integration of multiple functions such as high-efficiency sunscreen, good skin compatibility and antibacterial property in a sunscreen system.

Description

Technical Field

[0001] This invention relates to the field of bio-sunscreen nanomaterials technology, specifically to a multifunctional sunscreen nanoparticle that blocks ultraviolet light across the entire spectrum, its preparation method, and its applications. Background Technology

[0002] As people's understanding of skin damage caused by ultraviolet radiation (including acute sunburn, chronic photoaging, and the risk of skin cancer) deepens, the research and development of sunscreen products has gradually become a research hotspot of common concern in the fields of daily chemical industry and biomedicine. An ideal sunscreen should not only have the ability to block UVA and UVB across the entire spectrum of ultraviolet radiation, but also have good biocompatibility and low skin permeability to meet the market demand for multifunctional skincare products. Traditional organic sunscreens are prone to degradation under prolonged sun exposure, which not only reduces their protective effect but may also generate sensitizing photodegradation products, thus triggering skin allergies or phototoxic reactions. Inorganic sunscreens (such as nano-TiO2 and ZnO) have good ultraviolet shielding effects, but due to their photocatalytic properties, they can generate free radicals under ultraviolet light, causing skin damage. Furthermore, they are prone to aggregation, leading to uneven dispersion, unnatural white residue on the skin surface after application, and the potential transdermal absorption risk and resulting nanotoxicity issues associated with nano-sized particles.

[0003] Furthermore, currently available sunscreen materials are still functionally limited, making it difficult to simultaneously achieve multiple functions such as high-efficiency sun protection, good skin compatibility, and antibacterial properties within the same system. Therefore, developing a new type of sunscreen material that can achieve full-spectrum ultraviolet protection while also possessing biocompatibility and antibacterial properties, based on green and safe design principles, has significant scientific and practical value. Summary of the Invention

[0004] To address the problems existing in the background technology, the present invention provides a multifunctional sunscreen nanoparticle with full-band ultraviolet shielding, its preparation method and application. The sunscreen nanoparticle can achieve efficient shielding of full-band ultraviolet rays, and has antibacterial properties and good antioxidant capacity. It achieves the synergistic integration of multiple functions such as efficient sun protection, good skin compatibility and antibacterial properties in the sunscreen system.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides a multifunctional sunscreen nanoparticle that blocks ultraviolet light across the entire spectrum. The nanoparticle has a covalent cross-linked network formed by polyphenolic compounds and amino acids and their peptide derivatives through the Mannich reaction as its backbone, and antibacterial nanoparticles are composited in the backbone.

[0006] According to the above scheme, the amino acids and their peptide derivatives are one or more of natural proteins, recombinant proteins, polypeptides, oligopeptides, or amino acids.

[0007] According to the above scheme, the polyphenolic compound is any one or more of tannic acid, gallic acid, chlorogenic acid, quercetin, hesperidin, anthocyanin, epigallocatechin gallate, and pentagalloglucoside.

[0008] According to the above scheme, the antibacterial nanoparticles are at least one of metal antibacterial nanoparticles, metal oxide antibacterial nanoparticles, and carbon-based antibacterial nanoparticles.

[0009] According to the above scheme, the metal antibacterial nanoparticles are Ag, Au, or Cu; the metal oxide antibacterial nanoparticles are ZnO, TiO2, or CuO nanoparticles; and the carbon-based antibacterial nanoparticles are graphene quantum dots, carbon dots, or fullerenes.

[0010] According to the above scheme, the mass ratio of the polyphenolic compound to the amino acid and its peptide derivative is (0.1-8):1, preferably (0.25-5):1; the amount of antibacterial nanoparticles added is 0.1%-200% of the polyphenolic compound, preferably 25%-200%.

[0011] Secondly, the present invention provides a method for preparing the above-mentioned full-band ultraviolet shielding multifunctional sunscreen nanoparticles: when the antibacterial nanoparticles are generated by reduction of metal precursor salt, method A is used; when the antibacterial nanoparticles are pre-prepared antibacterial nanoparticles, method A or method B is used. Method A includes the following steps: S1. Activate polyphenolic compounds by reacting them with formaldehyde through hydroxymethylation; S2. Under weakly alkaline conditions, the activated polyphenols are reacted with amino acids and their peptide derivatives by the Mannich reaction to form a methylene bridge cross-linking network; S3. Add metal precursor salt, utilize the reducing properties of polyphenols to generate antibacterial nanoparticles in situ and anchor them in the cross-linked network, and then purify them; Method B includes the following steps: S1. Activate polyphenolic compounds by reacting them with formaldehyde through hydroxymethylation; S2. Under weakly alkaline conditions, amino acids and their peptide derivatives, along with pre-prepared antibacterial nanoparticles, are simultaneously added to an activated polyphenol solution to perform the Mannich reaction and physical encapsulation of the antibacterial nanoparticles, followed by purification.

[0012] According to the above scheme, in methods A and B, the pH value of the Mannich reaction is 8.0-10.0, the reaction temperature is 20-80℃, preferably 40-80℃, and the reaction time is 1-8 hours, preferably 1-6 hours.

[0013] According to the above scheme, the metal precursor salt in method A is one or more of silver nitrate, silver nitrate, chloroauric acid, zinc acetate, or copper sulfate.

[0014] Thirdly, this invention provides the application of the above-mentioned full-band ultraviolet shielding multifunctional sunscreen nanoparticles in the preparation of sunscreen products.

[0015] According to the above scheme, the sun protection product is sunscreen, sunscreen spray, sunscreen lotion, sunscreen gel, sunscreen film, or sunscreen textile finishing agent.

[0016] Polyphenolic compounds contain multiple polyphenolic hydroxyl active sites that can participate in the Mannich reaction. After activation by hydroxymethylation, hydroxymethylation intermediates are generated, which then undergo condensation reactions with amino groups in amino acids and their peptide derivatives to form stable methylene bridges, constructing a covalent cross-linked network. In this covalent cross-linked network, amino acids and their peptide derivatives form a uniform and dense coating layer on the surface of the polyphenolic compound, constructing a regular core-shell structure. Simultaneously, antibacterial nanoparticles are composited within the covalent cross-linked network, forming stable composite sunscreen nanoparticles.

[0017] The beneficial effects of this invention are: 1) This invention achieves stable aggregation of polyphenolic compounds and amino acids and their peptide derivatives into nanoparticles through covalent cross-linking. The amino acids and their peptide derivatives form a uniform and dense coating layer on the surface of the polyphenolic compounds, forming a regular shell-core structure. Antibacterial nanoparticles are composited in the covalent cross-linking network. The three components form stable nanoparticles through intermolecular interactions, and their synergistic effect achieves absorption and scattering of UVA and UVB bands, thereby achieving efficient shielding of ultraviolet rays across the entire spectrum. It also has antibacterial properties, good antioxidant capacity, and skin compatibility. In the sunscreen system, it achieves the synergistic integration of multiple functions such as efficient sun protection, good skin compatibility, and antibacterial properties. 2) The covalent cross-linking structure significantly enhances the photostability of sunscreen nanoparticles, effectively overcoming the defects of traditional organic sunscreens that are prone to photodegradation and failure, prolonging the product's protective efficacy, and overcoming the potential toxicity problems of traditional organic and inorganic sunscreens. 3) The covalent cross-linked structure prevents the dissociation of nanoparticles during storage and use, reduces the risk of transdermal absorption of polyphenolic compounds and amino acids and their peptide derivatives, and provides stable anchoring sites for antibacterial nanoparticles, inhibiting their migration and aggregation, and significantly improving product safety.

[0018] 4) This invention provides two methods for preparing sunscreen nanoparticles. The raw materials used, namely polyphenolic compounds and amino acids and their peptide derivatives, are all naturally derived biomacromolecules with good biodegradability and low immunogenicity. The entire preparation process is carried out in an aqueous phase without the use of organic solvents, surfactants, or other toxic reagents. Compared with the multi-step, expensive nanotechnology and complex formulations involved in existing technologies, this invention features a simple process, mild conditions, and ease of large-scale production, conforming to the principles of green chemistry and sustainable development. Attached Figure Description

[0019] Figure 1 These are TEM images of the sunscreen nanoparticles prepared in Examples 1 and 2 of the present invention, where (a) is Example 1 and (b) is Example 2; Figure 2 The UV absorption capacity of the sunscreen nanoparticles prepared in Example 1 and Comparative Example 1 of this invention; Figure 3 This refers to the UV absorption capacity of sunscreen nanoparticles in different proportions in Example 1 of the present invention; Figure 4 The antioxidant capacity of the sunscreen nanoparticles in Example 1 of the present invention is shown in (a) as the UV-Vis absorption spectrum of the sunscreen nanoparticles scavenging DPPH free radicals, (b) as the UV-Vis absorption spectrum of the sunscreen nanoparticles scavenging ABTS free radicals, and (c) as the scavenging efficiency of the sunscreen nanoparticles on DPPH and ABTS free radicals. Figure 5 The results of the sun protection ability of the sun protection nanoparticle pickerling emulsion in Example 1 of the present invention are shown, wherein (a) is the ultraviolet transmittance diagram of the sun protection nanoparticle pickerling emulsion, and (b) is the SPF and UVAPF of the sun protection nanoparticle. Figure 6 The results show the antibacterial ability of the sunscreen nanoparticles in Example 1 of this invention; Figure 7 The results of the cytotoxicity of the sunscreen nanoparticles in Example 1 of this invention; Figure 8 This describes the effect of sunscreen nanoparticles in Example 1 of the present invention on cytotoxicity caused by ultraviolet light irradiation. Figure 9 This illustrates the effect of sunscreen nanoparticles in Example 1 of the present invention on intracellular reactive oxygen species generated after ultraviolet light irradiation. Figure 10 The photoprotective properties of the sunscreen nanoparticles in Example 1 of this invention are shown in (a) and (b) are the results of H&E and Masson staining of animal skin after ultraviolet irradiation, respectively. Detailed Implementation

[0020] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0021] This invention provides a multifunctional sunscreen nanoparticle that blocks ultraviolet light across the entire spectrum. The nanoparticle uses a covalent cross-linked network formed by polyphenolic compounds and amino acids and their peptide derivatives through the Mannich reaction as its backbone, and incorporates antibacterial nanoparticles within the backbone.

[0022] Among them, amino acids and their peptide derivatives are one or more of natural proteins, recombinant proteins, polypeptides, oligopeptides, or amino acids.

[0023] Preferably, the polyphenolic compound is any one or more of tannic acid, gallic acid, chlorogenic acid, quercetin, hesperidin, anthocyanin, epigallocatechin gallate, and pentagalloglucoside.

[0024] Preferably, the antibacterial nanoparticles are at least one of metal antibacterial nanoparticles, metal oxide antibacterial nanoparticles, and carbon-based antibacterial nanoparticles. Optionally, the metal antibacterial nanoparticles are Ag, Au, or Cu; the metal oxide antibacterial nanoparticles are ZnO, TiO2, or CuO nanoparticles; and the carbon-based antibacterial nanoparticles are graphene quantum dots, carbon dots, or fullerenes.

[0025] Preferably, in the sunscreen nanoparticles, the mass ratio of polyphenolic compounds to amino acids and their peptide derivatives is (0.1-8):1, more preferably (0.25-5):1, and the amount of antibacterial nanoparticles added is 0.1%-200% of the polyphenolic compounds, more preferably 25%-200%.

[0026] This invention also provides two methods for preparing the above-mentioned multifunctional sunscreen nanoparticles that block ultraviolet rays across the entire spectrum: (1) When the antibacterial nanoparticles are generated by reduction of metal precursor salt, method A is used: in-situ generation method, the specific steps of which include: S1. Activate polyphenolic compounds by reacting them with formaldehyde through hydroxymethylation; S2. Under weakly alkaline conditions, the activated polyphenols are reacted with amino acids and their peptide derivatives by the Mannich reaction to generate a methylene bridge cross-linking network, forming a nanoparticle framework. S3. Add metal precursor salt, utilize the reducing properties of polyphenols to generate antibacterial nanoparticles in situ and anchor them in the cross-linked network, and then purify them; In step S1 of this method, polyphenolic compounds are dissolved in water or buffer solution, formaldehyde is added, and hydroxymethylation is performed by stirring at 20-60℃ for 0.5-4 hours. The concentration of polyphenolic compounds in the solution is 0.05-0.12 mol / L, and the molar ratio of formaldehyde to polyphenolic compounds is (0.1-3):1. In step S2, under weakly alkaline conditions (pH 8.0-10.0), the activated polyphenols and amino acids and their peptide derivatives are stirred at 40-80℃ for 1-6 hours to carry out the Mannich reaction. The concentration of amino acids and their peptide derivatives in the solution is 0.006-0.05 mol / L. In step S3, after adding the metal precursor salt, the reaction is carried out at 20-60℃ in the dark for 0.5-4 hours. Antibacterial nanoparticles are generated in situ using the reducing properties of polyphenols and anchored in the network. Then, the nanoparticles are obtained by centrifugation (5000-15000 rpm, 3-30 minutes) or dialysis. The concentration of the metal precursor salt in the solution is 0.04-0.16 mol / L.

[0027] The metal precursor salt in Method A is one or more of silver nitrate, silver nitrate, chloroauric acid, zinc acetate, or copper sulfate.

[0028] (2) When the antibacterial nanoparticles are pre-prepared antibacterial nanoparticles, method B: one-step doping method is adopted, including the following steps: S1. Activate polyphenolic compounds by reacting them with formaldehyde through hydroxymethylation; S2. Under weakly alkaline conditions, amino acids and their peptide derivatives, along with pre-prepared antibacterial nanoparticles, are simultaneously added to an activated polyphenol solution to perform the Mannich reaction and physical encapsulation of the antibacterial nanoparticles, followed by purification.

[0029] In method B, step S1 is the same as above. In step S2, the weakly alkaline conditions are pH 8.0-10.0. Amino acids and their peptide derivatives and antibacterial nanoparticles are simultaneously added to the activated polyphenol solution. The Mannich reaction is carried out by stirring at 20-80℃ for 1-8 hours, and the physical encapsulation and composite of antibacterial nanoparticles are carried out simultaneously. Then, the nanoparticles are obtained by centrifugation (5000-15000 rpm, 3-30 minutes) or dialysis purification.

[0030] The present invention provides that the above-mentioned multifunctional sunscreen nanoparticles with full-band ultraviolet shielding can be used to prepare sunscreen products, such as sunscreen cream, sunscreen spray, sunscreen lotion, sunscreen gel, sunscreen film or sunscreen textile finishing agent.

[0031] The following are specific examples: Example 1 This embodiment prepares a multifunctional sunscreen nanoparticle that blocks ultraviolet light across the entire spectrum. The framework consists of a covalently cross-linked network formed by epigallocatechin gallate (EGCG) and silk fibroin through the Mannich reaction, and silver nanoparticles are composited within the framework.

[0032] Its preparation method is as follows: 0.5 g of epigallocatechin gallate (EGCG) was dissolved in 50 mL of deionized water, and 0.2 mL of formaldehyde was added. The mixture was activated by stirring at 40 °C for 2 hours. Silk fibroin was added at a mass ratio of EGCG to silk fibroin of 2:1. The pH was adjusted to 9.0 with 0.1 M NaOH, and the mixture was stirred at 60 °C for 3 hours to induce Mannich cross-linking. 0.1 N AgNO3 solution was slowly added dropwise at a mass ratio of EGCG-silk fibroin to silver nitrate of 1:2. The reaction was carried out at room temperature in the dark for 1 hour. Polyphenols reduced AgNO3 to generate silver nanoparticles, which were then anchored to the covalently cross-linked network. The nanoparticles were purified by centrifugation and washing with deionized water.

[0033] Example 2 This embodiment prepares a multifunctional sunscreen nanoparticle that blocks ultraviolet light across the entire spectrum. The backbone is a covalently cross-linked network formed by EGCG and RGD peptides through the Mannich reaction, and zinc oxide nanoparticles are composited in the backbone.

[0034] Its preparation method is as follows: 0.5 g of EGCG was dissolved in 50 mL of deionized water, and 0.2 mL of formaldehyde was added. The mixture was activated by stirring at 40 °C for 2 hours. RGD peptide was added at a mass ratio of EGCG to arginine-glycine-aspartic peptide (RGD peptide) of 2:1. The pH was adjusted to 9.0 with 0.1 M NaOH, and the mixture was stirred at 60 °C for 3 hours to induce Mannich crosslinking. Zinc oxide nanoparticles were slowly added dropwise at a mass ratio of EGCG-RGD to zinc oxide nanoparticles of 1:4. The mixture was reacted at room temperature in the dark for 1 hour to anchor the zinc oxide nanoparticles onto the crosslinking network. The nanoparticles were washed by centrifugation with deionized water to obtain purified nanoparticles.

[0035] Comparative Example 1 To verify the effect of whether or not formaldehyde is used in the Mannich condensation reaction on the formation of sunscreen nanoparticles, this comparative example was set up.

[0036] The difference between Comparative Example 1 and Example 1 is that the Mannich reaction is not performed. The specific preparation method is as follows: Weigh 0.5 g of EGCG and dissolve it in 50 mL of deionized water. Add silk fibroin at a mass ratio of EGCG to silk fibroin of 2:1. Adjust the pH to 9.0 with 0.1 M NaOH and stir at 60 °C for 3 hours. Slowly add 0.1 N AgNO3 solution dropwise at a mass ratio of EGCG-silk fibroin to silver nitrate of 1:2. React at room temperature in the dark for 1 hour. Polyphenols reduce AgNO3 to generate silver nanoparticles, which are then anchored. Wash the precipitate with deionized water by centrifugation to obtain an insoluble macromolecular aggregate precipitate.

[0037] (1) Morphology of sunscreen nanoparticles The sunscreen nanoparticles prepared in Examples 1 and 2 were subjected to TEM analysis, and their TEM images are shown below. Figure 1 As shown, from Figure 1 As shown in (a), the nanoparticles have smooth surfaces without obvious depressions or damage, indicating that silk fibroin has successfully encapsulated the EGCG core, forming core-shell structured nanoparticles. On the surface and surrounding area of ​​the spherical carrier, numerous dark-colored particles with extremely small diameters and high electron density can be clearly observed; these dark-colored particles are the loaded silver nanoparticles. The silver nanoparticles are distributed on the surface of the EGCG-silk fibroin carrier without obvious aggregation, indicating that the silver nanoparticles have successfully bound to the outside of the carrier, forming a well-defined EGCG-silk fibroin-silk composite nanoparticle structure. Figure 1 In (b), the internal structure of the composite can be clearly observed. The nanoparticles exhibit obvious sandwich structure characteristics, with the dark areas with higher electron density corresponding to zinc oxide nanoparticles, while the gray areas with lower electron density correspond to the EGCG-RGD organic crosslinking network. The zinc oxide nanoparticles are uniformly coated inside the EGCG-RGD matrix, rather than simply adsorbed on the surface. This distribution provides direct evidence for the successful encapsulation of zinc oxide.

[0038] (2) UV absorption capacity of sunscreen nanoparticles ① The UV absorption capacity of the sunscreen nanoparticles prepared in Example 1 and Comparative Example 1 were tested respectively. The specific method is as follows: Use a UV-Vis spectrophotometer to perform UV-Vis absorption spectroscopy (UV-vis) analysis on the test solution. Before the test, accurately set the scanning wavelength range (200-400 nm) and scanning speed, and then measure the absorbance of the sample.

[0039] The results are as follows Figure 2As shown, the UV absorption curves of the sunscreen nanoparticles in Example 1 exhibit better absorption in the UVB band (280-320nm) (average absorbance 0.57 ± 0.07) and the UVA band (320-400nm) (average absorbance 0.36 ± 0.06). In contrast, Comparative Example 1 shows average absorbances of 0.28 ± 0.19 and 0.03 ± 0.02 in the UVB and UVA bands, respectively. This is because the Mannich reaction using formaldehyde forms a hydroxymethylated EGCG intermediate, which subsequently undergoes a condensation reaction with silk fibroin to form a covalently cross-linked network, generating stable, suspended nanoparticles with a higher degree of conjugation, contributing to stronger light signals in the UVB and UVA bands. Comparative Example 1, however, binds only through weaker physical interactions such as hydrogen bonds and hydrophobic interactions, lacking a new conjugated structure and suffering signal loss due to precipitation, resulting in lower absorbance.

[0040] ② Effect of the ratio of components in the sunscreen nanoparticles prepared in Example 1 on their ultraviolet absorption capacity. EGCG-silk fibroin network backbones were prepared according to EGCG to silk fibroin mass ratios of 1:2 and 2:1, respectively. Their UV absorption capacity was measured, and the results are as follows: Figure 4 As shown, when the mass ratio of EGCG to silk fibroin is 2:1, its absorption intensity in UVB and UVA is greater. This is because when the mass ratio of EGCG to silk fibroin is 2:1, the silk fibroin forms a more uniform and dense coating layer on the surface of the EGCG core. This regular core-shell structure not only enhances the absorption and scattering of UVA light by the particles, but may also form a more compact molecular stack through the interactions between EGCG and silk fibroin molecules (such as hydrogen bonds and hydrophobic interactions), thereby producing a synergistically enhanced ultraviolet absorption effect.

[0041] When introducing silver nanoparticles, the mass ratio of EGCG to silk fibroin was selected as 2:1. Sunscreen nanoparticles were prepared with EGCG-silk fibroin network framework and silver nitrate mass ratios of 1:1, 1:4, and 1:8, respectively. Their UV absorption capacity was then tested and compared. The results are as follows: Figure 3 As shown, the UV absorption curves of EGCG-silk fibroin-silver nanoparticles with different proportions exhibit the same peak shape as those of EGCG-silk fibroin nanoparticles. The binding of Ag nanoparticles enhances the UV absorption capacity of the sunscreen nanoparticles in the UVB (280-320 nm) and UVA (320-400 nm) bands, with a particularly significant increase in UVB absorption. This is attributed to the surface plasmon resonance effect of Ag nanoparticles and their scattering effect on incident light, both of which contribute to the light absorption in the UVB and UVA bands. Figure 3The 1:4 ratio sample exhibited the strongest ultraviolet absorption intensity in the UVB and UVA bands. This is because insufficient Ag resulted in unsaturated nanoparticle binding sites and incomplete coating, thus leading to the weakest absorption enhancement effect. Conversely, excessive Ag may cause local aggregation, reducing the effective absorption cross-section.

[0042] (3) Free radical scavenging ability of sunscreen nanoparticles The free radical scavenging ability of the sunscreen nanoparticles prepared in Example 1 was tested to evaluate the antioxidant capacity of the sunscreen nanoparticles provided by the present invention. Specifically, a free radical solution was set as a blank control group, a 10 mg / mL vitamin C solution as a positive control group, and the experimental group was set as a 10 mg / mL nanoparticle suspension.

[0043] To determine the ABTS free radical scavenging rate, ABTS (7 mM) and potassium persulfate (4.5 mM) were dissolved in deionized water and mixed at a 1:1 volume ratio. The precursor solution was then left to stand in the dark at room temperature for 12 hours to allow complete oxidation, yielding a deep blue-green ABTS. + Free radical solution. Then, 1 mL of this solution was diluted with 80 mL of water to achieve a UV absorbance of 0.8 ± 0.2 at 734 nm. Next, 2 mL of the diluted ABTS solution was added to the sample.

[0044] To determine the DPPH radical scavenging rate, 2.5 μg / mL of DPPH methanol stock solution was added to the sample. The mixture was then placed in a constant-temperature shaking incubator and incubated in the dark at 37 ℃ and 150 r / min for 60 minutes. After incubation, the sample was centrifuged at 8000 r / min for 8 minutes, and the supernatant was collected. The absorbance of the sample supernatant at wavelengths of 734 nm and 517 nm was measured using UV-Vis spectrophotometry, ABTS, and DPPH scavenging experiments, respectively.

[0045] Free radical scavenging efficiency = (absorbance of blank control group - absorbance of experimental group) / absorbance of blank control group × 100%. All experiments were repeated three times to ensure the accuracy and reproducibility of the results.

[0046] The results are as follows Figure 4As shown, the sunscreen nanoparticles prepared in this invention can achieve a scavenging rate of over 96% for DPPH free radicals and over 97% for ABTS free radicals, demonstrating excellent free radical scavenging ability, with antioxidant capacity comparable to vitamin C. EGCG, a classic polyphenolic antioxidant, possesses highly efficient free radical scavenging capabilities due to its abundant ortho-phenolic hydroxyl structures. Meanwhile, silk fibroin itself exhibits good reactive oxygen species (ROS) free radical scavenging activity, and its self-assembled β-sheet structure can exert antioxidant function by regulating oxidative stress pathways. The synergistic effect of EGCG and silk fibroin significantly enhances the antioxidant capacity after combining the two.

[0047] (4) The sun protection ability of sunscreen nanoparticles prepared into Pickering emulsion The nanoparticles prepared in Example 1 were used to prepare Pickering emulsions. The ultraviolet transmittance of EGCG-silk fibroin nanoparticles and EGCG-silk fibroin-silk nanoparticle Pickering emulsions in the 290-400 nm wavelength range was tested using an ultraviolet spectrophotometer with an integrating sphere. The sun protection factor (SPF) and UVA protection factor (UVAPF) of the two emulsions were calculated according to the formula to evaluate the feasibility of preparing the nanoparticles of the present invention into sunscreen emulsions and to demonstrate their sun protection performance.

[0048] The specific method is as follows: Nanoparticles are dispersed in an aqueous phase. The aqueous and oil phases are mixed at a ratio of 3:7 at 80°C. The mixture is stirred using a high-speed homogenizer at an appropriate speed to ensure complete emulsification, yielding a Pickering emulsion. The addition amount of either EGCG-silk fibroin or EGCG-silk fibroin-silk is 10%. No phase separation occurred in the emulsion within 48 hours under visual observation, indicating that the emulsion has a certain degree of stability. The emulsion is then subjected to a concentration of 2 mg / cm³. 2 The product was uniformly coated onto a quartz substrate and pressed to a certain thickness. High-resolution transmission spectra (1 nm resolution) were obtained in the UVB (290-320 nm) and UVA (320-400 nm) bands. The sun protection factor (SPF) and UVA protection factor (UVAPF) were calculated.

[0049]

[0050] . Where E(λ) is the relative erythema effect spectrum, and S(λ) is the solar spectral irradiance (W·m). -2 ·nm -1 T(λ) is the average transmittance of the sunscreen sample, and λ is the wavelength.

[0051] The results are as follows Figure 5As shown, the UV transmittance of the EGCG-silk fibroin-silk Pickering emulsion was significantly lower than that of the EGCG-silk fibroin emulsion with the same amount of added silver in both the UVB and UVA bands, demonstrating stronger UV protection capabilities. In Figure (a), the UV transmittance of the EGCG-silk fibroin-silk Pickering emulsion was significantly reduced in the UVA band, indicating that the introduction of silver nanoparticles improved the emulsion's broad-spectrum sun protection performance. Figure 5 As shown in (b), the EGCG-silk fibroin-silver Pickering emulsion has an SPF of over 35.5 and a UVAPF of over 17. This indicates that the introduction of silver nanoparticles significantly enhances the UV protection performance of EGCG-silk fibroin nanoparticles, especially greatly improving their UVA protection. This synergistic effect complements the UVB absorption capacity of EGCG-silk fibroin itself, giving the EGCG-silk fibroin-silver nanoparticle Pickering emulsion excellent broad-spectrum sun protection properties.

[0052] (4) Antibacterial ability of sunscreen nanoparticles The antibacterial activity of the sunscreen nanoparticles prepared in Example 1 against Escherichia coli and Staphylococcus aureus was tested.

[0053] The specific method is as follows: Nanoparticles and bacterial suspension were incubated together at 37 ℃ and 120 rpm for 4 hours with shaking. After incubation, bacterial suspensions from each group were serially diluted with sterile phosphate buffer, and 50 μL was evenly spread on the surface of ordinary agar plates and then incubated upside down in a 37 ℃ incubator overnight. After incubation, plate counting was performed, with 3 replicates for each group. Results are expressed as mean ± standard deviation, and the antibacterial rate was calculated based on the colony count of the blank control group: Antibacterial rate (%) = (colony count of control group - colony count of experimental group) / colony count of control group × 100%.

[0054] The results are as follows Figure 6 As shown, the sunscreen nanoparticles exhibit strong antibacterial activity. When the concentration reaches 1000 μg / mL, the number of Escherichia coli colonies is as low as 12 ± 2, with an antibacterial rate of up to 98%, and the number of Staphylococcus aureus colonies is as low as 53 ± 10, with an antibacterial rate of up to 96%.

[0055] (5) Cytotoxicity test of sunscreen nanoparticles The cytotoxicity of the sunscreen nanoparticles prepared in Example 1 was tested to evaluate their cell compatibility.

[0056] The specific method is as follows: Nanoparticles of different concentrations (0, 0.1, 1, 20, 50, 100 μg / mL) were incubated with cells for 24 hours, and the absorbance was measured at a wavelength of 570 nm. The relative cell viability (%) of each group was calculated as follows: (Absorbance of blank group - Absorbance of experimental group) / Absorbance of blank group × 100%.

[0057] The results are as follows Figure 7 As shown, the sunscreen nanoparticles exhibited good cell compatibility within a concentration range of 0-100 μg / mL. Experimental results indicate that even after treatment at a higher concentration (100 μg / mL) for 24 hours, cell viability remained at a high level, demonstrating excellent biocompatibility.

[0058] (6) Sunscreen nanoparticles' photoprotective properties The phototoxicity of the sunscreen nanoparticles prepared in Example 1 and the generation of intracellular reactive oxygen species were tested to evaluate the photoprotective effect of the sunscreen nanoparticles of the present invention on cells.

[0059] The specific steps were as follows: Cells were seeded into well plates and allowed to adhere to the plates. A quartz plate was then placed over the wells, and a Pickering emulsion containing 10% sunscreen nanoparticles was evenly coated onto the surface of the quartz plate. Subsequently, a full-wavelength ultraviolet lamp (UVA+UVB, 8 W) was placed 10 cm above the bottom of the wells for 15 minutes of irradiation. Cell viability was detected using the MTT assay, and the level of intracellular reactive oxygen species (ROS) production was visualized using a reagent kit combined with fluorescence microscopy. The fluorescence images were then semi-quantitatively analyzed using ImageJ software to evaluate the photoprotective properties of the emulsion. Cells not exposed to UV radiation served as the negative control group, while cells exposed to UV radiation but not protected on the quartz plate served as the positive control group.

[0060] After ultraviolet light irradiation, the results of cytotoxic activity were as follows: Figure 8 As shown, the cell viability protected by Pickering emulsion containing sunscreen nanoparticles reached 96%, with no significant difference compared to the negative control group and a significant difference compared to the positive control group. This indicates that Pickering emulsion containing nanoparticles can act as a physical barrier, almost completely blocking cell damage caused by ultraviolet radiation, and restoring cell survival rate to normal levels.

[0061] Intracellular reactive oxygen species levels after ultraviolet light irradiation, such as Figure 9 As shown, the average intracellular fluorescence intensity of cells protected by Pickering emulsion containing sunscreen nanoparticles decreased to 90, which was not significantly different from the negative control group, but significantly different from the positive control group. This indicates that the Pickering emulsion, as a physical barrier, can almost completely block the production of ultraviolet-induced intracellular reactive oxygen species.

[0062] (7) Animal experiments on the photoprotective properties of sunscreen nanoparticles The sunscreen nanoparticles prepared in Example 1 were applied to animals to evaluate their photoprotective ability after exposure to ultraviolet radiation.

[0063] Specifically, different formulations of nanoparticle sunscreens were topically applied to the dorsal skin of mice from the Cancer Research Institute for three days. Histological examination was then performed using hematoxylin-eosin (H&E) and Masson staining. Simultaneously, TUNEL and TNF-α staining using immunofluorescence techniques were employed to assess the direct effects of UV irradiation on mouse skin DNA damage and inflammation, as well as the indirect effects resulting from activation by the nanoparticle sunscreens. The experiment included a negative control group of mice not exposed to UV radiation and a positive control group of mice exposed to UV radiation but without the protection of the sunscreen nanoparticles.

[0064] TUNEL staining assesses the degree of apoptosis induced by ultraviolet (UV) exposure, while TNF-α staining uses immunofluorescence to label tumor necrosis factor-α in mouse skin to assess the skin inflammatory response induced by UV exposure.

[0065] The results of hematoxylin-eosin (H&E) and Masson staining are as follows: Figure 10 As shown in (a), the results show that the experimental group protected by the nanoparticle emulsion has a significant inhibitory effect on the epidermal thickening caused by ultraviolet radiation. Its epidermal thickness is not significantly different from that of the negative control group, while the positive control group shows obvious epidermal thickening (shown by the short black line) and inflammatory cell infiltration (arrows indicate keratin production), indicating that the sunscreen particle emulsion has a high anti-ultraviolet damage effect.

[0066] TUNEL staining and TNF-α staining, as shown Figure 10 As shown in (b), TUNEL staining revealed a large number of TUNEL-positive cells in the positive control group after UV irradiation (yellow boxes indicate apoptosis), while the experimental group showed lower apoptosis levels, with no significant difference compared to the negative control group, demonstrating excellent biocompatibility and skin safety characteristics, indicating that the sunscreen nanoparticles have a high UV protection effect. The positive control group showed a significant increase in TNF-α levels after UV irradiation, indicating that UV exposure can induce an inflammatory response. The experimental group showed a significant decrease in TNF-α expression in the skin, with levels comparable to the negative control group, indicating that the nanoparticle emulsion exhibits excellent efficacy in inhibiting UV-induced DNA damage and inflammatory responses.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multifunctional sunscreen nanoparticle that blocks ultraviolet rays across the entire spectrum, characterized in that, The nanoparticles use a covalent cross-linked network formed by polyphenolic compounds and amino acids and their peptide derivatives through the Mannich reaction as a framework, and antibacterial nanoparticles are composited in the framework.

2. The nanoparticles according to claim 1, characterized in that, The amino acids and their peptide derivatives are one or more of the following: natural proteins, recombinant proteins, polypeptides, oligopeptides, or amino acids.

3. The nanoparticles according to claim 1, characterized in that, The polyphenolic compounds are any one or more of tannic acid, gallic acid, chlorogenic acid, quercetin, hesperidin, anthocyanins, epigallocatechin gallate, and pentagalloglucoside.

4. The nanoparticles according to claim 1, characterized in that, The antibacterial nanoparticles are at least one of metal antibacterial nanoparticles, metal oxide antibacterial nanoparticles, and carbon-based antibacterial nanoparticles.

5. The nanoparticles according to claim 4, characterized in that, The metal antibacterial nanoparticles are Ag, Au, or Cu; the metal oxide antibacterial nanoparticles are ZnO, TiO2, or CuO nanoparticles; and the carbon-based antibacterial nanoparticles are graphene quantum dots, carbon dots, or fullerenes.

6. The nanoparticles according to any one of claims 1-5, characterized in that, The mass ratio of the polyphenolic compound to the amino acid and its peptide derivative is (0.1-8):1, and the amount of antibacterial nanoparticles added is 0.1%-200% of the polyphenolic compound.

7. The method for preparing multifunctional sunscreen nanoparticles with full-band ultraviolet shielding as described in any one of claims 1-6, characterized in that: When the antibacterial nanoparticles are generated by reduction of metal precursor salt, method A is used; when the antibacterial nanoparticles are pre-prepared antibacterial nanoparticles, method A or method B is used. Method A includes the following steps: S1. Activate polyphenolic compounds by reacting them with formaldehyde through hydroxymethylation; S2. Under weakly alkaline conditions, the activated polyphenols are reacted with amino acids and their peptide derivatives by the Mannich reaction to form a methylene bridge cross-linking network; S3. Add metal precursor salt, utilize the reducing properties of polyphenols to generate antibacterial nanoparticles in situ and anchor them in the cross-linked network, and then purify them; Method B includes the following steps: S1. Activate polyphenolic compounds by reacting them with formaldehyde through hydroxymethylation; S2. Under weakly alkaline conditions, amino acids and their peptide derivatives, along with pre-prepared antibacterial nanoparticles, are simultaneously added to an activated polyphenol solution to perform the Mannich reaction and physical encapsulation of the antibacterial nanoparticles, followed by purification.

8. The preparation method according to claim 7, characterized in that, The pH value of the Mannich reaction is 8.0-10.0, the reaction temperature is 20-80℃, and the reaction time is 1-8 hours.

9. The preparation method according to claim 7, characterized in that, The metal precursor salt in Method A is one or more of silver nitrate, chloroauric acid, zinc acetate, or copper sulfate.

10. The application of the multifunctional sunscreen nanoparticles with full-band ultraviolet shielding as described in any one of claims 1-6 in the preparation of sunscreen products.

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