A puerarin-loaded composite nanoparticle and a preparation method and application thereof

Abstract

The application discloses a puerarin-loaded composite nanoparticle and a preparation method and application thereof, relates to the technical field of nano drug delivery systems, and first prepares a zein-puerarin basic nanoparticle by adopting a reverse solvent precipitation method, then mixes a hyaluronic acid solution with the zein-puerarin basic nanoparticle solution by using an FNC125R flash nanoparticle preparation system, and finally obtains the puerarin-loaded composite nanoparticle. The application constructs a 'puerarin-zein-hyaluronic acid' nano synergistic system, can not only solve the problems of low oral bioavailability of puerarin and poor permeability of traditional external preparations, but also can significantly improve the permeability of puerarin in skin tissues, so as to realize local efficient drug delivery of the skin, and through synergistic effect, can enhance the inherent antioxidant and anti-inflammatory activities of puerarin, which provides a new efficient treatment idea for treating UVB-induced solar dermatitis.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery systems, and in particular to a composite nanoparticle loaded with puerarin, its preparation method, and its application. Background Technology

[0002] Puerarin, a bioactive isoflavone found in kudzu root and wild kudzu spp., possesses a variety of well-defined pharmacological activities, including antioxidant, anti-inflammatory, microcirculation-improving, and neuroprotective effects. It is widely used in alternative traditional Chinese medicine and has been found to be effective in treating chronic diseases such as cardiovascular disease, liver disease, gastric disease, respiratory disease, diabetes, Alzheimer's disease, and tumors. Studies have shown that puerarin can resist oxidative stress caused by various factors, inhibit inflammatory factors, promote collagen synthesis, and combat skin photoaging through multiple pathways, assisting in the repair of damage and moisturizing the skin, demonstrating promising applications in promoting skin repair. However, the poor water solubility, low skin permeability, and insufficient chemical stability of puerarin severely limit its transdermal delivery efficiency and therapeutic efficacy.

[0003] Photodermatitis, also known as sunburn or acute phototoxic dermatitis, is an acute photosensitive inflammatory reaction of the skin caused by excessive exposure to UVB (medium-wave ultraviolet) radiation. Its pathogenesis is closely related to oxidative stress and inflammatory responses. Clinical manifestations include erythema, edema, crusting, wrinkles, and decreased elasticity. Recurrent episodes can accelerate skin aging and increase the probability of skin cancer. Current research mainly focuses on exploring the preventive and therapeutic effects and mechanisms of natural products and traditional Chinese medicine. Novel formulations such as Cynomorium songaricum extract mixed with exosome hydrogels, glycyrrhizin gel cold compresses, and ruthenium-based artificial antioxidant enzymes have been developed, aiming to improve efficacy by regulating the microenvironment and targeting and clearing reactive oxygen species (ROS).

[0004] Nanoparticle-based drug delivery systems are drug delivery carriers constructed using nanotechnology, possessing core characteristics such as high specific surface area, strong modifiability, solubility, targeting, and controlled release. Due to the excellent permeability and low toxicity of nanoparticles, they have become highly efficient carriers for drug delivery in dermatological diseases. Therefore, they are widely used in nanocarrier formulations for dermatological conditions. However, the key to constructing nanoparticle-based drug delivery systems lies in how to construct suitable drug delivery carriers for specific drugs to maximize their bioavailability, permeability, and stability, thereby expanding their applications. Summary of the Invention

[0005] The purpose of this invention is to provide a composite nanoparticle loaded with puerarin, its preparation method and application, to solve the problems of low bioavailability, poor water solubility, difficulty in oral administration or poor transdermal efficacy of puerarin, and further to solve the problem of how to construct a suitable drug delivery system for puerarin.

[0006] To achieve the above objectives, the first aspect of the present invention provides a method for preparing composite nanoparticles loaded with puerarin, comprising the following steps: (1) Dissolve zein in an aqueous ethanol solution to obtain a zein solution; (2) Add puerarin to the zein solution to obtain a mixture; (3) Pour the mixture into the acid solution and stir. Then, remove the ethanol by rotary evaporation. At the same time, use the acid solution to compensate for the volume of vaporized ethanol until the ethanol is completely evaporated, and obtain the zein-puerarin-based nanoparticle solution. (4) Add hyaluronic acid to water and adjust the pH value to obtain a hyaluronic acid solution; (5) Using the FNC125R flash nanoparticle preparation system, hyaluronic acid solution and zein-puerarin-based nanoparticle solution were mixed to obtain a mixture. After centrifuging the mixture to remove large particles, composite nanoparticles were obtained.

[0007] Preferably, in step (1), the concentration of the ethanol aqueous solution is 80-90%, and the ratio of zein to ethanol aqueous solution is 1g:40-60mL. The concentration of the ethanol aqueous solution refers to the volume ratio of ethanol.

[0008] Preferably, in step (2), the mass ratio between puerarin and zein in the zein solution is 0.05~0.15:1.

[0009] Preferably, in step (3), the pH value of the acid solution is 4.0, and the ratio of the mixed solution to the acid solution is 10 mL: 30~50 mL. The acid solution in this invention is obtained by adjusting the pH to 4.0 by adding 0.1M hydrochloric acid to distilled water.

[0010] Preferably, in step (4), the concentration of the hyaluronic acid solution is 0.4~1.6 mg / mL and the pH value is 4.0.

[0011] More preferably, in step (4), the concentration of the hyaluronic acid solution is 1.0~1.6 mg / mL.

[0012] Preferably, in step (5), the specific process of mixing using the FNC125R flash nanoparticle preparation system is as follows: Channel A contains 2 mL of zein-puerarin basic nanoparticle solution with a discharge flow rate of 0.4 mL / s, and Channel B contains 2.5 mL of hyaluronic acid solution with a discharge flow rate of 0.5 mL / s. The solution flowing out of the instrument is discarded, and the large particles are collected and centrifuged to remove them. The supernatant is then taken to obtain composite nanoparticles.

[0013] This invention does not use ordinary stirring and mixing methods to mix hyaluronic acid solution with zein-puerarin-based nanoparticle solution. Instead, it uses a special device (FNC125R flash nanoparticle preparation system) to mix the two solutions, which can produce nanoparticles in a short time. The preparation method is efficient and fast, suitable for mass production. At least 50 mL of nanoparticles can be produced in 30 seconds, while traditional methods require stirring at 800 rpm for at least 30 minutes or even longer, resulting in a longer production time.

[0014] A second aspect of the present invention provides composite nanoparticles loaded with puerarin, which are prepared by the above-described preparation method.

[0015] A third aspect of this invention provides an application of puerarin-loaded composite nanoparticles in products for treating photodermatitis. These products include, but are not limited to, pharmaceuticals and cosmetics, with the cosmetic dosage forms including creams, gels, lotions, etc.

[0016] Therefore, the present invention employs the above-mentioned composite nanoparticles loaded with puerarin, its preparation method, and its application, which have the following beneficial effects: This invention focuses on enhancing the therapeutic effect of puerarin on UVB-induced photodermatitis. It proposes constructing a synergistic nanodelivery system based on zein and hyaluronic acid to help solve the problems of puerarin's solubility, skin retention, and targeting. This lays a theoretical and experimental foundation for developing efficient and stable transdermal puerarin formulations, enhances the drug's anti-inflammatory and antioxidant effects, and provides an innovative and efficient treatment option for the precise treatment of photodermatitis damage. It also promotes the application and transformation of puerarin in skin repair, anti-aging, and other fields.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 The particle size, PDI, and zeta potential of puerarin-loaded composite nanoparticles prepared for different polysaccharides were studied. (A) The effect of different concentrations of CMC-Na on the particle size and PDI of nanoparticles; (B) The effect of different concentrations of CMC-Na on the zeta potential of nanoparticles; (C) The effect of different concentrations of pectin on the particle size and PDI of nanoparticles; (D) The effect of different concentrations of pectin on the zeta potential of nanoparticles; (E) The effect of different concentrations of HA on the particle size and PDI of nanoparticles; (F) The effect of different concentrations of HA on the zeta potential of nanoparticles. Different letters indicate significant differences between groups. P <0.05), the same letter indicates no significant difference, n=3; Figure 2 TEM images of ZPH NPs; Figure 3 The FTIR spectra of Zein, PUE, HA and various nanoparticles (ZP and ZPH NPs) are shown. Figure 4 CD spectra of each nanoparticle (Zein, ZH, ZP and ZPH NPs); Figure 5 XRD spectra of Zein, PUE, HA and various nanoparticles (ZP and ZPH NPs); Figure 6 DSC spectra of Zein, PUE, HA and various nanoparticles (ZP and ZPH NPs); Figure 7 The effect of temperature on (A) particle size and (B) zeta potential of ZP NPs and ZPH NPs; Figure 8 The effect of pH on (A) particle size and (B) zeta potential of ZP NPs and ZPH NPs; Figure 9 The effects of NaCl on the particle size (A) and zeta potential (B) of ZP NPs and ZPH NPs; Figure 10 To investigate the toxicity of different concentrations of ZPH NPs, and compare them with the control group, ** P <0.01, *** P <0.001, n=3; Figure 11 Cell survival rates at different UVB modeling durations were compared with the control group. ### P <0.001, n=3; Figure 12 The efficacy of ZPH NPs on UVB-induced HaCaT cells is represented by different letters, indicating significant differences between groups. P <0.05), the same letter indicates no significant difference, n=3; Figure 13 ZPH NPs promote scratch healing of UVB-damaged HaCaT cells, where (A) is an image of HaCaT cell scratches; (B) is a quantitative map of HaCaT cell scratch healing rate; different letters indicate significant differences between groups. P <0.05), the same letter indicates no significant difference, n=3; Figure 14 The anti-inflammatory and antioxidant effects of ZPH NPs on UVB-induced HaCaT cells are shown in different letters, indicating significant differences between groups. P <0.05), the same letter indicates no significant difference, n=3; Figure 15 To improve the symptoms of photodermatitis in mice using ZPH NPs, the following diagrams are provided: (A) Flowchart of the UVB-induced photodermatitis experiment in mice; (B) Image of the skin condition on the back of mice; (C) Results of skin moisture content test; (D) Results of skin elasticity index test; (F) Severity score of skin damage. Different letters indicate significant differences between groups. P <0.05), the same letter indicates no significant difference, n=6; Figure 16 To improve the H&E staining and epidermal thickness statistics of UVB-induced photodermatitis by ZPH NPs, (A) H&E staining of mouse skin tissue; (B) Statistical graph of mouse skin epidermal thickness; different letters indicate significant differences between groups ( P <0.05), with the same letter indicating no significant difference.

[0019] Figure 17 Masson staining and collagen deposition quantification maps of ZPH NPs improving UVB-induced photodermatitis, where (A) is Masson staining of mouse skin tissue; (B) is a collagen deposition quantification map; different letters indicate significant differences between groups ( P <0.05), with the same letter indicating no significant difference.

[0020] Figure 18 The effects of ZPH NPs on oxidative stress indicators and inflammatory factors in the skin tissue of mice with photodermatitis are shown in the figure. (A) MDA content; (B) TNF-α content; (C) IL-1β content; (D) IL-6 content. Different letters indicate significant differences between groups. P <0.05), with the same letter indicating no significant difference. Detailed Implementation

[0021] The present invention will be further described below. It should be noted that this embodiment is based on the present technical solution and provides detailed implementation methods and specific operation processes, but the present invention is not limited to this embodiment.

[0022] Example 1 This embodiment provides a method for preparing composite nanoparticles loaded with puerarin, including the following steps: (1) Accurately weigh an appropriate amount of hyaluronic acid (HA) and prepare it into a 2 mg / mL HA solution. During the preparation process, stir continuously at 600 rpm for 2 hours to completely dissolve the hyaluronic acid. Adjust the pH of the resulting solution to 4.0 with 0.1 M HCl and set aside.

[0023] (2) Dissolve 1g of zein in 50mL of 85vt% ethanol aqueous solution, specifically by stirring at 600rpm for 2h to obtain a zein solution. Then, add puerarin (PUE) powder to 20mL of zein solution at a constant mass ratio of PUE / zein (0.1:1), and stir at 800rpm in the dark for 1h to obtain a PUE / Zein ethanol solution. Maintaining the stirring speed, pour 10mL of PUE / Zein ethanol solution into 40mL of acid solution and stir for 3min; remove the ethanol by rotary evaporation at 40℃ and 0.1MPa pressure, and compensate for the volume of vaporized ethanol with acid solution to obtain a zein-puerarin-based nanoparticle (ZP NPs) solution. The acid solution used in this example was obtained by adjusting the pH of distilled water to 4.0 with 0.1M hydrochloric acid.

[0024] (3) Dilute the HA solution in step (1) with acid solution to prepare HA solutions with concentrations of 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mg / mL for subsequent preparation; (4) Nanoparticles were prepared by high-speed mixing using the FNC125R flash nanoparticle preparation system (purchased from Siyi Scientific Instruments). Channel A contained 2 mL of ZP NPs with a discharge flow rate of 0.4 mL / s, and channel B contained 2.5 mL of diluted HA solution with a discharge flow rate of 0.5 mL / s. The first part of the solution was discarded and collected. The solution was centrifuged at 3000 rpm for 10 min to remove large particles. The supernatant was collected to obtain composite nanoparticles loaded with puerarin, which were denoted as zein-puerarin-hyaluronic acid nanoparticles (ZPH NPs).

[0025] Example 2 The difference between this embodiment and Example 1 is that the type of polysaccharide is different. In this embodiment, hyaluronic acid is replaced with pectin, and the resulting composite nanoparticles loaded with puerarin are denoted as zein-puerarin-pectin nanoparticles (ZPP NPs).

[0026] Example 3 The difference between this embodiment and Example 1 is that the type of polysaccharide is different. In this embodiment, hyaluronic acid is replaced with sodium carboxymethyl cellulose (CMC-Na), and the resulting composite nanoparticles loaded with puerarin are denoted as zein-puerarin-sodium carboxymethyl cellulose nanoparticles (ZPC NPs).

[0027] Comparative Example 1 The difference between this comparative example and Example 1 is that puerarin was not added during the preparation process. The specific steps are as follows: (1) Accurately weigh an appropriate amount of hyaluronic acid (HA) and prepare it into a 2 mg / mL HA solution. During the preparation process, stir continuously at 600 rpm for 2 hours to completely dissolve the hyaluronic acid. Adjust the pH of the resulting solution to 4.0 with 0.1 M HCl and set aside.

[0028] (2) Dissolve 1g of zein in 50mL of 85% ethanol aqueous solution, specifically by stirring at 600rpm for 2h to obtain a zein solution. Maintaining the stirring speed, pour 10mL of the zein solution into 40mL of acid solution and stir for 3min. Remove the ethanol by rotary evaporation at 40℃ and 0.1MPa pressure, and compensate for the volume of vaporized ethanol with the acid solution to obtain the zein NPs solution. The acid solutions used in this comparative example were all obtained by adjusting the pH of distilled water to 4.0 with 0.1M hydrochloric acid.

[0029] (3) Dilute the HA solution in step (1) with acid solution to prepare a 1.0 mg / mL HA solution for subsequent preparation; (4) Nanoparticles were prepared by high-speed mixing using the FNC125R flash nanoparticle preparation system (purchased from Siyi Scientific Instruments). Channel A contained 2 mL of Zein NPs solution with a discharge flow rate of 0.4 mL / s, and channel B contained 2.5 mL of diluted HA solution with a discharge flow rate of 0.5 mL / s. The first part of the solution was discarded and collected. The solution was centrifuged at 3000 rpm for 10 min to remove large particles. The supernatant was collected to obtain nanoparticles, which were denoted as zein-hyaluronic acid nanoparticles (ZH NPs).

[0030] Experimental Example 1 The composite nanoparticles prepared in Examples 1-3 were characterized.

[0031] (1) Measurement of particle size, PDI and Zeta potential The hydrodynamic particle size, polymer dispersibility index (PDI), and zeta potential of puerarin composite nanoparticles prepared from different polysaccharides were tested using the NanoBrook Omni assay. The results are as follows: Since the purpose of this invention is to prepare transdermal drug delivery formulations, nanoparticles with a particle size of less than 200 nm have higher transdermal flux and more uniform skin distribution. The stability of nanoparticles is directly proportional to the absolute value of their zeta potential. When the potential is between -30 and -50 mV, the nanoparticles balance stability and skin compatibility. Different types and concentrations of polysaccharides have a significant impact on the size of complex nanoparticles.

[0032] like Figure 1As shown in AB, due to the high viscosity of CMC-Na, increasing the concentration leads to an increase in viscosity, resulting in an increase in the particle size of the prepared ZPC NPs with increasing concentration. The particle size is generally greater than 200 nm, and the PDI is generally greater than 0.3. Although the zeta potential meets the requirements, the excessively large particle size leads to poor uniformity and significant particle size differences, making them unsuitable for subsequent experiments. Figure 1 As shown in CF, the viscosity of pectin and HA is moderate. Although the particle size of both ZPP NPs and ZPH NPs increases with increasing concentration, the PDI is less than 0.3, indicating good uniformity. Considering the requirements of skin formulations, the particle size and zeta potential of ZPH NPs prepared with 1.0-1.6 mg / mL HA solution meet the requirements. Therefore, subsequent experiments screened the optimal concentration from HA solutions of 1.0, 1.2, 1.4, and 1.6 mg / mL.

[0033] (2) Determination of the encapsulation efficiency of puerarin composite nanoparticles Chromatographic conditions: Durashell C18-AM (5 μm, 4.6 mm × 250 mm), mobile phase: methanol:water = 30:70 (v / v), flow rate: 1.0 mL / min, detection wavelength: 250 nm, column temperature: 30 ℃, injection volume: 10 μL.

[0034] Take 500 μL of PUE composite nanoparticles and centrifuge at 4℃ and 12000 rpm for 10 min. The lower layer is drug-loaded nanoparticles. Add ten times the amount of methanol to destroy the encapsulation structure of the nanoparticles and release the PUE, which is the total mass of PUE encapsulated (C1)

[92] . Take 500 μL of PUE composite nanoparticles and directly add ten times the amount of methanol to destroy the nanoparticle structure, which is the total amount of PUE (C2). After filtration, analyze by HPLC and calculate the encapsulation rate according to the formula.

[0035] , The test results are shown in Table 1. When the HA concentration is 1.0 mg / mL, the encapsulation efficiency is 86.87 ± 0.99%. Considering the three parameters of particle size, PDI and zeta potential, the nanoparticles prepared at this concentration have the best performance (particle size: 141.06 ± 0.56 nm, PDI: 0.175 ± 0.002, zeta potential: -30.65 ± 0.82 mV). Therefore, the puerarin composite nanoparticles (ZPH NPs) prepared with 1.0 mg / mL HA solution were selected for further research.

[0036] Table 1. Effect of different HA concentrations on encapsulation efficiency

[0037] (3) Morphological characterization by transmission electron microscopy (TEM) 1) Add 10 μL of nanoparticle solution to a copper grid and let it precipitate for 1 min. Then, use filter paper to absorb the floating liquid.

[0038] 2) Add 10 μL of uranium acetate (or phosphotungstic acid staining solution) to a copper grid to precipitate for 1 min, and then remove the floating liquid with filter paper.

[0039] 3) Dry at room temperature for several minutes.

[0040] 4) Electron microscopy is used for detection and imaging at 80-120kV.

[0041] Observe under a transmission electron microscope and collect and analyze images.

[0042] To further explore the surface morphology of ZPH NPs, electron microscopy was used to characterize the morphology of the nanoparticles. For example... Figure 2 As shown in the TEM, ZPH NPs are spherical particles with smooth surfaces, well-distributed, uniform in size and morphology, and with distinct particle boundaries. This phenomenon is attributed to the electrostatic repulsion and steric hindrance provided by the hydrophilic HA surface, which promotes cell internalization after drug administration and improves delivery efficiency.

[0043] (4) Fourier transform infrared (FTIR), circular dichroism (CD), and X-ray diffraction (XRD) structural characterization.

[0044] FTIR spectra of the nanoparticles were determined using FTIR, with a lyophilized sample to potassium bromide ratio of 1:100. The scanning range was 400 cm⁻¹. -1 -4000cm -1 Preparation process of freeze-dried samples: Place the solution of nanoparticles to be tested in an evaporating dish and seal it with plastic wrap. The solution should not exceed half the volume of the dish. Then place it at -20℃ for 24 hours and at -80℃ overnight to ensure sufficient pre-freezing. Finally, place the pre-frozen sample in a vacuum freeze dryer for 2 days to obtain the sample.

[0045] The CD spectrometer was used for determination: First, the concentration of zein in the nanoparticles was diluted to 0.20 mg / mL using hydrochloric acid solution at pH=4. Then, under a constant nitrogen flow, CD values ​​of different samples were obtained at a speed of 40 nm / min in the 190 nm-260 nm range, with a bandwidth of 1 nm and an ultraviolet path length of 0.10 cm. Finally, the obtained mdeg values ​​were converted to molar ellipticity [θ].

[0046] The crystal structure of the nanoparticles was determined by XRD, and the samples were lyophilized. The diffraction angle 2θ range was set to 5°-60°, and the scanning speed was set to 5° / min. The preparation process for the lyophilized samples was as follows: the solution of the nanoparticles to be tested was placed in an evaporating dish and sealed with plastic wrap, ensuring the solution did not exceed half the volume of the dish; then it was placed at -20°C for 24 hours and then at -80°C overnight to ensure sufficient pre-freezing; finally, the pre-frozen sample was lyophilized in a vacuum freeze dryer for 2 days.

[0047] To investigate the internal interactions of nanoparticles, structural characterization was performed using FTIR, CD, and XRD. The FTIR spectra are shown below. Figure 3 As shown, PUE is between 1513.85 and 1444.42 cm⁻¹. -1 The absorption peaks at these locations correspond to the C=C skeletal stretching vibrations of the benzene ring, at 890.95, 835.03, and 796.46 cm⁻¹. -1 The absorption peak at 1627.63 cm⁻¹ corresponds to the out-of-plane bending of the CH group of the benzene ring. -1 The peaks at this point represent C=O stretching vibrations, and these are all characteristic peaks of PUE. The zein peak is at 1641.13 cm⁻¹. -1 There is a typical amide peak at this point, corresponding to its C=O stretching vibration. After the formation of ZP NPs, this amide peak shifts to 1639.2 cm⁻¹. -1 This indicates a hydrophobic interaction between zein and PUE. After PUE is encapsulated in ZPH NPs, all characteristic peaks of PUE disappear, which may be due to the formation of hydrogen bonds or other non-covalent interactions between PUE and the excipients, ultimately leading to the formation of co-amorphous peaks.

[0048] The secondary structures of Zein, ZH, ZP, and ZPH NPs were characterized using circular dichroism (CD), such as... Figure 4 As shown in Figure A, all samples exhibited typical α-helix characteristics in the far-ultraviolet region, with a strong positive peak near 198 nm and characteristic negative peaks near 208 nm and 222 nm, indicating that the addition of HA and PUE did not disrupt the core secondary structure of zein.

[0049] like Figure 4 As shown in Figure B, quantitative analysis revealed targeted changes in the secondary structure of ZPH NPs compared to the original Zein NPs. The α-helix content decreased slightly from 35% to 31%, a smaller decrease than that of ZP NPs (29%), indicating that the hydrophobic core structure of the protein was well preserved after the addition of PUE and HA. The random coil content significantly increased to 41%, the highest among the four groups. This change may be due to the interaction between the hydrophilic segments of HA and the protein molecule, as well as the embedding of puerarin molecules, which together increased the flexibility and flexibility of the protein molecule surface.

[0050] In summary, ZPH NPs, while retaining the core α-helical hydrophobic framework, enhance the hydrophilicity and flexibility of the molecular surface by increasing the random coil content. This not only facilitates the stable dispersion of nanoparticles in aqueous environments but also provides a favorable structural basis for the encapsulation of puerarin and subsequent optimization of biocompatibility.

[0051] X-ray diffraction (XRD) is commonly used to assess the crystallinity of encapsulated compounds or biopolymer matrices. For example... Figure 5 As shown, PUE exhibits numerous sharp and strong diffraction peaks at multiple locations, displaying typical characteristics of a highly crystalline state. In contrast, zein shows two broad peaks at 9° and 20°, reflecting its amorphous nature. The characteristic peaks of PUE were not observed at all in the diffraction patterns of the nanoparticles (ZP NPs and ZPHNPs), indicating that PUE is in an amorphous morphology, confirming that PUE has been successfully encapsulated within ZPH NPs.

[0052] (5) DSC thermal stability of ZPH NPs Differential scanning calorimetry (DSC) stability characterization: The thermal behavior of the raw material and lyophilized nanoparticles was characterized by DSC. 4 mg of powder sample was sealed in an aluminum pot and heated from 25 °C to 300 °C at a rate of 10 °C / min, with a flow rate of 50 mL / min.

[0053] Preparation process of freeze-dried nanoparticles: Place the nanoparticle solution to be tested in an evaporating dish and seal it with plastic wrap. The solution should not exceed half the volume of the dish. Then place it at -20℃ for 24 hours and at -80℃ overnight to ensure sufficient pre-freezing. Finally, place the pre-frozen sample in a vacuum freeze dryer to freeze-dry for 2 days.

[0054] Figure 6 Differential scanning calorimetry (DSC) curves of PUE, Zein, HA, and nanoparticles (ZP and ZPH NPs) are shown. Zein, being an amorphous structure, did not exhibit a sharp endothermic peak, instead showing a smooth baseline. The broad endothermic peak of PUE at 124 °C is likely due to moisture loss, while the sharp endothermic peak at 213 °C corresponds to its melting point, indicating its crystalline state. After the formation of ZP NPs and ZPH NPs, this endothermic peak completely disappeared, and the characteristic endothermic peak of PUE also disappeared, resulting in a relatively flat curve overall, indicating that PUE exists in an amorphous and non-crystalline form within the nanoparticles. These results confirm a strong molecular interaction between PUE and the supports (Zein and HA), forming a homogeneous amorphous system, which contributes to improving the solubility and bioavailability of PUE.

[0055] (6) Thermal stability test Freshly prepared ZP NPs and ZPH NPs were heated in water baths at 25, 35, 50, 65, and 80 °C for 30 min. After heating, the nanoparticles were cooled to ambient temperature, and their particle size and zeta potential were measured.

[0056] Thermal stability can assess the resistance of nanoparticles to high temperatures. For example... Figure 7 As shown in AB, the particle size and zeta potential of the nanoparticles were measured after heating in a water bath at 25, 35, 50, 65, and 80 °C for 30 min. The particle size of ZP NPs and ZPH NPs did not increase significantly. Figure 7 A), indicating that the nanoparticles possess thermal stability within a temperature range of 25-80℃. Similarly, the zeta potential of the two types of nanoparticles did not change significantly ( Figure 7 (B) may be due to electrostatic repulsion resisting its polymerization.

[0057] (7) pH stability test The effect of pH on ZP NPs and ZPH NPs was evaluated. The pH of the samples was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 with 0.1M hydrochloric acid (diluted 10 times) or sodium hydroxide solution, and the samples were allowed to stand for 24 hours before measuring the particle size and zeta potential.

[0058] Nanoparticles may experience different pH environments during manufacturing, storage, and use; therefore, assessing pH stability is crucial. Due to the isoelectric point of zein (pI≈6.2), ZP NPs exhibit significant aggregation and a sharp increase in particle size at pH 6. Figure 8 A). ZP NPs with pH close to their isoelectric point have relatively low zeta potentials ( Figure 8 (B) The extremely weak electrostatic repulsion led to significant aggregation of nanoparticles. In contrast, as the pH increased from 4.0 to 8.0, the particle size of ZPH NPs remained relatively stable, while the zeta potential increased from -30.2 mV to -49.1 mV. This indicates that the addition of HA helps improve the pH stability of ZPH NPs.

[0059] (8) Ion strength stability test Equal volumes of ZP NPs, ZPH NPs, and NaCl solution were mixed to result in final NaCl concentrations of 0, 10, 20, 30, 40, and 50 mmol / L (0, 0.585, 1.170, 1.755, 2.340, and 2.925 mg / mL) in each dispersion. The mixtures were stirred at 500 rpm for 15 min and then allowed to stand at room temperature for 24 h. Finally, particle size and zeta potential were measured.

[0060] Ionic strength stability demonstrates the salt tolerance of the nanoparticles. ZP NPs are sensitive to NaCl concentration; the particle size increases rapidly upon the addition of NaCl solution. Figure 9 A), while the Zeta potential decreased significantly ( Figure 9 B). In contrast, the particle size of ZPH NPs was less affected, although the zeta potential decreased. This result indicates that the surface space barrier provided by the HA coating effectively suppressed the increase in particle size and improved ionic strength stability.

[0061] In summary, the addition of HA modification to nanoparticles significantly improves their environmental stability, especially in terms of pH-induced aggregation and high-salt-induced precipitation near the isoelectric point. The ZPH NPs prepared in this invention have smaller particle sizes and exhibit excellent stability over a wide range of temperatures, pH values, and ionic strengths, making them promising nanocarrier systems for drug delivery.

[0062] Experimental Example 2 The cells used in this experiment were immortalized human keratinocytes (HaCaT), purchased from Shanghai Jinyuan Biotechnology Co., Ltd.

[0063] In vitro efficacy evaluation of ZPH NPs against HaCaT cells: (1) Preparation of drugs Preparation of DMEM complete medium: Prepare DMEM blank medium: serum: penicillin-streptomycin solution in a ratio of 45:5:0.5, shake well, and store at 4°C for later use.

[0064] Preparation of ascorbic acid (VC): Weigh an appropriate amount of VC and dissolve it in DMSO, vortex to mix, and prepare a 100 mg / mL stock solution for subsequent cell experiments.

[0065] Preparation of ZPH NPs: ZPH NPs were prepared according to the methods in "Examples 1-3". Six concentrations of ZPH NPs (1, 2, 4, 8, 16, and 32 μg / mL) were prepared using DMEM blank medium and stored at 4°C in the dark. Before administration, the NPs were irradiated with ultraviolet light for 5 min.

[0066] Preparation of ZH NPs: ZH NPs were prepared according to the methods in "Examples 1-3" (without PUE). ZH NPs at a concentration of 16 μg / mL were prepared using DMEM blank medium and stored at 4°C in the dark. Before administration, the NPs were irradiated with ultraviolet light for 5 min.

[0067] (2) Culture of HaCaT cells HaCaT cells were cultured in DMEM complete medium containing 10% serum and placed in a 37°C, 5% CO2 incubator. When the cell density reached 70%, the medium was changed; when it reached 80%–90%, the cells were washed twice with PBS buffer, then digested with 1.5 mL of 0.25% trypsin for 5 min before passage. For cell cryopreservation, 1 mL of serum-free cryopreservation solution was added to healthy HaCaT cells, and the cells were gently pipetted and transferred to cryovials. The cells were first placed at -80°C for 24 h, and then stored in liquid nitrogen. For cell thawing, the HaCaT cells frozen in liquid nitrogen were quickly transferred to a 37°C water bath, gently thawed, and then cultured according to the above procedure. Subsequently, HaCaT cells in the logarithmic growth phase were selected for cell plating.

[0068] (3) Effect of ZPH NPs on HaCaT cell survival HaCaT cells were seeded at a density of 8 × 10³ cells / well in 100 μL of 96-well plates and incubated in a CO2 incubator for 24 h. When the cell confluence reached 70-80%, ZPH NPs (1, 2, 4, 8, 16, and 32 μg / mL) were prepared using DMEM blank medium, and the control group was also prepared using DMEM blank medium. Each group was divided into 6 replicates, with three parallel runs. After 24 h of culture, the culture medium was discarded, and 100 μL of DMEM complete medium containing 10% CCK-8 was added to each well. After incubation for 30 min, the absorbance (OD) at 450 nm was measured using a microplate reader.

[0069] , (4) Establishment of UVB-induced HaCaT cell model Following the HaCaT cell culture procedure described above, cells were seeded and cultured until they reached 70%–80% confluence before modeling. Two groups were established: a Control group and a Model group (both in DMEM blank medium). The Model group underwent UVB irradiation for 5, 10, 15, 20, 25, and 30 minutes, with six replicates per group and three parallel experiments. Before modeling, cells were washed twice with 100 μL of PBS, and 100 μL of DMEM blank medium was added to each well. The 96-well plate was opened and irradiated at a distance of 10 cm from the UVB light source. The Control group was not irradiated. After modeling, cells were incubated in a 5% CO2 incubator for 24 hours, the medium was discarded, and subsequent steps were the same as for HaCaT cell culture.

[0070] (5) Protective effect of ZPH NPs on UVB-induced HaCaT cells Following the HaCaT cell culture procedure described above, cells were seeded. The experiment was divided into four groups: Control group (DMEM blank medium), Model group (DMEM blank medium), ZH nanoparticle empty vector group (16 μg / mL), VC positive control group (ascorbic acid), and ZPHNPs administration groups (concentrations of 1, 2, 4, 8, and 16 μg / mL). Each group had 6 replicates, and the experiment was independently repeated 3 times. When the cell confluence reached 70%–80%, the culture medium in the wells was discarded, and the cells were washed twice with 100 μL PBS. The corresponding concentrations of ZPHNPs or VC were added for intervention. Except for the Control group, all other groups underwent UVB modeling. After modeling, the 96-well plates were incubated in a 5% CO2 cell culture incubator for 24 hours, the culture medium was discarded, and subsequent HaCaT cell culture procedures were performed.

[0071] (6) Effects of ZPH NPs on UVB-damaged HaCaT cell scratch healing HaCaT cells were used at a rate of 3 × 10 5 Cells were seeded at a density of 1 mL per well in 12-well plates and incubated in a CO2 incubator for 24 h until a 100% confluent monolayer was formed. A 10 μL pipette tip was used to streak the cells perpendicularly to the monolayer, and floating cells and debris were removed with PBS. Control, Model, ZH NPs, VC, and ZPH NPs treatment groups were established. All groups used 1% low-serum medium to prepare the drug. 1 mL of the corresponding drug or 1% low-serum medium was added according to the group. Except for the Control group, all other groups underwent UVB irradiation to induce modeling, with the light source 10 cm away from the 12-well plate for 5 min. After modeling, images were taken at a fixed position under an inverted microscope, recorded as 0 h. After 24 h of further culture, images were taken at the same position, with cell debris gently washed away with PBS before each image. The scratch images were analyzed using ImageJ software to measure the scratch area and calculate the scratch healing rate.

[0072] , (7) Detection of MDA and NO, TNF-α, IL-1β and IL-6 using kits HaCaT cells were used at a rate of 2 × 10 5Cells were seeded at a density of 1 mL per well in 12-well plates. Once cell confluence reached 70-80%, UVB induction was performed to establish a model. Control group, Model group, ZH NPs group (16 μg / mL), VC group (100 μg / mL), and ZPH NPs administration group (16 μg / mL) were set up. All groups used DMEM blank medium for culture. Except for the Control group, all other groups were irradiated with UVB for 15 min. After modeling, the 12-well plates were placed in a CO2 incubator. After 8 h, half of the supernatant was aspirated for the detection of TNF-α, IL-1β, and IL-6 inflammatory factors. After culturing for another 24 h, the remaining supernatant was aspirated for the detection of NO and MDA oxidative stress indicators, following the kit instructions.

[0073] The test results are as follows: (1) Effect of ZPH NPs on the proliferation of HaCaT cells like Figure 10 As shown, compared with the Control group, ZPH NPs significantly improved the metabolic activity of HaCaT cells in the concentration range of 1-32 μg / mL. Among them, 4 μg / mL ZPH NPs showed the highest cell viability. With the increase of the drug concentration, the cell viability decreased in a dose-dependent manner, but all concentration groups were significantly higher than the Control group, indicating that the above concentrations of ZPH NPs did not have toxic side effects on HaCaT cells.

[0074] (2) Establishment of UVB-induced HaCaT cell model HaCaT cells were divided into groups and irradiated with UVB for 5, 10, 15, 20, 25, and 30 minutes, respectively. Figure 11 As shown, the damage to HaCaT cells became increasingly severe with prolonged irradiation time. When the UVB irradiation time was 15 min, the cell survival rate reached 51.6%, therefore this time was selected for subsequent UVB modeling efficacy evaluation experiments.

[0075] (3) Protective effect of ZPH NPs on UVB-induced HaCaT cells The results are as follows Figure 12 As shown, ZPH NPs can significantly enhance cell viability in a dose-dependent manner within the concentration range of 1~32 μg / mL and effectively reverse UVB-induced cell damage, with the strongest protective effect at 16 μg / mL.

[0076] (4) ZPH NPs promote scratch healing of UVB-damaged HaCaT cells The results are as follows Figure 13As shown in the results, after UVB irradiation, the 24 h scratch healing rate of the Model group was significantly lower than that of the Control group, suggesting that UVB causes cell damage and scratch repair impairment. Empty ZH can significantly improve the scratch healing rate, verifying that the zein-hyaluronic acid carrier itself has a certain pro-repair effect, but the effect is weaker than that of the drug-loaded ZPH NPs group and the VC group. Intervention with ZPH NPs can significantly increase the scratch healing rate, which was significantly higher than that of the Model group at 24 h, and the repair effect is comparable to that of the Control group and superior to that of the VC group. The results show that ZPH NPs can effectively promote scratch healing in UVB-damaged HaCaT cells, restore the damage repair ability of cells, and enhance the healing-promoting efficacy, which provides functional support for the treatment of skin damage caused by solar dermatitis.

[0077] (5) Antioxidant and anti-inflammatory effects of ZPH NPs on UVB-induced HaCaT cells The results are shown in Figure 14 , ZPH NPs at 16 μg / mL can effectively reverse the increase of MDA, NO, TNF-α, IL-1β and IL-6 induced by UVB, indicating that it has the best antioxidant and anti-inflammatory effects at 16 μg / mL.

[0078] The protective effect of ZPH NPs is superior to that of ZH NPs (empty vehicle group) and 100 μg / mL VC, with a significant anti-UVB damage effect, which preliminarily verifies that ZPH NPs have anti-UVB, anti-inflammatory and antioxidant effects.

[0079] Test Example 3 Efficacy evaluation of ZPH NPs on UVB-induced solar dermatitis in mice: SPF-grade male C57BL / 6 mice aged 6-8 weeks, with a body weight of 18-22 g, were purchased from the Guangdong Medical Laboratory Animal Center (Laboratory Unit License No.: SYXK (Guangdong) 2022-0125). The experimental mice were housed in an SPF-grade laboratory of the Experimental Animal Center of Guangdong Pharmaceutical University, with a 12 h light / 12 h dark cycle, a temperature of 24 ± 2°C, a humidity of 55 ± 5%, and free access to food and water. All animal experiments were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University, and the animal ethics number is gdpulacspf20221002.

[0080] (1) Preparation of drugs Preparation of ascorbic acid (VC): Prepare it immediately before use. Weigh an appropriate amount of VC and dissolve it in normal saline, vortex and mix well to prepare a 0.5% VC solution, store it at 4°C away from light, and administer 300 μL per mouse according to a fixed area Preparation of ZPH NPs: Prepare a 1 mg / mL HA solution and adjust the pH to 4.0. Dissolve 1 g of zein in 50 mL of 85% ethanol solution, stirring at 600 rpm for 2 h to obtain a Zein solution. Then add PUE (Zein:PUE = 1:0.1, w / w) to the Zein solution and stir at 800 rpm in the dark for 1 h. Maintaining the stirring speed, pour 10 mL of the PUE / Zein ethanol solution into 40 mL of pH=4 hydrochloric acid solution and stir for 3 min. Remove the ethanol by rotary evaporation at 40 °C and 0.1 MPa, and compensate for the volume of vaporized ethanol with pH=4 hydrochloric acid solution to obtain ZP NPs. Use the FNC125R flash nanoparticle preparation system to rapidly mix the HA solution and ZP NPs solution (ZP NPs:HA = 1:1.25, v / v) to prepare and collect biopolymers. Large particles were removed by centrifugation at 3000 rpm for 10 min, and ZPH NPs were finally prepared (ZPH-H: containing 0.15 mg / mL PUE, ZPH-L: containing 0.075 mg / mL PUE). The NPs were stored at 4°C protected from light, and 300 μL was administered to each mouse according to a fixed area.

[0081] Preparation of ZH NPs: Prepare a 1 mg / mL HA solution and adjust the pH to 4.0. Dissolve 1 g of zein in 50 mL of 85% ethanol solution, specifically by stirring at 600 rpm for 2 h to obtain a Zein solution. Maintaining the stirring speed, pour 10 mL of the Zein solution into 40 mL of pH=4 hydrochloric acid solution and stir for 3 min. Remove the ethanol by rotary evaporation at 40 °C and 0.1 MPa pressure, and compensate for the volume of vaporized ethanol with pH=4 hydrochloric acid solution to obtain Zein NPs. Use an FNC125R fast flash nanoparticle preparation system to rapidly mix the HA solution and Zein NPs solution (Zein NPs:HA = 1:1.25, v / v) to prepare and collect biopolymers. Centrifuge at 3000 rpm for 10 min to remove large particles, finally preparing ZH NPs with a total mass concentration equivalent to ZPH-H, store at 4 °C protected from light, and administer 300 μL per mouse according to a fixed area.

[0082] (2) Experimental scheme After a week of acclimatization, all mice underwent hair removal on their backs. Twenty-four hours later, the mice were randomly divided into six groups (n=6 per group): Con group (saline), UV irradiation group (saline + UVB), empty vector group (ZH NPs + UVB, 300μL), ascorbic acid treatment group (VC + UVB, 300μL), and nanoparticle treatment group (ZPH-L: UVB + ZPH NPs, ZPH-H: UVB + ZPH NPs, 300μL). Mice in the Con group were deprived of light and given a 2×3cm saline dressing. The other groups received continuous UVB irradiation for three days at a daily dose of 1.2 J / cm². 2 After irradiation, a 2×3cm dressing containing a specific drug was applied. When removing the dressing, residual drug on the skin surface was wiped off with physiological saline. The condition of the mice's backs was observed and photographed daily. On the fifth day, skin elasticity and water content were measured using an instrument (RealBubee), and the severity of skin damage was scored. The mice were then deeply anesthetized using an isoflurane gas anesthesia machine, and blood was drawn from their hearts. After blood collection, the mice were euthanized by cervical dislocation. The back skin samples were collected, aliquoted, weighed, and rapidly frozen in liquid nitrogen, then stored at -80°C. The aliquoted skin samples were wrapped in aluminum foil to prevent curling and placed in tissue cell fixation solution (4% paraformaldehyde) at 4°C for 24 hours for subsequent HE and Masson staining.

[0083] (3) Sat Skin Condition Score for Photodermatitis Table 2. Scoring Criteria for Skin Damage Severity

[0084] (4) Skin tissue sections and H&E and Masson staining HE staining: Skin tissue was fixed in 4% paraformaldehyde for 24 h, followed by dehydration and paraffin embedding. The embedded skin tissue was sectioned using a microtome, dewaxed, and hydrated with graded ethanol before being stained with hematoxylin and eosin. The stained sections were then dehydrated, cleared, and mounted with neutral resin. Images were acquired using a scanner, and skin epidermal thickness was measured using ImageJ software.

[0085] Masson staining: The initial fixation, embedding, sectioning, and dewaxing procedures were consistent with those in 3.2.12(1). Subsequently, the tissue was stained with iron hematoxylin for 10 min, bluing for 1 min, rinsed with water, and stained with Ponceau S for 1 min. After rinsing again, it was treated with 1% phosphomolybdic acid solution for 5 min. The tissue was then rapidly dehydrated with a gradient of ethanol, cleared with xylene (5 min / time, 3 times), rinsed with PBS, blotted dry, and finally mounted with neutral resin. Images were observed and acquired using an optical microscope, and the amount of collagen deposition was quantitatively analyzed using ImageJ software.

[0086]

[0087] (5) Determination of MDA, TNF-α, IL-6 and IL-1β levels in mouse skin tissue Skin tissue was ground with liquid nitrogen, weighed, and aliquoted into centrifuge tubes. An appropriate amount of PBS containing protease inhibitors was added (skin weight: PBS volume = 1 g: 9 mL). The skin tissue was homogenized using a homogenizer, and the homogenate was collected after centrifugation. The supernatant was then used to detect the levels of MDA, TNF-α, IL-6, and IL-1β according to the ELISA kit instructions.

[0088] The test results are as follows: like Figure 15 Figure A shows the experimental flowchart for UVB-induced photodermatitis in mice. Macroscopic assessment of the skin condition on the back of the mice (…) Figure 15 In group B, the control group mice had smooth, flat skin on their backs without erythema or edema, and showed no signs of inflammation. In the model group mice, mild erythema appeared on day 1 after irradiation; by day 2, the erythema and edema significantly worsened, with disordered skin texture, roughness, thickening, pigmentation, and wrinkles. On day 3, the damage further aggravated, with crusting and a leathery feel – typical characteristics of UVB-induced acute photodermatitis. After drug intervention, all groups showed varying degrees of improvement in skin damage. The ZPH-H intervention showed the most significant effect, with milder erythema and edema, and skin texture and smoothness approaching that of the normal control group. The ZH unloaded group showed slight relief of erythema and edema, but the damage was still significant, and the protective effect was slightly weaker than that of the ZPH-H group.

[0089] To more intuitively assess skin condition, skin hydration and skin elasticity indices were measured. The results are as follows: Figure 15 As shown in Figure CE, compared with the Control group, the Model group mice showed significantly reduced skin moisture content and elasticity, and significantly increased skin lesion severity scores. Compared with the model group, ZPH-H significantly increased skin moisture content and elasticity, reduced skin lesion scores, and was comparable to the positive control group (VC). The empty ZH group showed slight improvement in the above indicators. These results indicate that ZPH NPs can dose-dependently repair skin barrier damage in mice with photodermatitis and alleviate skin inflammation symptoms.

[0090] Figure 16 H&E staining and epidermal thickness statistics of skin pathology sections, from Figure 16As shown in Figure A, the Control group exhibited intact overall skin structure, with a moderate epidermal layer thickness, tightly and regularly arranged collagen fibers in the dermis, and abundant accessory glands (hair follicles and sebaceous glands). No inflammatory cell infiltration was observed, indicating that the mouse skin tissue was in good condition, structurally intact, and undamaged. The Model group showed significant inflammatory pathological changes; yellow arrows indicated infiltration of numerous inflammatory cells such as neutrophils and macrophages. The epidermis was incomplete, the epidermal and dermal structures were loose and separated, accessory gland structures disappeared, and collagen fibers were broken and disordered. The ZPH-H group showed abundant new granulation tissue in the dermis, dense angiogenesis, vigorous and regularly arranged collagen fibers, intact accessory gland structure, and a significantly reduced number of inflammatory cells. Morphological results indicate that high-dose ZPH NPs can significantly improve epidermal hyperplasia, dermal inflammation, and structural damage in mice with photodermatitis, and repair skin tissue.

[0091] HE staining combined with epidermal thickness statistics Figure 16 B) indicates that ZPH NPs can improve the histopathology of mice with photodermatitis by inhibiting inflammatory cell infiltration, promoting the reconstruction of epidermal and dermal structures, and repairing skin accessory glands and collagen tissue, demonstrating the outstanding advantages of drug-loaded nanoparticles in improving skin inflammation and structural damage.

[0092] Figure 17 Masson staining and collagen deposition quantification of skin pathological sections were performed. The results showed that in the Control group, dermal collagen fibers were dark blue and densely and regularly arranged; in the Model group, collagen fibers were disordered, lightly stained blue, and the collagen volume fraction was significantly reduced, indicating that UVB successfully induced the destruction of skin collagen structure. In the VC group, ZPH-L group, and ZPH-H group, the collagen arrangement gradually became more regular, the blue staining deepened, and the collagen volume fraction significantly recovered. Among them, there was no significant difference between the ZPH-H group and the Control group, indicating that ZPH-H NPs can significantly repair skin collagen damage in mice with photodermatitis.

[0093] like Figure 18 The expression levels of inflammatory factors and oxidative stress markers in mouse skin damage were detected by ELISA. Compared with the control group, the levels of inflammatory factors IL-6, TNF-α, IL-1β, and the oxidative stress marker MDA in mice after modeling were significantly increased, indicating that severe skin damage and antioxidant system imbalance in mice induced by UVB stimulated the release of inflammatory factors. After drug administration, the above indicators decreased to varying degrees, with the ZPH-H group showing the most significant decrease in the expression levels of IL-6, TNF-α, IL-1β, and MDA, alleviating the inflammatory response and oxidative stress in a dose-dependent manner and reversing the pathological damage of UVB-induced photodermatitis. Puerarin-based composite nanoparticles ZPH NPs show promise as a potential protective agent against photodermatitis.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing composite nanoparticles loaded with puerarin, characterized in that: Includes the following steps: (1) Dissolve zein in an aqueous ethanol solution to obtain a zein solution; (2) Add puerarin to the zein solution to obtain a mixture; (3) Pour the mixture into the acid solution and stir. Then, remove the ethanol by rotary evaporation. At the same time, use the acid solution to compensate for the volume of vaporized ethanol until the ethanol is completely evaporated, and obtain the zein-puerarin-based nanoparticle solution. (4) Add hyaluronic acid to water and adjust the pH value to obtain a hyaluronic acid solution; (5) Using the FNC125R flash nanoparticle preparation system, hyaluronic acid solution and zein-puerarin-based nanoparticle solution were mixed to obtain a mixture. After centrifuging the mixture to remove large particles, composite nanoparticles were obtained.

2. The method for preparing composite nanoparticles loaded with puerarin according to claim 1, characterized in that: In step (1), the concentration of the ethanol aqueous solution is 80-90%, and the ratio of zein to ethanol aqueous solution is 1g:40-60mL.

3. The method for preparing composite nanoparticles loaded with puerarin according to claim 1, characterized in that: In step (2), the mass ratio between puerarin and zein in the zein solution is 0.05~0.15:

1.

4. The method for preparing composite nanoparticles loaded with puerarin according to claim 1, characterized in that: In step (3), the pH value of the acid solution is 4.0, and the ratio of the mixed solution to the acid solution is 10 mL: 30~50 mL.

5. The method for preparing composite nanoparticles loaded with puerarin according to claim 1, characterized in that: In step (4), the concentration of the hyaluronic acid solution is 0.4~1.6 mg / mL and the pH value is 4.

0.

6. The method for preparing composite nanoparticles loaded with puerarin according to claim 1, characterized in that: In step (5), the specific process of mixing using the FNC125R flash nanoparticle preparation system is as follows: Channel A contains 2 mL of zein-puerarin basic nanoparticle solution with a discharge flow rate of 0.4 mL / s, and Channel B contains 2.5 mL of hyaluronic acid solution with a discharge flow rate of 0.5 mL / s. The solution flowing out of the instrument is discarded, and the large particles are collected and centrifuged to remove them. The supernatant is then taken to obtain composite nanoparticles.

7. A composite nanoparticle loaded with puerarin, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the composite nanoparticles loaded with puerarin according to claim 7, characterized in that: Application of composite nanoparticles loaded with puerarin in anti-photodermatitis products.

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