Apigenin-assembled nanoparticles, methods of making and use in anti-photoaging products

Abstract

The application provides a kind of apigenin assembled nanoparticles, preparation method and its application in anti-photoaging product, it is related to nanometer delivery technical field, including apigenin and ferulic acid are co-assembled according to 1:1.2~1.2:1 molar ratio, form binary co-assembled nanoparticles;Binary co-assembled nanoparticles and exosome are directly mixed, and apigenin assembled nanoparticles are obtained by ultrasonic treatment.In 24h in vitro transdermal experiment, skin permeability and skin retention are increased by about 232% and 207% respectively compared with API, and are increased by about 23% and 25% respectively compared with binary co-assembled nanoparticles, with high skin permeability, high percutaneous bioavailability and follicular targeting, can promote the sustained release of API in skin, play long-acting effect for photoaging treatment.Solve the technical problems that apigenin in the prior art has very low water solubility, poor skin permeability, low percutaneous bioavailability and insufficient photoaging targeting, which restricts clinical transformation.

Description

Technical Field

[0001] This invention relates to the field of nanodelivery technology, and in particular to a apigenin-assembled nanoparticle, its preparation method, and its application in anti-photoaging products. Background Technology

[0002] Clinical manifestations of photoaging include wrinkles, loss of elasticity, sagging, rough texture, hyperpigmentation, and telangiectasia. It weakens the skin barrier function and increases the risk of skin tumors such as actinic keratosis and squamous cell carcinoma. Flavonoids can reduce the production of reactive oxygen species, alleviate oxidative stress, reduce DNA damage, promote collagen repair, and regulate inflammatory factors, thereby playing a role in combating photoaging.

[0003] Apigenin (API) is a typical flavonoid compound with three phenolic hydroxyl groups at the 5', 7', and 4' positions and a C=C double bond in its epoxide ring, giving it significant antioxidant and anti-inflammatory bioactivity. Literature reports that API can reduce free radicals, activate antioxidant signaling pathways, enhance the activity of intracellular antioxidant enzymes, and reversibly inhibit tyrosinase activity in melanin biosynthesis. However, API suffers from extremely low water solubility, a high oil-water partition coefficient, and low skin permeability and transdermal bioavailability, severely limiting its application in anti-photoaging.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for preparing apigenin-assembled nanoparticles, in order to solve the technical problems in the prior art that apigenin has extremely low water solubility, poor skin permeability, low transdermal bioavailability, and insufficient photoaging targeting, which restricts its clinical translation.

[0006] The second objective of this invention is to provide apigenin-assembled nanoparticles.

[0007] A third objective of this invention is to provide the application of apigenin-assembled nanoparticles prepared by the above-described preparation method or the above-described apigenin-assembled nanoparticles in the preparation of anti-photoaging products.

[0008] The fourth objective of this invention is to provide an anti-photoaging composition.

[0009] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a method for preparing apigenin-assembled nanoparticles, comprising the following steps: A. co-assembling apigenin and ferulic acid in a molar ratio of 1:1.2 to 1.2:1 to form binary co-assembled nanoparticles; B. The binary co-assembled nanoparticles and exosomes were directly mixed and ultrasonically treated to obtain apigenin-assembled nanoparticles; the mass ratio of the exosomes to the binary co-assembled nanoparticles was 7.5:2.5 to 8:2; the exosomes were licorice exosomes.

[0010] Furthermore, the co-assembly process involves dissolving apigenin and ferulic acid in a solvent, adjusting the pH to 7.7–8.5, removing the solvent, and then incubating at 60–70°C for 2 hours. Preferably, the solvent removal includes rotary evaporation to remove the solvent; Preferably, the solvent includes methanol.

[0011] Furthermore, the ultrasound session lasts for 8 to 13 minutes; Preferably, the power of the ultrasound is 120~180W.

[0012] Furthermore, the exosomes are prepared by differential centrifugation. Preferably, the differential centrifugation method includes centrifuging at 3000g for 40 min, centrifuging at 12000g for 1.5 h at 4°C, taking the supernatant and ultracentrifuging at 160000g for 2.5 h, resuspending the precipitate in phosphate buffer, and filtering to obtain exosomes.

[0013] Secondly, the present invention provides apigenin-assembled nanoparticles, which are prepared by the above-described preparation method.

[0014] Furthermore, the nanoparticles include binary co-assembled nanoparticles and exosomes encapsulated around the binary co-assembled nanoparticles.

[0015] Furthermore, the particle size of the nanoparticles is 200~300nm.

[0016] Thirdly, the present invention provides the application of apigenin-assembled nanoparticles prepared by the above-described preparation method or the above-described apigenin-assembled nanoparticles in the preparation of anti-photoaging products.

[0017] Furthermore, the product is a topical transdermal drug delivery formulation; Preferably, the dosage form of the formulation includes at least one of cream, gel, microemulsion, liposome dispersion or nanosuspension.

[0018] Fourthly, the present invention provides an anti-photoaging composition comprising apigenin-assembled nanoparticles prepared by the above-described preparation method or the above-described apigenin-assembled nanoparticles. Preferably, the composition further includes a pharmaceutically acceptable carrier.

[0019] This invention provides a method for preparing apigenin-assembled nanoparticles. The method involves forming binary co-assembled nanoparticles from API and FA, which are then encapsulated in exosomes. The three components synergistically improve the water solubility and stability of API, resulting in a clear and transparent liquid. FA not only synergistically enhances the water solubility and stability of API but also enhances its anti-photoaging effect. In a 24-hour in vitro transdermal experiment, the skin permeability and skin retention were increased by approximately 232% and 207% compared to API, respectively, and by approximately 23% and 25% compared to the binary co-assembled nanoparticles, respectively. This method exhibits high skin permeability, high transdermal bioavailability, and follicle targeting, promoting the sustained release of active API components within the skin and providing a long-lasting effect for photoaging treatment. This invention solves the technical problems of extremely low water solubility, poor skin permeability, low transdermal bioavailability, and insufficient photoaging targeting of apigenin in existing technologies, which have hindered clinical translation. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 A schematic diagram illustrating the preparation process of API-FA and API-FA@Exo nanoparticles provided in this embodiment of the invention; Figure 2 Comparative images of the appearance of API, API-FA, and API-FA@Exo formulations provided in embodiments of the present invention; Figure 3 Comparative images of the appearance of apigenin and chlorogenic acid, glycyrrhizic acid, cinnamic acid, gallic acid and caffeic acid after being co-assembled according to embodiments of the present invention; Figure 4 Morphological observation diagrams of Exo, API-FA, and API-FA@Exo provided for embodiments of the present invention; Figure 5 Comparison of the appearance of the co-assembled nanoparticles of apigenin, chlorogenic acid, and glycyrrhizic acid provided in this embodiment of the invention after being placed for 1 month; Figure 6 Morphological observation images of the co-assembled nanoparticles of API-FA with aloe vera exosomes and ginseng exosomes provided in the embodiments of the present invention; Figure 7 The following are observation diagrams of the skin penetration of API components in different formulations provided in the embodiments of the present invention at 3 hours, 6 hours and 9 hours. In the diagram, the red arrows represent hair follicles, and the scale bar is 100 μm. Figure 8 The results of β-galactosidase staining of HSF cells after treatment with different agents are provided in the embodiments of the present invention. In the figure, the red circles represent positive HSF cells stained blue, Bar=100 μm. Figure 9 The reactive oxygen species staining results of HSF cells treated with different agents are provided in the embodiments of the present invention, wherein Bar=100 μm; Figure 10 The following images illustrate the appearance, epidermal thickness, inflammatory infiltration, and arrangement of different groups of skin in embodiments of the present invention. A represents the timeline of ultraviolet irradiation and drug administration; B shows representative images of photoaged back skin at different time points during a 6-week period (different colored boxes represent different recovery states); C shows H&E staining of photoaged skin after 6 weeks, with black arrows representing the epidermis; D shows Masson staining; and E shows Sirius red staining of photoaged skin after 6 weeks, with scales of 400 μm or 100 μm and n=5. Figure 11 The following is a statistical chart of collagen content in different groups of skin provided in the embodiments of the present invention, wherein A is the epidermal thickness measured from HE staining images using Image J software; B is the transepidermal water loss (TEWL) of back skin after different treatments and C is the stratum corneum water content; D is the amount of collagen deposition calculated from Masson staining images using Image J software, n=5. Detailed Implementation

[0022] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0023] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0024] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] The present invention provides a method for preparing apigenin-assembled nanoparticles, comprising the following steps: A. co-assembling apigenin and ferulic acid in a molar ratio of 1:1.2 to 1.2:1 to form binary co-assembled nanoparticles; B. The binary co-assembled nanoparticles and exosomes were directly mixed and ultrasonically treated to obtain apigenin-assembled nanoparticles; the mass ratio of the exosomes to the binary co-assembled nanoparticles was 7.5:2.5 to 8:2; the exosomes were licorice exosomes.

[0026] The apigenin-assembled nanoparticles prepared in this invention form binary co-assembled nanoparticles through API and FA, which are then encapsulated by exosomes. The three components synergistically improve the water solubility and stability of API, resulting in a clear and transparent liquid. FA not only synergistically improves the water solubility and stability of API but also enhances its anti-photoaging effect. In a 24-hour in vitro transdermal experiment, the skin permeability and skin retention were increased by approximately 232% and 207% compared to API, respectively, and by approximately 23% and 25% compared to the binary co-assembled nanoparticles, respectively. This demonstrates high skin permeability, high transdermal bioavailability, and follicle targeting, promoting the sustained release of active API components within the skin and providing a long-lasting effect for photoaging treatment. This invention solves the technical problems of extremely low water solubility, poor skin permeability, low transdermal bioavailability, and insufficient photoaging targeting of apigenin in existing technologies, which have hindered clinical translation.

[0027] The molar ratio of apigenin to ferulic acid can be, but is not limited to, 1:1.2, 1:1.1, 1:1, 1.1:1 or 1.2:1, or any value between 1:1.2 and 1.2:1.

[0028] The mass ratio of exosomes to binary co-assembled nanoparticles can be, but is not limited to, 7.5:2.5, 7.6:2.4, 7.7:2.3, 7.8:2.2, 7.9:2.1 or 8:2, or any value between 7.5:2.5 and 8:2.

[0029] In some specific embodiments, the licorice exosomes may be selected from at least one of Ural licorice, inflatable licorice and glossy licorice, preferably Ural licorice.

[0030] In some specific embodiments, co-assembly involves dissolving apigenin and ferulic acid in a solvent, adjusting the pH to 7.7–8.5, removing the solvent, and incubating at 60–70°C for 2 hours. This method can stably maintain the particle size of apigenin-assembled nanoparticles at 200–300 nm and PDI ≤ 0.30.

[0031] The pH value can be, but is not limited to, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5, or any value between 7.7 and 8.5.

[0032] The incubation temperature can be, but is not limited to, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃ or 70℃, or any temperature between 60℃ and 70℃.

[0033] In some specific embodiments, the solvent removal includes rotary evaporation; in some specific embodiments, the solvent includes methanol. In some specific embodiments, the ultrasonic treatment lasts for 8 to 13 minutes; in some specific embodiments, the ultrasonic power is 120 to 180 W.

[0034] The duration of the ultrasound can be, but is not limited to, 8, 9, 10, 11, 12, or 13 minutes, or any time between 8 and 13 minutes.

[0035] In some specific embodiments, the exosomes are prepared by differential centrifugation. In some specific embodiments, the differential centrifugation includes centrifuging at 3000g for 40 min, centrifuging at 12000g for 1.5 h at 4°C, taking the supernatant and ultracentrifuging at 160000g for 2.5 h, resuspending the precipitate in phosphate buffer, and filtering to obtain exosomes.

[0036] According to another aspect of the present invention, apigenin-assembled nanoparticles are also provided, which are prepared by the above-described preparation method.

[0037] The apigenin-assembled nanoparticles exhibit a distinct bilayer membrane structure. In some specific embodiments, the nanoparticles include binary co-assembled nanoparticles and exosomes encapsulated around the binary co-assembled nanoparticles.

[0038] In some specific embodiments, the particle size of the nanoparticles is 200~300nm.

[0039] According to another aspect of the present invention, the application of apigenin-assembled nanoparticles prepared by the above-described preparation method or the above-described apigenin-assembled nanoparticles in the preparation of anti-photoaging products is also provided.

[0040] In some specific embodiments, the product is a topical transdermal drug delivery formulation; In some specific embodiments, the dosage form of the formulation includes at least one of cream, gel, microemulsion, liposome dispersion or nanosuspension.

[0041] According to another aspect of the present invention, an anti-photoaging composition is also provided, comprising apigenin assembled nanoparticles prepared by the above-described preparation method or the above-described apigenin assembled nanoparticles. In some specific embodiments, the composition further includes a pharmaceutically acceptable carrier.

[0042] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0043] Example I. Preparation of Apigenin-Assembled Nanoparticles 1. Preparation of licorice exosomes Fresh Ural licorice was washed, crushed, and ground before licorice exosomes were extracted using differential centrifugation. The specific steps are as follows: The juice was centrifuged at 3000 g for 40 min at 4°C, followed by centrifugation at 12000 g for 1.5 h. The supernatant was collected and centrifuged at 160000 g for 2.5 h using an ultracentrifuge to precipitate licorice exosomes. The precipitate was resuspended in phosphate buffer (pH 6–7.5), filtered through a 0.22 μm filter membrane, and the filtrate was the exosome solution, which was stored at -80°C. Licorice exosomes (Exo) were confirmed by high-performance liquid chromatography and transmission electron microscopy.

[0044] 2. Preparation of API-FA co-assembled nanoparticles Nanoparticles were prepared according to the single-factor levels in Table 1 and the single-factor combinations of each experimental group shown in Table 2. The specific methods are as follows: Ferulic acid and apigenin were dissolved in 2 mL of methanol at their corresponding molar ratios. 10% (M / V) NaOH was added to aid dissolution and the pH was adjusted to 7.7–8.5. The mixture was then added dropwise to 10 mL of preheated hot water (60°C), and the methanol was removed by rotary evaporation. The mixture was then incubated in a water bath for 2 hours to obtain API-FA nanoparticles.

[0045] The particle size distribution, polymer dispersibility index (PDI), and zeta potential of API-FA nanoparticles were determined using a Zetasizer Nano ZS microscope. The morphology and structure of the nanoparticles were observed using a transmission electron microscope (TEM, H-7650) at 60 kV. Successful co-assembly was confirmed by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction, ROESY two-dimensional nuclear magnetic resonance spectroscopy, and molecular dynamics simulations.

[0046] Table 1 Specific Single Factors

[0047] When the molar ratio of API to FA is 1:1, the amount of API is 13.01 mg and the amount of FA is 9.7 mg.

[0048] Table 2

[0049] 3. Preparation of API-FA / Exo co-assembled bilayer nanoparticles Nanoparticles were prepared according to the single-factor levels in Table 3 and the single-factor combinations of each experimental group shown in Table 4. API-FA nanoparticles were then coated with licorice exosomes using an ultrasonic method. The API-FA nanoparticles were the sample prepared in step A-2. The specific steps are as follows: API-FA and licorice exosomes were added according to the corresponding method and mass ratio, and then sonicated to prepare API-FA@Exo solution.

[0050] Table 3 Specific Single Factors

[0051] Table 4

[0052] II. Effect Verification 1. In vitro transdermal test In vitro transdermal experiments were conducted using fresh, intact porcine skin fitted into a TP-6 ​​Franz diffusion cell. Subcutaneous tissue was carefully removed, and the skin was fixed between the supply and receiving chambers, with the stratum corneum side facing upwards. The receiving chamber contained a receiving solution of PEG 400 and PBS (pH 7.4) at a volume ratio of 7:3, and the temperature was maintained at 32°C with stirring at 350 rpm. 0.3 mL of API-FA, API-FA@Exo, or API solution (as a control) was applied to the skin surface. 1 mL samples were taken from the receiving chamber at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h, and an equal volume of pre-warmed fresh receiving solution was immediately added. The concentration of API in the samples at different time points was determined by high-performance liquid chromatography (HPLC).

[0053] After the transdermal assay, skin samples were carefully removed and rinsed with saline. The stratum corneum and subcutaneous tissue were separated using an adhesive tape peeling method, and then minced and extracted using ultrasound. The cumulative amounts of API and FA in the stratum corneum and subcutaneous tissue were quantitatively analyzed by HPLC. Furthermore, the dynamic penetration behavior of API in the skin was visualized at 3h, 6h, and 9h time points using laser scanning confocal microscopy.

[0054] 2. Cellular anti-photoaging experiment HSF cells were loaded at a rate of 1×10 5 Seeds were planted at a density of cells / well in 12-well culture plates, followed by UVA irradiation (12 J / cm²). 2 A photoaging model was established, which was divided into a model group, a positive group, and a sample group.

[0055] Control group: HSF cells that have not undergone photoaging treatment; Model group: Photoaging model; Positive group (VC): photoaging model + VC; FA group: photoaging model + FA; API Group: Photoaging Model + API; API-FA group: Photoaging model + API-FA; API-FA@Exo Group: Aging Model + API-FA@Exo After treating different samples for 24 h, cells and supernatants were collected for analysis. β-galactosidase staining was used to observe cell senescence. The TNF-α content in the supernatant was detected by ELISA, and the mRNA expression levels of col1A1 and p16 in cell lysates were detected by RT-qPCR. ROS staining was performed to observe the antioxidant effects of different formulations.

[0056] 3. Animal in vivo anti-photoaging experiment 1. Animals: Six-week-old female Babl / c mice. After one week of acclimatization, the fur on the back of the mice was carefully removed.

[0057] 2. Grouping: Mice were randomly divided into six groups (n=5 per group), including: Control group: No irradiation; Photoaging model group (Model): UV; Positive control group: UV + Vitamin C; Treatment group 1: UV + API; Treatment group 2: UV + API-FA; Treatment group 3: UV+API-FA@Exo.

[0058] 3. Drug Administration: The photoaging experiment lasted for 6 weeks. The irradiation dose in the first and second weeks was one minimum erythema dose (MED), including UVA (1200 mJ / cm²) and UVB (200 mJ / cm²). The dose was doubled to two MEDs in weeks three through six. During the last four weeks of irradiation, the appropriate formulation (API, API-FA, API-FA@Exo, VC) or PBS (control group) was applied topically to the back skin once daily. Skin desquamation, wrinkles, leathery thickening, edema, and pigmentation were regularly photographed and recorded. After drug administration, skin tissue specimens were collected for H&E staining, Masson staining, and Sirius red staining.

[0059] III. Results 1. Results of the investigation on the API-FA binary co-assembly nanoparticle process The effects of the molar ratio of API to FA, pH, and incubation temperature on the particle size and PDI of API-FA co-assembled nanoparticles were investigated. Smaller particle size allows for easier penetration into the skin and deposition in hair follicles; a lower PDI value results in a narrower particle size distribution and more uniform particle size. The results are shown in Table 5. A 1:1 molar ratio of API to FA yields smaller particle size and PDI values; increasing the content of either API or FA significantly increases both particle size and PDI. The molar ratio of API to FA is the most significant factor affecting the API-FA particle size. A molar ratio between 1:1.2 and 1.2:1 keeps the particle size and PDI values ​​within a stable, relatively small range, with 1:1 being the preferred ratio.

[0060] When the solution pH is 8 and the incubation temperature is 60℃, the obtained particle size and PDI value are relatively small. Increasing or decreasing the solution pH, or increasing or decreasing the incubation temperature, will increase the particle size and PDI value of the nanoparticles. Adjusting the pH between 7.7 and 8.5 and the incubation temperature between 60℃ and 70℃ can keep the particle size and PDI value of the nanoparticles stable within a small range. The preferred pH is 8.0 and the incubation temperature is 60℃.

[0061] Table 5. Effects of different factors on particle size and PDI of API-FA co-assembled nanoparticles

[0062] 2. Results of the API-FA / Exo bilayer nanoparticle process investigation As shown in Table 6, when the two are directly mixed, the particle size and PDI value of the bilayer nanoparticles are relatively small. When Exo is added dropwise to API-FA, the particle size and PDI value of the bilayer nanoparticles increase; however, when API-FA is added to Exo, Exo cannot effectively encapsulate API-FA, making it prone to breakage. Therefore, the order of addition is the most significant factor affecting the particle size and PDI value of API-FA / Exo.

[0063] When the mass ratio of API-FA to Exo is 7.5:2.5 and 8:2, Exo can effectively encapsulate API-FA. However, when the mass ratio is 9:1, Exo cannot encapsulate API-FA, indicating that further increasing the Exo content actually increases the particle size of the composite nanoparticles. An ultrasonic time of 8–13 min is beneficial for the encapsulation of API-FA / Exo nanoparticles. Shorter ultrasonic times result in insufficient Exo encapsulation of API-FA, while increasing the ultrasonic time causes the initially encapsulated nanoparticles to break down. Therefore, when the mass ratio of API-FA to Exo is between 7.5:2.5 and 8:2, and the ultrasonic time is between 8 and 13 min, the particle size and PDI value of the bilayer nanoparticles can be stably maintained within a small range.

[0064] Table 6. Effects of different factors on particle size and PDI of API-FA / Exo co-assembled nanoparticles

[0065] 3. Effects of A-2 and D-3 co-assembled nanoparticles on API water solubility and morphology The preparation process diagrams of API-FA (A-1) and API-FA@Exo (D-3) are shown below. Figure 1 As shown. Figure 2 As shown, API has very low solubility in water, resulting in a turbid solution. After co-assembling with FA into nanoparticles, the solution becomes clear and transparent, without particles or precipitates, and the solubility increases significantly. This indicates that co-assembling API and FA into nanoparticles can significantly improve the water solubility of API. A control group was set up using chlorogenic acid, glycyrrhizic acid, cinnamic acid, gallic acid, and caffeic acid to replace ferulic acid in the preparation method A-2 to assemble nanoparticles. The results are as follows. Figure 3 As shown, only chlorogenic acid and glycyrrhizic acid can co-assemble to form a relatively clear and transparent solution; while cinnamic acid, gallic acid and caffeic acid cannot co-assemble to form nanoparticles, and the resulting solution is relatively turbid, indicating that apigenin cannot dissolve well in the solution.

[0066] like Figure 2 As shown, API-FA@Exo is lighter in color compared to API-FA, which is due to the encapsulation effect of exosomes. Transmission electron microscopy (TEM) observations are as follows... Figure 4 As shown, API-FA nanoparticles are approximately spherical, while API-FA@Exo nanoparticles exhibit a distinct bilayer membrane structure, indicating that API-FA@Exo bilayer nanoparticles were successfully prepared and that licorice exosomes can encapsulate API-FA.

[0067] 4. Stability of API co-assembled with other phenolic acids nanoparticles The stability of the co-assembled nanoparticles of API, chlorogenic acid (LYS), and glycyrrhizic acid (GCS) was compared after being placed at 25°C for one month.

[0068] The results are as follows Figure 5 As shown, after one month at 25°C, API-LYS exhibited particulate precipitation, while the API-GCS solution became cloudy, indicating poor stability of the nanoparticles co-assembled with API, LYS, and GCS, respectively. Conversely, the API-FA solution remained clear and transparent after one month, suggesting that co-assembly with API and FA improves the stability of the nanoparticles. Furthermore, the particle size and PDI of API-LYS were 304.24 nm and 0.34, respectively, while those of API-GCS were 339.15 nm and 0.38, respectively. Due to their relatively large particle size and PDI values, they are not suitable for transdermal application.

[0069] 5. Co-assembly of API-FA (A-2) and other plant-derived exosomes Following the preparation conditions of D-3, aloe vera exosomes (LH Exo) and ginseng exosomes (RS Exo) were used instead of licorice exosomes for co-assembly. The morphological observation results are as follows: Figure 6 As shown, aloe vera exosomes cannot co-assemble with API-FA (A-2) and cannot form co-assembled nanoparticles with a bilayer; while ginseng exosomes and licorice exosomes can form composite nanoparticles with a distinct bilayer with API-FA (A-2).

[0070] 6. Transdermal permeability of different binary co-assembled nanoparticles and exosome-coated bilayer nanoparticles Skin permeability tests were conducted to evaluate the transdermal performance of nanoparticles prepared using different methods. The results are shown in Table 7. Among the binary co-assembled nanoparticles, the 24-h skin permeation and skin retention of A-1~A-3, B-2~B-4, and C-3~C-4 were significantly increased, with A-2 showing the best results. This indicates that nanoparticles under suitable API-FA molar ratios, pH, and incubation temperature conditions can easily penetrate the stratum corneum. In the exosome-coated bilayer nanoparticles, compared with A-2, the 24-h skin permeation and skin retention of D-2, E-1, and F-5 were all reduced, while the skin permeation and skin retention of D-3, E-2, F-2, and F-4 were further increased, indicating that after exosome coating, exosomes and API-FA can synergistically improve the skin permeability of API. Moreover, the permeation and retention of API and FA in A-2 and D-3 were significantly higher than those of free API, indicating that the preparation of binary nanoparticles and their exosome-coated nanoparticles can significantly improve the transdermal performance of API.

[0071] It was found that when the molar ratio of API to FA in API-FA was between 1:1.2 and 1.2:1, the pH was between 7.7 and 8.5, and the incubation temperature was between 60℃ and 70℃, the skin penetration and retention of API did not change significantly. Furthermore, when the mass ratio of API-FA to Exo in API-FA@Exo was between 7.5:2.5 and 8:2, and the sonication time was between 8 and 13 minutes, the skin penetration and retention of API also did not change significantly.

[0072] Table 7. Skin permeability and skin retention in different groups (μg / (cm³)) 2 ·h))

[0073] 7. Dynamic permeation behavior of A-2 and D-3 co-assembled nanoparticles Fluorescence microscopy observation results as follows Figure 7 As shown, free APIs could hardly penetrate the skin within 3 hours, while API-FA@Exo and API-FA both achieved significant penetration after 3 hours. At this time, API-FA mainly accumulated in the epidermal layer, while API-FA@Exo penetrated into deeper skin layers. After 6 hours, the fluorescence intensity of API-FA@Exo reached its peak, distributed throughout the entire skin layer, and its intensity was significantly higher than that of API-FA and API. Furthermore, after 9 hours, no significant difference was observed in the fluorescence intensity of API-FA@Exo and API-FA, but both were significantly higher than API, consistent with the in vitro penetration results. Due to the tendency of 200-300 nm nanoparticles to deposit and accumulate in hair follicles, API-FA@Exo and API-FA mainly penetrated through the hair follicle pathway, and the fluorescence intensity in the hair follicle region was higher than that in the non-hair follicle region, indicating that both have hair follicle targeting. Hair follicles, as drug reservoirs, can promote the continuous release of active API components in the skin, playing a long-term role in the treatment of photoaging of the skin.

[0074] 8. Skin permeability of API-FA@RS Exo exosomes Compared to A-2, API-FA@RS Exo did not significantly increase the skin permeation and retention of A-2, and its effect was significantly lower than that of D-3, indicating that ginseng exosomes and API-FA do not have a synergistic effect in improving API skin permeability. Although ginseng exosomes can also encapsulate API-FA, they cannot improve the skin permeability of API-FA.

[0075] Table 8. Skin penetration and retention of API-FA@RS Exo

[0076] 9. Anti-photoaging effects of different binary nanoparticles and their exosome-coated nanoparticles on cells Photoaging mechanisms include oxidative stress, collagen degradation, inflammatory responses, and cellular senescence. This study evaluated the anti-photoaging effects of binary nanoparticles and their exosome-coated nanoparticles prepared using different methods by measuring the levels of p16 mRNA, TNF-α, and Col1A1 in different groups.

[0077] The results are shown in Table 9. Compared with the blank control group, the model group showed a significant increase in P16 mRNA level, a significant increase in TNF-α level, and a significant decrease in Col1A1 level, indicating that the photoaging cell model was successfully established. Compared with the model group, in API-FA, A-1~A-2, B-4, C-1, and C-3 all significantly decreased P16 mRNA expression level and TNF-α level, and increased Col1A1 content. Meanwhile, in the API-FA@Exo group, compared with A-2, D-3, E-2, and F-4 significantly decreased P16 mRNA expression level and TNF-α level, and increased Col1A1 content, indicating that the exosome-coated composite nanoparticles had a stronger anti-photoaging effect. In addition, A-2 and D-3 showed significantly better ability to improve P16, TNF-α, and Col1A1-related indicators than API or FA alone. The above suggests that, under optimal preparation processes, API-FA and API-FA@Exo also exhibit enhanced anti-collagen degradation, anti-inflammatory response, and cell aging effects, thereby significantly improving their anti-photoaging properties.

[0078] Furthermore, compared to A-2, API-FA@RS Exo could not further reduce P16 mRNA expression levels, TNF-α levels, or increase Col1A1 content; its effect was significantly lower than D-1, indicating that ginseng exosomes and API-FA do not have a synergistic effect in resisting photoaging. Therefore, even though ginseng exosomes can encapsulate API-FA, they cannot enhance the anti-photoaging effect of API-FA.

[0079] Table 9. Effects of different groups on the expression of p16 mRNA and inflammatory factor TNF-α in HSF cells.

[0080] 10. Cellular anti-photoaging effect of A-2 and D-1 co-assembled nanoparticles β-galactosidase staining, as a marker of cellular senescence, was used to detect photoaging-induced senescent cells. Results are as follows: Figure 8As shown, no positive blue staining was observed in the blank control group, while the model group exhibited dense, deep blue cells, indicating that the photoaging model was successfully constructed. Compared with the model group, the number of positive cells in the VC group was significantly reduced, and the blue staining degree in the API group was similar to that in the model group, confirming its weak anti-photoaging ability. The number of blue-stained cells in the API-FA group was significantly reduced, and its anti-photoaging effect was better than that of API alone; while in the API-FA@Exo group, only a very few lightly stained cells were observed, and their level almost returned to normal, indicating that the assembly of licorice exosomes, API, and FA has a synergistic effect on anti-photoaging.

[0081] Oxidative stress levels were assessed using reactive oxygen species (ROS) detection. Figure 9 As shown, photoaging induced a significant accumulation of intracellular reactive oxygen species (ROS), and all sample groups were able to reverse this effect. Among them, API-FA@Exo exhibited the strongest ROS inhibition and the best antioxidant effect. API-FA@Exo treatment significantly restored ROS generation levels, bringing them close to normal levels, and outperformed the positive control, indicating that the assembly of licorice exosomes, API, and FA has a synergistic effect in inhibiting ROS.

[0082] 11. In vivo anti-photoaging effects of PI and its co-assembled systems To investigate the in vivo anti-photoaging effects of APIs and their co-assembled systems, such as... Figure 10 As shown in Figure A, a skin photoaging model was established on the back skin of Babl / c mice using a combined UVA and UVB irradiation method.

[0083] like Figure 10 As shown in Figure B, after 6 weeks of UV irradiation, the model group's skin exhibited typical photoaging characteristics, including severe wrinkles, desquamation, dryness, roughness, and a leathery appearance. In the API-administered group, significant desquamation, roughness, and leathery changes were still observed, indicating poor skin permeability and failure to effectively reverse the photoaging phenotype. With the aid of API-FA@Exo or API-FA delivery, skin wrinkles and leathery texture were significantly reduced, and skin smoothness and elasticity were significantly improved, with the overall condition approaching that of the positive control group; however, no significant difference was observed between the API-FA@Exo group and the API-FA group. Transdermal water loss (TEWL) and skin hydration were subsequently measured. Figure 11 In groups B and C, the model group showed a significant increase in TEWL and a significant decrease in hydration compared to the control group, while the API treatment group showed no significant difference.

[0084] Skin samples were collected and subjected to H&E staining to assess epidermal thickness, degree of keratinization, and inflammatory infiltration. Figure 10 C and Figure 11As shown in Figure A, compared to the control group, the model group exhibited significantly increased epidermal thickness accompanied by marked hyperkeratosis, which is the cause of the leathery and rough appearance of the skin. Furthermore, significant inflammatory cell infiltration was observed in the dermis. After treatment with API-FA@Exo or API-FA, epidermal thickness was significantly reduced, hyperkeratosis was alleviated, inflammatory infiltration decreased, and the histological state approached that of the normal control group. Although API treatment alone improved epidermal thickening and inflammatory infiltration to some extent, its effect was significantly inferior to the co-assembled formulation of both APIs, confirming the synergistic effect of API and FA in restoring the tissue structure of photoaged skin.

[0085] In photoaged skin, type I collagen is the main structural protein, providing tension and support, while type III collagen maintains skin elasticity and suppleness. UV radiation upregulates the expression of matrix metalloproteinases, accelerating collagen degradation and inhibiting its synthesis, leading to a significant reduction in both types of collagen, with a more pronounced decrease in type I collagen. This results in an imbalance in the type I / III collagen ratio, leading to skin laxity and deepened wrinkles. Masson staining was used to assess the number, arrangement, and spatial distribution of collagen fibers in photoaged skin. Figure 10 As shown in D in the figure, the model group exhibited a significant reduction in collagen fiber content, with disordered arrangement and uneven distribution, resulting in skin laxity and wrinkles. After treatment with API-FA@Exo or API-FA, the arrangement and quantity of collagen fibers recovered to near-normal levels, indicating that it can improve collagen loss caused by photoaging. Figure 11 The results showed that API treatment alone failed to restore collagen content or arrangement in photoaged skin. Analysis using Sirius red staining, such as... Figure 10 As shown in Figure E, the levels of both type I and type III collagen in the model group were significantly decreased, with the reduction in type I collagen being more pronounced. Furthermore, the disordered arrangement and uneven distribution of type I and type III collagen were observed in both the model group and the API group, consistent with Masson staining results. Both API-FA@Exo and API-FA significantly increased the ratio of type I / III collagen, while API alone failed to effectively increase this ratio. In conclusion, A-2 and D-1 possess strong in vivo anti-photoaging effects.

[0086] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing apigenin-assembled nanoparticles, characterized in that, Includes the following steps: A. Apigenin and ferulic acid were co-assembled in a molar ratio of 1:1.2 to 1.2:1 to form binary co-assembled nanoparticles; B. Binary co-assembled nanoparticles and exosomes were directly mixed and ultrasonically treated to obtain apigenin-assembled nanoparticles. The mass ratio of the exosomes to the binary co-assembled nanoparticles is 7.5:2.5 to 8:2; The exosomes are licorice exosomes.

2. The preparation method according to claim 1, characterized in that, The co-assembly process involves dissolving apigenin and ferulic acid in a solvent, adjusting the pH to 7.7-8.5, removing the solvent, and then incubating at 60-70°C for 2 hours. The solvent removal includes solvent removal via rotary evaporation; The solvent includes methanol.

3. The preparation method according to claim 1, characterized in that, The ultrasound session lasted 8-13 minutes. The power of the ultrasound is 120~180W.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The exosomes were prepared by differential centrifugation. The differential centrifugation method includes centrifuging at 3000g for 40 min and 12000g for 1.5 h at 4°C, taking the supernatant and ultracentrifuging at 160000g for 2.5 h, resuspending the precipitate in phosphate buffer, and filtering to obtain exosomes.

5. A kind of apigenin-assembled nanoparticles, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 4.

6. The apigenin-assembled nanoparticles according to claim 5, characterized in that, The nanoparticles include binary co-assembled nanoparticles and exosomes encapsulated around the binary co-assembled nanoparticles.

7. The apigenin-assembled nanoparticles according to claim 6, characterized in that, The nanoparticles have a particle size of 200~300nm.

8. The application of the apigenin-assembled nanoparticles prepared by the preparation method according to any one of claims 1 to 4, or the apigenin-assembled nanoparticles according to any one of claims 5 to 7, in the preparation of anti-photoaging products.

9. The application according to claim 8, characterized in that, The product is a topical transdermal drug delivery formulation; The dosage form of the formulation includes at least one of cream, gel, microemulsion, liposome dispersion or nanosuspension.

10. An anti-photoaging composition, characterized in that, This includes apigenin-assembled nanoparticles prepared by the preparation method according to any one of claims 1 to 4, or apigenin-assembled nanoparticles according to any one of claims 5 to 7.

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