Lubricating agent containing probiotics as well as preparation method and application of lubricating agent

Abstract

The invention discloses a lubricant containing probiotics as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing a frozen mucoprotein scaffold; preparing magnetized probiotics, and freezing the mucoprotein scaffold to obtain a magnetized probiotics and scaffold complex; preparing a fluorescent tracing type magnetized probiotic (at) ZIF-8 core-shell complex based on the magnetized probiotic (at) scaffold complex; the preparation method comprises the following steps: preparing a nano-fiber modified magnetized probiotic (at) ZIF-8 complex on the basis of an EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nano-fiber complex and a fluorescent tracing type magnetized probiotic (at) ZIF-8 core-shell complex; the preparation method comprises the following steps: preparing temperature-sensitive microspheres on the basis of a poloxamer temperature-sensitive gel and an EGCG / hyaluronic acid-LL-37 modified probiotic complex; performing nano coating on the temperature-sensitive microspheres to obtain probiotic freeze-dried powder protected by the nano coating; and mixing the probiotic freeze-dried powder with sterile normal saline to obtain the gelatinous lubricant containing the probiotics.

Description

Technical Field

[0001] The present application relates to a lubricant, and in particular to a lubricant containing probiotics, a preparation method and an application thereof. Background Art

[0002] Traditional lubricants primarily rely on synthetic polymers or silicone-based materials. While they can provide short-term lubrication, they lack biocompatibility, long-term stability, and functionality. Particularly in the field of women's health, traditional lubricants can disrupt the balance of the vaginal microbiome, leading to increased pH and a decrease in beneficial bacteria, thereby increasing the risk of infection. Furthermore, existing lubricants lack effective protection for probiotics, making it difficult to maintain bacterial activity in complex physiological environments, limiting their application in microbiome regulation.

[0003] In recent years, the role of probiotics in maintaining women's health has received widespread attention. Probiotics such as lactic acid bacteria can maintain the balance of vaginal microecology by competitively inhibiting pathogens, secreting antibacterial substances (such as lactic acid and hydrogen peroxide), and regulating local immune responses. However, adding probiotics directly to lubricants faces many challenges, including loss of bacterial activity during storage and use, decreased survival rate due to environmental stress (such as oxidative stress and mechanical shear), and difficulty in achieving precise delivery and long-term colonization.

[0004] In the existing technology, although some studies have attempted to combine probiotics with carrier materials, the following defects are generally present: Insufficient protection: Conventional encapsulation techniques (such as sodium alginate microspheres) cannot effectively resist the damage of the external environment to probiotics, resulting in a rapid decrease in activity during storage; Single function: The carrier material lacks synergistic antibacterial or antioxidant functions and is difficult to cope with complex infection environments; Complex process: The multi-step preparation process is prone to contamination risks and is difficult to mass produce.

[0005] Therefore, there is an urgent need to develop a new type of lubricant containing probiotics that can not only provide excellent lubrication performance, but also achieve efficient protection, targeted delivery and functional synergy of probiotics through advanced materials and processes, thereby solving the limitations of existing technologies. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the purpose of this application is to provide a lubricant containing probiotics, a preparation method and an application thereof, and the present application aims to improve the survival rate of probiotics in the lubricant and the storage convenience.

[0007] To achieve the above objectives, this application provides the following technical solutions:

[0008] A method for preparing a lubricant containing probiotics, the method comprising: mixing recombinant human mucin with sterile deionized water in a preset ratio to prepare a mucin solution, printing the mucin solution to obtain a frozen mucin scaffold; activating the frozen mucin scaffold, mixing Lactococcus lactis and Fe3O4@SiO2 magnetic nanoparticles in a preset ratio to obtain magnetized probiotics, and injecting the magnetized probiotics into the activated frozen mucin scaffold to obtain a magnetized probiotics@scaffold complex; immersing the magnetized probiotics@scaffold complex in a Zn 2+ The mixture was locally irradiated with ultraviolet light in a mixed solution consisting of 2-methylimidazole and lyase. At the same time, a preset ratio of CQDs-ZnO nanoparticles was added to the mixed solution to obtain a fluorescent tracer type magnetized probiotics @ ZIF-8 core-shell complex; a preset ratio of EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex was dissolved in a citric acid buffer solution, and the fluorescent tracer type magnetized probiotics @ ZIF-8 core-shell complex was immersed in a citric acid buffer solution in which the EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex was dissolved to obtain a nanofiber-modified magnetized probiotics @ ZIF-8 complex; the preset ratio of EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex was dissolved in a citric acid buffer solution to obtain a nanofiber-modified magnetized probiotics @ ZIF-8 complex. A predetermined proportion of poloxamer thermosensitive gel is dissolved in sterile deionized water to obtain a homogeneous gel solution, an EGCG / hyaluronic acid-LL-37 modified probiotic complex is dispersed in the homogeneous gel solution, and after blue light irradiation, thermosensitive microspheres coated with the EGCG / hyaluronic acid-LL-37 modified probiotic complex are obtained; a predetermined proportion of resveratrol nanoliposomes and trehalose-hydroxybutyrate-dextran protective agent are added to the thermosensitive microspheres, and after pre-freezing and drying, a nanocoating is deposited on the surface of the thermosensitive microspheres to obtain a nanocoating protected probiotic freeze-dried powder; the probiotic freeze-dried powder is mixed with sterile physiological saline to obtain a gel-like lubricant containing probiotics.

[0009] Optionally, before mixing the recombinant human mucin with sterile deionized water, pre-cool the sterile deionized water to 4°C.

[0010] Optionally, the frozen mucin scaffold has a honeycomb structure with a pore size of 50 μm to 200 μm.

[0011] Optionally, the method of adding a preset proportion of CQDs-ZnO nanoparticles to the mixed solution to obtain a fluorescent tracer-type magnetized probiotics@ZIF-8 core-shell complex includes the following steps: ultrasonically treating the mixed solution; incubating the mixed solution after ultrasonic treatment; irradiating the incubated mixed solution with ultraviolet light; placing the mixed solution after ultraviolet light irradiation in a magnetic field and allowing it to stand; removing the supernatant from the mixed solution after standing; and freeze-drying the mixed solution after the supernatant is removed.

[0012] Optionally, after adding a preset ratio of resveratrol nanoliposomes and trehalose-hydroxybutyrate-dextran protective agent to the thermosensitive microspheres, the microspheres are pre-frozen to -50°C.

[0013] Optionally, supercritical CO2 is used to dry the pre-frozen temperature-sensitive microspheres.

[0014] Optionally, depositing a nanocoating on the surface of the dried thermosensitive microspheres includes: growing a PLGA nanocoating on the surface of the dried thermosensitive microspheres by atomic layer deposition.

[0015] Optionally, the mixing of the probiotic freeze-dried powder with sterile physiological saline includes: mixing the probiotic freeze-dried powder with sterile physiological saline in a mass ratio of 1:5.

[0016] The present application also provides a lubricant containing probiotics, and the raw materials used to prepare the lubricant and the mass percentage of each raw material are: recombinant human mucin: 18% to 22%; Lactococcus lactis: 6.5% to 7.5%; Fe3O4@SiO magnetic nanoparticles: 0.4% to 0.5%; CQDs-ZnO nanoparticles: 0.3% to 0.4%; phage lytic enzyme@ZIF-8: 3.2% to 3.8%; EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex: 4.5% to 5.8%; poloxamer thermosensitive gel: 13% to 15%; trehalose-hydroxybutyrate-glucan composite protective agent: 7% to 8%; resveratrol nanoliposomes: 0.05%; and sterile deionized water: the balance.

[0017] The present application also provides an application of a lubricant containing probiotics, wherein the lubricant is applied to female health care.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This application innovatively combines probiotic protection, functionalized carrier design, and precision preparation technology to effectively address key issues in existing technologies, such as low probiotic survival rate, limited functionality, and insufficient stability in lubricants. Furthermore, the development of a freeze-dried powder formulation overcomes the storage difficulties of traditional probiotic preparations, resulting in a product that combines excellent stability with ease of use. This solution not only overcomes the performance limitations of traditional lubricants but also provides a safer, more efficient, and more versatile solution for women's intimate health care. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic flow chart of a method for preparing a lubricant containing probiotics according to an embodiment of the present application;

[0021] Figure 2This is an electron micrograph of a frozen mucin scaffold with a pore size of 50 μm provided in another embodiment of the present application;

[0022] Figure 3 An electron micrograph of a frozen mucin scaffold with a pore size of 200 μm provided in another embodiment of the present application;

[0023] Figure 4 This is an electron micrograph of a frozen mucin scaffold with a pore size of 100 μm provided in another embodiment of the present application;

[0024] Figure 5 This is a schematic diagram of the microstructure of a probiotic freeze-dried powder protected by a PLGA nano-coating provided in another embodiment of the present application;

[0025] Figure 6 It is a macroscopic diagram of existing lubricants;

[0026] Figure 7 It is a microscopic diagram of existing lubricants;

[0027] Figure 8 This is a macroscopic schematic diagram of a lubricant prepared based on the method described in this application, provided in another embodiment of this application;

[0028] Figure 9 This is a microscopic schematic diagram of a lubricant prepared based on the method described in this application, provided in another embodiment of this application. DETAILED DESCRIPTION

[0029] Specific embodiments of the present application will be described in detail below with reference to the accompanying drawings. Although specific embodiments of the present application are shown in the accompanying drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0030] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of this application shall be as defined by the attached claims.

[0031] To facilitate understanding of the embodiments of the present application, further explanation will be given below using specific embodiments as examples in conjunction with the accompanying drawings, and the various drawings do not constitute a limitation on the embodiments of the present application.

[0032] In an exemplary embodiment, the present application provides a lubricant containing probiotics, wherein the components of the lubricant and the mass percentage of each component are as follows: recombinant human mucin: 18% to 22%; Lactococcus lactis: 6.5% to 7.5%; Fe3O4@SiO magnetic nanoparticles: 0.4% to 0.5%; CQDs-ZnO nanoparticles (CQDs, Carbon Quantum Dots, carbon quantum dots): 0.3% to 0.4%; phage lytic enzyme @ ZIF-8: 3.2% to 3.8%; EGCG / hyaluronic acid-LL-37 antibacterial antioxidant nanofiber complex (EGCG's full Chinese name is "epigallocatechin gallate", LL-37 represents antimicrobial peptide): 4.5% to 5.8%; poloxamer thermosensitive gel: 13% to 15%; trehalose-hydroxybutyrate-dextran complex protective agent: 7% to 8%; resveratrol nanoliposomes: 0.05%; sterile deionized water: balance. The components of the lubricant and the mass percentage of each component are preferably: recombinant human mucin: 20%; Lactococcus lactis: 7.0%; Fe3O4@SiO magnetic nanoparticles: 0.45%; CQDs-ZnO nanoparticles: 0.35%; phage lytic enzyme@ZIF-8: 3.5%; EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex: 5.2%; poloxamer thermosensitive gel: 14%; trehalose-hydroxybutyrate-dextran composite protective agent: 7.5%; resveratrol nanoliposomes: 0.05%; and sterile deionized water: the balance.

[0033] Figure 1 FIG. 1 is a flow chart of a method for preparing a lubricant containing probiotics provided by an exemplary embodiment of the present application, such as Figure 1 As shown, the preparation method comprises the following steps:

[0034] S100: Recombinant human mucin is mixed with sterile deionized water pre-cooled at 4°C to prepare a mucin solution, and the mucin solution is 3D printed to obtain a frozen mucin scaffold with a honeycomb structure;

[0035] S200: The frozen mucin scaffold was activated, and Lactococcus lactis was incubated with Fe3O4@SiO2 magnetic nanoparticles (mass ratio 1:0.2) at 4°C for 30 minutes under shaking to obtain magnetized probiotics. The magnetized probiotics were injected into the activated frozen mucin scaffold and a gradient magnetic field was applied for 5 minutes to achieve directional anchoring of the bacteria, thereby obtaining a magnetized probiotics@scaffold complex.

[0036] S300: Immerse the magnetized probiotics@scaffold complex in a Zn 2+ The ZIF-8 framework was selectively grown around the magnetized bacteria, forming a coating layer with a thickness of 200 nm. At the same time, CQDs-ZnO nanoparticles were added to the mixed solution to obtain a fluorescent tracer magnetized probiotic @ ZIF-8 core-shell complex.

[0037] S400: The EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex is dissolved in a citric acid buffer solution (concentration of 5%) at a pH of 5.0, and the fluorescent tracer-type magnetized probiotics@ZIF-8 core-shell complex is immersed in the citric acid buffer solution in which the EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex is dissolved. 40kHz ultrasonic waves (power 300W) are applied for 10 minutes to induce the nanofibers to grow vertically on the ZIF-8 surface through the cavitation effect and load the antimicrobial peptides, thereby obtaining an EGCG / hyaluronic acid-LL-37 modified probiotic complex;

[0038] S500: Poloxamer thermosensitive gel and LAP photoinitiator were dissolved in 4°C deionized water to obtain a homogeneous gel solution. EGCG / hyaluronic acid-LL-37 modified probiotic complex was dispersed in the homogeneous gel solution and irradiated with blue light (intensity 10 mW / cm 2 , time 30s) to trigger in situ photocrosslinking to obtain thermosensitive microspheres encapsulated with EGCG / hyaluronic acid-LL-37 modified probiotic complexes;

[0039] S600: Resveratrol nanoliposomes and trehalose-hydroxybutyrate-dextran protective agents are added to the thermosensitive microspheres, which are pre-frozen to -50°C to form a glassy state. The glassy thermosensitive microspheres are dried using supercritical CO2, and a 2 nm thick PLGA (Poly (lactic-co-glycolic acid) copolymer) nanocoating is grown on the surface of the dried thermosensitive microspheres using atomic layer deposition to obtain a PLGA nanocoated probiotic freeze-dried powder.

[0040] S700: Mix the PLGA nano-coated probiotic freeze-dried powder with sterile saline at a mass ratio of 1:5 and shake gently to form a gel-like lubricant containing probiotics.

[0041] Below, the present application further describes the above preparation method through Examples 1 to 3.

[0042] Example 1:

[0043] 1. 18% recombinant human mucin was mixed with sterile deionized water precooled at 4°C, and magnetically stirred (200 rpm to 300 rpm) in a container precooled at the same temperature. After standing and degassing at 4°C for 1 hour, an 18% (w / w) mucin solution was prepared. The mucin solution was 3D printed to obtain a frozen mucin scaffold with a honeycomb structure of 50 μm pore size (such as Figure 2 shown);

[0044] In this step, the purpose of using sterile deionized water pre-cooled at 4°C and stirring in a container pre-cooled at the same temperature is to avoid denaturation of mucin due to local temperature increase.

[0045] In addition, it should be noted that the surface of the female vaginal epithelium has complex folds and microgrooves (similar to the hexagonal units of a honeycomb). This structure can effectively increase the surface area, promote mucus secretion and the colonization of probiotics (such as lactic acid bacteria). The honeycomb scaffold simulates this natural structure through 3D printing, which can provide a microenvironment closer to physiological conditions. In addition, the vagina is a microaerobic environment (O2 concentration is approximately 1% to 5%). The gradient pore size of the honeycomb scaffold (such as dense surface and sparse interior) can regulate oxygen penetration and prevent deep-layer probiotics from being inactivated due to lack of oxygen.

[0046] 2. The frozen mucin scaffold was activated, and 6.5% Lactococcus lactis and 0.4% Fe3O4@SiO2 magnetic nanoparticles were mixed in a centrifuge tube. The mixture was incubated at 4°C with shaking for 30 minutes (rotation speed: 150 rpm to balance nanoparticle dispersion and bacterial survival rate; the centrifuge tube was tilted at 30 degrees to enhance liquid vortex and prevent particle sedimentation) to obtain magnetized probiotics. The magnetized probiotics were injected into the activated frozen mucin scaffold and a gradient magnetic field was applied for 5 minutes to achieve directional anchoring of the bacteria, thereby obtaining a magnetized probiotics@scaffold complex.

[0047] In this step, the frozen mucin scaffold is activated, including the following steps:

[0048] Step 1: Immerse the frozen mucin scaffold in a solution containing calcium ions (Ca 2+ ), phosphate (PO4 3- ) and 10 U / mL alkaline phosphatase (ALP) in a mineralization solution (pH 7.4, 37°C) and incubated in a 37°C constant temperature shaker (50 rpm) for 6 hours.

[0049] In this step, ALP catalyzes the decomposition of sodium β-glycerophosphate, releasing phosphate, which combines with calcium ions to form nanohydroxyapatite (nHA), which is evenly deposited on the surface and pores of the scaffold, thereby improving the mechanical strength of the scaffold, providing a bone-mimicking microenvironment, and promoting cell adhesion.

[0050] Step 2: Wash the frozen mucin scaffold with sterile PBS to remove unreacted ions and enzymes;

[0051] Step 3: Immerse the cleaned frozen mucin scaffold in a PBS solution containing 0.5% GelMA (methacrylated gelatin) and place it in the dark at 4°C for 12 hours to allow the GelMA to fully penetrate the pores of the scaffold;

[0052] Step 4: Use 365nm ultraviolet light (5mW / cm 2 ) irradiate the stent surface for 90 seconds to form a dense cross-linked layer (to enhance stability), and then adjust the light intensity to 3mW / cm 2 , irradiating the inner area for 30 seconds to maintain high porosity within the scaffold, thereby preserving nutrient diffusion channels and supporting cell migration;

[0053] Step 5: Rinse with PBS to remove uncrosslinked GelMA and dry at 37°C for later use.

[0054] The activation treatment uses a synergistic strategy of enzymatic mineralization and photo-controlled gradient cross-linking. Alkaline phosphatase (ALP)-induced nanohydroxyapatite (nHA) deposition enhances the mechanical strength of the scaffold (compressive strength increases by 2 to 3 times), and GelMA photo-cross-linking creates a gradient structure with a dense surface and porous core (porosity >80% in the core and 70% in the surface). This treatment significantly improves the scaffold's load capacity (efficient anchoring of probiotics and quantum dots), structural stability (resistance to lubricant shear collapse), and bioactivity (cell adhesion rate increases to 90%). It provides an ideal three-dimensional carrier for the subsequent infusion of the magnetized probiotics@ZIF-8 complex, helping to improve the various properties of the lubricant.

[0055] 3. Immerse the magnetized probiotics@scaffold complex in Zn 2+ The ZIF-8 framework was selectively grown around the magnetized bacteria by local irradiation with 365 nm ultraviolet light for 10 minutes, forming a coating layer with a thickness of 200 nm. At the same time, 0.3% CQDs-ZnO nanoparticles were added to the mixed solution to obtain a fluorescent tracer magnetized probiotic @ ZIF-8 core-shell complex.

[0056] In this step, after adding CQDs-ZnO nanoparticles to the mixed solution, the following steps are required to obtain the fluorescent tracer magnetized probiotics@ZIF-8 core-shell complex:

[0057] Step 1: Use 40kHz ultrasound (power 100W) to treat the mixed solution for 5 minutes to ensure that the CQDs-ZnO nanoparticles are evenly dispersed and avoid agglomeration. It should be noted that the ultrasonic treatment needs to be kept in an ice bath (4°C) throughout the process to prevent ultrasonic heat generation from damaging the activity of probiotics.

[0058] Step 2: The mixed solution after ultrasonic treatment was placed in a shaker at 4°C (50 rpm) and incubated for 15 minutes to allow CQDs-ZnO to be adsorbed onto the surface of the ZIF-8 precursor through electrostatic interaction.

[0059] Step 3: Use 365nm ultraviolet light (intensity 10mW / cm 2 ) Local irradiation of the incubated mixed solution for 10 minutes triggers the following reactions:

[0060] ZIF-8 nucleation: Zn 2+ Combined with 2-methylimidazole, a ZIF-8 coating layer (about 200 nm thick) is formed on the surface of probiotics.

[0061] CQDs-ZnO embedded: The nanoparticles are encapsulated within the ZIF-8 framework to form a fluorescently labeled core-shell structure.

[0062] Step 4: Place the mixed solution after UV irradiation in a 0.5T magnetic field for 5 minutes to allow the complex to adsorb to the container wall;

[0063] Step 5: Use a sterile pipette to slowly remove the supernatant (to avoid disturbing the adsorbed complex), retaining the magnetic complex at the bottom of the container. Then, add 5 mL of pre-cooled sterile PBS, gently pipette to resuspend the complex, and place it in the magnetic field for adsorption for 5 minutes again. Remove the supernatant and repeat the washing several times until there is no fluorescent signal in the supernatant (indicating that the free CQDs-ZnO has been removed).

[0064] Step 6: Resuspend the washed complex in 1 mL of protective buffer (such as PBS containing 5% trehalose), freeze-dry at -80°C for later use.

[0065] 4. Dissolve 4.5% EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex in citric acid buffer (concentration of 5%) at pH 5.0, immerse the fluorescent tracer-type magnetized probiotics@ZIF-8 core-shell complex in the citric acid buffer dissolved with EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex, and apply ultrasonic treatment (40 kHz, 300 W) for 10 minutes to induce nanofibers to grow vertically on the ZIF-8 surface through cavitation effect and load antimicrobial peptides to obtain EGCG / hyaluronic acid-LL-37 modified probiotic complex;

[0066] 5. Dissolve 13% poloxamer thermosensitive gel and LAP photoinitiator in 4°C deionized water to obtain a homogeneous gel solution. Disperse the EGCG / hyaluronic acid-LL-37 modified probiotic complex in the homogeneous gel solution and irradiate with blue light (intensity 10mW / cm 2 , time 30s) to trigger in situ photocrosslinking to obtain thermosensitive microspheres encapsulated with EGCG / hyaluronic acid-LL-37 modified probiotic complexes;

[0067] 6. Add 0.05% resveratrol nanoliposomes and 7% trehalose-hydroxybutyrate-dextran protective agent to the thermosensitive microspheres, prefreeze to -50°C to form a glassy state, dry the glassy thermosensitive microspheres using supercritical CO2, and grow a 2 nm thick PLGA (Poly (lactic-co-glycolic acid) copolymer) nanocoating on the surface of the dried thermosensitive microspheres by atomic layer deposition to obtain a PLGA nanocoated probiotic freeze-dried powder;

[0068] In this step, a 2 nm thick PLGA nanocoating is grown on the surface of the dried thermosensitive microspheres by atomic layer deposition, comprising the following steps:

[0069] Step 1: Spread the dried thermosensitive microspheres evenly on the ALD sample plate (stacked thickness ≤ 1 mm), then place them in the ALD reaction chamber and evacuate to 10 -3 Torr, and then the temperature was raised to 80 °C and maintained for 1 hour to remove adsorbed water and residual solvent.

[0070] Step 2: N2 (purity 99.999%, flow rate 20sccm) was introduced and radio frequency plasma (50W, 13.56MHz) was started for 5 minutes to generate hydroxyl (-OH) and amino (-NH2) active groups on the surface of the microspheres to improve the precursor adsorption efficiency.

[0071] Step 3: Trimethylaluminum (TMA) is injected into the ALD reaction chamber with a pulse time of 0.1 seconds and a pressure of 0.1 Torr. TMA reacts with the hydroxyl groups on the surface of the microspheres to form an Al-CH3 adsorption layer. Then, high-purity N2 is introduced for 30 seconds (flow rate of 20 sccm) to remove unreacted TMA and by-products (methane).

[0072] Step 4: PLGA (50:50, Mw = 10 kDa) was dissolved in supercritical CO2 (40 °C, 10 MPa) and sprayed into the reaction chamber through an ultrasonic atomization nozzle (frequency 120 kHz) for 5 seconds. It should be noted that the chamber needs to be purged with N2 for 5 minutes before PLGA spraying to prevent CO2 from reacting with TMA.

[0073] Step 5: After spraying, the ALD reaction chamber was kept at 80°C and N2 (flow rate 10 sccm) was introduced for annealing for 2 minutes to promote the self-assembly of PLGA molecular chains on the Al2O3 surface to form a film;

[0074] Step 6: Repeat 10 TMA sprays and 1 PLGA spray for 5 cycles until the thickness reaches 2 nm.

[0075] In addition, it should be noted that the reason why the thermosensitive microspheres need to be pre-frozen to -50°C to form a glassy state is that -50°C is far below the eutectic point of the protective agent mixture (usually -30°C), ensuring that all water is instantly vitrified and no ice crystals are formed, which is beneficial to improving the survival rate of probiotics (experiments have shown that the survival rate of probiotics treated in a -50°C glassy state is as high as 89%, while that frozen at -20°C is only 60% to 70%. If it is higher than -50°C, the water will form sharp ice crystals, which will pierce the probiotic cell membrane or destroy the ZIF-8 core-shell structure (causing the survival rate to drop by more than 30%). In addition, if the glassy state is not formed, the water will evaporate through the liquid during supercritical CO2 drying, which will cause the probiotics to lyse due to the sudden change in osmotic pressure, the microspheres to shrink and deform, and the nanocoating (PLGA) to adhere unevenly, thereby reducing the protective effect.

[0076] 7. Mix the PLGA nano-coated probiotic freeze-dried powder with sterile saline at a mass ratio of 1:5 and shake gently to form a gel lubricant containing probiotics.

[0077] In this step, the freeze-dried powder already contains poloxamer thermosensitive gel, whose gelation behavior depends on the water content. At a ratio of 1:5, water penetration can make the gel reach the critical concentration of sol-gel transition (about 20% to 25% polymer), forming a uniform gel with moderate viscosity. The shear-thinning properties of the lubricant at this ratio match the viscoelasticity of the vaginal mucosa, which is not easy to lose and can reduce the friction coefficient (suitable for human application). In addition, the ratio of 1:5 can ensure that the protective agent (such as trehalose-hydroxybutyrate-glucan) in the freeze-dried powder is fully hydrated, which is conducive to restoring the metabolic activity of probiotics. If the ratio is too low (for example, 1:3), incomplete hydration will result, and the bacteria will be lysed due to the sudden increase in osmotic pressure; if the ratio is too high (for example, 1:7), the probiotic concentration will be diluted, thereby reducing the colonization effect.

[0078] Example 2:

[0079] 1. Mix 22% recombinant human mucin with sterile deionized water precooled at 4°C, stir magnetically (200 rpm to 300 rpm) in a container precooled at the same temperature, and allow to stand at 4°C for 1 hour to degas. Then, a 22% (w / w) mucin solution was prepared. The mucin solution was 3D printed to obtain a frozen mucin scaffold with a honeycomb structure and a pore size of 200 μm (such as Figure 3 shown);

[0080] 2. The frozen mucin scaffold was activated, and 7.5% Lactococcus lactis and 0.5% Fe3O4@SiO2 magnetic nanoparticles were mixed in a centrifuge tube. The mixture was incubated at 4°C with shaking for 30 minutes (rotation speed: 150 rpm to balance nanoparticle dispersion and bacterial survival rate; the centrifuge tube was tilted at 30 degrees to enhance liquid vortex and prevent particle sedimentation) to obtain magnetized probiotics. The magnetized probiotics were injected into the activated frozen mucin scaffold and a gradient magnetic field was applied for 5 minutes to achieve directional anchoring of the bacteria, thereby obtaining a magnetized probiotics@scaffold complex.

[0081] 3. Immerse the magnetized probiotics@scaffold complex in Zn 2+ The ZIF-8 framework was selectively grown around the magnetized bacteria, forming a coating layer with a thickness of 200 nm. At the same time, 0.4% CQDs-ZnO nanoparticles were added to the mixed solution to obtain a fluorescent tracer magnetized probiotics@ZIF-8 core-shell complex.

[0082] 4. Dissolve 5.8% EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex in citric acid buffer (concentration of 5%) at pH 5.0, immerse the fluorescent tracer-type magnetized probiotics@ZIF-8 core-shell complex in the citric acid buffer dissolved with EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex, and apply ultrasonic treatment (40KHz, 300W) for 10 minutes to induce nanofibers to grow vertically on the ZIF-8 surface through cavitation effect and load antimicrobial peptides to obtain EGCG / hyaluronic acid-LL-37 modified probiotic complex;

[0083] 5. Dissolve 15% poloxamer thermosensitive gel and LAP photoinitiator in 4°C deionized water to obtain a homogeneous gel solution. Disperse the EGCG / hyaluronic acid-LL-37 modified probiotic complex in the homogeneous gel solution and irradiate with blue light (intensity 10mW / cm 2 , time 30s) to trigger in situ photocrosslinking to obtain thermosensitive microspheres encapsulated with EGCG / hyaluronic acid-LL-37 modified probiotic complexes;

[0084] 6. Add 0.05% resveratrol nanoliposomes and 8% trehalose-hydroxybutyrate-dextran protective agent to the thermosensitive microspheres, prefreeze to -50°C to form a glassy state, dry the glassy thermosensitive microspheres using supercritical CO2, and grow a 2 nm thick PLGA (Poly (lactic-co-glycolic acid) copolymer) nanocoating on the surface of the dried thermosensitive microspheres using atomic layer deposition to obtain a PLGA nanocoated probiotic freeze-dried powder;

[0085] 7. Mix the PLGA nano-coated probiotic freeze-dried powder with sterile saline at a mass ratio of 1:5 and shake gently to form a gel lubricant containing probiotics.

[0086] It should be noted that all processing steps in Example 2 are the same as those in Example 1, and therefore will not be described in detail.

[0087] Example 3:

[0088] 1. Mix 20% recombinant human mucin with sterile deionized water precooled at 4°C, stir magnetically (200 rpm to 300 rpm) in a container precooled at the same temperature, and let it stand at 4°C for 1 hour to degas. Then, a 20% (w / w) mucin solution was prepared. The mucin solution was 3D printed to obtain a frozen mucin scaffold with a honeycomb structure of 100 μm pore size (such as Figure 4 shown);

[0089] 2. The frozen mucin scaffold was activated, and 7.0% Lactococcus lactis and 0.45% Fe3O4@SiO2 magnetic nanoparticles were mixed in a centrifuge tube. The mixture was incubated at 4°C for 30 minutes with shaking (rotation speed: 150 rpm to balance nanoparticle dispersion and bacterial survival rate; the centrifuge tube was tilted at 30 degrees to enhance liquid vortex and prevent particle sedimentation) to obtain magnetized probiotics. The magnetized probiotics were injected into the activated frozen mucin scaffold and a gradient magnetic field was applied for 5 minutes to achieve directional anchoring of the bacteria, thereby obtaining a magnetized probiotics@scaffold complex.

[0090] In this step, this embodiment improves the activation treatment of the frozen mucin scaffold based on the previous embodiment. The improved activation treatment specifically includes the following steps:

[0091] Step 1: Place frozen mucin in a Tris-HCl mineralization solution (pH 8.5) containing 10 mM CaCl2, 5 mM sodium β-glycerophosphate, 20 U / mL alkaline phosphatase (ALP), and 0.1% polydopamine (PDA) as a mineralization inducer, and then incubate at 37°C in a constant temperature shaker (50 rpm) for 6 hours.

[0092] In this step, PDA is cross-linked with mucin through phenolic hydroxyl groups, and at the same time, ALP catalyzes the decomposition of sodium β-glycerophosphate to release PO4 3- , and Ca 2+ Nanohydroxyapatite (nHA) is formed, thereby forming a biomimetic bone matrix coating on the surface of the scaffold.

[0093] Step 2: Wash the frozen mucin scaffold with sterile PBS to remove unreacted ions;

[0094] Step 3: The cleaned frozen mucin scaffold was immersed in an ethanol suspension containing 0.5% TiO2@MoS2 (titanium dioxide-molybdenum disulfide heterojunction) and sonicated for 10 minutes (40kHz).

[0095] In this step, TiO2@MoS2 has near-infrared (NIR) responsiveness and can trigger reactive oxygen species (ROS) sterilization through light irradiation (such as vaginal endoscopy-assisted phototherapy) in subsequent use.

[0096] Step 4: Use 405nm blue light (10mW / cm 2 ) irradiated the stent surface for 5 minutes, so that the nanoparticles were stably anchored on the stent surface through the PDA adhesion layer.

[0097] Step 5: The irradiated scaffold was immersed in acetic acid solution (pH 5.0) containing 1% Fe3O4@chitosan (CS) and incubated at 4°C with shaking for 2 h.

[0098] In this step, chitosan is bound to the scaffold through electrostatic adsorption, which can impart pH responsiveness (enhanced adhesion in the acidic environment of the vagina).

[0099] Step 6: 0.1% genipin was used to cross-link the incubated scaffold for 10 minutes to form a gradient structure of dense surface and porous interior on the scaffold.

[0100] 3. Immerse the magnetized probiotics@scaffold complex in Zn 2+ The ZIF-8 framework was selectively grown around the magnetized bacteria, forming a coating layer with a thickness of 200 nm. At the same time, 0.35% CQDs-ZnO nanoparticles were added to the mixed solution to obtain a fluorescent tracer magnetized probiotics@ZIF-8 core-shell complex.

[0101] 4. 5.2% EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex was dissolved in a citric acid buffer solution with a pH of 5.0 (concentration of 5%). The fluorescent tracer-type magnetized probiotics@ZIF-8 core-shell complex was immersed in the citric acid buffer solution containing the EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex. Ultrasonic treatment was applied (40 kHz, 300 W) for 10 minutes to induce the nanofibers to grow vertically on the ZIF-8 surface through the cavitation effect and load the antimicrobial peptides to obtain the EGCG / hyaluronic acid-LL-37 modified probiotic complex.

[0102] 5. Dissolve 14% poloxamer thermosensitive gel and LAP photoinitiator in 4°C deionized water to obtain a homogeneous gel solution. Disperse the EGCG / hyaluronic acid-LL-37 modified probiotic complex in the homogeneous gel solution and irradiate with blue light (intensity 10mW / cm 2 , time 30s) to trigger in situ photocrosslinking to obtain thermosensitive microspheres encapsulated with EGCG / hyaluronic acid-LL-37 modified probiotic complexes;

[0103] 6. Add 0.05% resveratrol nanoliposomes and 7.5% trehalose-hydroxybutyrate-dextran protective agent to the thermosensitive microspheres, pre-freeze to -50°C to form a glassy state, use supercritical CO2 to dry the glassy thermosensitive microspheres, and use atomic layer deposition to grow a 2 nm thick PLGA (Poly (lactic-co-glycolic acid, poly (lactic acid - glycolic acid copolymer)) nanocoating on the surface of the dried thermosensitive microspheres to obtain a PLGA nanocoating-protected probiotic freeze-dried powder (such as Figure 5 shown);

[0104] 7. Mix the PLGA nano-coated probiotic freeze-dried powder with sterile saline at a mass ratio of 1:5 and shake gently to form a gel lubricant containing probiotics.

[0105] The present application conducted a performance comparison of the gel lubricants prepared in Examples 1 to 3, and the comparison results are shown in Table 1.

[0106] Table 1

[0107]

[0108]

[0109]

[0110]

[0111] In Table 1, compared with Example 1 and Example 2, Example 3, through formula optimization and advanced technology, performs best in key indicators such as probiotic activity retention rate (89%), antibacterial effect (inhibition zone 14.2±1.5mm), lubricant viscosity (900±80mPa·s) and storage stability (activity retention rate 82% after 30 days). At the same time, it has low water content (0.9±0.1%) and high biocompatibility (cell survival rate 95±1%), comprehensively balancing functionality, stability and safety to meet practical application needs.

[0112] Furthermore, the present application compares the lubricant prepared by the present method with the existing lubricant, and the comparison results are shown in Table 2:

[0113] Table 2

[0114]

[0115]

[0116]

[0117] Based on Table 2, it can be seen that the present application significantly outperforms the existing technology in core indicators such as probiotic activity, antibacterial property, and stability by implementing a multi-level protection strategy (ZIF-8 core-shell, PLGA coating), functional composite design (antimicrobial peptide + quantum dots), and advanced preparation technology (3D printing scaffold, atomic layer deposition). While taking into account biocompatibility and multifunctionality, it is suitable for application scenarios with high requirements for microecological balance (such as gynecological care).

[0118] Figure 6 It is a macroscopic diagram of existing lubricants; Figure 8 This is a macroscopic diagram of a lubricant prepared by the method described in this application, provided in another embodiment of this application. Table 3 provides a comparative illustration of the two:

[0119] Table 3

[0120]

[0121] By comparison Figure 6 and Figure 8 It can be seen that the lubricant prepared by this technical solution exhibits better gelation and wall adhesion properties on a macro scale. Although traditional lubricants are also in a gel state, they have a loose structure and strong fluidity, and are easily ineffective when flushed with body fluids.

[0122] Figure 7 It is a microscopic diagram of existing lubricants; Figure 9 This is a microscopic diagram of a lubricant prepared based on the method described in this application, provided in another embodiment of this application. Table 4 provides a comparative illustration of the two:

[0123] Table 4

[0124]

[0125]

[0126] By comparison Figure 7 and Figure 9 It can be seen that at the microscopic level, the lubricant prepared by this technical solution exhibits a distinct three-dimensional porous structure, forming channels for probiotic "entrapment" and "controlled release," consistent with the theory of honeycomb cryo-scaffolds. Existing lubricants, on the other hand, exhibit a loose, disordered network structure, lacking carriers or functional structures, and are unable to effectively support the stable release or adhesion of probiotics.

[0127] In another exemplary embodiment, the present application also provides an application of a lubricant containing probiotics, wherein the lubricant is applied to women's health care.

[0128] In the present embodiment, the female vaginal microecology is a complex microbial system dominated by lactic acid bacteria (such as Lactobacillus crispatus, Lactobacillus gasseri, etc.), and its health state is mainly reflected in: Acidic environment (pH 3.8 to 4.5): maintained by lactic acid produced by lactic acid bacteria metabolism, it can inhibit the excessive reproduction of pathogenic microorganisms (such as Gardnerella associated with bacterial vaginosis and Candida albicans associated with fungal vaginitis). Microbial balance: Probiotics prevent the colonization of pathogenic bacteria by competitive occupation, secretion of antibacterial substances (such as hydrogen peroxide, bacteriocins) and immune regulation. When the microecology is unbalanced (such as increased pH and decreased lactic acid bacteria), the following adverse reactions are likely to occur:

[0129] Bacterial vaginosis (BV): Excessive proliferation of anaerobic bacteria such as Gardnerella, leading to increased discharge and foul odor.

[0130] Vaginal candidiasis (VVC): Fungal infection such as Candida albicans causes itching and curd-like discharge.

[0131] Risk of recurrent infection: An unbalanced microenvironment may increase the risk of HPV infection or affect treatment effectiveness.

[0132] Common methods for maintaining the vaginal microbiome include oral or topical probiotic supplementation, which can be problematic due to low survival rates (destroyed by gastric acid), difficulty colonizing, and the need for frequent use. Acidic gels (such as lactic acid preparations) only temporarily adjust pH and fail to replenish active probiotics or inhibit pathogens. Antibiotic therapy, while effective in the short term, can further disrupt the microbial balance and lead to recurrence.

[0133] This application achieves long-term, proactive microecological maintenance through the following methods:

[0134] (1) Precision delivery and colonization of probiotics

[0135] ZIF-8 core-shell protection:

[0136] Encapsulates Lactococcus lactis to prevent it from being washed away by vaginal secretions or enzymatically hydrolyzed.

[0137] It slowly releases live bacteria in an acidic environment, prolonging the duration of action (experimental data shows continuous release within 72 hours).

[0138] Honeycomb mucin scaffold:

[0139] Simulates the structure of the vaginal mucosa, provides attachment sites for probiotics, and enhances colonization efficiency (colonization rate increased by 50% vs. free probiotics).

[0140] (2) Dynamic pH regulation and pathogen inhibition

[0141] Lactococcus lactis metabolism:

[0142] Continuously produces lactic acid, maintains pH ≤ 4.5, and inhibits the growth of pathogens.

[0143] Antimicrobial peptide LL-37 and quantum dots work together:

[0144] Directly kills Gardnerella (inhibition zone diameter ≥ 14 mm) and destroys Candida albicans biofilm (biofilm removal rate 80%).

[0145] 3) Mucosal repair and anti-oxidation

[0146] EGCG / hyaluronic acid nanofibers:

[0147] Hyaluronic acid promotes mucosal hydration and repair, while EGCG's antioxidant properties reduce inflammatory damage (the cellular inflammatory factor IL-6 decreases by 60%).

[0148] Resveratrol Liposomal:

[0149] Alleviates oxidative stress (ROS levels reduced by 40%) and protects probiotics and epithelial cells.

[0150] The probiotic-containing lubricant prepared in this application breaks through the passive regulation limitations of traditional methods through the four-in-one mechanism of "protection-colonization-antibacterial-repair", providing a long-term, safe and multifunctional solution for the daily maintenance of vaginal microecology. It is especially suitable for women at high risk of microecological imbalance (such as those in the active sexual period, perimenopausal period, and those with low immunity).

[0151] The above embodiments are intended only to illustrate the technical concepts and features of this application. Their purpose is to enable those familiar with the art to understand the content of this application and implement it accordingly. They are not intended to limit the scope of protection of this application. Any equivalent changes or modifications made in accordance with the spirit of this application shall be included in the scope of protection of this application.

Claims

1. A method for preparing a lubricant containing probiotics, characterized in that: The preparation method comprises: Recombinant human mucin is mixed with sterile deionized water in a preset ratio to prepare a mucin solution, and the mucin solution is printed to obtain a frozen mucin scaffold; The frozen mucin scaffold was activated, and Lactococcus lactis and Fe3O4@SiO2 magnetic nanoparticles were mixed and incubated in a preset ratio to obtain magnetized probiotics. The magnetized probiotics were then injected into the activated frozen mucin scaffold to obtain a magnetized probiotics@scaffold complex. The magnetized probiotics@scaffold complex was immersed in Zn 2+ The mixture of ZIF-8 and CQDs was locally irradiated with ultraviolet light. At the same time, a preset ratio of CQDs-ZnO nanoparticles was added to the mixture to obtain a fluorescent tracer magnetized probiotics@ZIF-8 core-shell complex. The EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex of a preset ratio was dissolved in a citric acid buffer solution, and the fluorescent tracer-type magnetized probiotics@ZIF-8 core-shell complex was immersed in the citric acid buffer solution containing the EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex to obtain the nanofiber-modified magnetized probiotics@ZIF-8 complex; A preset ratio of poloxamer thermosensitive gel was dissolved in sterile deionized water to obtain a homogeneous gel solution, and the EGCG / hyaluronic acid-LL-37 modified probiotic complex was dispersed in the homogeneous gel solution. After irradiation with blue light, thermosensitive microspheres encapsulated with the EGCG / hyaluronic acid-LL-37 modified probiotic complex were obtained; Adding a preset ratio of resveratrol nanoliposomes and trehalose-hydroxybutyrate-dextran protective agent to the thermosensitive microspheres, pre-freezing and drying, and then depositing a nanocoating on the surface of the thermosensitive microspheres to obtain a probiotic freeze-dried powder protected by the nanocoating; The probiotic freeze-dried powder is mixed with sterile physiological saline to obtain a gel-like lubricant containing probiotics.

2. The method according to claim 1, characterized in that Before mixing the recombinant human mucin with sterile deionized water, pre-cool the sterile deionized water at 4°C.

3. The method according to claim 1, characterized in that The frozen mucin scaffold is a honeycomb structure with a pore size of 50 μm to 200 μm.

4. The method according to claim 1, wherein The method of adding a preset ratio of CQDs-ZnO nanoparticles to the mixed solution to obtain a fluorescent tracer type magnetized probiotics@ZIF-8 core-shell complex comprises the following steps: The mixed solution is subjected to ultrasonic treatment; Incubating the mixed solution after ultrasonic treatment; The incubated mixed solution is irradiated with ultraviolet light; placing the mixed solution after ultraviolet light irradiation in a magnetic field and allowing it to stand; After the mixed solution has been allowed to stand, the supernatant is removed by aspiration; The mixed solution after the supernatant was removed was freeze-dried.

5. The method according to claim 1, wherein After adding a preset ratio of resveratrol nanoliposomes and trehalose-hydroxybutyrate-dextran protective agent to the thermosensitive microspheres, they were pre-frozen to -50°C.

6. The method according to claim 1, characterized in that The pre-frozen thermosensitive microspheres were dried using supercritical CO2.

7. The method according to claim 1, characterized in that The method of depositing a nano coating on the surface of the dried temperature-sensitive microspheres comprises: Atomic layer deposition was used to grow PLGA nanocoating on the surface of the dried thermosensitive microspheres.

8. The method according to claim 1, characterized in that The method of mixing the probiotic freeze-dried powder with sterile physiological saline comprises: The probiotic freeze-dried powder was mixed with sterile saline at a mass ratio of 1:

5.

9. A lubricant containing probiotics, characterized in that: The raw materials used to prepare the lubricant and the mass percentage of each raw material are: Recombinant human mucin: 18% to 22%; Lactococcus lactis: 6.5% to 7.5%; Fe3O4@SiO2 magnetic nanoparticles: 0.4% to 0.5%; CQDs-ZnO nanoparticles: 0.3% to 0.4%; bacteriophage lytic enzyme@ZIF-8: 3.2% to 3.8%; EGCG / hyaluronic acid-LL-37 antibacterial and antioxidant nanofiber complex: 4.5% to 5.8%; Poloxamer thermosensitive gel: 13% to 15%; Trehalose-hydroxybutyrate-dextran complex protective agent: 7% to 8%; Resveratrol nanoliposomes: 0.05%; Sterile deionized water: balance.

10. An application of a lubricant containing probiotics, characterized in that: The lubricant is used in women's health care.

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