A naringenin-antimicrobial peptide composite liposome hydrogel as well as a preparation method and application thereof
Patent Information
- Application Number
- CN202611082210.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-21
- Publication Date
- 2026-11-03
AI Technical Summary
Long-term use of traditional acne treatments can easily lead to skin irritation and bacterial resistance.
The naringenin-antimicrobial peptide complex liposome hydrogel is used. By co-loading naringenin and Bacillus subtilis antimicrobial peptide in carbomer hydrogel, the phospholipid bilayer structure of liposomes is used to promote drug retention and penetration in the skin, and combined with anti-inflammatory and bactericidal effects, a sustained-release effect is achieved.
It significantly reduces the risk of bacterial resistance, improves antibacterial efficacy, alleviates local inflammatory response, and enhances the treatment effect of acne and other inflammatory skin diseases, while also exhibiting good stability and skin adhesion.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of nanomedicine delivery and topical skin preparation technology, specifically relating to a naringenin-antimicrobial peptide composite liposome hydrogel, its preparation method and application. Background Technology
[0002] Acne is a common chronic inflammatory skin disease, and its occurrence is closely related to abnormal sebum secretion, follicular keratosis, proliferation of acne-associated pathogens, and local inflammatory response. Traditional topical treatments mainly include topical antibiotics, retinoids, and benzoyl peroxide, but long-term use can easily lead to adverse reactions such as skin irritation, itching, and peeling, as well as antibiotic-induced bacterial resistance. Summary of the Invention
[0003] In order to overcome the shortcomings of the prior art, the present invention aims to provide a naringenin-antimicrobial peptide complex liposome hydrogel, its preparation method and application, so as to solve the problem that long-term use of traditional acne treatment drugs is prone to skin irritation and bacterial resistance.
[0004] The technical solution adopted in this invention is: In a first aspect, the present invention provides a naringenin-antimicrobial peptide composite liposome hydrogel, wherein the hydrogel is composed of naringenin-Bacillus subtilis antimicrobial peptide co-loaded liposomes uniformly dispersed in a carbomer hydrogel matrix; wherein the co-loaded liposomes are based on soybean lecithin and cholesterol as membrane materials, and encapsulate the active ingredients naringenin and Bacillus subtilis antimicrobial peptides; wherein naringenin is distributed in the membrane phase of the phospholipid bilayer, and Bacillus subtilis antimicrobial peptides are encapsulated in the aqueous phase of the liposomes.
[0005] Furthermore, the average particle size of the co-loaded liposomes is 100-200 nm, and the polydispersity index (PDI) is ≤0.2.
[0006] Secondly, the present invention provides a method for preparing a naringin-antimicrobial peptide complex liposome hydrogel, comprising the following steps: Step 1: Under light-protected conditions, soybean lecithin, cholesterol, and naringenin are dissolved in a chloroform-methanol mixed solvent to obtain a homogeneous and transparent lipid phase solution, wherein the mass ratio of soybean lecithin to cholesterol is (4-6):1, and the mass ratio of naringenin to the mixture of soybean lecithin and cholesterol is 1:(12-20); the lipid phase solution is subjected to reduced pressure rotary evaporation at 40-50℃ to remove the organic solvent until a uniform lipid film is formed on the inner wall of the container, and then vacuum dried to completely remove the residual organic solvent, to obtain a dry lipid film loaded with naringenin; Step 2: Dissolve the Bacillus subtilis antimicrobial peptide in 10 mmol / L PBS buffer (pH 6.8) preheated to 35-39°C to prepare an antimicrobial peptide hydration solution; the concentration of the Bacillus subtilis antimicrobial peptide in the antimicrobial peptide hydration solution is 0.5-2 mg / mL; add the antimicrobial peptide hydration solution to the dried lipid film loaded with naringenin prepared in Step 1, and rotate to hydrate and completely detach the lipid film to obtain a crude liposome suspension; Step 3: Transfer the crude liposome suspension to a liposome extruder and granulate it step by step using a polycarbonate membrane under the condition of room temperature and protection from light throughout the process to obtain a naringenin-antimicrobial peptide co-loaded liposome suspension. Step 4: Disperse 1-4g of Carbomer 940 powder evenly in 30-70ml of purified water, seal and let it stand to swell to obtain a uniform and transparent gel matrix; then add 0.5-2g of humectant and 0.5-2g of preservative and mix well; then add neutralizing agent dropwise while stirring to adjust the pH of the system to 6.8-7.0 to obtain the neutralized gel matrix; Step 5: Under conditions of complete darkness and room temperature, the naringenin-antimicrobial peptide co-loaded liposome suspension prepared in step 3 is added dropwise to the neutralized gel matrix prepared in step 4 and mixed evenly. Then, PBS buffer solution with pH 6.8 is added to 100-200ml and stirred evenly to obtain naringenin-Bacillus subtilis antimicrobial peptide co-loaded liposome hydrogel.
[0007] Furthermore, the chloroform-methanol mixed solvent in step 1 is composed of chloroform and methanol in a volume ratio of 2:1, and the rotary evaporation under reduced pressure has a rotation speed of 80-100 rpm.
[0008] Furthermore, the Bacillus subtilis antimicrobial peptide described in step 2 is prepared by the following method: Bacillus subtilis was inoculated into LB liquid medium and cultured at 37℃ and 150-180 r / min for 20-28 h with shaking to obtain fermentation broth. The fermentation broth was centrifuged to remove bacterial cells, and the supernatant was collected. The supernatant was placed in an ice-water bath, and ammonium sulfate powder was slowly added with stirring until the ammonium sulfate saturation of the system was 75%-85%. After standing at 4℃ for 1-3 h, it was centrifuged at 8000-12000 r / min for 20-40 min, and the precipitate was collected. The precipitate was reconstituted with 10 mmol / L PBS buffer at pH 6.2, and then dialyzed through a dialysis bag with a molecular weight cutoff of 3-5 kDa to desalt it. The dialyzed antimicrobial peptide solution was freeze-dried to obtain Bacillus subtilis antimicrobial peptide lyophilized powder, which was stored at -20℃ in the dark for later use. The rotational hydration temperature in step 2 was 35-39℃, and the rotational hydration time was 30-60 min.
[0009] Furthermore, the step 3 operation of extruding and granulating the crude liposome suspension through a polycarbonate membrane is as follows: the crude liposome suspension is transferred to a liposome extruder, and granulated by extrusion through a polycarbonate membrane under the condition of room temperature and protection from light throughout the process: first, it is extruded 5 times through a 0.45μm pore size polycarbonate membrane for preliminary homogenization; then it is extruded 8-10 times through a 0.22μm pore size polycarbonate membrane; and finally, it is extruded 8-10 times through a 0.1μm pore size polycarbonate membrane.
[0010] Furthermore, the preservative in step 4 is selected from at least one of phenoxyethanol, parabens, and capryloyl hydroxamic acid; the moisturizer is selected from at least one of propylene glycol, glycerin, and PEG-400; and the neutralizer is selected from at least one of triethanolamine and tris(hydroxymethyl)aminomethane.
[0011] When adding the co-loaded liposome suspension dropwise to the neutralized gel matrix in step 5, maintain stirring at 150-300 r / min. After the addition is complete, continue stirring for 15-30 min until the system is homogeneous. The degassing time under reduced pressure is 3-10 min.
[0012] Thirdly, the present invention provides the application of naringenin-antimicrobial peptide complex liposome hydrogel in the treatment of inflammatory or bacterial skin infections.
[0013] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a naringenin-antimicrobial peptide composite liposome hydrogel. Firstly, pharmacologically, the natural antimicrobial substance derived from Bacillus subtilis possesses broad-spectrum antimicrobial activity and is less likely to induce drug resistance. Compared with traditional topical antibiotic preparations, it can significantly reduce the risk of bacterial resistance during long-term use. Simultaneously, both naringenin and antimicrobial peptides are naturally derived active ingredients with good biocompatibility. Combining the anti-inflammatory and antioxidant effects of naringenin with the membrane-disrupting and bactericidal effects of antimicrobial peptides, their mechanisms of action are complementary—naringenin focuses on inhibiting the release of inflammatory factors, reducing local inflammatory responses, and assisting in antimicrobial action, while the antimicrobial peptide exerts a rapid bactericidal effect by disrupting the cell membrane integrity of pathogenic bacteria such as Propionibacterium acnes. This creates a synergistic effect in both antimicrobial and anti-inflammatory aspects, which is beneficial for improving the local treatment efficacy of acne, folliculitis, and other inflammatory and bacterial skin diseases.
[0014] This invention co-loads naringenin and antimicrobial peptides into a liposome system, resulting in composite liposomes with a particle size of 100–200 nm, a polydispersity index of less than 0.14, uniform particle size distribution, and good system stability, effectively reducing the risk of active ingredient aggregation and leakage. The phospholipid bilayer structure of the liposomes promotes drug retention and penetration in the skin and endows the formulation with certain sustained-release properties.
[0015] This invention uses carbomer 940 as the gel matrix, and the pH of the formulation is controlled at 6.8-7.0, which is compatible with the slightly acidic physiological environment of the skin surface and has low irritation. The gel has a uniform and delicate texture, with good spreadability and skin adhesion, and no obvious greasy feeling or foreign body sensation after administration. The liposome nanoparticle size can promote the retention of drugs in the stratum corneum of the skin and improve the local administration efficiency, making it suitable for topical treatment of local skin conditions such as acne. Detailed Implementation
[0016] The structural and working principles of the present invention will be further explained in detail below.
[0017] Example 1: A method for preparing a naringin-antimicrobial peptide complex liposome hydrogel, comprising the following steps: Step 1: Under light-protected conditions, soybean lecithin, cholesterol, and naringenin were dissolved in a chloroform-methanol mixed solvent, wherein the volume ratio of chloroform to methanol was 2:1, to obtain a homogeneous and transparent lipid phase solution. The mass ratio of soybean lecithin to cholesterol was 4:1, and the mass ratio of naringenin to the mixture of soybean lecithin and cholesterol was 1:15. The lipid phase solution was subjected to rotary evaporation under reduced pressure at 45°C and 90 rpm to remove the organic solvent until a uniform lipid film formed on the inner wall of the container. After the rotary evaporation was completed, the solution was dried under vacuum for 2 hours to completely remove the residual organic solvent, resulting in a dried lipid film loaded with naringenin. Step 2: Dissolve the Bacillus subtilis antimicrobial peptide in 10 mmol / L PBS buffer (pH 6.8, preheated to 35°C) to prepare an antimicrobial peptide hydration solution; the concentration of the Bacillus subtilis antimicrobial peptide in the antimicrobial peptide hydration solution is 1 mg / mL; add the antimicrobial peptide hydration solution to the dried lipid film loaded with naringenin prepared in Step 1, and place it in a 35°C water bath shaker at 100 rpm for 30 min to hydrate completely and remove the lipid film, obtaining a crude liposome suspension; The Bacillus subtilis antimicrobial peptide was prepared by the following method: Bacillus subtilis was inoculated into LB liquid medium and cultured at 37°C and 150 rpm for 28 h with shaking to obtain fermentation broth. The fermentation broth was centrifuged to remove bacterial cells, and the supernatant was collected. The supernatant was placed in an ice-water bath, and ammonium sulfate powder was slowly added with stirring until the ammonium sulfate saturation of the system was 75%. After standing at 4°C for 1 h, it was centrifuged at 8000 rpm for 40 min, and the precipitate was collected. The precipitate was reconstituted with 10 mmol / L PBS buffer (pH 6.2), and then dialyzed through a dialysis bag with a molecular weight cutoff of 3 kDa to remove salts. The dialyzed antimicrobial peptide solution was freeze-dried to obtain Bacillus subtilis antimicrobial peptide lyophilized powder, which was stored at -20°C in the dark for later use.
[0018] Step 3: Transfer the crude liposome suspension to a liposome extruder and extrude it stepwise through a polycarbonate membrane under ambient temperature and light-protected conditions: first, extrude it 5 times through a 0.45μm pore size polycarbonate membrane for initial homogenization; then, extrude it 8 times through a 0.22μm pore size polycarbonate membrane; finally, extrude it 10 times through a 0.1μm pore size polycarbonate membrane to obtain a naringenin-antimicrobial peptide co-loaded liposome suspension. Step 4: Weigh 2g of carbomer 940 powder and disperse it evenly in 40ml of ultrapure water. After sealing, place it in a 4℃ refrigerator and let it swell to obtain a uniform and transparent gel matrix. Then add 1g of propylene glycol and 1g of phenoxyethanol and stir at 150r / min at room temperature until the mixture is uniform. Then add triethanolamine dropwise while stirring to adjust the pH of the system to 6.8. Continue stirring until the system is uniform to obtain the neutralized gel matrix. Step 5: Under conditions of complete protection from light and room temperature, the naringenin-antimicrobial peptide co-loaded liposome suspension prepared in Step 3 was added dropwise to the neutralized gel matrix prepared in Step 4 and mixed evenly. The mixture was stirred at 150 r / min, and after the addition was complete, the mixture was stirred for another 30 min until the system was homogeneous. Then, PBS buffer solution with pH 6.8 was added to 100 ml and stirred evenly to obtain naringenin-Bacillus subtilis antimicrobial peptide co-loaded liposome hydrogel, which was stored at 4℃ in the dark.
[0019] Example 2: A method for preparing a naringin-antimicrobial peptide complex liposome hydrogel, comprising the following steps: Step 1: Under light-protected conditions, soybean lecithin, cholesterol, and naringenin are dissolved in a chloroform-methanol mixed solvent, wherein the volume ratio of chloroform to methanol is 2:1, to obtain a homogeneous and transparent lipid phase solution. The mass ratio of soybean lecithin to cholesterol is 5:1, and the mass ratio of naringenin to the mixture of soybean lecithin and cholesterol is 1:12. The lipid phase solution is subjected to rotary evaporation under reduced pressure at 40°C and 100 rpm to remove the organic solvent until a uniform lipid film forms on the inner wall of the container. After rotary evaporation, the solution is dried under vacuum for 2 hours to completely remove residual organic solvent, resulting in a dried lipid film loaded with naringenin. Step 2: Dissolve the Bacillus subtilis antimicrobial peptide in 10 mmol / L PBS buffer (pH 6.8, preheated to 36°C) to prepare an antimicrobial peptide hydration solution; the concentration of the Bacillus subtilis antimicrobial peptide in the antimicrobial peptide hydration solution is 0.5 mg / mL; add the antimicrobial peptide hydration solution to the dried lipid film loaded with naringenin prepared in Step 1, and place it in a 36°C water bath shaker at 100 rpm for 45 min to completely detach the lipid film, obtaining a crude liposome suspension; The Bacillus subtilis antimicrobial peptide was prepared by the following method: Bacillus subtilis was inoculated into LB liquid medium and cultured at 37°C and 180 rpm for 20 h with shaking to obtain fermentation broth. The fermentation broth was centrifuged to remove bacterial cells, and the supernatant was collected. The supernatant was placed in an ice-water bath, and ammonium sulfate powder was slowly added with stirring until the ammonium sulfate saturation of the system was 85%. After standing at 4°C for 1.5 h, it was centrifuged at 12000 rpm for 20 min, and the precipitate was collected. The precipitate was reconstituted with 10 mmol / L PBS buffer (pH 6.2), and then dialyzed through a dialysis bag with a molecular weight cutoff of 4 kDa to remove salts. The dialyzed antimicrobial peptide solution was freeze-dried to obtain Bacillus subtilis antimicrobial peptide lyophilized powder, which was stored at -20°C in the dark for later use.
[0020] Step 3: Transfer the crude liposome suspension to a liposome extruder and extrude it stepwise through a polycarbonate membrane under ambient temperature and light-protected conditions: first, extrude it 5 times through a 0.45μm pore size polycarbonate membrane for initial homogenization; then, extrude it 10 times through a 0.22μm pore size polycarbonate membrane; finally, extrude it 9 times through a 0.1μm pore size polycarbonate membrane to obtain a naringenin-antimicrobial peptide co-loaded liposome suspension. Step 4: Weigh 4g of carbomer 940 powder and disperse it evenly in 70ml of ultrapure water. After sealing, place it in a 4℃ refrigerator and let it swell to obtain a uniform and transparent gel matrix. Then add a mixture of 1.5g of propylene glycol and glycerin, and a mixture of 1.8g of phenoxyethanol and parabens. Stir at 300r / min at room temperature until the mixture is uniform. Then, while stirring, add tris(hydroxymethyl)aminomethane dropwise to adjust the pH of the system to 6.9. Continue stirring until the system is uniform to obtain the neutralized gel matrix. Step 5: Under conditions of complete protection from light and room temperature, the naringenin-antimicrobial peptide co-loaded liposome suspension prepared in Step 3 was added dropwise to the neutralized gel matrix prepared in Step 4 and mixed evenly. The mixture was stirred at 300 r / min, and after the addition was complete, the mixture was stirred for another 15 min until the system was homogeneous. Then, PBS buffer solution with pH 6.8 was added to 200 ml and stirred evenly to obtain naringenin-Bacillus subtilis antimicrobial peptide co-loaded liposome hydrogel, which was stored at 4℃ in the dark.
[0021] Example 3: A method for preparing a naringin-antimicrobial peptide complex liposome hydrogel, comprising the following steps: Step 1: Under light-protected conditions, soybean lecithin, cholesterol, and naringenin were dissolved in a chloroform-methanol mixed solvent, wherein the volume ratio of chloroform to methanol was 2:1, to obtain a homogeneous and transparent lipid phase solution. The mass ratio of soybean lecithin to cholesterol was 6:1, and the mass ratio of naringenin to the mixture of soybean lecithin and cholesterol was 1:20. The lipid phase solution was subjected to rotary evaporation under reduced pressure at 50°C and 80 rpm to remove the organic solvent until a uniform lipid film formed on the inner wall of the container. After the rotary evaporation was completed, the solution was dried under vacuum for 2 hours to completely remove the residual organic solvent, resulting in a dried lipid film loaded with naringenin. Step 2: Dissolve the Bacillus subtilis antimicrobial peptide in 10 mmol / L PBS buffer (pH 6.8, preheated to 39°C) to prepare an antimicrobial peptide hydration solution; the concentration of the Bacillus subtilis antimicrobial peptide in the antimicrobial peptide hydration solution is 2 mg / mL; add the antimicrobial peptide hydration solution to the dried lipid film loaded with naringenin prepared in Step 1, and place it in a 37°C water bath shaker at 100 rpm for 50 min to completely detach the lipid film, obtaining a crude liposome suspension; The Bacillus subtilis antimicrobial peptide was prepared by the following method: Bacillus subtilis was inoculated into LB liquid medium and cultured at 37°C and 160 rpm for 25 h with shaking to obtain fermentation broth. The fermentation broth was centrifuged to remove bacterial cells, and the supernatant was collected. The supernatant was placed in an ice-water bath, and ammonium sulfate powder was slowly added with stirring until the ammonium sulfate saturation of the system was 80%. After standing at 4°C for 2 h, it was centrifuged at 9000 rpm for 25 min, and the precipitate was collected. The precipitate was reconstituted with 10 mmol / L PBS buffer (pH 6.2), and then dialyzed through a dialysis bag with a molecular weight cutoff of 5 kDa to remove salts. The dialyzed antimicrobial peptide solution was freeze-dried to obtain Bacillus subtilis antimicrobial peptide lyophilized powder, which was stored at -20°C in the dark for later use.
[0022] Step 3: Transfer the crude liposome suspension to a liposome extruder and extrude it stepwise through a polycarbonate membrane under ambient temperature and light-protected conditions: first, extrude it 5 times through a 0.45μm pore size polycarbonate membrane for initial homogenization; then, extrude it 9 times through a 0.22μm pore size polycarbonate membrane; finally, extrude it 8 times through a 0.1μm pore size polycarbonate membrane to obtain a naringenin-antimicrobial peptide co-loaded liposome suspension. Step 4: Weigh 1g of carbomer 940 powder and disperse it evenly in 30ml of ultrapure water. After sealing, place it in a 4℃ refrigerator and let it swell to obtain a homogeneous and transparent gel matrix. Then add 0.5g of glycerol and a mixture of 0.8g of parabens and capryloyl hydroxamic acid. Stir at 200r / min at room temperature until the mixture is homogeneous. Then, while stirring, add a mixture of triethanolamine and tris(hydroxymethyl)aminomethane dropwise to adjust the pH of the system to 7. Continue stirring until the system is homogeneous to obtain the neutralized gel matrix. Step 5: Under conditions of complete protection from light and room temperature, the naringenin-antimicrobial peptide co-loaded liposome suspension prepared in Step 3 was added dropwise to the neutralized gel matrix prepared in Step 4 and mixed evenly. The mixture was stirred at 200 r / min, and after the addition was complete, the mixture was stirred for another 20 min until the system was homogeneous. Then, PBS buffer solution with pH 6.8 was added to 150 ml and stirred evenly to obtain naringenin-Bacillus subtilis antimicrobial peptide co-loaded liposome hydrogel, which was stored at 4℃ in the dark.
[0023] Example 4: A method for preparing a naringin-antimicrobial peptide complex liposome hydrogel, comprising the following steps: Step 1: Under light-protected conditions, soybean lecithin, cholesterol, and naringenin were dissolved in a chloroform-methanol mixed solvent, wherein the volume ratio of chloroform to methanol was 2:1, to obtain a homogeneous and transparent lipid phase solution. The mass ratio of soybean lecithin to cholesterol was 4:1, and the mass ratio of naringenin to the mixture of soybean lecithin and cholesterol was 1:18. The lipid phase solution was subjected to rotary evaporation under reduced pressure at 45°C and 100 rpm to remove the organic solvent until a uniform lipid film was formed on the inner wall of the container. After the rotary evaporation was completed, the solution was dried under vacuum for 2 hours to completely remove the residual organic solvent, resulting in a dried lipid film loaded with naringenin. Step 2: Dissolve the Bacillus subtilis antimicrobial peptide in 10 mmol / L PBS buffer (pH 6.8, preheated to 37°C) to prepare an antimicrobial peptide hydration solution; the concentration of the Bacillus subtilis antimicrobial peptide in the antimicrobial peptide hydration solution is 1.5 mg / mL; add the antimicrobial peptide hydration solution to the dried lipid film loaded with naringenin prepared in Step 1, and place it in a 39°C water bath shaker at 100 rpm for 60 min to hydrate completely and remove the lipid film, obtaining a crude liposome suspension; The Bacillus subtilis antimicrobial peptide was prepared by the following method: Bacillus subtilis was inoculated into LB liquid medium and cultured at 37°C and 170 rpm for 20 h with shaking to obtain fermentation broth. The fermentation broth was centrifuged to remove bacterial cells, and the supernatant was collected. The supernatant was placed in an ice-water bath, and ammonium sulfate powder was slowly added with stirring until the ammonium sulfate saturation of the system reached 82%. After standing at 4°C for 3 h, it was centrifuged at 10000 rpm for 30 min, and the precipitate was collected. The precipitate was reconstituted with 10 mmol / L PBS buffer (pH 6.2), and then dialyzed through a dialysis bag with a molecular weight cutoff of 4 kDa to desalt it. The dialyzed antimicrobial peptide solution was freeze-dried to obtain Bacillus subtilis antimicrobial peptide lyophilized powder, which was stored at -20°C in the dark for later use.
[0024] Step 3: Transfer the crude liposome suspension to a liposome extruder and extrude it stepwise through a polycarbonate membrane under ambient temperature and light-protected conditions: first, extrude it 5 times through a 0.45μm pore size polycarbonate membrane for initial homogenization; then, extrude it 8 times through a 0.22μm pore size polycarbonate membrane; finally, extrude it 8 times through a 0.1μm pore size polycarbonate membrane to obtain a naringenin-antimicrobial peptide co-loaded liposome suspension. Step 4: Weigh 3g of carbomer 940 powder and evenly disperse it in 50ml of ultrapure water. After sealing, place it in a 4℃ refrigerator and let it swell to obtain a homogeneous and transparent gel matrix. Then add 2g of a mixture of glycerol and PEG-400, as well as 1.5g of parabens. Stir at 180r / min at room temperature until the mixture is homogeneous. Then add triethanolamine dropwise while stirring to adjust the pH of the system to 7. Continue stirring until the system is homogeneous to obtain the neutralized gel matrix. Step 5: Under conditions of complete protection from light and room temperature, the naringenin-antimicrobial peptide co-loaded liposome suspension prepared in Step 3 was added dropwise to the neutralized gel matrix prepared in Step 4 and mixed evenly. The mixture was stirred at 250 r / min, and after the addition was complete, the mixture was stirred for another 25 min until the system was homogeneous. Then, PBS buffer solution with pH 6.8 was added to 180 ml, and the mixture was stirred evenly to obtain the naringenin-Bacillus subtilis antimicrobial peptide co-loaded liposome hydrogel, which was stored at 4℃ in the dark.
[0025] Comparative Example 1: Preparation of Naringenin Monopeptide Hydrogel The steps 1 and 4 are the same as in Example 1, except for steps 2, 3 and 5, as follows: Step 2: Measure 100 mL of 10 mmol / L PBS buffer (pH 6.2) preheated to 35 °C as the hydration solution; then place it in a 35 °C water bath shaker for 30 min to rotate and hydrate until the lipid film is completely detached, and obtain a crude liposome suspension. Step 3: Transfer the crude liposome suspension to a liposome extruder and extrude it stepwise through a polycarbonate membrane under ambient temperature and light-protected conditions: first, extrude it 5 times through a 0.45μm pore size polycarbonate membrane for initial homogenization; then, extrude it 8 times through a 0.22μm pore size polycarbonate membrane; finally, extrude it 10 times through a 0.1μm pore size polycarbonate membrane to obtain a naringenin monocarrier liposome suspension. Step 5: Under conditions of complete darkness and room temperature, the naringenin mono-loaded liposome suspension prepared in Step 3 is added dropwise to the neutralized gel matrix prepared in Step 4 and mixed evenly. The mixture is stirred at 300 r / min. After the addition is complete, the mixture is stirred for another 15 min until the system is homogeneous. Then, PBS buffer solution with pH 6.8 is added to 100 ml and stirred evenly to obtain naringenin mono-loaded liposome hydrogel.
[0026] Comparative Example 2: Preparation of Antimicrobial Peptide Monopeptide Hydrogel Step 1: Under light-protected conditions, soybean lecithin and cholesterol are dissolved in a chloroform-methanol mixed solvent, wherein the volume ratio of chloroform to methanol is 2:1, to obtain a homogeneous and transparent lipid phase solution, wherein the mass ratio of soybean lecithin to cholesterol is 5:1; the lipid phase solution is subjected to rotary evaporation under reduced pressure at 40°C and 100 rpm to remove the organic solvent until a uniform lipid film is formed on the inner wall of the container. After the rotary evaporation is completed, the solution is dried under vacuum for another 2 hours to completely remove the residual organic solvent, resulting in a blank dried lipid film; In step 2, the process is the same as in Example 1, except that the antimicrobial peptide hydration solution is added to the blank dry lipid film prepared in step 1, and the film is placed in a 39°C water bath shaker for 40 min to completely detach the lipid film, resulting in a crude liposome suspension. Step 3: Transfer the crude liposome suspension to a liposome extruder and extrude it stepwise through a polycarbonate membrane under ambient temperature and light-protected conditions: first, extrude it 5 times through a 0.45μm pore size polycarbonate membrane for initial homogenization; then, extrude it 9 times through a 0.22μm pore size polycarbonate membrane; finally, extrude it 8 times through a 0.1μm pore size polycarbonate membrane to obtain an antimicrobial peptide monopeptide liposome suspension. Step 4: Exactly the same as step 4 in Example 1; Step 5: Under conditions of complete darkness and room temperature, the antimicrobial peptide monopeptide suspension prepared in Step 3 is added dropwise to the neutralized gel matrix prepared in Step 4 and mixed evenly. The mixture is stirred at 150 r / min. After the addition is complete, the mixture is stirred for another 30 min until the system is homogeneous. Then, PBS buffer solution with pH 6.8 is added to 200 ml and stirred evenly to obtain the antimicrobial peptide monopeptide hydrogel.
[0027] Experimental section: 1. Antibacterial test The antibacterial activity of Example 1 and the comparative examples against *Propionibacterium acnes* was evaluated using the agar diffusion method. Activated *Propionibacterium acnes* was inoculated into liquid culture medium and anaerobically cultured at 37°C for 24 h. The bacterial concentration was adjusted to approximately 1 × 10^8 CFU / mL for later use. The culture was then evenly spread onto an agar surface, and 200 μL of sample was added using a pipette. This included: (1) Naringenin-antibacterial lipopeptide complex liposome hydrogel prepared in Example 1; (3) Comparative Example 1: Preparation of naringenin liposome hydrogel; (4) The antimicrobial peptide liposome hydrogel prepared in Comparative Example 2; (5) Blank hydrogel.
[0028] The above plates were placed in an anaerobic culture device and incubated at 37°C for 48 h.
[0029] After the culture was completed, the diameter of the inhibition zone formed by each group of samples was measured with vernier calipers. Each group was measured in parallel 3 times. The results are expressed as mean ± standard deviation, as shown in Table 2.
[0030] Table 1. Results of inhibition zone diameter measurement for Propionibacterium acnes in each sample group. The inhibitory effects of each sample group on Staphylococcus aureus were determined using the same method, including: (1) Naringenin-antibacterial lipopeptide complex liposome hydrogel prepared in Example 1; (3) Comparative Example 1: Preparation of naringenin liposome hydrogel; (4) The antimicrobial peptide liposome hydrogel prepared in Comparative Example 2; (5) Blank hydrogel.
[0031] Table 2. Results of inhibition zone diameter determination for Staphylococcus aureus in each sample group. Experimental results show that, compared with the comparative examples, the naringenin-antimicrobial peptide complex liposome hydrogel prepared in Example 1 has the largest antibacterial zone diameter, indicating that it has superior antibacterial activity against acne-associated bacteria.
[0032] The reason for this may be that naringenin can exert anti-inflammatory and auxiliary antibacterial effects, while antimicrobial peptides can directly disrupt the bacterial cell membrane structure. The synergistic effect of the two enhances the antibacterial ability of the system. At the same time, liposomes improve the enrichment and sustained release effect of active ingredients around the bacteria, further improving the antibacterial efficiency. Therefore, the composite delivery system constructed in this invention can achieve antibacterial effects superior to single-component systems.
[0033] 2. Storage stability study The formulation of Example 1 was sealed in vials and stored at 4°C in the dark for 14 days. Samples were taken on days 0, 1, 3, 7, and 14, with each measurement performed in triplicate to simultaneously assess the physical and chemical stability of the formulation. The results are shown in Table 3. The specific measurement method is as follows: Liposome particle size variation determination: Accurately weigh 0.5 g of the hydrogel sample to be tested, add 4.5 mL of PBS buffer (10 mmol / L, pH 6.2), and vortex at low speed for 30 s to fully swell and disperse the gel, releasing liposomes; after appropriate dilution with buffer, the average particle size, PDI, and Zeta potential of the liposomes were measured using a dynamic light scattering instrument. The measurement results are shown in Table 3. Table 3. Results of stability evaluation of the samples As can be seen from Table 3, at 4℃, the product exhibits small particle size increase, excellent dispersion uniformity, and small zeta potential increase after 14 days of storage, indicating strong storage stability.
[0034] 3. HaCat cell viability assay HaCaT cells were seeded at a density of 10,000 cells per well in 96-well plates, with 100 μL of culture medium added to each well. Sterile PBS was used as a humidity barrier around the wells, and the plates were pre-cultured for 24 hours. When cell adhesion reached over 95%, the old culture medium was discarded, and the culture medium for the test samples was added to each well. A blank control group was also included, and the plates were cultured for another 24 hours. Afterward, the drug-containing culture medium was aspirated, and each well was washed once with 100 μL of sterile PBS. Then, 100 μL of LMTT working solution (0.5 mg / mL, prepared with DMEM) was precisely added, and the plates were incubated for 4 hours. After incubation, the liquid in the wells was aspirated, and 100 μL of LDMSO was added to each well. The plates were shaken at room temperature for 30 minutes to fully dissolve the formazan crystals. Finally, the absorbance of each well was measured at 490 nm using a microplate reader, and the cell viability was calculated using the formula "Cell viability = OD sample group / OD blank group × 100%", as shown in Table 4.
[0035] Table 4. Cell viability test results HaCat cell viability assays showed that, compared to the blank control group, the cell viability of HaCat cells in all sample groups was above 95% after 24 hours of treatment. Specifically, Example 1, which simultaneously loaded naringenin and antimicrobial lipopeptides, achieved a cell viability of 96.7%, higher than Comparative Example 1 (95.3%) loaded only with naringenin and Comparative Example 2 (95.3%) loaded only with antimicrobial lipopeptides. These results indicate that the active ingredients and excipients used in this product have no significant cytotoxicity to HaCat cells, demonstrating that this formulation is safe and non-toxic to HaCat cells.
Claims
1. A naringin-antimicrobial peptide complex liposome hydrogel, characterized in that, The hydrogel is composed of naringenin-Bacillus subtilis antimicrobial peptide co-loaded liposomes uniformly dispersed in a carbomer hydrogel matrix. The co-loaded liposomes use soybean lecithin and cholesterol as the base membrane material and encapsulate the active ingredients naringenin and Bacillus subtilis antimicrobial peptides; wherein naringenin is distributed in the membrane phase of the phospholipid bilayer and Bacillus subtilis antimicrobial peptides are encapsulated in the aqueous phase of the liposomes.
2. The naringenin-antimicrobial peptide composite liposome hydrogel according to claim 1, characterized in that, The co-loaded liposomes have an average particle size of 100-200 nm and a polydispersity index (PDI) ≤ 0.
2.
3. The method for preparing a naringenin-antimicrobial peptide complex liposome hydrogel according to claim 1 or 2, characterized in that, Includes the following steps: Step 1: Under light-protected conditions, soybean lecithin, cholesterol, and naringenin are dissolved in a chloroform-methanol mixed solvent to obtain a homogeneous and transparent lipid phase solution, wherein the mass ratio of soybean lecithin to cholesterol is (4-6):1, and the mass ratio of naringenin to the mixture of soybean lecithin and cholesterol is 1:(12-20); the lipid phase solution is subjected to reduced pressure rotary evaporation at 40-50℃ to remove the organic solvent until a uniform lipid film is formed on the inner wall of the container, and then vacuum dried to completely remove the residual organic solvent, to obtain a dry lipid film loaded with naringenin; Step 2: Dissolve the Bacillus subtilis antimicrobial peptide in 10 mmol / L PBS buffer (pH 6.8) preheated to 35-39°C to prepare an antimicrobial peptide hydration solution; the concentration of the Bacillus subtilis antimicrobial peptide in the antimicrobial peptide hydration solution is 0.5-2 mg / mL; add the antimicrobial peptide hydration solution to the dried lipid film loaded with naringenin prepared in Step 1, and rotate to hydrate and completely detach the lipid film to obtain a crude liposome suspension; Step 3: Transfer the crude liposome suspension to a liposome extruder and granulate it step by step using a polycarbonate membrane under room temperature and light-protected conditions to obtain a naringenin-antimicrobial peptide co-loaded liposome suspension, which is then stored in a sealed container at 4°C in the dark. Step 4: Disperse 1-4g of Carbomer 940 powder evenly in 30-70ml of purified water, seal and let it stand to swell to obtain a uniform and transparent gel matrix; then add 0.5-2g of humectant and 0.5-2g of preservative and mix well; then add neutralizing agent dropwise while stirring to adjust the pH of the system to 6.8-7.0 to obtain the neutralized gel matrix; Step 5: Under conditions of complete darkness and room temperature, the naringenin-antimicrobial peptide co-loaded liposome suspension prepared in step 3 is added dropwise to the neutralized gel matrix prepared in step 4 and mixed evenly. Then, PBS buffer solution with pH 6.8 is added to 100-200ml and stirred evenly to obtain naringenin-Bacillus subtilis antimicrobial peptide co-loaded liposome hydrogel.
4. The method for preparing a naringin-antimicrobial peptide complex liposome hydrogel according to claim 3, characterized in that, The chloroform-methanol mixed solvent in step 1 is composed of chloroform and methanol in a volume ratio of 2:1, and the rotary evaporation under reduced pressure has a rotation speed of 80-100 rpm.
5. The method for preparing a naringin-antimicrobial peptide composite liposome hydrogel according to claim 3, characterized in that, The Bacillus subtilis antimicrobial peptides described in step 2 were prepared using the following method: Bacillus subtilis was inoculated into LB liquid medium and cultured at 37℃ and 150-180 r / min for 20-28 h with shaking to obtain fermentation broth. The fermentation broth was centrifuged to remove bacterial cells, and the supernatant was collected. The supernatant was placed in an ice-water bath, and ammonium sulfate powder was slowly added with stirring until the ammonium sulfate saturation of the system was 75%-85%. After standing at 4℃ for 1-3 h, it was centrifuged at 8000-12000 r / min for 20-40 min, and the precipitate was collected. The precipitate was reconstituted with 10 mmol / L PBS buffer (pH 6.2), and then dialyzed through a dialysis bag with a molecular weight cutoff of 3-5 kDa to desalt it. The dialyzed antimicrobial peptide solution was freeze-dried to obtain Bacillus subtilis antimicrobial peptide lyophilized powder, which was stored at -20℃ in the dark for later use. The temperature for the rotary hydration in step 2 is 35-39℃, and the rotational hydration time is 30-60 min.
6. The method for preparing a naringin-antimicrobial peptide complex liposome hydrogel according to claim 3, characterized in that, The step granulation operation of the crude liposome suspension via polycarbonate membrane in step 3 is as follows: the crude liposome suspension is transferred to a liposome extruder, and granulation is performed via polycarbonate membrane extrusion under the condition of room temperature and protection from light throughout the process: first, it is extruded 5 times through a 0.45μm pore size polycarbonate membrane for preliminary homogenization; then, it is extruded 8-10 times through a 0.22μm pore size polycarbonate membrane; and finally, it is extruded 8-10 times through a 0.1μm pore size polycarbonate membrane.
7. The method for preparing a naringin-antimicrobial peptide composite liposome hydrogel according to claim 3, characterized in that, The preservative in step 4 is selected from at least one of phenoxyethanol, parabens, and capryloyl hydroxamic acid; the humectant is selected from at least one of propylene glycol, glycerin, and PEG-400; and the neutralizer is selected from at least one of triethanolamine and tris(hydroxymethyl)aminomethane.
8. The method for preparing a naringin-antimicrobial peptide complex liposome hydrogel according to claim 3, characterized in that, When adding the co-loaded liposome suspension dropwise to the neutralized gel matrix in step 5, keep stirring at 150-300 r / min. After the addition is complete, continue stirring for 15-30 min until the system is homogeneous. The degassing time is 3-10 minutes.
9. The application of the naringenin-antimicrobial peptide complex liposome hydrogel as described in claim 1 in the treatment of inflammatory or bacterial skin infections.