An in-situ foam gel containing exosomes, its preparation method and application

Abstract

This invention belongs to the field of biomedical technology, and relates to an in-situ foam gel containing exosomes, its preparation method, and its application. The raw materials for preparing the in-situ foam gel include an oil phase, a surfactant, surfactant auxiliaries, a gel matrix, a bioactive agent, exosomes, and a solvent. The surfactant includes any one or a combination of at least two of Tween 80, vitamin E polyethylene glycol succinate, or lecithin. The in-situ foam gel containing exosomes of this invention can form porous foam in situ within the uterine cavity, cervix, and vagina, and is used to prevent postoperative intrauterine adhesions, promote endometrial regeneration, and treat bacterial and mixed vaginitis, especially suitable for repair treatment after hysteroscopic surgery or curettage.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an in-situ foam gel containing exosomes, its preparation method and application. Background Technology

[0002] Abortion surgery carries the risk of damage to the basal layer of the endometrium. The repair process is influenced by factors such as the degree and extent of damage, intrauterine inflammation, and hormonal fluctuations. Intrauterine injury often leads to destruction of the basal layer of the endometrium, triggering inflammatory responses, infection, and fibrin deposition, resulting in thin endometrium and intrauterine adhesions (IUA). Clinically, this manifests as reduced menstrual flow or even amenorrhea, and in severe cases, can lead to decreased fertility. The goal of endometrial repair is primarily to promote the restoration of the anatomical structure of the uterine cavity and the structure of the endometrial tissue (endometrial thickness ≥8 mm on the day of ovulation), enabling the endometrium to provide a suitable implantation environment for the fertilized egg and protecting fertility. Barrier materials physically isolate the damaged tissue surface, preventing abnormal adhesion to surrounding tissues during the critical healing period, thus preventing postoperative IUA. An ideal barrier material, in addition to physical isolation, should also meet the requirements of promoting endometrial repair, increasing intrauterine retention time, completely covering the critical period of endometrial repair, being completely degradable and absorbable within an appropriate period, and being safe and non-toxic. However, existing barrier materials (such as intrauterine devices and intrauterine support balloons) are mostly inert materials, which can only provide physical isolation and lack the function of promoting endometrial repair. Currently, commonly used anti-adhesion products in clinical practice include hyaluronic acid gel and carboxymethyl cellulose membranes, which have the following main shortcomings: (1) short retention time, which makes them easy to be washed away by intrauterine secretions; (2) no active treatment function, which cannot regulate inflammation or promote regeneration; (3) poor adhesion, which makes it difficult to cover irregular wounds.

[0003] Bacterial vaginosis (BV) is a vaginal infection caused by a decrease or disappearance of lactobacilli that normally produce hydrogen peroxide in the vagina, and an increase in facultative anaerobic bacteria and anaerobic bacteria. Mixed vaginitis is a vaginal inflammation caused by two or more pathogenic microorganisms, and is often accompanied by vulvar and cervical infections. The mainstream treatment is oral and topical antibiotics and antifungal drugs. Existing vaginal preparations (such as suppositories and gels) have the following problems: (1) uneven drug distribution and short retention time; (2) inability to deliver multiple types of antibacterial / antifungal drugs at the same time; (3) inability to cover vaginal folds.

[0004] While thermosensitive gels (such as poloxamer) have been reported in recent years, they lack the ability to spontaneously form foam structures, failing to achieve a high-coverage, low-pressure physical barrier. Furthermore, drugs with poor water solubility, such as nintedanib, dexamethasone, clotrimazole, and miconazole, exhibit low loading and rapid release in aqueous gels, leading to uneven drug distribution and uncontrollable concentration and duration of action. Therefore, there is an urgent need for a novel repair formulation for use in the uterine cavity, cervix, and vagina that combines self-emulsification, in-situ foam formation, and sustained-release function. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an in-situ foam gel containing exosomes, its preparation method, and its application.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an in-situ foam gel containing exosomes, wherein the raw materials for preparing the in-situ foam gel include an oil phase, a surfactant, a surfactant auxiliary, a gel matrix, a bioactive agent, exosomes, and a solvent;

[0008] The surfactant includes any one or a combination of at least two of Tween 80, vitamin E polyethylene glycol succinate, or lecithin;

[0009] The oil phase comprises medium-chain triglycerides;

[0010] The surfactant additives include diethylene glycol monoethyl ether and / or propylene glycol;

[0011] The gel matrix comprises any one or a combination of at least two of poloxamer, chitosan, or β-glycerophosphate disodium salt hydrate;

[0012] The bioactive agent includes sodium hyaluronate;

[0013] The exosomes include animal exosomes or plant exosomes;

[0014] The solvent includes water.

[0015] This invention provides a composition that can be injected via catheter and spontaneously emulsifies and forms a stable foam gel upon contact with bodily fluids in the uterine cavity, cervix, or vagina. Specifically, it involves injecting the liquid precursor into the uterine cavity or cervix using a 5Fr catheter, or injecting the liquid precursor into the vagina using a vaginal syringe. Upon contact with the endometrial layer at body temperature (37°C), a foam-like gel forms within 30 seconds, covering the wound. This achieves: (1) efficient coverage of wounds and folds with a relatively long retention time; (2) loading and sustained-release of drugs; (3) complete degradation within an appropriate period, safe and non-toxic inhibition of adhesions; (4) filling the extracellular matrix (ECM) of the endometrium, improving water content and capillary circulation, restoring the balance of extracellular flora, and promoting endometrial repair and regeneration. In this composition, vitamin E polyethylene glycol succinate plays a dual role as both a surfactant and an oil phase. When this substance is added as a surfactant, other oil phase components may not need to be added separately in specific formulations.

[0016] Preferably, the raw materials for preparing the in-situ foam gel include, by weight, 1-15 parts of oil phase, 1-10 parts of surfactant, 1-10 parts of surfactant auxiliaries, 0.5-20 parts of gel matrix, 0.5-2 parts of bioactive agent, and 43-96 parts of solvent, and by concentration, 10 parts of exosomes. 7 -10 12 per mL.

[0017] Preferably, the concentration of the exosomes is 10. 9 -10 11 per mL.

[0018] The mass fractions of the oil phase can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 parts, etc. The mass fractions of the surfactant can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 parts, etc. The mass fractions of the surfactant auxiliaries can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 parts, etc. The mass fractions of the gel matrix can be selected from 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 parts, etc. The quantities available are 12, 13, 14, 15, 16, 17, 18, 19, and 20 parts; the bioactive agent can be selected in quantities of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2 parts; the solvent can be selected in quantities of 43, 45, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 96 parts; and the exosome concentration can be selected as 1×10⁻⁶. 7 cells / mL, 2×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL, 2×10 8 cells / mL, 5×10 8 cells / mL, 1×10 9 cells / mL, 2×10 9 cells / mL, 5×10 9 cells / mL, 8×10 9 cells / mL, 1×10 10 cells / mL, 2×10 10 cells / mL, 5×10 10 cells / mL, 8×10 10 cells / mL, 1×10 11 cells / mL, 2×10 11 cells / mL, 5×10 11 cells / mL, 8×10 11 cells / mL, 1×10 12 The number of cells / mL, etc., can be selected from other specific point values ​​within the above range, which will not be elaborated here.

[0019] Preferably, the plant exosomes are prepared by a method comprising the following steps:

[0020] (1) Mix the plant with sodium chloride to obtain an exudate, and then desalinate the exudate to obtain a dialysis solution;

[0021] (2) After dialysis and filtration, centrifuge the supernatant and then perform ultracentrifugation to collect the precipitate.

[0022] Preferably, the mass ratio of the plant to sodium chloride is (1-4):1. The specific values ​​in (1-4) can be selected from 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0023] Preferably, the temperature at which the plant is mixed with sodium chloride is 2-8°C and the time is 2-24 h.

[0024] Temperatures can be selected from 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., and time can be selected from 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0025] Preferably, the mass percentage of sodium chloride in the dialysate is less than 0.01%, for example, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0026] Preferably, the filtration includes sequential filtration using mPVDF cup filters of 0.8μm, 0.45μm and 0.22μm.

[0027] Preferably, the centrifugal force is 1500-2500 g, the temperature is 2-8℃, and the time is 10-30 min.

[0028] The centrifugal force can be selected from 1500 g, 1800 g, 2000 g, 2200 g, 2500 g, etc., the temperature can be selected from 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., and the time can be selected from 10 min, 12 min, 15 min, 18 min, 20 min, 22 min, 25 min, 28 min, 30 min, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0029] Preferably, the centrifugal force of the ultracentrifugation is 80,000-120,000g, the temperature is 2-8℃, and the time is 2-4 h.

[0030] The centrifugal force can be selected from 80,000 g, 85,000 g, 90,000 g, 95,000 g, 100,000 g, 105,000 g, 110,000 g, 115,000 g, 120,000 g, etc., the temperature can be selected from 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., and the time can be selected from 2 h, 2.2 h, 2.5 h, 2.8 h, 3 h, 3.2 h, 3.5 h, 3.8 h, 4 h, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0031] Preferably, the plant exosomes include motherwort exosomes and / or potato exosomes.

[0032] Preferably, the animal exosomes include stem cell exosomes.

[0033] Preferably, the plant exosomes include drug-loaded plant exosomes.

[0034] This invention loads drugs into exosomes as active drug components in self-emulsifying foam gels, solving the problem of uneven distribution of poorly water-soluble drugs in gels, while enhancing the sustained-release effect of drugs and significantly prolonging the residence time of drugs at the site of action.

[0035] Preferably, the drug content in the drug-loaded plant exosomes is 0.1-20% by mass, more preferably 1-10%.

[0036] For example, 0.1%, 0.2%, 0.5%, 0.8%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0037] Preferably, the drug comprises any one or a combination of at least two of dexamethasone, metronidazole, or clotrimazole.

[0038] Preferably, the drug-loaded plant exosomes are prepared by a method comprising the following steps:

[0039] (1) Dissolve plant exosomes in buffer solution to obtain exosome buffer solution;

[0040] (2) Mix the exosome buffer with the drug, electroporate, and centrifuge to obtain the drug-loaded plant exosomes.

[0041] Preferably, the buffer solution comprises 200-400 mM sucrose, 200-300 mM glucose, and 150-250 nM trehalose in molar concentration, and 0.01-0.1% dipotassium ethylenediaminetetraacetate in mass percentage, and the solvent is PBS solution.

[0042] The molar concentration of sucrose can be selected from 200 mM, 220 mM, 250 mM, 280 mM, 300 mM, 320 mM, 350 mM, 380 mM, 400 mM, etc.; the molar concentration of glucose can be selected from 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, etc.; the molar concentration of trehalose can be selected from 150 nM, 180 nM, 200 nM, 220 nM, 250 nM, etc.; and the mass percentage of dipotassium ethylenediaminetetraacetate can be selected from 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.08%, 0.1%, etc. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0043] Preferably, the concentration of exosomes in the exosome buffer solution is 10. 9 -10 11 cells / mL, for example 1×10 9 cells / mL, 2×10 9 cells / mL, 3×10 9 cells / mL, 4×10 9 cells / mL, 5×10 9 cells / mL, 6×10 9 cells / mL, 7×10 9 cells / mL, 8×10 9 cells / mL, 9×10 9 cells / mL, 1×10 10 cells / mL, 2×10 10 cells / mL, 5×10 10 cells / mL, 8×10 10 cells / mL, 1×10 11 The number of cells / mL, etc., can be selected from other specific point values ​​within the above range, which will not be elaborated here.

[0044] Preferably, the voltage of the electric rotary switch is 200-300 V, the capacitance is 900-1200 μF, and the pulse square wave is 2-5 ms.

[0045] The voltage can be selected from 200 V, 220 V, 250 V, 280 V, 300 V, etc., and the capacitor can be selected from 900 μF, 920 μF, 950 μF, 980 μF, 1000 μF, 1020 μF, 1050 μF, 1080 μF, 1100 μF, 1120 μF, 1150 μF, 1180 μF, 1200 μF, etc. The square wave can be selected from 2 ms, 3 ms, 4 ms, 5 ms, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0046] Preferably, the centrifugation speed is 80,000-120,000 g, the temperature is 2-8℃, and the time is 2-4 h.

[0047] The rotation speed can be selected from 80,000 g, 85,000 g, 90,000 g, 95,000 g, 100,000 g, 105,000 g, 110,000 g, 115,000 g, 120,000 g, etc., the temperature can be selected from 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., and the time can be selected from 2h, 2.2h, 2.5h, 2.8h, 3h, 3.2h, 3.5h, 3.8h, 4h, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0048] In a second aspect, the present invention provides a method for preparing an in-situ foam gel containing exosomes according to the first aspect, the preparation method comprising:

[0049] (1) Mix the solvent with the gel matrix, and then mix with the bioactive agent to obtain a cold gel solution;

[0050] (2) Mix the oil phase, surfactant, surfactant auxiliaries, and exosomes to obtain a self-emulsifying phase;

[0051] (3) Add the self-emulsifying phase to the cold gel solution, stir, and filter to obtain the gel.

[0052] Preferably, the temperature for mixing the solvent and the gel matrix in step (1) is 2-8°C and the time is 1-16 h.

[0053] Temperatures can be selected from 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., and time can be selected from 1 h, 2 h, 5 h, 8 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0054] Preferably, the temperature for mixing with the bioactive agent is 2-8°C, and the time is 1-3 h.

[0055] Temperatures can be selected from 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., and time can be selected from 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, 2.8 h, 3 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0056] Preferably, the mixing temperature in step (2) is 2-8°C and the time is 1-3 hours.

[0057] Temperatures can be selected from 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc., and time can be selected from 1 h, 1.2 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, 2.8 h, 3 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0058] Preferably, the dropping rate of the self-emulsifying phase in step (3) is 0.5-1.5 mL / min, such as 0.5 mL / min, 0.8 mL / min, 1 mL / min, 1.2 mL / min, 1.5 mL / min, etc. Other specific point values ​​within the above range can be selected, and will not be elaborated here.

[0059] Preferably, the stirring temperature in step (3) is 2-8℃, the stirring speed is 100-300 rpm, and the stirring time is 20-40 min.

[0060] Temperatures can be selected from 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc.; rotation speeds can be selected from 100 rpm, 120 rpm, 150 rpm, 180 rpm, 200 rpm, 220 rpm, 250 rpm, 280 rpm, 300 rpm, etc.; and time can be selected from 20 min, 22 min, 25 min, 28 min, 30 min, 32 min, 35 min, 38 min, 40 min, etc. Other specific values ​​within the above ranges can also be selected, which will not be elaborated here.

[0061] Thirdly, the present invention provides the use of the exosome-containing in-situ foam gel according to the first aspect in the preparation of a drug for preventing postoperative intrauterine adhesions, a drug for promoting endometrial regeneration, a drug for treating bacterial and mixed vaginitis, or a drug for treating hemorrhoids.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] The gel prepared by this invention spontaneously forms a foam structure in situ, adhering to irregular wounds and reducing tissue pressure. Self-emulsification of the gel is achieved through the addition of a self-emulsifying agent, significantly improving the hydrophobic drug loading capacity and stability. The gel prepared by this invention is a thermosensitive gel, which can prolong the intrauterine retention time (>5 days). The gel possesses triple functions of anti-inflammatory, anti-adhesion, and regeneration promotion. The gel is a low-temperature liquid with low injection force, making it compatible with existing hysteroscopic instruments. Furthermore, all components are pharmaceutical excipients, ensuring high safety and allowing for the treatment of mixed-type vaginitis. It can simultaneously deliver hydrophobic / hydrophilic antibacterial drugs, and the foam structure enhances vaginal fold coverage, significantly superior to traditional suppositories or solutions. Detailed Implementation

[0064] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0065] Preparation Example 1

[0066] This preparation example provides a method for preparing Leonurus japonicus exosomes, the preparation method comprising:

[0067] (1) After washing the motherwort leaves, use distilled water to ultrasonically rinse until clear and colorless, soak for 1 hour, and disinfect by soaking in 70% ethanol aqueous solution for 5 minutes. Repeat three times, then take them out and put them in a sterilization oven with HEPA filtration system at 4°C and 8m / s to dry the surface until there are no visible water droplets.

[0068] (2) Gently mix 1 kg of Leonurus japonicus leaves with 350 g of sodium chloride, seal and store at 4°C for 12 hours to obtain exudate, desalt using a dialysis bag until the mass percentage of sodium chloride is <0.01% to obtain dialysate; filter the dialysate sequentially using 0.8 μm, 0.45 μm and 0.22 μm mPVDF cup filters to obtain filtrate, centrifuge the filtrate at 2,000 g for 30 min at 4°C, and take the supernatant; transfer the supernatant to a new centrifuge tube, centrifuge at 100,000 g for 120 min at 4°C, and take the precipitate.

[0069] Preparation Example 2

[0070] This preparation example provides a method for preparing Leonurus japonicus exosomes loaded with dexamethasone, the preparation method comprising:

[0071] (1) Preparation of Leonurus japonicus exosome buffer, wherein the buffer comprises, by molar concentration, 300 mM sucrose, 250 mM glucose, and 200 nM trehalose, and by mass percentage, 0.1% dipotassium ethylenediaminetetraacetate (K2-EDTA), and 10 tbsp of Leonurus japonicus exosomes prepared in Example 1. 11Cells / mL, solvent is 0.1×PBS;

[0072] (2) At 35℃, 5g of dexamethasone (Sigma-Aldrich, #D4902) was added to 10 mL of Leonurus japonicus exosome buffer and mixed for 30 min at 4℃ and 200 rpm. After mixing, samples were taken and examined under a 400× optical microscope. No aggregates larger than 20 μm were found.

[0073] (3) Set the parameters of the electroporator (Thermo Fisher Neon NxT) to 250 V, 1000 μF, square wave 3ms, pulse 1 time. Use the exosome extraction reagent (SBI# EXOquick-TC) to extract exosomes. After lysis, use liquid chromatography-evaporative light scattering detection (HPLC-ELSD) to quantify the dexamethasone released by exosome lysis. If the content is less than 20%, take out 2 mL of solution and pulse the remaining exosome solution once with a square wave of 1ms. Repeat this process and detection until the content of dexamethasone in the exosomes exceeds 20%. Dilute the mass percentage of dexamethasone in the exosomes to 20% by calculation. Collect the exosomes loaded with dexamethasone by ultracentrifugation at 4℃, 100,000×g for 2 hours.

[0074] Preparation Example 3

[0075] This preparation example provides a method for preparing potato exosomes, the method comprising:

[0076] (1) After washing the potatoes, rinse them three times with pure water, soak them for 1 hour, peel them, and soak them in 70% ethanol aqueous solution for 5 minutes. Repeat the sterilization process three times. Place them in a sterilization oven with a HEPA filter system and blow them at a temperature of 4℃ and a wind speed of 8 m / s until there are no visible water droplets left on the surface.

[0077] (2) At 4℃, using tools that have been sterilized in advance, 1 kg of potatoes were shredded into small particles of 3 mm × 3 mm × (3-10) mm, 350 g of sodium chloride was added, mixed evenly and then put into 50 mL centrifuge tubes, nitrogen gas was introduced and the mixture was left to stand for 2 hours, and then centrifuged at 2,000 g at 4℃ for 30 min. The supernatant was taken and desalted using a dialysis bag until the mass percentage of sodium chloride was <0.01% to obtain the dialysate. The dialysate was then filtered sequentially through 0.8 μm, 0.45 μm and 0.22 μm mPVDF cup filters. The filtrate was centrifuged at 100,000 g at 4℃ for 2 hours to precipitate potato exosomes.

[0078] Preparation Example 4

[0079] This preparation example provides a method for preparing potato exosomes loaded with metronidazole, the preparation method comprising:

[0080] (1) Preparation of potato exosome buffer, wherein the buffer comprises 300 mM sucrose, 250 mM glucose, and 200 nM trehalose in molar concentration, and 0.1% K2-EDTA in mass percentage, and 10g of potato exosomes prepared in Example 3. 11 Cells / mL, solvent is 0.1×PBS;

[0081] (2) At 35℃, add 6g of metronidazole (Hubei Hongyuan Pharmaceutical Technology Co., Ltd.) to 10mL of potato exosome buffer, mix for 30min at 4℃ and 200rpm using a mixer, and take samples for testing under a 400× optical microscope. No aggregates larger than 20μm were found.

[0082] (3) Set the parameters of the electroporator (Thermo Fisher Neon NxT) to 250 V, 1000 μF, square wave 3ms, and 2 pulses. Use the exosome extraction reagent (SBI# EXOquick-TC) to extract and lyse exosomes respectively. Use liquid chromatography-evaporative light scattering detection (HPLC-ELSD) to quantify the drug released by exosome lysis. If the content is less than 20%, take out 2 mL of solution and pulse the remaining exosome solution once with a square wave of 1ms. Repeat this process and detection until it is slightly more than 20%. Calculate the dilution to 20%. Centrifuge at 4℃, 4,000×g for 15 minutes to remove the residual drug that is not completely dissolved. Take the supernatant and centrifuge at 4℃, 100,000×g for 2 hours to collect the exosomes loaded with metronidazole.

[0083] Preparation Example 5

[0084] This preparation example provides a method for preparing potato exosomes loaded with clotrimazole. The only difference between this method and preparation example 4 is that step (2) is "at 35°C, add 6g of clotrimazole (Changzhou Aide Biotechnology Co., Ltd.) to 10mL of potato exosome buffer and mix for 30min at 4°C and 200rpm using a mixer. After mixing, take a sample and examine it under a 400× optical microscope to detect no aggregates larger than 20μm". Other operations remain unchanged.

[0085] Preparation Example 6

[0086] This preparation example provides a method for preparing potato exosomes loaded with sodium hyaluronate. The only difference between this method and preparation example 4 is that step (2) is "at 35°C, add 6g of sodium hyaluronate (Bloomage Biotech, #NanoHA) to 10mL of potato exosome buffer and mix for 30min at 4°C and 200rpm using a mixer. After mixing, take a sample and examine it under a 400× optical microscope to detect no aggregates larger than 20μm". Other operations remain unchanged.

[0087] Example 1

[0088] This embodiment provides an in-situ foam gel, the preparation method of which includes:

[0089] (1) Cool 70g of water for injection to 4°C, add 18g of poloxamer 407 (Sigma-Aldrich, #16758), and stir at 4°C for 12 h; add 2g of cross-linked sodium hyaluronate (Bloomage Biotech, #HA-CL-M), and continue stirring for 2 h to prepare a cold gel solution.

[0090] (2) 6g of MCT (medium-chain triglycerides, Zhejiang Wumart Biotechnology Co., Ltd., #GTCC), 3g of Tween 80 (Beyotime Biotechnology, #ST2789), 2g of Transcutol (diethylene glycol monoethyl ether, French Gaffars, #Transcutol P) and 1g of Leonurus japonicus exosomes loaded with dexamethasone prepared in Preparation Example 2 were mixed at 4°C for 2 h to obtain an emulsion phase;

[0091] (3) The self-emulsifying phase was added dropwise to the cold gel at a rate of 1 mL / min and stirred at 300 rpm for 30 min at 4℃; filtered through a 0.22 μm filter membrane, dispensed, and stored at 4℃.

[0092] Example 2

[0093] This embodiment provides an in-situ foam gel, the preparation method of which includes:

[0094] (1) Cool 68g of water for injection to 4°C, add 2g of chitosan (Sigma-Aldrich, #448877), 10g of 10% Pluronic® F-68 solution (Poloxamer 188, Shanghai Maokang Biotechnology Co., Ltd., #MS4305) and stir at 4°C for 1h; add 8g of sodium β-glycerophosphate (Sigma-Aldrich, #50020), continue stirring at 4°C for 1h, add 2g of cross-linked sodium hyaluronate (Bloomage Biotechnology, #HA-CL-M), and continue stirring for 2h to prepare a cold gel solution.

[0095] (2) Mix 8g of vitamin E TPGS (vitamin E polyethylene glycol succinate, Sigma-Aldrich, #57668), 2g of 1,2-propanediol (SKC Korea, #pharmaceutical grade 1,2-propanediol) and 2g of stem cell culture medium exosomes (Suzhou Yuanyi Stem Cell Technology Co., Ltd.) at 4℃ for 2 h to obtain an emulsion phase;

[0096] (3) The self-emulsifying phase was added dropwise to the cold gel at a rate of 1 mL / min and stirred at 300 rpm for 30 min at 4℃; filtered through a 0.22 μm filter membrane, dispensed, and stored at 4℃.

[0097] Example 3

[0098] This embodiment provides an in-situ foam gel, the preparation method of which includes:

[0099] (1) Cool 70g of water for injection to 4°C, add 18g of poloxamer 407 (Sigma-Aldrich, #16758), and stir at 4°C for 12 h; add 2g of cross-linked sodium hyaluronate (Bloomage Biotech, #HA-CL-M), and continue stirring for 2 h to prepare a cold gel solution.

[0100] (2) Mix 6g MCT, 3g Tween 80, 2g Transcutol, 3g potato exosomes loaded with metronidazole prepared in Example 4, and 1g potato exosomes loaded with clotrimazole prepared in Example 5 at 4°C for 2 h to obtain a self-emulsifying phase.

[0101] (3) The self-emulsifying phase was added dropwise to the cold gel at a rate of 1 mL / min, stirred at 300 rpm for 30 min at 4°C, and the pH was adjusted to 4.4 with glacial acetic acid; filtered through a 0.22 μm filter membrane, dispensed, and stored at 4°C.

[0102] Example 4

[0103] This embodiment provides an in-situ foam gel, which differs from Example 1 only in that step (2) is to "mix 6g MCT, 3g Tween 80, 2g Transcutol and 0.8g of Leonurus japonicus exosomes prepared in Example 1 with 0.2g dexamethasone at 4°C for 2 h to obtain an emulsion phase", while other operations remain unchanged.

[0104] Example 5

[0105] This embodiment provides an in-situ foam gel, which differs from Example 3 only in that step (2) is to "mix 6g MCT, 3g Tween 80, 2g Transcutol and 3.2g potato exosomes prepared in Example 3, 0.6g metronidazole and 0.2g clotrimazole at 4°C for 2 h to obtain an emulsion phase", while other operations remain unchanged.

[0106] Example 6

[0107] This embodiment provides an in-situ foam gel, which differs from Example 3 only in that step (2) is "mixing 6g MCT, 3g Tween 80, 2g Transcutol, and 1g of potato exosomes loaded with clotrimazole in Preparation Example 5 at 4°C for 2 h to obtain a self-emulsifying phase", while other operations remain unchanged.

[0108] Example 7

[0109] This embodiment provides an in-situ foam gel, which differs from Example 1 only in that step (2) is "mixing 6g MCT, 3g Tween 80 and 2g Transcutol at 4℃ for 2 h to obtain an emulsion phase", while other operations remain unchanged.

[0110] Example 8

[0111] This embodiment provides an in-situ foam gel, which differs from Example 3 only in that step (2) is "mixing 6g MCT, 3g Tween 80 and 2g Transcutol at 4℃ for 2 h to obtain a self-emulsifying phase", while other operations remain unchanged.

[0112] Example 9

[0113] This embodiment provides a self-emulsifying liquid, which is prepared by mixing the in-situ foam gel prepared in Example 7 and the motherwort exosomes prepared in Example 1 at 4°C for 2 h. The concentrations of each component in the resulting emulsion, except for dexamethasone, are consistent with those of the in-situ foam gel in Example 1.

[0114] Example 10

[0115] This embodiment provides a self-emulsifying liquid, which is prepared by mixing the in-situ foam gel prepared in Example 8 and the potato exosomes prepared in Example 3 at 4°C for 2 h. The concentration of each component in the resulting emulsion, except for clotrimazole, is the same as that of the in-situ foam gel in Example 6.

[0116] Example 11

[0117] This embodiment provides an in-situ foam gel, which differs from Example 2 only in that step (2) is "to mix 8g of vitamin E TPGS (vitamin E polyethylene glycol succinate, Sigma-Aldrich, #57668), 2g of 1,2-propanediol (SKC Korea, #pharmaceutical grade 1,2-propanediol) and 2g of potato exosomes loaded with sodium hyaluronate prepared in Example 6 to obtain an emulsion phase", while other operations remain unchanged.

[0118] Comparative Example 1

[0119] This comparative example provides an in-situ foam gel, which differs from Example 1 only in that step (2) is "mixing 6g MCT, 3g sodium cocoyl glycinate (Shanghai Yuanye Biotechnology Co., Ltd.), 2g Transcutol and 1g of Leonurus japonicus exosomes loaded with dexamethasone prepared in Example 2 at 4°C for 2 h to obtain an emulsion phase", while other operations remain unchanged.

[0120] Comparative Example 2

[0121] This embodiment provides an in-situ foam gel, which differs from Example 1 only in that step (2) is to "mix 6g MCT, 3g polyoxyethylene lauryl ether (Brij-35, Dalian Meilun Biotechnology Co., Ltd.), 2g Transcutol and 1g of Leonurus japonicus exosomes loaded with dexamethasone prepared in Example 2 at 4°C for 2 h to obtain an emulsion phase", while other operations remain unchanged.

[0122] Comparative Example 3

[0123] This embodiment provides an in-situ foam gel, which differs from Example 1 only in that step (2) is to "mix 6g MCT, 3g Tween 80, 2g glycerol (glycerol, Hunan Guangshengyuan Pharmaceutical Technology Co., Ltd., #medical grade pure glycerol) and 1g of Leonurus japonicus exosomes loaded with dexamethasone prepared in Example 2 at 4°C for 2 h to obtain an emulsion phase", while other operations remain unchanged.

[0124] Comparative Example 4

[0125] This embodiment provides an in-situ foam gel, which differs from Example 1 only in that step (2) is to "mix 6g MCT, 3g Tween 80, 2g PEG 600 (Shanghai Test, #30150628) and 1g of Leonurus japonicus exosomes loaded with dexamethasone prepared in Example 2 at 4°C for 2 h to obtain an emulsion phase", while other operations remain unchanged.

[0126] Test Example 1

[0127] The test items and test methods are shown in Table 1.

[0128] Table 1

[0129]

[0130] The formulation and process of this invention meet all the requirements for the use of gels in the Chinese Pharmacopoeia, indicating that this formulation can be used for clinical purposes. Example 2 did not involve drug encapsulation; therefore, there are no data on drug encapsulation rate or in vitro release.

[0131] Comparative Examples 2 and 4 remained liquid aqueous solutions when heated to 37°C, while Comparative Example 3 showed stratification after standing for 4 hours, and neither could form a gel.

[0132] Test Example 2

[0133] Thirty c57bl / 6 mice (weighing 16.9g-19.3g) were randomly divided into 5 groups (n=6 / group). Bilateral cornual mechanical curettage was performed. Immediately after the procedure, the following were injected into the uterine cavity: (A) Group: 10μL of foam gel from Example 2 of this invention, without other drugs; (B) Group: 10μL of foam gel from Example 11 of this invention, without other drugs; (C) Group: 10μL of commercially available cross-linked sodium hyaluronate gel for uterine cavity (Changzhou Bairuiji, #Gongankang); (D) Group: 10μL of 2% stem cell culture medium exosome solution; (E) Group: 10μL of PBS. Histological analysis was performed 14 days after the procedure. The degree of intrauterine adhesions was scored according to the "Chinese Expert Consensus on the Diagnosis and Treatment of Intrauterine Adhesions with Integrated Traditional Chinese and Western Medicine (2024 Edition)". The score was based on the adhesion range index, with higher scores indicating more severe adhesions.

[0134] Table 2

[0135]

[0136] The results of intrauterine adhesion scoring, endometrial thickness, and gland count are shown in Table 2. Table 2 shows that, in terms of endometrial thickness, commercially available cross-linked sodium hyaluronate gel alone only improves adhesions and does not have a significant effect on endometrial repair. The exosome-containing foam gel of this invention, compared to commercially available cross-linked sodium hyaluronate gel, has a comparable effect in improving intrauterine adhesions, but produces a significantly thicker endometrium and a greater number of glands, indicating a significant improvement in functional repair of the intrauterine endometrium. Comparing groups A and B, the plant exosomes loaded with sodium hyaluronate showed a slightly better repair effect than the stem cell exosome group. Comparing groups D and E, which are stem cell exosome aqueous solutions without foam gel formulation, there was no significant difference compared to the negative control PBS.

[0137] Test Example 3

[0138] Fifteen c57bl / 6 mice (weighing 16.8g-18.1g) were randomly divided into 5 groups (n=3 / group). Four experimental groups underwent bilateral cornual mechanical curettage, while one control group received no intervention. Immediately after the procedure, the experimental groups received the following intrauterine injections: (A) Group: 10μL of foam gel from Example 1; (B) Group: 10μL of foam gel from Example 4; (C) Group: 10μL of foam gel from Comparative Example 1; (D) Group: 10μL of PBS. Histological analysis was performed 14 days postoperatively, and intrauterine adhesions were scored according to the "Chinese Expert Consensus on the Diagnosis and Treatment of Intrauterine Adhesions Using Integrated Traditional Chinese and Western Medicine (2024 Edition)".

[0139] Table 3

[0140]

[0141] The results of intrauterine adhesion scoring, endometrial thickness, and glandular count are shown in Table 3. Table 3 shows that animal modeling was successful compared to the control group and group D. Comparing groups A, B, and C, it can be seen that the self-emulsifying foam gel loaded with dexamethasone into exosomes has a significantly better therapeutic effect on intrauterine adhesions than the self-emulsifying foam gel simply mixed with exosomes and dexamethasone. Comparing groups A and C, after loading dexamethasone, the self-emulsifying gel using sodium cocoyl glycinate as a surfactant showed significantly worse endometrial repair than the self-emulsifying gel using Tween 80 as a surfactant. Combined with the experimental results of Comparative Examples 2, 3, and 4, which failed to form a gel, it is evident that different surfactants and co-surfactants have a significant impact on the structure and function of exosome self-emulsifying gels.

[0142] Test Example 4

[0143] Twenty-four c57bl / 6 mice (weighing 18.1g-20.4g) were randomly divided into four groups (n=6 / group) and vaginally inoculated with 5μL of 1×10 8 CFU / mL Candida albicans (ATCC 10231, Ningbo Mingzhou Biotechnology Co., Ltd., #B310989), 5μL 1×10 9 CFU / mL Gardnerella vaginalis (ATCC 14019, Ningbo Mingzhou Biotechnology Co., Ltd., #B280029). (A) Group: Example 3 foam gel, 10 μL per dose; (B) Group: Example 5 foam gel, 10 μL per dose; (C) Group: 1.4 g of clotrimazole vaginal tablets (Bayer Pharmaceuticals, Germany, Canesten, each tablet contains 0.5 g clotrimazole) powder (containing 0.05 g clotrimazole), 1.6 g of metronidazole vaginal effervescent tablets (Shanghai Xinyi Pharmaceutical Factory, each tablet contains 0.2 g metronidazole) powder (containing 0.02 g metronidazole), mixed evenly with 100 μL of intrauterine cross-linked sodium hyaluronate gel (Changzhou Bairuiji, #Gongankang), 10 μL per dose; (D) Group: No infection, no medication.

[0144] The first dose was administered 24 hours post-surgery, once daily for 5 consecutive days. On day 7, the pathogen load in vaginal secretions was detected by real-time fluorescence PCR (Suzhou Chuanglan Biotechnology, Vaginitis Triple Detection Kit, National Medical Device Registration Certificate 20253401378).

[0145] Table 4

[0146]

[0147] The loading results of Candida and Gardnerella vaginalis are shown in Table 4. Animal model data show that, compared with groups A, B, and C, the foam gel of this invention can effectively inhibit the growth of vaginal pathogens, achieve simultaneous delivery of hydrophobic and hydrophilic antibacterial drugs, and enhance the coverage of vaginal folds with its foam structure. Compared with traditional suppositories or solutions, it has a significantly stronger anti-infective effect. Compared with groups A and B, the self-emulsifying foam gel loaded with drugs into exosomes has a significantly better anti-infective effect than the self-emulsifying foam gel mixed with exosomes and drugs. It can be seen that the drug loading into exosomes further enhances the improved dispersion effect of the self-emulsifying gel.

[0148] Test Example 5

[0149] Based on the guidelines for solubility measurement in Chinese Pharmacopoeia 2025 edition 9023, the solubility determination method was used to evaluate the solubility of dexamethasone and clotrimazole in aqueous solutions, self-emulsified solutions, exosome suspensions, and exosome self-emulsified suspensions.

[0150] The assessment was divided into a dexamethasone group and a clotrimazole group.

[0151] In the dexamethasone group, the self-emulsifying liquid is the emulsifying liquid described in Example 9, the exosome suspension is the Leonurus japonicus exosome suspension loaded with dexamethasone described in Preparation Example 2, and the exosome self-emulsifying suspension is the in-situ foam gel suspension described in Example 1.

[0152] In the clotrimazole group, the self-emulsifying liquid is the emulsifying liquid described in Example 10, the exosome suspension is the Leonurus japonicus exosome suspension loaded with clotrimazole described in Preparation Example 5, and the exosome self-emulsifying suspension is the in-situ foam gel suspension described in Example 6.

[0153] Test method:

[0154] (1) Three replicates were prepared for each group. Excess dexamethasone or clotrimazole powder was added to 1 mL of water and self-emulsifying solution respectively until obvious precipitation appeared after standing, resulting in supersaturated drug aqueous solution and self-emulsifying solution. Exosomes containing dexamethasone or clotrimazole and in situ foam gel containing dexamethasone or clotrimazole were resuspended in 1 mL of water and self-emulsifying solution respectively with a pipette until the exosome precipitate could not be dispersed by pipetting, resulting in supersaturated exosome suspension and exosome self-emulsifying suspension.

[0155] (2) Shake at 37℃ and 100 rpm for 24 hours;

[0156] (3) Centrifuge at 23℃, 4,000g for 15 minutes, take the supernatant, and add lysis agent to the group containing exosomes at a volume ratio of 1:1;

[0157] (4) Determine the concentration of dexamethasone or clotrimazole in the supernatant.

[0158] Table 5

[0159]

[0160] The concentrations of dexamethasone or clotrimazole in the supernatant are shown in Table 5. As can be seen from Table 5, the solubility of the exosome self-emulsifying suspension group of this invention is significantly higher than the sum of the individual effects of the self-emulsifying liquid group and the exosome suspension group, indicating a synergistic solubilizing effect between the two solubilizing methods, rather than a simple additive effect. Furthermore, clotrimazole has lower solubility and a greater solubilization factor, demonstrating the significant advantage of this invention for highly hydrophobic drugs.

[0161] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An in situ foaming gel comprising exosomes, characterized in that, The raw materials for preparing the in-situ foam gel include an oil phase, surfactants, surfactant auxiliaries, a gel matrix, bioactive agents, exosomes, and solvents. The surfactant includes any one or a combination of at least two of Tween 80, vitamin E polyethylene glycol succinate, or lecithin; The oil phase comprises medium-chain triglycerides; The surfactant additives include diethylene glycol monoethyl ether and / or propylene glycol; The gel matrix comprises any one or a combination of at least two of poloxamer, chitosan, or β-glycerophosphate disodium salt hydrate; The bioactive agent includes sodium hyaluronate; The exosomes include animal exosomes or plant exosomes; The solvent includes water.

2. The exosome-containing in situ foaming gel according to claim 1, characterized in that, The raw materials for preparing the in-situ foam gel include, in mass parts, 1-15 parts of oil phase, 1-10 parts of surfactant, 1-10 parts of surfactant aid, 0.5-20 parts of gel matrix, 0.5-2 parts of bioactive agent, and 43-96 parts of solvent, and include, in concentration, 10 7 -10 12 exosomes / mL.

3. The exosome-containing in situ foaming gel of claim 1, wherein, The plant exosomes include drug-loaded plant exosomes; The drug includes any one or a combination of at least two of dexamethasone, metronidazole, or clotrimazole; The drug-loaded plant exosomes contain 0.1-20% by mass of the drug.

4. The in-situ foam gel containing exosomes according to claim 3, characterized in that, The drug-loaded plant exosomes were prepared by a method comprising the following steps: (1) Dissolve plant exosomes in buffer solution to obtain exosome buffer solution; (2) Mix the exosome buffer with the drug, electroporate, and centrifuge to obtain the drug-loaded plant exosomes.

5. The in-situ foam gel containing exosomes according to claim 4, characterized in that, The buffer solution comprises 200-400 mM sucrose, 200-300 mM glucose, and 150-250 nM trehalose in molar concentration, and 0.01-0.1% dipotassium ethylenediaminetetraacetate in mass percentage, and the solvent is PBS solution; The concentration of the exosomes in the exosome buffer is 10 9 -10 11 individuals / mL; The voltage of the electric rotary switch is 200-300 V, the capacitance is 900-1200 μF, and the pulse square wave is 2-5 ms.

6. The method for preparing an in-situ foam gel containing exosomes according to any one of claims 1-5, characterized in that, The preparation method includes: (1) Mix the solvent with the gel matrix, and then mix with the bioactive agent to obtain a cold gel solution; (2) Mix the oil phase, surfactant, surfactant auxiliaries, and exosomes to obtain a self-emulsifying phase; (3) Add the self-emulsifying phase to the cold gel solution, stir, and filter to obtain the gel.

7. The preparation method according to claim 6, characterized in that, In step (1), the solvent and gel matrix are mixed at a temperature of 2-8°C for 1-16 hours. The temperature for mixing with the bioactive agent is 2-8℃, and the time is 1-3 h; The mixing temperature in step (2) is 2-8℃, and the time is 1-3h; The dropping rate of the self-emulsifying phase in step (3) is 0.5-1.5 mL / min; The stirring temperature in step (3) is 2-8℃, the stirring speed is 100-300 rpm, and the stirring time is 20-40 min.

8. The use of the exosome-containing in-situ foam gel according to any one of claims 1-5 in the preparation of a drug for preventing postoperative intrauterine adhesions, a drug for promoting endometrial regeneration, a drug for treating bacterial and mixed vaginitis, or a drug for treating hemorrhoids.

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