Anti-acne hydrogel and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to anti-acne hydrogel and a preparation method thereof. The anti-acne hydrogel is prepared from metal-polyphenol network nanoparticles and a hydrogel base material, the metal-polyphenol network nanoparticles comprise a first polyphenol compound, a second polyphenol compound and metal ions, the first polyphenol compound is a tannic acid compound, and the second polyphenol compound is a flavonoid compound; the hydrogel base material comprises a hydrophilic high-molecular polymer and a cationic polymer. The metal-polyphenol network nano-particles are creatively utilized, the anti-inflammatory and anti-oxidation activity of flavonoid compounds, the convergence and protein binding characteristic of tannin and the antibacterial activity of metal ions are integrated at the same time, and the vicious circle of acne can be intervened at the same time from multiple links of antibacterial, anti-inflammatory, anti-oxidation and keratinization adjustment; and the defect of single action target spot of the existing medicine is overcome.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-acne hydrogel and its preparation method. Background Technology

[0002] Acne is a globally prevalent chronic inflammatory pilosebaceous disease with a complex pathological mechanism, mainly involving excessive sebum secretion, abnormal follicular keratinization, excessive proliferation of Propionibacterium acnes, and the ensuing inflammatory response and oxidative stress.

[0003] Current topical medications for acne treatment (such as benzoyl peroxide, retinoids, and topical antibiotics) generally suffer from problems such as single target, strong skin irritation, and easy development of drug resistance. In recent years, nanomedicine delivery systems (such as liposomes and polymer nanoparticles) combined with hydrogels have shown potential, but still face challenges such as complex preparation processes, limited drug loading capacity, and limited functionality.

[0004] Metal-polyphenol networks, as an emerging nanomaterial, can self-assemble through the coordination of metal ions and polyphenol compounds, integrating the antibacterial activity of metal ions with the antioxidant and anti-inflammatory activities of polyphenols. However, how to precisely design metal-polyphenol network nanoparticles to target the complex pathological processes of acne and combine them with suitable delivery systems to develop a novel, highly effective, and low-allergenic acne treatment formulation remains a pressing technical challenge in this field. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an anti-acne hydrogel with multiple effects including antibacterial, anti-inflammatory, and antioxidant properties, and good biocompatibility.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned anti-acne hydrogel.

[0007] Another object of the present invention is to provide the use of the above-mentioned anti-acne hydrogel in the preparation of acne treatment drugs or cosmetics.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides an anti-acne hydrogel, comprising metal-polyphenol network nanoparticles and a hydrogel substrate; the metal-polyphenol network nanoparticles comprise a first polyphenol compound, a second polyphenol compound, and metal ions, wherein the first polyphenol compound is a tannic acid compound, and the second polyphenol compound is a flavonoid compound; the hydrogel substrate comprises a hydrophilic polymer and a cationic polymer. The multiple phenolic hydroxyl groups contained in tannin molecules are excellent hydrogen donors, capable of effectively scavenging reactive oxygen species such as superoxide anion radicals and hydroxyl radicals, protecting the body's biomolecules from oxidative damage.

[0010] Preferably, the mass ratio of the metal-polyphenol network nanoparticles to the hydrogel substrate is 1-10:1-2. For example, it can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 2:1, 2:2, 3:2, 4:2, 5:2, 6:2, 7:2, 8:2, 9:2, or 10:2. This ratio is beneficial for maintaining gel stability and the release rate of active ingredients. When the proportion of nanoparticles is 1-10, it can ensure that sufficient active ingredients can exert antibacterial and anti-inflammatory effects. When the proportion of substrate is 1-2, it can maintain the stable structure of the gel and good moisturizing adhesion, avoiding particle aggregation. The two work synergistically to improve bioavailability and user experience.

[0011] Preferably, the second polyphenol compound is selected from one or more of luteolin, baicalin, apigenin, quercetin, and kaempferol. More preferably, it is luteolin. This type of flavonoid, as a second polyphenol compound, is naturally derived and, compared to traditional anti-acne ingredients such as salicylic acid and retinoic acid, is less irritating to the skin barrier, making it suitable for people with sensitive skin and acne. More preferably, the second polyphenol compound is luteolin, which has more outstanding comprehensive anti-acne properties compared to the other flavonoid compounds mentioned above: On the one hand, luteolin has stronger antibacterial activity against Propionibacterium acnes, with a minimum inhibitory concentration (MIC) as low as 1.25 μg / mL, significantly lower than baicalein (2.5 μg / mL) and quercetin (5 μg / mL), and can rapidly reduce the number of pathogenic bacteria; on the other hand, in terms of anti-inflammation, luteolin can inhibit the activity of transcription factor NF-κB in immune cells such as macrophages, thereby reducing the production of key pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, it can also exert anti-inflammatory activity by affecting the HMGB1-NF-κB signaling pathway. The astringent properties of tannins (i.e., their ability to bind with proteins to form insoluble complexes) help form a protective film on the surface of inflamed or damaged tissues, reducing local exudation. Furthermore, it can alleviate inflammatory responses by reducing the release of inflammatory mediators. In terms of antioxidant activity, the ortho-phenolic hydroxyl groups in the chemical structure of luteolin (especially the ortho-dihydroxyl groups on the B ring) are the basis for its powerful antioxidant capacity, directly providing hydrogen atoms to neutralize free radicals. Simultaneously, it can promote the entry of nuclear factor E2-related factor 2 (Nrf2) into the cell nucleus, binding to antioxidant response elements (AREs), thereby enhancing the activity of in vivo antioxidant enzyme systems such as catalase and superoxide dismutase. In terms of antibacterial activity, luteolin can disrupt the integrity of bacterial cell walls, increasing cell membrane permeability. It can also inhibit the activity of bacterial DNA topoisomerases, affecting the synthesis of bacterial nucleic acids and proteins, thereby inhibiting bacterial proliferation. Tannins can alter membrane permeability and disrupt physiological functions through interactions with bacterial cell membrane proteins and cell walls. It can also chelate with metal ions (such as copper ions) required for microbial growth, or inhibit the activity of bacterial metabolic enzymes, thereby inhibiting bacterial growth.

[0012] Preferably, the metal ion is selected from one or more of copper ions, zinc ions, and iron ions. Copper ions are more preferred. Copper ions exhibit outstanding targeted antibacterial activity against Propionibacterium acnes, can catalyze the production of reactive oxygen species from polyphenols to enhance bactericidal activity, are stable in coordination with polyphenols, can also promote collagen synthesis in the skin, synergistically reduce inflammation, and accelerate wound repair, demonstrating superior overall efficacy compared to zinc and iron ions.

[0013] Preferably, the hydrophilic polymer is one or more of polyvinyl alcohol, polyethylene glycol, and polyacrylamide; the cationic polymer is one or more of quaternized chitosan, polyethyleneimine, and chitosan. Polyvinyl alcohol has strong hydrophilicity and excellent water retention, and can stably form a gel network, providing good structural support for the system; quaternized chitosan is positively charged, which can anchor negatively charged polyphenol-metal nanoparticles, and also has antibacterial properties and good biocompatibility. The two work synergistically to enhance gel stability and anti-acne efficacy.

[0014] Preferably, the molar ratio of the first polyphenol compound, the second polyphenol compound, and the metal ion is 1-4:4-8:5-10. More preferably, the molar ratio of luteolin:tannin:copper chloride is 2:8:10. This ratio results in nanoparticles with uniform size, good dispersibility, and a negative surface potential, exhibiting excellent stability.

[0015] Secondly, the present invention provides a method for preparing the anti-acne hydrogel as described above, comprising the following steps:

[0016] Step 1: Preparation of a metal-polyphenol network nanoparticle suspension;

[0017] Step 2: Prepare the first hydrogel substrate solution and the second hydrogel substrate solution;

[0018] Step 3: Mix the nanoparticle suspension obtained in Step 1 with the first hydrogel substrate solution and the second hydrogel substrate solution obtained in Step 2, and stir to form an anti-acne hydrogel.

[0019] Preferably, in step one, the method for preparing the nanoparticle suspension includes: dissolving a first polyphenol compound, a second polyphenol compound, and a metal salt in water, adjusting the pH to 8-10, stirring the mixture at room temperature in the dark for 4-12 hours at a stirring speed of 1000-1500 rpm, and then centrifuging and washing to obtain the nanoparticle suspension at a centrifugation speed of 4500-5000 rpm for 8-12 minutes. More preferably, the second polyphenol compound is dissolved using a 2 M sodium hydroxide solution and with the aid of ultrasound.

[0020] Preferably, in step two, the method for preparing the hydrogel substrate solution includes:

[0021] Step 1: Add the hydrophilic polymer to deionized water, let it swell, and then heat it to 80-95℃ to dissolve it, thus obtaining the first hydrogel substrate solution;

[0022] Step 2: Add the cationic polymer to deionized water, swell it, and then heat it to 30-50°C to dissolve it, thus obtaining the second hydrogel substrate solution; the cationic polymer preferably has a degree of substitution of 90%.

[0023] Preferably, in step three, the volume ratio of the added nanoparticle suspension to the hydrogel substrate solution is 1-7 ml : 40-50 ml, the stirring speed is 1000-2000 rpm, and the stirring time is 10-30 minutes.

[0024] Thirdly, the present invention provides the use of the anti-acne hydrogel as described above in the preparation of medicaments, medical devices or cosmetics for the prevention or treatment of acne.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) Multi-target synergistic treatment: This invention creatively utilizes metal-polyphenol network nanoparticles, which simultaneously integrate the anti-inflammatory and antioxidant activities of flavonoids, the astringent and protein binding properties of tannins, and the antibacterial activities of metal ions. It can simultaneously intervene in the vicious cycle of acne from multiple aspects such as antibacterial, anti-inflammatory, antioxidant and keratinization regulation, overcoming the shortcomings of existing drugs with single targets.

[0027] (2) High efficiency and safety: Most of the selected active ingredients are of natural origin and have good biocompatibility. Through nano-sizing and hydrogel encapsulation, the stability of the ingredients is improved, the skin irritation that may be caused by a single ingredient is reduced, and a sustained-release effect can be achieved, prolonging the duration of action.

[0028] (3) Excellent formulation properties: The hydrogel based on polyvinyl alcohol and quaternized chitosan has high water content, good bioadhesion and film-forming properties. Quaternized chitosan itself has antibacterial properties and can effectively bind to negatively charged nanoparticles and skin through electrostatic interaction, making the product skin-friendly, easy to apply, non-greasy, and less likely to stain clothing.

[0029] (4) Simple preparation process: The preparation process of metal-polyphenol network nanoparticles is simple and mild. The formation of hydrogel is based on physical mixing and electrostatic cross-linking, without the need for complex chemical synthesis or harsh reaction conditions, and is easy to scale up for production. Attached Figure Description

[0030] Figure 1 shows the MBC determination results of metal-polyphenol nanoparticles on Propionibacterium acnes. In Figure 1, the sample in Figure 2 is the metal-polyphenol nanoparticle of Comparative Example 2, and the sample in Figure 3 is the metal-polyphenol nanoparticle of Comparative Example 3.

[0031] Figure 2 shows the appearance of the hydrogel substrate. In Figure A, the sample is the hydrogel substrate of Comparative Example 4, and in Figure B, the sample is the hydrogel substrate of Comparative Example 2.

[0032] Figure 3 The particle size distribution of the metal-polyphenol network nanoparticles in Comparative Example 1 is shown.

[0033] Figure 4 The potential distribution diagram is shown for the metal-polyphenol network nanoparticles in Comparative Example 1.

[0034] Figure 5 Scanning electron microscope image of the metal-polyphenol network nanoparticles in Comparative Example 1;

[0035] Figure 6 This is a schematic image of the appearance of the anti-acne hydrogel in Example 1;

[0036] Figure 7 shows schematic images of the minimum bactericidal concentration (MBC) determination experiments for Escherichia coli, Staphylococcus aureus, and Propionibacterium acnes in Examples 1, 1, and 4, respectively. In Figures A-C, the experimental subjects are all Escherichia coli, and the samples from top to bottom are Comparative Example 1, Comparative Example 4, and Example 1, respectively. In Figures D-F, the experimental subjects are all Staphylococcus aureus, and the samples from top to bottom are Comparative Example 1, Comparative Example 4, and Example 1, respectively. In Figures G-I, the experimental subjects are all Propionibacterium acnes, and the samples from top to bottom are Comparative Example 1, Comparative Example 4, and Example 1, respectively.

[0037] Figure 8 shows the effect of the samples on the viability of HaCaT cells. In Figure A, the sample is Comparative Example 1; in Figure B, the sample is Comparative Example 4; and in Figure C, the sample is Comparative Example 5.

[0038] Figure 9 The results of the oxidative stress modeling experiment using 2,2'-azobis(2-amidinylpropane) dihydrochloride (AAPH) in HaCaT cells are shown. Figure A shows the detection of reactive oxygen species (ROS) in HaCaT cells after AAPH-induced oxidative stress; Figure B shows the detection of superoxide dismutase (SOD) in HaCaT cells after AAPH-induced oxidative stress; Figure C shows the detection of malondialdehyde (MDA) in HaCaT cells after AAPH-induced oxidative stress; and Figure D shows the detection of glutathione peroxidase (GSH-PX) in HaCaT cells after AAPH-induced oxidative stress.

[0039] Figure 10 shows the results of the effect on RAW264.7 cell viability. In Figure A, the sample is Comparative Example 1; in Figure B, the sample is Comparative Example 4; and in Figure C, the sample is Comparative Example 5.

[0040] Figure 11 shows the detection results of nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) levels after an inflammation model was constructed in RAW264.7 cells induced by lipopolysaccharide (LPS, concentration 1 μg / ml) in Example 1. In Figure 1, nitric oxide (NO) levels are detected; interleukin-6 and TNF-α levels are detected; interleukin-10 levels are detected; and transforming growth factor-β levels are detected. Detailed Implementation

[0041] The following provides a further detailed description of this application.

[0042] Example 1

[0043] Preparation of an anti-acne hydrogel:

[0044] Step 1: Weigh 2 mM luteolin into a beaker, add 20 mL of deionized water, and use 2 M sodium hydroxide solution with sonication to promote dissolution until the solution turns into a clear orange liquid. Weigh 10 mM tannin into another beaker, add 10 mL of deionized water, and sonicate until completely dissolved. Weigh 10 mM copper chloride, add 10 mL of deionized water, and sonicate until completely dissolved to obtain a metal ion solution. Mix the above three solutions, adjust the pH to 9 with 2 M sodium hydroxide solution, and carry out the reaction at room temperature, in the dark, with magnetic stirring (1400 rpm) for 8 hours. The resulting solution is centrifuged and washed with water (5000 rpm, 10 min) until the supernatant is clear to obtain a metal-polyphenol network nanoparticle suspension.

[0045] Step 2: Weigh 3 g of polyvinyl alcohol (degree of hydrolysis 92-94%), add 24 mL of deionized water, allow it to swell completely, and then heat it in a 90°C constant temperature water bath until the solution is clear and transparent, obtaining the first hydrogel substrate solution. Weigh 1.5 g of quaternized chitosan (degree of substitution 90%), add 20 mL of deionized water, allow it to swell at room temperature, and then heat it in a 50°C constant temperature water bath until it is completely dissolved and the solution is clear and transparent, obtaining the second hydrogel substrate solution.

[0046] Step 3: While stirring, slowly add 6 mL of metal-polyphenol network nanoparticle suspension to the first hydrogel substrate solution, and magnetically stir at 1500 rpm for 10 minutes to ensure uniform dispersion of the nanoparticles. Then add the second hydrogel substrate solution and continue stirring at 1500 rpm for 20 minutes to obtain the anti-acne hydrogel.

[0047] Example 2

[0048] Unlike Example 1, the second polyphenol compound used in this example is quercetin.

[0049] The rest is the same as in Example 1, and will not be repeated here.

[0050] Example 3

[0051] Unlike Example 1, the second polyphenol compound used in this example is epigallocatechin gallate. The rest is the same as in Example 1 and will not be repeated here.

[0052] The rest is the same as in Example 1, and will not be repeated here.

[0053] Comparative Example 1

[0054] 2 mM luteolin was weighed and placed in a beaker, 20 mL of deionized water was added, and sonication was used to promote dissolution with 2 M sodium hydroxide solution until the solution turned into a clear orange liquid. 10 mM tannin was weighed and placed in another beaker, 10 mL of deionized water was added, and sonication was performed until completely dissolved. 10 mM copper chloride was weighed and placed in 10 mL of deionized water, and sonication was performed until completely dissolved to obtain a metal ion solution. The three solutions were mixed, and the pH was adjusted to 9 with 2 M sodium hydroxide solution. The reaction was carried out at room temperature, in the dark, and with magnetic stirring (1400 rpm) for 8 hours. The resulting solution was centrifuged and washed with water (5000 rpm for 10 min) until the supernatant was clear, yielding metal-polyphenol network nanoparticles.

[0055] Comparative Example 2

[0056] 2 mM quercetin was weighed into a beaker and 20 mL of deionized water was added. Dissolution was accelerated using 2 M sodium hydroxide solution and sonication until the solution turned into a clear orange liquid. 10 mM tannin was weighed into another beaker and 10 mL of deionized water was added. The solution was sonicated until completely dissolved. 10 mM copper chloride was weighed into a beaker and 10 mL of deionized water was added. The solution was sonicated until completely dissolved, yielding a metal ion solution. The three solutions were mixed, and the pH was adjusted to 9 using 2 M sodium hydroxide solution. The mixture was stirred for 8 hours at room temperature, in the dark, and with magnetic stirring (1400 rpm). The resulting solution was centrifuged and washed with water (5000 rpm for 10 min) until the supernatant became clear, yielding metal-polyphenol network nanoparticles.

[0057] Comparative Example 3

[0058] Weigh 2 mM EGCG into a beaker, add 20 mL of deionized water, and promote dissolution with 2 M sodium hydroxide solution and sonication until the solution turns into a clear orange liquid. Weigh 10 mM tannin into another beaker, add 10 mL of deionized water, and sonicate until completely dissolved. Weigh 10 mM copper chloride, add 10 mL of deionized water, and sonicate until completely dissolved to obtain a metal ion solution. Mix the above three solutions, adjust the pH to 9 with 2 M sodium hydroxide solution, and carry out the reaction at room temperature, in the dark, with magnetic stirring (1400 rpm) for 8 hours. The resulting solution is centrifuged and washed with water (5000 rpm, 10 min) until the supernatant is clear to obtain metal-polyphenol network nanoparticles.

[0059] Comparative Example 4

[0060] Step 1: Weigh 3 g of polyvinyl alcohol (degree of hydrolysis 92-94%), add 24 mL of deionized water, allow it to swell completely, and then heat it in a 90°C constant temperature water bath until the solution is clear and transparent, obtaining a hydrophilic polymer solution. Weigh 1.5 g of quaternized chitosan (degree of substitution 90%), add 20 mL of deionized water, allow it to swell at room temperature, and then heat it in a 50°C constant temperature water bath until it is completely dissolved and the solution is clear and transparent, obtaining a cationic polymer solution.

[0061] Step 2: Add the cationic polymer solution to the hydrophilic polymer solution and stir at 30°C and 300 rpm to form a hydrogel substrate.

[0062] Comparative Example 5

[0063] Step 1: Weigh 3 g of polyacrylamide, add 24 mL of deionized water, allow it to swell completely, then heat it in a constant temperature water bath until the solution is clear and transparent, yielding a hydrophilic polymer solution. Weigh 1.5 g of chitosan, add 20 mL of deionized water, allow it to swell at room temperature, then heat it in a 50°C constant temperature water bath until it is completely dissolved and the solution is clear and transparent, yielding a cationic polymer solution.

[0064] Step 2: Add the cationic polymer solution to the hydrophilic polymer solution and stir at 30°C and 300 rpm to form a hydrogel substrate.

[0065] Test method:

[0066] The antibacterial effects of nanoparticles with different formulations were compared, and the optimal composition and ratio of nanoparticles were selected as the conditions for subsequent experiments. The composition of the hydrogel substrate was chosen by comparing the appearance and stability of different formulations. Finally, the physicochemical characterization of the nanoparticles and the physicochemical and biological characterization of the hydrogel were performed to determine the anti-acne ability of the anti-acne hydrogel.

[0067] The following are the specific operating steps.

[0068] 1. Visually inspect the samples from the examples and comparative examples to compare their stability.

[0069] 2. Particle size distribution and zeta potential test: The particle size distribution and zeta potential of the metal-polyphenol network nanoparticles diluted 5000 times were tested using a dynamic light scattering particle size analyzer.

[0070] 3. Electron Microscopy Test: An appropriate amount of metal-polyphenol network nanoparticle suspension diluted 5000 times was used as the test solution and uniformly transferred to the surface of clean tin foil. The sample was then allowed to dry naturally at room temperature and normal pressure. The dried tin foil sample was then subjected to gold sputtering treatment, and the treated sample was then placed on the sample stage of a field emission scanning electron microscope for observation.

[0071] 4. Determination of the minimum bactericidal concentration (MBC) of Escherichia coli:

[0072] Preparation and calibration of bacterial culture: Under aseptic conditions, single colonies of preserved *E. coli* were picked and inoculated into sterile LB liquid medium and statically cultured at 37°C for 24 h to obtain seed culture; the seed culture was centrifuged (8000-10000 rpm, 5-8 min), the supernatant was discarded, and the bacterial cells were resuspended in sterile physiological saline. This process was repeated twice to remove residual culture medium; the bacterial concentration was adjusted to... ,spare.

[0073] Construction of antimicrobial system: In a sterile environment, 5.0 ml of sterile LB liquid medium was added to each 15 ml sterile centrifuge tube, and 100 μl of the above standardized bacterial solution was accurately added. Then, the samples of the example and comparative examples were added respectively. Blank control (LB medium + sterile physiological saline) and bacterial solution control (LB medium + bacterial solution) were set up respectively, and each group was repeated in 3 parallels.

[0074] Culture and MBC determination: Invert the centrifuge tubes horizontally and incubate at 37°C for 24 hours. Then, take 50 μl of culture from each tube and spread it onto an LB solid agar plate. Incubate at 37°C for 16-24 hours and observe colony growth. The concentration corresponding to the lowest sample addition amount with no visible colony growth is the MBC.

[0075] 5. Determination of the minimum bactericidal concentration (MBC) of Staphylococcus aureus:

[0076] Preparation and calibration of bacterial culture: Under aseptic conditions, preserved Staphylococcus aureus was inoculated onto LB broth and incubated at 37°C for 24 hours. The cultured bacterial culture was then centrifuged and resuspended to adjust the bacterial concentration to the specified level. l, for backup.

[0077] Construction of the antibacterial system: Under aseptic conditions, add 5 ml of LB liquid culture medium to a sterile test tube, then add 100 μl of the above concentration. The bacterial culture was then added to different groups of the examples and comparative samples.

[0078] Culture and MBC determination: After incubating the test tubes horizontally upside down in a 37℃ incubator for 24 hours, take 50 μl of culture medium from each tube and spread it on an agar plate; observe the bacterial growth on the agar plate. The concentration of nanoparticles corresponding to no bacterial growth is the MBC concentration of LTC against Staphylococcus aureus.

[0079] 6. Determination of the minimum bactericidal concentration (MBC) of Propionibacterium acnes:

[0080] Preparation and calibration of bacterial suspension: Under sterile conditions, preserved Propionibacterium acnes was inoculated into brain and heart extract liquid culture medium and incubated at 37°C for 24 hours; the cultured bacterial suspension was then centrifuged and resuspended to adjust the bacterial concentration to the specified level. ,spare.

[0081] Construction of the antibacterial system: Under aseptic conditions, add 5 ml of brain and heart extract liquid culture medium to a sterile test tube, and then add 100 μl of the above concentration (3 × 10⁻⁶). 8 The bacterial culture was prepared at CFU / ml, and then nanoparticles of 0.9 mg, 1.8 mg, 2.7 mg, 3.6 mg, 4.5 mg, and 5.4 mg were added respectively.

[0082] Culture and MBC determination: After incubating the test tubes horizontally upside down in a 37℃ incubator for 24 hours, take 50μl of culture medium from each tube and spread it on an agar plate; observe the bacterial growth on the agar plate. The concentration of nanoparticles corresponding to no bacterial growth is the MBC concentration of LTC against Propionibacterium acnes.

[0083] 7. Detection of HaCaT cell viability:

[0084] Cell plating and pre-culture: HaCaT cells were plated at a cell density of 10,000 cells / well, with 100 μL of culture medium added to each well. Sterile PBS buffer (pH 7.4, as a humidity barrier) was injected into the peripheral wells, and the plated cells were placed in a cell culture incubator for 24 h.

[0085] Sample processing: When the cell adhesion rate is greater than 95%, the old culture medium in the well is aspirated, and the basal culture medium solution containing gradient concentrations of the example sample and the comparative sample is added to each well. Five replicate wells are set up for each concentration. At the same time, a blank control group (containing only culture medium) is set up. The treated cells are placed in a cell culture incubator and cultured for another 24 hours.

[0086] MTT incubation: Aspirate the old culture medium after culture, wash each well once with 100 μL sterile PBS, and then accurately add 100 μL of MTT working solution (concentration of 0.5 mg / mL, dissolved in DMEM) to each well. Place the culture plate in a cell culture incubator and incubate for 4 hours.

[0087] Absorbance detection and result calculation: After incubation, remove the culture medium from the wells and try to dry the remaining liquid in the wells. Add 100 μL of DMSO to each well and place the culture plate on a shaker at room temperature for 30 min. After the formazan generated by the MTT reaction is completely dissolved, use a microplate reader to measure the absorbance value (OD value) of each well at a wavelength of 490 nm. Calculate the cell proliferation rate using the formula "Cell proliferation rate = OD sample group / OD blank group × 100%".

[0088] 8. Methods for establishing an AAPH-induced oxidative damage model in HaCaT cells:

[0089] Cell seeding and adherent culture: according to HaCaT cells in logarithmic growth phase were seeded into 96-well plates at a cell density of 100 μL per well. The 96-well plates were then incubated in an incubator at 37°C and 5% CO2 for 24 h.

[0090] AAPH treatment: Add 100 μL of culture medium containing different concentrations (0 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.7 mg / mL, 3.4 mg / mL, 6.8 mg / mL, 13.6 mg / mL, 27.1 mg / mL) of AAPH to the culture wells. Set up 3 parallel experimental wells for each concentration. Incubate the culture plate in the above incubator for 24 h.

[0091] MTT assay and model concentration screening: After 24 h of incubation, discard the old culture medium in each well, add 100 μL of 0.5 mg / mL medium containing MTT to each well, and continue incubation in the incubator for 4 h; after incubation, discard the culture medium, add 100 μL of DMSO solution to each well, shake to mix for 10 min, and measure the absorbance (OD value) of each well at a wavelength of 490 nm using a microplate reader; calculate the relative activity of cells at each AAPH concentration according to the formula "cell proliferation rate = OD (AAPH group) / OD (blank group) × 100%", and screen out the optimal AAPH concentration for constructing the HaCaT cell oxidative stress injury model.

[0092] 9. Detection of cellular superoxide dismutase (SOD) levels:

[0093] Cell seeding and culture: Cell density of 1 cell / well was determined. HaCaT cells in logarithmic growth phase were seeded into 6-well culture plates (2 mL per well) and incubated at 37°C in a 5% CO2 incubator for 24 h.

[0094] Oxidative damage modeling: When the cell adhesion rate is greater than 95%, remove the old culture medium from the wells, add 100 μL of basal culture medium containing 13.6 mg / ml AAPH, set up a blank control group (containing only culture medium), set up 3 replicate wells for each group, and place the culture plate in the above incubator for 24 h for adhesion culture.

[0095] Sample intervention: Discard the old culture medium after modeling, and add culture medium containing metal-polyphenol network nanoparticles (concentrations of 5.625 μg / ml, 11.25 μg / ml, and 22.5 μg / ml) and anti-acne hydrogel (concentration groups of 0.08038 mg / ml, 0.1675 mg / ml, and 0.3125 mg / ml) to each well. Set up a blank control group (containing only culture medium) and a positive control group (containing 0.1 mg / ml vitamin C culture medium). Place the culture plate in a cell culture incubator and incubate for 24 h.

[0096] SOD level detection: Discard the old culture medium after culture, add 1 ml of phosphate-buffered saline (PBS) to each well and wash three times. Follow the instructions of the superoxide dismutase (SOD) kit (Beyotime) to detect the intracellular SOD level.

[0097] Detection of cellular malondialdehyde (MDA) levels:

[0098] The cell seeding, oxidative damage modeling, and sample intervention steps are the same as those described above in this test method, and will not be repeated here.

[0099] MDA level detection: Discard the old culture medium after culture, add 1 ml of phosphate buffered saline (PBS) to each well and wash three times. Follow the instructions of the malondialdehyde (MDA) detection kit (Beyotime) to determine the intracellular MDA level.

[0100] Detection of cellular glutathione peroxidase (GSH-PX) levels:

[0101] The cell seeding, oxidative damage modeling, and sample intervention steps are the same as those described above in this test method, and will not be repeated here.

[0102] GSH-PX level detection: Discard the old culture medium after culture, add 1 ml of phosphate-buffered saline (PBS) to each well and wash three times. Follow the instructions of the glutathione peroxidase (GSH-PX) kit (Nanjing Jiancheng) to measure the intracellular GSH-PX level.

[0103] 10. Methods for detecting RAW264.7 cell viability:

[0104] Cell seeding and pre-culture: RAW264.7 cells were seeded at a density of 10,000 cells / well, with 100 μL of culture medium added to each well. Sterile PBS buffer (pH 7.4, as a humidity barrier) was injected into the peripheral wells, and the seeded cells were placed in a cell culture incubator for 24 h.

[0105] Sample preparation: When the cell adhesion rate was greater than 95%, the old culture medium in the wells was aspirated, and basal culture medium solutions containing gradient concentrations of metal-polyphenol network nanoparticles (0 μg / ml, 5.625 μg / ml, 11.25 μg / ml, 22.5 μg / ml, 45 μg / ml, 90 μg / ml, 180 μg / ml), hydrogel substrate (0 mg / ml, 0.15625 mg / ml, 0.3125 mg / ml, 0.625 mg / ml, 1.25 mg / ml, 2.5 mg / ml), and anti-acne hydrogel (0 mg / ml, 0.15625 mg / ml, 0.3125 mg / ml, 0.625 mg / ml, 1.25 mg / ml, 2.5 mg / ml) were added to each well. Five replicate wells were set up for each concentration. A blank control group (containing only culture medium) was also set up. The treated cells were placed in a cell culture incubator and cultured for another 24 h.

[0106] MTT incubation: Aspirate the old culture medium after culture, wash each well once with 100 μl sterile PBS, and then accurately add 100 μL of MTT working solution (concentration of 0.5 mg / mL, dissolved in DMEM) to each well. Place the culture plate in a cell culture incubator and incubate for 4 h.

[0107] Absorbance detection and result calculation: After incubation, the culture medium in the wells was removed, and the residual liquid in the wells was dried as much as possible. 100 μL of DMSO was added to each well, and the culture plate was placed on a shaker at room temperature and shaken for 30 min. After the formazan generated by the MTT reaction was completely dissolved, the absorbance value (OD value) of each well was measured using a microplate reader at a wavelength of 490 nm. The cell proliferation rate was calculated using the formula "Cell proliferation rate = OD sample group / OD blank group × 100%" to determine the effect of LTC and H on the viability of RAW264.7 cells.

[0108] 11. Detection methods for LPS-induced inflammation-related markers in RAW264.7 cells:

[0109] Cell seeding and pre-culture: RAW264.7 cells were seeded into culture plates at a density of 10,000 cells / well, 100 μL of culture medium was added to each well, and sterile phosphate-buffered saline (PBS, pH 7.4, as a humidity barrier) was injected into the peripheral wells. The culture plates were then placed in a cell culture incubator and cultured for 24 h.

[0110] Inflammation model establishment: When the cell adhesion rate is greater than 95%, the old culture medium in the well is removed, and a basal culture medium solution containing 1 μg / ml LPS is added to each well. Five replicate wells are set up for each group. At the same time, a blank control group (containing only culture medium) is set up. The culture plate is placed in a cell culture incubator and cultured for 24 hours.

[0111] Sample intervention: Discard the old culture medium after modeling, and add culture medium containing nanoparticles (metal-polyphenol network nanoparticles, concentrations of 5.625 μg / ml, 11.25 μg / ml, and 22.5 μg / ml) and anti-acne polymers (concentrations of 0.08038 mg / ml, 0.1675 mg / ml, and 0.3125 mg / ml) to each well. At the same time, a blank control group (containing only culture medium), a model group (containing 1 μg / ml LPS), and a positive control group (containing 0.1 μg / ml dexamethasone (DEX) in culture medium) are set up. The culture plates are placed in a cell culture incubator and cultured for 24 h.

[0112] Inflammatory marker detection: After culture, the cell supernatant of each well was aspirated and centrifuged at 8000 rpm for 15 min. The levels of each inflammatory marker in the supernatant were measured according to the instructions of the nitric oxide (NO) assay kit, interleukin-6 (IL-6) assay kit, tumor necrosis factor-α (TNF-α) assay kit, interleukin-10 (IL-10) assay kit, and transforming growth factor-β (TGF-β) assay kit.

[0113] The test results of the examples and comparative examples are shown in the table below.

[0114] Table 1

[0115]

[0116] As shown in Figure 1, the minimum bactericidal concentrations of the nanoparticles in Examples 2 and 3 against Propionibacterium acnes (8.5 mg / ml and 16.5 mg / ml, respectively) are significantly higher than those of the nanoparticles in Example 1.

[0117] As shown in Figure 2, the water separation in the hydrogel substrate of Comparative Example 2 indicates that the properties of the substrate of Comparative Example 2 are unstable.

[0118] Depend on Figure 3 Analysis of the particle size distribution spectrum and Zeta potential spectrum shows that the metal-polyphenol network nanoparticles of Comparative Example 1 have a spherical or near-spherical microstructure and a uniform particle size distribution; at the same time, the nanoparticles have a clear negative charge characteristic on their surface.

[0119] Depend on Figure 4The electron electron microscopy images show that the metal-polyphenol network nanoparticles in Comparative Example 1 have a spherical morphology and their size meets the characteristic requirements of composite nanoscale materials.

[0120] As shown in Figure 7, the anti-acne hydrogel of Example 1, the metal-polyphenol network nanoparticles of Comparative Example 1, and the hydrogel substrate of Comparative Example 4 all have excellent antibacterial effects against Escherichia coli, Staphylococcus aureus, and Propionibacterium acnes, the pathogenic bacterium of acne.

[0121] As shown in Figure 8, the anti-acne hydrogel of Example 1, the metal-polyphenol network nanoparticles of Comparative Example 1, and the hydrogel substrate of Comparative Example 4 all have good biocompatibility with Hacat cells.

[0122] Depend on Figure 9 It can be seen that the anti-acne hydrogel of Example 1, the metal-polyphenol network nanoparticles of Comparative Example 1, and the hydrogel substrate of Comparative Example 4 all have good antioxidant effects on Hacat cells under AAPH oxidative stress modeling.

[0123] As shown in Figure 10, the anti-acne hydrogel of Example 1, the metal-polyphenol network nanoparticles of Comparative Example 1, and the hydrogel substrate of Comparative Example 4 all have good biocompatibility with RAW cells.

[0124] As shown in Figure 11, the anti-acne hydrogel of Example 1, the metal-polyphenol network nanoparticles of Comparative Example 1, and the hydrogel substrate of Comparative Example 4 all have good anti-inflammatory effects on the construction of an inflammation model of RAW264.7 cells induced by lipopolysaccharide (LPS, concentration of 1 μg / ml).

[0125] As shown in Figures 7 to 11, the anti-acne hydrogel of Example 1, the metal-polyphenol network nanoparticles of Comparative Example 1, and the hydrogel substrate of Comparative Example 4 all exhibit superior performance. However, while nanoparticles can penetrate hair follicles, they are easily and quickly removed from the skin surface due to sebum and sweat secretion, resulting in a short duration of action. In contrast, when nanoparticles are firmly loaded into the hydrogel substrate, their tiny size allows them to effectively carry drugs deep into the pilosebaceous unit, directly acting on Propionibacterium acnes and the inflamed area. This not only allows for prolonged adhesion to the acne area but also enables intelligent sustained-release of the nanoparticles and their encapsulated drugs through responses to environmental pH, enzymes, etc., ensuring the continuous and stable action of the therapeutic components on the lesion. Furthermore, the hydrogel substrate not only provides a moist healing environment but also has cooling and soothing effects on inflammation. The combination of these two elements forms a tiered treatment model: "hydrogel controls the overall situation (macro-inflammatory environment), nanoparticles precisely target (micro-pathogens)." This synergistic effect makes the composite hydrogel significantly more effective than any single component in inhibiting bacterial growth and reducing the release of pro-inflammatory factors (such as TNF-α and IL-6).

[0126] In terms of results, the anti-acne hydrogel demonstrates an overwhelming advantage:

[0127] For example, in the antibacterial experiment diagram against Propionibacterium acnes ( Figure 7-G , Figure 7-H , Figure 7-I In the study, it was clearly observed that the anti-acne hydrogel group showed a lower bactericidal concentration compared to the individual nanoparticle group and hydrogel substrate group, demonstrating its stronger antibacterial efficacy.

[0128] Table 2 shows the experimental sources for this invention.

[0129] Table 2

[0130]

[0131] The above are all embodiments of this application and are not intended to limit the scope of protection of this application. Any solutions that can be easily conceived by those skilled in the art after reading this application are also the protection solutions claimed by this application. All equivalent changes made to the structure, shape, and principle of this application should be covered within the scope of protection of this application.

Claims

1. An anti-acne hydrogel, characterized in that, The invention comprises metal-polyphenol network nanoparticles and a hydrogel substrate; the metal-polyphenol network nanoparticles comprise a first polyphenol compound, a second polyphenol compound, and metal ions, wherein the first polyphenol compound is a tannic acid compound, and the second polyphenol compound is a flavonoid compound; the hydrogel substrate comprises a hydrophilic polymer and a cationic polymer.

2. The anti-acne hydrogel according to claim 1, characterized in that, The mass ratio of the metal-polyphenol network nanoparticles to the hydrogel substrate is 1-10:1-2.

3. The anti-acne hydrogel according to claim 1, characterized in that, The flavonoids are selected from one or more of luteolin, baicalin, epigallocatechin gallate, quercetin, and kaempferol.

4. The anti-acne hydrogel according to claim 1, characterized in that, The metal ions are selected from one or more of copper ions, zinc ions, and iron ions.

5. The anti-acne hydrogel according to claim 1, characterized in that, The hydrophilic polymer is one or more of polyvinyl alcohol, polyethylene glycol, and polyacrylamide; the cationic polymer is one or more of quaternized chitosan, polyethyleneimine, and chitosan.

6. The anti-acne hydrogel according to claim 1, characterized in that, The molar ratio of the first polyphenol compound, the second polyphenol compound, and the metal ion is 1-4:4-8:5-10.

7. A method for preparing an anti-acne hydrogel according to any one of claims 1-6, characterized in that, Includes the following steps: Step 1: Preparation of a metal-polyphenol network nanoparticle suspension; Step 2: Prepare the first hydrogel substrate solution and the second hydrogel substrate solution respectively; Step 3: Mix the nanoparticle suspension obtained in Step 1 with the first hydrogel substrate solution and the second hydrogel substrate solution obtained in Step 2, and stir to form an anti-acne hydrogel.

8. The method for preparing the anti-acne hydrogel according to claim 7, characterized in that, The preparation method of the metal-polyphenol network nanoparticle suspension includes: dissolving a first polyphenol compound, a second polyphenol compound and a metal salt in water, adjusting the pH to 8-10, stirring and reacting at room temperature in the dark for 4-12 hours, and centrifuging and washing to obtain the nanoparticle suspension.

9. The method for preparing the anti-acne hydrogel according to claim 7, characterized in that, The preparation method of the hydrogel substrate solution includes the following steps: Step 1: Add the hydrophilic polymer to deionized water, let it swell, and then heat it to 80-95℃ to dissolve it, thus obtaining the first hydrogel substrate solution; Step 2: Add the cationic polymer to deionized water, let it swell, and then heat it to 30-50℃ to dissolve, thus obtaining the second hydrogel substrate solution.

10. The method for preparing the anti-acne hydrogel according to claim 7, characterized in that, In step three, the ratio of the amount of nanoparticle suspension added to the hydrogel substrate solution is 1-7 ml: 40-50 ml, the stirring speed is 1000-2000 rpm, and the stirring time is 10-30 minutes.

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