Collagenase monoclonal antibody, cosmetic composition of collagenase monoclonal antibody combined with exosome and application of cosmetic composition

Abstract

The invention discloses a collagenase monoclonal antibody, a cosmetic composition with the collagenase monoclonal antibody combined with exosome and application of the collagenase monoclonal antibody and the cosmetic composition. The sequence of a heavy chain variable region of the collagenase monoclonal antibody is shown as SEQ ID NO.1, and the sequence of a light chain variable region of the collagenase monoclonal antibody is shown as SEQ ID NO.2; the collagenase monoclonal antibody disclosed by the invention can be better combined with collagenase and has excellent affinity; besides, the exosome and the collagenase monoclonal antibody are combined for use, the pectinase monoclonal antibody is specifically combined with the pectinase to inhibit the activity of the pectinase, the exosome is assisted to promote the generation of collagen, and in addition, the exosome also has certain anti-oxidation and moisturizing effects, so that the anti-wrinkle and anti-aging purposes are achieved.

Description

Technical Field

[0001] The present invention relates to the field of cosmetic technology, and in particular to a collagenase monoclonal antibody and a cosmetic composition in combination with exosomes and applications thereof. Background Art

[0002] With the progress of society and the improvement of people's living standards, people are paying more and more attention to skin problems. The skin plays a very important role in protecting individuals from external influences, the so-called barrier function. The barrier function is a defense against various external stimuli (chemical substances, air pollutants, dry environment, ultraviolet rays, etc.) and a protective function to prevent excessive dissipation of body water through the skin. This protective function can only maintain its function when the stratum corneum composed of keratinocytes has been formed normally.

[0003] Collagen is a biopolymer, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein, and even up to 80% or more in some organisms. In addition, collagen is an important component of human skin, and many skin care products contain collagen. Collagen can moisturize the skin, and also has a certain effect of tightening the skin and fading acne marks.

[0004] The chemical name of collagenase is collagenase, which can specifically hydrolyze the three-dimensional helical structure of natural collagen under physiological pH and temperature conditions without damaging other proteins and tissues. Therefore, by inhibiting the activity of collagenase, it is helpful to slow down the degradation rate of collagen and achieve the purpose of increasing the collagen content in the skin.

[0005] In addition, exosomes refer to small membrane vesicles (30-150nm) that contain complex RNA and proteins. Nowadays, they specifically refer to disc-shaped vesicles with a diameter of 40-100nm. Exosomes can regulate the biological activity of recipient cells through the proteins, nucleic acids, lipids, etc. they carry.

[0006] At present, in response to various skin problems, various products such as anti-wrinkle, anti-aging, whitening, wrinkle removal, and moisturizing have appeared on the market, but the effects of existing products are uneven and cannot better meet people's needs. Summary of the invention

[0007] The object of the present invention is to provide a collagenase monoclonal antibody and a cosmetic composition and application thereof in combination with exosomes. The cosmetic composition of the present invention combines exosomes and collagenase monoclonal antibodies, utilizes the pectinase monoclonal antibody to specifically bind to pectinase to inhibit the activity of pectinase, and then promotes the production of collagen with the aid of exosomes, thereby achieving the purpose of anti-wrinkle and anti-aging.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] In a first aspect, the present invention provides a collagenase monoclonal antibody, wherein the heavy chain variable region sequence of the collagenase monoclonal antibody is shown as SEQ ID NO.1, and the light chain variable region sequence is shown as SEQ ID NO.2.

[0010] The second aspect of the present invention provides a cosmetic composition, which comprises umbilical cord mesenchymal stem cell exosomes, the collagenase monoclonal antibody and a base liquid.

[0011] Preferably, the content of the umbilical cord mesenchymal stem cell exosomes is 0.01-10 mg / ml.

[0012] Preferably, the content of the collagenase monoclonal antibody is 0.001-0.1 mg / ml.

[0013] Preferably, the base fluid comprises physiological saline.

[0014] The third aspect of the present invention provides a use of the above-mentioned beauty dissolving composition in the preparation of anti-wrinkle and / or anti-aging products.

[0015] A fourth aspect of the present invention provides an anti-wrinkle beauty serum, comprising the beauty composition.

[0016] A fifth aspect of the present invention provides an anti-aging beauty solution, which includes the cosmetic composition.

[0017] Compared with the prior art, the beneficial effects of the present invention include at least:

[0018] The collagenase monoclonal antibody of the present invention can better bind to collagenase and has excellent affinity; in addition, the present invention combines exosomes and collagenase monoclonal antibodies, utilizes the pectinase monoclonal antibody to specifically bind to pectinase, inhibits the activity of pectinase, and then promotes the production of collagen with the aid of exosomes. In addition, exosomes also have certain antioxidant and moisturizing effects, thereby achieving the purpose of anti-wrinkle and anti-aging. DETAILED DESCRIPTION

[0019] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0020] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0021] The embodiment of the present invention is a collagenase monoclonal antibody, the heavy chain variable region sequence of the collagenase monoclonal antibody is shown in SEQ ID NO.1, specifically EATRSQWSEASQKLMIVCNDGFFRESAKFDIQEWNVCMDFGHASESEDDSAALQWIIYTNVCSPLADKEWQCVGTPIYWELMQNAPSDGRSSWWPINVCFER CTCSPEMQELSS; the light chain variable region sequence is shown in SEQ ID NO.2, specifically RVVQPWYCRDPESLPWSHIQTWVWPENESHFVMSIDTICFTALGWVDEQWSRSDYRHYHPDRQAVMCWEVDSITGQYLEEDKSVPKSFNPCGFGNDPDAWSE SYLLAH.

[0022] Still another embodiment of the present invention provides a cosmetic composition, comprising umbilical cord mesenchymal stem cell exosomes, the above-mentioned collagenase monoclonal antibody and a base solution.

[0023] In one embodiment, the content of the umbilical cord mesenchymal stem cell exosomes may be 0.01-10 mg / ml.

[0024] In one embodiment, the content of the collagenase monoclonal antibody may be 0.001-0.1 mg / ml.

[0025] In one embodiment, the base fluid comprises physiological saline.

[0026] Another embodiment of the present invention provides a use of the above-mentioned beauty dissolving composition in the preparation of anti-wrinkle and / or anti-aging products.

[0027] Another embodiment of the present invention provides an anti-wrinkle beauty serum, which includes the beauty composition.

[0028] Another embodiment of the present invention provides an anti-aging beauty solution, wherein the anti-aging beauty solution includes the cosmetic composition.

[0029] The technical solution of the present invention is further described in detail below through specific embodiments.

[0030] Example 1

[0031] This embodiment is a method for preparing collagenase monoclonal antibodies, which comprises the following steps:

[0032] Collagenase (type I collagenase, Sigma) was used as the immunogen and physiological saline was used to prepare a 100 μg / ml collagenase solution. Then, the collagenase solution was emulsified with an equal volume of Freund's complete adjuvant and 50 μg of collagenase was injected subcutaneously at multiple points into each BALB / c mouse (15 mice) for initial immunization. Subsequently, every 3 weeks, the collagenase solution was emulsified with an equal volume of Freund's incomplete adjuvant and 30 μg of collagenase was injected subcutaneously at multiple points into each BALB / c mouse (15 mice) for booster immunization. After the third immunization, the collagenase solution was diluted with an equal volume of physiological saline and 50 μg of collagenase was injected subcutaneously at multiple points into each BALB / c mouse (15 mice) for booster immunization. On the 5th day after the 4th immunization, blood was collected from the tail vein of the mice, and then the serum antibody titer was measured by ELIS.

[0033] Select the mice with the highest titer for cell fusion and positive cell line screening:

[0034] Preparation of myeloma cells: Myeloma cells SP2 / 0 were resuscitated 6 days before cell fusion.

[0035] Cell fusion: Before fusion, BALB / c mice aged 6 to 7 weeks were taken and killed. Peritoneal macrophages were taken as feeder cells under sterile operation and spread on 6 96-well culture plates. One day later, spleen cells were obtained from the immunized mice, and myeloma cells were collected for in vitro culture. The cells were washed three times with DMEM basal medium, spleen cells and tumor cells were mixed at a ratio of 6:1, centrifuged and washed once, the supernatant was removed, and then the precipitate was dispersed. 1 ml of 50% PEG was added to the precipitate under stirring conditions. 1450 , gently suck the mixed cells into the pipette and then slowly drip into the centrifuge tube. After all the cells have dripped, use DMEM medium to terminate the effect of PEG. After centrifugation and discarding the supernatant, the cells are resuspended in an appropriate volume of HAT medium, and the fused cells are added to the 96-well cell culture plate with feeder cells at 100u1 / well, and placed in a 37°C, 5% CO2 incubator for culture. Starting from the 7th day, add one drop of HT medium to each well.

[0036] Positive cell line screening:

[0037] Coating ELISA plate: Dilute collagenase to 1ug / ml with 0.05M carbonate buffer, pH 9.6, add 100ul to each well of a 96-well ELISA plate, coat the ELISA plate overnight at 4°C, and wash the plate once with 0.15M phosphate buffered saline, pH 7.4, containing 0.05% Tween-20 (PBST);

[0038] Add culture supernatant sample: 80ul per culture well, add to the coated wells, incubate at 37℃ for 60min, wash the plate once with PBST;

[0039] Add enzyme-labeled secondary antibody: add 100ul of goat anti-mouse-HRP diluted 1:5000 to each well, incubate at 37°C for 30min, and wash the plate three times with PBST;

[0040] Add substrate for color development: Add H2O2-TMB substrate to each well, develop color for 5 minutes in the dark, add 50ul 2M sulfuric acid to each well to terminate the reaction, and measure the absorbance at 450nm in an enzyme reader.

[0041] Positive cell lines with high absorbance values ​​were selected and cloned by limiting dilution method. After the fourth cloning, the positive rate of culture wells was 100%, and a total of 8 hybridoma cells were obtained. The monoclonal hybridoma cells were expanded and cultured, the culture medium was aspirated, and cell freezing solution was added to each well to resuspend the cells, and the cells were frozen for later use.

[0042] The monoclonal antibodies were mass-produced by the induction method in syngeneic mice. Two weeks before injection, 0.5 ml of paraffin oil was injected into the peritoneal cavity of mice. 5×10 cells of the above 8 hybridoma strains were injected into the peritoneal cavity of BALB / c mice. 6 After 14 days, the ascites was collected, the solid components were removed by centrifugation, and then purified by caprylic acid-ammonium sulfate precipitation method to obtain collagenase monoclonal antibody.

[0043] The AMC sensor was used to calculate the affinity of the purified 8 antibodies (respectively denoted as anti-1 to anti-8) with collagenase, and the calculation results are shown in Table 1;

[0044] Table 1

[0045]

[0046]

[0047] Combined with the data in Table 1;

[0048] Antibody 4 has the best affinity with the antigen and was selected for subsequent experiments.

[0049] The SBA Clonotyping System-HRP kit was used to measure the antibody Ig class and subclass, and the result was IgG1.

[0050] The sequence of the above antibody 4 was determined by using an antibody sequence determination method, and the determination was as follows:

[0051] Heavy chain variable region sequence (SEQ ID NO. 1): EATRSQWSEASQKLMIVCNDGFFRESAKFDIQEWNVCMDFGHASESEDDSA ALQWIIYTNVCSPLADKEWQCVGTPIYWELMQNAPSDGRSSWWPINVCFERCTCSPEMQELSS;

[0052] Light chain variable region sequence (SEQ ID NO. 2): RVVQPWYCRDPESLPWSHIQTWVWPENESHFVMSIDTICFTALGWVDEQWS RSDYRHYHPDRQAVMCWEVDSITGQYLEEDKSVPKSFNPCGFGNDPDAWSESYLLAH.

[0053] Example 2

[0054] This embodiment is a method for preparing umbilical cord mesenchymal stem cell exosomes, and the preparation method comprises the following steps:

[0055] 1. Instruments and consumables, D-MEM / F12 culture medium, fetal bovine serum, inverted microscope, clean bench, CO2 incubator, centrifuge, cytokines, ITS and PBs, etc.;

[0056] 2. Reagent configuration

[0057] 1. D-MEM / F12 medium configuration: D-MEM / F12 medium containing 10% fetal bovine serum, one cytokine, and 5ml ITS;

[0058] 2. Cytokines: containing 20ng / ml EGF, 5ng / ml bFGF;

[0059] 3. Preparation before the experiment

[0060] 5 pairs of scissors, 5 pairs of hemostats, 3 pairs of tissue forceps, 3 pairs of ordinary forceps, 2 kidney-shaped dishes, 3 50ml beakers, 2 medicine spoons (about 20cm), 500ml PBS autoclaved at 121℃ for 30 minutes;

[0061] 4. Experimental operation

[0062] (1) Expose the clean bench to ultraviolet light for 30 minutes and wipe the surface;

[0063] (2) Place the sterilized laboratory equipment on the workbench, light the alcohol lamp, take out a kidney-shaped dish, pour half of 75% alcohol into it, place the umbilical cord into the kidney-shaped dish, and sterilize for 30 seconds;

[0064] (3) Take a 50 ml beaker, pour half of the sterilized PBS into it, put the umbilical cord into the beaker, and wash it three times;

[0065] (4) Take out another kidney-shaped dish, put the umbilical cord in it, pour in an appropriate amount of PBS, take scissors in the right hand and ordinary tweezers in the left hand, and cut the umbilical cord into small pieces of about 2 cm;

[0066] (5) Use the hemostatic forceps in your left hand and the tissue forceps in your right hand to slowly remove the outer skin and blood vessels of the umbilical cord;

[0067] (6) Place the remaining tissue in a clean beaker and cut it into pieces of about 3 mm with scissors;

[0068] (7) Use a medicine spoon to spread the tissue blocks in the T75 bottle, with about 100 tissue blocks in each bottle, until all the tissue blocks are spread;

[0069] (8) Slowly add 15 ml of culture medium to one side of the T75 bottle and place in a CO2 incubator for incubation;

[0070] (9) Change the medium every 48 hours. After one week, observe the crawling of stem cells. If the crawling is good, discard the tissue block and add culture medium.

[0071] (10) After 48 h, the cells were digested by adding 1 ml of trypsin for digestion, and the cells were collected and centrifuged (1200 rpm, 5 min). The supernatant was discarded, and the cells were resuspended in culture medium. The cells were divided into 4 groups according to the number of cells (1×10 6 ) cells are inoculated, which is the P1 generation;

[0072] (11) After 48 hours, digest and centrifuge, count, and passage to obtain the P2 generation;

[0073] (12) Two days later, digest and centrifuge the supernatant (to test for sterility, mycoplasma, and endotoxin), and count the cells at 4.5 × 10 6 Cryopreservation;

[0074] (13) Resuscitate P2 cells according to production needs, transfer to P3 cells and collect supernatant;

[0075] (14) Centrifuge the supernatant at 4000 × g to remove cells and cell debris;

[0076] (15) Add exosome precipitation reagent to the supernatant, mix thoroughly, let stand, and then centrifuge at 10,000 × g for 30 min to collect the precipitate;

[0077] (16) The precipitate was resuspended in PBS and placed in a purification column, which was then placed in a collection tube. The collection tube was centrifuged at 4000 × g for 10 min at 4°C, and the solution in the collection tube was freeze-dried to obtain umbilical cord mesenchymal stem cell exosomes.

[0078] Example 3

[0079] This embodiment is an anti-wrinkle experiment;

[0080] The experiment was conducted with 6-week-old SPF-grade female hairless mice (HR-1);

[0081] After one week of feeding, the mice were randomly divided into 4 groups, namely, exosome group, monoclonal antibody group, exosome + monoclonal antibody group and normal saline group, with 5 mice in each group;

[0082] Mice were irradiated with ultraviolet light for 8 weeks, 4 times a week for the first 4 weeks, 30 minutes each time, with an irradiation intensity of 80 mJ / cm 2 ; irradiation was performed 5 times a week for the next four weeks, each irradiation lasted 40 minutes, and the irradiation intensity was 120 mJ / cm 2 ;

[0083] Within four weeks after irradiation, the mice in each group were treated as follows: the mice in the exosome + monoclonal antibody group were subcutaneously injected with a mixture of exosomes and monoclonal antibodies (exosome concentration was 0.1 mg / ml, monoclonal antibody concentration was 0.002 mg / ml) on the back, with an injection volume of 0.5 ml; the mice in the exosome group were subcutaneously injected with exosome solution (exosome concentration was 0.1 mg / ml) on the back, with an injection volume of 0.5 ml; the mice in the monoclonal antibody group were subcutaneously injected with monoclonal antibody solution (monoclonal antibody concentration was 0.002 mg / ml) on the back, with an injection volume of 0.5 ml; the mice in the normal saline group were subcutaneously injected with an equal amount of normal saline on the back; the mice in each group were injected once every 1 day.

[0084] After the irradiation, the maximum wrinkle depth of the mouse back skin (with the injection site as the center and a diameter of 1.5 cm) was measured; and the average value of each group of mice was calculated. The calculation results are shown in Table 2;

[0085] Table 2

[0086] Group Maximum wrinkle depth Normal saline group 236.5μm Exosome group 167.3μm Monoclonal antibody group 192.7μm Exosomes + monoclonal antibody group 86.1μm

[0087] As shown in Table 1, compared with the saline group, the other groups were able to reduce the maximum depth of wrinkles on the mouse skin, indicating that both exosomes and monoclonal antibodies have certain anti-wrinkle effects. Compared with the exosomes and monoclonal antibody groups, the combined use of exosomes and monoclonal antibodies can achieve better anti-wrinkle effects.

[0088] Obtain the 1.5 cm aging of the back of each group of mice 2 Skin tissue (skin tissue centered at the injection site) was cleaned and dried, and then the skin tissue was cut into pieces. The skin and saline were mixed and homogenized at a mass ratio of 1:19, centrifuged at 9000 rpm for 12 min at 4°C, filtered, and the supernatant was collected. Then, the supernatant MDA and Hyp content were detected using a kit. The test results are shown in Table 2.

[0089] Table 2

[0090] Group Hyp (μg / mg) MDA (nmol / mg) Normal saline group 0.71±0.17 7.15±0.36 Exosome group 1.28±0.23 4.31±0.32 Monoclonal antibody group 1.93±0.19 5.62±0.41 Exosomes + monoclonal antibody group 2.53±0.26 3.27±0.37

[0091] From Table 2, we can see that:

[0092] Hyp is a unique amino acid in collagen, and its content can reflect the metabolism of collagen. During the skin aging process, a variety of products are produced, and the change in the content of MDA can determine the skin aging condition. Combined with the data in Table 2, it can be seen that the monoclonal antibody and exosomes of the technical solution of the present application can increase the Hyp content, which further indicates that the degradation of collagen in the skin can be prevented, and at the same time the MDA content can be reduced, indicating that the monoclonal antibody and exosomes of the technical solution of the present application can inhibit the generation of MDA and have anti-skin aging performance.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A collagenase monoclonal antibody, characterized in that: The heavy chain variable region sequence of the collagenase monoclonal antibody is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.

2.

2. A cosmetic composition, characterized in that The cosmetic composition comprises umbilical cord mesenchymal stem cell exosomes, the collagenase monoclonal antibody according to claim 1 and a base liquid.

3. The cosmetic composition according to claim 2, characterized in that The content of the umbilical cord mesenchymal stem cell exosomes is 0.01-10 mg / ml.

4. The cosmetic composition according to claim 2, characterized in that The content of the collagenase monoclonal antibody is 0.001-0.1 mg / ml.

5. The cosmetic composition according to claim 1, characterized in that The base fluid includes physiological saline.

6. Use of the beauty dissolving composition according to any one of claims 2 to 5 in the preparation of anti-wrinkle and / or anti-aging products.

7. An anti-wrinkle beauty serum, characterized in that: The anti-wrinkle beauty serum comprises the beauty composition according to any one of claims 2 to 5.

8. An anti-aging beauty solution, characterized in that: The anti-aging beauty solution comprises the beauty composition according to any one of claims 2 to 5.

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