A stem cell-derived nanoprotein and a preparation method and application thereof

Abstract

The present application relates to a kind of stem cell-derived nano-proteins with inflammation regulation and tissue repair effect, the stem cell-derived nano-proteins are prepared by microfluidic process from bioactive substance derived from stem cells and soluble calcium salt.The stem cell-derived nano-proteins prepared by the present application have moderate particle size, uniform distribution, stable and controllable quality, simple preparation process operation, and excellent effect in the repair and regeneration of inflammation damage type tissue organs and cosmetic repair.

Description

Technical Field

[0001] This invention belongs to the field of regenerative medicine, specifically relating to a stem cell-derived nanoprotein for treating inflammation and tissue repair. Background Technology

[0002] With the development of regenerative medicine, stem cell therapy, as a treatment method that directly intervenes in the repair process, has received widespread attention from researchers and has been extensively studied in the areas of inflammation regulation and tissue repair. The potential of stem cell therapy lies in its ability to directly promote wound tissue regeneration or to regulate inflammation and promote tissue repair at the molecular level through nutritional and paracrine effects.

[0003] Mesenchymal stem cells (MSCs) are adult stem cells derived from the mesodermal mesenchyme of tissues, possessing multi-lineage differentiation and self-renewal potential. They exhibit typical trilineage differentiation potential and are widely used in preclinical and clinical research for various diseases. However, as a cell therapy, the homing ability and therapeutic efficacy of MSCs after subcutaneous or intravenous transplantation are often limited due to their large particle size and certain immunogenicity. Furthermore, MSC preparations present challenges in preservation and transportation, with cell viability often significantly decreasing from cell culture to clinical application. More critically, stem cells may pose a potential tumorigenic risk when used in vivo. These factors limit the promotion and practical application of MSCs in product development and clinical practice.

[0004] With the deepening development of cell therapy research, researchers have discovered that mesenchymal stem cells (MSCs) exert significant therapeutic effects by releasing therapeutic cytokines and extracellular vesicles carrying their bioactive components through paracrine pathways. For example, anti-inflammatory cytokines such as interleukin-4 (IL-4) and interleukin-10 (IL-10) secreted by MSCs can effectively regulate inflammatory responses; simultaneously, they secrete basic fibroblast growth factor (bFGF) and transforming growth factor β1 (TGF-β1), which can induce fibroblast proliferation and differentiation, collagen deposition, and promote tissue repair. Furthermore, exosomes secreted by MSCs can effectively regulate inflammation, promote cell proliferation and migration, and angiogenesis by modulating intercellular communication. Studies have confirmed that stem cell culture supernatants contain a large number of therapeutic growth factors produced by stem cells through paracrine pathways, as well as extracellular vesicles containing their bioactive components, which have high application value in diseases involving damage repair.

[0005] Although stem cell culture supernatants show potential for repairing inflammatory diseases, their clinical application still faces several key technical challenges. First, most therapeutic cytokines produced by stem cell paracrine secretion are macromolecules, susceptible to enzymatic degradation and exhibiting poor stability. Second, these macromolecular drugs have limited membrane permeability, making it difficult to target intracellular molecules. Furthermore, the production, storage, transportation, and quality control of stem cell-derived extracellular vesicles present significant bottlenecks, hindering stable large-scale production. These factors severely limit the mass production and clinical application of stem cell-derived drugs.

[0006] There is an urgent need to develop a protein nanoparticle containing stem cell-derived bioactive components that is simple to prepare, highly stable, and easy to control in order to solve the problem of high difficulty in the clinical translation of stem cell-derived drugs. Summary of the Invention

[0007] In view of the above-mentioned technical defects, namely, the therapeutic cytokines produced by stem cell paracrine secretion are easily degraded by enzymes and have poor stability; the membrane permeability of cytokine drugs is limited, making it difficult to act on intracellular targets; and the secreted cell vesicles cannot be mass-produced, standardized, quality-controlled, and stably preserved. The purpose of this invention is to provide stem cell-derived nanoproteins (SCNPs) that can be mass-produced and quality-controlled, are easy to industrialize, have better stability, and at the same time have stronger repair and inflammation regulation functions, as well as their preparation method and applications.

[0008] To address the aforementioned technical problems, a first aspect of the present invention provides a stem cell-derived nanoprotein with inflammation regulation and tissue repair functions, wherein the stem cell-derived nanoprotein is prepared from bioactive substances derived from stem cells and soluble calcium salts.

[0009] Preferably, the average particle size of the stem cell-derived nanoprotein is 50-200 nm. More preferably, the average particle size of the nanoprotein is 50-150 nm. Even more preferably, the average particle size of the nanoprotein is 80-150 nm. Even more preferably, the average particle size of the nanoprotein is 80-120 nm.

[0010] Preferably, the polydispersity index (PDI) of the stem cell-derived nanoprotein is 0.05-0.30. More preferably, the polydispersity index (PDI) of the stem cell-derived nanoprotein is 0.05-0.25. Even more preferably, the polydispersity index (PDI) of the stem cell-derived nanoprotein is 0.05-0.20. Even more preferably, the polydispersity index (PDI) of the stem cell-derived nanoprotein is 0.05-0.15. Even more preferably, the polydispersity index (PDI) of the stem cell-derived nanoprotein is 0.1-0.15.

[0011] Preferably, the stem cell-derived nanoprotein can be filtered through a 0.22 μm filter membrane.

[0012] Preferably, the stem cell-derived nanoprotein has a spherical morphology and a uniform texture.

[0013] Preferably, the stem cell-derived nanoprotein is prepared by mixing a solution of bioactive substances derived from stem cells with a soluble calcium salt solution using a microfluidic device.

[0014] Preferably, the flow rate ratio of the stem cell-derived bioactive substance solution to the soluble calcium salt solution in the microfluidic device is 1:2 to 1:6. More preferably, the flow rate ratio of the stem cell-derived bioactive substance solution to the soluble calcium salt solution in the microfluidic device is 1:3 to 1:5. Even more preferably, the flow rate ratio of the stem cell-derived bioactive substance solution to the soluble calcium salt solution in the microfluidic device is 1:3 to 1:4.

[0015] Preferably, the flow rate of the stem cell-derived bioactive substance solution in the microfluidic device is 1-10 mL / min, and the flow rate of the soluble calcium salt solution is 1-20 mL / min. More preferably, the flow rate of the stem cell-derived bioactive substance solution in the microfluidic device is 1-5 mL / min, and the flow rate of the soluble calcium salt solution is 5-10 mL / min. Even more preferably, the flow rate of the stem cell-derived bioactive substance solution in the microfluidic device is 2-4 mL / min, and the flow rate of the soluble calcium salt solution is 8-10 mL / min. Even more preferably, the flow rate of the stem cell-derived bioactive substance solution in the microfluidic device is 3 mL / min, and the flow rate of the soluble calcium salt solution is 9 mL / min.

[0016] Preferably, the soluble calcium salt is selected from one or more of calcium chloride, calcium gluconate, calcium lactate, calcium gluconate, calcium phosphate, calcium bromide, calcium iodide, calcium nitrate, calcium sulfate, calcium acetate, and calcium oxalate. More preferably, the soluble calcium salt is selected from one or more of calcium chloride, calcium gluconate, calcium lactate, calcium gluconate, and calcium phosphate. Even more preferably, the soluble calcium salt is calcium chloride.

[0017] Preferably, the concentration of the soluble calcium salt is 10-60 mg / mL. More preferably, the concentration of the soluble calcium salt is 30-60 mg / mL. Even more preferably, the concentration of the soluble calcium salt is 30-50 mg / mL. Even more preferably, the concentration of the soluble calcium salt is 30-40 mg / mL.

[0018] Preferably, the protein concentration in the stem cell-derived bioactive substance solution is 0.1-10 mg / mL, and the total nucleic acid concentration is 5-500 μg / mL. More preferably, the protein concentration in the stem cell-derived bioactive substance solution is 1-10 mg / mL, and the total nucleic acid concentration is 10-200 μg / mL. Preferably, the protein concentration in the stem cell-derived bioactive substance solution is 1-10 mg / mL, and the total nucleic acid concentration is 50-200 μg / mL. More preferably, the protein concentration in the stem cell-derived bioactive substance solution is 2 mg / mL, and the total nucleic acid concentration is 100 μg / mL.

[0019] Preferably, the preparation method of the solution of bioactive substances derived from stem cells is as follows: the stem cells are subjected to low-temperature homogenization treatment, the bioactive components derived from stem cells are extracted and collected, and impurities are removed by centrifugation to obtain the solution.

[0020] Preferably, the method for preparing the solution of bioactive substances derived from stem cells is as follows:

[0021] (1) Culture stem cells in a culture medium and collect the cells when the cell confluence reaches 70-90%.

[0022] (2) The collected cell suspension was passed through a low-temperature homogenization device to harvest a solution of bioactive components derived from stem cells;

[0023] (3) Remove cell debris and cell vesicles from the bioactive component solution obtained in step (2) by ultra-high speed centrifugation, and take the impurity-free stem cell-derived supernatant to obtain the product.

[0024] Preferably, the stem cells in step (1) are mesenchymal stem cells derived from umbilical cord, placenta, adipose tissue, bone marrow, dental pulp, menstrual blood, umbilical cord blood, induced pluripotent stem cells, epidermal stem cells, or endometrial stem cells. More preferably, the stem cells are mesenchymal stem cells derived from human umbilical cord.

[0025] Preferably, the culture medium in step (1) is DMEM / F12 culture medium supplemented with 5% human platelet lysis buffer (HPL).

[0026] Preferably, the ultra-high speed centrifugation in step (3) is performed at 300,000g under 4°C conditions.

[0027] A second aspect of the present invention provides a method for preparing stem cell-derived nanoproteins, the method comprising the following steps:

[0028] (1) Culture stem cells in a culture medium and collect the cells when the cell confluence reaches 70-90%.

[0029] (2) The collected cell suspension was passed through a low-temperature homogenization device to harvest a solution of bioactive components derived from stem cells;

[0030] (3) Remove cell debris and cell vesicles from the bioactive component solution obtained in step (2) by ultra-high speed centrifugation, and take the impurity-free stem cell-derived supernatant.

[0031] (4) Stem cell-derived nanoproteins were prepared by combining the obtained impurity-free supernatant with a soluble calcium salt solution using a microfluidic device.

[0032] Preferably, the stem cells in step (1) are mesenchymal stem cells derived from umbilical cord, placenta, adipose tissue, bone marrow, dental pulp, menstrual blood, umbilical cord blood, induced pluripotent stem cells, epidermal stem cells, or endometrial stem cells. More preferably, the stem cells are mesenchymal stem cells derived from human umbilical cord.

[0033] Preferably, the culture medium in step (1) is DMEM / F12 culture medium supplemented with 5% human platelet lysis buffer (HPL).

[0034] Preferably, the ultra-high speed centrifugation in step (3) is performed at 300,000g under 4°C conditions.

[0035] Preferably, the soluble calcium salt in step (4) is selected from one or more of calcium chloride, calcium gluconate, calcium lactate, calcium gluconate, calcium phosphate, calcium bromide, calcium iodide, calcium nitrate, calcium sulfate, calcium acetate, and calcium oxalate. More preferably, the soluble calcium salt in step (4) is selected from one or more of calcium chloride, calcium gluconate, calcium lactate, calcium gluconate, and calcium phosphate. Even more preferably, the soluble calcium salt in step (4) is calcium chloride.

[0036] Preferably, the concentration of the soluble calcium salt in step (4) is 10-60 mg / mL. More preferably, the concentration of the soluble calcium salt in step (4) is 30-60 mg / mL. Even more preferably, the concentration of the soluble calcium salt in step (4) is 40-60 mg / mL. Even more preferably, the concentration of the soluble calcium salt in step (4) is 40 mg / mL, 50 mg / mL, or 60 mg / mL.

[0037] Preferably, in step (4), the protein concentration in the bioactive substance solution derived from stem cells is 0.1–10 mg / mL, and the total nucleic acid concentration is 5–500 μg / mL. More preferably, in step (4), the protein concentration in the bioactive substance solution derived from stem cells is 2 mg / mL, and the total nucleic acid concentration is 100 μg / mL.

[0038] Preferably, the flow rate ratio of the impurity-free supernatant to the soluble calcium salt in step (4) is 1:2 to 1:6. More preferably, the flow rate ratio of the impurity-free supernatant to the soluble calcium salt in step (4) is 1:3 to 1:5. Even more preferably, the flow rate ratio of the impurity-free supernatant to the soluble calcium salt in step (4) is 1:3 to 1:4.

[0039] Preferably, in step (4), the flow rate of the impurity-free supernatant is 1-10 mL / min, and the flow rate of the soluble calcium salt solution is 1-20 mL / min. More preferably, in step (4), the flow rate of the impurity-free supernatant is 1-5 mL / min, and the flow rate of the soluble calcium salt solution is 5-10 mL / min. Further preferably, in step (4), the flow rate of the impurity-free supernatant is 2-4 mL / min, and the flow rate of the soluble calcium salt solution is 8-10 mL / min. Further preferably, in step (4), the flow rate of the impurity-free supernatant is 3 mL / min, and the flow rate of the soluble calcium salt solution is 9 mL / min.

[0040] A third aspect of the present invention provides the use of the above-described stem cell-derived nanoprotein in the preparation of medicaments for the prevention or treatment of diseases selected from genetic diseases, cancer, immune diseases, central nervous system diseases, inflammatory diseases, cardiovascular diseases, or infectious diseases.

[0041] Preferably, the disease is an inflammatory disease.

[0042] The fourth aspect of the present invention provides the use of the above-mentioned stem cell-derived nanoprotein in the preparation of medical aesthetic products, wherein the medical aesthetic products have one or more effects selected from hair growth, whitening, wrinkle removal, firming, freckle removal, improvement of dark circles, and improvement of rosacea.

[0043] The fifth aspect of the present invention provides the use of the above-described stem cell-derived nanoprotein in the preparation of a drug that inhibits the secretion of pro-inflammatory cytokines.

[0044] Preferably, the pro-inflammatory cytokines are IL-6 and / or TNF-α.

[0045] The sixth aspect of the present invention provides the use of the above-described stem cell-derived nanoprotein in the preparation of a medicament that promotes the secretion of repair-type cytokines.

[0046] Preferably, the repair cytokine is IL-4 and / or IL-10.

[0047] The seventh aspect of the present invention provides the use of the above-described stem cell-derived nanoprotein in the preparation of a medicament that promotes the proliferation of human dermal cells (HDF).

[0048] The eighth aspect of the present invention provides the use of the above-described stem cell-derived nanoprotein in the preparation of a medicament that promotes collagen synthesis.

[0049] Preferably, the ability to promote collagen synthesis is the ability to promote type I collagen synthesis.

[0050] The beneficial effects of this invention are:

[0051] 1. The stem cell-derived nanoprotein of the present invention is in the form of nanoparticles with a particle size that can be stably controlled between 50-200 nm. It is uniform in size and has typical nanoparticle morphology and function. It can induce inflammatory macrophages to polarize into anti-inflammatory and repair-type macrophages, reduce the secretion of pro-inflammatory factors, and release a large amount of repair-type cytokines. Compared with stem cell exosomes and transforming growth factor β1, it has a more powerful immunomodulatory and repair function.

[0052] 2. The stem cell-derived nanoprotein provided by this invention has superior stability compared to stem cell exosomes and transforming growth factor β1, is not easily degraded, and its nanostructure makes it easier to cross the cell membrane barrier and effectively act on intracellular targets.

[0053] 3. The stem cell-derived nanoprotein of the present invention contains calcium ions (Ca). 2 It can directly participate in the regulation of cell metabolism, promote the biosynthesis of collagen and elastin, the main components of the extracellular matrix (ECM), and work synergistically with the bioactive components derived from stem cells to endow stem cell-derived nanoproteins with a strong ability to promote collagen secretion and extracellular matrix synthesis. At the same time, it can activate cell cycle-related signaling pathways, promote cell mitosis, and accelerate the process of tissue regeneration.

[0054] 4. The stem cell-derived nanoprotein of the present invention is a non-living cell and cell vesicle preparation, which can avoid the safety risks associated with the use of exogenous cell transplantation when applied in vivo.

[0055] 5. The stem cell-derived nanoprotein preparation process of the present invention is simple, highly operable, and produces stable and controllable product quality, thus possessing significant industrialization value. Attached Figure Description

[0056] Figure 1 This is an image showing the particle size distribution of the stem cell-derived nanoprotein of the present invention;

[0057] Figure 2 This is a transmission electron microscope image of the stem cell-derived nanoprotein of the present invention;

[0058] Figure 3 The results of the inflammation regulation experiment of stem cell-derived nano proteins (NPs), stem cell exosomes (Exo), and transforming growth factor-β1 (TGF-β1) of this invention are as follows;

[0059] Figure 4 The results of cell proliferation experiments using stem cell-derived nano proteins (NPs), stem cell exosomes (Exo), and transforming growth factor-β1 (TGF-β1) of this invention are as follows:

[0060] Figure 5 The results of the collagen synthesis-promoting experiments of stem cell-derived nano proteins (NPs), stem cell exosomes (Exo), and transforming growth factor-β1 (TGF-β1) of this invention are as follows:

[0061] Figure 6 The results of the stability test of the stem cell-derived nanoprotein of this invention are shown. Detailed Implementation

[0062] The experimental materials, reagents, and instruments used in the experimental examples of this invention are all commercially available. The invention will be further described in detail below with reference to the experimental examples and accompanying drawings, but the embodiments of this invention are not limited thereto. Unless otherwise specified, the significance symbols in the accompanying drawings are defined as follows: * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0063] Experimental Example 1: Screening Experiment of Calcium Ion Concentration in the Preparation of Stem Cell-Derived Nanoproteins

[0064] 1. Test Methods

[0065] Third-generation human umbilical cord-derived mesenchymal stem cells (hU-MSCs) were seeded in cell culture dishes and cultured in DMEM / F12 medium supplemented with 5% human platelet lysis buffer (HPL). When the cells reached 90% confluence, they were collected and lysed using a low-temperature homogenization device to extract their bioactive substances. The concentrated bioactive substance solution was centrifuged at 300,000g at 4°C to remove cell vesicles and other particulate impurities. Subsequently, the impurity-free concentrated supernatant (i.e., bioactive substances derived from stem cells with a biological protein concentration of 2 mg / mL and a total nucleic acid concentration of 100 μg / mL) was mixed with 10, 20, 30, 40, 50, and 60 mg / mL of injectable calcium chloride using a microfluidic device. The total flow rate was fixed at 12 mL / min, and the flow rate ratio was 1:3. This allowed the two substances to combine through electrostatic and calcium complexation interactions, resulting in stem cell-derived nanoproteins of suitable size and uniform distribution. Take an appropriate amount of stem cell-derived nanoprotein solution and use a Malvern particle size potential analyzer to detect its particle size distribution.

[0066] 2. Test Results

[0067] Calcium ion concentration plays a regulatory role in the preparation of stem cell-derived nanoproteins. As shown in Table 1, under microfluidic conditions with a fixed flow rate ratio (1:3), a calcium ion concentration of 10 mg / mL effectively binds to the bioactive components derived from stem cells to form nanoparticles. These nanoparticles exhibit an ideal particle size distribution (approximately 80 nm), but the yield is low. With increasing calcium ion concentration, the yield of stem cell-derived nanoproteins increases in a concentration-dependent manner, while the particle size gradually increases, and the polydispersity index slightly increases. Notably, when the calcium ion concentration exceeds 40 mg / mL, the nanoparticle yield exhibits a plateau effect, no longer significantly increasing with further increases in calcium ion concentration. At this point, the increase in calcium ion concentration mainly results in an increase in nanoparticle size, rather than a further increase in yield. Given the requirements for nanoparticle size in the terminal membrane sterilization process, a concentration range of 30-50 mg / mL was ultimately determined to be the suitable calcium ion concentration range for preparing stem cell-derived nanoproteins. This concentration range ensures both high yield of stem cell-derived nanoproteins and maintains good particle size uniformity and membrane permeability. In subsequent experimental examples, a calcium ion concentration of 40 mg / mL was used to prepare stem cell-derived nanoproteins.

[0068] Table 1. Particle size, PDI, and particle number concentration (n=3) of NPs prepared with different concentrations of calcium ions.

[0069]

[0070] Experimental Example 2: Flow Rate Ratio Screening Experiment in the Preparation of Stem Cell-Derived Nanoproteins

[0071] 1. Test Methods

[0072] Third-generation human umbilical cord-derived mesenchymal stem cells (hU-MSCs) were seeded in cell culture dishes and cultured in DMEM / F12 medium supplemented with 5% human platelet lysis buffer (HPL). When the cells reached 90% confluence, they were collected and lysed using a low-temperature homogenization device to extract bioactive substances. The concentrated bioactive substance solution was then centrifuged at 300,000g at 4°C to remove cell vesicles and other particulate impurities. The impurity-free supernatant (i.e., a biological protein concentration of 2 mg / mL) was concentrated using a microfluidic device. A mixture of stem cell-derived bioactive substances with a total nucleic acid concentration of 100 μg / mL and injectable calcium chloride at a concentration of 40 mg / mL was prepared. The total flow rate was fixed at 12 mL / min, with flow ratios of 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6 (e.g., a flow ratio of 1:2 means the stem cell-derived bioactive substance solution was flowed at 4 mL / min, and the injectable calcium chloride at 8 mL / min). This allowed the two substances to bind together through electrostatic and calcium complexation interactions, resulting in stem cell-derived nanoproteins of suitable size and uniform distribution. An appropriate amount of the stem cell-derived nanoprotein solution was taken, and its particle size distribution was analyzed using a Malvern particle size potential analyzer.

[0073] 2. Test Results

[0074] As shown in Table 2, at a flow rate ratio of 1:1, calcium ions cannot effectively bind with stem cell-derived bioactive components to self-assemble into nanoparticles. As the flow rate ratio increases to 1:2, nanoparticles with suitable size and uniform distribution can be obtained, but the particle number concentration and yield are low. Further increasing the flow rate ratio to 1:3 and 1:4, the number of stem cell-derived nanoprotein particles gradually increases, with particle sizes around 110 nm and a PDI less than 0.2, indicating that an appropriate flow rate ratio helps stem cell-derived bioactive components effectively bind with calcium ions, thereby assembling into nanoparticles and improving yield. However, with further increases in the flow rate ratio, the number of stem cell-derived nanoprotein particles gradually decreases, and the particle size and distribution differences of the prepared nanoproteins significantly increase. These results indicate that when the mixing flow rate ratio is too small, it is difficult for calcium ions to efficiently bind with stem cell-derived bioactive components, preventing self-assembly into nanoparticles. Conversely, an excessively large flow rate ratio easily leads to excessively large particle sizes and increased distribution differences in the prepared stem cell-derived nanoproteins, resulting in a decrease in yield. Therefore... Only by selecting an appropriate flow rate ratio can the formation of stem cell-derived nanoproteins be facilitated.

[0075] like Figure 1As shown, the stem cell-derived nanoproteins prepared with a flow rate ratio of 1:3 exhibited uniform particle size distribution, with an average particle size of 115 nm and a PDI of 0.13, indicating that nanoscale protein particles could be successfully prepared under these conditions. The stem cell-derived nanoproteins used in subsequent experiments 3-7 were all prepared using this process.

[0076] Table 2. Particle size, PDI, and particle number concentration (n=3) of NPs prepared at different flow rate ratios.

[0077]

[0078]

[0079] Note: " / " in the table indicates that no relevant detection data could be obtained because nanoparticles were not formed.

[0080] Experimental Example 3: Morphological Characterization of Stem Cell-Derived Nanoprotein Structures

[0081] 1. Test Methods

[0082] Take 2-3 drops of the stem cell-derived nanoprotein suspension prepared in Experiment Example 2 and drop it onto a copper grid. Let it stand at room temperature for 10 minutes to settle. Wash with deionized water, absorb excess liquid with filter paper, add uranium acetate staining solution for negative staining, dry at room temperature, and then place the copper grid under a 120kV transmission electron microscope to observe the morphology and structure of the stem cell-derived nanoprotein and take pictures to record.

[0083] 2. Test Results

[0084] like Figure 2 As shown in the transmission electron microscope (TEM) images, the stem cell-derived nanoprotein particles exhibit a near-spherical structure and uniform texture. The particle size observed by TEM is consistent with the particle size measurement results.

[0085] Experiment 4: Investigation of the Inflammation Regulation Capacity of Stem Cell-Derived Nanoproteins

[0086] 1. Preparation of test samples

[0087] The main methods for preparing exosomes include ultracentrifugation, density gradient centrifugation, ultrafiltration, precipitation reagent method, immunoaffinity capture method, and microfluidic technology, among which ultracentrifugation is the most commonly used basic method.

[0088] In this experimental example, stem cell exosomes were extracted and prepared as a comparative example using ultracentrifugation. The specific method is as follows: 5 × 10⁶ cells / day were taken... 6The culture supernatant of stem cells was centrifuged at 300g and 2000g for 10 min to remove dead cells. The supernatant was then centrifuged at 10000g for 30 min to remove large vesicles. The processed cell supernatant was collected and filtered through a 0.22μm filter membrane. The processed cell supernatant was then transferred into an ultracentrifuge tube and centrifuged at 100000g for 70 min at 4℃. The supernatant was removed, and the exosome precipitate was resuspended in an appropriate amount of PBS buffer to obtain the final product.

[0089] Transforming growth factor β1 (TGF-β1), used as a comparative example, was purchased from Suzhou Nearshore Protein Technology Co., Ltd. The preparation method of stem cell-derived nanoproteins is described in Experimental Example 2.

[0090] 2. Test methods

[0091] RAW264.7 cells were loaded at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density in 12-well cell culture plates and cultured at 37°C for 24 hours. After removing the culture medium, the cells were incubated for 4 hours in DMEM medium containing stem cell-derived nanoproteins (NPs), stem cell exosomes (Exo), and transforming growth factor β1 (TGF-β1) (the total protein concentration of the drugs in each experimental group was 40 μg / mL). Then, LPS was added to the culture medium to a final concentration of 5 μg / mL. After culturing for another 24 hours, the levels of TNF-α, IL-6, IL-4, and IL-10 in the cells of each group were detected by ELISA kit (n=3).

[0092] 3. Test Results

[0093] Persistent inflammation is a common clinical manifestation of inflammatory injuries and general skin diseases, mainly due to the release of inflammatory factors and other inflammatory mediators, which hinders wound healing and interferes with treatment and repair. Therefore, inhibiting the inflammatory response and regulating inflammatory factors are crucial for the treatment of such diseases. To investigate the effects of the stem cell-derived nanoprotein prepared in this invention on these cytokines, this experiment used stem cell exosomes (Exo) and transforming growth factor β1 (TGF-β1) as controls, and evaluated the inflammatory regulatory capacity of the stem cell-derived nanoprotein using an ELISA kit.

[0094] When RAW264.7 cells were stimulated with lipopolysaccharide (LPS), the expression levels of pro-inflammatory cytokines IL-6 and TNF-α were significantly increased compared to the control group, while the expression levels of repair cytokines IL-4 and IL-10 showed no significant change. After co-culturing with the stem cell-derived nanoprotein described in this invention, the secretion of pro-inflammatory cytokines IL-6 and TNF-α in RAW264.7 cells was significantly decreased, and significantly lower than other treatment groups, while the secretion of repair cytokines IL-4 and IL-10 was significantly higher than other treatment groups (see...). Figure 3 ).

[0095] The above experimental results show that the stem cell-derived nanoprotein prepared in this invention can effectively reduce the secretion of inflammatory cytokines and promote the secretion of repair cytokines. Furthermore, the inflammation regulation ability of the stem cell-derived nanoprotein is significantly better than that of the stem cell exosome group and the transforming growth factor β1 group. This indicates that nanoparticleization of bioactive components derived from stem cells can significantly enhance their therapeutic effects, providing an efficient and precise treatment solution for inflammatory tissue damage and general skin diseases.

[0096] Experimental Example 5: Investigation of the Cell Proliferation-Promoting Ability of Stem Cell-Derived Nanoproteins

[0097] 1. Test Methods

[0098] Human dermal cells (HDF) in the logarithmic growth phase and in good growth condition were collected. Adherent cells were digested with 0.25% trypsin for 5 min, digestion was stopped by adding complete culture medium, and the cells were centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the bottom cells were resuspended in 1 mL of culture medium and counted. Cells were then cultured at 2 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of [number] cells / well in 96-well cell culture plates. After cell attachment, the culture medium was discarded, and stem cell-derived nanoproteins (NPs), stem cell exosomes (Exo), and transforming growth factor β1 (TGF-β1) were added to each well for drug administration (sample source or preparation method was the same as in Experiment 4, and the total protein concentration of each drug in each experimental group was 40 μg / mL). The plates were then incubated at 37°C for further culture. At 0h, 12h, 24h, and 48h after drug administration, the culture medium was removed, and 100 μL of fresh culture medium containing 10 μL of CCK-8 reagent was gently added to each well, avoiding the formation of air bubbles. The plates were then incubated at 37°C for another 4h, and the absorbance (OD) of each sample at 450 nm was measured using a multi-mode microplate reader.

[0099] 2. Test Results

[0100] In this embodiment, the proliferative effects of stem cell-derived nanoproteins and control drugs on HDF cells were investigated at different time points using the CCK-8 reagent. The results are as follows: Figure 4 As shown, compared with the control group (PBS group), all three drugs (TGF-β1, Exo and NPs) can promote HDF cell proliferation to a certain extent. However, the cell proliferation-promoting effect of stem cell-derived nanoprotein is significantly better than that of Exo group and TGF-β1 group. This is consistent with the inflammatory regulation ability of stem cell-derived nanoprotein, which further confirms the excellent effect of stem cell-derived nanoprotein prepared in this invention.

[0101] Experiment 6: Investigation of the ability of stem cell-derived nanoproteins to promote collagen synthesis

[0102] 1. Test Methods

[0103] L929 cells (mouse fibroblasts) were used at a rate of 2 × 10⁻⁶. 5 The cells were seeded at a density in 6-well plates and cultured at 37°C for 24 hours. After changing the culture medium, stem cell-derived nanoproteins (NPs), stem cell exosomes (Exo), and transforming growth factor β1 (TGF-β1) were added for drug administration (sample source or preparation method was the same as in Experiment 4, and the total protein concentration of the drugs in each experimental group was 40 μg / mL). After culturing for another 24 hours, the expression level of COL1A1 (α1-1 type collagen) in each group of cells was detected by qPCR (n=3). RNA was extracted from L929 cells using a total RNA extraction kit, and its concentration was detected and purified using Nanodrop2000. Different volumes of each sample were taken according to their concentration and added to PCR tubes, with 500 ng RNA per sample. 4 μL of 4×g DNAwiper mix was added to remove genomic DNA contamination, and DEPC was added to a total volume of 16 μL. The mixture was gently pipetted and incubated at 42°C for 2 min. Then, 4 μL of 5×HiScriptⅡqRT Super MixⅡ reverse transcriptase was added. The samples were placed in a gradient PCR instrument and incubated at 50℃ for 15 min, followed by incubation at 85℃ for 5 s for reverse transcription to obtain cDNA from each sample. The cDNA samples obtained from reverse transcription were diluted 5-fold with DEPC water and then subjected to qRT-PCR. 2 μL of cDNA template, 10 μL of 2×ChamQ SYBR qPCR Master Mix, 0.4 μL each of 50×ROX Reference DyeⅠ fluorescent probe, forward primer (10 μM), and reverse primer (10 μM) were added to the PCR plate. DEPC was added to bring the total volume to 20 μL. The wells of the PCR plate were covered with sealing film and placed in an automated real-time PCR system. The qRT-PCR reaction was performed according to the following steps:

[0104] Pre-denaturation: Reaction at 95℃ for 30s (1.6℃ / s)

[0105] Cyclic amplification: 95℃ reaction for 10s — 60℃ reaction for 30s — signal acquisition

[0106] Melting curve: 95℃ reaction for 15s — 60℃ reaction for 60s — 95℃ reaction for 15s — Signal acquisition

[0107] The Ct value was obtained based on the amplification curve, and ΔT and ΔΔT values ​​were calculated using the following formulas to analyze the COL1A1 and COL1A2 mRNA levels in each group (GAPDH was used as an internal reference gene):

[0108] mRNA expression level (%) = 2 -ΔΔT ×100

[0109] 2. Test Results

[0110] COL1A1 is a major component of type I collagen, and increased COL1A1 mRNA expression directly reflects enhanced type I collagen synthesis. This study used qPCR to quantitatively detect changes in COL1A1 mRNA expression levels in L929 cells after treatment with stem cell-derived nanoproteins (NPs), stem cell exosomes (Exo), and transforming growth factor β1 (TGF-β1), thus investigating the collagen-promoting ability of stem cell-derived nanoproteins.

[0111] The results are as follows Figure 5 As shown, compared with the TGF-β1 group and the Exo group, the expression level of COL1A1 mRNA in L929 cells treated with stem cell-derived nanoprotein group was significantly increased, indicating that the calcium ions (Ca ions) loaded in the nanoprotein group were significantly increased. 2 +) It can directly participate in the regulation of cell metabolism and promote the biosynthesis of cellular collagen. In synergy with the bioactive components derived from stem cells, stem cell-derived nanoproteins have a strong effect on promoting collagen and extracellular matrix (ECM) synthesis, which is crucial for tissue damage repair and skin care.

[0112] Experiment 7: Stability Study of Stem Cell-Derived Nanoproteins

[0113] 1. Test Methods

[0114] Stem cell-derived nanoproteins, stem cell exosomes, and TGF-β1 (sample source or preparation method same as in Example 4) were incubated in DMEM cell culture medium containing 10% fetal bovine serum (FBS) at 37°C. Samples were taken at 0, 4, 8, 12, and 24 h, and the protein concentration changes of the above drugs were determined using a BCA kit.

[0115] 2. Test Results

[0116] In this experiment, Exo, TGF-β1, and stem cell-derived nanoproteins (the total protein concentration of each experimental group was 40 μg / mL) were incubated with DMEM cell culture medium containing 10% fetal bovine serum (FBS) in a 37°C water bath. The stability of each group of samples was then examined by continuously monitoring the changes in protein concentration.

[0117] The experimental results show (see) Figure 6The concentration of TGF-β1 in cell culture medium gradually decreased with prolonged storage time, indicating poor stability and significantly limiting its therapeutic effect. In contrast, stem cell-derived nanoproteins showed better stability than stem cell exosomes, demonstrating excellent stability and significant potential for in vivo application.

[0118] Although specific embodiments of the invention have been described, those skilled in the art will recognize that various changes and modifications can be made to the invention without departing from its scope or spirit. Therefore, the invention is intended to cover all such changes and modifications falling within the scope of the appended claims and their equivalents.

Claims

1. A stem cell-derived nanoprotein with anti-inflammatory and tissue repair functions, characterized in that, The stem cell-derived nanoprotein is prepared from bioactive substances derived from stem cells and soluble calcium salts; the preparation method of the stem cell-derived nanoprotein includes the following steps: (1) Culture stem cells in a culture medium and collect the cells when the cell confluence reaches 70-90%. (2) The collected cell suspension was passed through a low-temperature homogenization device to harvest a solution of bioactive components derived from stem cells; (3) Remove cell debris and cell vesicles from the bioactive component solution obtained in step (2) by ultra-high speed centrifugation, and take the impurity-free stem cell-derived supernatant. (4) The obtained impurity-free supernatant and soluble calcium salt solution were used to prepare stem cell-derived nanoproteins through a microfluidic device; The soluble calcium salt is calcium chloride. The protein concentration in the bioactive substance solution derived from stem cells is 0.1-10 mg / mL, the total nucleic acid concentration is 5-500 μg / mL, the concentration of the soluble calcium salt is 10-60 mg / mL, the flow rate ratio of the bioactive substance solution derived from stem cells to the soluble calcium salt solution in the microfluidic device is 1:2-1:6, and the stem cells are mesenchymal stem cells derived from human umbilical cord.

2. The stem cell-derived nanoprotein according to claim 1, characterized in that, The average particle size of the stem cell-derived nanoprotein is 50-200 nm.

3. The stem cell-derived nanoprotein according to claim 1, characterized in that, The polydispersity index of the stem cell-derived nanoprotein is 0.05-0.

30.

4. The stem cell-derived nanoprotein according to claim 1, characterized in that, The flow rate ratio of the stem cell-derived bioactive substance solution to the soluble calcium salt solution in the microfluidic device is 1:3-1:

5.

5. The stem cell-derived nanoprotein according to claim 4, characterized in that, The flow rate ratio of the stem cell-derived bioactive substance solution to the soluble calcium salt solution in the microfluidic device is 1:3-1:

4.

6. The method for preparing stem cell-derived nanoproteins according to any one of claims 1-5, characterized in that, The preparation method includes the following steps: (1) Culture stem cells in a culture medium and collect the cells when the cell confluence reaches 70-90%. (2) The collected cell suspension was passed through a low-temperature homogenization device to harvest a solution of bioactive components derived from stem cells; (3) Remove cell debris and cell vesicles from the bioactive component solution obtained in step (2) by ultra-high speed centrifugation, and take the impurity-free stem cell-derived supernatant. (4) The obtained impurity-free supernatant and soluble calcium salt solution were used to prepare stem cell-derived nanoproteins through a microfluidic device; The soluble calcium salt is calcium chloride. The protein concentration in the bioactive substance solution derived from stem cells is 0.1-10 mg / mL, the total nucleic acid concentration is 5-500 μg / mL, the concentration of the soluble calcium salt is 10-60 mg / mL, the flow rate ratio of the bioactive substance solution derived from stem cells to the soluble calcium salt solution in the microfluidic device is 1:2-1:6, and the stem cells are mesenchymal stem cells derived from human umbilical cord.

7. The use of the stem cell-derived nanoprotein according to any one of claims 1-5 in the preparation of medical aesthetic products, characterized in that, The medical aesthetic products have one or more effects selected from hair growth, whitening, wrinkle removal, firming, freckle removal, improvement of dark circles, and improvement of redness.

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