Azelaic acid nanocrystals, their preparation methods, and their application in the preparation of topical acne treatments.

Abstract

This application relates to the field of nanoparticle technology, and in particular to an azelaic acid nanocrystal, its preparation method, and its application in the preparation of topical acne treatment formulations. The method uses ethanol and propylene glycol as solvents and PVA as a stabilizer to prepare spherical microcrystalline AZANPs via a pH-controlled phase inversion method. These AZANPs have a small hydrated particle size, good dispersion uniformity, and good transdermal penetration. The in vitro transdermal transdermal absorption of the prepared 5% nano-ointment is approximately 2.5 times that of a 12% active pharmaceutical ingredient ointment. In an acne model, it achieves efficacy comparable to a 10% commercially available ointment with half the drug loading, and exhibits good safety.

Description

Technical Field

[0001] This application relates to the field of nanoparticle technology, and in particular to an azelaic acid nanocrystal, its preparation method, and its application in the preparation of topical acne treatments. Background Technology

[0002] Azelaic acid (AZA) is a naturally occurring saturated straight-chain nine-carbon dicarboxylic acid found in plants and animals. It possesses multiple pharmacological activities, including antibacterial, anti-inflammatory, and competitive inhibition of tyrosinase activity, and is widely used for the topical treatment of skin diseases such as acne and rosacea. However, due to its highly crystalline molecular structure and poor water-oil solubility, azelaic acid has significant limitations in transdermal absorption, making it difficult to penetrate the dense stratum corneum to reach the target site in the dermal pilosebaceous unit. Commercially available azelaic acid preparations (such as 15% gel and 20% cream) typically require extremely high drug concentrations to achieve effective antibacterial and anti-inflammatory effects. However, high concentrations of azelaic acid preparations are often accompanied by adverse reactions such as local burning sensation, erythema, and skin irritation, severely impacting patient compliance and treatment efficacy.

[0003] Nanocrystal technology, by reducing drug particle size to the nanoscale (typically 100–1000 nm), can significantly improve the dissolution rate and transdermal penetration of poorly soluble drugs without introducing large amounts of organic solvents. The pH-shift method is a simple and efficient nanocrystal preparation technique that induces phase inversion of the drug through pH changes, achieving controlled growth of nanocrystals under the control of stabilizers. However, no studies have yet reported on the preparation of azelaic acid nanocrystals using this technique for transdermal delivery in the treatment of acne. Summary of the Invention

[0004] Based on this, the purpose of this application is to overcome the defects of poor transdermal absorption of azelaic acid and strong irritation of high-concentration formulations in the prior art, and to provide azelaic acid nanocrystals and their preparation method, azelaic acid nanocrystal ointment and its application in the preparation of topical acne treatment formulations, in order to achieve transdermal delivery efficiency and therapeutic effect comparable to or better than high-concentration commercially available formulations under the condition of reducing drug loading.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] First, this application provides a method for preparing azelaic acid nanocrystals, comprising the following steps:

[0007] S1. Azelaic acid powder, polyvinyl alcohol aqueous solution, and organic mixed solvent are mixed and the azelaic acid powder is dissolved under stirring to form a homogeneous solution; the organic mixed solvent is ethanol and propylene glycol, and the volume ratio of ethanol to propylene glycol is 6~7:3~4; in the homogeneous solution, the mass concentration of azelaic acid is 5~20% w / v; the mass concentration of polyvinyl alcohol is 0.1~5.0% w / v;

[0008] S2. Under stirring conditions, add a pH adjuster dropwise to the homogeneous solution until the homogeneous solution turns light blue and the Tyndall effect can be observed. Stop the titration when the pH value of the resulting system is 5-6, and obtain azelaic acid nanocrystal suspension.

[0009] S3. The azelaic acid nanocrystal suspension is placed at 50-60℃ for 6-8 hours to allow the ethanol to fully evaporate, and then freeze-dried to obtain azelaic acid nanocrystals.

[0010] Preferably, the mass concentration of azelaic acid in the homogeneous solution obtained in step S1 is 15%~20% w / v.

[0011] Preferably, the mass concentration of polyvinyl alcohol in the homogeneous solution obtained in step S1 is 0.15~2.5% w / v.

[0012] Preferably, in step S1, the mass ratio of azelaic acid powder to polyvinyl alcohol is 1~200:1. More preferably, the mass ratio of azelaic acid powder to polyvinyl alcohol is 35~45:1.

[0013] Preferably, the mass concentration of polyvinyl alcohol in the polyvinyl alcohol aqueous solution is 0.5-25% w / v; more preferably, it is 0.5-5% w / v.

[0014] Based on a general inventive concept, this application provides azelaic acid nanocrystals, which are prepared according to the above-described preparation method.

[0015] Preferably, the hydrated particle size of the azelaic acid nanocrystals is 200–400 nm; the dispersion coefficient is ≤0.2. Further, the hydrated particle size of the azelaic acid nanocrystals is 200–300 nm.

[0016] Based on a general inventive concept, this application provides the application of azelaic acid nanocrystals in the preparation of topical acne treatment formulations.

[0017] Preferably, the topical preparation is a topical ointment, which includes an ointment base and an active ingredient, wherein the active ingredient includes azelaic acid nanocrystals.

[0018] Preferably, the azelaic acid nanocrystals in the topical ointment have a mass content of 5%.

[0019] Preferably, the ointment base comprises an aqueous phase component and an oil phase component; the aqueous phase component includes one or more of glycerin, propylene glycol, hyaluronic acid, and allantoin; the oil phase component includes one or more of cetearyl alcohol, glyceryl monostearate, caprylic / capric triglyceride, and polydimethylsiloxane.

[0020] Compared with the prior art, this application has the following beneficial effects:

[0021] This application uses ethanol and propylene glycol as solvents and PVA as a stabilizer to prepare spherical microcrystalline AZANPs via a pH-controlled phase inversion method. These AZANPs exhibit small hydrated particle size, good dispersion uniformity, and excellent transdermal penetration. The in vitro transdermal transdermal capacity of the prepared 5% nano-ointment is approximately 2.5 times that of a 12% active pharmaceutical ingredient ointment. In an acne model, it achieves efficacy comparable to a 10% commercially available ointment with half the drug loading, demonstrating good safety. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some implementation methods of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0023] Figure 1 The hydrated particle size distribution map and TEM image of AZANPs prepared in Example 1 are shown. Figure 1 a is the hydrated particle size distribution diagram. Figure 1 b is a TEM image of AZANPs (scale bar 500 nm). Figure 1 c is a TEM image of AZANPs (scale bar 50 nm).

[0024] Figure 2 The results of UV-Vis and infrared absorption spectroscopy tests for AZANPs and AZA are shown below. Figure 2 a is the ultraviolet-visible spectrum. Figure 2 b is the standard curve showing the relationship between the ultraviolet absorption and concentration of AZANPs. Figure 2 c is the infrared spectrum.

[0025] Figure 3 X-ray powder diffraction patterns of AZANPs and AZA.

[0026] Figure 4 The figure shows the effect of PVA concentration and AZA dosage on the hydrated particle size and PDI of AZANPs. Figure 4 Figure a shows the effect of PVA concentration on the hydrated particle size and PDI of AZANPs. Figure 4Figure b shows the effect of AZA dosage on the hydrated particle size and PDI of AZANPs.

[0027] Figure 5 The figure shows the effect of different mixed solvent ratios on the preparation of AZANPs. Figure 5 a is a macroscopic image of the reaction system under different ethanol-propylene glycol ratios; Figure 5 Figure b shows the results of the comparative analysis of the average hydrated particle size of the successfully assembled systems (7:3 and 6:4) (n=3).

[0028] Figure 6 The images show the in vitro transdermal penetration and intradermal retention effects of different ointment components. Figure 6 a represents the in vitro permeability-time change curve. Figure 6 b represents the cumulative in vitro permeability. Figure 6 c represents skin permeability. Figure 6 d represents the amount of drug retained intradermally (n=3).

[0029] Figure 7 The results of the evaluation of the acne treatment effects of different ointments in Experiment Example 5 are as follows: Figure 7 'a' represents the animal experiment procedure. Figure 7 b shows the morphological monitoring of acne in each group of mice. Figure 7 c represents the dynamic change curve of lesion area (n=6). ***P<0.001. (G1: 5% AZANPs ointment group; G2: 10% commercially available azelaic acid ointment group; G3: 12% AZA raw material group; G4: blank matrix group; G5: PBS group).

[0030] Figure 8 The images show H&E staining of skin tissue from mice in each group after 7 days of continuous drug administration in Experiment Example 6 (n=3).

[0031] Figure 9 Masson staining images of skin tissue from mice in each group after 7 days of continuous drug administration in Experiment Example 6 (n=3).

[0032] Figure 10 Immunofluorescence staining images of IL-1β, TNF-α, MMP-9 and PPAR-γ in the skin tissue of mice in each group of Experiment Example 6 (G1: 5% AZANPs ointment group; G2: 10% commercially available azelaic acid ointment group; G3: 12% AZA raw material group; G4: blank matrix group; G5: PBS group; n=6).

[0033] Figure 11 The results show the semi-quantitative levels of IL-1β, TNF-α, MMP-9, and PPAR-γ in the skin tissues of mice in each group of Experiment 6. Figure 11 a, Figure 11 b、 Figure 11 c. Figure 11 d represents the semi-quantitative results of IL-1β, TNF-α, MMP-9, and PPAR-γ, respectively. (G1: 5% AZANPs ointment group; G2: 10% commercially available azelaic acid ointment group; G3: 12% AZA raw material group; G4: blank matrix group; G5: PBS group; n=6).

[0034] Figure 12 The images and quantitative analysis results of Ki67 and CK14 co-localization immunofluorescence staining in the skin tissues of mice in each group of Experiment Example 6 are shown below. Figure 12 Image a shows the co-localization immunofluorescence staining images of Ki67 and CK14 in the skin tissue of mice in each group. Figure 12 b represents the quantitative analysis results of the Ki67 positivity rate. Figure 12 c represents the quantitative analysis results of epidermal thickness in the skin tissue of mice in each group. (G1: 5% AZANPs ointment group; G2: 10% commercially available azelaic acid ointment group; G3: 12% AZA raw material group; G4: blank matrix group; G5: PBS group; n=6).

[0035] Figure 13 The results of the antibacterial activity study in Experiment Example 7 are as follows. Figure 13 a represents the morphology of acne 5 days after treatment (G1: 5% AZANPs ointment group; G2: 10% azelaic acid ointment group; G3: 12% AZA raw material group; G4: blank matrix group; G5: PBS group). Figure 13 b shows the results of homogenization of mouse skin tissue in each group (n=6).

[0036] Figure 14 The curves showing the changes in body weight of mice in each group during the drug administration period in Experiment Example 8 are shown (G1: 5% AZANPs ointment group; G2: 10% commercially available azelaic acid ointment group; G3: 12% AZA raw material group; G4: blank matrix group; G5: PBS group; n=6).

[0037] Figure 15 The images show the H&E staining of the major organs of mice in each group in Experiment 8 (G1: 5% AZANPs ointment group; G2: 10% commercially available azelaic acid ointment group; G3: 12% AZA raw material group; G4: blank matrix group; G5: PBS group).

[0038] Figure 16 The relative levels of liver and kidney indicators in each group of mice in Experiment 8 are shown below. Figure 16 a~ Figure 16 d represents the relative levels of BUN, CREA, ALT, and ASTL (G1: 5% AZANPs ointment group; G2: 10% commercially available azelaic acid ointment group; G3: 12% AZA raw material group; G4: blank matrix group; G5: PBS group; n=6). Detailed Implementation

[0039] The embodiments described in this specification are merely for explaining this application and are not intended to limit this application.

[0040] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range, just as any upper limit can be combined with any other upper limit to form an undefined range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an undefined range.

[0041] Those skilled in the art will understand that the order in which the steps are written in the various embodiments or examples does not imply a strict execution order and does not limit the implementation process in any way. The detailed execution order of each step should be determined by its function and possible internal logic. Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, but sequentially is preferred.

[0042] The present application is further illustrated below with reference to embodiments. It should be understood that these embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0043] Example 1: Preparation of azelaic acid nanocrystals.

[0044] (1) Solution preparation: Mix ethanol and 1,3-propanediol thoroughly in a volume ratio of 6:4 to form a mixed solvent; prepare 2.5% (w / v) PVA aqueous solution and 0.1 mol / L NaOH aqueous solution using ultrapure water for later use.

[0045] (2) Nano-sizing: Azelaic acid powder (AZA), PVA aqueous solution and ethanol-propylene glycol mixed solvent were added sequentially to a 5 mL centrifuge tube; in this reaction system, the mass concentration of azelaic acid was 200 mg / mL (20% w / v) and the mass concentration of PVA was 0.5% w / v. The magnetic stirrer was set to a stirring speed of 1200 rpm and stirred continuously to make AZA fully dissolve in the mixed solvent to form a milky white, clear and homogeneous solution. At the same time, 0.1 mol / L NaOH aqueous solution was slowly added dropwise during the stirring process to adjust the pH of the system until the system changed from milky white to transparent light blue and obvious Tyndall effect could be observed. The titration was stopped when the endpoint pH was measured to be about 5.5, and a coarse suspension of azelaic acid nanocrystals was obtained.

[0046] (3) Transfer the crude nano-suspension obtained from the above operations to an open container and place it in a 50°C constant temperature water bath for 8 hours to promote the full evaporation of ethanol and reduce the interference of residual solvent on subsequent applications or characterization. After the reaction is completed, cool the nano-suspension to room temperature to obtain the final AZANPs suspension, which can be stored at room temperature for later use. All the above operations should be carried out in the dark.

[0047] Example 1: Characterization of the physicochemical properties of AZANPs.

[0048] The main purpose of this experiment is to examine the physicochemical properties of the azelaic acid nanocrystals prepared in Example 1.

[0049] I. Particle size and morphology characterization

[0050] (1) The ABZNPs suspension prepared in Example 1 was used as the measurement sample, and the hydrated particle size of the sample was determined using a Nano-ZS90 Malvern particle size potential analyzer.

[0051] (2) Take 100 μL of the ABZNPs suspension prepared in Example 1 and add the sample to the copper mesh in 2 to 3 times. Then set the oven temperature to 60°C and put the copper mesh into the oven to dry until the sample on the copper mesh is fully dry. Then observe and record the micromorphology of the nano-formulation using a transmission electron microscope.

[0052] Particle size results of AZANPs ( Figure 1 a) The hydrated particle size of AZANPs is 262.6 ± 12.7 nm. (Transmission electron microscopy observation results) Figure 1 b、 Figure 1 c) The AZANP nanoparticles are spherical and uniform in size, with network adhesion between particles caused by solvents and stabilizers, and good dispersibility in the liquid phase. Due to differences in testing principles, and the fact that weak electrolyte drugs form a thicker solvation shell in alcohol solvents, there will be a significant difference between the hydrodynamic particle size measured by DLS and the dry particle size measured by TEM.

[0053] The formation mechanism of spherical AZANPs nanoparticles is as follows: From a thermodynamic perspective, during the pH-controlled phase transition-induced supersaturation process, AZA molecules are uniformly precipitated and self-assembled into thermodynamically most stable spherical particles under the influence of surface tension. The "network adhesion" phenomenon observed in the TEM image may be due to the long-chain network effect of PVA and the PVA-mediated bridging effect. First, the flexible PVA segments adsorbed on the surface of adjacent AZA nanocrystals will interpenetrate and entangle in a highly concentrated local environment, forming a physical cross-linked structure similar to a "semi-interpenetrating network". Second, during the negative staining and high-vacuum drying process of TEM copper mesh sample preparation, free or weakly adsorbed PVA will be concentrated into a film as water evaporates, producing a bridging effect, and finally forming a continuous stabilizer film layer in the dry state.

[0054] II. Ultraviolet-Visible Spectroscopy and Solubility Analysis

[0055] Take 100 μL of AZANPs suspension, dilute it with ethanol solution to an appropriate factor, use pure ethanol as a blank control to subtract the absorbance blank value, and use AZA raw material powder as a control sample. Scan the ultraviolet-visible absorption spectrum of the sample in the wavelength range of 200~800 nm to obtain the ultraviolet absorption spectrum.

[0056] like Figure 2 The UV spectroscopy results (a) show that both AZA raw material and AZANPs exhibit the same terminal characteristic absorption peak near 210 nm, proving that azelaic acid maintained its complete molecular framework structure during solvent evaporation and pH-controlled nano-sizing. Based on the UV absorption standard concentration curve of azelaic acid (… Figure 2 b) The concentration of azelaic acid in the AZANPs suspension was calculated to be approximately 254.5 mg / mL (>20% w / v), which is in line with expectations and can be used for the subsequent preparation of AZANPs ointment.

[0057] III. Infrared Spectroscopy Analysis

[0058] Take the AZANPs suspension and freeze it at -20℃ for more than 12 hours. After pre-freezing, transfer it to a vacuum freeze dryer and continue drying for 24 to 48 hours to obtain AZANPs powder. Use AZA raw material powder as a control sample. Take an appropriate amount of dried KBr powder, grind it into fine powder, mix it evenly with AZANPs powder and AZA powder respectively, and then compress it into tablets. After tableting, scan the tablets and subtract the blank background of KBr to obtain the infrared absorption spectra of HNPs and Hemin.

[0059] The results are as follows Figure 2 As shown in c, in the spectrum of AZA active pharmaceutical ingredient, the CH stretching vibration peak of the methylene group (-CH2-) in the AZA molecular structure appears at 2934.16 cm⁻¹.-1 and 2848.83cm -1 Location, 1686.78cm -1 The peak at 1280.61 cm⁻¹ is the characteristic strong absorption peak of the carbonyl group (C=O) on the free carboxyl group of azelaic acid. -1 and 1208.03cm -1 The peak corresponds to the CO stretching vibration. In the spectra of AZANPs, the core framework characteristic peaks of AZA are all preserved, proving that the pH-controlled nanostructuring method did not destroy the chemical structure of azelaic acid. Furthermore, AZA exhibits peaks at 3000–3500 cm⁻¹. -1 The hydroxyl absorption band in the region overlaps with the hydroxyl (-OH) absorption band of PVA, and the C=O stretching vibration peak is observed at 1686.78 cm⁻¹. -1 The redshift reached 1699.37cm. -1 This indicates that during the self-assembly of nanoparticles, the carboxyl groups at the azelaic acid end group may form strong intermolecular hydrogen bonds with a large number of hydroxyl groups on the PVA chain. This not only restricts the free crystallization of azelaic acid molecules but also effectively enhances the adsorption strength of PVA on the nanocrystal surface, thereby providing greater steric hindrance to stabilize the drug.

[0060] IV. X-ray Diffraction Analysis

[0061] To evaluate the impact of general nanoparticle preparation processes on the solid-state physical properties (crystal form and crystallinity) of drugs, X-ray powder diffraction was used to determine the lyophilized powders of the active pharmaceutical ingredient, polymer stabilizer, and their corresponding nanoparticles.

[0062] Figure 3 X-ray powder diffraction (XRD) patterns of AZANPs, AZA, and PVP K30 are shown. The results show that the AZA active pharmaceutical ingredient exhibits multiple sharp, high-intensity characteristic diffraction peaks at diffraction angles of approximately 9°, 19°, 23°, and 27°, indicating that the original azelaic acid molecule possesses a highly ordered crystalline structure. The PVA polymer stabilizer, however, shows typical broad amorphous diffuse peaks around 20°. In the diffraction patterns of AZANPs, the main characteristic diffraction peaks of AZA are present, and the peak positions do not show significant shifts, indicating that the basic lattice framework of the azelaic acid molecule remains unchanged. Furthermore, the absolute intensities of all characteristic diffraction peaks of AZANPs show a significant decrease, and the baseline exhibits slight broadening. This suggests that during the pH-controlled nano-sizing process, the volatilization of ethanol led to a supersaturated state, while the adsorption of PVA effectively limited the ordered growth of AZA crystals, ultimately transforming azelaic acid into a microcrystalline state.

[0063] Experimental Example 2: Effect of PVA concentration on the particle size of AZANP nanocrystals.

[0064] AZANPs were prepared according to the method in Example 1, with only the concentration of PVA in the reaction system changed to investigate the effect of PVA concentration on the particle size of AZANPs. Specifically, 0.1 mol / L NaOH aqueous solution was slowly added dropwise during stirring to adjust the pH of the system until the system abruptly changed from milky white to a transparent light blue and a clear Tyndall effect was observed. The titration was stopped at this point, and the endpoint pH was measured to be approximately 5-6. The mixture was then reacted in a constant temperature water bath for 8 hours to promote the complete evaporation of ethanol, yielding an azelaic acid nanocrystal suspension. Particle size analysis was performed directly on the AZANPs suspension, and the relevant data were recorded.

[0065] Figure 4 a demonstrates the effect of PVA concentration on the nanocrystal size of AZANPs. From Figure 4 As can be seen, with the increase of PVA concentration, the particle size of AZANPs nanocrystals generally shows a trend of first decreasing and then increasing. When the PVA concentration increases from 0.1% w / v to 0.5% w / v, the particle size gradually decreases, indicating that an appropriate amount of PVA reduces the solid-liquid surface tension, provides sufficient steric hindrance, and effectively inhibits Oswald ripening. When the PVA concentration continues to increase, the increase in solution viscosity will cause non-specific aggregation between particles, leading to a renewed increase in particle size. When the PVA concentration is 0.5% w / v, the hydrated particle size is measured to be around 260 nm, and the PDI value is less than 0.2.

[0066] Experimental Example 3: Effect of AZA dosage on the particle size of AZANPs nanocrystals.

[0067] AZANPs were prepared according to the method in Example 1, with only the concentration of AZA in the reaction system being varied. The effect of AZA dosage on the particle size of AZANPs was investigated. Specifically, 0.1 mol / L NaOH aqueous solution was slowly added dropwise during stirring to adjust the pH of the system until the system abruptly changed from milky white to a transparent light blue, and a clear Tyndall effect was observed. The titration was stopped at this point, with the endpoint pH measured to be approximately 5-6. The mixture was then reacted in a constant temperature water bath for 8 hours to promote the complete evaporation of ethanol, yielding an azelaic acid nanocrystal suspension. The particle size of the AZANPs suspension was directly analyzed, and the relevant data were recorded.

[0068] Figure 4 b shows the effect of AZA dosage on the particle size of AZANPs. As the dosage increases, the AZA particle size decreases. When the dosage is 200 mg / mL, the prepared nanocrystals have a particle size of about 280 nm and a PDI value of less than 0.2.

[0069] Experimental Example 4: Investigation of the ratio of mixed solvents.

[0070] AZANPs were prepared according to the preparation method in Example 1, with only the volume ratio of the mixed solvents ethanol and acetone changed. The preparation effect of AZANPs was observed, and the colloidal characteristics, nucleation state, and solvent residue in the AZANPs suspension after water bath evaporation were recorded. The specific results are shown in Table 1.

[0071] Table 1. Statistical table of the effect of volume ratio of ethanol and acetone on the preparation effect of AZANPs.

[0072]

[0073] In the table, colloidal characteristics "-" indicates no Tyndall effect, and "+" indicates Tyndall effect; nucleation characteristics "-" indicates obvious particles under transmission electron microscopy, and "+" indicates no obvious particles under transmission electron microscopy.

[0074] Residual ethanol content refers to the mass content of ethanol in the AZANPs suspension after evaporation in a constant temperature water bath; residual propylene glycol content refers to the mass content of propylene glycol in the AZANPs suspension after evaporation in a water bath.

[0075] Figure 5 Table 1 shows the macroscopic appearance of the reaction system under different ethanol-propanediol ratios. Based on the results in Table 1, during the preparation process, when the volume ratio of ethanol to 1,3-propanediol was too high (8:2) or too low (5:5), the prepared AZANPs suspensions appeared milky white and slightly turbid or clear and without opalescence, respectively, and the Tyndall effect was barely observed, indicating that stable nanocolloids were not formed in the system. This may be due to the rapid evaporation of high proportions of ethanol in the system leading to crystal aggregation, or the increased viscosity of the system preventing the crystals from reaching the supersaturation nucleation threshold. When the volume ratio of ethanol to 1,3-propanediol was adjusted to 7:3 and 6:4, the system successfully nucleated and exhibited a characteristic light blue opalescence. Figure 5 a) DLS results are as follows Figure 5 As shown in b, when the volume ratio is 6:4, the average hydrated particle size of the drug particles is approximately 262.6 ± 12.7 nm, slightly smaller than the average hydrated particle size of approximately 277.67 ± 32.51 nm in the 7:3 group. Furthermore, considering the safety requirements and low solvent residue requirements for topical formulations, the residual ethanol content in the 6:4 group is 9.60%, significantly lower than the 22.50% in the 7:3 group. Therefore, taking into account nucleation state, particle size, and residual solvent content, a mixed solvent of ethanol and 1,3-propanediol in a volume ratio of 6:4 was selected to prepare AZANP nanoparticles.

[0076] Example 2: Application of azelaic acid nanocrystals.

[0077] The AZANPs suspension prepared in Example 1 was pre-frozen at -20°C for more than 12 hours, and then freeze-dried for 48 hours to obtain a dried AZANPs sample. It was then mixed with an ointment matrix (Table 2) by a professional institution to prepare a 5% azelaic acid nanoparticle ointment (G1). Similarly, the AZA active pharmaceutical ingredient powder was uniformly dispersed in the same matrix using a physical grinding method to obtain a 12% azelaic acid active pharmaceutical ingredient ointment (G3). G2 was a 10% commercially available azelaic acid ointment.

[0078] Table 2 Ointment Base Components

[0079]

[0080] Experimental Example 5: Investigation of in vitro percutaneous osmotic dynamics and intradermal retention.

[0081] Thaw frozen pigskin at room temperature for 5 minutes, soak in physiological saline for 1 hour, and equilibrate with PBS (pH=7.4) for 30–60 minutes. Use sterile PBS (pH=7.4) as the receiving solution in the Franz diffusion cell receiving chamber, with a volume of approximately 7 mL and an effective diffusion area of ​​1.76 cm². Fix the skin with the stratum corneum facing upwards between the supply and receiving chambers. Maintain a constant temperature water bath at 32°C and magnetically stir at 300 rpm. Accurately weigh 30 mg of ointment (approximately 17 mg / cm²). 2 The sample was evenly applied to the skin surface. At 2, 4, 6, 8, 10, 12, and 24 hours, 7 mL of the receiving solution was collected, filtered through a 0.45 µm filter, sealed, and stored, with an equal volume of preheated PBS added. After the 24-hour experiment, any remaining ointment on the skin surface was wiped off with a cotton swab, and skin tissue was cut and soaked in methanol at -20°C for storage to determine intradermal retention. The receiving solution and skin tissue at each time point were pretreated as follows, and the azelaic acid content was determined by HPLC to calculate the cumulative permeation and intradermal retention (n=3).

[0082] Sample pretreatment methods:

[0083] (1) Received liquid sample: Accurately transfer 7.0 mL of the filtered received liquid, add a small amount of phosphoric acid to adjust the pH to 2~3, add an equal volume of ethyl acetate and vortex vigorously for 3 min, centrifuge at 3000 rpm for 5 min, take the upper organic phase and blow it dry with nitrogen in a 40℃ water bath, redissolve the residue with the mobile phase, centrifuge at 13000 rpm for 5 min and filter through a 0.45 μm microporous membrane to obtain the test solution.

[0084] (2) Intradermal retention sample: Skin tissue was extracted by vortexing and water bath sonication for 1 hour, centrifuged at 10,000 rpm for 10 min, the supernatant was taken and nitrogen blown out, then reconstituted with the mobile phase, centrifuged at 13,000 rpm for 5 min, and filtered through a 0.45 μm microporous membrane to obtain the intradermal retention test solution.

[0085] Chromatographic conditions:

[0086] Chromatographic column: C18 column (4.6 mm × 250 mm, 5 μm), with a pre-protection column connected before the column; mobile phase: 30% acetonitrile + 70% phosphoric acid aqueous solution (0.3%), filtered through a 0.45 μm filter membrane and degassed by sonication; column temperature: 40℃; flow rate: 1.0 mL / min; injection volume: 100 μL; detection wavelength: 210 nm; single run time: 25 min.

[0087] The results are as follows Figure 6 As shown, the transdermal penetration of azelaic acid gradually increased within 24 hours. The transdermal penetration behavior of the 5% AZANPs ointment group was significantly better than that of the 12% AZA raw material ointment group, and showed similar transdermal penetration behavior to the 10% commercially available group. Figure 6 a). The cumulative penetration of the AZANPs ointment group over 24 hours was 52.5 ± 3.25 μg / cm³. 2 It was approximately 2.5 times that of the raw material control group, and compared to the commercially available group (60.36±2.62μg / cm). 2 Basically unchanged ( Figure 6 b); The cumulative permeability is 6.16 ± 0.38% ( Figure 6 c), significantly higher than the control group and the commercially available group. Regarding intradermal retention ( Figure 6 d), the AZANPs ointment group reached 14.53±0.78μg / cm. 2 The concentration was significantly higher than that of the active pharmaceutical ingredient group, compared with the commercially available group (17.60±0.67μg / cm). 2 It is comparable to other drugs, and can maintain an effective antibacterial and anti-inflammatory concentration under low drug loading conditions, while significantly reducing the risk of skin irritation.

[0088] Experimental Example 6: Evaluation of the in vivo anti-acne efficacy of AZANPs ointment.

[0089] like Figure 7 As shown in a, this embodiment establishes a mouse dorsal acne infection model by intradermal injection of Propionibacterium acnes (C. acnes) to systematically evaluate the in vivo anti-acne efficacy of AZANPs ointment.

[0090] 1. Laboratory animals and grouping

[0091] Experimental animals: BALB / c mice (SPF grade), male, 6 weeks old, 20-25g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and entrusted to the SPF Grade Laboratory Animal Center of Xiangya School of Medicine, Central South University for foster care; the experimental protocol was reviewed and approved by the ethics committee (ethics approval number: APU-2025-0311).

[0092] Group design: Six mice were selected and their back hair was removed. They were then injected intradermally with 20 μL of sterile PBS solution as the Normal group. The model group was randomly divided into 5 groups (n=6): G1 (5% AZANPs ointment), G2 (10% commercially available azelaic acid ointment), G3 (12% AZA raw material ointment), G4 (blank matrix), and G5 (PBS).

[0093] 2. Acne Model Construction

[0094] The fur on the back of the mouse was removed using a shaver and depilatory cream, exposing a 3×3cm area. 2 Exposed areas. Cultivate C. acnes to the logarithmic growth phase (OD). 600 =0.3), concentrated 3 times, washed with PBS to remove residual culture medium, and 20 μL of bacterial solution was injected intradermally into the back of mice using an insulin injector to induce an acne model. Skin lesion changes were continuously monitored before administration.

[0095] 3. Drug administration and morphology monitoring

[0096] Drug administration began 24 hours after modeling. Each group received 20 μL of the corresponding test substance evenly daily for 7 consecutive days (Day 1 to Day 7). The length (L) and width (W) of the lesions were measured daily using calipers. The acne area was calculated according to S=πLW / 4, and a dynamic change curve of the lesion area was plotted and photographed for recording.

[0097] The results show that ( Figure 7 In the PBS group (G5) and the blank matrix group (G4), the inflammatory nodules resolved very slowly, with persistent redness, swelling, and crusting. In the 12% raw material group (G3), due to insufficient penetration and high-concentration stimulation, the deep nodules did not shrink significantly. The 5% AZANPs ointment group (G1) and the 10% commercially available ointment group (G2) both showed strong anti-inflammatory capabilities, with nodule volume shrinking rapidly. The healing process in group G1 was stable; by day 7, skin texture had largely returned to normal, and scarring was relatively faint. Group G2 showed significant tissue damage and pigmentation. Figure 7 b). Quantitative analysis ( Figure 7 c) indicates that the area reduction curves of G1 and G2 basically overlap (P=ns), and G1 achieves the same acne treatment effect with only half the drug loading of G2 (***P<0.001 vs G3, G4, G5).

[0098] Experimental Example 7: Histopathological analysis of acne skin tissue.

[0099] H&E staining, Masson staining, and immunofluorescence staining were performed on the skin tissues of mice in each group of Experiment Example 6 to evaluate the therapeutic effect of AZANPs ointment from multiple dimensions, including histopathology, collagen remodeling, inflammatory factors, and barrier repair.

[0100] 1. H&E staining

[0101] Fixed acne skin tissue was taken, and after dehydration, paraffin infiltration, embedding, sectioning (5μm), dewaxing, hydration, H&E staining, dehydration, and mounting, the histopathological morphology was observed and images were acquired under an optical microscope.

[0102] result( Figure 8 The Normal group showed intact skin structure, a fine epidermis, and normal hair follicles. The G5 (PBS) and G4 (blank matrix) groups showed abnormally increased dermal and epidermal thickness, extensive inflammatory cell infiltration (red arrows), and ruptured hair follicle structures (blue arrows). The G3 group (12% API) had the worst pathology, with a large inflammatory abscess in the deep dermis containing a large number of neutrophils, accompanied by severe epidermal necrosis and crusting. This demonstrates that ordinary powder formulations, due to insufficient penetration, cannot effectively reach deep infection sites, and high concentrations actually exacerbate tissue damage. The G2 group (10% commercially available) showed a slight decrease in epidermal thickness, but a small amount of inflammatory cell infiltration remained in the deep dermis. The G1 group (5% AZANPs ointment) showed epidermal thickness restored to physiological levels, almost complete disappearance of dermal inflammatory cells, intact and orderly hair follicle structure, and tissue morphology closest to the Normal group.

[0103] 2. Masson staining

[0104] Skin tissue sections were stained with Masson's stain after following the same procedure as in 6.1, and the distribution and arrangement of collagen fibers were observed under an optical microscope. Figure 9 ).

[0105] Results: In the Normal group, collagen fibers were neatly arranged and evenly distributed; in the G5 and G4 groups, dermal collagen fibers were disordered and had a large amount of abnormal deposition, showing pathological changes of fibrosis; in the G3 group, fibrosis improvement was limited; in the G2 group, collagen arrangement was regular and fibrotic lesions were significantly improved; in the G1 group, collagen fiber arrangement was basically restored to normal, abnormal deposition was significantly reduced, and the tissue repair effect was better than that of the G3 and G2 groups.

[0106] 3. Immunofluorescence staining (IL-1β, TNF-α, MMP-9, PPAR-γ)

[0107] Paraffin sections were prepared, and immunofluorescence staining for IL-1β, TNF-α, MMP-9, and PPAR-γ was performed. Images were acquired using a laser confocal microscope, and the average optical density (AOD) was measured using ImageJ software. Figure 10 , Figure 11 ).

[0108] result( Figure 10 , Figure 11In the G5 and G3 groups, the expression levels of IL-1β, TNF-α, and MMP-9 were the highest. In the G4 group, due to the presence of soothing components in the matrix, the levels of TNF-α and MMP-9 were slightly lower than those in the G5 group, but IL-1β remained high. The high concentration of powder in the G3 group caused secondary chemical stimulation to the lesion skin, leading to a higher level of pro-inflammatory mediator release. In the G1 group (5% AZANPs ointment), the AOD values ​​of IL-1β, TNF-α, and MMP-9 were all downregulated to near the physiological baseline level of the Normal group, which was better than that of the G2 group. The expression of PPAR-γ in the pilosebaceous unit of the G1 group was significantly upregulated, reaching the same level as the G2 group under the condition of half dosage, and significantly higher than that of the G3 group, which was consistent with the results of the in vitro transdermal experiment.

[0109] 4. Evaluation of skin histological remodeling (Ki67 / CK14 double staining)

[0110] Each group of slides was subjected to Ki67 and CK14 immunofluorescence double staining, and images were acquired using a laser confocal microscope. Image J was used to measure the epidermal thickness and Ki67 positivity rate of each group. Figure 12 ).

[0111] result( Figure 12 In groups G5 and G4, epidermal acanthosis was significant, CK14-positive areas were highly expanded, and Ki67-positive cells were abundantly aggregated. In group G3, the Ki67 positivity rate was higher than that in groups G1 and G2, indicating that high-concentration drug physicochemical stimulation exacerbated epidermal stress-induced proliferation. In groups G1 and G2, the thickness of CK14-positive areas was significantly reduced, and the Ki67 positivity rate decreased. In group G1, with only half the drug loading of group G2, the skin recovered to a normal, slender, band-like structure, and the Ki67 positivity rate returned to normal physiological homeostasis. This indicates that 5% AZANPs nano-ointment can more effectively regulate epidermal cell proliferation and differentiation under low-dose conditions, promoting the recovery of damaged skin to a normal state.

[0112] Experimental Example 8: Study on the in vivo antibacterial activity of AZANPs ointment.

[0113] In this embodiment, an acne model was constructed by intradermal injection of C. acnes. Five days after administration, skin tissue homogenate was extracted and spread onto blood agar plates to quantitatively evaluate the antibacterial activity of each group.

[0114] 1. Experimental Methods

[0115] Exposed 3×3cm after hair removal treatment 2For the skin area, C. acnes bacterial suspension in the logarithmic growth phase (OD600=0.3) was concentrated 2-fold, washed with PBS, and injected intradermally at 20 μL. Five injection sites were spaced apart in the same area for each mouse. 24 h after modeling, the mice were grouped (G1-G5, n=6). 20 μL of the test substance was evenly applied to each injection site daily for 5 consecutive days, after which the mice were euthanized. Acne-affected skin tissue of equal area was excised, homogenized at 65 Hz for 90 s, and evenly spread on blood agar plates. The plates were then incubated anaerobically at 37°C for 24 h, and the colony growth was photographed and recorded. Figure 13 ).

[0116] 2. Experimental Results

[0117] result( Figure 13 In the G5 (PBS) and G4 (blank matrix) groups, the skin was significantly red and swollen, and the tissue homogenate showed dense bacterial colonies, indicating that the model was established and bacterial proliferation was not effectively inhibited. In the G3 (12% API) group, some skin lesions improved, but there was still a lot of bacterial growth, and the bacterial count was significantly lower than that in the G4 and G5 groups. In the G1 (5% AZANPs ointment) and G2 (10% commercially available) groups, the acne basically subsided, and there was a small amount of bacterial growth. The bacterial load was significantly lower than that in the G3, G4 and G5 groups, indicating that the 5% AZANPs ointment can significantly inhibit the excessive proliferation of C. acnes in the deep layers of the skin, and the antibacterial effect is comparable to that of the 10% commercially available group.

[0118] Experimental Example 9: In vivo safety evaluation of AZANPs ointment.

[0119] Based on the in vivo efficacy experiment in Experiment 6, the in vivo biosafety of AZANPs ointment was systematically evaluated through weight monitoring, H&E staining of major organs, and detection of serum biochemical indicators.

[0120] 1. Weight changes

[0121] During the 7-day drug administration period, the body weight of mice in each group was measured daily using the same balance, and a body weight change curve was plotted. Figure 14 ).

[0122] result( Figure 14 ): After modeling (Day-1), the mice experienced a slight decrease in body weight due to local acute inflammation caused by intradermal injection of C. acnes. During the subsequent 7-day administration period, the body weight of each group steadily recovered and stabilized without abnormal fluctuations, indicating that the local application of AZANPs nano-ointment had no significant negative impact on the overall physiological state of the mice.

[0123] 2. Pathological analysis of major organs

[0124] Mice were euthanized after treatment, and their hearts, livers, spleens, lungs, and kidneys were harvested. After washing with PBS, the organs were fixed in 4% paraformaldehyde, paraffin sections were prepared, and after H&E staining, the histopathological morphology of the organs was observed under an optical microscope. Figure 15 ).

[0125] result( Figure 15 The major organs of mice in each treatment group, such as heart, liver, spleen, lung, and kidney, remained intact, and no pathological damage such as tissue degeneration, necrosis, or inflammatory cell infiltration was found. This indicates that the local application of AZANPs nano-ointment did not cause organ damage, and AZANPs has excellent biocompatibility.

[0126] 3. Serum biochemical index analysis

[0127] Blood was collected from the eyeballs to obtain serum, which was centrifuged at 3000 rpm for 10 min. The supernatant serum was collected and stored at -80°C. Liver function indicators (ALT, AST) and kidney function indicators (BUN, CRE) were measured using a biochemical assay kit. Figure 16 ).

[0128] result( Figure 16 The ALT, AST, BUN, and CRE levels in each group were all within the normal physiological range, and there were no statistically significant differences between the treatment groups and the control group. This indicates that the topical application of AZANPs nano-ointment does not cause significant damage to the liver and kidneys and has good biocompatibility.

[0129] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing azelaic acid nanocrystals, characterized in that, Includes the following steps: S1. Azelaic acid powder, polyvinyl alcohol aqueous solution, and organic mixed solvent are mixed and the azelaic acid powder is dissolved under stirring to form a homogeneous solution; the organic mixed solvent is ethanol and propylene glycol, and the volume ratio of ethanol to propylene glycol is 6~7:3~4; in the homogeneous solution, the mass concentration of azelaic acid is 5~20% w / v; the mass concentration of polyvinyl alcohol is 0.1~5.0% w / v; S2. Under stirring conditions, add a pH adjuster dropwise to the homogeneous solution until the homogeneous solution turns light blue and the Tyndall effect can be observed. Stop the titration when the pH value of the resulting system is 5-6, and obtain azelaic acid nanocrystal suspension. S3. The azelaic acid nanocrystal suspension is placed at 50-60℃ for 6-8 hours to allow the ethanol to fully evaporate, and then freeze-dried to obtain azelaic acid nanocrystals.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of azelaic acid powder to polyvinyl alcohol is 1~200:

1.

3. The preparation method according to claim 1, characterized in that, The polyvinyl alcohol concentration in the aqueous solution is 0.5~25.0% w / v.

4. An azelaic acid nanocrystal, characterized in that, It is prepared according to any one of claims 1 to 3.

5. The azelaic acid nanocrystals according to claim 4, characterized in that, The hydrated particle size of the azelaic acid nanocrystals is 200–400 nm; the dispersion coefficient is ≤0.

2.

6. The use of azelaic acid nanocrystals prepared by the preparation method according to any one of claims 1 to 3, or azelaic acid nanocrystals according to any one of claims 4 to 5, in the preparation of topical acne treatment formulations.

7. The application according to claim 6, characterized in that, The topical preparation is a topical ointment, which includes an ointment base and an active ingredient. The active ingredient includes azelaic acid nanocrystals prepared according to any one of claims 1 to 3 or azelaic acid nanocrystals according to any one of claims 4 to 5.

8. The application according to claim 7, characterized in that, The azelaic acid nanocrystals in the topical ointment contain 5% by mass.

9. The application according to claim 7, characterized in that, The ointment base comprises an aqueous phase and an oil phase; the aqueous phase comprises one or more of glycerin, propylene glycol, hyaluronic acid, and allantoin; the oil phase comprises one or more of cetearyl alcohol, glyceryl monostearate, caprylic / capric triglyceride, and polydimethylsiloxane.

Images