Pickering emulsion with stable protein-polyphenol coating modified particles as well as preparation method and application of Pickering emulsion

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a Pickering emulsion with stable protein-polyphenol coating modified particles and a preparation method and application thereof.The preparation method comprises the following steps that protein and polyphenol are used for modifying solid particles, and the protein-polyphenol coating modified particles are obtained; dispersing the protein-polyphenol coating modified particles in water to obtain a dispersion liquid of the protein-polyphenol coating modified particles; and mixing the dispersion liquid with an oil phase, and carrying out high-speed homogenization or ultrasonic emulsification to obtain the Pickering emulsion. By adopting the preparation method to effectively modify the solid particles, not only can the inherent properties of the solid particles and the structure and function of protein be maintained, but also the Pickering emulsion prepared from the modified particles is uniform in droplet size and has excellent storage stability, and the service life of the Pickering emulsion is prolonged. The antioxidant and anti-inflammatory characteristics of polyphenol and the catalysis functions of protein are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a protein-polyphenol coated modified particle-stabilized Pickering emulsion, its preparation method, and its application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Pickering emulsions are widely used as delivery systems in biomedicine and other fields. Solid particles adsorb at the oil-water interface, acting as a physical barrier to protect emulsion droplets from aggregation. These solid particles not only function as emulsifiers but also provide functionality, such as efficient loading of mesoporous silica nanoparticles, the photothermal and magnetic properties of iron oxide nanoparticles, and the hemostatic and antidiarrheal functions of halloysite clay nanotubes. However, due to the high hydrophilicity or high hydrophobicity of these solid particles, they cannot be used directly as emulsifiers and require wetting property regulation.

[0004] Currently, the strategies for regulating particle wettability usually involve surface chemical modification or adsorption of surfactants. These strategies require particles to have specific reactive groups or surfactants to have specific properties, which lack versatility and may lead to health and environmental safety issues, thus limiting the application of Pickering emulsions in the biopharmaceutical field.

[0005] Natural polyphenols are widely sourced natural compounds composed of two or more phenolic structural units, possessing excellent antioxidant, anti-inflammatory, antibacterial, and immunomodulatory biological activities. Polyphenols can mediate abundant covalent and non-covalent interactions, exhibiting strong affinity for a variety of surfaces. Furthermore, coating strategies using polyphenols as assembly units are simple, versatile, and suitable for surface modification of various materials. However, the hydrophilic nature of these assembly units limits the application of polyphenol-based coating strategies in Pickering emulsions. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a protein-polyphenol coated modified particle-stabilized Pickering emulsion, its preparation method, and its applications. This preparation method effectively modifies solid particles, preserving their inherent properties (such as mesoporous silica loading capacity and the hemostatic and antidiarrheal effects of halloysite clay nanotubes), the structure and function of proteins (catalysis, cell targeting, fluorescence imaging, etc.), and the antioxidant and anti-inflammatory properties of polyphenols. The modified particle-stabilized Pickering emulsion is safe and non-toxic, exhibits excellent stability, and possesses the functionality of both polyphenols and proteins, making it applicable to fields such as biomedicine.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a method for preparing a protein-polyphenol coated modified particle-stabilized Pickering emulsion, comprising the following steps: Protein and polyphenols are used to modify solid particles to obtain protein-polyphenol coated modified particles. The solid particles are selected from at least one of polystyrene microspheres, mesoporous silica nanoparticles, silica nanoparticles, halloysite clay nanotubes, attapulgite, white calcium phosphogypsum, or hydroxyapatite. The mass ratio of protein, polyphenols and solid particles is 1:1-5:1-5. Protein-polyphenol coated modified particles were dispersed in water to obtain a dispersion of protein-polyphenol coated modified particles. The dispersion is mixed with the oil phase and homogenized at high speed or ultrasonically emulsified to obtain a Pickering emulsion. The concentration of the protein-polyphenol coating modified particles in the Pickering emulsion is 0.1-15 mg / mL.

[0008] Protein-polyphenol coated modified particles use solid particles (such as mesoporous silica, halloysite clay nanotubes, etc.) as a substrate, forming a coating on their surface. This allows them to retain the inherent properties of the solid particles (such as the loading capacity of mesoporous silica and the hemostatic and antidiarrheal effects of halloysite). In contrast, protein-polyphenol composite particles do not have a solid particle substrate and cannot obtain these additional functions. The coating modification reaction conditions are mild (20-40℃), do not require specific reactive groups, and can maintain the structural functions of proteins (catalysis, cell targeting, etc.) and the antioxidant and anti-inflammatory properties of polyphenols. The direct composite process of composite particles may damage the structure of proteins or polyphenols, leading to a decrease in activity.

[0009] Coating modification can effectively regulate particle wettability (e.g., the contact angle of HNTs@BSA-EGCG is increased to 60.6±2.2°), making the particles more easily adsorbed at the oil-water interface, and the prepared emulsion has excellent storage stability; however, the wettability of protein-polyphenol composite particles is difficult to regulate, and the emulsion is prone to demulsification.

[0010] Coating modification is not limited by the material or shape of solid particles and is applicable to various particles such as polystyrene microspheres and silica; the preparation of composite particles is limited by the compounding conditions of proteins and polyphenols, and the range of applicable materials is narrow. In addition, coating-modified particles combine the functions of solid particles, proteins, and polyphenols (such as catalysis, antioxidation, and targeting), and emulsions can simultaneously possess multiple biological activities (such as anti-inflammatory, antibacterial, and cell protection); composite particles only contain the functions of proteins and polyphenols, and their functionality is singular.

[0011] When the active ingredient is a drug and the prepared Pickering emulsion is a drug, when the drug is taken orally, the protein-polyphenol coating modified particles in the Pickering emulsion adhere to the surface of the emulsion droplets. This not only stabilizes the emulsion, but the polyphenols in the emulsion also have antioxidant properties, which can protect the active substances in the droplets and prevent the active ingredient from becoming ineffective prematurely, thus ensuring its therapeutic effect.

[0012] In some embodiments, a mixed aqueous solution of protein and polyphenol is mixed with a dispersion of solid particles to be modified, and the mixture is stirred at 20-40 °C for 1-20 h to allow the protein and polyphenol to adhere to the surface of the solid particles. The solid and liquid are then separated to obtain protein-polyphenol coated modified particles. Alternatively, the protein is first mixed with a dispersion of the solid particles to be modified and reacted for 4-16 h to obtain solid particles with a protein coating; then the solid particles with the protein coating are reacted with a polyphenol solution for 1-6 h to obtain protein-polyphenol modified particles. The temperature of the above reactions is 20-40 ℃.

[0013] In some embodiments, the protein is selected from bovine serum albumin, sodium caseinate, zein, horseradish peroxidase, glucose oxidase, β-lactoglobulin, and ovalbumin, preferably bovine serum albumin.

[0014] In some embodiments, the polyphenol is selected from at least one of epigallocatechin gallate, tannic acid, proanthocyanidins, ellagitannins, catechins, caffeic acid, and gallic acid; preferably epigallocatechin gallate.

[0015] In some embodiments, the solid particles are halloysite clay nanotubes.

[0016] In some embodiments, the reaction temperature during the modification of solid particles with proteins and polyphenols is 20-40 °C, preferably 36-38 °C, specifically 37 °C.

[0017] In some embodiments, solid-liquid separation is performed after the reaction is complete. The centrifugation speed is 7000-9000 rpm and the centrifugation time is 4-10 min.

[0018] Preferably, the rotation speed for solid-liquid separation is 7500-8500 rpm.

[0019] Preferably, after solid-liquid separation, the method further includes washing the prepared protein-polyphenol modified particles with deionized water.

[0020] In some embodiments, the oil phase is selected from olive oil, medium-chain triglycerides, ethyl acetate, toluene, n-heptane, corn oil, squalene, and isopropyl myristate, preferably olive oil.

[0021] In some embodiments, the Pickering emulsion further includes a pharmaceutically active ingredient, which is a mixture of a hydrophobic active ingredient and an amphiphilic active ingredient.

[0022] Preferably, the hydrophobic active ingredient is castor oil or carvacrol; the amphiphilic active ingredient is hydroxytyrosol, proanthocyanidins, tannic acid, chlorogenic acid or caffeic acid.

[0023] Further preferred, the hydrophobic active ingredient is carvacrol; the amphiphilic active ingredient is hydroxytyrosol.

[0024] Carvacrol provides antibacterial and anti-inflammatory effects, inhibiting the growth of harmful bacteria; hydroxytyrosol exerts strong antioxidant and anti-inflammatory effects, scavenging free radicals and regulating the expression of inflammatory factors; olive oil itself contains auxiliary active ingredients such as olive polyphenols, which synergistically enhance antioxidant capacity with hydroxytyrosol.

[0025] Experiments revealed that hydroxytyrosol has very low solubility in olive oil, making it difficult to fully exert its anti-inflammatory and antioxidant effects. Dissolving carvacrol and hydroxytyrosol together before dissolving them in olive oil allows for the dissolution of more hydroxytyrosol. Furthermore, the synergistic effect of hydroxytyrosol and carvacrol provides a better anti-inflammatory effect and can be more effective in treating colitis.

[0026] The oil phase environment of olive oil and the structure of Pickering emulsion work together to protect carvacrol and hydroxytyrosol from external factors (such as oxidation and light), thus extending the shelf life of the active ingredients.

[0027] During the treatment of colitis, the protein-polyphenol coating modified particles in Pickering emulsion adhere to the surface of the emulsion droplets, which not only stabilize the emulsion, but also provide antioxidant protection for the active substances in the droplets, thus ensuring the therapeutic effect.

[0028] Furthermore, experimental verification has shown that the prepared Pickering emulsion preferentially adheres to the site of inflammatory colitis, which helps to enhance the treatment effect.

[0029] More preferably, the mass ratio of hydroxytyrosol, carvacrol and olive oil is 1:15-25:500-600, and more preferably 1:19-20:520-540.

[0030] In some embodiments, the emulsification method is ultrasonic treatment, with an ultrasonic power of 50-400 W and an ultrasonic time of 1-30 min.

[0031] Preferably, the ultrasonic treatment power is 100-200 W and the ultrasonic time is 1-3 min.

[0032] In some embodiments, the high-speed homogenization process is performed at a rotation speed of 5000-15000 rpm for a time of 0.5-5 min.

[0033] Preferably, the high-speed homogenization process is performed at a rotation speed of 8000-12000 W for 1-2 minutes.

[0034] In some embodiments, the concentration of the protein-polyphenol coated modified particles in the Pickering emulsion is 4-8 mg / mL.

[0035] Preferably, the amount of the oil phase is 5%-85% of the total volume of oil and water in the Pickering emulsion, and more preferably 20%-60%.

[0036] Secondly, a Pickering emulsion stabilized with the aforementioned protein-polyphenol coating modified particles is provided.

[0037] Thirdly, the present invention provides the use of the Pickering emulsion in the preparation of intestinal inflammation drugs.

[0038] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: (1) This invention combines polyphenol-based coating with Pickering emulsion system, and uses the amphiphilic or hydrophobic properties of proteins to improve the hydrophobicity of the coating on the particle surface, thus solving the problem that the modified particles are not suitable for Pickering emulsion system due to the hydrophilic properties of the assembly units of polyphenol-based coating.

[0039] (2) The reaction conditions are mild, not limited by the material or shape, and do not require specific reaction sites, making it widely applicable. Moreover, it can maintain the inherent properties of the material (such as the loading capacity of mesoporous silica, the hemostatic and antidiarrheal effects of halloysite clay nanotubes), the structure and function of proteins (catalysis, cell targeting, fluorescence imaging, etc.), and the antioxidant and anti-inflammatory properties of polyphenols.

[0040] (3) The prepared emulsion is safe and non-toxic, has excellent stability, and has broad application prospects. Attached Figure Description

[0041] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0042] Figure 1 The contact angle of HNTs@BSA-EGCG obtained in Example 1 of this invention; Figure 2 The contact angle of the MS@CS-TA nanoparticles in Example 2 of this invention; Figure 3 The antioxidant results of HNTs@BSA-EGCG prepared in Example 1 of this invention; Figure 4 The images show the UV absorption spectra of the MS@CS-TA nanoparticles in Example 2 and the MS@TS-CS-TA nanoparticles in Example 4 of this invention. Figure 5These are fluorescence images of MS@CS-TA particles prepared in Example 2 and MS@TS-CS-TA particles obtained in Example 4 of the present invention for scavenging intracellular reactive oxygen species. Figure 6 The results show the experimental effects of MS@CS-TA particles prepared in Example 2 and MS@TS-CS-TA particles obtained in Example 4 on the ultraviolet protection of cells under ultraviolet irradiation. Figure 7 Images of the Pickering emulsion and the emulsion after centrifugation stabilization of the homogenized emulsion samples in Comparative Examples 2, 3, and 5 of this invention are shown below. Specifically, A represents the left image of the Pickering emulsion and the right image of the emulsion after centrifugation stabilization of the homogenized emulsion sample in Comparative Example 2; B represents the left image of the Pickering emulsion and the right image of the emulsion after centrifugation stabilization of the homogenized emulsion sample in Comparative Example 3; and C represents the left image of the Pickering emulsion and the right image of the emulsion after centrifugation stabilization of the homogenized emulsion sample in Example 5. Figure 8 The images shown are photographs and microscopic images of the samples after ultrasonic emulsification in Comparative Example 4 and Example 9 of the present invention. Among them, A is a photograph of the sample after ultrasonic emulsification in Comparative Example 4 after being placed at room temperature for 1 day; B is a photograph and microscopic image of the sample after ultrasonic emulsification in Example 9 after being placed for 1 day; and C is a photograph and microscopic image of the sample after ultrasonic emulsification in Example 9 after being placed for 60 days. Figure 9 The images show catalytic test results of MS@HRP-TA nanoparticles in Example 3 and Pickering emulsion after ultrasonic emulsification in Example 11 of this invention. In the image, A is a schematic diagram and B is a comparison diagram of catalytic performance. Figure 10 The results of cell protection experiments on the ultrasonically emulsified Pickering emulsions of Examples 8 and 10 of the present invention under ultraviolet irradiation; Figure 11 The images show the adhesion of the Pickering emulsion prepared in Examples 5-7 of this invention in the intestines of healthy mice and mice with colitis, where A is a fluorescence image and B is the quantitative statistical analysis of the corresponding fluorescence intensity. Figure 12 This is a comparative diagram showing the protective effect of Pickering emulsions prepared in Examples 5-7 of this invention on cell viability under oxidative stress conditions; Figure 13 The anti-inflammatory effect of the Pickering emulsion prepared in Examples 5-7 of this invention is shown in the RT-PCR detection comparison of pro-inflammatory cytokines (A: TNF-α, B: IL-1β and C: IL-6) and anti-inflammatory cytokines (D: IL-10). Figure 14 Statistical analysis of flow cytometry results of the proportion of M1(A) and M2(B) macrophages to assess the immunomodulatory effect of the Pickering emulsions prepared in Examples 5-7 of this invention; Figure 15 The therapeutic effect of the PCHE emulsion prepared in Example 7 of this invention on inflammatory colitis is shown in the following figures: A represents the animal experimental procedure; B represents the body weight of C57BL / 6 mice during treatment; C represents the change in the disease activity index (DAI); D represents colon images of each group; and E represents the corresponding colon length statistics. Detailed Implementation

[0043] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0044] The following detailed description of embodiments is intended to provide further illustration of the present invention, but is not intended to limit the invention.

[0045] Example 1 A method for preparing protein-polyphenol coated modified particles includes the following steps: 50 mg of halloysite clay nanotubes were added to 20 mL of ultrapure water, followed by 5 mL of bovine serum albumin (BSA) aqueous solution (10 mg / mL). The mixture was sonicated for 30 s to ensure homogeneity. The mixture was then reacted in a shaker for 12 h at 37 °C and 100 rpm. After the reaction, the mixture was centrifuged at 8000 rpm for 5 min and washed three times with deionized water to obtain BSA-modified halloysite clay nanotubes. 20 mL of ultrapure water and 0.5 mL of epigallocatechin gallate aqueous solution (55 mg / mL) were then added to the centrifuged precipitate. The mixture was then reacted in a shaker for 4 h at 37 °C and 100 rpm. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 5 min and washed three times with deionized water to obtain halloysite clay nanotubes (HNTs@BSA-EGCG) modified with bovine serum albumin-epigallocatechin gallate coating.

[0046] Example 2 A method for preparing protein-polyphenol coated modified particles includes the following steps: 50 mg of mesoporous silica nanoparticles were added to 20 mL of ultrapure water, followed by 5 mL of sodium caseinate aqueous solution (10 mg / mL). The mixture was sonicated for 30 s to ensure homogeneity. The mixture was then reacted in a shaker for 12 h at 37 °C and 100 rpm. After the reaction, the mixture was centrifuged at 8000 rpm for 5 min and washed three times with deionized water to obtain sodium caseinate-modified mesoporous silica (MS@CS). Next, 20 mL of ultrapure water and 0.5 mL of tannic acid aqueous solution (40 mg / mL) were added to the centrifuged precipitate. The mixture was then reacted in a shaker for 4 h at 37 °C and 100 rpm. After the reaction, the mixture was centrifuged at 8000 rpm for 5 min and washed three times with deionized water to obtain sodium caseinate-tannic acid-coated modified mesoporous silica nanoparticles (MS@CS-TA).

[0047] Example 3 A method for preparing protein-polyphenol coated modified particles differs from Example 2 in that the proteins are replaced with bovine serum albumin, zein, horseradish peroxidase, and glucose oxidase, respectively, at concentrations of 10 mg / mL, 10 mg / mL, 5 mg / mL, and 5 mg / mL. The zein is dissolved in a 75% ethanol solution. Other steps and conditions are the same as in Example 2, yielding bovine serum albumin-tannic acid coated modified nanoparticles, zein-tannic acid coated modified mesoporous silica nanoparticles, horseradish peroxidase-tannic acid coated modified mesoporous silica nanoparticles (MS@HRP-TA), and glucose oxidase-tannic acid coated modified mesoporous silica nanoparticles.

[0048] Example 4 A method for preparing protein-polyphenol coated modified particles includes the following steps: 50 mg of MS nanoparticles were added to 20 mL of ultrapure water. 6 mg of the UV filter bis-ethylhexyloxyphenol methoxyphenyl triazine (TS) was added to 3 mL of dimethyl sulfoxide, and then added to the MS dispersion. The mixture was stirred for 30 min, centrifuged at 8000 rpm for 5 min, and washed twice with deionized water to obtain the UV filter-loaded MS nanoparticles (MS@TS). The precipitate was resuspended in 20 mL of ultrapure water, and then 5 mL of CS aqueous solution (10 mg / mL) was added. The mixture was sonicated for 30 s to ensure homogeneity. The mixture was then reacted on a shaker for 12 h at 37 °C and 100 rpm. After the reaction, the mixture was centrifuged at 8000 rpm for 5 min and washed three times with deionized water. Finally, 20 mL of ultrapure water and 0.5 mL of TA aqueous solution (40 mg / mL) were added to the centrifuged precipitate. The mixture was then placed in a shaker and reacted for another 4 hours at a temperature of 37 °C and a rotation speed of 100 rpm. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 5 minutes and washed three times with deionized water to obtain mesoporous silica nanoparticles (MS@TS-CS-TA) modified with sodium caseinate-tannic acid coating and loaded with UV filter.

[0049] Example 5 A method for preparing a Pickering emulsion includes the following steps: Disperse 80 mg of HNTs@BSA-EGCG obtained in Example 1 into 4 mL of ultrapure water, add it to a 20 mL glass bottle, then add 6 mL of olive oil, and homogenize at 10000 rpm for 2 min to obtain Pickering emulsion (PE).

[0050] Example 6 A method for preparing a Pickering emulsion includes the following steps: Take 80 mg of HNTs@BSA-EGCG prepared in Example 1, disperse it in 4 mL of ultrapure water, and add it to a 20 mL glass bottle to obtain HNTs@BSA-EGCG dispersion; Add 200 μL of carvacrol (CAR) to 5.8 mL of olive oil and mix well to obtain the oil phase; The oil phase was added to the HNTs@BSA-EGCG dispersion and homogenized at high speed for 2 min at 10,000 rpm to obtain the Pickering emulsion (PCE) loaded with the active ingredient CAR.

[0051] Example 7 A method for preparing a Pickering emulsion includes the following steps: Take 80 mg of HNTs@BSA-EGCG prepared in Example 1, disperse it in 4 mL of ultrapure water, and add it to a 20 mL glass bottle to obtain HNTs@BSA-EGCG dispersion; Dissolve 10 mg of hydroxytyrosol (HT) in 200 μL of carvacrol, then add it to 5.8 mL of olive oil and mix well to obtain the oil phase; The oil phase was added to the HNTs@BSA-EGCG dispersion and homogenized at high speed for 2 min at 10,000 rpm to obtain the Pickering emulsion (PCHE) loaded with the active ingredients CAR and HT.

[0052] Example 8 A method for preparing a Pickering emulsion includes the following steps: Disperse 18 mg of MS@CS-TA obtained in Example 2 into 1.5 mL of water, add it to a 5 mL glass bottle, then add 1.5 mL of medium-chain triglycerides, and sonicate at 200 W for 2 min to obtain Pickering emulsion (MS@CS-TA E).

[0053] Example 9 A method for preparing a Pickering emulsion includes the following steps: 12 mg of MS@CS-TA prepared in Example 2 was dispersed in 0.6 mL of water, added to a 5 mL glass bottle, and then 2.4 mL of medium-chain triglycerides were added. The mixture was then sonicated at 200 W for 2 min to obtain Pickering emulsion.

[0054] Example 10 A method for preparing a Pickering emulsion includes the following steps: Disperse 18 mg of MS@TS-CS-TA obtained in Example 4 into 1.5 mL of water, add it to a 5 mL glass bottle, then add 1.5 mL of medium-chain triglycerides, and sonicate at 200 W for 2 min to obtain Pickering emulsion (MS@TS-CS-TA E).

[0055] Example 11 A method for preparing Pickering emulsion, which differs from Example 9 in that the particles are MS@HRP-TA obtained in Example 3, while the remaining steps are the same as in Example 9.

[0056] Comparative Example 1 To compare the effect of BSA-EGCG on particle properties, Comparative Example 1 was set up. Comparative Example 1 consisted of BSA-modified halloysite clay nanotubes (HNTs@BSA). The difference between Comparative Example 1 and Example 1 was that after obtaining HNTs@BSA, no further EGCG aqueous solution was added. Other steps and conditions were the same as in Example 1.

[0057] Comparative Example 2 To compare the effect of the BSA-EGCG coating on the emulsification properties of HNTs, Comparative Example 2 was set up. The difference between Comparative Example 2 and Example 5 is that the particles in Comparative Example 2 are HNTs whose surface has not been modified with the BSA-EGCG coating, while the other steps and conditions are the same as in Example 5.

[0058] Comparative Example 3 To compare the effect of the BSA-EGCG coating on the emulsifying properties of HNTs, Comparative Example 3 was set up. The difference between Comparative Example 3 and Example 5 is that the particles in Comparative Example 3 are HNTs@BSA, while the other steps and conditions are the same as in Example 5.

[0059] Comparative Example 4 To compare the effect of CS-TA coating on the emulsification performance of particles, Comparative Example 4 was set up. The difference between Comparative Example 4 and Example 9 is that the particles are MS nanoparticles without CS-TA coating modification on the surface, while the other steps and conditions are the same as in Example 9.

[0060] Performance characterization of protein-polyphenol coated modified particles: Contact angle tests were performed on unmodified HNTs, HNTs@BSA-EGCG from Example 1, and HNTs@BSA from Comparative Example 1. Figure 1 As can be seen, the contact angles of HNTs, HNTs@BSA, and HNTs@BSA-EGCG are 14.5 ± 0.46°, 37.5 ± 1.6°, and 60.6 ± 2.2°, respectively. This indicates that the hydrophobicity of HNTs is significantly increased after the introduction of the BSA-EGCG coating. This may be due to the interaction between the hydroxyl groups of EGCG and the hydroxyl or amino groups of BSA to form hydrogen bonds, which reduces the exposure of water-soluble groups of BSA. It can also affect the conformation of BSA and expose more hydrophobic groups.

[0061] Contact angle tests were performed on unmodified MS and MS@CS-TA from Example 2. The results showed that the CS-TA coating also improved the hydrophobicity of MS, such as... Figure 2 As shown.

[0062] Performance characterization of protein-polyphenol coated modified particles: Antioxidant performance tests were performed on HNTs@BSA-EGCG in Example 1 and HNTs@BSA in Comparative Example 3: Particles with a concentration of 2 mg / mL were incubated with 0.2 mM 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) ethanol solution in the dark for 30 min. After incubation, the mixture was centrifuged at 8000 rpm for 5 min to obtain the supernatant. The UV absorbance of the supernatant at 517 nm was measured using a UV-Vis spectrophotometer. Figure 3 As shown, HNTs@BSA-EGCG has an antioxidant capacity of up to 92.5%, while HNTs@BSA has an antioxidant capacity of only 6.6%, which is not significantly different from the unmodified HNTs, indicating that the antioxidant capacity is provided by the EGCG in the coating.

[0063] The UV protection performance of MS@CS-TA in Example 2 and MS@TA-CS-TA in Example 4 was tested respectively. The UV absorption of dispersions of the same concentration in the 200-400 nm range was measured using a UV spectrophotometer. Figure 4 As shown, the UV filter bis-ethylhexyloxyphenol methoxyphenyl triazine (TS) has UV absorption in the range of 290~370 nm, while MS@TA-CS-TA nanoparticles also have a wide UV absorption range.

[0064] Cellular-level antioxidant tests were performed on the MS@CS-TA nanoparticles obtained in Example 2 and the MS@TS-CS-TA nanoparticles obtained in Example 4 of this invention: mouse embryonic fibroblasts were subjected to 1.5 × 10⁻⁶ saturated plasma. 5 Cells were seeded at a density of / wells into 24-well plates. After 12 h of culture, cells were divided into six groups: non-UV irradiation group, UV irradiation group, UV filter bis-ethylhexyloxyphenol methoxyphenyl triazine (TS) plus UV irradiation group, MS plus UV irradiation group, MS@CS-TA plus UV irradiation group, and MS@TS-CS-TA plus UV irradiation group, with a particle concentration of 500 µg / mL, and cultured for another 4 h. Subsequently, cells in the non-UV irradiation group were covered with a black opaque film, while cells in the UV irradiation group were exposed to UV light (UVB, 8 W, 6 min). After 2 h of further culture, cells were washed three times with PBS, and then stained with 2',7'-dichlorofluorescein diacetate (1 μL / mL, 200 μL) to detect reactive oxygen species in the cells. The stained cells were imaged using an inverted fluorescence microscope. The results are shown below. Figure 5 As shown, under ultraviolet (UV) irradiation, the fluorescence intensity of the UV-irradiated group and the MS plus UV irradiation group were stronger than that of the non-UV irradiated group, indicating that the reactive oxygen species (ROS) levels increased in both groups under UV irradiation. However, the fluorescence intensity of the MS@CS-TA plus UV irradiation group and the MS@TS-CS-TA plus UV irradiation group were close to that of the non-UV irradiated group, indicating lower ROS levels. This is because the TA in the CS-TA coating can act as an effective antioxidant to scavenge ROS and prevent oxidative stress damage caused by UV irradiation.

[0065] UV protection cell experiments were conducted on MS, MS@CS, MS@CS-TA obtained in Example 2, the UV filter bis-ethylhexyloxyphenol methoxyphenyl triazine (TS), and the MS@TS-CS-TA nanoparticles obtained in Example 4. Mouse embryonic fibroblasts were used in a 1×10⁻⁶ saturated atmosphere. 4 Cells were seeded at a density of / wells into 96-well plates. After 12 h of culture, cells were divided into seven groups: no UV irradiation, UV irradiation, MS plus UV irradiation, MS@CS nanoparticles plus UV irradiation, MS@CS-TA nanoparticles plus UV irradiation, UV filter plus UV irradiation, and MS@TS-CS-TA nanoparticles plus UV irradiation. The concentration of added particles was 500 µg / mL, and the cells were cultured for another 4 h. Subsequently, cells in the no-UV irradiation group were covered with a black opaque film, while cells in the UV irradiation group were exposed to UV light (UVB, 8 W, 6 min). Cells were cultured for another 24 h, and cell viability was assessed using the MTT assay. Figure 6 As shown, the cell groups without TA particles in the coating and the UV filter group showed no significant difference in cell survival rate under UV irradiation compared to the UV-irradiated group, but were significantly lower than the non-UV-irradiated group. However, MS@CS-TA nanoparticles significantly improved cell survival rate, showing no significant difference compared to the non-UV-irradiated group. In particular, MS@TS-CS-TA nanoparticles indicated that TA can scavenge reactive oxygen species, reduce oxidative stress damage caused by UV irradiation, and synergistically improve cell survival rate with the UV protection effect of UV filters.

[0066] Performance characterization of Pickering emulsion: Stability tests were performed on the samples from Comparative Examples 2, 3, and 5 of this invention after high-speed homogenization and emulsification: The Pickering emulsions were photographed and recorded. 3 mL of the emulsion was then transferred from a 1 mL pipette to a 10 mL centrifuge tube and centrifuged at 8000 rpm for 10 min. The results are as follows: Figure 7 As shown in Figures A and B, Comparative Examples 1 and 4 exhibit distinct oil layers, which demulsify after centrifugation, while... Figure 7 As shown in Figure C, no obvious oil layer was observed in the homogenized emulsified sample of Example 5, and it exhibited centrifugal stability. The results indicate that the BSA-EGCG coating improved the emulsifying ability of HNTs, and the prepared Pickering emulsion showed good stability.

[0067] Stability tests were conducted on the ultrasonically emulsified samples (placed at room temperature) of Comparative Example 4 and Example 9 of this invention: The samples were photographed and recorded. Sample 10 was pipetted onto a glass slide, and its morphology was observed and photographed under an optical microscope. The results are as follows: Figure 8As shown in Figure A, a distinct oil layer appeared in Comparative Example 4, indicating that the unmodified MS nanoparticles used did not have emulsifying ability; while a distinct emulsified layer was observed in Example 9, as shown in Figure A. Figure 8 As shown in left image B, the microscopic image reveals that the Pickering emulsion has a compact microstructure, such as... Figure 8 As shown in B (right). After the emulsion obtained in Example 9 was left at room temperature for 60 days, the height of the emulsion layer ( Figure 8 (C-left) and micromorphology ( Figure 8 (C on the right) showed no significant change. The results indicate that the CS-TA coating improved the emulsifying ability of MS nanoparticles, and the prepared Pickering emulsion exhibited good stability.

[0068] Catalytic tests were performed on horseradish peroxidase (HRP), MS@HRP-TA nanoparticles from Example 3 of this invention, and the Pickering emulsion prepared in Example 11. In the presence of hydrogen peroxide (H2O2), HRP was able to oxidize fluorescent red dye (AR) to the fluorescent red product rose red (RS). Hydrogen peroxide (40 μL; 20 mM) and AR (2 μL; 1 mg / mL) were added to phosphate-buffered saline (PBS; 10 mM, pH 7.4), followed by the addition of HRP, MS@HRP-TA nanoparticles from Example 3, and the Pickering emulsion prepared in Example 11, respectively. The results were immediately measured using a UV spectrophotometer. The results are as follows: Figure 9 As shown in Figures A and B, MS@HRP-TA nanoparticles and stable emulsions still retain the catalytic activity of HRP.

[0069] UV protection tests were performed on the emulsion (MS@CS-TA E) prepared in Example 8 and the Pickering emulsion (MS@TS-CS-TA E) prepared in Example 10 of this invention: mouse embryonic fibroblasts were subjected to a UV protection test at 1×10⁻⁶. 4 Cells were seeded at a density of / wells in 96-well plates. After 12 h of culture, the plates were covered with quartz plates and divided into four groups: no UV exposure, UV exposure, MS@CS-TA E with UV exposure, and MS@TS-CS-TA E with UV exposure. Cells in the no UV exposure group were covered with a black opaque film, while cells in the UV exposure group were exposed to UV light (UVB, 8 W, 6 min). MS@CS-TA E and MS@TS-CS-TA E were coated onto quartz plates and then exposed to UV light (UVB, 8 W, 6 min). Cells were then cultured for another 24 h, and cell viability was assessed using the MTT assay. The results are shown below. Figure 10As shown, the cell survival rate of the MS@TS-CS-TA E emulsion protection group was similar to that of the group without UV irradiation, but much higher than that of the cell survival rate exposed to UV light, and also higher than that of the MS@CS-TA E emulsion protection group. This indicates that the UV filter encapsulated in MS plays an important role in UV protection.

[0070] In vivo adhesion experiments of Pickering emulsions prepared in Examples 5, 6, and 7: Each C57BL / 6 mouse in the healthy group and the inflammatory colitis group was administered 100 μL of PCHE (pill-in-the-valley olive oil with added lipophilic fluorescent dye DiD) by gavage. Eight hours later, the mice were dissected without washing, and the colon was imaged using an IVIS fluorescence imaging system. The fluorescence intensity was statistically analyzed. Results are as follows: Figure 11 As shown in Figures A and B, the fluorescence intensity was higher in the inflammatory colitis group, indicating that PCHE preferentially adheres to the site of inflammatory colitis, which is beneficial for enhancing the treatment effect.

[0071] Examples 5-7: Cell protection experiments using Pickering emulsion under oxidative stress: Mouse monocyte / macrophage RAW 264.7 cells were seeded at 8000 cells / well in 96-well plates and cultured for 12 h. Then, 600 μM H2O2 was added and mixed with culture medium containing PE, PCE, and PCHE (1.25 mg / mL) for 24 h. MTT assay was performed, and cell viability was calculated. Figure 12 As shown, cell survival rates increased in both the PCE emulsion group and the PCHE emulsion group, especially in the PCHE group, where the survival rate reached over 90%. This is attributed to the synergistic antioxidant effects of carvacrol and hydroxytyrosol, which effectively scavenged reactive oxygen species and protected cells from oxidative stress damage.

[0072] In vitro anti-inflammatory activity experiments of Pickering emulsions prepared in Examples 5, 6, and 7: RAW264.7 cells were injected at a concentration of 1×10⁻⁶ cells / mL. 5 Macrophages were seeded per well in 24-well plates. After adhesion, inflammation was induced by stimulation with 500 ng / mL lipopolysaccharide (LPS) for 12 h. The plates were then cultured for another 24 h in culture medium, PE, PCE, and PCHE (1.25 mg / mL), respectively. After removing the culture medium, the plates were gently washed with PBS, and total RNA was extracted. Reverse transcription of RNA yielded cDNA, and RT-PCR was performed to detect the expression of inflammation-related genes. Under oxidative stress, macrophages secrete large amounts of pro-inflammatory factors, further exacerbating the inflammation of inflammatory bowel disease. PE, PCE, and PCHE have anti-inflammatory effects, promoting the expression of the anti-inflammatory factor interleukin-10 (IL-10) and reducing the expression of pro-inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6)). Figure 13As shown in Figures A, B, C, and D, the PCHE group exhibited the best anti-inflammatory effect.

[0073] In vitro immunomodulatory experiments of Pickering emulsions prepared in Examples 5, 6, and 7: First, RAW264.7 cells were cultured at 6 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of [insert density here] in confocal dishes and stimulated with lipopolysaccharide (LPS) at 500 ng / mL for 12 h. They were then co-incubated with PE, PCE, and PCHE (1.25 mg / mL) for 24 h, respectively, followed by incubation with anti-F4 / 80-FITC and anti-CD86-PE antibodies for 30 min. Cells were then fixed with 4% paraformaldehyde and permeabilized with Triton X-100 cell permeabilization agent, followed by incubation with anti-CD206-APC antibody for 30 min. Quantitative analysis of stained cells was performed by flow cytometry. Converting macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type can improve the intestinal microenvironment and may be helpful in treating enteritis. Figure 14 As shown in Figures A and B, PCHE exhibits immunomodulatory effects, inhibiting M1-type polarization. Compared to the lipopolysaccharide (LPS) stimulation group, the proportion of CD86 macrophages (M1 type) decreased from 24.93% to 18.78%, while the proportion of CD206 macrophages (M2 type) increased from 3.89% to 12.41%. Moreover, the proportion of CD206 macrophages in the PCHE group was significantly higher than that in the PCE group. This is because the enhanced antioxidant capacity of hydroxytyrosol promotes macrophage polarization towards the M2 type, effectively regulating macrophages towards the anti-inflammatory M2 type.

[0074] Example 7: Therapeutic effect of PCHE emulsion prepared in mice with inflammatory bowel disease: A C57BL / 6 mouse model of inflammatory bowel disease was established using sodium dextran sulfate (DSS). 200 μL of the emulsion was administered daily by gavage. Weight changes, fecal consistency, and fecal bleeding were monitored, and the severity of symptoms was assessed using the Disease Activity Index (DAI). On the last day of treatment, the mice were sacrificed, and colonic tissue was collected to measure its length. Figure 15 In the middle group (A), except for the healthy group, all other groups of mice showed weight loss and increased DAI scores, indicating successful model establishment. After 5 days of emulsion treatment, the weight loss of colitis mice decreased, and the DAI scores were lower ( Figure 15 China B and Figure 15 (C). The colon in the healthy group was longer than that in the colitis group, while the emulsion treatment alleviated the colonic shortening caused by inflammation, especially in the PCHE group, which showed no significant difference from the healthy group. This indicates that PCHE emulsion can reduce colitis-induced tissue damage. Figure 15 China D and Figure 15 (E).

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for the preparation of a protein-polyphenol coating modified Pickering emulsion of particles, characterized in that: The method comprises the following steps: The protein and the polyphenol are used to modify the solid particles to obtain protein-polyphenol coating modified particles, the solid particles are selected from at least one of polystyrene microspheres, mesoporous silica nanoparticles, silica nanoparticles, halloysite clay nanotubes, attapulgite, white phosphor calcium stone or hydroxyapatite; the mass ratio of the protein, the polyphenol and the solid particles is 1:1-5:1-5; The protein-polyphenol coating modified particles are dispersed in water to obtain a dispersion of the protein-polyphenol coating modified particles; The dispersion is mixed with an oil phase, and high-speed homogenization or ultrasonic emulsification is performed to obtain a Pickering emulsion, and the concentration of the protein-polyphenol coating modified particles in the Pickering emulsion is 0.1-15 mg / mL.

2. The method of preparing a protein-polyphenol coating modified Pickering emulsion particles according to claim 1, characterized in that: A mixed aqueous solution of the protein and the polyphenol is uniformly mixed with a dispersion of the solid particles to be modified, and the mixture is continuously stirred and reacted at 20-40 ℃ for 1-20 h to allow the protein and the polyphenol to adhere to the surface of the solid particles, and then solid-liquid separation is performed to obtain protein-polyphenol coating modified particles. Alternatively, the protein is first mixed with a dispersion of the solid particles to be modified and reacted for 4-16 h to obtain solid particles with a protein coating; and then the solid particles with the protein coating are reacted with a polyphenol solution for 1-6 h to obtain protein-polyphenol modified particles, and the temperature of the above reactions is 20-40 ℃.

3. The method of preparing a protein-polyphenol coating modified Pickering emulsion particle according to claim 1, characterized in that: The protein is selected from at least one of bovine serum albumin, sodium caseinate, zein, horseradish peroxidase, glucose oxidase, beta-lactoglobulin and ovalbumin; Alternatively, the polyphenol is selected from at least one of epigallocatechin gallate, tannic acid, procyanidin, tannin, catechin, caffeic acid and gallic acid; Alternatively, the oil phase is selected from olive oil, medium-chain triglyceride, ethyl acetate, toluene, n-heptane, corn oil, squalene and isopropyl myristate.

4. The method of preparing a protein-polyphenol coating modified Pickering emulsion particle according to claim 3, characterized in that: The Pickering emulsion further comprises a pharmaceutical active ingredient, and the pharmaceutical active ingredient is a mixture of a hydrophobic active ingredient and an amphiphilic active ingredient; the hydrophobic active ingredient is ricin oil or carvacol; and the amphiphilic active ingredient is hydroxytyrosol, procyanidin, tannic acid, chlorogenic acid or caffeic acid.

5. The method of preparing a protein-polyphenol coating modified Pickering emulsion particle according to claim 4, characterized in that: The hydrophobic active ingredient is carvacol, and the amphiphilic active ingredient is hydroxytyrosol; and the mass ratio of hydroxytyrosol, carvacol and olive oil is 1:15-25:500-600.

6. The method of preparing a protein-polyphenol coating modified Pickering emulsion particle according to claim 1, characterized in that: The method of emulsification is ultrasonic treatment, the power of the ultrasonic treatment is 50-400 W, and the ultrasonic time is 1-30 min.

7. The method of preparing a protein-polyphenol coating modified Pickering emulsion particle according to claim 1, characterized in that: The rotation speed of high-speed homogenization is 5000-15000 rpm, and the time is 0.5-5 min.

8. The method for preparing the protein-polyphenol coated modified particle-stabilized Pickering emulsion according to claim 1, characterized in that: The amount of the oil phase is 5%-85% of the total volume of the oil and water in the Pickering emulsion.

9. A protein-polyphenol coating modified particle stabilized Pickering emulsion characterized by: Prepared by the preparation method of any one of claims 1-8.

10. Use of the protein-polyphenol coating modified particle stabilized Pickering emulsion of claim 9 in the preparation of an intestinal inflammation drug.

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