Lycopene-loaded targeted nano NK cell exosome gel and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a targeted nano NK cell exosome gel loaded with lycopene and application thereof, the targeted nano NK cell exosome gel loaded with lycopene is prepared from the following components in percentage by mass: 0.1 to 1 weight percent of lycopene oleoresin, 0.5 to 5 weight percent of NK cell exosome, 60 to 80 weight percent of gel system and the balance of ultrapure water; the lycopene oleoresin is prepared by the following steps: firstly, adding a eutectic solvent into a tomato fermentation extracting solution, carrying out ultrasonic extraction on lycopene, and then uniformly mixing the lycopene, sophorolipid and vegetable oil. According to the application, the NK cell exosome and the lycopene are organically matched, the NK cell exosome directly acts on the local part of the cervix uteri through the NK cell, and meanwhile, the lycopene can repair the damaged cervix uteri cell and restore the damaged cervix uteri cell to a normal structure, so that the purposes of removing viruses and preventing and treating cervical cancer are achieved.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological medicines, and more particularly to a lycopene-loaded targeted nano NK cell exosome gel and an application thereof. BACKGROUND

[0002] NK cells, as a kind of natural anti-virus cells without antigen presentation, play an important role in anti-tumor and virus clearance. NK cells have the ability to produce a large amount of interferon-γ (IFN-γ), and also achieve the purpose of virus clearance by lysing target cells.

[0003] Exosomes are endosome-derived lipid bilayer extracellular vesicles, belonging to small extracellular vesicles, with a size of dozens to hundreds of nanometers. They can load bioactive substances to target cells in specific tissues to achieve the purpose of targeted therapy. NK cell-derived exosomes (NK-Exos) are nanoscale vesicles (30-150 nm) secreted by NK cells, carrying bioactive substances such as proteins, nucleic acids (such as miRNA, mRNA) and lipids of the parent cell. These exosomes not only retain part of the immune function of NK cells, but also have unique biological characteristics, and play an important role in immune regulation, anti-tumor, anti-infection and tissue repair. For example, NK-Exos expresses activation receptors (such as NKG2D, perforin, granzyme, etc.) on the surface of NK cells, which can directly recognize and kill target cells without MHC restriction. NK-Exos can inhibit virus replication by delivering anti-virus proteins or miRNA, or directly kill infected cells.

[0004] Lycopene can promote the secretion of pro-inflammatory cytokines (such as IL-2 and IFN-γ), which are key signaling molecules for NK cell activation and proliferation. At the same time, lycopene can up-regulate the expression of NK cell surface activation receptors (such as NKG2D and NKp46), thereby improving the recognition and killing efficiency of target cells. Lycopene can also indirectly release the inhibition of NK cell function by inhibiting immune suppressor factors such as TGF-β.

[0005] Lactobacillus in the female vagina is crucial to maintaining the health of the female reproductive tract. Unhealthy lifestyles (such as smoking, staying up late, and alcohol abuse) can destroy the vaginal microenvironment, leading to a decrease in lactobacillus that secretes lactic acid and hydrogen peroxide, and the destruction of the normal environment of the vagina and cervix. Some anaerobes will overgrow, eventually leading to bacterial inflammation of the vagina and cervix, mycoplasma infection, and mycotic vaginitis. Patients with vaginal and cervical inflammation are more susceptible to HPV infection, and inflammation can exacerbate cervical lesions.

[0006] HPV is a recognized clear pathogenic factor that can cause cervical cancer, and early intervention can effectively prevent the occurrence of cervical cancer by removing HPV virus.

[0007] At present, the research on lycopene is mainly concentrated in the health product industry, and the main effects are antioxidant, immune enhancement, etc. There is no public report on the combination of lycopene and NK cell exosomes into a nano external use gel for the treatment of HPV infection. Based on the above statements, the application provides a lycopene-loaded targeted nano NK cell exosome gel and its application. SUMMARY

[0008] In order to solve the above technical problems, the application provides a lycopene-loaded targeted nano NK cell exosome gel and its application.

[0009] In a first aspect, the application provides a lycopene-loaded targeted nano NK cell exosome gel, which adopts the following technical solution: A lycopene-loaded targeted nano NK cell exosome gel, comprising the following components by mass fraction: lycopene oleoresin 0.1-1wt%, NK cell exosome 0.5-5wt%, gel system 60-80wt%, and the balance is ultrapure water. The lycopene oleoresin is prepared by adding a eutectic solvent to a tomato fermentation extract, ultrasonic extraction of lycopene, and then uniformly mixing lycopene, sophorolipid and vegetable oil.

[0010] Preferably, the mass ratio of the tomato fermentation extract and the eutectic solvent is 1:1-5; and the mass ratio of the lycopene, sophorolipid and vegetable oil is 1-5:15-20:75-85.

[0011] Preferably, the tomato fermentation liquid is prepared by the following method: The tomato juice is inoculated into a fermentation device containing Lactobacillus acidophilus and Lactobacillus rhamnosus, and after standing and fermentation, centrifugation, taking the supernatant, and filtration, a tomato fermentation extract is obtained.

[0012] Preferably, the mass ratio of the Lactobacillus acidophilus, Lactobacillus rhamnosus and tomato juice is 7-9:6-10:100.

[0013] Preferably, the tomato juice is obtained by crushing fresh tomatoes and mixing with deionized water.

[0014] Preferably, the fermentation conditions are: temperature 36.5-37.5℃, time 16-24h, and pH 3.50-3.90.

[0015] Preferably, the eutectic solvent comprises betaine and malic acid in a molar ratio of 1:1-4, and the water content is 10-15wt%.

[0016] Preferably, the ultrasonic extraction conditions are: temperature 35-45℃, time 10-20min, and power 300-400W.

[0017] Preferably, the plant oil is one or more of soybean oil, jatropha oil, palm oil, castor oil, coconut oil, sunflower oil, rapeseed oil, and olive oil.

[0018] Preferably, the NK cell exosome is prepared by the following method: S1, NK cell culture: fresh umbilical cord is made into a homogenate, filtered to remove debris, and cell suspension is collected; the cell suspension is cultured in a basic medium containing exosome-free serum; S2, separation and purification of NK cell exosome: the NK cell exosome is collected by differential centrifugation, and then washed with PBS to obtain high-purity NK cell exosome; S3, identification of NK cell exosome: the exosome is labeled with CD63 or NKG2D antibody by flow cytometry.

[0019] Preferably, the basic medium containing exosome-free serum includes exosome-free fetal bovine serum, IL-2, IL-15, and IL-12, and the cell density is (0.5-1) x 10 6 / mL.

[0020] Preferably, the basic medium containing exosome-free serum includes, per 100mL, exosome-free fetal bovine serum 10-15mL, IL-2 50-200 IU / mL, IL-15 10-50 ng / mL, and IL-12 1-10 ng / mL, and the cell density is (0.5-1) x 10 6 / mL.

[0021] Preferably, the gel system includes carboxymethyl chitosan, poloxamer, and cinnamaldehyde in a mass ratio of 1-10:20-30:6-15.

[0022] Preferably, the preparation method of the lycopene-loaded targeted nano NK cell exosome gel includes the following steps: The poloxamer is added to ultrapure water, and stirred overnight, and then the lycopene oleoresin, NK cell exosome, carboxymethyl chitosan, and cinnamaldehyde are added in sequence and stirred uniformly to obtain the lycopene-loaded targeted nano NK cell exosome gel.

[0023] In a second aspect, the application provides a use of a lycopene-loaded targeted nano NK cell exosome gel, which adopts the following technical solution: Application of a lycopene-loaded targeted nano NK cell exosome gel in the treatment of HPV infection.

[0024] In summary, the present application has the following beneficial effects: (1) The lycopene in the gel of the present application is obtained by first fermenting fresh tomatoes and then ultrasonic extraction with a deep eutectic solvent, and has the characteristics of high purity and strong activity; the gel system includes carboxymethyl chitosan, poloxamer and cinnamaldehyde, has antibacterial effect, good compatibility with vaginal mucosa, no irritation, and can also increase skin permeability, which helps deep sterilization; the NK cell exosome has higher biological stability and lower immunogenicity; the components in the gel of the present application interact with each other, can clear HPV virus, prevent the process of invading normal cells, and achieve the purpose of preventing the occurrence of cervical cancer.

[0025] (2) The lycopene in the gel of the present application can protect lactic acid bacteria from oxidative damage, balance the microenvironment of the vagina and cervix, prevent the occurrence of vaginitis and cervicitis caused by excessive reproduction of anaerobes, and directly kill anaerobes, mycoplasma and fungi that have reproduced excessively, and restore normal tissue structure; and can also effectively protect NK cells from oxidation, thereby exerting biological efficacy, and the NK cells can directly kill HPV viruses in the cervix, reduce viral load, and ultimately clear various types of HPV viruses to prevent and treat cervical malignancy.

[0026] (3) The gel of the present application can treat bacterial, mycoplasma and fungal vaginitis and cervicitis caused by various anaerobes, mycoplasma and fungi; effectively clear various genotypes of HPV (whether high-risk or low-risk) in the female cervix and male reproductive system, treat HPV-positive patients; and normal people can use it to maintain the balance of microbial flora in the reproductive tract, prevent inflammation and HPV infection. DETAILED DESCRIPTION

[0027] The present application is further described below in conjunction with examples.

[0028] Examples 1-3 provide a lycopene-loaded targeted nano NK cell exosome gel and a preparation method thereof.

[0029] Example 1: A lycopene-loaded targeted nano NK cell exosome gel, comprising the following components by mass fraction: lycopene oleoresin 0.1 wt%, NK cell exosome 0.5 wt%, gel system 60 wt%, and the balance is ultrapure water; The lycopene oleoresin is prepared by the following method: Step (1) Fresh tomatoes are crushed and mixed with deionized water (the mass ratio of fresh tomatoes to deionized water is 1:2) to obtain tomato juice; the tomato juice is inoculated into a fermentation device containing Lactobacillus acidophilus and Lactobacillus rhamnosus (the mass ratio of Lactobacillus acidophilus, Lactobacillus rhamnosus and tomato juice is 7:6:100), and then placed in an environment with a temperature of 36.5°C and a pH of 3.50 for 16 hours of static fermentation; after centrifugation, the supernatant is taken, filtered, and a tomato fermentation extract is obtained; Step (2) The tomato fermentation extract and the deep eutectic solvent (including betaine and malic acid with a molar ratio of 1:1, and containing 10wt% of water) are mixed in a mass ratio of 1:1, and the deep eutectic solvent is added to the tomato fermentation extract; ultrasonic extraction is performed at a temperature of 35°C and a power of 300W for 10 minutes to obtain lycopene; Step (3) The lycopene, sophorolipid and vegetable oil (soybean oil) are uniformly mixed in a mass ratio of 1:15:84 to obtain a lycopene oleoresin; The NK cell exosome is prepared by the following method: S1, NK cell culture: Freshly derived umbilical cords are made into homogenate, filtered to remove debris, and cell suspension is collected; the cell suspension is cultured in a basic medium containing no exosome serum (100mL, including no exosome fetal bovine serum 10mL, IL-2 50IU / mL, IL-15 50ng / mL, IL-12 1ng / mL, and cell density 0.5×10 6 / mL); S2, separation and purification of NK cell exosome: NK cell exosome is collected by differential centrifugation, and then washed with PBS to obtain high-purity NK cell exosome; S3, identification of NK cell exosome: exosome is labeled by CD63 or NKG2D antibody by flow cytometry; The gel system comprises carboxymethyl chitosan, poloxamer (poloxamer 188) and cinnamaldehyde in a mass ratio of 1:20:6; A preparation method of a lycopene-loaded targeted nano NK cell exosome gel, comprising the following steps: Poloxamer (poloxamer 188) is added to ultrapure water, stirred overnight, and then lycopene oleoresin, NK cell exosome, carboxymethyl chitosan and cinnamaldehyde are added in sequence and stirred uniformly to obtain a lycopene-loaded targeted nano NK cell exosome gel.

[0030] Example 2: A lycopene-loaded targeted nano NK cell exosome gel comprises the following components in mass fraction: lycopene oleoresin 0.5wt%, NK cell exosome 2.5wt%, gel system 70wt%, and the balance is ultrapure water. Lycopene oil resin, prepared by the following method: Step (1) Fresh tomatoes are crushed and mixed with deionized water (the mass ratio of fresh tomatoes to deionized water is 1:2) to obtain tomato juice; the tomato juice is inoculated into a fermentation device containing Lactobacillus acidophilus and Lactobacillus rhamnosus (wherein the mass ratio of Lactobacillus acidophilus, Lactobacillus rhamnosus and tomato juice is 8:9:100), and is allowed to stand for fermentation at a temperature of 37℃ and a pH of 3.75 for 20h, after which it is centrifuged, the supernatant is taken, filtered, and a tomato fermentation extract is obtained; Step (2) The tomato fermentation extract is added with a deep eutectic solvent (including betaine and malic acid at a molar ratio of 1:3, with a water content of 13wt%) at a mass ratio of 1:3, and is subjected to ultrasonic extraction at a temperature of 40℃ and a power of 350W for 15min to obtain lycopene; Step (3) The lycopene, sophorolipid and vegetable oil (coconut oil) are uniformly mixed at a mass ratio of 3:18:79 to obtain lycopene oil resin; NK cell exosome, prepared by the following method: S1, NK cell culture: Freshly derived umbilical cord is made into a homogenate, filtered to remove debris, and cell suspension is collected; the cell suspension is cultured in a basic medium containing no exosome serum (for 100mL, including no exosome fetal bovine serum 13mL, IL-2 100IU / mL, IL-15 30ng / mL, IL-12 5ng / mL, and a cell density of 0.8x10 6 / mL); S2, separation and purification of NK cell exosome: the NK cell exosome is collected by differential centrifugation, and then rinsed with PBS to obtain high-purity NK cell exosome; S3, identification of NK cell exosome: the exosome is labeled with CD63 or NKG2D antibody by flow cytometry; Gel system, including carboxymethyl chitosan, poloxamer (poloxamer 407) and cinnamaldehyde at a mass ratio of 5:25:11; A preparation method of a lycopene-loaded targeted nano NK cell exosome gel, comprising the following steps: Poloxamer (poloxamer 407) is added to ultrapure water, stirred overnight, and then lycopene oil resin, NK cell exosome, carboxymethyl chitosan and cinnamaldehyde are added in sequence and stirred uniformly to obtain a lycopene-loaded targeted nano NK cell exosome gel.

[0031] Example 3: A lycopene-loaded targeted nano NK cell exosome gel, comprising the following mass fractions of components: lycopene oleoresin 1 wt%, NK cell exosome 5 wt%, gel system 80 wt%, and the balance being ultrapure water; The lycopene oleoresin is prepared by the following method: Step (1) Fresh tomatoes are crushed and mixed with deionized water (the mass ratio of fresh tomatoes to deionized water is 1:2) to obtain tomato juice; the tomato juice is inoculated into a fermentation device containing Lactobacillus acidophilus and Lactobacillus rhamnosus (wherein the mass ratio of Lactobacillus acidophilus, Lactobacillus rhamnosus and tomato juice is 9:10:100), and is allowed to stand for fermentation at a temperature of 37.5°C and a pH of 3.90 for 24 hours, after which it is centrifuged, the supernatant is taken, filtered, and a tomato fermentation extract is obtained; Step (2) The tomato fermentation extract is added with a deep eutectic solvent (including betaine and malic acid at a molar ratio of 1:4, and containing 15 wt% of water) at a mass ratio of 1:5, and is subjected to ultrasonic extraction at a temperature of 45°C and a power of 400W for 20 minutes to obtain lycopene; Step (3) The lycopene, sophorolipid and vegetable oil (olive oil) are uniformly mixed at a mass ratio of 5:20:75 to obtain lycopene oleoresin; The NK cell exosome is prepared by the following method: S1. Culturing of NK cells: Freshly derived umbilical cords are made into homogenate, filtered to remove debris, and cell suspension is collected; the cell suspension is cultured in a basic medium containing no exosome serum (for 100 mL, including 15 mL of exosome-free fetal bovine serum, 200 IU / mL of IL-2, 10 ng / mL of IL-15, and 10 ng / mL of IL-12, with a cell density of 1×10 6 / mL); S2. Separation and purification of NK cell exosomes: NK cell exosomes are collected by differential centrifugation, and then rinsed with PBS to obtain high-purity NK cell exosomes; S3. Identification of NK cell exosomes: Exosomes are labeled with CD63 or NKG2D antibodies by flow cytometry; The gel system comprises carboxymethyl chitosan, poloxamer (poloxamer 188) and cinnamaldehyde at a mass ratio of 10:30:15; A preparation method of a lycopene-loaded targeted nano NK cell exosome gel, comprising the following steps: Poloxamer (poloxamer 188) is added to ultrapure water, and stirred overnight; then lycopene oleoresin, NK cell exosomes, carboxymethyl chitosan and cinnamaldehyde are added in sequence and stirred uniformly to obtain a lycopene-loaded targeted nano NK cell exosome gel.

[0032] To verify the comprehensive performance of the lycopene-loaded targeted nano NK cell exosome gel prepared in Embodiment 1-3 of the present application, the applicant sets up Comparative Examples 1-4, as follows: Comparative Example 1: Comparative Example 1 is different from Embodiment 1 only in that the same mass of lycopene oleoresin is used to replace the NK cell exosome to provide a lycopene-loaded gel and a preparation method thereof, as follows: A lycopene-loaded gel comprises the following components by mass fraction: lycopene oleoresin 0.6 wt%, a gel system 60 wt%, and the balance being ultrapure water; The lycopene oleoresin is prepared by the following method: Step (1) Fresh tomatoes are crushed and mixed with deionized water (the mass ratio of fresh tomatoes to deionized water is 1:2) to obtain tomato juice; the tomato juice is inoculated into a fermentation device containing Lactobacillus acidophilus and Lactobacillus rhamnosus (wherein the mass ratio of Lactobacillus acidophilus, Lactobacillus rhamnosus and tomato juice is 7:6:100), and is allowed to stand for fermentation at a temperature of 36.5°C and a pH of 3.50 for 16 hours, after which it is centrifuged, the supernatant is taken, filtered, and a tomato fermentation extract is obtained; Step (2) The tomato fermentation extract is added with a deep eutectic solvent (including betaine and malic acid at a molar ratio of 1:1, with a water content of 10 wt%) at a mass ratio of 1:1, and ultrasonic extraction is performed for 10 minutes at a temperature of 35°C and a power of 300W to obtain lycopene; Step (3) The lycopene, sophorolipid and vegetable oil (soybean oil) are uniformly mixed at a mass ratio of 1:15:84 to obtain lycopene oleoresin; The gel system comprises carboxymethyl chitosan, poloxamer (poloxamer 188) and cinnamaldehyde at a mass ratio of 1:20:6; A preparation method of a lycopene-loaded gel comprises the following steps: The poloxamer (poloxamer 188) is added to ultrapure water, and is fully stirred overnight, and then the lycopene oleoresin, carboxymethyl chitosan and cinnamaldehyde are added in sequence and stirred uniformly to obtain a lycopene-loaded gel.

[0033] Comparative Example 2: Comparative Example 2 is different from Embodiment 1 only in that the same mass of NK cell exosome is used to replace the lycopene oleoresin to provide a targeted nano NK cell exosome-containing gel and a preparation method thereof, as follows: A targeted nano NK cell exosome-containing gel comprises the following components by mass fraction: NK cell exosome 0.6 wt%, a gel system 60 wt%, and the balance being ultrapure water; NK cell exosome, prepared by the following method: S1, culture of NK cells: fresh umbilical cord is made into homogenate, filtered to remove debris, and cell suspension is collected; the cell suspension is cultured in a basic medium containing no exosome serum (10 mL of exosome-free fetal bovine serum, 100 IU / mL of IL-2, 50 ng / mL of IL-15, 1 ng / mL of IL-12, and a cell density of 0.5 x 10 6 / mL) per 100 mL; S2, separation and purification of NK cell exosome: NK cell exosome is collected by differential centrifugation, and then washed with PBS to obtain high-purity NK cell exosome; S3, identification of NK cell exosome: exosome is labeled by CD63 or NKG2D antibody by flow cytometry; A gel system comprising carboxymethyl chitosan, poloxamer (poloxamer 188), and cinnamyl aldehyde at a mass ratio of 1:20:6; A preparation method of a targeted nano NK cell exosome gel, comprising the following steps: Poloxamer (poloxamer 188) is added to ultrapure water, and stirred overnight, then NK cell exosome, carboxymethyl chitosan, and cinnamyl aldehyde are added in sequence, and stirred uniformly to obtain a targeted nano NK cell exosome gel.

[0034] Comparative Example 3: Comparative Example 3 is different from Example 1 only in that the preparation method of lycopene oleoresin is different, and a targeted nano NK cell exosome gel loaded with lycopene and a preparation method thereof are provided, which are as follows: A targeted nano NK cell exosome gel loaded with lycopene, comprising the following components by mass fraction: lycopene oleoresin 0.1 wt%, NK cell exosome 0.5 wt%, gel system 60 wt%, and the balance being ultrapure water; Lycopene oleoresin, prepared by the following method: Step (1) Fresh tomatoes are crushed and mixed with deionized water (mass ratio of fresh tomatoes to deionized water is 1:2) to obtain tomato juice; Step (2) Lycopene is obtained by adding a eutectic solvent (including betaine and malic acid at a molar ratio of 1:1, with a water content of 10 wt%) to the tomato fermentation extract at a mass ratio of 1:1 of tomato juice to eutectic solvent, and ultrasonic extraction is carried out at a temperature of 35°C and a power of 300W for 10 min; Step (3) Lycopene, sophorolipid, and vegetable oil (soybean oil) are uniformly mixed at a mass ratio of 1:15:84 to obtain lycopene oleoresin; NK cell exosome, prepared by the following method: S1, culture of NK cells: fresh umbilical cord is made into a homogenate, filtered to remove debris, and cell suspension is collected; the cell suspension is cultured in a basic medium containing no exosome serum (10 mL of exosome-free fetal bovine serum, 100 IU / mL of IL-2, 50 ng / mL of IL-15, and 1 ng / mL of IL-12 per 100 mL, with a cell density of 0.5 x 10 6 / mL); S2, separation and purification of NK cell exosome: NK cell exosome is collected by differential centrifugation, and then washed with PBS to obtain high-purity NK cell exosome; S3, identification of NK cell exosome: exosome is labeled by flow cytometry using CD63 or NKG2D antibody; A gel system comprising carboxymethyl chitosan, poloxamer (poloxamer 188), and cinnamyl aldehyde in a mass ratio of 1:20:6; A preparation method of a lycopene-loaded targeted nano NK cell exosome gel, comprising the following steps: Poloxamer (poloxamer 188) is added to ultrapure water, and stirred overnight, then lycopene oleoresin, NK cell exosome, carboxymethyl chitosan, and cinnamyl aldehyde are added in sequence and stirred uniformly to obtain a lycopene-loaded targeted nano NK cell exosome gel.

[0035] Comparative Example 4: Comparative Example 4 is the same as Example 1 except that the gel system is different, and a lycopene-loaded targeted nano NK cell exosome gel and a preparation method thereof are provided, as follows: A lycopene-loaded targeted nano NK cell exosome gel comprises the following components in mass fraction: lycopene oleoresin 0.1 wt%, NK cell exosome 0.5 wt%, gel system 60 wt%, and the balance is ultrapure water; Lycopene oleoresin, prepared by the following method: Step (1): fresh tomatoes are crushed and mixed with deionized water (mass ratio of fresh tomatoes to deionized water is 1:2) to obtain tomato juice; Step (2): lycopene is obtained by adding a deep eutectic solvent (including betaine and malic acid in a molar ratio of 1:1, with a water content of 10 wt%) to the tomato fermentation extract at a mass ratio of 1:1, and ultrasonic extraction is performed at a temperature of 35°C and a power of 300W for 10 min; Step (3): lycopene, sophorolipid, and vegetable oil (soybean oil) are uniformly mixed at a mass ratio of 1:15:84 to obtain lycopene oleoresin; NK cell exosome, prepared by the following method: S1, culture of NK cells: fresh umbilical cord is made into a homogenate, filtered to remove debris, and cell suspension is collected; the cell suspension is cultured in a basic medium containing no exosome serum (10 mL of exosome-free fetal bovine serum, 50 IU / mL of IL-2, 50 ng / mL of IL-15, and 1 ng / mL of IL-12 per 100 mL, and the cell density is 0.5 x 10 6 / mL); S2, separation and purification of NK cell exosome: the NK cell exosome is collected by differential centrifugation, and then washed with PBS to obtain high-purity NK cell exosome; S3, identification of NK cell exosome: the exosome is labeled by CD63 or NKG2D antibody by flow cytometry; The gel system is poloxamer (poloxamer 188); A preparation method of a lycopene-loaded targeted nano NK cell exosome gel, comprising the following steps: Poloxamer (poloxamer 188) is added to ultrapure water, and stirred overnight, and then lycopene oleoresin and NK cell exosome are added in sequence and stirred uniformly to obtain a lycopene-loaded targeted nano NK cell exosome gel.

[0036] Performance test The efficacy of the gels prepared in Examples 1-3 and Comparative Examples 1-4 of the present application is tested respectively.

[0037] Tester screening: 140 HPV-positive women are screened by fluorescent quantitative PCR, and genotyping is determined, and the testers are randomly divided into 7 groups, 20 people in each group, and are recorded as Examples 1-3 and Comparative Examples 1-4 respectively. On the basis of not distinguishing the genotypes, the gels prepared in Examples 1-3 and Comparative Examples 1-4 are respectively applied to the cervix every day using a dosing device, and the gel is applied uniformly and completely to the cervix while rotating, once a day, for 15-45 days. After 45 days of use, cervical exfoliative cells are collected, fluorescent quantitative PCR is used to detect HPV virus and determine whether it is negative, positive cases are determined for genotyping, and the negative conversion rate is recorded in Table 1 below.

[0038] Table 1 Negative conversion rate of testers in Examples 1-3 and Comparative Examples 1-4 ; As shown by the data in Table 1 above, the HPV negative conversion rate of the lycopene-loaded targeted nano NK cell exosome gel in Examples 1-3 of the present application is significantly higher than that in Comparative Examples 1-4.

[0039] As can be seen from the comparison between Comparative Example 1 and Comparative Examples 1 and 2, the organic combination of NK cell exosomes and lycopene can achieve an effect of 1+1>2, greatly improving the HPV negative conversion rate; once the cervix is damaged and a gap appears, HPV virus will infect host cells; NK cell exosomes carrying lycopene, due to the nanoscale particle size, will target damaged cervical cells, kill HPV-infected cervical cells, and lycopene can repair damaged cervical cells to restore normal structure.

[0040] As can be seen from the comparison between Comparative Example 1 and Comparative Example 3, the lycopene of the application is obtained by first fermenting fresh tomatoes and then ultrasonic extraction with a low eutectic solvent, has higher purity and stronger activity, can effectively protect NK cell exosomes from oxidation, make NK cell exosomes fully exert biological efficacy, and thus help to improve the HPV negative conversion rate.

[0041] As can be seen from the comparison between Comparative Example 1 and Comparative Example 4, the gel system of the application includes carboxymethyl chitosan, poloxamer and cinnamaldehyde, has excellent antibacterial effect, improves the compatibility of the gel with the vaginal mucosa, and makes NK cell exosomes and lycopene better exert efficacy.

[0042] Within the scope of the application, those skilled in the art can make modifications to the present embodiment without creative contribution after reading the present specification, but as long as it is within the scope of the claims of the present application, it is protected by the Patent Law.

Claims

1. A lycopene-loaded targeted nano-NK cell exosome hydrogel, characterized in that, Comprise the following components by mass fraction: lycopene oleoresin 0.1-1wt%, NK cell exosome 0.5-5wt%, gel system 60-80wt%, the balance is ultrapure water; The lycopene oleoresin is prepared by adding a eutectic solvent to a tomato fermentation extract, ultrasonic extraction of lycopene, and then uniformly mixing lycopene, sophorolipid and plant oil.

2. The lycopene-loaded targeted nano-NK cell exosome hydrogel of claim 1, characterized in that, The mass ratio of the tomato fermentation extract to the eutectic solvent is 1:1-5; the mass ratio of the lycopene, sophorolipid and plant oil is 1-5:15-20:75-85.

3. The lycopene-loaded targeted nano-NK cell exosome hydrogel of claim 1, characterized in that, The tomato fermentation liquid is prepared by the following method: The tomato juice is inoculated into a fermentation device containing Lactobacillus acidophilus and Lactobacillus rhamnosus, and after standing and fermentation, centrifugation, taking the supernatant, and filtration, a tomato fermentation extract is obtained.

4. The lycopene-loaded targeted nano-NK cell exosome hydrogel of claim 1, characterized in that, The eutectic solvent comprises betaine and malic acid in a molar ratio of 1:1-4, and the water content is 10-15wt%.

5. The lycopene-loaded targeted nano-NK cell exosome hydrogel of claim 1, characterized in that, The plant oil is one or more of soybean oil, jatropha oil, palm oil, castor oil, coconut oil, sunflower oil, rapeseed oil, and olive oil.

6. The lycopene-loaded targeted nano-NK cell exosome hydrogel of claim 1, wherein, The NK cell exosome is prepared by the following method: S1, culture of NK cells: fresh umbilical cord is made into a homogenate, filtered to remove debris, and cell suspension is collected; the cell suspension is cultured in a basic medium containing no exosome serum; S2, separation and purification of NK cell exosomes: NK cell exosomes are collected by differential centrifugation, and then washed with PBS to obtain high-purity NK cell exosomes; S3, identification of NK cell exosomes: exosomes are labeled with CD63 or NKG2D antibodies by flow cytometry.

7. The lycopene-loaded targeted nano-NK cell exosome hydrogel of claim 6, characterized in that, The exosome-free serum containing basal medium includes exosome-free fetal bovine serum, IL-2, IL-15, IL-12, and the cell density is (0.5-1) x 10 6 / mL.

8. The lycopene loaded targeted nano NK cell exosome hydrogel of claim 1, wherein, The gel system comprises carboxymethyl chitosan, poloxamer and cinnamaldehyde in a mass ratio of 1-10:20-30:6-15.

9. The lycopene-loaded targeted nano-NK cell exosome hydrogel of claim 8, characterized in that, The preparation method of the lycopene-loaded targeted nano NK cell exosome gel comprises the following steps: Poloxamer is added to ultrapure water, stirred overnight, and then lycopene oleoresin, NK cell exosomes, carboxymethyl chitosan and cinnamaldehyde are added in sequence and stirred uniformly to obtain the lycopene-loaded targeted nano NK cell exosome gel.

10. Use of the lycopene-loaded targeted nano NK cell exosome gel according to any one of claims 1-9 in the treatment of HPV infection.