A gynecological antibacterial gel and a preparation method thereof

Abstract

The application discloses a gynecological antibacterial gel and a preparation method thereof, relates to the technical field of biological medicine manufacturing, and belongs to the patent classification A61K38 / 47. The preparation method comprises the following steps: mixing methylparaben, propylparaben and propylene glycol, and stirring and heating until completely dissolved to obtain component B; adding carbomer into deionized water, stirring and dissolving, then adding lysozyme sustained-release composite microspheres, continuously stirring and uniformly dispersing, and then adding cocamidopropyl betaine, cocoglycoside, lauryl glucoside, poloxamer, glycerol, disodium ethylenediaminetetraacetate and triethanolamine, stirring and uniformly dissolving to obtain component A; and adding component B into component A, stirring and uniformly mixing to obtain the gynecological antibacterial gel. The application can delay the release speed of lysozyme in the gynecological antibacterial gel, so that the antibacterial performance of the gynecological antibacterial gel can be stably exerted for a long time.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicine manufacturing, belongs to the patent classification A61K38 / 47, and specifically relates to a gynecological antibacterial gel and a preparation method thereof. BACKGROUND

[0002] In the modern medical field, antibacterial gel as an important external preparation is widely used in the prevention and treatment of infections at skin, mucosa and other parts. Its main function is to form a protective barrier at the applied part to prevent bacteria from multiplying and spreading, and promote wound healing and recovery. Common antibacterial gels include silver ion antibacterial gel, iodine ion antibacterial gel, polymer antibacterial gel and the like. These different types of antibacterial gels have the functions of antibacterial, anti-inflammatory, hemostatic and the like, and can be used for treating various diseases and wounds such as burns, incisions, cuts and lacerations. In the gynecological field, the antibacterial gel is used for preventing and treating bacterial infections at the female vagina, cervix and other parts, and plays an important role in maintaining the health of the female reproductive system.

[0003] Lysozyme is a natural alkaline protein existing in body fluids such as human tears and saliva, belongs to the innate immune system, and is mainly produced by monocytes and neutrophils. It can specifically recognize and act on the bacterial cell wall, hydrolyze the peptidoglycan component in it, cause the bacterial cell wall to rupture, and the bacteria lose protection and die, so as to achieve the effect of dissolving bacteria, inhibiting the growth and reproduction of bacteria. At the same time, in the immune system of the body, lysozyme can also act as an immune regulator, enhance the phagocytic function of phagocytes, promote the activity of immune cells, and improve the resistance of the body to bacterial infection. Therefore, lysozyme is widely used in drug preparations, and is added to gel to make lysozyme antibacterial gel for local treatment of skin or mucosa. Related research shows that lysozyme has a good inhibition rate on multiple drug-resistant bacteria such as Staphylococcus aureus, and because of its unique mechanism of directly destroying the bacterial cell wall, no drug resistance report has been reported so far. Adding lysozyme to the gynecological antibacterial gel can endow the gel with good antibacterial performance, effectively resist common bacteria causing gynecological inflammation, and help prevent and treat gynecological inflammation.

[0004] However, there is a key problem in the application of lysozyme to gynecological antibacterial gel at present, that is, the release of lysozyme in the gel system is too fast. The fast release makes it impossible for lysozyme to maintain an effective concentration at the action site for a long time, and cannot continuously and stably exert the antibacterial effect. In the treatment of gynecological inflammation, since the bacteria at the inflammation site persist and are easy to breed repeatedly, if the lysozyme is released rapidly, it is difficult to effectively inhibit and kill the subsequent breeding bacteria, resulting in incomplete treatment of inflammation, easy recurrence, and inability to achieve the ideal purpose of treating and preventing gynecological infection. Therefore, how to control the release speed of lysozyme in the gynecological antibacterial gel so that it can exert the antibacterial performance for a long time and stably becomes a problem to be solved. SUMMARY

[0005] The purpose of the present application is to provide a gynecological antibacterial gel and a preparation method thereof to solve the technical problem of fast release of lysozyme proposed in the above background art. The present application can delay the release speed of lysozyme in the gynecological antibacterial gel, so that it can exert the antibacterial performance for a long time and stably.

[0006] To achieve the above purpose, the present application provides the following technical solutions:

[0007] A gynecological antibacterial gel comprises A component and B component.

[0008] The A component comprises the following components by weight:

[0009] 20-30 parts of lysozyme sustained-release composite microspheres, 40-50 parts of cocamidopropyl 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 ethylenediaminetetraacetate, 0.5-1 part of triethanolamine, and 700-800 parts of deionized water.

[0010] The B component comprises the following components by weight:

[0011] 30-40 parts of propylene glycol, 1-5 parts of methyl paraben, and 1-5 parts of propyl paraben.

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

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

[0014] S1, uniformly mixing D,L-lactide and glycolide to obtain a monomer mixture, adding polyethylene glycol to the monomer mixture, vacuum dehydrating, then adding stannous octoate catalyst, heating to complete reaction under nitrogen protection, and purifying and drying to obtain a triblock copolymer;

[0015] S2, the triblock copolymer is added into dichloromethane, stirred and dissolved to obtain an organic phase solution; the lysozyme, trehalose and sodium chloride are added into deionized water, stirred and dissolved to obtain an internal aqueous phase solution;

[0016] S3, the internal aqueous phase solution is slowly added into the organic phase solution under ice bath condition, ultrasonic emulsification is performed to form a primary emulsion;

[0017] S4, the polyvinyl alcohol is added into deionized water, heated, stirred and dissolved to obtain an external aqueous phase solution;

[0018] S5, the primary emulsion is slowly added into the external aqueous phase solution, stirred until the solvent is completely volatilized to form a stable multiple emulsion, then the lysozyme sustained-release composite microspheres are obtained through centrifugal separation, washing and drying.

[0019] In the technical scheme of the present application, the PLGA-PEG-PLGA triblock copolymer is synthesized by ring-opening polymerization, in which the PEG (polyethylene glycol) segment is combined with the lysozyme molecule through hydrogen bond to form a preliminary sustained-release mechanism, and the PLGA segment realizes the sustained release of the drug through the degradation rate control. In the process of preparing the microspheres, the internal aqueous phase solution containing lysozyme and the polymer organic phase solution are emulsified under low temperature condition by multiple emulsion method to form a stable W / O / W type multiple emulsion, and then the solvent is volatilized to obtain microspheres with compact structure. This method can protect the biological activity of lysozyme, and by adding trehalose as a protective agent and combining with ice bath operation condition, the active conformation of lysozyme is effectively maintained. The obtained microspheres have significant sustained-release characteristics, and the preliminary sustained release is realized through the hydrogen bond between PEG and lysozyme at the initial stage, and the sustained release is realized by the degradation of PLGA at the later stage. At the same time, the PEG segment enriched on the surface of the microspheres significantly enhances the adhesion of the microspheres to the mucosal tissue, prolonging the local residence time.

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

[0021] In the technical scheme of the present application, as described above, by embedding polyethylene glycol into PLGA to form a PLGA-PEG-PLGA triblock copolymer, the polyethylene glycol segment is combined with the lysozyme molecule through hydrogen bonding to form a preliminary lysozyme release mechanism. To achieve a good initial release mechanism, a sufficient amount of polyethylene glycol must be embedded in the PLGA-PEG-PLGA triblock copolymer. Therefore, the present application controls the polyethylene glycol addition amount to be greater than 12% of the mass of the monomer mixture. With the continued increase in the amount of polyethylene glycol, until the polyethylene glycol addition amount is greater than 16% of the mass of the monomer mixture, the present application team unexpectedly found that the strength of the lysozyme sustained-release composite microspheres prepared suddenly decreased significantly. In the subsequent external force stirring mixing process, the composite microspheres are easily broken. After research, it was found that this is because polyethylene glycol is a hydrophilic polymer, and when it is added in excess to PLGA, it will destroy the hydrophobic interaction and van der Waals force between the PLGA molecular chains. Due to the limited compatibility of PEG with PLGA, excessive PEG will form micro zone separation in the PLGA matrix, resulting in phase interface defects. These micro zones not only reduce the entanglement density of the PLGA molecular chain, but also cause stress concentration points to appear inside the material. When the microspheres are subjected to external force, these defect sites are prone to crack propagation, ultimately causing the microspheres to lose structural integrity and break. This phenomenon is particularly evident when the PEG addition amount exceeds 16%, which is manifested as a significant decrease in the mechanical strength of the microspheres. Therefore, the present application strictly controls the polyethylene glycol addition amount within the specified range, thereby balancing the release effect of lysozyme and the mechanical strength of the composite microspheres.

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

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

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

[0025] A preparation method of a gynecological antibacterial gel, comprising the following steps:

[0026] Mixing nipagin methyl ester, nipagin propyl ester and propylene glycol, heating to 70℃, stirring until completely dissolved, to obtain component B;

[0027] Add carbomer to deionized water, stir and dissolve, then add lysozyme sustained-release composite microspheres, continue to stir and disperse uniformly, then add cocamidopropyl betaine, cocoyl glucoside, lauryl glucoside, poloxamer, glycerol, disodium ethylenediaminetetraacetate and triethanolamine, heat to 40℃, stir and dissolve uniformly to obtain component A;

[0028] The B component is added into the A component, and the mixture is stirred and mixed uniformly to obtain the gynecological antibacterial gel.

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

[0030] 1. The application synthesizes the PLGA-PEG-PLGA triblock copolymer by a ring-opening polymerization method, and prepares the lysozyme sustained-release composite microspheres by a multiple emulsion method, so that the multi-stage release mechanism of lysozyme is realized. In the early stage, the hydrogen bond between the PEG segment and lysozyme is used to form preliminary sustained release, and in the later stage, the controllable degradation of PLGA is used to realize sustained release, so that the gel can stably play the antibacterial activity in the vaginal environment for a long time, and the problems of the traditional lysozyme preparation, such as too fast release and short action time, are effectively solved.

[0031] 2. In the preparation process of the microspheres, trehalose is added as a protective agent and combined with ice bath operation to effectively maintain the active conformation of lysozyme.

[0032] 3. The application successfully balances the sustained release effect of lysozyme and the mechanical strength of the composite microspheres by strictly controlling the addition amount of polyethylene glycol in the specified range. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0034] Embodiment 1

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

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

[0037] Lysozyme sustained-release composite microspheres 28 parts, cocamidopropyl betaine 47 parts, cocoglucoside 49 parts, lauryl glucoside 28 parts, poloxamer (type 407) 9 parts, glycerol 28 parts, carbomer (type 940) 9 parts, disodium ethylenediaminetetraacetate 2.5 parts, triethanolamine 0.8 parts, and deionized water 780 parts.

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

[0039] Propylene glycol 37 parts, methyl paraben 4 parts, and propyl paraben 4 parts.

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

[0041] Step S1: weigh D, L-lactide 7.2 g, glycolide 2.8 g, mix uniformly, then transfer to a dry three-necked flask, add polyethylene glycol (PEG-2000) 1.5 g. Install condenser, thermometer and vacuum interface, replace air with nitrogen for 3 times. Turn on the vacuum pump, vacuum dehydration at 100℃ for 2 hours, continuously stirring during the process. After dehydration, turn off the vacuum, add stannous octoate 0.01 g, continue to protect with nitrogen, heat to 180℃ for 12 hours. After the reaction is completed, cool to room temperature, dissolve the product with dichloromethane, slowly drop into ice ethyl ether for precipitation, filter and vacuum dry (40℃, 12 hours) to obtain PLGA-PEG-PLGA triblock copolymer.

[0042] Step S2: add PLGA-PEG-PLGA triblock copolymer 10 g to 350 mL dichloromethane, magnetically stir (300 rpm) at room temperature until completely dissolved to obtain an organic phase solution; weigh lysozyme (20000 U / mg) 2.5 g, trehalose 0.6 g, sodium chloride 0.08 g, add to 130 mL deionized water, stir to dissolve, then ultrasonic degassing for 5 minutes to obtain an inner aqueous phase solution.

[0043] Step S3: place the organic phase solution in an ice bath, slowly drop the inner aqueous phase solution into the organic phase, and emulsify for 40 seconds using an ultrasonic cell disruptor (power 200 W) to form a stable W / O primary emulsion.

[0044] Step S4: weigh polyvinyl alcohol and add to 200 mL deionized water, heat to 80℃ and stir to dissolve, cool to room temperature, then filter to prepare a polyvinyl alcohol outer aqueous phase solution with a concentration of 1.5 wt%.

[0045] Step S5: slowly drop the primary emulsion into the outer aqueous phase solution, magnetically stir (600 rpm) at room temperature for 6 hours to completely volatilize dichloromethane. Transfer the re-emulsion to a centrifuge tube, centrifuge at 5000 rpm for 20 minutes, discard the supernatant, and wash the precipitate with deionized water 3 times. Finally, disperse the microspheres in 50 mL trehalose solution (5 wt%), freeze-dry (-80℃ pre-freeze for 4 hours, vacuum dry for 24 hours) to obtain lysozyme sustained-release composite microspheres.

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

[0047] Mix nipagin methyl ester, nipagin propyl ester and propylene glycol, heat to 70℃, stir (300 rpm) until completely dissolved to obtain component B;

[0048] Carbomer was added into deionized water, stirred and dissolved (400 rpm), then lysozyme sustained-release composite microspheres were added, and stirring was continued to disperse uniformly, then cocamidopropyl betaine, cocoglycoside, lauryl glucoside, poloxamer, glycerol, disodium ethylenediaminetetraacetate and triethanolamine were added, heated to 40°C, and stirred and dissolved uniformly to obtain component A;

[0049] Component B was added into component A, stirred and mixed for 15 minutes (800 rpm) to ensure uniform mixing. After standing and degassing for 30 minutes, a gynecological antibacterial gel was obtained.

[0050] Example 2

[0051] A gynecological antibacterial gel comprising component A and component B, the mass ratio of component A to component B being 10:1.5.

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

[0053] Lysozyme sustained-release composite microspheres 23 parts, cocamidopropyl betaine 42 parts, cocoglycoside 42 parts, lauryl glucoside 21 parts, poloxamer (type 407) 6 parts, glycerol 21 parts, carbomer (type 940) 6 parts, disodium ethylenediaminetetraacetate 2 parts, triethanolamine 0.6 parts, deionized water 720 parts.

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

[0055] Propylene glycol 32 parts, methyl paraben 2 parts, propyl paraben 2 parts.

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

[0057] Step S1: 7.2 g of D, L-lactide and 2.8 g of glycolide were weighed, mixed uniformly, and then transferred to a dry three-necked flask. A condenser tube, a thermometer and a vacuum interface were installed, and nitrogen was introduced to replace air for 3 times. A vacuum pump was turned on, and vacuum dehydration was carried out at 100°C for 2 hours with continuous stirring. After dehydration, the vacuum was closed, 0.01 g of stannous octoate was added, and nitrogen protection was continued. The temperature was raised to 180°C and reacted for 12 hours. After the reaction was completed, the product was dissolved with dichloromethane, slowly dropped into ice ethyl ether for precipitation, filtered and vacuum dried (40°C, 12 hours) to obtain a PLGA-PEG-PLGA triblock copolymer.

[0058] Step S2: PLGA-PEG-PLGA triblock copolymer 10 g was added to 350 mL dichloromethane, and magnetically stirred (300 rpm) at room temperature until completely dissolved to obtain an organic phase solution; lysozyme (20000 U / mg) 1.5 g, trehalose 0.6 g, sodium chloride 0.08 g were weighed into 130 mL deionized water, stirred and dissolved, and then ultrasonically degassed 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 dropped into the organic phase while being emulsified for 40 seconds using an ultrasonic cell disruptor (power 200 W) to form a stable W / O primary emulsion.

[0060] Step S4: Polyvinyl alcohol was weighed into 200 mL deionized water, heated to 80°C and stirred until dissolved, then cooled to room temperature and filtered to prepare a polyvinyl alcohol outer aqueous phase solution with a concentration of 1.5 wt%.

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

[0062] A method for preparing a gynecological antibacterial gel, comprising the following steps:

[0063] Nipagin methyl ester, nipagin propyl ester and propylene glycol were mixed and heated to 70°C, and stirred (300 rpm) until completely dissolved to obtain component B;

[0064] Carbomer was added to deionized water and stirred (400 rpm) until dissolved, then the lysozyme sustained-release composite microspheres were added and stirred until uniformly dispersed, and then cocamidopropyl betaine, cocoglycoside, lauryl glucoside, poloxamer, glycerol, disodium ethylenediaminetetraacetate and triethanolamine were added, heated to 40°C and stirred until dissolved to obtain component A;

[0065] Component B was added to component A and stirred for 15 minutes (800 rpm) to ensure uniform mixing. After standing for 30 minutes, a gynecological antibacterial gel was obtained.

[0066] Example 3

[0067] A gynecological antibacterial gel comprising component A and component B, the mass ratio of component A to component B being 10:1.5.

[0068] The component A comprises the following components by weight:

[0069] Lysostaphin sustained-release composite microspheres 25 parts, cocamidopropyl betaine 45 parts, cocoglycoside 45 parts, lauryl glucoside 25 parts, poloxamer (type 407) 7 parts, glycerol 25 parts, carbomer (type 940) 8 parts, disodium ethylenediaminetetraacetate 2 parts, triethanolamine 0.8 parts, deionized water 750 parts.

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

[0071] Propylene glycol 35 parts, methyl nipagin 3 parts, propyl nipagin 3 parts.

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

[0073] Step S1: weigh D, L-lactide 7.2 g, glycolide 2.8 g, mix uniformly and transfer to a dry three-necked flask, add polyethylene glycol (PEG-2000) 1.4 g. Install condenser, thermometer and vacuum interface, replace air with nitrogen for 3 times. Turn on the vacuum pump, vacuum dehydrate at 100℃ for 2 hours, continuously stir during the process. After dehydration, turn off the vacuum, add stannous octoate 0.01 g, continue to protect with nitrogen, heat to 180℃ and react for 12 hours. After the reaction is completed, cool to room temperature, dissolve the product with dichloromethane, slowly drop into ice ethyl ether for precipitation, filter and vacuum dry (40℃, 12 hours) to obtain PLGA-PEG-PLGA triblock copolymer.

[0074] Step S2: add PLGA-PEG-PLGA triblock copolymer 10 g to 350 mL dichloromethane, magnetically stir (300 rpm) at room temperature until completely dissolved to obtain an organic phase solution; weigh lysostaphin (20000 U / mg) 2 g, trehalose 0.6 g, sodium chloride 0.08 g, add to 130 mL deionized water, stir to dissolve, then ultrasonic degassing for 5 minutes to obtain an inner aqueous phase solution.

[0075] Step S3: place the organic phase solution in an ice bath, slowly drop the inner aqueous phase solution into the organic phase, and use an ultrasonic cell disruptor (power 200 W) to emulsify for 40 seconds to form a stable W / O type primary emulsion.

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

[0077] Step S5: The primary emulsion was slowly dropped into the external aqueous phase solution, and magnetic stirring (600 rpm) was performed at room temperature for 6 hours to completely volatilize dichloromethane. The re-emulsion was transferred to a centrifuge tube, centrifuged at 5000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was washed with deionized water for 3 times. Finally, the microspheres were dispersed in 50 mL of a trehalose solution (5 wt%), and freeze-drying was performed (pre-freezing at -80℃ for 4 hours, and vacuum drying for 24 hours) to obtain the lysozyme sustained-release composite microspheres.

[0078] A method for preparing a gynecological antibacterial gel, comprising the following steps:

[0079] Nipagin methyl ester, nipagin propyl ester and propylene glycol were mixed, heated to 70℃, and stirred (300 rpm) until completely dissolved to obtain component B;

[0080] Carbomer was added to deionized water, stirred and dissolved (400 rpm), then the lysozyme sustained-release composite microspheres were added, and stirring was continued to disperse uniformly, then cocamidopropyl betaine, cocoglycoside, lauryl glucoside, poloxamer, glycerol, disodium ethylenediaminetetraacetate and triethanolamine were added, heated to 40℃, and stirred and dissolved uniformly to obtain component A;

[0081] Component B was added to component A, and stirring was performed for 15 minutes (800 rpm) to ensure uniform mixing. Degassing was performed at room temperature for 30 minutes to obtain the gynecological antibacterial gel.

[0082] Example 4

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

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

[0085] Lysozyme sustained-release composite microspheres 30 parts, cocamidopropyl betaine 50 parts, cocoglycoside 50 parts, lauryl glucoside 30 parts, poloxamer (type 407) 10 parts, glycerol 30 parts, carbomer (type 940) 10 parts, disodium ethylenediaminetetraacetate 3 parts, triethanolamine 1 part, and deionized water 800 parts.

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

[0087] Propylene glycol 40 parts, nipagin methyl ester 5 parts, and nipagin propyl ester 5 parts.

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

[0089] Step S1: weigh D, L-lactide 7.2 g, glycolide 2.8 g, mix uniformly, then transfer to a dry three-necked flask, add polyethylene glycol (PEG-2000) 1.6 g. Install condenser, thermometer and vacuum interface, replace air with nitrogen for 3 times. Turn on the vacuum pump, vacuum dehydration at 100℃ for 2 hours, continuously stirring during the process. After dehydration, turn off the vacuum, add stannous octoate 0.01 g, continue to protect with nitrogen, heat to 180℃ for 12 hours. After the reaction is completed, cool to room temperature, dissolve the product with dichloromethane, slowly drop into ice ethyl ether for precipitation, filter and vacuum dry (40℃, 12 hours) to obtain PLGA-PEG-PLGA triblock copolymer.

[0090] Step S2: add PLGA-PEG-PLGA triblock copolymer 10 g to 350 mL dichloromethane, magnetically stir (300 rpm) at room temperature until completely dissolved to obtain an organic phase solution; weigh lysozyme (20000 U / mg) 3 g, trehalose 0.6 g, sodium chloride 0.08 g, add to 130 mL deionized water, stir to dissolve, then ultrasonic degassing for 5 minutes to obtain an inner aqueous phase solution.

[0091] Step S3: place the organic phase solution in an ice bath, slowly drop the inner aqueous phase solution into the organic phase, and emulsify for 50 seconds using an ultrasonic cell disruptor (power 200 W) to form a stable W / O primary emulsion.

[0092] Step S4: weigh polyvinyl alcohol and add to 200 mL deionized water, heat to 80℃ and stir to dissolve, cool to room temperature, then filter to prepare a polyvinyl alcohol outer aqueous phase solution with a concentration of 2 wt%.

[0093] Step S5: slowly drop the primary emulsion into the outer aqueous phase solution, magnetically stir (600 rpm) at room temperature for 6 hours to completely volatilize dichloromethane. Transfer the re-emulsion to a centrifuge tube, centrifuge at 5000 rpm for 20 minutes, discard the supernatant, and wash the precipitate with deionized water 3 times. Finally, disperse the microspheres in 50 mL trehalose solution (5 wt%), freeze-dry (-80℃ pre-freeze for 4 hours, vacuum dry for 24 hours) to obtain lysozyme sustained-release composite microspheres.

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

[0095] Mix nipagin methyl ester, nipagin propyl ester and propylene glycol, heat to 70℃, stir (300 rpm) until completely dissolved to obtain component B;

[0096] Carbomer was added into deionized water, stirred and dissolved (400 rpm), then lysozyme sustained-release composite microspheres were added, and stirring was continued to disperse uniformly, then cocamidopropyl betaine, cocoglycoside, lauryl glucoside, poloxamer, glycerol, disodium ethylenediaminetetraacetate and triethanolamine were added, heated to 40℃, and stirred and dissolved uniformly to obtain component A;

[0097] Component B was added into component A, stirred and mixed for 15 minutes (800 rpm) to ensure uniform mixing. After standing and degassing for 30 minutes, a gynecological antibacterial gel was obtained.

[0098] Example 5

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

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

[0101] Lysozyme sustained-release composite microspheres 20 parts, cocamidopropyl betaine 40 parts, cocoglycoside 40 parts, lauryl glucoside 20 parts, poloxamer (type 407) 5 parts, glycerol 20 parts, carbomer (type 940) 5 parts, disodium ethylenediaminetetraacetate 1 part, triethanolamine 0.5 part, deionized water 700 parts.

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

[0103] Propylene glycol 30 parts, methyl paraben 1 part, propyl paraben 1 part.

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

[0105] Step S1: 7.2 g of D, L-lactide and 2.8 g of glycolide were weighed, mixed uniformly, and then transferred to a dry three-necked flask. A condenser tube, a thermometer and a vacuum interface were installed, and nitrogen was introduced to replace air for 3 times. A vacuum pump was turned on, and vacuum dehydration was carried out at 100℃ for 2 hours with continuous stirring. After dehydration, the vacuum was closed, 0.01 g of stannous octoate was added, and nitrogen protection was continued. The temperature was raised to 180℃ and reacted for 12 hours. After the reaction was completed, the product was dissolved with dichloromethane, slowly dropped into ice ethyl ether for precipitation, filtered and vacuum dried (40℃, 12 hours) to obtain a PLGA-PEG-PLGA triblock copolymer.

[0106] Step S2: PLGA-PEG-PLGA triblock copolymer 10 g was added to 350 mL dichloromethane, and magnetically stirred (300 rpm) at room temperature until completely dissolved to obtain an organic phase solution; lysozyme (20000 U / mg) 1 g, trehalose 0.6 g, sodium chloride 0.08 g were weighed and added to 130 mL deionized water, and after stirring and dissolving, ultrasonic degassing was performed 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 dropped into the organic phase while using an ultrasonic cell disruptor (power 200 W) for emulsification for 30 seconds to form a stable W / O type primary emulsion.

[0108] Step S4: Polyvinyl alcohol was weighed and added to 200 mL deionized water, heated to 80°C and stirred to dissolve, and after cooling to room temperature, filtered to prepare a polyvinyl alcohol outer aqueous phase solution with a concentration of 1 wt%.

[0109] Step S5: The primary emulsion was slowly dropped into the outer aqueous phase solution, and magnetically stirred (600 rpm) at room temperature for 6 hours to completely volatilize the dichloromethane. The re-emulsion was transferred to a centrifuge tube and centrifuged at 5000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was washed with deionized water 3 times. Finally, the microspheres were dispersed in 50 mL trehalose solution (5 wt%), freeze-dried (-80°C pre-freezing for 4 hours, vacuum drying for 24 hours) to obtain lysozyme sustained-release composite microspheres.

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

[0111] Nipagin methyl ester, nipagin propyl ester and propylene glycol were mixed and 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), then the lysozyme sustained-release composite microspheres were added, and the stirring was continued to disperse uniformly, and then cocamidopropyl betaine, cocoglycoside, lauryl glucoside, poloxamer, glycerol, disodium ethylenediaminetetraacetate and triethanolamine were added, and heated to 40°C, stirred and dissolved uniformly to obtain component A;

[0113] Component B was added to component A and stirred for 15 minutes (800 rpm) to ensure uniform mixing. After standing and degassing for 30 minutes, the gynecological antibacterial gel was obtained.

[0114] Comparative Example 1

[0115] Comparative Example 1 differs from Example 1 in that the lysozyme sustained-release composite microspheres are replaced by lysozyme, and the amount of lysozyme is kept the same, and the remaining steps are the same.

[0116] Comparative Example 2

[0117] Comparative Example 2 differs from Example 1 in that no polyethylene glycol is added in step S1 in the preparation of the lysozyme sustained-release composite microspheres, and the remaining steps are the same.

[0118] Comparative Example 3

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

[0120] Comparative Example 4

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

[0122] Performance test:

[0123] 1. Lysozyme sustained-release performance test: using a dialysis bag method (molecular weight cutoff 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 constant temperature oscillation (100 rpm). 5 mL of sample was taken at 1 h, 4 h, 12 h, 24 h, 3 d, and 7 d (replenished with an equal amount of fresh PBS), and the absorbance at 280 nm was measured by ultraviolet-visible spectrophotometry. The cumulative release rates at 24 h and 7 d were calculated. The release rate at 24 h was used to represent the initial sustained-release effect of lysozyme, and the release rate at 7 d was used to represent the overall sustained-release effect of lysozyme. The test results are shown in Table 1.

[0124] 2. Lysozyme activity retention rate test: using the micrococcus turbidity method, the microsphere dissolution solution (containing 1 mg of lysozyme) was mixed with the micrococcus suspension (OD600 = 0.5) and reacted at 37°C for 10 minutes. The OD600 drop value was measured, and the activity retention rate (%) was calculated by comparing with the activity of free lysozyme (activity retention rate (%) = (activity of lysozyme in microspheres / activity of free lysozyme) x 100%). The test results are shown in Table 1.

[0125] Table 1:

[0126]

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

[0128] 4. Mucosal adhesion test: Franz diffusion cell method was used to prepare porcine vaginal mucosa tissue, and the gel was applied to the mucosa surface (2 mg / cm 2 ), and the time for the gel to completely fall off was recorded. The test results are shown in Table 2.

[0129] 5. Lysozyme sustained-release composite microsphere mechanical strength test: Microsphere crushing pressure test method was used, 10 mg of freeze-dried microsphere powder was placed in a cylindrical mold with a diameter of 5 mm, a microhardness tester (FISHER HM-200) was used to apply a vertical pressure to the microspheres at a rate of 0.1 mm / s, and the maximum pressure value (unit: mN) when the microspheres were broken was recorded. Each group was tested for 20 microspheres, and the average value and standard deviation were taken. The test results are shown in Table 2.

[0130] Table 2:

[0131]

[0132] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacements for part of the technical features, and any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A gynecological antibacterial gel, characterized in that, The A component and the B component are included; The A component includes the following components by weight parts: Lysostaphin sustained-release composite microspheres 20-30 parts, cocamidopropyl betaine 40-50 parts, cocoyl glucoside 40-50 parts, lauryl glucoside 20-30 parts, poloxamer 5-10 parts, glycerol 20-30 parts, carbomer 5-10 parts, disodium ethylenediaminetetraacetate 1-3 parts, triethanolamine 0.5-1 part, and deionized water 700-800 parts; The preparation method of the lysostaphin sustained-release composite microspheres includes the following steps: S1, uniformly mix D, L-lactide and glycolide to obtain a monomer mixture, add polyethylene glycol to the monomer mixture, the polyethylene glycol is added in an amount of 12-16% of the mass of the monomer mixture, vacuum dewatering is performed, then stannous octoate catalyst is added, heating is performed under nitrogen protection until complete reaction, purification and drying are performed, and a triblock copolymer compound is obtained; S2, the triblock copolymer compound is added to dichloromethane to be stirred and dissolved to obtain an organic phase solution; lysostaphin, trehalose and sodium chloride are added to deionized water to be stirred and dissolved to obtain an inner aqueous phase solution; S3, under ice bath conditions, the inner aqueous phase solution is slowly added to the organic phase solution, ultrasonic emulsification is performed, and an initial emulsion is formed; S4, polyvinyl alcohol is added to deionized water, heated and stirred to be dissolved to obtain an outer aqueous phase solution; S5, the initial emulsion is slowly added to the outer aqueous phase solution, stirring is performed until the solvent is completely volatilized, a stable multiple emulsion is formed, then centrifugal separation, washing and drying are performed, and lysostaphin sustained-release composite microspheres are obtained; The B component includes the following components by weight parts: Propylene glycol 30-40 parts, methylparaben 1-5 parts, and propylparaben 1-5 parts.

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

3. The gynecological antiseptic gel according to claim 1, wherein In the step S2, the mass ratio of the triblock copolymer compound to lysostaphin is 10:1-3.

4. The gynecological antiseptic gel of claim 1, wherein, In the step S3, the ultrasonic emulsification time is 30-50 s.

5. The gynecological antiseptic gel of claim 1, wherein, In the step S4, the concentration of polyvinyl alcohol in the aqueous phase solution is 1-2 wt%.

6. A process for the preparation of the gynaecological antiseptic gel as claimed in any one of claims 1 to 5, characterized in that, The following steps are included: Methylparaben, propylparaben and propylene glycol are mixed, heated to 70°C, and stirred until completely dissolved to obtain the B component; Carbomer is added to deionized water, stirred and dissolved, then lysostaphin sustained-release composite microspheres are added, stirring is continued to disperse uniformly, then cocamidopropyl betaine, cocoyl glucoside, lauryl glucoside, poloxamer, glycerol, disodium ethylenediaminetetraacetate and triethanolamine are added, heated to 40°C, and stirred and dissolved uniformly to obtain the A component; The B component is added to the A component, stirred and mixed uniformly to obtain the gynecological antibacterial gel.

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