Anti-human papilloma virus gel and preparation method thereof
By combining NK cell exosomes into lyophilized powder and gel, the problem of NK cell exosomes inactivation at room temperature is solved, effective HPV virus killing and vaginal mucosal repair are achieved, avoiding drug resistance and high costs.
Patent Information
- Application Number
- CN202510250153.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-04
- Publication Date
- 2025-07-11
AI Technical Summary
The existing anti-HPV gels rely on physical isolation methods and have long efficacy and high cost. NK cell exosomes cannot exist stably at room temperature, and cells cannot be directly used to make gynecological gel products.
The NK cell exosomes are made into lyophilized powder and gels composed of carbomer, β-glucan, triethanolamine, methyl nepaper and propyl nepaperate. The lyophilized powder is packaged separately from sterile water and mixed when used.
Maintain NK cell exosome activity to the greatest extent, promote wound healing, eliminate viruses, improve vaginal flora environment, avoid drug resistance, and improve resistance.
Smart Images
Figure CN120284847A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of anti-human papillomavirus, and particularly relates to an anti-human papillomavirus gel and a preparation method thereof. Background Art
[0002] Human papillomavirus (HPV) is a spherical DNA virus that widely exists in nature. It has humans as the only host, is highly resistant to drying and can be stored for a long time. Currently, only more than 150 known HPV subtypes can be divided into low-risk types and high-risk types according to the severity of the diseases they cause.
[0003] Leukocytes (English name: leukocyte, white blood cell, abbreviation: WBC), formerly known as white blood cells, are a type of cell in the blood. Leukocytes are also commonly referred to as immune cells, and their main function is to phagocytose bacteria and defend against diseases. Natural killer cells (NK) are a type of leukocyte, which are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity reactions and autoimmune diseases in some cases. NK cells in peripheral blood are separated by magnetic beads and amplified with a suitable culture medium, then the culture is collected, and after filtration and impurity removal, ultrafiltration and purification are carried out to obtain NK cell exosomes. The main components of the product obtained by this method are exosomes and some large-molecular-weight proteins, etc. Its uses mainly include enhancing immunity, anti-inflammatory, antibacterial, etc., and there are reports indicating that NK cells also have significant effects on improving skin conditions.
[0004] Although NK cells, as immune cells, also show powerful functions in the beauty field, cells cannot be directly used to make gynecological gel products, not only because cells cannot survive for a long time outside a specific environment, but also because cells cannot directly act on human skin. The effective components secreted by cells, such as exosomes and cytokines, also cannot exist stably at room temperature and will inactivate and lose their efficacy in a short time. Currently, many anti-HPV gels have been successively launched on the market, but most rely on physical isolation methods supplemented with components that promote wound healing, and have a long treatment course and high cost. Summary of the Invention
[0005] In order to solve the problems in the prior art, the present invention provides an anti-human papillomavirus gel and a preparation method thereof, and the NK cell exosomes are made into freeze-dried powder to maximize the purpose of maintaining the activity of the effective components of NK cell exosomes.
[0006] The present invention solves its technical problems by adopting the following technical solutions:
[0007] The object of the present invention is to provide an anti-human papillomavirus gel, which comprises NK cell supernatant exosomes and a gel. The gel comprises carbomer, β-glucan, triethanolamine, methylparaben and propylparaben.
[0008] Further, the concentration of carbomer is 1% - 10%, the concentration of β-glucan is 0.5 - 1.5%, the concentration of triethanolamine is 0.2 - 1.5%, the concentration of methylparaben is 0.1 - 0.5%, and the concentration of propylparaben is 0.1 - 0.5%.
[0009] Further, the NK cell exosomes are purified with a 100 kDa ultrafiltration tube, frozen at -80 - -45 °C and then dried to form a lyophilized powder.
[0010] Further, the total protein content of the NK cell exosomes is 20 - 100 ng / 100 g of the gel.
[0011] A preparation method of an anti-human papillomavirus gel comprises the following steps:
[0012] S1. Preparation of NK cell exosomes: Centrifuge to separate NK cells and the supernatant medium, collect the medium for standby; after the collected NK cell precipitate is processed, mix it with the supernatant, then perform preliminary impurity removal and detection. After passing the detection, the NK cell culture is immediately purified and concentrated with a 100 kDa ultrafiltration tube and the total protein content is detected;
[0013] S2. Preparation of the lyophilized powder: Aliquot the NK cell exosomes prepared in S1 into ampoules, 5 - 10 mL per bottle; after freezing, form a lyophilized powder, and extract one bottle of the lyophilized powder to detect whether the total protein content meets the expectation. Those that meet the expectation are aliquoted into the lyophilized powder chamber, and the freeze-drying chamber containing the lyophilized powder is stored temporarily in the refrigerator;
[0014] S3: Preparation of the gel: Weigh carbomer, soak it in ultrapure water to dissolve and then sterilize; weigh β-glucan to dissolve, then add methylparaben and propylparaben to β-glucan and mix evenly. Mix the carbomer, triethanolamine and β-glucan mixed solution evenly, add sterile water to make up the volume to form a gel, and fill it into a gel tube.
[0015] Further, in S1, the NK cells are treated with a PBS buffer containing 0.3% Triton X-100 for 10 minutes and then mixed with the supernatant, and are preliminarily purified with a 0.22 μm filter, and endotoxin and mycoplasma are detected. After passing the detection, the NK cell culture is immediately purified and concentrated with a 100 kDa ultrafiltration tube and the total protein content of the collected exosomes is detected.
[0016] Further, in S2, a sterile water chamber filled with sterile water is also prepared. The sterile water chamber is filled with 100 μL of sterile water, and the sterile water chamber is detachably connected to the freeze-drying chamber and then stored temporarily in the refrigerator at 4 °C.
[0017] Furthermore, the preparation method further includes perfusion of the sterile water in the sterile water tank into the freeze-drying chamber, shaking the freeze-drying chamber to dissolve the freeze-dried powder of NK cell exosomes, and then perfusion of the dissolved freeze-dried powder of NK cell exosomes into the gel tube filled with gel, and using a syringe to assist in drug administration.
[0018] Compared with the prior art, the beneficial technical effects of the present invention are as follows:
[0019] The present invention provides an anti-human papillomavirus gel containing NK cell exosomes. Making the NK cell exosomes into freeze-dried powder can maximize the preservation of the activity of its active ingredients. β-glucan also has a good effect in promoting wound healing. The combination of NK cell exosomes and β-glucan repairs the damaged vaginal mucosa and eliminates the virus at the same time, improving the types of microbial flora. Carbomer forms a barrier on the surface of the vaginal mucosa, hindering the contact of pathogenic microorganisms. The present invention repairs the damaged vaginal mucosa while killing the virus, rather than relying on the method of killing and removing epidermal cells to achieve the treatment purpose. The present invention uses bioactive substances as raw materials, does not produce drug resistance, improves the vaginal flora environment, enhances resistance, and avoids re-infection with the virus.
[0020] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly, it can be implemented according to the content of the specification. And in order to make the above content, its purpose, features and advantages of the present invention more obvious and understandable, the following specifically illustrates the specific embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 It is a schematic structural diagram of the packaging and drug applicator of the anti-human papillomavirus gel containing NK cell exosomes of the present invention.
[0022] Figure 2 It is a graph showing the results of the efficacy verification of the gel containing NK cell exosomes of the present invention against HPV6, 11, 16, 18, 31, 33, 45, 52 and 58 pseudoviruses.
[0023] Figure 3 It is a graph showing the results of the efficacy verification of whether the gel containing NK cell exosomes of the present invention directly acts on HPV6, 11, 16, 18, 31, 33, 45, 52 and 58 pseudoviruses.
[0024] Figure 4 It is a graph showing the results of the efficacy verification of whether the gel containing NK cell exosomes of the present invention promotes epidermal cell growth and wound healing.
[0025] 1 - gel tube, 2 - freeze-drying chamber, 3 - sterile water tank, 4 - syringe, 5 - pull ring. DETAILED DESCRIPTION OF THE EMBODIMENTS
[0026] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to illustrate and explain the present invention exemplarily, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection aimed by the present invention.
[0027] In addition, unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention can be obtained through market purchase or prepared by existing methods.
[0028] Example 1
[0029] An anti-human papillomavirus gel containing NK cell exosomes, comprising freeze-dried powder of NK cell exosomes and a gel. The gel components are carbomer, β-glucan, triethanolamine, methylparaben, and propylparaben. The concentration of carbomer is 1%, the concentration of β-glucan is 1.5%, the concentration of triethanolamine is 1.5%, the concentration of methylparaben is 0.5%, and the concentration of propylparaben is 0.5%.
[0030] The freeze-dried powder of NK cell exosomes is the freeze-dried powder prepared from NK cell exosomes. The anti-human papillomavirus gel also includes sterile water for dissolving the freeze-dried powder of NK cell exosomes. The NK cell exosomes are quantified by the total protein concentration, and the concentration is 100 ng / 100 g gel.
[0031] Example 2
[0032] An anti-human papillomavirus gel containing NK cell exosomes, comprising freeze-dried powder of NK cell exosomes and a gel. The gel components are carbomer, β-glucan, triethanolamine, methylparaben, and propylparaben. The concentration of carbomer is 10%, the concentration of β-glucan is 0.5%, the concentration of triethanolamine is 0.2%, the concentration of methylparaben is 0.1%, and the concentration of propylparaben is 0.1%.
[0033] The freeze-dried powder of NK cell exosomes is the freeze-dried powder prepared from NK cell exosomes. The anti-human papillomavirus gel also includes sterile water for dissolving the freeze-dried powder of NK cell exosomes. The NK cell exosomes are quantified by the total protein concentration, and the concentration is 20 ng / 100 g gel.
[0034] Example 3
[0035] An anti-human papillomavirus gel containing NK cell exosomes, comprising freeze-dried powder of NK cell exosomes and a gel. The gel components are carbomer, β-glucan, triethanolamine, methylparaben, and propylparaben. The concentration of carbomer is 5%, the concentration of β-glucan is 1%, the concentration of triethanolamine is 0.8%, the concentration of methylparaben is 0.3%, and the concentration of propylparaben is 0.3%.
[0036] The NK cell supernatant exosome is a freeze-dried powder prepared from NK cell exosomes. The anti-human papillomavirus gel also includes sterile water for dissolving the freeze-dried powder of NK cell exosomes. The NK cell exosomes are quantified by the total protein concentration, and the concentration is 60 ng / 100 g gel.
[0037] Example 4
[0038] An applicator for anti-human papillomavirus gel, as Figure 1 shown, includes a gel tube 1 filled with gel, a freeze-drying chamber 2 filled with freeze-dried powder of NK cell exosomes, a sterile water chamber 3 filled with sterile water, and a pusher 4. The sterile water chamber 3 is detachably connected to the freeze-drying chamber 2. One end of the sterile water chamber 3 is provided with a pull ring 5. One end of the freeze-drying chamber 2 and one end of the gel tube 1 both have detachable lids. The end of the freeze-drying chamber 2 with the lid is sealed with an aluminum film. The end of the gel tube 1 with the lid may be provided with a sharp part for piercing the aluminum film at the bottom of the freeze-drying chamber 2.
[0039] Usage method of the applicator:
[0040] 1. Remove the pull ring 5, press the sterile water chamber 3 to pour the sterile water into the freeze-dried powder area in the freeze-drying chamber 2, and gently shake the freeze-drying chamber 2 to dissolve the freeze-dried powder of NK cell exosomes.
[0041] 2. Remove the lids of the freeze-drying chamber 2 and the gel tube 1, invert the freeze-drying chamber 2, and use the sharp part of the gel tube 1 to pierce the aluminum film at the bottom of the freeze-drying chamber 2 and fasten it.
[0042] 3. Shake to make the liquid part pour into the gel, and use the pusher 4 to assist in applying the medicine.
[0043] The NK cell exosomes are rich in various active ingredients, but they are not easy to store in daily life. Placing them at room temperature for a long time in a liquid state will cause the inactivation of the active ingredients and the weakening of the efficacy. In order to make up for this shortcoming, the present invention selects to make the cell exosomes into freeze-dried powder, which is separately packaged in different containers from the sterile water. The two different containers are connected together, and pressing can release the sterile water to dissolve the freeze-dried powder. After the freeze-dried powder part and the gel part are buckled together, the reconstituted NK cell exosomes are poured into the gel, shaken and mixed evenly, and then the medicine is applied using the pusher 4.
[0044] Example 5
[0045] Production process of the anti-HPV gel containing NK cell exosomes:
[0046] 1 Weighing and batching, raw and auxiliary materials treatment
[0047] 1.1 Preparation of NK cell exosomes
[0048] 1.1.1 Centrifuge and separate NK cells and supernatant medium, and collect the supernatant for standby.
[0049] 1.1.2 The collected NK cells were treated with PBS buffer containing 0.3% Triton X-100 for 10 minutes and then mixed with the supernatant.
[0050] 1.1.3 Detect the content of cytokines such as human interferon and epidermal growth factor (EGF) in NK cell culture, and test the content of endotoxin and mycoplasma. The endotoxin content is required to be lower than 2.5EU / mL, and mycoplasma should not be detected.
[0051] After passing the test in 1.1.3, the NK cell culture was initially purified using a 0.22 μm filter.
[0052] 1.1.4 After the impurities are removed, the liquid is purified and concentrated using a 100 kDa ultrafiltration tube, and the collected exosomes are washed 3-5 times with PBS buffer, and the total protein content of the collected exosomes is detected.
[0053] 1.2 Preparation of freeze-dried powder
[0054] 1.2.1. Collect NK cell exosomes according to the batch production instructions and add 10 mL to each ampoule.
[0055] 1.2.2. Place it at -80-45℃ overnight. After it is completely frozen, use a freeze-drying machine to make freeze-dried powder. Take out one bottle and test whether the total protein content meets expectations.
[0056] 1.2.4. According to the total protein content and production requirements, the exosome freeze-dried powder that meets the expected requirements is packaged into the freeze-dried powder compartment 2, and 100 μL of sterile water is filled into the sterile water compartment. The sterile water compartment 3 and the freeze-dried compartment 2 are spliced and temporarily stored in a refrigerated place at 4°C.
[0057] 1.3 Gel preparation (taking 1000g gel as an example)
[0058] 1.3.1. According to the batch production instructions, weigh a sufficient amount of carbomer (concentration of 10%), soak it in ultrapure water, let it stand overnight, and sterilize it after all the carbomer powder is dissolved.
[0059] 1.3.2. According to the batch production instructions, weigh 5g of β-glucan and dissolve it. Add 0.2% of methylparaben and propylparaben respectively according to the final volume and mix well.
[0060] 1.3.3. Mix the carbomer, triethanolamine and β-glucan mixed solution, add sterile water to make up to volume, the final concentration of carbomer is 8%, and the final volume is 1000g.
[0061] 1.3.4. Refrigerate or store at room temperature temporarily.
[0062] 1.3.5. Filling: Take gel tube 1 and dispense 3g of gel.
[0063] 2. Seal
[0064] Seal each part with a lid or aluminum foil, and seal it after bagging each set.
[0065] Test Example 1: Test the anti-HPV virus efficacy of the NK cell exosome gel of the present invention using HEK-293T cells and HPV pseudovirus
[0066] 1. When the confluence of 293T cells reaches 85%, digest and collect the cell precipitate. After collecting the cell precipitate, resuspend it with a small amount of medium without antibiotics. Take 10 μL of the cell suspension for counting, and dilute the cell suspension to 7.5×10 3 / 100 μL according to the counting result, and inoculate the cells into a 96-well plate at 100 μL of cell suspension per well.
[0067] 2. Dilute the pseudovirus into a suitable working solution, and the NK cell exosome gel containing the NK cell exosome is diluted in gradients; mix the pseudovirus and the diluted NK cell exosome gel in equal volumes, incubate at room temperature for 30 min, or incubate in a 4°C refrigerator for 1 h.
[0068] 3. After incubation, add the pseudovirus-containing NK cell exosome gel mixture to the cells and place them in a carbon dioxide incubator.
[0069] 4. After 72 h, take out and observe the GFP expression, take pictures of appropriate fields of view, analyze the fluorescence area with Image J software, and perform statistical analysis with GraphPad Prism 8. (Refer to Appendix Figure 2 )
[0070] Test Example 2: Whether the NK cell exosome gel of the present invention directly acts on the HPV virus
[0071] 1. When the confluence of 293T cells reaches 85%, digest and collect the cell precipitate. After collecting the cell precipitate, resuspend it with a small amount of medium without antibiotics. Take 10 μL of the cell suspension for counting, and dilute the cell suspension to 7.5×10 3 / 100 μL according to the counting result, and inoculate the cells into a 96-well plate at 100 μL of cell suspension per well.
[0072] 2. Dilute the pseudovirus into a suitable working solution, and the NK cell exosome gel containing the NK cell exosome is diluted in gradients; mix the pseudovirus and the diluted NK cell exosome in equal volumes, incubate at room temperature for 30 min, or incubate in a 4°C refrigerator for 1 h. As a control group, do not incubate and directly add it to the adherent 293T cells.
[0073] 3. After incubation, add the pseudovirus-containing NK cell exosome gel mixture to the cells and place them in a carbon dioxide incubator.
[0074] After 4.72 h, take out and observe the GFP expression, take pictures of appropriate fields of view, analyze the fluorescence area with Image J software, and perform statistical analysis with GraphPad Prism 8. (Refer to the appendix Figure 3 )
[0075] Conclusion: The NK cell exosome-containing gel of the present invention has a killing effect on HPV virus, and there is no need to secrete immune factors after cell absorption to exert antiviral function. It can directly inactivate the virus in vitro.
[0076] Experimental Example 3: Whether the NK cell exosome-containing gel of the present invention promotes epidermal cell growth and scratch healing
[0077] 1. When the confluence of Hucat cells reaches 85%, digest and collect the cell precipitate, and divide the cells onto 6-well plates with the same number of cells in each well.
[0078] 2. When the confluence of cells in the 6-well plate reaches 70%, starve the cells with a medium without fetal bovine serum for 12 hours.
[0079] 3. After the starvation treatment, use a 10 μL pipette tip to draw lines, 3 lines in each well; wash away the floating cells with PBS buffer, add exosome-containing media with different total protein concentrations, take pictures and record as 0. After taking pictures, put them into the incubator.
[0080] 4. After 16 hours, take out and observe and take pictures, record as 16, compare the pictures taken at 0 and 16 hours, analyze the scratch width with Image J software, and perform statistical analysis with GraphPad Prism 8. (Refer to the appendix Figure 4 )
[0081] Conclusion: The NK cell exosome-containing gel of the present invention can promote the proliferation of epidermal cells Hucat and scratch healing.
[0082] The serial numbers of the above-mentioned embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.
[0083] The above describes the embodiments of the present invention in conjunction with the drawings, but the present invention is not limited to the above specific embodiments. The above specific embodiments are only illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the claims of the present invention. These all belong to the protection scope of the present invention.
Claims
1. An anti-human papillomavirus gel, characterized in that, It includes freeze-dried powder of NK cell exosomes and gel. The gel includes carbomer, β-glucan, triethanolamine, methylparaben and propylparaben.
2. The anti-human papillomavirus gel according to claim 1, wherein: The concentration of carbomer is 1% - 10%, the concentration of β-glucan is 0.5 - 1.5%, the concentration of triethanolamine is 0.2 - 1.5%, the concentration of methylparaben is 0.1 - 0.5%, and the concentration of propylparaben is 0.1 - 0.5%.
3. The anti-human papillomavirus gel according to claim 1, wherein: The freeze-dried powder of NK cell exosomes is made from exosomes purified from the culture supernatant of NK cells. The anti-human papillomavirus gel also includes sterile water for dissolving the freeze-dried powder of NK cell exosomes.
4. The anti-human papillomavirus gel according to claim 3, wherein: The total protein content of NK cell exosomes is 20 - 100 ng / 100 g gel.
5. The preparation method of a human papillomavirus-resistant gel according to any one of claims 1-4, characterized in that: It includes the following steps: S1. Preparation of NK cell exosomes: Centrifuge to separate NK cells and the supernatant medium, and collect the medium for later use; the collected NK cells are processed and then mixed with the supernatant, followed by preliminary impurity removal and detection. After passing the detection, the NK cell culture is immediately purified and concentrated, and the total protein content of the collected exosomes is detected. S2. Preparation of freeze-dried powder: The NK cell exosomes prepared in S1 are dispensed into bottles, and after freezing, they are made into freeze-dried powder. The qualified freeze-dried powder is taken and dispensed into the freeze-dried powder chamber, and the freeze-dried chamber containing the freeze-dried powder is stored refrigerated temporarily. S3: Preparation of gel: Weigh carbomer, soak it in ultrapure water, dissolve it and sterilize it; weigh β-glucan and dissolve it, then add methylparaben and propylparaben to β-glucan and mix well. Mix the mixed solution of carbomer, triethanolamine and β-glucan well, add sterile water to make up the volume to form a gel, and fill it into the gel tube.
6. The preparation method of an anti-human papillomavirus gel according to claim 5, characterized in that: In S1, the NK cells are treated with PBS buffer containing 0.3% Triton X-100 for 10 minutes and then mixed with the supernatant, and are preliminarily purified with a 0.22 μm filter, and the endotoxin and mycoplasma are detected. After passing the detection, the NK cell culture is immediately purified and concentrated with a 100 kDa ultrafiltration tube and the total protein content of the collected exosomes is detected.
7. The preparation method of an anti-human papillomavirus gel according to claim 5, wherein: In S2, a sterile water chamber filled with sterile water is also prepared. The sterile water chamber is filled with 100 μL of sterile water. After the sterile water chamber is detachably connected to the freeze-dried chamber, it is stored refrigerated at 4°C temporarily.
8. The preparation method of a human papillomavirus-resistant gel according to claim 7, characterized in that: The preparation method also includes pouring the sterile water in the sterile water chamber into the freeze-dried chamber, shaking the freeze-dried chamber to dissolve the freeze-dried powder of NK cells, and then pouring the dissolved freeze-dried powder of NK cells into the gel tube containing the gel, and using a syringe to assist in drug use.