Stem cell supernatant liposome and preparation method, encapsulation efficiency detection method and application thereof

Abstract

The invention belongs to the technical field of biological medicine and medical beauty, and particularly relates to stem cell supernatant liposome as well as a preparation method, an encapsulation efficiency detection method and application thereof. The preparation method of the stem cell supernatant liposome comprises the following steps: culturing spermatogonial stem cells, and collecting spermatogonial stem cell supernatant; dissolving sitosterol and soya bean lecithin by using an organic solvent to form an organic phase; and mixing the spermatogonial stem cell supernatant with the organic phase by adopting a solvent injection method, a film dispersion method or a reverse evaporation method to obtain the stem cell supernatant liposome. According to the stem cell supernatant liposome preparation method provided by the invention, the stem cell supernatant liposome with high encapsulation efficiency and stable structure can be prepared, and due to the double-layer protection effect of lipid, the degradation degree of protein drugs can be reduced, so that the effect of improving the relative stability of active substances of the protein drugs is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and medical aesthetics, and specifically relates to a stem cell supernatant liposome and a preparation method, an encapsulation rate detection method and application thereof. Background Art

[0002] The use of stem cell supernatant in cosmetics has certain advantages. Advantages include its possible anti-aging effects, as stem cell supernatant contains a variety of growth factors and cytokines, which are believed to promote the regeneration and repair of skin cells. In addition, it may help improve the elasticity and firmness of the skin and reduce the appearance of wrinkles and fine lines. A mixture of various bioactive substances secreted by stem cells into the culture medium. These substances include growth factors, cytokines, proteins, metabolites, etc., which play an important role in cell growth, differentiation and tissue repair. The contents contain a variety of MicoRNAs, proteins and growth factors, such as miR126, miR125a, miR124a, cell signaling protein wnt4, and EGF, etc., which play an important role in cell growth, differentiation and tissue repair. However, it also has obvious disadvantages, such as the high cost of extracting and processing stem cell supernatant, and the addition of this ingredient to cosmetics may result in expensive products. Secondly, its effect may not be as significant as advertised, because the concentration of stem cell supernatant in cosmetics is usually low and may not be enough to produce the expected skin care effect. Moreover, there may be problems with the stability of stem cell supernatant, which is easily affected by external factors such as temperature and light, resulting in inactivation of active ingredients.

[0003] Therefore, there is a need to provide a simple and effective method to improve the stability of stem cell supernatant in cosmetic applications. Summary of the invention

[0004] Based on this, the purpose of the present invention is to provide a stem cell supernatant liposome and its preparation method, encapsulation rate detection method and application, which have solved the problems in the prior art.

[0005] To achieve the above object, the present invention adopts the following technical solution.

[0006] The present invention provides a method for preparing stem cell supernatant liposomes, comprising:

[0007] Cultivating spermatogonial stem cells and collecting spermatogonial stem cell supernatant;

[0008] Using an organic solvent to dissolve sitosterol and soybean lecithin to form an organic phase;

[0009] The spermatogonial stem cell supernatant is mixed with the organic phase by a solvent injection method, a thin film dispersion method or a reverse evaporation method to obtain stem cell supernatant liposomes.

[0010] Liposomes are tiny vesicles composed of one or more layers of phospholipid bilayers that can encapsulate drugs or other substances and are used in drug delivery, gene therapy, cosmetics and other fields. The application of liposomes in cosmetics is mainly reflected in their use as carriers of drugs or active ingredients, which can improve the stability, bioavailability and skin permeability of these ingredients. Since the structure of liposomes is similar to that of cell membranes, they can fuse with skin cell membranes to promote the absorption of active ingredients. In cosmetics, liposomes can be used to deliver moisturizers, antioxidants, sunscreens, etc. to enhance their effects. In addition, liposomes can also be used as sustained-release systems to prolong the action time of active ingredients in cosmetics and improve the efficacy of cosmetics. Due to their good biocompatibility and low toxicity, they are less irritating to the skin and are therefore widely used in the cosmetics industry.

[0011] The membrane material of liposome is usually composed of phospholipids and additives. In the present invention, soybean lecithin is selected as the phospholipid material. Compared with other phospholipids, it has higher purity and stability, and has price advantages compared with other phospholipids such as egg yolk lecithin. In the present invention, the additive is phytosterol. Soybean lecithin and plants are used as raw materials for liposomes to prepare stem cell supernatant liposomes. The prepared liposome material can reduce the degradation degree of protein drugs, thereby achieving the effect of improving the relative stability of their active substances.

[0012] In some embodiments, the mass volume ratio of the sitosterol, soybean lecithin and organic solvent is: 15mg-25mg: 75mg-85mg: 5mL-25mL. When the amount of sitosterol used is lower than this range, the fluidity of the phospholipid bilayer membrane cannot be effectively adjusted, resulting in a decrease in the stability of the membrane and the ability to resist changes in external conditions; when the amount of sitosterol used is higher than this range, the liposome membrane is too rigid, affecting its flexibility, thereby reducing the stability of the liposome; when the amount of soybean lecithin used is lower than this range, the drug cannot be effectively encapsulated, and the liposome membrane may become fragile and more susceptible to the external environment and rupture; when the amount of soybean lecithin used is higher than this range, the viscosity of the liquid system will increase, affecting the stability of the liposome; when the amount of organic solvent used is lower than this range, the organic solvent will dissolve the membrane material slowly or fail to fully encapsulate the phospholipid molecules, thereby affecting the dispersion of the phospholipid molecules in the aqueous phase; when the amount of organic solvent used is higher than this range, it will mainly increase unnecessary costs, and too much may also cause environmental pollution.

[0013] In some embodiments, the protein concentration in the spermatogonial stem cell supernatant is 2 mg / mL to 2.5 mg / mL.

[0014] There are many methods for protein determination, among which the commonly used determination methods include Kjeldahl nitrogen determination method, biuret method, Bradford method, Lowry method, ultraviolet absorption method and BCA method. Kjeldahl nitrogen determination method estimates protein content by measuring the nitrogen content in the sample. The biuret method uses biuret reagent to react with protein to produce a purple complex, and the protein content is determined by colorimetry. The Bradford method uses Coomassie Brilliant Blue G-250 dye to bind to protein and determine protein concentration by color change. The Lowry method is based on the complex generated by the reaction of protein with copper ions, which then reacts with Folin reagent to produce a blue product, and the protein content is determined by colorimetry. The ultraviolet absorption method uses the characteristic absorption of protein at a wavelength of 280nm to determine its concentration. The BCA method (bicinchoninic acid method) is a modified Lowry method that uses BCA reagent to react with protein to generate a purple complex and determines the protein content by colorimetry. The above methods each have their own advantages and disadvantages. Among them, the BCA method for determining protein concentration is not affected by the chemical substances in most samples, and is compatible with up to 5% SDS, 5% Triton X-100, and 5% Tween20, 60, and 80 in the sample. At the same time, this method is not affected by the type of protein composition. Therefore, the present invention adopts the BCA method to determine the protein concentration.

[0015] In some embodiments, the organic solvent includes: methanol, ethanol, ether and chloroform. Preferably, the organic solvent is ether and chloroform.

[0016] In some embodiments, the solvent injection method comprises: adding PBS to the spermatogonial stem cell supernatant and mixing to form an aqueous phase;

[0017] Then, the organic phase is added to the aqueous phase, the stirring speed is set to 400r / min to 500r / min, and the organic phase and the aqueous phase are merged; then the stirring speed is set to 400r / min to 500r / min, and the stirring time is 2h to 3h, so that the organic solvent is volatilized to obtain the stem cell supernatant liposome; the organic solvent is ether. In the solvent injection method, too low a stirring speed will lead to aggravated aggregation of liposome particles, while too high a stirring speed will lead to uneven particle size distribution, which will affect the stability of the liposome.

[0018] In some embodiments, the stirring speed is set to 400 r / min, 450 r / min or 500 r / min to merge the organic phase and the aqueous phase; the stirring speed is set to 400 r / min, 450 r / min or 500 r / min, and the stirring time is set to 2 h, 2.5 h or 3 h to volatilize the organic solvent.

[0019] In some embodiments, the steps of the thin film dispersion method include: subjecting the organic phase to reduced pressure rotary evaporation to form a thin film on the wall of the flask, then adding a mixture of the spermatogonial stem cell supernatant and PBS, and continuing to rotary evaporation to dissolve the thin film to obtain the stem cell supernatant liposomes; the organic solvent is chloroform.

[0020] In some embodiments, the volume ratio of the spermatogonial stem cell supernatant to PBS is 3 mL to 5 mL: 3 mL to 5 mL.

[0021] In some embodiments, the temperature of the reduced pressure rotary evaporation is 32°C to 35°C, and the speed of the reduced pressure rotary evaporation is 25r / min to 40r / min. For example, in some embodiments, the temperature of the reduced pressure rotary evaporation is 32°C, 33°C, 34°C or 35°C, and the speed of the reduced pressure rotary evaporation is 25r / min, 30r / min, 35r / min or 40r / min. Preferably, in the thin film dispersion method, the speed of the reduced pressure rotary evaporation is 30r / min.

[0022] In some embodiments, the reverse evaporation method comprises: taking the supernatant of the spermatogonial stem cells as an aqueous phase, adding the aqueous phase to the organic phase, and obtaining colostrum by ultrasound; the organic solvent in the organic phase is ether; the volume ratio of the aqueous phase to the organic phase is 1:2-5;

[0023] The organic solvent in the colostrum is removed by using a rotary evaporator, and the rotary evaporation is continued to form a thin film, and then PBS is added to completely dissolve the thin film to obtain the stem cell supernatant liposome.

[0024] Preferably, the volume ratio of the aqueous phase to the organic phase is 1:4.

[0025] In some embodiments, in the reverse evaporation method, the temperature of the reduced pressure rotary evaporation is 32°C to 35°C, and the speed of the reduced pressure rotary evaporation is 25r / min to 40r / min. For example, in some embodiments, the temperature of the reduced pressure rotary evaporation is 32°C, 33°C, 34°C or 35°C, and the speed of the reduced pressure rotary evaporation is 25r / min, 30r / min, 35r / min or 40r / min.

[0026] Preferably, in the reverse evaporation method, the rotation speed of the reduced pressure rotary evaporation is 30 r / min.

[0027] In some embodiments, the volume ratio of the PBS to the spermatogonial stem cell supernatant is 3 mL to 5 mL: 13 mL to 15 mL.

[0028] The present invention also provides stem cell supernatant liposomes prepared by the preparation method.

[0029] Liposome quality evaluation includes encapsulation efficiency, particle size distribution, surface charge, morphology observation, stability, in vitro release, etc. These evaluation indicators together constitute a complete system for liposome quality control to ensure the safety and effectiveness of liposomes as drug carriers.

[0030] Common methods for determining the liposome encapsulation rate include dialysis, ultracentrifugation, gel permeation chromatography, fluorescent probe method, etc. The dialysis method is to place the liposomes in a dialysis bag, remove the free drug by dialysis, and then measure the concentration difference of the drug before and after dialysis to calculate the encapsulation rate. The ultracentrifugation method is to use an ultracentrifuge to separate the liposomes from the free drug, and calculate the encapsulation rate by measuring the concentration of the drug in the supernatant and subtracting the concentration of the supernatant from the total concentration. The gel permeation chromatography method uses a gel column to separate liposomes and free drugs, and calculates the encapsulation rate by detecting the content of the drug in different components. The fluorescent probe method uses fluorescently labeled liposomes or drugs to calculate the encapsulation rate by measuring the fluorescence intensity. However, the above methods have their own advantages and disadvantages. For example, the advantages of the dialysis method are simple operation, low cost, and suitable for laboratory-scale encapsulation rate determination. The disadvantage is that the determination time is long, and the dialysis may be incomplete for small molecules, affecting the accuracy of the determination results. The advantage of the ultracentrifugation method is high separation efficiency, which can better distinguish liposomes from unencapsulated drugs. The disadvantages are that it requires expensive equipment, complicated operation, and the liposome structure may be destroyed during high-speed centrifugation, affecting the determination of encapsulation efficiency. The advantages of gel permeation chromatography are good separation effect, the ability to simultaneously determine liposomes and unencapsulated drugs, and less damage to liposomes. The disadvantages are that it requires specific chromatographic columns and equipment, which is expensive, and the detection sensitivity for some drugs may be insufficient.

[0031] In summary, in the field of liposome encapsulation rate determination, there are problems such as operational complexity (multiple steps), time consumption, destructiveness (destroying liposome integrity) and cost, and selectivity (different determination methods will affect the encapsulation rate results).

[0032] The present invention also provides a method for detecting the liposome encapsulation rate of the stem cell supernatant, comprising: a high-speed centrifugation method or a membrane breaking method.

[0033] In some embodiments, the high-speed centrifugation method comprises: centrifuging the stem cell supernatant liposome solution at 10,000 r / min to 15,000 r / min and 4° C. for 30 to 45 min, then determining the total protein content in the liposome solution before centrifugation and the protein content in the supernatant after centrifugation, and calculating the encapsulation efficiency according to equation 1:

[0034] Equation 1: Encapsulation efficiency = (C total - C free) / C total × 100%;

[0035] Wherein, Cfree is the free protein content in the supernatant after centrifugation, and Ctotal is the total protein in the liposome solution before centrifugation.

[0036] In some specific embodiments, in the high-speed centrifugation method, the centrifugal speed is 10000r / min, 11000r / min, 12000r / min, 13000r / min, 14000r / min or 15000r / min; the centrifugal time is 30min, 35min, 40min or 45min. Preferably, in the high-speed centrifugation method, the centrifugal condition is 15000r / min, 30min.

[0037] In some embodiments, the membrane breaking method includes: taking 2 mL of the stem cell supernatant liposome solution, dividing it into 2 parts, diluting one part by 2 times, and adding Triton X-100 after diluting one part by 2 times, wherein the volume fraction of Triton X-100 in the stem cell supernatant liposome solution is 0.2% to 0.5%, and vortexing to break the emulsion. The total protein content in the liposome solution before centrifugation and the protein content in the supernatant after centrifugation are calculated according to the protein determination method, and the encapsulation efficiency is calculated according to equation 1. Preferably, the volume fraction of Triton X-100 is 0.3%.

[0038] The present invention also provides the use of the stem cell supernatant liposome in preparing cosmetics for anti-aging or skin regeneration and repair.

[0039] Based on the technical solution of the present invention, compared with the prior art, the present invention has the following beneficial effects:

[0040] Liposomes are tiny vesicles composed of a phospholipid bilayer that can mimic the structure of a cell membrane. Through modification, proteins can be fixed on the surface of liposomes or embedded in the lipid bilayer, thereby imparting new functions or improving their stability. The method provided by the present invention combines the advantages of spermatogonial stem cell supernatant and liposomes, and while exerting their respective functions, they can also complement each other. When SCCS stem cell supernatant is encapsulated in liposomes, due to the protective effect of the lipid bilayer, the degree of degradation of protein drugs can be reduced, thereby achieving the effect of enhancing the relative stability of its active substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the standard curve of the standard protein solution.

[0042] Figure 2 It is a bar graph showing the statistical results of the encapsulation efficiency of the stem cell supernatant of Example 1, Example 5, and Example 8 measured by high-speed centrifugation and membrane disruption methods. DETAILED DESCRIPTION

[0043] The experimental methods in the following examples of the present invention without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

[0044] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0045] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0046] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0047] The following describes the invention in conjunction with specific embodiments.

[0048] Experimental Materials:

[0049] Mouse spermatogonial stem cells: purchased from Sean Biotechnology, catalog number: SNPM-M212.

[0050] Phytosterols: purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S24012.

[0051] Example 1: Preparation of stem cell supernatant liposomes

[0052] 1. Activation and culture of spermatogonial stem cells (SCCS): Shake and thaw the cryotube containing 1 mL of cell suspension quickly in a 37°C water bath, add 4 mL of DMEM-F12 culture medium and mix well. Centrifuge at 1000 rpm for 4 minutes, discard the supernatant, add 1-2 mL of DMEM-F12 culture medium and blow evenly. Then add all the cell suspensions to a culture bottle containing DMEM-F12 culture medium and culture overnight (or add the cell suspension to a 10 cm dish, add about 8 mL of DMEM-F12 culture medium, and culture overnight). Change the medium the next day and check the cell density until the primary cells reach a cell density of 80% to 90%. Digest with 0.25% trypsin and pass at 1:3. Overnight culture conditions: 37°C, 5% CO2.

[0053] 2. Extraction of stem cell supernatant: Take the fourth-generation SCCS with good growth condition and use 1×10 6 The density of cells was inoculated into a culture flask of DMEM-F12 medium. When the cell confluence reached 60%, the culture medium containing cells was placed in a centrifuge and centrifuged at 6000rpm for 30min. The supernatant was then collected and filtered at 0.22μm to obtain SCCS stem cell supernatant. The protein concentration in the SCCS stem cell supernatant was determined for the subsequent preparation of stem cell supernatant liposomes. The method for determining the protein concentration of the SCCS stem cell supernatant is described as follows:

[0054] Take 1 mL of the above SCCS stem cell supernatant and put it in a centrifuge tube, add PBS to dilute it to 5 mL, and determine the protein concentration by BCA method, that is, prepare 0.5 mg / mL standard protein solution, add 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μL, which is equivalent to the standard concentrations of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, respectively.

[0055] Take 20 μL of the diluted SCCS stem cell supernatant and place it in the sample well. Add 200 μL of BCA working solution to all the sample wells. Bake at 37°C until the color of the standard wells shows a clear gradient. Use an ELISA reader to measure the absorbance at 562 nm. See the attached standard curve. Figure 1 According to the calibration curve, the sample well concentration was calculated to be C = 0.449 mg / mL, that is, the protein concentration of the SCCS stem cell supernatant was C = 2.25 mg / mL.

[0056] 3. Preparation of SCCS liposomes: Using the solvent injection method, weigh 20 mg of sitosterol and 80 mg of soybean lecithin and add 5 mL of ether to dissolve to form an organic phase; take 5 mL of SCCS stem cell supernatant with a protein concentration of 2.25 mg / mL, add 15 mL of PBS and mix well, then slowly add the organic phase and set the stirring speed to 500 r / min during the process to stir continuously to completely merge the organic phase and the aqueous phase. Set the stirring speed of the magnetic stirrer to 500 r / min at room temperature and stir for 2 hours to evaporate the organic phase to obtain SCCS liposomes.

[0057] Example 2: Preparation of stem cell supernatant liposomes

[0058] Compared with Example 1, the difference is that the preparation method of SCCS liposomes is different, and the remaining steps are the same. Specifically, the preparation of SCCS liposomes is as follows: using the solvent injection method, 15 mg of sitosterol and 75 mg of soybean lecithin are weighed and added to 10 mL of ether to dissolve to form an organic phase; 5 mL of SCCS stem cell supernatant with a protein concentration of 2 mg / mL is taken, 15 mL of PBS is added to mix, and then the organic phase is slowly added dropwise and during this process, the stirring speed is set to 400 r / min and stirred continuously to completely merge the organic phase and the aqueous phase. Under normal temperature conditions, the stirring speed is set to 400 r / min with a magnetic stirrer, stirred for 3 hours, and the organic phase is evaporated to obtain SCCS liposomes.

[0059] Example 3: Preparation of stem cell supernatant liposomes

[0060] Compared with Example 1, the difference is that the preparation method of SCCS liposomes is different, and the remaining steps are the same. Specifically, the preparation of SCCS liposomes is as follows: using the solvent injection method, weigh 25mg of sitosterol and 85mg of soybean lecithin, add 20mL of ether to dissolve, and form an organic phase; take 5mL of SCCS stem cell supernatant with a protein concentration of 2.3mg / mL, add 15mL of PBS to mix, and then slowly drip the organic phase and in this process, set the stirring speed to 500r / min and stir continuously to make the organic phase and the aqueous phase completely merge. Set the stirring speed to 500r / min with a magnetic stirrer at room temperature, stir for 2h, evaporate the organic phase, and obtain SCCS liposomes.

[0061] Example 4: Preparation of stem cell supernatant liposomes

[0062] Compared with Example 1, the difference is that the preparation method of liposomes is different, and the remaining steps are the same. Specifically, the preparation method of SCCS liposomes is as follows: using the solvent injection method, weighing 20 mg of sitosterol and 80 mg of soybean lecithin, adding 5 mL of ether to dissolve, forming an organic phase; taking 5 mL of SCCS stem cell supernatant with a protein concentration of 2.5 mg / mL, adding 15 mL of PBS to mix, and then slowly dropping the organic phase and in this process, setting the stirring speed to 500 r / min and stirring continuously to completely merge the organic phase and the aqueous phase. Under normal temperature conditions, the stirring speed is set to 500 r / min with a magnetic stirrer, stirring for 2 hours, and the organic phase is completely volatilized to obtain SCCS liposomes.

[0063] Example 5: Preparation of stem cell supernatant liposomes

[0064] 1. Activation and culture of spermatogonial stem cells (SCCS): Shake and thaw the cryotube containing 1 mL of cell suspension quickly in a 37°C water bath, add 4 mL of DMEM-F12 culture medium and mix well. Centrifuge at 1000 rpm for 4 minutes, discard the supernatant, add 1-2 mL of DMEM-F12 culture medium and blow evenly. Then add all the cell suspensions to a culture bottle containing DMEM-F12 culture medium and culture overnight (or add the cell suspension to a 10 cm dish, add about 8 mL of DMEM-F12 culture medium, and culture overnight). Change the medium the next day and check the cell density until the primary cells reach a cell density of 80% to 90%. Digest with 0.25% trypsin and pass at 1:3. Overnight culture conditions: 37°C, 5% CO2.

[0065] 2. Extraction of stem cell supernatant: Take the fourth-generation SCCS with good growth condition and use 1×10 6 The density of cells was inoculated into a culture flask of DMEM-F12 medium. When the cell confluence reached 60%, the culture medium containing cells was placed in a centrifuge and centrifuged at 6000rpm for 30min. The supernatant was then collected and filtered at 0.22μm to obtain SCCS stem cell supernatant. The protein concentration in the SCCS stem cell supernatant was determined for the subsequent preparation of stem cell supernatant liposomes. The method for determining the protein concentration of the SCCS stem cell supernatant is described as follows:

[0066] Take 1 mL of the above SCCS stem cell supernatant and put it in a centrifuge tube, add PBS to dilute it to 5 mL, and determine the protein concentration by BCA method, that is, prepare 0.5 mg / mL standard protein solution, add 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μL, which is equivalent to the standard concentrations of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, respectively.

[0067] Take 20 μL of the diluted SCCS stem cell supernatant and place it in the sample well. Add 200 μL of BCA working solution to all the sample wells. Bake at 37°C until the color of the standard wells shows a clear gradient. Use an ELISA reader to measure the absorbance at 562 nm. See the attached standard curve. Figure 1 According to the calibration curve, the sample well concentration was calculated to be C = 0.449 mg / mL, that is, the protein concentration of the SCCS stem cell supernatant was C = 2.25 mg / mL.

[0068] 2. Preparation of SCCS liposomes: Using the thin film dispersion method, 20 mg of sitosterol and 80 mg of soybean lecithin were weighed and added into 5 mL of chloroform to dissolve to form an organic phase. At 35 °C, the speed was set to 30 r / min and the reduced pressure rotary evaporation (pressure equipment parameter 100 HPa) was performed until a uniform thin film appeared on the wall of the flask. Then, 5 mL of SCCS and 5 mL of PBS mixture were added and the rotary evaporation was continued to dissolve the thin film (without reducing pressure or light pressure). The thin film on the wall could be seen to fall off obviously, and the rotary evaporation was stopped to obtain SCCS liposomes.

[0069] Example 6: Preparation of stem cell supernatant liposomes

[0070] Compared with Example 5, the difference is that the preparation method of SCCS liposomes is different, and the remaining steps are the same. Specifically, the preparation of SCCS liposomes is as follows: using a thin film dispersion method, 17 mg of sitosterol and 76 mg of soybean lecithin are weighed and added to 7 mL of chloroform to dissolve to form an organic phase, and at 32 ° C, the speed is set to 40 r / min, and the pressure is reduced and rotary evaporated (pressure equipment parameter 100 HPa) until a uniform film appears on the wall of the flask, and then 3 mL of SCCS supernatant and 3 mL of PBS mixed solution are added to continue rotary evaporation to dissolve the film (without decompression or light pressure), and the film on the wall can be seen to fall off obviously, and the rotary evaporation is stopped to obtain SCCS liposomes.

[0071] Example 7: Preparation of stem cell supernatant liposomes

[0072] Compared with Example 5, the difference is that the preparation method of SCCS liposomes is different, and the remaining steps are the same. Specifically, the preparation of SCCS liposomes is as follows: using a thin film dispersion method, 24 mg of sitosterol and 83 mg of soybean lecithin are weighed and added to 9 mL of chloroform to dissolve to form an organic phase, and at 35 ° C, the speed is set to 35 r / min, and the pressure is reduced and rotary evaporated (pressure equipment parameter 100 HPa) until a uniform film appears on the wall of the flask, and then 4 mL of SCCS supernatant and 4 mL of PBS mixed solution are added to continue rotary evaporation to dissolve the film (without decompression or light pressure), and the film on the wall can be seen to fall off obviously, and the rotary evaporation is stopped to obtain SCCS liposomes.

[0073] Example 8: Preparation of stem cell supernatant liposomes

[0074] 1. Activation and culture of spermatogonial stem cells (SCCS): Shake and thaw the cryotube containing 1 mL of cell suspension quickly in a 37°C water bath, add 4 mL of DMEM-F12 culture medium and mix well. Centrifuge at 1000 rpm for 4 minutes, discard the supernatant, add 1-2 mL of DMEM-F12 culture medium and blow evenly. Then add all the cell suspensions to a culture bottle containing DMEM-F12 culture medium and culture overnight (or add the cell suspension to a 10 cm dish, add about 8 mL of DMEM-F12 culture medium, and culture overnight). Change the medium the next day and check the cell density until the primary cells reach a cell density of 80% to 90%. Digest with 0.25% trypsin and pass at 1:3. Overnight culture conditions: 37°C, 5% CO2.

[0075] 2. Extraction of stem cell supernatant: Take the fourth-generation SCCS with good growth condition and use 1×10 6 The density of cells was inoculated into a culture flask of DMEM-F12 medium. When the cell confluence reached 60%, the culture medium containing cells was placed in a centrifuge and centrifuged at 6000rpm for 30min. The supernatant was then collected and filtered at 0.22μm to obtain SCCS stem cell supernatant. The protein concentration in the SCCS stem cell supernatant was determined for the subsequent preparation of stem cell supernatant liposomes. The method for determining the protein concentration of the SCCS stem cell supernatant is described as follows:

[0076] Take 1 mL of the above SCCS stem cell supernatant and put it in a centrifuge tube, add PBS to dilute it to 5 mL, and determine the protein concentration by BCA method, that is, prepare 0.5 mg / mL standard protein solution, add 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, and 20 μL of the standard to the standard wells of a 96-well plate, and add standard diluent to make up to 20 μL, which is equivalent to the standard concentrations of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, respectively.

[0077] Take 20 μL of the diluted SCCS stem cell supernatant and place it in the sample well. Add 200 μL of BCA working solution to all the sample wells. Bake at 37°C until the color of the standard wells shows a clear gradient. Use an ELISA reader to measure the absorbance at 562 nm. See the attached standard curve. Figure 1 According to the calibration curve, the sample well concentration was calculated to be C = 0.449 mg / mL, that is, the protein concentration of the SCCS stem cell supernatant was C = 2.25 mg / mL.

[0078] 2. Preparation of SCCS liposomes: Using the reverse evaporation method, weigh 20 mg of sitosterol and 80 mg of soybean lecithin and add 25 mL of ether to dissolve to form an organic phase; take 5 mL of SCCS, slowly inject the aqueous phase into the organic phase under magnetic force at room temperature, obtain colostrum by ultrasound, set the speed to 30 r / min in a 35°C water bath, and remove the organic solvent by reduced pressure evaporation in a rotary evaporator (pressure parameter set to 900 HPa), and continue rotary evaporation to form a colloidal substance and a small amount of film. Then add 15 mL of PBS and continue rotary evaporation for about 5 minutes to make the film completely fall off and dissolve to obtain SCCS liposomes.

[0079] Example 9: Preparation of stem cell supernatant liposomes

[0080] Compared with Example 8, the difference is that the preparation method of SCCS liposomes is different, and the remaining steps are the same. Specifically, the preparation of SCCS liposomes is as follows: using the reverse evaporation method, 16 mg of sitosterol and 78 mg of soybean lecithin are weighed and added to 23 mL of ether to dissolve to form an organic phase; 5 mL of SCCS is taken, and the aqueous phase is slowly injected into the organic phase under magnetic force at room temperature, and colostrum is obtained by ultrasound. Under a 35 ° C water bath, the speed is set to 35r / min, and the organic solvent is removed by reduced pressure evaporation in a rotary evaporator (the pressure parameter is set to 900HPa), and the colloid substance and a small part of the film are continuously evaporated. Then 15 mL of PBS is added, and the evaporation is continued for about 5 minutes, so that the film is completely dropped and dissolved to obtain SCCS liposomes.

[0081] Example 10: Preparation of stem cell supernatant liposomes

[0082] Compared with Example 8, the difference is that the preparation method of SCCS liposomes is different, and the remaining steps are the same. Specifically, the preparation of SCCS liposomes is as follows: adopt the reverse evaporation method, weigh 23mg of sitosterol, 82mg of soybean lecithin, add 25mL of ether to dissolve, and form an organic phase; take 5mL SCCS, slowly inject the aqueous phase into the organic phase under normal temperature magnetic force, ultrasonically obtain colostrum, set the speed to 35r / min under 35°C water bath, and remove the organic solvent by reduced pressure evaporation in a rotary evaporator (pressure parameter is set to 900HPa), and continue to rotary evaporation to form a colloidal substance and a small part of the film. Add 15mL PBS, continue rotary evaporation for about 5min, so that the film is completely dropped and dissolved, and obtain SCCS liposomes.

[0083] Example 11: Method for determining the liposome encapsulation efficiency of stem cell supernatant

[0084] The encapsulation efficiency of the SCCS liposomes prepared in Examples 1 to 10 was measured by high-speed centrifugation and membrane disruption, respectively. The specific methods are as follows:

[0085] 1. Determination of encapsulation efficiency by high-speed centrifugation:

[0086] Take 1 mL of each SCCS liposome solution of Example 1 to Example 10, and centrifuge at 15000 r / min and 4° C. for 30 min. According to the protein determination method provided in Example 1, the total protein content in the liposome solution before centrifugation and the protein content in the supernatant after centrifugation were calculated, and the encapsulation efficiency was calculated according to Equation 1. The calculation results are shown in Table 1.

[0087] Equation 1: Encapsulation efficiency = (C total - C free) / C total × 100%,

[0088] Wherein, Cfree is the free protein content in the supernatant after centrifugation, and Ctotal is the total protein in the liposome solution before centrifugation.

[0089] 2. Determination of encapsulation efficiency by membrane rupture method: Take 2 mL of each liposome solution of Example 1 to Example 10, divide it into 2 parts, dilute one part by 2 times, add Triton X-100 with a final concentration of 0.3% and vortex mix to break the emulsion. Calculate the total protein content in the liposome solution before centrifugation and the protein content in the supernatant after centrifugation according to the protein determination method provided in Example 1, calculate the encapsulation efficiency according to equation 1, and calculate the encapsulation efficiency. The calculation results are shown in Table 1.

[0090] Table 1 Encapsulation efficiency measurement results of Examples 1 to 9

[0091] Group High-speed centrifugation results Membrane permeabilization assay results Example 1 17.96％ 44.16％ Example 2 17.37％ 42.31％ Example 3 16.22％ 37.7％ Example 4 16.9％ 40.3％ Example 5 7.33％ 25.09％ Example 6 6.50％ 19.9％ Example 7 7.01％ 22.46％ Example 8 13.00％ 34.61％ Example 9 12.14％ 29.22％ Example 10 11.23％ 30.4％

[0092] It can be seen that Examples 1 to 10 use different preparation methods to prepare stem cell supernatant liposomes, among which the solvent injection method has a higher encapsulation rate than other methods. The reverse evaporation method can also be used as a preparation method for the supernatant liposomes. The film method has the lowest encapsulation rate, which may be due to the fact that the uniformity of the film of the film method is also difficult to control, resulting in a low encapsulation rate. If the film method is to be used, further optimization is required. When the solvent injection method is used for preparation, the group with the best encapsulation rate effect is Example 1; when the film dispersion method is used for preparation, the group with the best encapsulation rate effect is Example 5; when the reverse evaporation method is used for preparation, the group with the best encapsulation rate effect is Example 8.

[0093] In Examples 1 to 10, three groups with the highest encapsulation efficiency were selected and the encapsulation efficiency was measured using different methods. The results are as follows: Figure 2 As shown in the figure, the encapsulation efficiency determined by the membrane disruption method is higher than that by the high-speed centrifugation method in the two detection methods. The reason may be that the high-speed centrifugation method will destroy the liposome structure and release more free protein. It can be seen from the calculation formula that the more free protein, the lower the encapsulation efficiency value. Therefore, the membrane disruption method is considered to be the most suitable method for detecting the encapsulation efficiency of the stem cell supernatant liposome prepared in the present invention.

[0094] Comparative Example 1

[0095] Compared with Example 1, the difference is that the preparation method of liposomes is different, and the remaining steps are the same. Specifically, in Comparative Example 1, the preparation method of SCCS liposomes is: using the solvent injection method, weighing 20 mg of sitosterol and 80 mg of soybean lecithin, adding 5 mL of ether to dissolve, forming an organic phase; taking 5 mL of SCCS stem cell supernatant with a protein concentration of 2.25 mg / mL, adding 15 mL of PBS to mix, and then slowly dropping the organic phase and in this process, setting the stirring speed to 800 r / min and stirring continuously to completely merge the organic phase and the aqueous phase. Under normal temperature conditions, the stirring speed is set to 800 r / min with a magnetic stirrer, stirring for 2 hours, so that the organic phase is completely volatilized, and SCCS liposomes are obtained.

[0096] The encapsulation efficiency of the liposomes prepared in Comparative Example 1 was measured by the membrane rupture method, and the results showed that the encapsulation efficiency was 32.2%. It can be seen that in Comparative Example 1, during the fusion process of the aqueous phase and the organic phase and the volatilization process of the organic phase, the stirring speed was too high, resulting in uneven particle size distribution, which affected the stability of the liposomes and made the encapsulation efficiency of the liposomes too low.

[0097] Comparative Example 2

[0098] Compared with Example 1, the difference is that the preparation method of liposomes is different, and the remaining steps are the same. Specifically, in Comparative Example 2, the preparation method of SCCS liposomes is: using the solvent injection method, weighing 10 mg of sitosterol and 70 mg of soybean lecithin, adding 5 mL of ether to dissolve, forming an organic phase; taking 5 mL of SCCS stem cell supernatant with a protein concentration of 2.25 mg / mL, adding 15 mL of PBS to mix, and then slowly dripping the organic phase and in this process, setting the stirring speed to 500 r / min and stirring continuously to completely merge the organic phase and the aqueous phase. Under normal temperature conditions, the stirring speed is set to 500 r / min with a magnetic stirrer, stirring for 2 hours, so that the organic phase is completely volatilized, and SCCS liposomes are obtained.

[0099] The encapsulation efficiency of the liposomes prepared in Comparative Example 2 was measured by the membrane disruption method, and the results showed that the encapsulation efficiency was 37.1%. It can be seen that when the amount of sitosterol is used, the fluidity of the phospholipid bilayer membrane may not be effectively regulated, resulting in a decrease in the stability of the membrane and the ability to resist changes in external conditions, thereby reducing the encapsulation efficiency.

[0100] Comparative Example 3

[0101] Compared with Example 4, the difference is that the preparation method of liposomes is different, and the other steps are the same. Specifically, in Comparative Example 3, the preparation method of SCCS liposomes is: using a thin film dispersion method, weighing 20 mg of sitosterol and 80 mg of soybean lecithin, adding 5 mL of chloroform to dissolve, forming an organic phase, at 35 ° C, setting the speed to 50 r / min, and reducing pressure and rotary evaporation (pressure equipment parameter 100 HPa) until a uniform film appears on the wall of the flask, then adding 5 mL of SCCS and 5 mL of PBS mixed solution to continue rotary evaporation to dissolve the film (without reducing pressure or light pressure), it can be seen that the film on the wall falls off obviously, and the rotary evaporation is stopped to obtain SCCS liposomes.

[0102] The encapsulation efficiency of the liposomes prepared in Comparative Example 4 was measured by the membrane breaking method, and the results showed that the encapsulation efficiency was 20.7%. It can be seen that during the rotary evaporation process, when the rotation speed is too high, the uniform distribution of the liquid will be affected, and the uneven liquid distribution will lead to differences in the thickness and properties of each region after the film is formed, and bulges or holes may appear, resulting in uneven film formation, which will deteriorate the formation and drug loading performance of the liposomes.

[0103] Comparative Example 4

[0104] Compared with Example 4, the difference is that the preparation method of liposomes is different, and the other steps are the same. Specifically, in Comparative Example 4, the preparation method of SCCS liposomes is: using a thin film dispersion method, weighing 20 mg of sitosterol and 80 mg of soybean lecithin, adding 5 mL of chloroform to dissolve, forming an organic phase, at 40 ° C, setting the speed to 30 r / min, and reducing pressure and rotary evaporation (pressure equipment parameter 100 HPa) until a uniform film appears on the wall of the flask, then adding 5 mL of SCCS and 5 mL of PBS mixed solution to continue rotary evaporation to dissolve the film (without reducing pressure or light pressure), it can be seen that the film on the wall falls off obviously, and the rotary evaporation is stopped to obtain SCCS liposomes.

[0105] The encapsulation efficiency of the liposomes prepared in Comparative Example 4 was measured by the membrane rupture method, and the results showed that the encapsulation efficiency was 22.3%. It can be seen that when the temperature is too high during the rotary evaporation process, the organic solvent will be affected to evaporate uniformly, resulting in uneven film formation, which will deteriorate the formation and drug loading performance of the liposomes.

[0106] Comparative Example 5

[0107] Compared with Example 7, the difference is that the preparation method of liposomes is different, and the other steps are the same. Specifically, in Comparative Example 5, 25 mg of sitosterol and 85 mg of soybean lecithin are weighed and added to 20 mL of ether to dissolve to form an organic phase by reverse evaporation method; 10 mL of SCCS is taken, and the aqueous phase is slowly injected into the organic phase under magnetic force at room temperature, and colostrum is obtained by ultrasound. The organic solvent is removed by reduced pressure evaporation in a rotary evaporator (pressure parameter is set to 900 HPa) under a 35°C water bath, and the rotary evaporation is continued to form a colloidal substance and a small part of the film. Then 15 mL of PBS is added, and the rotary evaporation is continued for about 5 minutes to make the film completely fall and dissolve, and the SCCS liposomes are obtained.

[0108] The encapsulation efficiency of the liposomes prepared in Comparative Example 5 was measured by the membrane rupture method, and the results showed that the encapsulation efficiency was 26.4%. It can be seen that in the reverse evaporation process, an emulsion is formed first, and then the organic solvent is evaporated. When the volume of the inner aqueous phase is too high, the emulsification effect of the organic phase and the aqueous phase is affected, and fusion and rupture are more likely to occur, so that the encapsulation efficiency of the liposome is reduced.

[0109] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0110] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing stem cell supernatant liposomes, characterized in that: include: Cultivating spermatogonial stem cells and collecting spermatogonial stem cell supernatant; Using an organic solvent to dissolve sitosterol and soybean lecithin to form an organic phase; The spermatogonial stem cell supernatant is mixed with the organic phase by a solvent injection method, a thin film dispersion method or a reverse evaporation method to obtain stem cell supernatant liposomes.

2. The preparation method according to claim 1, characterized in that The mass volume ratio of the sitosterol, soybean lecithin and organic solvent is: 15mg-25mg: 75mg-85mg: 5mL-25mL; the protein concentration in the spermatogonial stem cell supernatant is 2mg / mL-2.5mg / mL.

3. The preparation method according to claim 2, characterized in that: The organic solvent includes methanol, ethanol, ether or chloroform.

4. The preparation method according to claim 2, characterized in that: The steps of the solvent injection method include: adding PBS to the supernatant of the spermatogonial stem cells and mixing to form an aqueous phase; The organic phase is then added to the aqueous phase, the stirring speed is set to 400 r / min to 500 r / min, and the organic phase and the aqueous phase are merged, and then the stirring speed is set to 400 r / min to 500 r / min, and stirred for 2 h to 3 h to volatilize the organic solvent to obtain the stem cell supernatant liposomes; the organic solvent is ether.

5. The preparation method according to claim 2, characterized in that: The steps of the thin film dispersion method include: performing reduced pressure rotary evaporation on the organic phase to form a thin film on the wall of the flask, then adding a mixture of the spermatogonial stem cell supernatant and PBS, and continuing to rotary evaporate to dissolve the thin film to obtain the stem cell supernatant liposomes; the organic solvent is chloroform; the temperature of the reduced pressure rotary evaporation is 32°C to 35°C, and the speed of the reduced pressure rotary evaporation is 25r / min to 40r / min.

6. The preparation method according to claim 2, characterized in that: The reverse evaporation method comprises the following steps: taking the supernatant of the spermatogonial stem cells as an aqueous phase, adding the aqueous phase into the organic phase, and ultrasonically obtaining colostrum; the organic solvent in the organic phase is ether; the volume ratio of the aqueous phase to the organic phase is 1:2-5; The organic solvent in the colostrum is removed by using a rotary evaporator, and the rotary evaporation is continued to form a thin film, and then PBS is added to completely dissolve the thin film to obtain the stem cell supernatant liposome.

7. The preparation method according to claim 6, characterized in that: The volume ratio of the PBS to the supernatant of the spermatogonial stem cells is 3 mL to 5 mL: 13 mL to 15 mL.

8. The stem cell supernatant liposome prepared by the preparation method according to any one of claims 1 to 7.

9. The method for detecting the liposome encapsulation efficiency of stem cell supernatant according to claim 8, characterized in that: include: High-speed centrifugation or membrane disruption.

10. Use of the stem cell supernatant liposome according to claim 8 in the preparation of cosmetics for anti-aging or skin regeneration and repair.

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