Nano-formulation for clearing senescent cells, preparation method therefor, and use thereof

Abstract

A nano-formulation for clearing senescent cells, a preparation method therefor, and use thereof. A gingerenone A-loaded aminated dendritic mesoporous silica nano-formulation is constructed. Dendritic mesoporous silica having a unique central radial pore structure is selected as a drug carrier; the drug carrier is further modified with amino groups, and gingerenone A is loaded onto the aminated dendritic mesoporous silica via non-covalent interactions. The obtained nano-formulation has a uniform particle size, and exhibits the characteristics of high drug loading, high encapsulation efficiency, high biocompatibility, rapid cellular uptake, and low cytotoxicity. Moreover, the nano-formulation can significantly enhance the ability of gingerenone A to clear senescent cells, thereby improving the bioavailability of gingerenone A, and broadening the clinical application of gingerenone A. The nano-formulation has good application prospects in functional foods, pharmaceuticals, and cosmetics.

Description

A nano preparation for removing senescent cells and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, and more particularly to a nano preparation for removing senescent cells and a preparation method and application thereof. BACKGROUND

[0002] Currently, the problem of population aging is increasingly prominent worldwide, making the field of anti-aging a global focus. Aging is a complex and irreversible process characterized by cell cycle arrest and functional impairment. Senescent cells often exhibit cell cycle arrest, functional decline, and secretion of senescence-associated secretory phenotype (SASP) factors, among others. Over time, new cells no longer arise, and old cells continue to die, leading to functional impairment of various tissues and organs. As more and more cells become senescent, the overall function of the body declines, ultimately leading to death. In addition, senescent cells secrete harmful substances such as SASP, which can damage surrounding tissues and promote inflammation and cancer development. Therefore, effectively removing senescent cells can reduce the release of harmful substances, improve tissue function, and reduce the risk of various aging-related diseases, including cancer, heart disease, Alzheimer's disease, and diabetes, thereby achieving the effect of delaying aging. Therefore, effectively removing senescent cells is of great significance.

[0003] Gingerone A (GA) is a natural plant chemical extracted from ginger, which has broad medicinal value, including controlling blood sugar, anti-inflammatory, anti-viral antioxidant, anti-cancer, anti-aging, etc. Existing studies have shown that gingerone A can improve glucose metabolism and control hyperglycemia by inhibiting the S6K1 target, activate the AMPK receptor in adipocytes to inhibit obesity and inflammation, and directly bind to IL-17RA to inhibit inflammation signaling. In addition, the study by Moaddel R et al. has proven that gingerone A can reduce the secretion levels of pro-inflammatory factors IL-6, CCL2 (MCP-1), and interferon gamma-induced protein 10 (IP-10), and increase the expression of anti-inflammatory cytokines IL-10 and IL-13, thereby inducing an increase in the pro-apoptotic marker caspase-3 in senescent cells, leading to apoptosis of senescent cells.

[0004] However, the low solubility of gingerone A in aqueous solution leads to poor bioavailability, greatly limiting the application of gingerone A.

[0005] SUMMARY

[0006] The present application aims to overcome the above-mentioned defects existing in the prior art, and provides a nano preparation for removing senescent cells, a preparation method and application thereof, wherein amino-modified dendritic mesoporous silica is selected as a drug carrier, and zingerone A is loaded on the drug carrier through non-covalent force, so that the ability of zingerone A to remove senescent cells can be significantly enhanced, the bioavailability of zingerone A can be improved, and the clinical application of zingerone A can be widened.

[0007] To achieve the above-mentioned object, the technical scheme of the present application is as follows:

[0008] The present application further discloses a nano preparation for removing senescent cells, which comprises amino-modified dendritic mesoporous silica and zingerone A loaded on the amino-modified dendritic mesoporous silica.

[0009] The present application further discloses a preparation method of the nano preparation for removing senescent cells, which comprises the following steps: dissolving zingerone A in a solvent to obtain a zingerone A solution, and adding the zingerone A solution and amino-modified dendritic mesoporous silica into a buffer solution to react, so that the zingerone A is loaded on the amino-modified dendritic mesoporous silica, and the nano preparation is obtained.

[0010] The present application further discloses the nano preparation for removing senescent cells or the nano preparation for removing senescent cells prepared by the preparation method, wherein the nano preparation for removing senescent cells has a more significant effect on galactosidase in rat plasma than zingerone A and has a longer time effect.

[0011] The present application further discloses an application of the nano preparation for removing senescent cells or the nano preparation for removing senescent cells prepared by the preparation method in a drug delivery system.

[0012] The present application further discloses an application of the nano preparation for removing senescent cells or the nano preparation for removing senescent cells prepared by the preparation method in the preparation of a functional food, a medicine or a cosmetic for removing senescent cells.

[0013] The present application has the following beneficial effects:

[0014] (1) The application first constructs a gingerenone A loaded aminated dendritic mesoporous silica nano preparation, selects dendritic mesoporous silica with a unique central radial pore structure as a drug carrier, and has a dendritic cavity structure, so that the dendritic mesoporous silica has higher pore permeability, larger pore volume, multiple surface functions and good biocompatibility compared with traditional mesoporous silica; and the dendritic mesoporous silica is further modified by amino, and gingerenone A is loaded on the aminated dendritic mesoporous silica by non-covalent force, so that high loading of gingerenone A is realized. The obtained nano preparation has the characteristics of uniform particle size, high drug loading, high encapsulation rate, high biocompatibility, rapid cell uptake and low cytotoxicity, has low toxicity to human dermal fibroblasts (HDF-a), human keratinocytes (Hacat), human liver cells (HepG2) and human embryonic lung cells (WI-38), can significantly enhance the ability of gingerenone A to remove senescent cells, improve the bioavailability of gingerenone A, and broaden the clinical application of gingerenone A. It is a slow-release system that takes advantage of its own advantages and overcomes the drawbacks of drugs. The preparation process is simple, safe, non-toxic and has no side effects, and is easy to industrialize, and has good application prospect in drug delivery system, functional food, medicine and cosmetics.

[0015] (2) The application first designs a gingerenone A loaded aminated dendritic mesoporous silica slow-release system. The aminated dendritic mesoporous silica has good biocompatibility, can be adsorbed on the cell surface and be rapidly taken up by the cell, continuously releases the loaded drug after entering the senescent cell, and the carrier is simultaneously degraded, so that safe and efficient drug delivery is realized, so as to achieve the purpose of removing senescent cells, and the drug can be rapidly released in the environment of senescent cells to avoid premature release in the body circulation and normal tissues.

[0016] (3) The application optimizes the preparation conditions to accurately control the particle size, drug loading rate and encapsulation rate. The prepared nano preparation has uniform particle size, uniform distribution and high encapsulation rate, and has high stability. The preparation process is simple, safe, non-toxic and easy to industrialize. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is an assembly schematic diagram of the GA@AMSN nano preparation of the embodiment of the application.

[0018] Figure 2 is a transmission electron microscope graph of MSN, AMSN and GA@AMSN of the embodiment of the application (A. MMSN; B. AMSN; C. GA@AMSN).

[0019] Figure 3 is a WI-38 and L-929 cytotoxicity graph of AMSN of the embodiment of the application.

[0020] Figure 4 is a diagram of the construction of the HDF-a cell aging model of the embodiment of the present application (A. SA-β-gal staining result diagram of HDF-a cells before ionizing radiation; B. SA-β-gal staining result diagram of HDF-a cells after ionizing radiation).

[0021] Figure 5 is a SA-β-gal staining result diagram of the effect of different concentrations of GA and GA@AMSN on the removal of senescent cells of the embodiment of the present application.

[0022] Figure 6 is an MTT result diagram of the toxicity of different concentrations of GA and GA@AMSN on proliferating HDF-a cells and their removal effect on senescent HDF-a cells (A. Toxicity of zingiberone A on proliferating HDF-a cells and its removal effect on senescent HDF-a cells; B. Toxicity of zingiberone A nano-preparation on proliferating HDF-a cells and its removal effect on senescent HDF-a cells). DETAILED DESCRIPTION

[0023] The present application is further described below in conjunction with specific embodiments, but in no way limits the present application.

[0024] Since zingiberone A has the problems of poor water solubility and low utilization rate, it is particularly important to select a suitable drug delivery body for effective intracellular delivery of zingiberone A. Based on this, the present inventors have conducted in-depth research on nano-drug sustained release systems, and found that mesoporous silica, as an inorganic material, has a regular nanoscale pore structure, presents high pore volume and large specific surface area, and has ordered and controllable pore size, and can be selectively functionalized on the particle surface. In addition, compared with other traditional materials, mesoporous silica has good biocompatibility, low toxicity, and degradability, indicating that mesoporous silica is a potential drug sustained release carrier.

[0025] Therefore, the present application obtained by the present inventors according to these insights is as follows.

[0026] The present application discloses a nano-preparation for removing senescent cells, which comprises amino-functionalized dendritic mesoporous silica and zingiberone A loaded on the amino-functionalized dendritic mesoporous silica.

[0027] Specifically, the dendritic mesoporous silica is selected as the drug carrier, has a dendritic cavity structure, and thus has higher pore permeability, larger pore volume, various surface functions and good biocompatibility compared with traditional mesoporous silica; and the dendritic mesoporous silica is further modified with amino groups, so that high loading of zingerone A is realized by non-covalent force, the obtained nano-preparation has uniform particle size, and has the characteristics of high drug loading, high encapsulation rate, high biocompatibility and low toxicity, can significantly enhance the ability of zingerone A to remove senescent cells, can make zingerone A more effectively play the anti-aging effect, improve the bioavailability of zingerone A, and broaden the clinical application of zingerone A.

[0028] The application further discloses a preparation method of the nano-preparation for removing senescent cells.

[0029] In a specific embodiment, the mass ratio of zingerone A to the amino-functionalized dendritic mesoporous silica in the zingerone A solution is (1-5):1.

[0030] In a specific embodiment, the nano-preparation has ideal drug loading and encapsulation effect, the loading amount of zingerone A in the nano-preparation is 15%-30%, and the encapsulation rate of zingerone A in the nano-preparation is 11%-35%.

[0031] In a specific embodiment, the nano-preparation has a particle size of 50 nm-200 nm, and a specific surface area of 800 m 2 / g-1000 m 2 / g. Specifically, the nano-preparation has a particle size of not more than 200 nm, is easy to enter the inside of a cell, slowly releases the drug in the cell environment, and has the advantages of uniform particle size distribution and large specific surface area.

[0032] In a specific embodiment, the zingerone A is a crystalline or solid powder natural product.

[0033] In a specific embodiment, the buffer solution comprises one or two or more of a phosphate buffer, a borate buffer solution, a citrate buffer solution, an acetate buffer, an imidazole salt buffer solution and a carbonate buffer solution.

[0034] In an embodiment, the pH of the buffer solution is 5.0-7.4.

[0035] In an embodiment, the concentration of zingerone A in the zingerone A solution is 2 mg / mL-6 mg / mL.

[0036] In an embodiment, the solvent includes any one of acetone, acetonitrile, ethanol, methanol, dimethyl sulfoxide, and water. Preferably, the solvent is acetonitrile.

[0037] In an embodiment, the reaction temperature is 25°C-80°C, and the reaction time is 6 h-36 h.

[0038] Specifically, the amino-functionalized dendritic mesoporous silica and zingerone A are mixed in a suitable addition ratio to prepare a nano-preparation in which zingerone A is loaded on the carrier, and the nano-preparation has uniform particle size, ensuring the stability of the nano-preparation, and has high drug loading and high encapsulation efficiency, and can significantly enhance the ability of zingerone A to remove senescent cells, so that zingerone A can more effectively play an anti-aging role.

[0039] In an embodiment, the method for preparing the amino-functionalized dendritic mesoporous silica includes the following steps:

[0040] (1) The template agent and the catalyst are added to water to form an aqueous phase.

[0041] (2) The silicon source is mixed with chlorobenzene to form an oil phase.

[0042] (3) The oil phase and the aqueous phase are subjected to a homogeneous reaction, and after the reaction is completed, centrifugation, drying, and calcination are performed to obtain dendritic mesoporous silica.

[0043] (4) The dendritic mesoporous silica is added to an acetonitrile solution and subjected to amino-functionalization modification with an ammonia source to achieve amino-functionalization modification, thereby obtaining the amino-functionalized dendritic mesoporous silica.

[0044] In an embodiment, the template agent includes one or two of cetyltrimethylammonium chloride, octadecyltrimethylammonium chloride, cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, cetylpyridinium chloride, and dodecyltrimethylammonium bromide. Preferably, the template agent is cetyltrimethylammonium chloride.

[0045] In an embodiment, the catalyst includes one or two or more of triethanolamine, tripropylamine, diethanolamine, ethanolamine, and ammonia. Preferably, the catalyst is triethanolamine.

[0046] In an embodiment, the silicon source includes one or more of tetraethyl orthosilicate, tetramethyl orthosilicate, tetra(2-methoxy-1-methylethyl) orthosilicate, tetraisopropyl orthosilicate, tetrabutyl orthosilicate, and tetraisopropyl orthosilicate. Preferably, the silicon source is tetraethyl orthosilicate.

[0047] In an embodiment, the ammonia source includes one or more of 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, and γ-aminopropyldiethoxymethylsilane. Preferably, the ammonia source is 3-aminopropyltriethoxysilane.

[0048] In an embodiment, the solvent includes one or more of chlorobenzene, cyclohexane, chloroform, dichloromethane, and water.

[0049] In an embodiment, the method for preparing the aminated dendritic mesoporous silica includes: adding a template agent and a catalyst into water and mixing at 40-60°C for 10-30 min to form an aqueous phase, and adding a chlorobenzene solution containing a silicon source, wherein the mass ratio of the template agent, the catalyst, and the silicon source is (20-30):(0.5-1.2):(6-12), mixing at 40-60°C for 12-36 h, centrifuging and drying, and calcining at 300-600°C for 4-8 h to obtain dendritic mesoporous silica; adding the dendritic mesoporous silica and an ammonia source into an acetonitrile solution according to a mass ratio of (0.08-0.1):(50-100), and reacting at 60-85°C for 8-16 h to achieve amination modification, thereby obtaining the aminated dendritic mesoporous silica.

[0050] Specifically, the present application first prepares dendritic mesoporous silica having a unique central radial pore structure in a water / oil two-phase system, and then modifies amino groups on the surface thereof to enable non-covalent binding with zingiberene A into a nano preparation. The present application optimizes the preparation conditions to regulate the structure of the mesoporous silica nanomaterial, including particle size, pore size, and specific surface area, which is conducive to forming a mesoporous structure with a good pore size distribution.

[0051] In an embodiment, the aminated dendritic mesoporous silica prepared by the present application has a particle size of 50-200 nm, a pore size of 2-10 nm, and a specific surface area of 300-1000 m 2 / g. 2 / g.

[0052] In an embodiment, the present application does not have special limitations on the mixing method, and the raw materials can be mixed uniformly.

[0053] The application also discloses application of the nano-preparation for removing senescent cells according to any embodiment of the application in a drug delivery system.

[0054] The application also discloses application of the nano-preparation for removing senescent cells according to any embodiment of the application in preparation of a functional food, a medicine or a cosmetic having the effect of removing senescent cells.

[0055] In a specific embodiment, the nano-preparation can be directly or after a conventional step added in the functional food, the medicine or the cosmetic.

[0056] Specifically, the application first constructs a gingerenone A-loaded aminated dendritic mesoporous silica sustained-release system. The aminated dendritic mesoporous silica has good biocompatibility, can be adsorbed on the cell surface and rapidly taken up by cells, continuously releases the loaded drug after entering the senescent cells, and realizes safe and efficient drug delivery by simultaneously degrading the carrier, so as to achieve the purposes of removing senescent cells and effectively reducing galactosidase activity, can avoid premature release of the drug in the body circulation and normal tissues and rapid release in the senescent cell environment, has superior biocompatibility, rapid cell uptake and low cytotoxicity, is a sustained-release system which can exert its own advantages and overcome the disadvantages of drugs; meanwhile, the preparation process is simple, safe and has no toxic side effects, is easy to industrialize, and has good application prospects in drug delivery systems, functional foods, medicines and cosmetics.

[0057] The specific surface area and pore structure of the sample of the application are tested by using a full-automatic adsorption instrument of Micromeritics TriStar II Plus. The sample is pretreated at 250 DEG C under high vacuum for 2 hours, and then the BET specific surface area and pore size distribution are measured by N2 adsorption at-196 DEG C under liquid nitrogen. The specific surface area of the material is calculated by using the data of the adsorption branch and then by Barrett-Emmett-Teller (BET) method; the pore size distribution of the material is calculated by using the data of the desorption branch and then by Berret-Joyner-Halenda (BJH) model; the pore volume of the material can be obtained by calculating the nitrogen adsorption amount at P / P0=0.9908.

[0058] The following are specific embodiments

[0059] Preparation of GA@AMSN in Example 1

[0060] Preparation of MSN: 30 g of N-hexadecyltrimethylammonium chloride (CTAC) was dissolved in 300 mL of ultrapure water in a 500 mL round-bottom flask. Then, 1.0 g of triethanolamine was added to the dissolved CTAC solution and stirred thoroughly to form an upper aqueous phase. Next, 12 mL of tetraethyl silicate was thoroughly mixed with 100 mL of solvent (60 mL cyclohexane + 40 mL chlorobenzene) in a beaker, and this was used as the oil phase. The oil phase was transferred to a flask to form a two-phase reaction system. The reaction was carried out at 60 °C with stirring at 300 rpm / min for 12 h. The resulting milky white liquid was removed and centrifuged (10000 rpm, 15 min) to obtain a white solid product. The product was washed three times with ethanol to remove the solvent, and then dried in air to obtain a white powder. The white powder was placed in a muffle furnace and calcined at 550℃ for 6 hours to remove CTAC. The resulting product was washed once with deionized water and once with ethanol, and then vacuum dried to obtain MSN, which had a specific surface area of ​​926.3128 m². 2 / g, pore size 8.5612nm, pore volume 3.374020cm³ 3 / g.

[0061] Preparation of AMSN: 200 mg MSN was added to 40 mL of acetonitrile and ultrasonically dispersed. 200 μl of 3-aminopropyltriethoxysilane (APTE) was added to the reaction system, and the mixture was stirred at 80 °C and 600 rpm for 12 h. After the reaction was completed, the liquid was centrifuged, and the resulting sample was washed twice with water and twice with ethanol to obtain AMSN.

[0062] Preparation of GA@AMSN: 40 mg AMSN was added to 5 mL of GA in acetonitrile solution (5 mg / mL, 10 mg / mL, 15 mg / mL). The mixture was ultrasonically dispersed and incubated at room temperature for 8 h with stirring. Then, the mixture was centrifuged, the supernatant was removed, the product was collected, washed three times with deionized water, and dried under vacuum to obtain GA@AMSN.

[0063] Comparative Example 1: Preparation of GA@MSN

[0064] The difference between this comparative example and Example 1 is that the carrier is not aminated. The specific preparation method of MSN@GA in this comparative example is as follows: After preparing MSN according to the method in Example 1, 40 mg of MSN was added to 5 mL of GA in acetonitrile solution (5 mg / mL, 10 mg / mL, 15 mg / mL). The mixture was ultrasonically dispersed and incubated at room temperature for 8 h. Then, it was centrifuged, the supernatant was removed, the product was collected, washed three times with deionized water, and dried under vacuum to obtain GA@MSN.

[0065] Example 2: Analysis of Drug Loading Effects of MSN and AMSN

[0066] The drug loading rate and encapsulation rate of GA@MSN and GA@AMSN were analyzed to investigate the effect of amino modification of the carrier. Specifically, 5 mg of AMSN and 5 mg of MSN were weighed into centrifuge tubes, 500 μl of 10 mg / ml gingerone A solution was added to the centrifuge tubes, the centrifuge tubes were placed in a reverse shaking instrument and shaken for 12 h, the supernatant was detected by HPLC after centrifugation, the concentration of gingerone A in the supernatant was obtained according to the standard curve of gingerone A, and the drug loading rate and encapsulation rate of gingerone A nano-preparation were calculated using the following formula. The results are shown in Table 1.

[0067] Table 1 Drug loading rate and encapsulation rate of GA@MSN and GA@AMSN

[0068] According to the results in Table 1, the drug loading rate of GA@AMSN is 23.71%, and the encapsulation rate is 31.08%; and the carrier without amino functional group modification cannot successfully adsorb GA. The particle size and morphology of the prepared MSN, AMSN and GA@AMSN were observed by transmission electron microscopy, and the results are shown in Figure 2. The MSN is a uniform dispersed sphere with a particle size of about 120 nm, the surface is rough and loose, and the pore structure is complete, proving that the MSN is successfully synthesized (Figure 2A). After amino modification and loading of gingerone A, the morphology and particle size of AMSN and GA@AMSN remain basically unchanged, and the pore structure remains complete, indicating that the modification and loading process does not damage the structure of MSN (Figures 2B and C). The potential of AMSN is +20 mv, and the potential of GA@AMSN after adsorbing gingerone A is 0. The above results show that, compared with the dendritic mesoporous silica MSN without amino modification, the introduction of amino groups on the surface of the carrier enhances the non-covalent force between the carrier and gingerone A molecules, enabling high loading of gingerone A. The obtained nano-preparation has uniform particle size, high drug loading and high encapsulation rate. Therefore, AMSN is finally determined as the best drug delivery body for gingerone A.

[0069] Example 3 Screening of the Optimal Dose Proportion of GA@AMSN Nano-Preparation

[0070] In view of the fact that the ratio of different drugs to the carrier will further affect the drug loading rate and encapsulation efficiency of the preparation and the stability of the preparation, the present application synthesizes nanometer preparations with different drug-to-carrier ratios, and the optimal drug-to-carrier ratio has been determined. Specifically, the following is prepared: nanometer preparations with different drug-to-carrier ratios (GA:AMSN (w / w) = 1:2, 1:1, 3:2, 2:1, 5:2) are prepared. The specific preparation process is as follows: first, AMSN is prepared according to the preparation method of Example 1, and then the corresponding amount of AMSN and GA acetone solution is ultrasonically dispersed according to the required molar ratio, and the mixture is incubated at room temperature for 8 h. Then, centrifugation is performed and the supernatant is removed, and ethanol is washed three times and dried by vacuum drying to obtain a light yellow powder, i.e. GA@AMSN with different drug-to-carrier ratios. The particle size, drug loading rate and encapsulation efficiency of the prepared nanometer preparation are shown in Table 2.

[0071] Table 2 Particle size, drug loading rate and encapsulation efficiency of GA@AMSN with different drug-to-carrier ratios

[0072] Example 4 Cytotoxicity of GA@AMSN

[0073] In order to ensure the biological safety of AMSN material, WI-38 and L-929 were used as cell models respectively, and the cytotoxicity of AMSN on WI-38 and L-929 was investigated by MTT method. In a 96-well assay plate, WI-38 and L929 cells were cultured at a cell density of 1.2x10 5 After 24 hours of culture, the original culture medium was removed, and culture medium containing AMSN at concentrations of 0 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L and 1000 μmol / ml was added respectively, and incubated for 24 h, and detected by MTT method. 100 μL of MTT solution was added to each well, and then the plate was incubated in the incubator for 4 h. After incubation, 100 μL of DMSO was added to lyse the cells. The absorbance of formazan was measured at 490 nm using a microplate reader. Each treatment group was repeated three times. As shown in Figure 3, AMSN had no significant effect on the proliferation of the above-mentioned cells within the concentration range of 0 μg / mL to 1000 μg / mL, and the cell viability remained above 90%, indicating that AMSN had high biological safety and could be further used in cell anti-aging experiments.

[0074] Example 5 Construction of HDF-a human dermal fibroblast aging model

[0075] The proliferating human dermal fibroblasts were constructed into an aging model by ionizing radiation. HDF-a human dermal fibroblasts were seeded in three T175 culture bottles, each containing 2x10 6The cells were resuscitated overnight using DMEM complete medium. After resuscitation, the cells were divided into a control group and an irradiation group. After the cells were cultured to an appropriate cell concentration, the cells in the irradiation group were subjected to ionizing radiation (10 Gy). After irradiation, the original culture medium was replaced with fresh complete medium, and the medium was changed every two days until the control cells were treated after irradiation for 7 days. On the 8th day, the control cells were placed in an X-ray room for the same amount of time as the irradiated cells. Subsequently, the cells in the control and irradiation groups were cultured for two more days until the 10th day, at which point the cells in the irradiation group were completely senescent. The results are shown in FIG. 4. The SA-β-gal staining results indicate that the human dermal fibroblasts after irradiation were stained darker than before irradiation, indicating that the cells after irradiation were significantly senescent, i.e., the model was successfully constructed.

[0076] Example 6 SA-β-gal staining to detect cell senescence

[0077] This example further compares the removal of senescent HDF-a cells by GA and GA@AMSN. Specifically, after the cells treated with AMSN and GA@AMSN were digested, centrifuged, and resuspended, they were seeded in a 6-well plate at a concentration of 2 x 10 5 The cells were washed once with PBS buffer, 1 mL of fixing solution was added to each well, and the cells were fixed at room temperature for 15 min. After fixing, the fixing solution was aspirated, and the cells were washed with PBS for 3 min x 3 times. The staining working solution was prepared according to the reagent instructions, 1 mL of the staining working solution was added to each well, and the cells were incubated overnight at 37°C in a CO2-free incubator. The next day, the staining was observed under an inverted microscope. The results in FIG. 5 show that GA@AMSN (5 μM, 10 μM, 20 μM) can remove senescent HDF-a cells. Compared with GA, which can only remove part of the senescent HDF-a cells at a concentration of 5 μM, GA@AMSN has a significant effect on removing senescent HDF-a cells at a concentration of 5 μM. Therefore, GA@AMSN can enhance the removal of senescent HDF-a cells by GA

[0078] Example 7 Removal of senescent cells by AMSN and GA@AMSN

[0079] The toxicity of GA and GA@AMSN on proliferative HDF-a cells and their clearance effect on senescent HDF-a cells were further compared by MTT experiment. Specifically, proliferative HDF-a cells and senescent HCoEpic cells were cultured in 96-well assay plates using a cell density of 1.2 x 105, and after 24 hours of culture, the original culture medium was removed, and culture medium containing GA at concentrations of 0 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L and 25 μmol / ml, and culture medium containing GA-loaded preparations at concentrations of 0 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L and 25 μmol / L were added, respectively, for a total incubation time of 24 h, and MTT method was used for detection. 100 μL of MTT solution was added to each well, and then the plate was incubated in an incubator for 4 h, and after incubation, 100 μL of DMSO was added to lyse the cells. The absorbance of methylene blue was measured at 490 nm using a microplate reader. Each treatment group was repeated three times. The results shown in Figure 9 show that low concentrations (5 μM and 10 μM) of GA and GA@AMSN have no obvious toxicity on proliferative HDF-a cells, however, 10 μM of GA@AMSN can significantly reduce the viability of senescent HDF-a cells, and even stronger than the clearance effect of 20 μM GA on senescent HDF-a cells, indicating that the clearance ability of GA@AMSN on senescent cells is significantly higher than that of GA compound alone.

[0080] Example 8 Galactosidase activity experiment of GA@AMSN nano-preparation

[0081] SD rats (200-220 g) were randomly divided into 3 groups to explore the effect of GA and GA@AMSN on the activity of rat plasma galactosidase. GA and GA@AMSN (at a concentration of 5 mg / kg of GA) were injected into the tail vein of rats, and the blank control group was injected with an equal amount of solvent. After the rats were injected with the drug through the tail vein, blood samples were collected at predetermined time points of 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 10 h, 24 h, and 48 h, centrifuged (3500 rpm, 10 min) to obtain plasma, and the galactosidase concentration was determined using a kit. The results are shown in Table 3.

[0082] Table 3 Effect of GA and GA@AMSN on galactosidase concentration (n=3)

[0083] As can be seen from the results of Table 3, the concentration of galactosidase in the GA and GA@AMSN groups is obviously lower than that in the blank group after administration, which is related to the antioxidant effect of GA. The concentration of galactosidase in the GA@AMSN group is more greatly decreased than that in the GA group, and the sustained action time reaches 8-10 hours, which is much higher than 4 hours in the GA group, proving that GA@AMSN can greatly increase the metabolic time of GA in vivo and effectively improve the bioavailability of GA.

[0084] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but should not be understood as limiting the scope of the patent application. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the present patent application should be subject to the appended claims.

Claims

1. A nano-formulation for eliminating senescent cells, characterized in that, The nanoformulation comprises aminated dendritic mesoporous silica and gingerone A supported on the aminated dendritic mesoporous silica.

2. The nano-formulation for clearing senescent cells according to claim 1, characterized in that, The loading of gingerone A in the nano-formulation is 15% to 30%; The encapsulation rate of gingerone A in the nano-formulation is 11% to 35%; The particle size of the nano-formulation is 50nm to 200nm; The specific surface area of ​​the nano-formulation is 800 m². 2 / g~1000m 2 / g.

3. The nano-formulation for clearing senescent cells according to claim 1, characterized in that, The particle size of the aminated dendritic mesoporous silica is 50 nm to 200 nm. The pore size of the aminated dendritic mesoporous silica is 2 nm to 10 nm. The specific surface area of ​​the aminated dendritic mesoporous silica is 300 m². 2 / g~1000m 2 / g.

4. A method for preparing a nano-formulation for clearing senescent cells as described in any one of claims 1-3, characterized in that, include: Shogaol A is dissolved in a solvent to obtain a shogaol A solution. The shogaol A solution and aminated dendritic mesoporous silica are added to a buffer solution to react and load the shogaol A onto the aminated dendritic mesoporous silica to obtain the nano-formulation.

5. The method for preparing the nano-formulation for clearing senescent cells according to claim 4, characterized in that, The mass ratio of gingerone A to aminated dendritic mesoporous silica in the gingerone A solution is (1-5):1; The buffer solutions include phosphate buffer, borate buffer, citrate buffer, phosphate and trimethylolpropane, acetate buffer, imidazole buffer, and carbonate buffer. One or more; The pH of the buffer solution is 5.0–7.4; The concentration of gingerone A in the gingerone A solution is 2 mg / mL to 6 mg / mL; The solvent includes any one of acetone, acetonitrile, ethanol, methanol, and dimethyl sulfoxide; The reaction temperature is 25℃~80℃; the reaction time is 6h~36h.

6. The method for preparing the nano-formulation for clearing senescent cells according to claim 4, characterized in that, The method for preparing the aminated dendritic mesoporous silica includes the following steps: The template agent and catalyst are added to water and mixed to form an aqueous phase; The silicon source is mixed with a solvent to form an oil phase; The oil phase and the aqueous phase are subjected to a homogeneous reaction. After the reaction is completed, the mixture is centrifuged, dried and then calcined to obtain dendritic mesoporous silica. The dendritic mesoporous silica was added to an acetonitrile solution and aminated with an ammonia source to obtain the aminated dendritic mesoporous silica.

7. The method for preparing the nano-formulation for clearing senescent cells according to claim 6, characterized in that, The template agent includes one or more of hexadecyltrimethylammonium chloride, octadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, hexadecyltrimethylammonium chloride, hexadecylpyridinium chloride, and dodecyltrimethylammonium bromide; The catalyst includes one or more of triethanolamine, tripropanolamine, diethanolamine, ethanolamine, and ammonia. The silicon source includes one or more of the following: tetraethyl orthosilicate, methyl orthosilicate, tetra(2-methoxy-1-methylethyl) silicate, tetraisopropyl orthosilicate, tetrabutyl orthosilicate, and tetraisopropyl orthosilicate. The ammonia source includes one or more of 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, and γ-aminopropyldiethoxymethylsilane; The solvent includes one or more of chlorobenzene, cyclohexane, chloroform, dichloromethane, and water; The mass ratio of the template agent, the silicon source, and the catalyst is (20-30):(0.5-1.2):(6-12); The mass ratio of the dendritic mesoporous silica to the ammonia source is (0.08–0.1):(50–100); The calcination temperature is 300℃~600℃; the calcination time is 4h~8h; The amination modification temperature is 60℃~85℃; the amination modification time is 8h~16h.

8. A nano-formulation for clearing senescent cells as described in any one of claims 1-3, or a nano-formulation for clearing senescent cells prepared by the preparation method described in any one of claims 4-7, characterized in that, Compared to gingerone A, the nano-formulation used to clear senescent cells has a more significant and longer-lasting effect on galactosidase in rat plasma.

9. The application of a nano-formulation for clearing senescent cells as described in any one of claims 1-3, or a nano-formulation for clearing senescent cells prepared by the preparation method described in any one of claims 4-7, in a drug delivery system.

10. The use of a nano-formulation for clearing senescent cells as described in any one of claims 1-3, or a nano-formulation for clearing senescent cells prepared by the preparation method described in any one of claims 4-7, in the preparation of functional foods, pharmaceuticals, or cosmetics that clear senescent cells.

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