Gynecological antibacterial gel and preparation method thereof

Abstract

The invention discloses gynecological antibacterial gel and a preparation method thereof, relates to the technical field of biological medicine manufacturing, and belongs to the patent classification number A61K38 / 47. The preparation method comprises the following steps: mixing the methylparaben, the propylparaben and the propylene glycol, heating and stirring until the methylparaben, the propylparaben and the propylene glycol are completely dissolved to obtain a component B; carbomer is added into deionized water and stirred to be dissolved, then lysozyme slow-release composite microspheres are added and continuously stirred to be dispersed uniformly, then cocamidopropyl betaine, coco-glucoside, lauryl glucoside, poloxamer, glycerin, disodium ethylene diamine tetraacetate and triethanolamine are added and heated, stirred and dissolved uniformly, and a component A is obtained; and adding the component B into the component A, and uniformly stirring and mixing to obtain the gynecological antibacterial gel. According to the invention, the release speed of the lysozyme in the gynecological antibacterial gel can be delayed, so that the antibacterial performance can be stably exerted for a long time.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical manufacturing, belongs to patent classification number A61K38 / 47, and specifically relates to a gynecological antibacterial gel and a preparation method thereof. Background Art

[0002] In the modern medical field, antibacterial gel, as an important topical preparation, is widely used in the prevention and treatment of infections in the skin, mucous membranes and other parts of the body. Its main function is to form a protective barrier at the application site to prevent the reproduction and spread of pathogens, while promoting wound healing and recovery. Common antibacterial gels include silver ion antibacterial gel, iodine ion antibacterial gel, polymer antibacterial gel, etc. These different types of antibacterial gels have antibacterial, anti-inflammatory, and hemostatic effects, and can be used to treat various diseases and trauma, such as burns, incisions, cuts, and lacerations. In the field of gynecology, antibacterial gel is used to prevent and treat bacterial infections in the female vagina, cervix and other parts of the body, and plays an important role in maintaining the health of the female reproductive system.

[0003] Lysozyme is a naturally occurring alkaline protein found in bodily fluids such as tears and saliva. It belongs to the innate immune system and is primarily produced by monocytes and neutrophils. It specifically recognizes and targets bacterial cell walls, hydrolyzing the peptidoglycan component, causing the cell wall to rupture and destroy the bacteria, leading to bacterial death and lysis. Lysozyme also acts as an immunomodulatory factor in the immune system, enhancing the phagocytic function of phagocytes, promoting immune cell activity, and improving the body's resistance to bacterial infection. For this reason, lysozyme is widely used in pharmaceutical formulations. Lysozyme is incorporated into gels to create lysozyme antibacterial gels for topical treatment of the skin or mucous membranes. Studies have shown that lysozyme has a high inhibitory rate against multidrug-resistant bacteria, such as Staphylococcus aureus. Due to its unique mechanism of directly destroying bacterial cell walls, no reports of drug resistance have been reported to date. Adding lysozyme to gynecological antibacterial gels imparts excellent antibacterial properties, effectively combating common pathogens that cause gynecological inflammation, helping to prevent and treat gynecological inflammation.

[0004] However, there is a key problem when currently applying lysozyme to gynecological antibacterial gels: the release of lysozyme in the gel system is too rapid. Excessive release makes it impossible for lysozyme to maintain an effective concentration at the site of action for a long time, and it cannot exert its antibacterial effect continuously and stably. When treating gynecological inflammation, since the pathogens at the site of inflammation persist and are prone to repeated breeding, if lysozyme is released quickly, it will be difficult to effectively inhibit and kill the pathogens that subsequently breed, resulting in incomplete treatment of the inflammation, easy recurrence, and failure to achieve the ideal purpose of treating and preventing gynecological infections. Therefore, how to control the release rate of lysozyme in gynecological antibacterial gels so that it can exert its antibacterial properties for a long time and stably has become a problem that needs to be solved urgently. Summary of the Invention

[0005] The present invention aims to provide a gynecological antibacterial gel and a preparation method thereof to address the technical problem of overly rapid lysozyme release mentioned in the above-mentioned background art. The present invention can slow the release rate of lysozyme in the gynecological antibacterial gel, allowing it to exert its antibacterial properties for a long time and stably.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A gynecological antibacterial gel, comprising component A and component B;

[0008] The A component includes the following components in parts by weight:

[0009] 20-30 parts of lysozyme sustained-release composite microspheres, 40-50 parts of cocoamidopropyl betaine, 40-50 parts of cocoyl glucoside, 20-30 parts of lauryl glucoside, 5-10 parts of poloxamer, 20-30 parts of glycerol, 5-10 parts of carbomer, 1-3 parts of disodium edetate, 0.5-1 part of triethanolamine, and 700-800 parts of deionized water.

[0010] The B component includes the following components in parts by weight:

[0011] Propylene glycol 30-40 parts, methylparaben 1-5 parts, propylparaben 1-5 parts.

[0012] As a preference, the mass ratio of component A to component B is 10:1-2.

[0013] Preferably, the preparation method of the lysozyme sustained-release composite microspheres comprises the following steps:

[0014] S1. D,L-lactide and glycolide are uniformly mixed to obtain a monomer mixture, polyethylene glycol is added to the monomer mixture, vacuum dehydration is performed, and then a stannous octoate catalyst is added. The mixture is heated under nitrogen protection until the reaction is complete, and the mixture is purified and dried to obtain a triblock copolymer compound;

[0015] S2, adding the triblock copolymer compound to dichloromethane, stirring and dissolving, to obtain an organic phase solution; adding lysozyme, trehalose and sodium chloride to deionized water, stirring and dissolving, to obtain an inner aqueous phase solution;

[0016] S3. In an ice bath, slowly add the inner aqueous phase solution dropwise to the organic phase solution to perform ultrasonic emulsification to form a primary emulsion;

[0017] S4, adding polyvinyl alcohol to deionized water, heating and stirring to dissolve, to obtain an external aqueous phase solution;

[0018] S5. Slowly add the primary emulsion dropwise to the external aqueous phase solution and stir until the solvent is completely evaporated to form a stable secondary emulsion, which is then centrifuged, washed and dried to obtain lysozyme sustained-release composite microspheres.

[0019] In the technical solution of the present invention, a PLGA-PEG-PLGA triblock copolymer is first synthesized by ring-opening polymerization. The PEG (polyethylene glycol) segments bind to the lysozyme molecules through hydrogen bonding, forming a preliminary sustained-release mechanism, while the PLGA segments achieve sustained drug release through degradation rate control. During the microsphere preparation process, the internal aqueous phase solution containing lysozyme and the polymer organic phase solution are emulsified at low temperatures using a double emulsion method to form a stable W / O / W double emulsion. The solvent is then evaporated to obtain densely structured microspheres. This method protects the biological activity of lysozyme. The addition of trehalose as a protective agent, combined with ice bath operation conditions, effectively maintains the active conformation of lysozyme. The resulting microspheres exhibit significant sustained-release properties, with initial sustained release achieved through hydrogen bonding between PEG and lysozyme, and later sustained release achieved through degradation of PLGA. Furthermore, the enrichment of PEG segments on the microsphere surface significantly enhances adhesion to mucosal tissues, prolonging local retention time.

[0020] Preferably, in step S1, the amount of polyethylene glycol added is 12-16% of the mass of the monomer mixture.

[0021] In the technical solution of the present invention, as described above, polyethylene glycol is embedded in PLGA to form a PLGA-PEG-PLGA triblock copolymer. Initially, the polyethylene glycol segments are hydrogen-bonded to the lysozyme molecules to form a preliminary sustained-release mechanism for the lysozyme. To achieve a good initial sustained-release mechanism, a sufficient amount of polyethylene glycol must be embedded in the PLGA-PEG-PLGA triblock copolymer. Therefore, the present invention controls the amount of polyethylene glycol added to be greater than 12% by weight of the monomer mixture. As the amount of polyethylene glycol continued to increase, until it reached a polyethylene glycol addition amount greater than 16% by weight of the monomer mixture, the present invention team unexpectedly discovered that the strength of the prepared lysozyme sustained-release composite microspheres suddenly and significantly decreased, and the composite microspheres were easily broken during subsequent external stirring and mixing. After research, it was found that this is because polyethylene glycol is a hydrophilic polymer. When it is added to PLGA in excess, it will destroy the hydrophobic interaction and van der Waals force between the PLGA molecular chains. Due to the limited compatibility of PEG and PLGA, excessive PEG will form microdomain separation in the PLGA matrix, resulting in phase interface defects. These microdomains not only reduce the entanglement density of the PLGA molecular chains, but also lead to stress concentration points inside the material. When the microspheres are subjected to external forces, these defective sites are prone to crack propagation, ultimately causing the microspheres to lose their structural integrity and break. This phenomenon is particularly obvious when the PEG addition exceeds 16%, which is manifested as a significant decrease in the mechanical strength of the microspheres. Therefore, the present invention strictly controls the amount of polyethylene glycol added within a specified range to balance the sustained release effect of lysozyme and the mechanical strength of the composite microspheres.

[0022] Preferably, in step S2, the mass ratio of the triblock copolymer compound to lysozyme is 10:1-3.

[0023] Preferably, in step S3, the ultrasonic emulsification time is 30-50 seconds.

[0024] Preferably, in step S4, the concentration of polyvinyl alcohol in the aqueous solution is 1-2 wt%.

[0025] A method for preparing a gynecological antibacterial gel comprises the following steps:

[0026] Methylparaben, propylparaben and propylene glycol were mixed, heated to 70° C., and stirred until completely dissolved to obtain component B;

[0027] Carbomer was added to deionized water and stirred to dissolve, and then lysozyme sustained-release composite microspheres were added and continued to be stirred and dispersed uniformly, followed by the addition of cocamidopropyl betaine, cocoyl glucoside, lauryl glucoside, poloxamer, glycerol, disodium edetate and triethanolamine, and the mixture was heated to 40° C. and stirred to dissolve uniformly to obtain component A.

[0028] Add component B to component A, stir and mix evenly to obtain gynecological antibacterial gel.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. This invention synthesizes PLGA-PEG-PLGA triblock copolymers through ring-opening polymerization and prepares lysozyme sustained-release composite microspheres using a double emulsion method, achieving a multi-stage release mechanism for lysozyme. Initial sustained release is achieved through hydrogen bonding between PEG segments and lysozyme, while sustained release is achieved through controlled degradation of PLGA. This allows the gel to exert its antibacterial activity stably and for a long time in the vaginal environment, effectively resolving the issues of rapid release and short duration of action associated with conventional lysozyme preparations.

[0031] 2. During the preparation of microspheres, the active conformation of lysozyme was effectively maintained by adding trehalose as a protective agent and combining it with ice bath operation.

[0032] 3. The present invention successfully balances the sustained-release effect of lysozyme and the mechanical strength of the composite microspheres by strictly controlling the amount of polyethylene glycol added within a specified range. DETAILED DESCRIPTION

[0033] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the implementation regulations described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0034] Example 1

[0035] A gynecological antibacterial gel comprises a component A and a component B, wherein the mass ratio of the component A to the component B is 10:1.5.

[0036] The A component includes the following components in parts by weight:

[0037] 28 parts of lysozyme sustained-release composite microspheres, 47 parts of cocoamidopropyl betaine, 49 parts of cocoyl glucoside, 28 parts of lauryl glucoside, 9 parts of poloxamer (type 407), 28 parts of glycerol, 9 parts of carbomer (type 940), 2.5 parts of disodium edetate, 0.8 parts of triethanolamine, and 780 parts of deionized water.

[0038] The B component includes the following components in parts by weight:

[0039] Propylene glycol 37 parts, methylparaben 4 parts, propylparaben 4 parts.

[0040] Preparation of lysozyme sustained-release composite microspheres:

[0041] Step S1: Weigh 7.2g of D,L-lactide and 2.8g of glycolide, mix them evenly and transfer them to a dry three-necked flask, and add 1.5g of polyethylene glycol (PEG-2000). Install a condenser, thermometer and vacuum interface, and introduce nitrogen to replace the air 3 times. Turn on the vacuum pump and dehydrate under vacuum at 100°C for 2 hours while stirring continuously. After dehydration, turn off the vacuum, add 0.01g of stannous octoate, continue to introduce nitrogen protection, and heat to 180°C for reaction for 12 hours. After the reaction is completed, cool to room temperature, dissolve the product with dichloromethane, slowly add ice ether to precipitate, filter and vacuum dry (40°C, 12 hours) to obtain PLGA-PEG-PLGA triblock copolymer.

[0042] Step S2: Add 10 g of PLGA-PEG-PLGA triblock copolymer to 350 mL of dichloromethane and stir magnetically (300 rpm) at room temperature until completely dissolved to obtain an organic phase solution; weigh 2.5 g of lysozyme (20,000 U / mg), 0.6 g of trehalose, and 0.08 g of sodium chloride, add them to 130 mL of deionized water, stir to dissolve, and then ultrasonically degas for 5 minutes to obtain an inner aqueous phase solution.

[0043] Step S3: The organic phase solution was placed in an ice bath, and the inner aqueous phase solution was slowly dripped into the organic phase, while an ultrasonic cell disruptor (power 200 W) was used for emulsification for 40 seconds to form a stable W / O type primary emulsion.

[0044] Step S4: Weigh polyvinyl alcohol and add it into 200 mL of deionized water, heat it to 80° C. and stir to dissolve it, cool it to room temperature and filter it to prepare a polyvinyl alcohol external aqueous phase solution with a concentration of 1.5 wt %.

[0045] Step S5: The primary emulsion was slowly dripped into the external aqueous phase solution and magnetically stirred (600 rpm) at room temperature for 6 hours to completely evaporate the dichloromethane. The secondary emulsion was transferred to a centrifuge tube and centrifuged at 5000 rpm for 20 minutes. The supernatant was discarded and the precipitate was washed three times with deionized water. Finally, the microspheres were dispersed in 50 mL of trehalose solution (5 wt%) and freeze-dried (pre-freezing at -80°C for 4 hours and vacuum drying for 24 hours) to obtain lysozyme sustained-release composite microspheres.

[0046] The preparation method of gynecological antibacterial gel comprises the following steps:

[0047] Methylparaben, propylparaben and propylene glycol were mixed, heated to 70° C., and stirred (300 rpm) until completely dissolved to obtain component B;

[0048] Carbomer was added to deionized water and stirred to dissolve (400 rpm), and then lysozyme sustained-release composite microspheres were added and continued to be stirred and dispersed uniformly. Cocoamidopropyl betaine, cocoyl glucoside, lauryl glucoside, poloxamer, glycerol, disodium edetate and triethanolamine were added, and the mixture was heated to 40° C. and stirred to dissolve uniformly to obtain component A.

[0049] Add component B to component A, stir and mix for 15 minutes (800 rpm) to ensure uniform mixing, and let it stand for 30 minutes to degas, thereby obtaining a gynecological antibacterial gel.

[0050] Example 2

[0051] A gynecological antibacterial gel comprises a component A and a component B, wherein the mass ratio of the component A to the component B is 10:1.5.

[0052] The A component includes the following components in parts by weight:

[0053] 23 parts of lysozyme sustained-release composite microspheres, 42 parts of cocoamidopropyl betaine, 42 parts of cocoyl glucoside, 21 parts of lauryl glucoside, 6 parts of poloxamer (407 type), 21 parts of glycerol, 6 parts of carbomer (940 type), 2 parts of disodium edetate, 0.6 parts of triethanolamine, and 720 parts of deionized water.

[0054] The B component includes the following components in parts by weight:

[0055] 32 parts of propylene glycol, 2 parts of methylparaben, 2 parts of propylparaben.

[0056] Preparation of lysozyme sustained-release composite microspheres:

[0057] Step S1: Weigh 7.2g of D,L-lactide and 2.8g of glycolide, mix them evenly and transfer them to a dry three-necked flask, and add 1.3g of polyethylene glycol (PEG-2000). Install a condenser, thermometer and vacuum interface, and introduce nitrogen to replace the air 3 times. Turn on the vacuum pump and dehydrate under vacuum at 100°C for 2 hours while stirring continuously. After dehydration, turn off the vacuum, add 0.01g of stannous octoate, continue to introduce nitrogen protection, and heat to 180°C for 12 hours. After the reaction is completed, cool to room temperature, dissolve the product with dichloromethane, slowly add ice ether to precipitate, filter and vacuum dry (40°C, 12 hours) to obtain PLGA-PEG-PLGA triblock copolymer.

[0058] Step S2: Add 10 g of PLGA-PEG-PLGA triblock copolymer to 350 mL of dichloromethane and stir magnetically (300 rpm) at room temperature until completely dissolved to obtain an organic phase solution; weigh 1.5 g of lysozyme (20,000 U / mg), 0.6 g of trehalose, and 0.08 g of sodium chloride, add them to 130 mL of deionized water, stir to dissolve, and then ultrasonically degas for 5 minutes to obtain an inner aqueous phase solution.

[0059] Step S3: The organic phase solution was placed in an ice bath, and the inner aqueous phase solution was slowly dripped into the organic phase, while an ultrasonic cell disruptor (power 200 W) was used for emulsification for 40 seconds to form a stable W / O type primary emulsion.

[0060] Step S4: Weigh polyvinyl alcohol and add it into 200 mL of deionized water, heat it to 80° C. and stir to dissolve it, cool it to room temperature and filter it to prepare a polyvinyl alcohol external aqueous phase solution with a concentration of 1.5 wt %.

[0061] Step S5: The primary emulsion was slowly dripped into the external aqueous phase solution and magnetically stirred (600 rpm) at room temperature for 6 hours to completely evaporate the dichloromethane. The secondary emulsion was transferred to a centrifuge tube and centrifuged at 5000 rpm for 20 minutes. The supernatant was discarded and the precipitate was washed three times with deionized water. Finally, the microspheres were dispersed in 50 mL of trehalose solution (5 wt%) and freeze-dried (pre-freezing at -80°C for 4 hours and vacuum drying for 24 hours) to obtain lysozyme sustained-release composite microspheres.

[0062] The preparation method of gynecological antibacterial gel comprises the following steps:

[0063] Methylparaben, propylparaben and propylene glycol were mixed, heated to 70° C., and stirred (300 rpm) until completely dissolved to obtain component B;

[0064] Carbomer was added to deionized water and stirred to dissolve (400 rpm), and then lysozyme sustained-release composite microspheres were added and continued to be stirred and dispersed uniformly. Cocoamidopropyl betaine, cocoyl glucoside, lauryl glucoside, poloxamer, glycerol, disodium edetate and triethanolamine were added, and the mixture was heated to 40° C. and stirred to dissolve uniformly to obtain component A.

[0065] Add component B to component A, stir and mix for 15 minutes (800 rpm) to ensure uniform mixing, and let it stand for 30 minutes to degas, thereby obtaining a gynecological antibacterial gel.

[0066] Example 3

[0067] A gynecological antibacterial gel comprises a component A and a component B, wherein the mass ratio of the component A to the component B is 10:1.5.

[0068] The A component includes the following components in parts by weight:

[0069] 25 parts of lysozyme sustained-release composite microspheres, 45 parts of cocoamidopropyl betaine, 45 parts of cocoyl glucoside, 25 parts of lauryl glucoside, 7 parts of poloxamer (407 type), 25 parts of glycerol, 8 parts of carbomer (940 type), 2 parts of disodium edetate, 0.8 parts of triethanolamine, and 750 parts of deionized water.

[0070] The B component includes the following components in parts by weight:

[0071] 35 parts of propylene glycol, 3 parts of methylparaben, 3 parts of propylparaben.

[0072] Preparation of lysozyme sustained-release composite microspheres:

[0073] Step S1: Weigh 7.2g of D,L-lactide and 2.8g of glycolide, mix them evenly and transfer them to a dry three-necked flask, and add 1.4g of polyethylene glycol (PEG-2000). Install a condenser, thermometer and vacuum interface, and introduce nitrogen to replace the air 3 times. Turn on the vacuum pump and dehydrate under vacuum at 100°C for 2 hours while stirring continuously. After dehydration, turn off the vacuum, add 0.01g of stannous octoate, continue to introduce nitrogen protection, and heat to 180°C for reaction for 12 hours. After the reaction is completed, cool to room temperature, dissolve the product with dichloromethane, slowly add ice ether to precipitate, filter and vacuum dry (40°C, 12 hours) to obtain PLGA-PEG-PLGA triblock copolymer.

[0074] Step S2: Add 10 g of PLGA-PEG-PLGA triblock copolymer to 350 mL of dichloromethane and stir magnetically (300 rpm) at room temperature until completely dissolved to obtain an organic phase solution; weigh 2 g of lysozyme (20,000 U / mg), 0.6 g of trehalose, and 0.08 g of sodium chloride, add them to 130 mL of deionized water, stir to dissolve, and then ultrasonically degas for 5 minutes to obtain an inner aqueous phase solution.

[0075] Step S3: The organic phase solution was placed in an ice bath, and the inner aqueous phase solution was slowly dripped into the organic phase, while an ultrasonic cell disruptor (power 200 W) was used for emulsification for 40 seconds to form a stable W / O type primary emulsion.

[0076] Step S4: Weigh polyvinyl alcohol and add it into 200 mL of deionized water, heat it to 80° C. and stir to dissolve it, cool it to room temperature and filter it to prepare a polyvinyl alcohol external aqueous phase solution with a concentration of 1.5 wt %.

[0077] Step S5: The primary emulsion was slowly dripped into the external aqueous phase solution and magnetically stirred (600 rpm) at room temperature for 6 hours to completely evaporate the dichloromethane. The secondary emulsion was transferred to a centrifuge tube and centrifuged at 5000 rpm for 20 minutes. The supernatant was discarded and the precipitate was washed three times with deionized water. Finally, the microspheres were dispersed in 50 mL of trehalose solution (5 wt%) and freeze-dried (pre-freezing at -80°C for 4 hours and vacuum drying for 24 hours) to obtain lysozyme sustained-release composite microspheres.

[0078] The preparation method of gynecological antibacterial gel comprises the following steps:

[0079] Methylparaben, propylparaben and propylene glycol were mixed, heated to 70° C., and stirred (300 rpm) until completely dissolved to obtain component B;

[0080] Carbomer was added to deionized water and stirred to dissolve (400 rpm), and then lysozyme sustained-release composite microspheres were added and continued to be stirred and dispersed uniformly. Cocoamidopropyl betaine, cocoyl glucoside, lauryl glucoside, poloxamer, glycerol, disodium edetate and triethanolamine were added, and the mixture was heated to 40° C. and stirred to dissolve uniformly to obtain component A.

[0081] Add component B to component A, stir and mix for 15 minutes (800 rpm) to ensure uniform mixing, and let it stand for 30 minutes to degas, thereby obtaining a gynecological antibacterial gel.

[0082] Example 4

[0083] A gynecological antibacterial gel comprises a component A and a component B, wherein the mass ratio of the component A to the component B is 10:2.

[0084] The A component includes the following components in parts by weight:

[0085] 30 parts of lysozyme sustained-release composite microspheres, 50 parts of cocoamidopropyl betaine, 50 parts of cocoyl glucoside, 30 parts of lauryl glucoside, 10 parts of poloxamer (type 407), 30 parts of glycerol, 10 parts of carbomer (type 940), 3 parts of disodium edetate, 1 part of triethanolamine, and 800 parts of deionized water.

[0086] The B component includes the following components in parts by weight:

[0087] 40 parts of propylene glycol, 5 parts of methylparaben, 5 parts of propylparaben.

[0088] Preparation of lysozyme sustained-release composite microspheres:

[0089] Step S1: Weigh 7.2g of D,L-lactide and 2.8g of glycolide, mix them evenly and transfer them to a dry three-necked flask, and add 1.6g of polyethylene glycol (PEG-2000). Install a condenser, thermometer and vacuum interface, and introduce nitrogen to replace the air 3 times. Turn on the vacuum pump and dehydrate under vacuum at 100°C for 2 hours while stirring continuously. After dehydration, turn off the vacuum, add 0.01g of stannous octoate, continue to introduce nitrogen protection, and heat to 180°C for reaction for 12 hours. After the reaction is completed, cool to room temperature, dissolve the product with dichloromethane, slowly add it dropwise into ice ether to precipitate, filter and vacuum dry (40°C, 12 hours) to obtain PLGA-PEG-PLGA triblock copolymer.

[0090] Step S2: 10 g of PLGA-PEG-PLGA triblock copolymer was added to 350 mL of dichloromethane and magnetically stirred (300 rpm) at room temperature until completely dissolved to obtain an organic phase solution; 3 g of lysozyme (20,000 U / mg), 0.6 g of trehalose, and 0.08 g of sodium chloride were weighed and added to 130 mL of deionized water. After stirring to dissolve, ultrasonic degassing was performed for 5 minutes to obtain an inner aqueous phase solution.

[0091] Step S3: The organic phase solution was placed in an ice bath, and the inner aqueous phase solution was slowly dripped into the organic phase, while an ultrasonic cell disruptor (power 200 W) was used for emulsification for 50 seconds to form a stable W / O type primary emulsion.

[0092] Step S4: Weigh polyvinyl alcohol and add it into 200 mL of deionized water, heat it to 80° C. and stir to dissolve it, cool it to room temperature and filter it to prepare a polyvinyl alcohol external aqueous phase solution with a concentration of 2 wt %.

[0093] Step S5: The primary emulsion was slowly dripped into the external aqueous phase solution and magnetically stirred (600 rpm) at room temperature for 6 hours to completely evaporate the dichloromethane. The secondary emulsion was transferred to a centrifuge tube and centrifuged at 5000 rpm for 20 minutes. The supernatant was discarded and the precipitate was washed three times with deionized water. Finally, the microspheres were dispersed in 50 mL of trehalose solution (5 wt%) and freeze-dried (pre-freezing at -80°C for 4 hours and vacuum drying for 24 hours) to obtain lysozyme sustained-release composite microspheres.

[0094] The preparation method of gynecological antibacterial gel comprises the following steps:

[0095] Methylparaben, propylparaben and propylene glycol were mixed, heated to 70° C., and stirred (300 rpm) until completely dissolved to obtain component B;

[0096] Carbomer was added to deionized water and stirred to dissolve (400 rpm), and then lysozyme sustained-release composite microspheres were added and continued to be stirred and dispersed uniformly. Cocoamidopropyl betaine, cocoyl glucoside, lauryl glucoside, poloxamer, glycerol, disodium edetate and triethanolamine were added, and the mixture was heated to 40° C. and stirred to dissolve uniformly to obtain component A.

[0097] Add component B to component A, stir and mix for 15 minutes (800 rpm) to ensure uniform mixing, and let it stand for 30 minutes to degas, thereby obtaining a gynecological antibacterial gel.

[0098] Example 5

[0099] A gynecological antibacterial gel comprises a component A and a component B, wherein the mass ratio of the component A to the component B is 10:1.

[0100] The A component includes the following components in parts by weight:

[0101] 20 parts of lysozyme sustained-release composite microspheres, 40 parts of cocoamidopropyl betaine, 40 parts of cocoyl glucoside, 20 parts of lauryl glucoside, 5 parts of poloxamer (type 407), 20 parts of glycerol, 5 parts of carbomer (type 940), 1 part of disodium edetate, 0.5 parts of triethanolamine, and 700 parts of deionized water.

[0102] The B component includes the following components in parts by weight:

[0103] 30 parts of propylene glycol, 1 part of methylparaben, 1 part of propylparaben.

[0104] Preparation of lysozyme sustained-release composite microspheres:

[0105] Step S1: Weigh 7.2g of D,L-lactide and 2.8g of glycolide, mix them evenly and transfer them to a dry three-necked flask, and add 1.2g of polyethylene glycol (PEG-2000). Install a condenser, thermometer and vacuum interface, and introduce nitrogen to replace the air 3 times. Turn on the vacuum pump and dehydrate under vacuum at 100°C for 2 hours while stirring continuously. After dehydration, turn off the vacuum, add 0.01g of stannous octoate, continue to introduce nitrogen protection, and heat to 180°C for reaction for 12 hours. After the reaction is completed, cool to room temperature, dissolve the product with dichloromethane, slowly add ice ether to precipitate, filter and vacuum dry (40°C, 12 hours) to obtain PLGA-PEG-PLGA triblock copolymer.

[0106] Step S2: Add 10 g of PLGA-PEG-PLGA triblock copolymer to 350 mL of dichloromethane and stir magnetically (300 rpm) at room temperature until completely dissolved to obtain an organic phase solution; weigh 1 g of lysozyme (20,000 U / mg), 0.6 g of trehalose, and 0.08 g of sodium chloride, add them to 130 mL of deionized water, stir to dissolve, and then ultrasonically degas for 5 minutes to obtain an inner aqueous phase solution.

[0107] Step S3: The organic phase solution was placed in an ice bath, and the inner aqueous phase solution was slowly dripped into the organic phase, while an ultrasonic cell disruptor (power 200 W) was used for emulsification for 30 seconds to form a stable W / O type primary emulsion.

[0108] Step S4: Weigh polyvinyl alcohol and add it into 200 mL of deionized water, heat it to 80° C. and stir to dissolve it, cool it to room temperature and filter it to prepare a polyvinyl alcohol external aqueous phase solution with a concentration of 1 wt %.

[0109] Step S5: The primary emulsion was slowly dripped into the external aqueous phase solution and magnetically stirred (600 rpm) at room temperature for 6 hours to completely evaporate the dichloromethane. The secondary emulsion was transferred to a centrifuge tube and centrifuged at 5000 rpm for 20 minutes. The supernatant was discarded and the precipitate was washed three times with deionized water. Finally, the microspheres were dispersed in 50 mL of trehalose solution (5 wt%) and freeze-dried (pre-freezing at -80°C for 4 hours and vacuum drying for 24 hours) to obtain lysozyme sustained-release composite microspheres.

[0110] The preparation method of gynecological antibacterial gel comprises the following steps:

[0111] Methylparaben, propylparaben and propylene glycol were mixed, heated to 70° C., and stirred (300 rpm) until completely dissolved to obtain component B;

[0112] Carbomer was added to deionized water and stirred to dissolve (400 rpm), and then lysozyme sustained-release composite microspheres were added and continued to be stirred and dispersed uniformly. Cocoamidopropyl betaine, cocoyl glucoside, lauryl glucoside, poloxamer, glycerol, disodium edetate and triethanolamine were added, and the mixture was heated to 40° C. and stirred to dissolve uniformly to obtain component A.

[0113] Add component B to component A, stir and mix for 15 minutes (800 rpm) to ensure uniform mixing, and let it stand for 30 minutes to degas, thereby obtaining a gynecological antibacterial gel.

[0114] Comparative Example 1

[0115] The difference between Comparative Example 1 and Example 1 is that the lysozyme sustained-release composite microspheres are replaced with lysozyme, the amount of lysozyme is kept the same, and the other steps are the same.

[0116] Comparative Example 2

[0117] The difference between Comparative Example 2 and Example 1 is that in the preparation process of lysozyme sustained-release composite microspheres, polyethylene glycol is not added in step S1, and the remaining steps are the same.

[0118] Comparative Example 3

[0119] The difference between Comparative Example 3 and Example 4 is that in the preparation process of lysozyme sustained-release composite microspheres, in step S1, the amount of polyethylene glycol added is 17% of the mass of the monomer mixture, and the other steps are the same.

[0120] Comparative Example 4

[0121] The difference between Comparative Example 4 and Example 4 is that in the preparation process of lysozyme sustained-release composite microspheres, in step S1, the amount of polyethylene glycol added is 18% by mass of the monomer mixture, and the remaining steps are the same.

[0122] Performance testing:

[0123] 1. Lysozyme sustained-release performance test: Using the dialysis bag method (molecular weight cut-off 8000), 10 mg of microspheres were placed in a dialysis bag and immersed in 50 mL of PBS buffer (pH 7.4, containing 0.1% Tween-80) at 37°C with constant shaking (100 rpm). 5 mL samples were taken at 1 hour, 4 hours, 12 hours, 24 hours, 3 days, and 7 days (supplemented with an equal amount of fresh PBS). The absorbance at 280 nm was measured using a UV-visible spectrophotometer, and the cumulative release rate over 24 hours and 7 days was calculated. The 24-hour release rate was used to characterize the initial sustained-release effect of lysozyme, while the 7-day release rate was used to characterize the overall sustained-release effect of lysozyme. The test results are shown in Table 1.

[0124] 2. Lysozyme Activity Retention Test: Using the micrococcal turbidimetric method, a microsphere solution (containing 1 mg of lysozyme) was mixed with a micrococcal suspension (OD600 = 0.5) and incubated at 37°C for 10 minutes. The decrease in OD600 was measured and compared with the activity of free lysozyme. The activity retention (%) was calculated as (lysozyme activity in microspheres / free lysozyme activity) × 100%. The test results are shown in Table 1.

[0125] Table 1:

[0126]

[0127] 3. Antibacterial performance test: Escherichia coli (E.coli ATCC 25922) and Staphylococcus aureus (S.aureusATCC 25923) bacterial solution (10 5 CFU / mL) was spread on LB agar plates, 50 μL of gel sample was placed in an Oxford cup, and the diameter of the inhibition zone was measured after incubation at 37°C for 24 h. The test results are shown in Table 2.

[0128] 4. Mucosal adhesion test: Using the Franz diffusion cell method, prepare the pig vaginal mucosal tissue and apply the gel to the mucosal surface (2 mg / cm 2 ), flushed with saline at a flow rate of 20 mL / min, and recorded the time it took for the gel to completely fall off. The test results are shown in Table 2.

[0129] 5. Mechanical Strength Testing of Lysozyme Sustained-Release Composite Microspheres: A microsphere crushing pressure test was performed. 10 mg of freeze-dried microsphere powder was placed in a 5 mm diameter cylindrical mold. A microhardness tester (FISHER HM-200) was used to apply vertical pressure to the microspheres at a rate of 0.1 mm / s. The maximum pressure at which the microspheres ruptured was recorded (unit: mN). Twenty microspheres were tested in each group, and the average and standard deviation were calculated. The test results are shown in Table 2.

[0130] Table 2:

[0131]

[0132] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A gynecological antibacterial gel, characterized in that: It includes component A and component B; The A component includes the following components in parts by weight: 20-30 parts of lysozyme sustained-release composite microspheres, 40-50 parts of cocoamidopropyl betaine, 40-50 parts of cocoyl glucoside, 20-30 parts of lauryl glucoside, 5-10 parts of poloxamer, 20-30 parts of glycerol, 5-10 parts of carbomer, 1-3 parts of disodium edetate, 0.5-1 part of triethanolamine, and 700-800 parts of deionized water. The B component includes the following components in parts by weight: Propylene glycol 30-40 parts, methylparaben 1-5 parts, propylparaben 1-5 parts.

2. A gynecological antibacterial gel according to claim 1, characterized in that: The mass ratio of component A to component B is 10:1-2.

3. The gynecological antibacterial gel according to claim 1, characterized in that: The preparation method of the lysozyme sustained-release composite microspheres comprises the following steps: S1. D,L-lactide and glycolide are uniformly mixed to obtain a monomer mixture, polyethylene glycol is added to the monomer mixture, vacuum dehydration is performed, and then a stannous octoate catalyst is added. The mixture is heated under nitrogen protection until the reaction is complete, and the mixture is purified and dried to obtain a triblock copolymer compound; S2, adding the triblock copolymer compound to dichloromethane, stirring and dissolving, to obtain an organic phase solution; adding lysozyme, trehalose and sodium chloride to deionized water, stirring and dissolving, to obtain an inner aqueous phase solution; S3. In an ice bath, slowly add the inner aqueous phase solution dropwise to the organic phase solution to perform ultrasonic emulsification to form a primary emulsion; S4, adding polyvinyl alcohol to deionized water, heating and stirring to dissolve, to obtain an external aqueous phase solution; S5. Slowly add the primary emulsion dropwise to the external aqueous phase solution and stir until the solvent is completely evaporated to form a stable secondary emulsion, which is then centrifuged, washed and dried to obtain lysozyme sustained-release composite microspheres.

4. The gynecological antibacterial gel according to claim 3, characterized in that: In the step S1, the amount of polyethylene glycol added is 12-16% of the mass of the monomer mixture.

5. The gynecological antibacterial gel according to claim 3, characterized in that: In the step S2, the mass ratio of the triblock copolymer compound to lysozyme is 10:1-3.

6. The gynecological antibacterial gel according to claim 3, characterized in that: In step S3, the ultrasonic emulsification time is 30-50 seconds.

7. The gynecological antibacterial gel according to claim 3, characterized in that: In step S4, the concentration of polyvinyl alcohol in the aqueous solution is 1-2 wt%.

8. A method for preparing the gynecological antibacterial gel according to any one of claims 1 to 7, characterized in that: The following steps are involved: Methylparaben, propylparaben and propylene glycol were mixed, heated to 70° C., and stirred until completely dissolved to obtain component B; Carbomer was added to deionized water and stirred to dissolve, and then lysozyme sustained-release composite microspheres were added and continued to be stirred and dispersed uniformly, followed by the addition of cocamidopropyl betaine, cocoyl glucoside, lauryl glucoside, poloxamer, glycerol, disodium edetate and triethanolamine, and the mixture was heated to 40° C. and stirred to dissolve uniformly to obtain component A. Add component B to component A, stir and mix evenly to obtain a gynecological antibacterial gel.

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