Engineered stem cell based on tyrosine-based material wrapping as well as construction method and application of engineered stem cell

Abstract

The invention belongs to the technical field of biological medicines, and discloses a construction method of engineered stem cells based on tyrosine-based material wrapping, which comprises the following steps: carrying out first incubation, washing and resuspension on stem cells in a TCEP solution, adding a PEG modified tyrosinase solution, carrying out second incubation, washing and resuspension, and carrying out second incubation, washing and resuspension on the stem cells to obtain the engineered stem cells based on tyrosine-based material wrapping. And adding a tyrosine solution or a tyrosine-based material solution, and carrying out third incubation. The engineered stem cells constructed by the invention can resist damage of factors such as pathological microenvironment oxidative stress and the like to cells, and stably maintain a paracrine treatment function; and the survival time and colonization efficiency of cells in vivo can be remarkably improved through a physical barrier, and immunological rejection after allograft is reduced. The engineered stem cells constructed by the invention can specifically solve the disease treatment problem according to different disease characteristics. The design thought of engineering cell construction also provides reference for engineering transformation of other cells, and has definite practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an engineered stem cell based on tyrosine-based material encapsulation, its construction method, and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) have become a research hotspot in disease treatment due to their potential for immune regulation and tissue repair. However, low homing efficiency after intravenous injection and rapid cell apoptosis caused by harsh microenvironments (reactive oxygen species, shear stress, etc.) severely limit their clinical translational efficacy. Constructing engineered stem cells by microencapsulating them on the cell surface using polymerized materials can significantly improve some of the problems encountered in their use.

[0003] Although the oxidative polymerization of dopamine can proceed under relatively mild conditions (pH 8.5), mammalian cells lose viability during this process. To address this issue, tyrosinase-catalyzed polymerization has been proposed. However, there are currently no reports on the use of tyrosinase-catalyzed encapsulation of tyrosine materials to construct engineered stem cells. Furthermore, single tyrosine materials cannot be used to specifically construct engineered stem cells based on disease characteristics, thus failing to systematically address the problem of disease treatment.

[0004] Therefore, it is necessary to develop a tyrosine-based platform that can address disease treatment issues in a targeted manner based on the characteristics of different diseases. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for constructing engineered stem cells based on tyrosine-based materials, which addresses the shortcomings of the prior art.

[0006] Another technical problem that this invention aims to solve is to provide an engineered stem cell based on a tyrosine-based material.

[0007] The final technical problem to be solved by this invention is to provide the application of the engineered stem cells based on tyrosine-based materials in the preparation of drugs for treating liver fibrosis.

[0008] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0009] The first aspect of the present invention provides a method for constructing engineered stem cells based on tyrosine-based materials, comprising the following steps: incubating stem cells in TCEP solution for a first time, washing and resuspending them, adding PEG-modified tyrosinase solution for a second time, washing and resuspending them, adding tyrosine solution or tyrosine-based material solution for a third time, thereby obtaining the desired product.

[0010] The tyrosine-based material is any one of arginine-modified tyrosine, tyrosine-modified hyaluronic acid, folic acid-modified tyrosine, and dopamine phenylboronic acid-modified tyrosine.

[0011] The method for preparing the arginine-modified tyrosine includes the following steps:

[0012] Step 1: Dissolve Boc-Tyr-OH, condensing agent and peptide protectant in solvent and carry out the first reaction to obtain the first reaction solution. Add the first reaction solution to ethylenediamine solution to carry out the second reaction. The resulting second reaction solution is separated and purified to obtain ethylenediamine-modified Boc-protected tyrosine Boc-Tyr-EDA.

[0013] Step 2: The Boc-Tyr-EDA obtained in Step 1 is dissolved in a solvent along with Boc-Arg(Pbf)-OH, a condensing agent, a polypeptide protectant, and an organic base catalyst, and a third reaction is carried out. The resulting third reaction solution is separated and purified to obtain Boc-Tyr-EDA-Arg(Pbf)-Boc, which undergoes a deprotection reaction under the action of a deprotection reagent. The resulting deprotection reaction solution is then separated and purified to obtain the arginine-modified tyrosine.

[0014] In step 1, the condensing agent is EDC.HCl; the peptide protecting agent is HOBt; the solvent is DMSO; the solvent for the ethylenediamine solution is DMSO; the molar ratio of Boc-Tyr-OH, the condensing agent, and the peptide protecting agent is 1:1.2~2:1.2~2, preferably 1:1.5:1.2; the first reaction is carried out under a nitrogen atmosphere with stirring in an ice bath for 20~40 min; the molar ratio of Boc-Tyr-OH to ethylenediamine is 1:4~8, preferably 1:5; the second reaction is carried out under a nitrogen atmosphere with stirring at room temperature for 24~48 h.

[0015] Preferably, in step 1, the separation and purification method of the second reaction solution is as follows: ethyl acetate is added to the second reaction solution, and it is washed 2 to 3 times with saturated NaCl aqueous solution to remove the used saturated NaCl aqueous solution. A large amount of white precipitate appears in the ethyl acetate. The white precipitate is collected by centrifugation and dried under vacuum at 30°C to obtain Boc-Tyr-EDA.

[0016] In step 2, the condensing agent is EDC.HCl; the peptide protecting agent is HOBt; the organic base catalyst is DIEA; the solvent is DMSO; the molar ratio of Boc-Tyr-EDA, Boc-Arg(Pbf)-OH, condensing agent, peptide protecting agent, and organic base catalyst is 1:1~1.5:1.2~2:1.2~2:3~6, preferably 1:1:1.2:1.2:3.4; the third reaction is carried out under a nitrogen atmosphere with stirring at room temperature for 24~48 h; the deprotecting reagent is trifluoroacetic acid; the molar ratio of Boc-Tyr-EDA-Arg(Pbf)-Boc to the deprotecting reagent is 1:20~40, preferably 1:30; the solvent used in the deprotection reaction is chloroform and / or dichloromethane; the deprotection reaction is carried out with stirring at room temperature for 12~ 24 hours.

[0017] Preferably, in step 2, the separation and purification method of the third reaction solution is as follows: add dichloromethane to the third reaction solution, wash it 2 to 3 times each with saturated NaHCO3 aqueous solution, 1 mol / L HCl aqueous solution and saturated NaCl aqueous solution, remove the above three detergents with a separatory funnel, dry the washed reaction solution with anhydrous magnesium sulfate and concentrate it by rotary evaporation to remove dichloromethane, use ethyl acetate as the developing solvent, and obtain Boc-Tyr-EDA-Arg(Pbf)-Boc by column chromatography.

[0018] Preferably, in step 2, the separation and purification method of the deprotection reaction solution is as follows: after removing dichloromethane and trifluoroacetic acid by rotary evaporation, anhydrous diethyl ether is added to the obtained concentrated product, a white precipitate is collected, and the precipitate is dried under vacuum at room temperature to obtain Tyr-EDA-Arg.

[0019] There are no special requirements for the amount of solvent used in steps 1 and 2; it is sufficient to dissolve and / or disperse the raw materials evenly.

[0020] The method for preparing the tyrosine-modified hyaluronic acid includes the following steps:

[0021] Step I: Boc-Tyr-OH, condensing agent, and peptide protectant are dissolved in a solvent and subjected to a first reaction to obtain a first reaction solution. This first reaction solution is then added to an ethylenediamine solution to carry out a second reaction. The resulting second reaction solution is separated and purified to obtain ethylenediamine-modified Boc-protected tyrosine Boc-Tyr-EDA. Under the action of a deprotecting reagent, the deprotection reaction solution is subjected to a deprotection reaction to obtain Tyr-EDA.

[0022] Step II: Hyaluronic acid is activated under the action of EDC.HCl and NHS to obtain activated hyaluronic acid, which is then subjected to an amidation reaction with Tyr-EDA obtained in Step I. The resulting amidation reaction solution is separated and purified to obtain the tyrosine-modified hyaluronic acid.

[0023] In step I, the condensing agent is EDC.HCl; the peptide protecting agent is HOBt; the solvent is DMSO; the solvent for the ethylenediamine solution is DMSO; the molar ratio of Boc-Tyr-OH, the condensing agent, and the peptide protecting agent is 1:1.2~2:1.2~2, preferably 1:1.5:1.2; the first reaction is carried out under a nitrogen atmosphere at room temperature with stirring for 20~40 min; the molar ratio of Boc-Tyr-OH to ethylenediamine is 1:4~8, preferably 1:5; the second reaction is carried out under a nitrogen atmosphere at room temperature with stirring for 24~48 h; the deprotecting reagent is trifluoroacetic acid; the molar ratio of Boc-Tyr-EDA to the deprotecting reagent is 1:10~20, preferably 1:10; the solvent used in the deprotection reaction is chloroform and / or dichloromethane; the deprotection reaction is carried out at room temperature with stirring for 12~ 24 hours.

[0024] Preferably, in step I, the separation and purification method of the second reaction solution is as follows: ethyl acetate is added to the second reaction solution, and it is washed 2 to 3 times with saturated NaCl aqueous solution to remove the used saturated NaCl aqueous solution. A large amount of white precipitate appears in the ethyl acetate. The white precipitate is collected by centrifugation and dried under vacuum at 30°C to obtain Boc-Tyr-EDA.

[0025] Preferably, in step I, the separation and purification method of the deprotection reaction solution is as follows: after removing dichloromethane and trifluoroacetic acid by rotary evaporation, anhydrous diethyl ether is added to the obtained concentrated product, a white precipitate is collected, and the precipitate is dried under vacuum at room temperature to obtain Tyr-EDA.

[0026] In step II, the molar ratio of carboxyl groups, EDC.HCl, and NHS in the hyaluronic acid is 1:4 to 8:4 to 8, preferably 1:5.28:5.28; the activation is carried out under ice bath stirring conditions for 20 to 40 minutes; the molar ratio of carboxyl groups in the hyaluronic acid to Tyr-EDA is 1:4 to 8, preferably 1:5.28; the amidation reaction is carried out at room temperature with stirring for 48 to 72 hours. The hyaluronic acid is commercially available, and there are no special requirements for its weight-average molecular weight. Preferably, the weight-average molecular weight of the hyaluronic acid is 57 kDa.

[0027] Preferably, in step II, the method for activating the hyaluronic acid under the action of EDC.HCl and NHS is as follows: first, dissolve the hyaluronic acid in MES buffer to obtain a hyaluronic acid solution, and then add the EDC.HCl and NHS to it for activation. Preferably, the concentration of the MES buffer is 0.01 mol / L and the pH value is 5.5.

[0028] Preferably, in step II, the purification method of the amidation reaction solution is as follows: the reaction solution is placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed with deionized water for 3 days, and the deionized water is replaced every 6 to 8 hours. The dialysis product is then freeze-dried to obtain HA-EDA-Tyr.

[0029] There are no special requirements for the amount of solvent used in steps I and II; it is sufficient to dissolve and / or disperse the raw materials evenly.

[0030] The method for preparing folic acid-modified tyrosine includes the following steps:

[0031] Step i: Boc-Tyr-OH, condensing agent and peptide protectant are dissolved in solvent and subjected to a first reaction to obtain a first reaction solution. This first reaction solution is then added to an ethylenediamine solution to carry out a second reaction. The resulting second reaction solution is then separated and purified to obtain ethylenediamine-modified Boc-protected tyrosine Boc-Tyr-EDA.

[0032] Step ii: The Boc-Tyr-EDA obtained in step i is dissolved in a solvent with FA-PEG-NH2, EDC.HCl and NHS and subjected to a third reaction. The resulting third reaction solution is separated and purified to obtain Boc-Tyr-PEG-FA, which undergoes a deprotection reaction under the action of a deprotection reagent. The resulting deprotection reaction solution is then separated and purified to obtain the folic acid-modified tyrosine.

[0033] In step i, the condensing agent is EDC.HCl; the peptide protecting agent is HOBt; the solvent is DMSO; the solvent of the ethylenediamine solution is DMSO; the molar ratio of Boc-Tyr-OH, the condensing agent, and the peptide protecting agent is 1:1.2~2:1.2~2, preferably 1:1.5:1.2; the first reaction is carried out under a nitrogen atmosphere with stirring in an ice bath for 20~40 min; the molar ratio of Boc-Tyr-OH to ethylenediamine is 1:4~8, preferably 1:5; the second reaction is carried out under a nitrogen atmosphere with stirring at room temperature for 24~48 h.

[0034] Preferably, in step i, the separation and purification method of the second reaction solution is as follows: add ethyl acetate to the second reaction solution, wash with saturated NaCl aqueous solution 2 to 3 times, remove the used saturated NaCl aqueous solution, a large amount of white precipitate appears in the ethyl acetate, collect the white precipitate by centrifugation, and dry it under vacuum at 30°C to obtain Boc-Tyr-EDA.

[0035] In step ii, the solvent is DMSO; the weight-average molecular weight of the polyethylene glycol segments in FA-PEG-NH2 is 2000-5000 Da, preferably 2000 Da; the molar ratio of FA-PEG-NH2, Boc-Tyr-EDA, EDC.HCl and NHS is 1:1.2-2:1.2-2, preferably 1:1.5:1.5; the third reaction is carried out under nitrogen atmosphere with stirring at room temperature for 24-48 h; the deprotection reagent is trifluoroacetic acid; the ratio of Boc-Tyr-PEG-FA to the deprotection reagent is 1 g:0.5-2 mL, preferably 1 g:1 mL; the solvent used in the deprotection reaction is chloroform and / or dichloromethane; the deprotection reaction is carried out with stirring at room temperature for 12-24 h.

[0036] Preferably, in step ii, the separation and purification method of the third reaction solution is as follows: the third reaction solution is placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed with deionized water for 3 days, the deionized water is replaced every 6 to 8 hours, and the dialysis product is freeze-dried to obtain Boc-Tyr-PEG-FA.

[0037] Preferably, in step ii, the separation and purification method of the deprotection reaction solution is as follows: after removing dichloromethane and trifluoroacetic acid by rotary evaporation, anhydrous diethyl ether is added to the obtained concentrated product, a white precipitate is collected, and the product is dried under vacuum at room temperature to obtain the folic acid-modified tyrosine.

[0038] There are no special requirements for the amount of solvent used in steps i and ii; it is sufficient to dissolve and / or disperse the raw materials evenly.

[0039] The preparation method of the dopamine phenylboronic acid modified tyrosine includes the following steps:

[0040] Step a: Dissolve Boc-Tyr-OH, condensing agent and peptide protectant in solvent and carry out the first reaction to obtain the first reaction solution. Add the first reaction solution to ethylenediamine solution and carry out the second reaction. The resulting second reaction solution is separated and purified to obtain ethylenediamine-modified Boc-protected tyrosine Boc-Tyr-EDA.

[0041] Step b: 4-hydroxymethylphenylboronic acid (HPBA) and N,N'-carbonyldiimidazole (CDI) are dissolved in a solvent to carry out the third reaction. The resulting third reaction solution is then separated and purified to obtain HPBA-CDI.

[0042] Step c: The Boc-Tyr-EDA obtained in step a and the HPBA-CDI obtained in step b are dissolved in a solvent to carry out the fourth reaction. The resulting fourth reaction solution is separated and purified to obtain Boc-Tyr-EDA-HPBA, which undergoes a deprotection reaction under the action of a deprotection reagent. The resulting deprotection reaction solution is separated and purified to obtain Tyr-EDA-HPBA.

[0043] Step d: The Tyr-EDA-HPBA obtained in step c is dissolved in a solvent with dopamine hydrochloride and an organic base catalyst to carry out the fifth reaction. The resulting fifth reaction solution is separated and purified to obtain the dopamine phenylboronic acid modified tyrosine.

[0044] In step a, the condensing agent is EDC.HCl; the peptide protecting agent is HOBt; the solvent is DMSO; the solvent of the ethylenediamine solution is DMSO; the molar ratio of Boc-Tyr-OH, the condensing agent, and the peptide protecting agent is 1:1.2~2:1.2~2, preferably 1:1.5:1.2; the first reaction is carried out under a nitrogen atmosphere with stirring in an ice bath for 20~40 min; the molar ratio of Boc-Tyr-OH to ethylenediamine is 1:4~8, preferably 1:5; the second reaction is carried out under a nitrogen atmosphere with stirring at room temperature for 24~48 h.

[0045] Preferably, in step a, the separation and purification method of the second reaction solution is as follows: add ethyl acetate to the second reaction solution, wash with saturated NaCl aqueous solution 2 to 3 times, remove the used saturated NaCl aqueous solution, a large amount of white precipitate appears in the ethyl acetate, collect the white precipitate by centrifugation, and dry under vacuum at 30°C to obtain Boc-Tyr-EDA.

[0046] In step b, the solvent is DMSO; the molar ratio of 4-hydroxymethylphenylboronic acid (HPBA) to N,N'-carbonyldiimidazole (CDI) is 1:1.2~2, preferably 1:1.5; the third reaction is carried out under nitrogen atmosphere with stirring at room temperature for 24~48 hours.

[0047] Preferably, in step b, the separation and purification method of the third reaction solution is as follows: add dichloromethane to the third reaction solution, wash it 2 to 3 times with saturated sodium chloride aqueous solution, dry it with anhydrous sodium sulfate, filter to remove anhydrous sodium sulfate, and remove dichloromethane by rotary evaporation to obtain HPBA-CDI.

[0048] In step c, the solvent is DMSO; the molar ratio of HPBA-CDI to Boc-Tyr-EDA is 1:1.2~2, preferably 1:1.2; the fourth reaction is carried out under nitrogen atmosphere with stirring at room temperature for 24~48 h; the deprotection reagent is trifluoroacetic acid; the molar ratio of Boc-Tyr-EDA-HPBA to the deprotection reagent is 1:10~20, preferably 1:10; the solvent used in the deprotection reaction is chloroform and / or dichloromethane; the deprotection reaction is carried out with stirring at room temperature for 12~24 h.

[0049] Preferably, in step c, the separation and purification method of the fourth reaction solution is as follows: dichloromethane is added to the fourth reaction solution, and the solution is washed 2 to 3 times each with saturated NaHCO3 aqueous solution, 1 mol / L HCl aqueous solution and saturated NaCl aqueous solution, dried with anhydrous magnesium sulfate, and concentrated by rotary evaporation to remove dichloromethane. The concentrated product is purified by column chromatography using ethyl acetate and methanol in a ratio of 30:1 as the developing solvent to obtain Boc-Tyr-EDA-HPBA.

[0050] Preferably, in step c, the separation and purification method of the deprotection reaction solution is as follows: after removing dichloromethane and trifluoroacetic acid by rotary evaporation, anhydrous diethyl ether is added to the obtained concentrated product, a white precipitate is collected, and the precipitate is dried under vacuum at room temperature to obtain Tyr-EDA-HPBA.

[0051] In step d, the solvent is DMF; the organic base catalyst is DIEA; the molar ratio of Tyr-EDA-HPBA, dopamine hydrochloride and the organic base catalyst is 1:1 ~ 1.1:1 ~ 1.1, preferably 1:1:1; the fifth reaction is carried out under a nitrogen atmosphere at room temperature with stirring for 12 ~ 24 h.

[0052] Preferably, in step d, the separation and purification method of the fifth reaction solution is as follows: the fifth reaction solution is rotary evaporated to remove DMF, diethyl ether is added to the obtained concentrated product, a white precipitate is collected, and the product is dried under vacuum at room temperature to obtain the dopamine phenylboronic acid modified tyrosine.

[0053] There are no special requirements for the amount of solvent used in steps a to d; it is sufficient to dissolve and / or disperse the raw materials evenly.

[0054] The preparation method of the PEG-modified tyrosinase solution includes the following steps: reacting MAL-PEG-NHS with tyrosinase in a buffer solution to obtain the solution.

[0055] The MAL-PEG-NHS contains polyethylene glycol segments with a weight-average molecular weight of 2000-5000 Da, preferably 2000 Da; the mass ratio of MAL-PEG-NHS to tyrosinase is 1:60; the buffer solution is phosphate buffer; the mass ratio of tyrosinase to buffer volume is 0.12 g:1 mL; the reaction is carried out under nitrogen atmosphere with stirring at room temperature for 20-40 min; the amount of PEG-modified tyrosinase solution used is 4 × 10⁻⁶ g / mL. 6 ~ 5×10 6 One stem cell uses 1 mL.

[0056] Preferably, the tyrosinase has an enzyme activity of 500 U / mg.

[0057] The stem cells are mesenchymal stem cells; the concentration of the TCEP solution is 0.5 ~ 1 mM, preferably 1 mM, and the dosage is 4 × 10⁻⁶ m³ / s. 6 ~ 5×10 6 Each stem cell uses 6 mL; the first incubation is at room temperature for 10-20 minutes, gently shaking the centrifuge tube every 3-5 minutes; the second incubation is at 37°C in a cell culture incubator for 20-40 minutes, gently shaking the centrifuge tube every 3-5 minutes; the concentration of the tyrosine-based material solution or tyrosine solution is 0.5-2 mM; the solvent for the tyrosine-based material solution or tyrosine solution is DMEM / F12 complete culture medium; the amount of the tyrosine-based material solution or tyrosine solution used is 4 × 10⁻⁶ mL per centrifuge tube. 6 ~ 5×10 6 4 mL of centrifuge tubes were used per stem cell; the third incubation was performed in a cell culture incubator at 37°C for 3 to 5 hours, with the centrifuge tubes gently shaken every 3 to 5 minutes; the solutions used for washing and resuspending were phosphate buffer.

[0058] Preferably, when the tyrosine-based material is arginine-modified tyrosine, the concentration of the tyrosine-based material solution is 1 mM; when the tyrosine-based material is tyrosine-modified hyaluronic acid, folic acid-modified tyrosine, or dopamine phenylboronic acid-modified tyrosine, the concentration of the tyrosine-based material solution is 2 mM; and the concentration of the tyrosine solution is 2 mM.

[0059] The second aspect of the present invention provides engineered stem cells based on tyrosine-based materials encapsulated and constructed by the above-described construction method.

[0060] The third aspect of this invention provides the application of the engineered stem cells encapsulated in tyrosine-based materials in the preparation of drugs for treating liver fibrosis.

[0061] Beneficial effects:

[0062] The engineered stem cells constructed in this invention can resist cell damage caused by factors such as oxidative stress in the pathological microenvironment and stably maintain paracrine therapeutic function. They can significantly improve cell survival time and colonization efficiency in vivo through physical barriers, reducing immune rejection after allogeneic transplantation. Furthermore, by encapsulating engineered stem cells with tyrosine materials modified with targeted molecules, local retention and targeted enrichment of cells at lesions can be achieved; by modifying tyrosine materials with drug molecules, an integrated system of cell therapy and controlled drug release can be constructed; and by encapsulating engineered stem cells with tyrosine materials modified with other amino acid molecules, synergistic functions such as NO release can be endowed. The engineered stem cells constructed in this invention can address disease treatment problems in a targeted manner according to different disease characteristics. The design concept of engineered cell construction will also provide a reference for the engineering modification of other cells, possessing clear practical application value. Attached Figure Description

[0063] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0064] Figure 1 The image shows the hydrogen nuclear magnetic resonance spectrum of Tyr-EDA-Arg in Example 1.

[0065] Figure 2 The hydrogen nuclear magnetic resonance spectrum of HA-EDA-Tyr in Example 2 is shown.

[0066] Figure 3 The hydrogen nuclear magnetic resonance spectrum of Tyr-EDA-HPBA-DA in Example 3 is shown.

[0067] Figure 4 The image shows the hydrogen nuclear magnetic resonance spectrum of Tyr-PEG-FA in Example 4.

[0068] Figure 5 The images show tyrosine-coated MSCs prepared in Example 5 and ordinary MSCs; the left image shows tyrosine-coated MSCs and the right image shows ordinary MSCs.

[0069] Figure 6 The figures show a comparison of cell viability and proliferation capacity between tyrosine-coated MSCs and ordinary MSCs in Example 6; where figure a is a calcein AM / propidium iodide staining image; figure b is a statistical graph of cell viability detection results; and figure c is a statistical graph of cell proliferation capacity.

[0070] Figure 7The image shows the results of induced differentiation staining of tyrosine-encapsulated MSCs and ordinary MSCs in Example 7.

[0071] Figure 8 The cell viability of tyrosine-coated MSCs and ordinary MSCs in Example 8 under three stress conditions: H2O2, trypsin, and cationic PEI; Figure a shows the calcein AM / propidium iodide staining pattern; Figure b shows the statistical results of cell viability detection.

[0072] Figure 9 The NO release from MSCs encapsulated with tyrosine-based materials and ordinary MSCs in Example 9 under H2O2 stimulation is shown.

[0073] Figure 10 The image shows DCFH-DA fluorescence staining of HA-EDA-Tyr-encapsulated MSCs and HA-EDA-Tyr / PB-encapsulated MSCs in Example 10 under H2O2 stimulation.

[0074] Figure 11 The images show the targeting and homing effects of MSCs encapsulated with tyrosine-based material and ordinary MSCs in Example 11. Image a shows in vivo imaging of mice on days 1, 3, 5, and 7 after injection of MSCs encapsulated with tyrosine-based material and ordinary MSCs, respectively. Image b shows in vivo imaging of the heart, liver, spleen, lung, and kidney of mice on day 7 after injection of MSCs encapsulated with tyrosine-based material and ordinary MSCs, respectively. Image c shows CD90 staining in liver tissue sections of mice on day 7 after injection of MSCs encapsulated with tyrosine-based material and ordinary MSCs, respectively. Image d shows the fluorescence intensity statistics of mice on days 1, 3, 5, and 7 after injection of MSCs encapsulated with tyrosine-based material and ordinary MSCs, respectively. Image e shows the fluorescence intensity statistics of the liver of mice on day 7 after injection of MSCs encapsulated with tyrosine-based material and ordinary MSCs, respectively.

[0075] Figure 12 Figure 12 shows the experimental results of improving liver fibrosis in mice by encapsulating MSCs with tyrosine-based materials and ordinary MSCs. Figure a shows the H&E, Masson, and Sirius red staining of liver tissue sections from each group of mice; Figure b shows the statistical results of blood alanine aminotransferase (ALT) in each group of mice; Figure c shows the statistical results of blood aspartate aminotransferase (AST) in each group of mice; Figure d shows the statistical results of blood total bilirubin in each group of mice; Figure e shows the statistical results of collagen fiber area stained with Masson; and Figure f shows the statistical results of collagen fiber area stained with Sirius red. Detailed Implementation

[0076] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0077] Example 1 Synthesis of the arginine-modified tyrosine material Tyr-EDA-Arg

[0078] (1) Preparation of Boc-Tyr-EDA: ① Weigh 1.000 g Boc-Tyr-OH (3.56 mmol), 0.819 g EDC.HCl (5.33 mmol, 1.5 times the amount of Boc-Tyr-OH, denoted as 1.5 eq), and 0.576 g HOBt (4.27 mmol, 1.2 eq) into a reaction flask, evacuate and purge with nitrogen. Under nitrogen conditions, add 10 mL of anhydrous DMSO, stir to dissolve the above compounds, and activate by stirring in an ice bath for 30 min to obtain the reaction solution. The purpose of this step is to activate the carboxyl group in Boc-Tyr-OH. ② Take another reaction flask, evacuate and purge with nitrogen, and under nitrogen conditions, add 1.2 mL of ethylenediamine (17.78 mmol, 5 eq) and 10 mL of anhydrous DMSO. ③ Take all the reaction solution obtained in step ① and add it dropwise to the reaction flask in step ② under nitrogen atmosphere. Stir the reaction at room temperature for 24 h to obtain the reaction solution. ④ Add 100 mL of ethyl acetate to the reaction solution obtained in step ③ and wash it three times with saturated NaCl aqueous solution. Remove the washed NaCl solution using a separatory funnel. A large amount of white precipitate appears in the ethyl acetate. Collect the white precipitate by centrifugation and dry it under vacuum at 30℃ to obtain Boc-Tyr-EDA.

[0079] (2) Preparation of Boc-Tyr-EDA-Arg(Pbf)-Boc: Weigh 1.000 g Boc-Tyr-EDA (3.09 mmol), 1.629 g Boc-Arg(Pbf)-OH (3.09 mmol, 1 eq, the amount of Boc-Tyr-EDA), 0.711 g EDC.HCl (3.711 mmol, 1.2 eq) and 0.501 g HOBt (3.711 mmol, 1.2 eq) into a reaction flask, evacuate and purge with nitrogen, add 20 mL of anhydrous DMSO under nitrogen conditions, stir until the above compounds dissolve, add 1.7 mL DIEA (10.51 mmol, 3.4 eq), and stir at room temperature for 24 h. After the reaction was completed, 100 mL of dichloromethane was added to the resulting reaction solution. The solution was washed three times successively with saturated NaHCO3 aqueous solution, 1 mol / L HCl aqueous solution, and saturated NaCl aqueous solution. The detergent was removed by a separatory funnel. The washed reaction solution was dried with anhydrous magnesium sulfate and concentrated by rotary evaporation to remove dichloromethane. Boc-Tyr-EDA-Arg(Pbf)-Boc was obtained by column chromatography using ethyl acetate as the developing solvent.

[0080] (3) Preparation of Tyr-EDA-Arg: Weigh 1.000 g of Boc-Tyr-EDA-Arg(Pbf)-Boc (1.20 mmol), dissolve it in 2.7 mL of dichloromethane, and add 2.7 mL of trifluoroacetic acid (36.06 mmol, 30 times the amount of Boc-Tyr-EDA-Arg(Pbf)-Boc, denoted as 30 eq). Stir the reaction at room temperature for 12 h. After the reaction is complete, remove dichloromethane and trifluoroacetic acid by rotary evaporation. Add anhydrous diethyl ether to the concentrated product, and a white precipitate appears. Collect the white precipitate and dry it under vacuum at room temperature to obtain Tyr-EDA-Arg, whose chemical structure is shown in Formula I.

[0081] 10 mg of the Tyr-EDA-Arg prepared in this example was dissolved in 0.6 mL of (CD3)2SO4, and the sample was recorded using a nuclear magnetic resonance spectroscopy instrument. 1 H NMR spectra, results as follows Figure 1 As shown in the Tyr-EDA-Arg NMR spectrum, multiple signal peaks appear around δ=6.7-7.1 ppm, which are NMR peaks on the tyramine benzene ring. Multiple signal peaks also appear around δ=1.4-1.7 ppm, which are NMR peaks of the arginine methylene group. This confirms that the arginine modification was successful.

[0082]

[0083] Formula I

[0084] Example 2 Synthesis of tyrosine-modified hyaluronic acid material HA-EDA-Tyr

[0085] (1) Preparation of Tyr-EDA: Weigh 1.000 g of Boc-Tyr-EDA (3.09 mmol) prepared in Example 1, dissolve it in 2.3 mL of dichloromethane, and add 2.3 mL of trifluoroacetic acid (30.92 mmol, 10 times the amount of Boc-Tyr-EDA, denoted as 10 eq) dropwise. Stir the reaction at room temperature for 12 h. After the reaction is complete, remove dichloromethane and trifluoroacetic acid by rotary evaporation. Add anhydrous diethyl ether to the concentrated product, and a white precipitate appears. Collect the white precipitate and dry it under vacuum at room temperature to obtain Tyr-EDA.

[0086] (2) Preparation of HA-EDA-Tyr: Weigh 0.100 g of hyaluronic acid (HA, weight average molecular weight 57 kDa, carboxyl content 0.25 mmol), 0.593 g of Tyr-EDA (1.32 mmol, 5.28 times the carboxyl content of hyaluronic acid, denoted as 5.28 eq), 0.252 g of EDC.HCl (1.32 mmol, 5.28 eq), and 0.151 g of NHS (1.32 mmol, 5.28 eq). Dissolve hyaluronic acid in MES buffer (0.01 mol / L, pH=5.5), add EDC.HCl and NHS, stir in an ice bath for 30 min to activate, add Tyr-EDA to the reaction solution, and react for 48 h. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed with deionized water. The deionized water was changed every 8 hours. After three days, the dialyzed product was freeze-dried in a freeze dryer (-50℃, 85 Pa) to obtain HA-EDA-Tyr.

[0087] 10 mg of the HA-EDA-Tyr prepared in this example was dissolved in 0.6 mL of D2O, and the sample was recorded using a nuclear magnetic resonance spectroscopy instrument. 1 H NMR spectra, results as follows Figure 2 As shown in the NMR spectrum of HA-EDA-Tyr, multiple signal peaks appear around δ=6.7-7.1 ppm, which are NMR peaks on the benzene ring of tyrosine, proving that the tyrosine grafting was successful.

[0088] Example 3 Synthesis of Tyr-EDA-HPBA-DA, a dopamine phenylboronic acid modified tyrosine material

[0089] (1) Preparation of HPBA-CDI: Weigh 1.000 g HPBA (6.58 mmol) and 1.601 g CDI (9.87 mmol, 1.5 times the amount of HPBA, denoted as 1.5 eq) into a reaction flask, evacuate and purge with nitrogen. Under nitrogen conditions, add 20 mL of anhydrous DMSO and stir at room temperature for 24 h. After the reaction is complete, add 200 mL of dichloromethane to the reaction solution, wash three times with saturated sodium chloride aqueous solution, dry with anhydrous sodium sulfate, filter to remove anhydrous sodium sulfate, and remove dichloromethane by rotary evaporation to obtain HPBA-CDI.

[0090] (2) Preparation of Boc-Tyr-EDA-HPBA: Weigh 1.00 g HPBA-CDI (4.07 mmol) and 1.577 g Boc-Tyr-EDA (4.88 mmol, 1.2 times the amount of HPBA-CDI, denoted as 1.2 eq) into a reaction flask, evacuate and purge with nitrogen, add 20 mL of anhydrous DMSO under nitrogen conditions, and stir at room temperature for 24 h. After the reaction is complete, add 200 mL of dichloromethane to the product, wash three times successively with saturated NaHCO3 aqueous solution, 1 mol / L HCl aqueous solution and saturated NaCl aqueous solution, dry with anhydrous magnesium sulfate, concentrate by rotary evaporation to remove dichloromethane, and purify the concentrated product by column chromatography using ethyl acetate and methanol in a ratio of 30:1 as the developing solvent to obtain Boc-Tyr-EDA-HPBA.

[0091] (3) Preparation of Tyr-EDA-HPBA: Weigh 1.000 g of Boc-Tyr-EDA-HPBA (1.20 mmol), add 0.92 mL of dichloromethane, and then add 0.92 mL of trifluoroacetic acid (12 mmol, 10 eq). Stir the reaction mixture at room temperature for 12 h. Remove dichloromethane and trifluoroacetic acid from the reaction solution by rotary evaporation. Concentrate the product and precipitate it with diethyl ether to obtain Tyr-EDA-HPBA.

[0092] (4) Preparation of Tyr-EDA-HPBA-DA: Weigh 1.00 g Tyr-EDA-HPBA (1.94 mmol) and 0.368 g dopamine hydrochloride (1.94 mmol) into a reaction flask, evacuate and purge with nitrogen. Under nitrogen conditions, add 20 mL of anhydrous DMF and 0.3 mL of DIEA (1.94 mmol), and stir at room temperature for 12 h. After the reaction is complete, remove DMF from the reaction solution by rotary evaporation, add diethyl ether to the concentrate, and precipitate a white powder. Collect the powder and dry it under vacuum at room temperature to obtain the product Tyr-EDA-HPBA-DA, whose chemical structure is shown in Formula III.

[0093] 10 mg of the Tyr-EDA-HPBA-DA prepared in this example was dissolved in 0.6 mL of D2O, and the sample was recorded using a nuclear magnetic resonance spectroscopy instrument. 1 H NMR spectra, results as follows Figure 3 As shown in the NMR spectrum of Tyr-EDA-HPBA-DA, multiple signal peaks appear around δ=6.7-7.1 ppm, which are NMR peaks on the benzene ring of tyrosine and dopamine, and a signal peak appears around δ=5.1 ppm, which is the methylene group on 4-hydroxymethylphenylboronic acid, proving the successful synthesis of Tyr-EDA-HPBA-DA.

[0094]

[0095] Formula III

[0096] Example 4 Synthesis of folic acid-modified tyrosine material Tyr-PEG-FA

[0097] (1) Preparation of Boc-Tyr-PEG-FA: Weigh 1.00 g of FA-PEG 2000 -NH2 (purchased from Shanghai Pengshuo Biotechnology Co., Ltd., catalog number PS2-NFA-2K, polyethylene glycol segment weight average molecular weight of 2000 Da, 0.5 mmol), 0.144 g EDC.HCl (0.75 mmol, FA-PEG) 2000 1.5 times the amount of -NH2 (denoted as 1.5 eq), 0.086 g NHS (0.75 mmol, 1.5 eq), and 0.253 g Boc-Tyr-EDA (0.75 mmol, 1.5 eq) were placed in a reaction flask, evacuated and purged with nitrogen. Under nitrogen atmosphere, 10 mL of anhydrous DMSO was added, and the mixture was stirred at room temperature for 24 h. After the reaction was complete, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed with deionized water. The deionized water was changed every 8 h. After three days, the dialyzed product was freeze-dried (-50℃, 85 Pa) to obtain Boc-Tyr-PEG-FA.

[0098] (2) Preparation of Tyr-PEG-FA: Weigh 1.00 g of Boc-Tyr-PEG-FA, dissolve it in 1 mL of dichloromethane, add 1 mL of trifluoroacetic acid dropwise, and stir the reaction at room temperature for 12 h. After the reaction is complete, remove dichloromethane and trifluoroacetic acid from the reaction solution by rotary evaporation, add anhydrous diethyl ether to the concentrated product, and a white precipitate appears. Collect the white precipitate, dry it to obtain Tyr-PEG-FA, whose structural formula is shown in Formula IV.

[0099] 10 mg of the Boc-Tyr-PEG-FA prepared in this example was dissolved in 0.6 mL of (CD3)2SO4, and the sample was recorded using a nuclear magnetic resonance spectroscopy instrument. 1 H NMR spectra, results as follows Figure 4 As shown in the NMR spectrum of Boc-Tyr-PEG-FA, the signal peaks around δ=1.9-2.1 ppm are the methylene peaks on folic acid, and the signal peaks around δ=4.5 ppm are the methylene peaks on folic acid and tyrosine, proving the successful synthesis of Boc-Tyr-PEG-FA.

[0100]

[0101] Formula IV

[0102] Example 5: Enzyme-catalyzed encapsulation of mesenchymal stem cells with tyrosine-based materials

[0103] MSCs were cultured in T75 culture flasks until the cell confluence reached 80%. 2 mL of 0.25% trypsin-EDTA solution (containing 2.5 g / L trypsin and 0.2 g / L EDTA-2Na) was added to digest the cells. After 2 minutes, 2 mL of DMEM / F12 complete medium was added to terminate the digestion. MSCs (approximately 4 × 10⁻⁶ cells / year) were collected by centrifugation in 15 mL centrifuge tubes. 6 ~ 5×10 6 (1 stem cell). Add 6 mL of 1 mM TCEP solution to a centrifuge tube and incubate for 20 minutes, gently shaking the centrifuge tube every 5 minutes. After incubation, wash twice with PBS buffer. Then, add 1 mL of PEG-modified tyrosinase solution and incubate for 30 minutes, gently shaking the centrifuge tube every 5 minutes. After incubation, wash twice with phosphate buffer. Add 4 mL of tyrosine-based material solutions of different concentrations (0.5, 1, 2 mM) (tyrosine and the four tyrosine-based materials prepared in Examples 1-4, using DMEM / F12 complete medium), incubate for 3 h, gently shaking the centrifuge tube every 5 minutes, and wash the cells three times with PBS buffer after incubation. Five different tyrosine-based materials were used to encapsulate MSCs, named Tyr-MSCs-0.5 / 1 / 2, Tyr-EDA-Arg-MSCs-0.5 / 1 / 2, HA-EDA-Tyr-MSCs-0.5 / 1 / 2, Tyr-EDA-HPBA-DA-MSCs-0.5 / 1 / 2, and Tyr-PEG-FA-MSCs-0.5 / 1 / 2. Figure 5 As shown, MSCs coated with tyrosine appear black, indicating that tyrosinase can successfully catalyze the coating of MSCs with tyrosine materials.

[0104] The preparation method of the above-mentioned PEG-modified tyrosinase solution is as follows: Weigh 0.004 g MAL-PEG2000-NHS and 0.24 g tyrosinase (500 U / mg) into a reaction flask, evacuate and purge with nitrogen, add 2 mL PBS under nitrogen conditions, stir at room temperature for 30 minutes to obtain the PEG-modified tyrosinase solution.

[0105] Example 6: Evaluation of the effect of tyrosine-based material encapsulation on MSCs viability and proliferation

[0106] The Tyr-MSCs-2 cells and uncoated MSCs (control) prepared in Example 5 were stained using the Calcein-AM / PI staining method to assess the effect of tyrosine coating on MSC survival. Results are as follows: Figure 6 As shown in a.

[0107] The CCK-8 cell viability assay was used to assess the viability of MSCs and evaluate the effect of tyrosine coating on MSC viability. Tyr-MSCs-0.5 / 1 / 2 (sample) and uncoated MSCs (control) prepared in Example 5 were collected by centrifugation. 800 μL of pre-prepared CCK-8 solution was added to each tube, and the cells were incubated for 1 h. 200 μL of the supernatant was transferred to a 96-well plate by centrifugation, and the absorbance of each well was measured at 450 nm using a microplate reader. The relative cell viability was calculated using formula (1). The results are as follows: Figure 6 As shown in b.

[0108] The CCK-8 cell viability assay was used to assess the proliferation capacity of MSCs and evaluate the effect of tyrosine-based material coating on MSC proliferation. Tyr-MSCs-2 (sample) prepared in Example 5 and uncoated MSCs (control) were seeded into 96-well plates containing cell culture medium (density: 1 × 10⁴ cells / well). After culturing for 0, 1, 2, and 3 days, the cell culture medium in each well was removed, and 200 μL of pre-prepared CCK-8 solution was added to each well. The plates were then incubated at 37°C in the dark for 2 h. The absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was calculated using formula (1) to characterize cell proliferation. The results are as follows: Figure 6 As shown in c.

[0109] The above experimental results indicate that tyrosinase-catalyzed encapsulation of tyrosine-based materials has little effect on the activity and proliferation of MSCs.

[0110]

[0111] Example 7: Effect of Tyrosine-based material coating on the dryness characteristics of MSCs

[0112] Adipocyte induction experiment: Tyr-MSCs-2 prepared in Example 5 and unencapsulated MSCs were incubated at 2×10⁻⁶ cells / year. 4 Cells were seeded at a density of 1 / well in 12-well plates and cultured at 37°C with 5% CO2. When the cell confluence reached 100%, the culture medium was carefully aspirated from the wells, and 2 mL of adipogenic induction medium was added. After 3 days of culture, the adipogenic induction medium was replaced with adipogenic maintenance medium, and thereafter the adipogenic maintenance medium was replaced every 3 days. After 12 days, the culture medium was removed, the cells were washed once with PBS buffer, fixed with 4% paraformaldehyde for 30 min, and washed three times with PBS buffer. The prepared Oil Red O staining solution was added, and the cells were stained at room temperature for 1 h. The cells were washed three times with 60% isopropanol and observed under a microscope.

[0113] Osteoblast induction experiment: Tyr-MSCs-2 prepared in Example 5 and uncoated MSCs were incubated at 2×10⁻⁶ cells / year. 4 Cells / well were seeded at a density of 12-well plates and cultured at 37°C and 5% CO2. When the cell confluence reached 70%, the culture medium was carefully aspirated from the wells, and 2 mL of osteogenic induction medium was added. The osteogenic induction medium was changed every 3 days. After 2 weeks, the culture medium was removed, the cells were washed once with PBS buffer, fixed with 4% paraformaldehyde for 30 min, and washed three times with PBS buffer. Alizarin Red staining solution was added, and staining was performed at room temperature for 1 h. The cells were washed three times with PBS buffer and observed under a microscope.

[0114] Chondrogenic induction experiment: Tyr-MSCs-2 prepared in Example 5 and uncoated MSCs were respectively prepared into cells with a density of 2×10⁻⁶. 7 Cell suspensions of cells / mL were prepared by dropping 10 μL of the cell suspension into 12-well plates containing complete culture medium and incubating at 37°C and 5% CO2 for 2 h to allow the cells to clump together. The complete culture medium was then removed from the wells, and 2 mL of chondrogenic differentiation induction medium was added. The culture medium was changed every 3 days. After 3 weeks, the culture medium was removed, and the cells were fixed with 4% paraformaldehyde for 30 min and washed 3 times with PBS buffer. The cell clumps were then embedded in paraffin, sectioned, stained with alexandrite blue for 1 h, and observed under a microscope.

[0115] Staining results as follows Figure 7 As shown, the results indicate that the induced differentiation of MSCs coated with tyrosine-based materials was similar to that of uncoated MSCs, suggesting that coating with tyrosine-based materials had no significant effect on the differentiation ability of MSCs.

[0116] Example 8: Protective effect of tyrosine-based material encapsulation on MSCs

[0117] The protective effect of tyrosine-based material encapsulation on MSCs was detected under three adverse conditions: H2O2, trypsin, and cationic PEI, using CCK-8 cell viability assays and CalceinAM / PI live / dead cell staining assays.

[0118] The protective effect of tyrosine-based material encapsulation on MSCs under stress was detected using Calcein AM / PI live / dead staining assays. The experiment consisted of three main groups, each with two subgroups: an encapsulated MSCs group (Tyr-MSCs-2 prepared in Example 5) and an unencapsulated MSCs group. The first group was set up in an H2O2 environment: 2 mL of 2 mmol H2O2 was added to both subgroups of MSCs and incubated for 4 h. The second group was set up in a trypsin environment: 2 mL of 0.25% trypsin solution was added to both subgroups of MSCs and incubated for 4 h. The third group was set up in a cationic PEI environment: 2 mL of cationic PEI solutions at different concentrations (40 μg / mL) were added to both subgroups of MSCs and incubated for 30 min. After incubation in each group, the cells were centrifuged and washed twice with PBS buffer. Each group was then incubated with 1 mL of pre-prepared Calcein AM / PI live / dead staining solution for 15 min in the dark. The supernatant was removed by centrifugation, and the cells were washed twice with PBS buffer to prepare a cell suspension. 300 μL of the cell suspension was added to a 24-well plate, and cell viability was observed using an inverted fluorescence microscope. The experimental results are as follows: Figure 8 As shown in a.

[0119] The protective effect of tyrosine-based material encapsulation on MSCs under stress was detected using the CCK-8 cell viability assay. The experiment was divided into three main groups, each with two subgroups: an encapsulated MSCs group (Tyr-MSCs-2 prepared in Example 5) and an unencapsulated MSCs group. The first group was set up in an H2O2 environment: 2 mL of H2O2 aqueous solution at different concentrations (0.5, 1, 2 mM) was added to both subgroups of MSCs, and incubation was carried out for 4 h. The second group was set up in a trypsin environment: 2 mL of 0.25% (2.5 g / L) trypsin solution was added to both subgroups of MSCs, and incubation was carried out for different times (1, 2, 3, 4 h). The third group was set up in a cationic PEI environment: 2 mL of cationic PEI solution at different concentrations (10, 20, 40 μg / mL) was added to both subgroups of MSCs, and incubation was carried out for 30 min. After incubation in each group, the cells were centrifuged and washed twice with PBS buffer. 800 μL of pre-prepared CCK-8 solution was added, and the cells were incubated at 37°C in the dark for 1 h. 200 μL of the supernatant was centrifuged and transferred to a 96-well plate. The absorbance of each well was measured at 450 nm using a microplate reader. The relative cell viability was calculated using formula (1). The experimental results are as follows: Figure 8 As shown in b.

[0120] The above experimental results show that MSCs encapsulated with tyrosine-based materials can survive better and have better activity under the adverse environment of hydrogen peroxide, trypsin, and cationic PEI, proving that tyrosine-based material encapsulation has a protective effect on MSCs under adverse environment.

[0121] Example 9: Effect of Tyrosine-based material encapsulation on NO release from MSCs

[0122] The experiment included two groups of MSCs: one encapsulated with tyrosine-based material (Tyr-EDA-Arg-MSCs-1 prepared in Example 5) and the other without encapsulation. Both groups of cells were freeze-dried and stimulated with 100 μM H2O2 for 72 h. NO release from the MSCs in each group was detected every 12 h using the Griess method.

[0123] Experimental results are as follows Figure 9 As shown, the results indicate that Tyr-EDA-Arg-encapsulated MSCs can release more NO signaling molecules under hydrogen peroxide conditions. NO can act as a signaling molecule to achieve disease treatment.

[0124] Example 10: Cell protection effect of HA-EDA-Tyr / Prussian blue nanozyme material (PB) encapsulation

[0125] The changes in intracellular ROS levels under H2O2 conditions were detected using DCFH-DA fluorescence staining. The experiment consisted of two groups: HA-EDA-Tyr-encapsulated MSCs and HA-EDA-Tyr / PB-encapsulated MSCs. Each group was incubated with 2 mL of 1 mmol H2O2 for 1 h. After incubation, the H2O2 was removed, and the cells were washed twice with PBS buffer. The PBS buffer was removed by centrifugation. Both groups were then incubated with pre-prepared DCFH-DA staining reagent at 37°C in the dark for 20 min, followed by Hoechst staining for 10 min. Finally, the cells were washed three times with PBS buffer and imaged under a fluorescence microscope.

[0126] The method for constructing HA-EDA-Tyr / PB-encapsulated MSCs described above is the same as the method for encapsulating mesenchymal stem cells with enzyme-catalyzed tyrosine-based material in Example 5, except that the tyrosine-based material solution in Example 5 is replaced with an HA-EDA-Tyr / PB solution. The HA-EDA-Tyr / PB solution is prepared as follows: Weigh 1 mg of PB and 50 mg of HA-EDA-Tyr material, and dissolve them separately in 5 mL of DMEM / F2 complete culture medium.

[0127] Experimental results are as follows Figure 10 As shown, the results indicate that the addition of PB to the HA-EDA-Tyr material can reduce the intracellular ROS level under H2O2 conditions.

[0128] Example 11 Effect of Tyrosine-based material encapsulation on MSCs targeted homing

[0129] To observe the localization of MSCs in mice, MSCs were labeled with the near-infrared fluorescent anthocyanin dye DiR. First, MSCs cultured in culture flasks were digested according to the method in Example 5, and collected by centrifugation in 15 mL centrifuge tubes. 6 mL of 4 μM DiR staining solution was added to the centrifuge tubes, and the cells were incubated in an incubator for 20 minutes. The cells were then gently washed twice with PBS to remove unbound dye. Then, DiR-labeled MSCs coated with tyrosine-based material were obtained according to the method in Example 1 (the concentration of the tyrosine-based material solution used in the preparation process was 2 mM).

[0130] (1) Construction of a mouse liver fibrosis model

[0131] Healthy male ICR mice with SPF grade of 20-23 grams were selected. After one week of acclimatization, the mice were intraperitoneally injected with a mixture of olive oil and CCl4 (CCl4 to olive oil volume ratio of 3:7) twice a week at a dose of 1.7 μL / g for 6 consecutive weeks to establish a mouse model of liver fibrosis.

[0132] (2) Mice with liver fibrosis were randomly divided into four groups: PBS group, MSCs group, Tyr-MSCs group, and FA-Tyr-MSCs group. A control group of healthy mice was also included. Mice in each group were fixed using a tail vein injection device. Mice in the MSCs group, Tyr-MSCs group, and FA-Tyr-MSCs group were injected with 7.5 × 10⁻⁶ mg of PBS via the tail vein. 6 MSCs, Tyr-MSCs, and Tyr-PEG-FA-MSCs were administered in a 100 μL injection per mouse; mice in the PBS group received a 100 μL PBS injection via the tail vein; healthy mice received no treatment.

[0133] On days 1, 3, 5, and 7 post-injection, fluorescence intensity was observed and recorded using a small animal in vivo imaging system. The results are as follows: Figure 11 As shown in a and 11d. Seven days after injection, the heart, liver, spleen, lung, and kidney of the mice were harvested. The fluorescence intensity of the liver was observed and recorded using a small animal imaging system. The results are shown in the figures below. Figure 11 As shown in b and 11e. Seven days after injection, mouse livers were collected and fixed in 4% paraformaldehyde solution. The tissues were embedded and sectioned in paraffin. CD90 immunohistochemistry (resulting in a brownish color) was performed using a 3,3'-diaminobenzidine (DAB) and horseradish peroxidase (HRP) combination staining method. The expression of CD90 in the liver was observed under a microscope, and photographs were taken and recorded. The results are shown in the figures. Figure 11 As shown in c.

[0134] Experimental results showed that the fluorescence signal intensity in the livers of mice encapsulated with Tyr-PEG-FA-MSCs (FA-Tyr-MSCs) and Tyr-MSCs was higher than that in the MSCs group, with the FA-Tyr-MSCs group exhibiting the highest fluorescence signal intensity. On day 7 post-stem cell injection, the number of CD90+ cells in liver slices from mice in both the FA-Tyr-MSCs and Tyr-MSCs groups was higher than that in the MSCs group, with the FA-Tyr-MSCs group showing the highest number of CD90+ cells in liver slices. These results indicate that MSCs encapsulated with tyrosine-based materials can better reside in the liver, and that MSCs encapsulated with Tyr-PEG-FA-MSCs material have a certain liver-targeting effect.

[0135] Example 12 Evaluation of the effect of MSCs encapsulated with tyrosine-based materials on improving liver fibrosis

[0136] The liver fibrosis mice constructed in Example 11 were randomly divided into four groups: PBS group, MSCs group, Tyr-MSCs group, and Tyr-HD-MSCs group, with a control group of healthy mice. Mice in each group were fixed using a tail vein injection device. Mice in the MSCs group, Tyr-MSCs group, and Tyr-HD-MSCs group were injected via tail vein with 7.5 × 10⁻⁶ ppm. 6 In Example 11, fluorescently labeled MSCs, Tyr-MSCs, and Tyr-EDA-HPBA-DA were constructed. Each mouse was injected with 100 μL of the MSCs, once a week for a total of 2 weeks. Mice in the PBS group were injected with 100 μL of PBS via the tail vein once a week for a total of 2 weeks. Healthy mice were not treated in any way.

[0137] Two weeks after treatment, blood was collected from mice and kept at room temperature for 2 hours. The coagulated blood was then centrifuged at 4°C, 3000 rpm for 15 minutes. The supernatant was collected, and alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were measured using an automated blood biochemistry analyzer to assess liver damage. The results are shown below. Figure 12 As shown in b, 12c and 12d.

[0138] Two weeks after treatment, mouse livers were collected and fixed in 4% paraformaldehyde solution. The water in the tissue was gradually replaced with a gradient of alcohols, and the dehydrated tissue was immersed in a clearing agent for 1 hour. The tissue was then transferred into molten paraffin and rapidly cooled to solidify into a wax block. Paraffin sections were prepared using a microtome and dried in a 60°C oven for 30 minutes to ensure firm adhesion. The sections were then stained with H&E, Masson's red, and Sirius red. The staining results were observed and photographed under a microscope, as shown below. Figure 12 As shown in Figure a. ImageJ software was used to statistically analyze collagen fiber area data; the results are shown below. Figure 12 As shown in e and 12f.

[0139] The above experimental results indicate that, based on serum biochemical indicators (ALT, AST, and TBIL), MSCs encapsulated with tyrosine-based materials have a better reversal effect on liver fibrosis. HE, Masson, and SiriusRed staining of liver tissue sections showed that MSC treatment reduced the disordered structure of liver lobules and significantly decreased the content of collagen fibers. Compared to the unencapsulated group, the encapsulated group showed a greater reduction in collagen fiber content, demonstrating that tyrosine-based material encapsulation plays a protective role for MSCs in vivo. Among them, the MSCs group encapsulated with Tyr-EDA-HPBA-DA material (Tyr-HD-MSCs) had the lowest content of collagen fibers in mouse livers. This demonstrates that the Tyr-HD-MSCs group had the best effect in reversing liver fibrosis.

[0140] This invention provides an idea and method for constructing and applying engineered stem cells based on tyrosine-based materials. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for constructing engineered stem cells based on tyrosine-based material encapsulation, characterized in that, The process includes the following steps: first incubation of stem cells in TCEP solution, followed by washing and resuspending, then addition of PEG-modified tyrosinase solution for second incubation, followed by washing and resuspending, then addition of tyrosine solution or tyrosine-based material solution for third incubation, thus obtaining the final product.

2. The construction method according to claim 1, characterized in that, The tyrosine-based material is any one of arginine-modified tyrosine, tyrosine-modified hyaluronic acid, folic acid-modified tyrosine, and dopamine phenylboronic acid-modified tyrosine.

3. The construction method according to claim 2, characterized in that, The method for preparing the arginine-modified tyrosine includes the following steps: Step 1: Dissolve Boc-Tyr-OH, condensing agent and peptide protectant in solvent and carry out the first reaction to obtain the first reaction solution. Add the first reaction solution to ethylenediamine solution and carry out the second reaction. The obtained second reaction solution is separated and purified to obtain ethylenediamine-modified Boc-protected tyrosine Boc-Tyr-EDA. Step 2: The Boc-Tyr-EDA obtained in Step 1 is dissolved in a solvent along with Boc-Arg(Pbf)-OH, a condensing agent, a polypeptide protectant, and an organic base catalyst, and a third reaction is carried out. The resulting third reaction solution is separated and purified to obtain Boc-Tyr-EDA-Arg(Pbf)-Boc, which undergoes a deprotection reaction under the action of a deprotection reagent. The resulting deprotection reaction solution is then separated and purified to obtain the arginine-modified tyrosine.

4. The construction method according to claim 2, characterized in that, The method for preparing the tyrosine-modified hyaluronic acid includes the following steps: Step I: Boc-Tyr-OH, condensing agent and peptide protectant are dissolved in solvent and subjected to a first reaction to obtain a first reaction solution. This first reaction solution is then added to an ethylenediamine solution to carry out a second reaction. The resulting second reaction solution is separated and purified to obtain ethylenediamine-modified Boc-protected tyrosine Boc-Tyr-EDA. Under the action of a deprotection reagent, the deprotection reaction is carried out. The resulting deprotection reaction solution is then separated and purified to obtain Tyr-EDA. Step II: Hyaluronic acid is activated under the action of EDC.HCl and NHS to obtain activated hyaluronic acid, which is then subjected to an amidation reaction with Tyr-EDA obtained in Step I. The resulting amidation reaction solution is separated and purified to obtain the tyrosine-modified hyaluronic acid.

5. The construction method according to claim 2, characterized in that, The method for preparing the folic acid-modified tyrosine includes the following steps: Step i: Dissolve Boc-Tyr-OH, condensing agent and peptide protectant in solvent and carry out the first reaction to obtain the first reaction solution. Add the first reaction solution to ethylenediamine solution and carry out the second reaction. The obtained second reaction solution is separated and purified to obtain ethylenediamine-modified Boc-protected tyrosine Boc-Tyr-EDA. Step ii: The Boc-Tyr-EDA obtained in step i is dissolved in a solvent with FA-PEG-NH2, EDC.HCl and NHS and subjected to a third reaction. The resulting third reaction solution is separated and purified to obtain Boc-Tyr-PEG-FA, which undergoes a deprotection reaction under the action of a deprotection reagent. The resulting deprotection reaction solution is then separated and purified to obtain the folic acid-modified tyrosine.

6. The construction method according to claim 2, characterized in that, The preparation method of the dopamine phenylboronic acid modified tyrosine includes the following steps: Step a: Dissolve Boc-Tyr-OH, condensing agent and peptide protectant in solvent and carry out the first reaction to obtain the first reaction solution. Add the first reaction solution to ethylenediamine solution and carry out the second reaction. The obtained second reaction solution is separated and purified to obtain ethylenediamine-modified Boc-protected tyrosine Boc-Tyr-EDA. Step b: 4-hydroxymethylphenylboronic acid HPBA and N,N'-carbonyldiimidazole CDI are dissolved in a solvent to carry out the third reaction. The resulting third reaction solution is separated and purified to obtain HPBA-CDI. Step c: Dissolve the Boc-Tyr-EDA obtained in step a and the HPBA-CDI obtained in step b in a solvent to carry out the fourth reaction. The resulting fourth reaction solution is separated and purified to obtain Boc-Tyr-EDA-HPBA. Under the action of a deprotection reagent, it undergoes a deprotection reaction. The resulting deprotection reaction solution is separated and purified to obtain Tyr-EDA-HPBA. Step d: The Tyr-EDA-HPBA obtained in step c is dissolved in a solvent with dopamine hydrochloride and an organic base catalyst to carry out the fifth reaction. The resulting fifth reaction solution is separated and purified to obtain the dopamine phenylboronic acid modified tyrosine.

7. The construction method according to claim 1, characterized in that, The preparation method of the PEG-modified tyrosinase solution includes the following steps: reacting MAL-PEG-NHS with tyrosinase in a buffer solution to obtain the solution.

8. The construction method according to claim 7, characterized in that, The weight-average molecular weight of the polyethylene glycol segments in the MAL-PEG-NHS is 2000-5000 Da; the mass ratio of MAL-PEG-NHS to tyrosinase is 1:60; the buffer solution is phosphate buffer; the mass ratio of tyrosinase to solvent volume is 0.12 g: 1 mL; the reaction is carried out under nitrogen atmosphere with stirring at room temperature for 20-40 min.

9. The construction method according to claim 1, characterized in that, The stem cells are mesenchymal stem cells; the concentration of the TCEP solution is 0.5-1 mM; the first incubation is at room temperature for 10-20 minutes; the second incubation is at 37°C for 20-40 minutes; the concentration of the tyrosine-based material solution or tyrosine solution is 0.5-2 mM; the third incubation is at 37°C for 3-5 hours; the washing and resuspension solutions are both phosphate buffered saline.

10. Engineered stem cells based on tyrosine-based materials encapsulated and constructed by the construction method according to any one of claims 1 to 9.

11. The use of engineered stem cells based on tyrosine-based materials as described in claim 10 in the preparation of drugs for treating liver fibrosis.

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