Antitumor stem cell binding negative hydrogen ion preparation and preparation method thereof

Abstract

The application discloses an anti-tumor stem cell combined negative hydrogen ion preparation and a preparation method thereof, and belongs to the field of biological medicines. The preparation comprises an engineered stem cell dispersion liquid, a negative hydrogen ion carrier and a pharmaceutically acceptable carrier. The engineered stem cell dispersion liquid is a lung interstitial mesenchymal stem cell dispersion liquid with down-regulated expression of a gene for inhibiting macrophage phagocytosis, up-regulated expression of a gene for promoting macrophage phagocytosis and connected PAR1 protein. The negative hydrogen ion carrier is a core-shell structure. The shell part is polylactic acid-glycolic acid copolymer, and the core part comprises the following components in parts by mass: cyclodextrin 30-50 parts, hyaluronic acid 15-25 parts, Adoxaceae extract 20-40 parts and negative hydrogen ion powder 5-15 parts. The application solves the problem of low drug delivery efficiency caused by low targeting efficiency of stem cells on a tumor microenvironment in the prior art, and has the potential to be applied to the preparation of a lung cancer treatment drug.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an anti-tumor stem cell-based negative hydrogen ion preparation and its preparation method. Background Technology

[0002] Lung cancer is one of the leading causes of cancer death worldwide, with over one million new cases each year, the vast majority of which are related to smoking. Lung cancer is divided into two main types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), the latter including several subtypes. Adenocarcinoma is the most common subtype of NSCLC, and its incidence is increasing annually; it is also the most common type of lung cancer among women and non-smokers. Despite recent advancements in the diagnosis and treatment of lung cancer, the overall survival rate for lung cancer patients remains below 15%.

[0003] Stem cells are a type of cell with differentiation potential, immune regulation, and homing characteristics, and have the potential to serve as carriers for tumor drug delivery, but their targeting efficiency in the tumor microenvironment is low. Macrophages can phagocytose or take up and clear cancer cells, but cancer cells usually highly express CD47 molecules, which inhibits the phagocytic activity of macrophages, thereby escaping immune clearance.

[0004] Therefore, there is an urgent need for a highly targeted stem cell preparation that can enhance the function of the human immune system to solve the above problems. Summary of the Invention

[0005] Therefore, the present invention provides an anti-tumor stem cell combined with negative hydrogen ion preparation and its preparation method, in order to solve the problem of low drug delivery efficiency caused by the low targeting efficiency of stem cells to the tumor microenvironment in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, an antitumor stem cell-based negative hydrogen ion preparation is provided, the preparation comprising an engineered stem cell dispersion, a negative hydrogen ion carrier, and a pharmaceutically acceptable carrier.

[0007] Furthermore, the engineered stem cell dispersion is a lung mesenchymal stem cell dispersion that has been induced to downregulate the expression of genes that inhibit macrophage phagocytosis, upregulate the expression of genes that promote macrophage phagocytosis, and is linked to the PAR1 protein, with a viable cell count of 1×10⁻⁶. 5 -1×10 7 The number of samples per mL is specified; the pharmaceutically acceptable carrier is physiological saline, phosphate buffer, or a medical biocompatible matrix.

[0008] Furthermore, the gene that inhibits macrophage phagocytosis is CD47, and the gene that promotes macrophage phagocytosis is CRT; among them, the inducer that downregulates CD47 gene expression is catechin; and the inducer that upregulates CRT gene expression is butadiene.

[0009] Furthermore, the negative hydrogen ion carrier has a core-shell structure; wherein the shell part is a polylactic acid-glycolic acid copolymer, and the core part includes the following components in parts by weight: 30-50 parts of cyclodextrin, 15-25 parts of hyaluronic acid, 20-40 parts of American plum blossom extract, and 5-15 parts of negative hydrogen ion powder.

[0010] Furthermore, the volume ratio of the engineered stem cell dispersion to the pharmaceutically acceptable carrier is 1:(1-5), the volume-to-mass ratio of the engineered stem cell dispersion to the negative hydrogen ion carrier is 1mL:(0.2-1)mg, and the volume-to-mass ratio of the pharmaceutically acceptable carrier to the negative hydrogen ion carrier is (1-10)mL:1mg; the dosage form of the preparation is an injection, and the injection method is intravenous injection.

[0011] According to a second aspect of the present invention, a method for preparing an anti-tumor stem cell-based negative hydrogen ion preparation is provided, which specifically includes the following steps: S1. Mesenchymal stem cells cultured to the third generation were seeded into 96-well plates and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2 for 12 hours. After culturing, they were cultured in DMEM medium containing catechin and then in DMEM medium containing butamin for 24-48 hours to obtain stem cells that promote macrophage phagocytosis. After both cultures, the stem cells were washed out for later use. Linkage of S2 and PAR1 proteins: N-hydroxysuccinimide palmitate was dissolved in dimethyl sulfoxide and added to phosphate buffer containing PAR1 protein. The mixture was reacted at room temperature for 1-4 hours, dialyzed with deionized water for 24 hours, and then lyophilized to obtain PA-PAR1. Stem cells that promote macrophage phagocytosis are cultured in DMEM medium containing PA-PAR1 for 5-15 min; the culture conditions are 37℃ and 5% CO2 atmosphere, and engineered stem cells are obtained after elution; the engineered stem cell dispersion is obtained after dispersion with physiological saline. S3. Preparation of negative hydrogen ion carriers: Cyclodextrin, negative hydrogen ion powder, hyaluronic acid, and Western mountain plum extract were mixed in a certain proportion, then soaked in a polylactic acid-hydroxyacetic acid copolymer solution for encapsulation, the solvent was removed and freeze-dried to obtain a negative hydrogen ion carrier. S4. Mix and disperse the engineered stem cell dispersion, negative hydrogen ion carrier and pharmaceutically acceptable carrier in proportion, and stir at room temperature at 50-100 rpm to obtain an anti-tumor stem cell-based negative hydrogen ion preparation.

[0012] Furthermore, in step S1, the concentrations of catechin and buteavorin in the DMEM medium are 30-70 μM and 5-10 μM, respectively.

[0013] Further, in step S2, the concentration of N-hydroxysuccinimide palmitate dissolved in dimethyl sulfoxide is 3-10 mg / mL, the concentration of PAR1 protein in phosphate buffer is 0.1 mg / mL, and the concentration of PA-PAR1 in DMEM medium containing PA-PAR1 is 1-20 μg / mL.

[0014] Furthermore, step S3 specifically includes the following steps: Cyclodextrin, negative hydrogen ion powder, American plum blossom extract and hyaluronic acid were mixed in proportion and then soaked in an ethyl acetate solution of polylactic acid-hydroxyacetic acid copolymer with a final concentration of 1-5 mg / mL. After being mixed evenly at room temperature, the mixture was vacuum rotary evaporated in a water bath at 30-40℃ to solidify the copolymer and uniformly coat the surface of the pre-coated body. The negative hydrogen ion carrier was obtained by freeze drying. The preparation of the extract from the Western Mountain plum blossom includes the following steps: Fresh plum blossoms from Xiyang Mountain are broken into small pieces of 1-2 cm, washed with clean water, and then mixed with deionized water at a mass-to-volume ratio of 1 g: (5-10) mL. The pH is adjusted to 4.0-6.0, and 1-2% of a compound hydrolytic enzyme is added to the mixture. The mixture is then hydrolyzed at 40-50℃ for 4-8 hours. After hydrolysis, the temperature is raised to 90-95℃ to inactivate the enzyme, yielding the hydrolysate. The compound hydrolytic enzyme is a mixture of cellulase and ligninase at a mass ratio of 4:1. The enzymatic hydrolysate was microfiltered through a filter membrane with a pore size of 0.1-0.2 μm to remove macromolecular fibers, and the permeate was collected. The permeate was concentrated and then spray-dried to obtain the extract of Prunus cerasifera flower.

[0015] According to a third aspect of the present invention, an application of an anti-tumor stem cell combined with a negative hydrogen ion preparation in the preparation of a drug for treating lung cancer is provided.

[0016] The present invention has the following advantages: 1. This invention induces mesenchymal stem cells (MSCs) with catechin and buteacon respectively, downregulating CD47 expression and upregulating CRT expression. This enables the MSCs to be efficiently recognized and phagocytosed by macrophages. The engineered MSCs themselves target and survive on tumors. In the tumor microenvironment, due to the high expression of CRT and the low expression of CD47, they are recognized and phagocytosed by tumor-associated macrophages (TAMs), thereby releasing negative hydrogen ion carriers in situ within the tumor for precise delivery. Furthermore, this invention connects the PAR1 protein to the surface of MSCs, responding to the high expression of MMP1 protein in the lung cancer microenvironment. Combined with the first-pass effect of the lung and the phagocytic activity of macrophages, this synergistically enhances the targeted delivery efficiency of the formulation.

[0017] 2. This invention encapsulates negative hydrogen ion powder in a core-shell structured carrier, avoiding the activity decay of negative hydrogen ions during in vivo circulation and prolonging their continuous effect time at the tumor site; and after the carrier and stem cells are phagocytosed by macrophages, the strong reducing properties of negative hydrogen ions can polarize the cancer-promoting M2 type cells into cancer-suppressing M1 type cells, thereby promoting lung cancer treatment. Detailed Implementation

[0018] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] According to a first aspect of the present invention, an antitumor stem cell-binding negative hydrogen ion preparation is provided, the preparation comprising an engineered stem cell dispersion, a negative hydrogen ion carrier, and a pharmaceutically acceptable carrier.

[0020] The engineered stem cell dispersion is a lung mesenchymal stem cell dispersion that has been induced to downregulate gene expression that inhibits macrophage phagocytosis, upregulate gene expression that promotes macrophage phagocytosis, and is linked to the PAR1 protein, with a viable cell count of 1×10⁻⁶. 5 -1×10 7 Cells / mL; pharmaceutically acceptable carriers are physiological saline, phosphate buffer, or medical biocompatible matrix.

[0021] Among them, the gene that inhibits macrophage phagocytosis is CD47, and the gene that promotes macrophage phagocytosis is CRT; among them, the inducer that induces downregulation of CD47 gene expression is catechin; and the inducer that induces upregulation of CRT gene expression is buteacon.

[0022] The negative hydrogen ion carrier has a core-shell structure; the shell part is a polylactic acid-glycolic acid copolymer, and the core part includes the following components by weight: 30-50 parts of cyclodextrin, 15-25 parts of hyaluronic acid, 20-40 parts of American plum blossom extract, and 5-15 parts of negative hydrogen ion powder.

[0023] The volume ratio of engineered stem cell dispersion to pharmaceutically acceptable carrier is 1:(1-5), the volume-to-mass ratio of engineered stem cell dispersion to negative hydrogen ion carrier is 1mL:(0.2-1)mg, and the volume-to-mass ratio of pharmaceutically acceptable carrier to negative hydrogen ion carrier is (1-10)mL:1mg. The dosage form of the preparation is an injection, and the injection method is intravenous injection.

[0024] According to a second aspect of the present invention, a method for preparing an anti-tumor stem cell-based negative hydrogen ion preparation is provided, which specifically includes the following steps: S1. Mesenchymal stem cells cultured to the third generation were seeded into 96-well plates and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2 for 12 hours. After culturing, they were cultured in DMEM medium containing catechin and then in DMEM medium containing butamin for 24-48 hours to obtain stem cells that promote macrophage phagocytosis. After both cultures, the stem cells were washed out for later use. Linkage of S2 and PAR1 proteins: N-hydroxysuccinimide palmitate was dissolved in dimethyl sulfoxide and added to phosphate buffer containing PAR1 protein. The mixture was reacted at room temperature for 1-4 hours, dialyzed with deionized water for 24 hours, and then lyophilized to obtain PA-PAR1. Stem cells that promote macrophage phagocytosis are cultured in DMEM medium containing PA-PAR1 for 5-15 min; the culture conditions are 37℃ and 5% CO2 atmosphere, and engineered stem cells are obtained after elution; the engineered stem cell dispersion is obtained after dispersion with physiological saline. S3. Preparation of negative hydrogen ion carriers: Cyclodextrin, negative hydrogen ion powder, hyaluronic acid, and Western mountain plum extract were mixed in a certain proportion, then soaked in a polylactic acid-hydroxyacetic acid copolymer solution for encapsulation, the solvent was removed and freeze-dried to obtain a negative hydrogen ion carrier. S4. Mix and disperse the engineered stem cell dispersion, negative hydrogen ion carrier and pharmaceutically acceptable carrier in proportion, and stir at room temperature at 50-100 rpm to obtain an anti-tumor stem cell-based negative hydrogen ion preparation.

[0025] In step S1, the concentrations of catechin and buteavorin in the DMEM medium were 30-70 μM and 5-10 μM, respectively.

[0026] In step S2, the concentration of N-hydroxysuccinimide palmitate dissolved in dimethyl sulfoxide is 3-10 mg / mL, the concentration of PAR1 protein in phosphate buffer is 0.1 mg / mL, and the concentration of PA-PAR1 in DMEM medium containing PA-PAR1 is 1-20 μg / mL.

[0027] Step S3 specifically includes the following steps: Cyclodextrin, negative hydrogen ion powder, American plum blossom extract and hyaluronic acid were mixed in proportion and then soaked in an ethyl acetate solution of polylactic acid-hydroxyacetic acid copolymer with a final concentration of 1-5 mg / mL. After being mixed evenly at room temperature, the mixture was vacuum rotary evaporated in a water bath at 30-40℃ to solidify the copolymer and uniformly coat the surface of the pre-coated body. The negative hydrogen ion carrier was obtained by freeze drying. The preparation of the extract from the Western Mountain plum blossom includes the following steps: Fresh plum blossoms from Xiyang Mountain are broken into small pieces of 1-2 cm, washed with clean water, and then mixed with deionized water at a mass-to-volume ratio of 1 g: (5-10) mL. The pH is adjusted to 4.0-6.0, and 1-2% of a compound hydrolytic enzyme is added to the mixture. The mixture is then hydrolyzed at 40-50℃ for 4-8 hours. After hydrolysis, the temperature is raised to 90-95℃ to inactivate the enzyme, yielding the hydrolysate. The compound hydrolytic enzyme is a mixture of cellulase and ligninase at a mass ratio of 4:1. The enzymatic hydrolysate was microfiltered through a filter membrane with a pore size of 0.1-0.2 μm to remove macromolecular fibers, and the permeate was collected. The permeate was concentrated and then spray-dried to obtain the extract of Prunus cerasifera flower.

[0028] According to a third aspect of the present invention, an application of an anti-tumor stem cell combined with a negative hydrogen ion preparation in the preparation of a drug for treating lung cancer is provided.

[0029] To better illustrate the technical path of the present invention, the following embodiments and comparative examples are provided.

[0030] Example 1 S1. Human lung mesenchymal stem cells (purchased from Hebei Beike Biotechnology Co., Ltd.) cultured to the third generation were seeded into 96-well plates and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2 atmosphere for 12 h. After culturing, they were cultured for 36 h in DMEM medium containing catechin and buteacon (purchased from Shanghai Maclean Biotechnology Co., Ltd.) to obtain stem cells that promote macrophage phagocytosis. After both cultures, the cells were washed and used for later use. The concentrations of catechin and buteacon in DMEM medium were 50 μM and 8 μM, respectively. S2. Dissolve 315 mg of N-hydroxysuccinimide palmitate (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS: 14464-31-4) in 45 mL of dimethyl sulfoxide, add it to 5 mL of phosphate buffer containing 0.1 mg / mL of PAR1 recombinant protein (purchased from Shanghai Zeye Biotechnology Co., Ltd.), react at room temperature for 2 h, dialyze with deionized water for 24 h, and then freeze dry to obtain PA-PAR1; Macrophage-promoting stem cells were cultured in DMEM medium containing 20 μg / mL PA-PAR1 for 10 min; the culture conditions were 37℃ and 5% CO2 atmosphere. After elution, engineered stem cells were obtained; they were dispersed with physiological saline to a viable cell count of 1×10⁶. 6 The engineered stem cell dispersion was obtained after the number of cells / mL was reduced; S3. Preparation of negative hydrogen ion carriers: 80 mg cyclodextrin, 40 mg hyaluronic acid, 60 mg American plum blossom extract, and 20 mg negative hydrogen ion powder (purchased from Rizhao Life Valley Biotechnology Development Co., Ltd.) were mixed in proportion, then soaked in a polylactic acid-glycolic acid copolymer solution for encapsulation, the solvent was removed and the mixture was freeze-dried to obtain a negative hydrogen ion carrier. The preparation of the extract from the Western Mountain plum blossom includes the following steps: 100g of fresh Western mountain plum blossoms were broken into 1cm pieces, washed with clean water, mixed with 1L of deionized water, and the pH was adjusted to 5.0. 8.8g of cellulase and 2.2g of ligninase were added, and the mixture was enzymatically hydrolyzed at 45℃ for 8 hours. After the enzymatic hydrolysis was completed, the temperature was raised to 95℃ to inactivate the enzyme, and the enzymatic hydrolysate was obtained. The enzymatic hydrolysate was microfiltered through a 0.1 μm pore size filter membrane to remove macromolecular fibers, and the permeate was collected. The permeate was concentrated and then spray-dried to obtain the extract of Prunus cerasifera flower; S4. Mix and disperse 100 mL of engineered stem cell dispersion, 100 mg of negative hydrogen ion carrier and 300 mL of physiological saline in proportion, and stir at room temperature at 50 rpm to obtain an anti-tumor stem cell combined with negative hydrogen ion preparation.

[0031] Example 2 This embodiment is based on Example 1, except that the concentrations of catechin and buteavorin in DMEM culture medium in step S1 are 30 and 5 μM, respectively, while the other specific parameters are the same as in Example 1.

[0032] Example 3

[0033] This embodiment is based on Example 1, except that the concentrations of catechin and buteavorin in DMEM culture medium in step S1 are 70 and 10 μM, respectively, while the other specific parameters are the same as in Example 1.

[0034] Example 4

[0035] This embodiment is based on Example 1, except that in step S2, the amount of N-hydroxysuccinimide palmitate added is 480 mg, dissolved in 48 mL of dimethyl sulfoxide, i.e., the concentration is 10 mg / mL; the amount of phosphate buffer containing PAR1 protein added is 12 mL, and the other specific parameters are the same as in Example 1.

[0036] Example 5

[0037] This embodiment is based on Embodiment 1, except that in step S4, the masses of cyclodextrin, negative hydrogen ion powder, hyaluronic acid and American plum blossom extract are 100mg, 10mg, 50mg and 40mg respectively, while the other specific parameters are the same as in Embodiment 1.

[0038] Example 6

[0039] This embodiment is based on Embodiment 1, except that in step S4, the masses of cyclodextrin, negative hydrogen ion powder, hyaluronic acid and American plum blossom extract are 60mg, 30mg, 30mg and 80mg respectively, while the other specific parameters are the same as in Embodiment 1.

[0040] Comparative Example 1

[0041] This comparative example is based on Example 1, except that step S1 is not performed, i.e., the induction culture of catechin and buteavorin is not performed. The other specific parameters are the same as in Example 1.

[0042] Comparative Example 2

[0043] This comparative example is based on Example 1, except that step S2 is not performed, i.e., the PAR1 protein is not connected. All other specific parameters are the same as in Example 1.

[0044] Comparative Example 3

[0045] This comparative example is based on Example 1, except that negative hydrogen ion powder is not added in step S3, but is replaced by an equal mass of cyclodextrin. The other specific parameters are the same as in Example 1.

[0046] Comparative Example 4

[0047] This comparative example is based on Example 1, except that the negative hydrogen ion carrier in step S3 specifically includes the following components: 160 mg of cyclodextrin and 40 mg of hyaluronic acid, that is, negative hydrogen ion powder and American plum blossom extract are not added, and the other specific parameters are the same as in Example 1.

[0048] Test Example 1

[0049] This invention used a human PAR1 ELISA kit (purchased from Shanghai Zeye Biotechnology Co., Ltd.), a human CRT ELISA kit (purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., model: ml105623), and a human CD47 ELISA kit (purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., model: ml057410) to determine the contents of PAR1, CD47, and CRT proteins in the preparations prepared in Examples 1-4 and Comparative Examples 1-2. Human lung mesenchymal stem cells that have not undergone any treatment and have been passaged to the third generation were used as the control group. The results are recorded in Table 1.

[0050] Table 1. PAR1, CD47, and CRT protein content in each group Example 1 154.2±6.3 335.2±20.7 44.2±2.7 Example 2 147.5±5.8 307.1±16.8 40.8±2.5 Example 3 159.7±7.0 351.4±22.5 46.6±3.0 Example 4 179.8±7.4 330.6±19.9 43.7±3.1 Comparative Example 1 150.9±6.0 99.3±5.4 94.6±3.9 Comparative Example 2 98.7±4.9 287.1±14.2 43.5±2.7 As shown in Table 1, in Examples 1-4, after sequential induction with catechin and buteacon, the expression level of CD47 in mesenchymal stem cells was significantly downregulated to 40.8%-46.6% of the control group, while the expression level of CRT was upregulated to 307.1%-351.4% of the control group, indicating that catechin and buteacon can effectively regulate the expression levels of CD47 and CRT proteins. In Comparative Example 1, the expression levels of the two proteins showed no significant change compared to the control group, further confirming the inducing effect of the two inducers. In Comparative Example 2, without PAR1 protein ligation, the expression levels of CRT and CD47 proteins showed no significant difference compared to the Example groups, indicating that PAR1 protein ligation does not affect the expression of genes promoting macrophage phagocytosis. Furthermore, in Examples 1-4, the expression level of PAR1 increased by 47.5%-79.8% compared to the control group, indicating that PA-PAR1 successfully ligated to the stem cell surface. The above results indicate that inducing mesenchymal stem cell culture with catechins and butea superba can achieve upregulation and downregulation of CD47 and CRT proteins, and this induction shows a concentration-dependent effect; in addition, a higher concentration of palmitic acid N-hydroxysuccinimide ester is beneficial to improving the connection efficiency of PAR1 protein.

[0051] Test Example 2 This invention uses mouse macrophages (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) as a model to determine the polarization ability of the preparation prepared in this invention on macrophages, specifically including the following steps: Mouse RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin antibiotics at 37°C and 5% CO2 for 24 h. The cultured macrophages and LLC cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were seeded in 6-well plates at a cell ratio of 1:1 and co-cultured at 37°C and 5% CO2 for 24 hours. The medium was replaced with DMEM containing 5% (v / v) of the formulation prepared in this invention, and cultured for another 24 h. RAW264.7 macrophages without added reagents and without co-culture with LLC cells were set as a blank group; macrophages co-cultured only with LLC cells but without added formulations were set as a control group. After culture, RAW264.7 cells were collected, washed twice with phosphate-buffered saline (PBS) pre-cooled to 4°C, and then fixed with 4% paraformaldehyde in the dark for 15 min. After washing, the cells were resuspended in PBS, permeabilized and blocked for 45 min, and then PE-labeled anti-mouse CD206 antibody and FITC-labeled anti-mouse iNOS antibody were added. The cells were incubated at 4°C in the dark for 30 min. After washing, the ratio of M1 / M2 macrophages was detected by flow cytometry. The results are recorded in Table 2. Among them, iNOS was the marker for M1 cells and CD206 was the marker for M2 cells.

[0052] Table 2. Percentage of M1 macrophages, percentage of M2 macrophages, and M1 / M2 ratio in each group. Example 1 61.3±2.9 31.4±1.3 1.95 Example 2 60.0±2.2 35.0±1.5 1.71 Example 3 64.8±3.1 29.7±1.2 2.18 Example 4 65.0±3.2 32.7±1.4 1.98 Example 5 64.9±3.0 31.6±1.4 2.05 Example 6 59.9±2.7 32.5±1.3 1.84 Comparative Example 1 48.1±1.6 46.7±2.2 1.02 Comparative Example 2 57.2±2.1 41.8±2.1 1.36 Comparative Example 3 39.8±1.4 57.7±3.2 0.68 Comparative Example 4 50.5±2.0 48.8±2.2 1.03 control group 28.7±1.4 70.5±3.5 0.41 Blank group 69.4±3.4 23.6±1.0 2.94 As can be seen, in this test, the M1 / M2 ratio of the untreated blank group was 2.94, while that of the control group, which was not treated and co-cultured with LLC cells, was only 0.41. This indicates that the cell model was successful and that the macrophages were affected by the lung cancer cells, thus causing a change in subtype.

[0053] As shown in Table 2, after adding the formulation prepared in the examples of this invention, the M1 / M2 ratio of macrophages was ≥1.7, significantly higher than that of the comparative and control groups, and close to that of the blank group. This indicates that the complete stem cell formulation can promote the polarization of M2 macrophages towards M1, thus possessing the potential to treat lung cancer. Compared with the examples, the M1 / M2 ratios of comparative examples 1-4 were only 0.68-1.36. Among them, comparative example 2 showed the highest ratio. This is because the main function of PAR1 is to respond to the highly expressed MMP protein in the lung cancer environment, thereby improving the homing efficiency of stem cells, rather than directly participating in the polarization regulation of macrophages. The reason why the ratio of the control group without catechin and buteacon induction was significantly reduced is that the control group did not undergo CD47 downregulation and CRT upregulation treatment. The stem cell surface still maintained a high level of CD47 expression and a low level of CRT expression. Therefore, the efficiency of its phagocytosis by macrophages was significantly reduced, resulting in the inability of the negative hydrogen ion carrier to be effectively delivered into the macrophage. The efficiency of M2 to M1 subtype polarization was greatly reduced. This indicates that the CD47 / CRT phenotypic editing induced by catechin and buteacon is the basic step for the preparation of this invention to promote macrophage reprogramming.

[0054] Comparative Example 3 exhibited the lowest M1 / M2 ratio among the comparative examples. This is because negative hydrogen ions reverse the polarization shift of macrophages towards the M2 type in the tumor microenvironment by inhibiting the CCL2 / CCR2 signaling axis and upregulate the expression of the M1 marker iNOS. Since negative hydrogen ions were not added in Comparative Example 3, this polarization signal could not be triggered, indicating that negative hydrogen ions are a core component of the formulation prepared in this invention for achieving macrophage reprogramming and thus inhibiting tumors. Furthermore, *Prunus armeniaca* extract is rich in flavonoids and other polyphenolic compounds, possessing antioxidant and anti-inflammatory effects. It reduces the content of reactive oxygen species inside macrophages, creating a favorable microenvironment for M1 polarization. The M1 / M2 ratio of only 1.03 in Comparative Example 4, which lacked *Prunus armeniaca* extract, confirms this, also indicating that negative hydrogen ions and *Prunus armeniaca* extract have a synergistic effect on the polarization shift of macrophages from the M2 type to the M1 type in the formulation prepared in this invention.

[0055] Test Example 3

[0056] To verify the therapeutic effect of the formulation prepared in this invention on lung cancer, mice were used as a model animal for verification, specifically including the following steps: Fifty-five C57BL / 6 mice were randomly divided into 11 groups of five mice each, with one group serving as the model group. Lewis cells in the logarithmic growth phase were collected from each group of mice and 0.2 mL of Lewis cell suspension was subcutaneously injected into the right axilla of each mouse. The concentration of the cell suspension was 1 × 10⁻⁶. 7 1 / mL; 7 days after inoculation, millet-sized nodules can be felt. When they grow to a diameter of 3-6 mm that can be touched under the skin, the model is considered to be successful. After successful modeling, mice in all groups except the model group were injected daily via the tail vein with the preparations prepared in each example and comparative example at a dose of 5 μL / g body weight for 15 consecutive days. Mice in the model group were injected with an equal volume of physiological saline. During this period, all mice were fed normally. After the last injection, all mice were fasted and deprived of water for 12 hours, then sacrificed, and the tumors were removed and weighed. The results are recorded in Table 3.

[0057] Table 3 Tumor quality in each group of mice Example 1 4.3±0.19 Example 2 4.8±0.20 Example 3 3.7±0.17 Example 4 4.0±0.20 Example 5 4.1±0.19 Example 6 4.6±0.21 Comparative Example 1 7.0±0.28 Comparative Example 2 6.3±0.25 Comparative Example 3 8.2±0.37 Comparative Example 4 7.2±0.30 Model group 9.0±0.58 It can be seen that the negative hydrogen ion-stem cell preparation prepared in this invention has a good inhibitory effect on lung cancer. In Examples 1-6, after injection of the preparation, the tumor mass of mice decreased by 46.67%-55.56% compared to the model group, a significantly better effect than the comparative group. The tumor mass of Comparative Examples 1-4 decreased by 22.22%, 30.00%, 8.89%, and 20.00% respectively compared to the model group. Mechanistically, PAR1 mediates efficient homing of stem cells to the lung cancer microenvironment, and CD47 downregulation combined with CRT upregulation promotes the phagocytosis and uptake of engineered stem cells by macrophages. After phagocytosis, the negative hydrogen ions released by the stem cells synergistically drive macrophage polarization from M2 to M1 type with the extract of *Prunus armeniaca*, thereby improving the tumor immune microenvironment and inhibiting tumor growth. The tumor mass decrease in Comparative Example 3 was only 8.89%, indicating that negative hydrogen ions are the core functional component for the anti-tumor effect of this preparation. Experimental results show that the inhibitory effect of the preparation prepared in this invention on lung cancer tumors depends on the high response of PAR1-MMP protein, the downregulation of CD47 expression and the upregulation of CRT expression to enhance the phagocytic capacity of macrophages on stem cells, and the release of negative hydrogen ions inside stem cells after macrophage phagocytosis and the release of Prunus cerasifera extract to polarize macrophages from M2 type to M1 type, thereby inhibiting tumors.

[0058] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A stem cell-based antitumor preparation combined with negative hydrogen ions, characterized in that, The formulation includes engineered stem cell dispersions, negative hydrogen ion carriers, and pharmaceutically acceptable carriers.

2. The formulation according to claim 1, characterized in that, The engineered stem cell dispersion is a lung mesenchymal stem cell dispersion that has been induced to downregulate gene expression that inhibits macrophage phagocytosis, upregulate gene expression that promotes macrophage phagocytosis, and is linked to PAR1 protein, with a viable cell count of 1×10⁵-1×10⁷ cells / mL; the pharmaceutically acceptable carrier is physiological saline, phosphate buffer, or a medical biocompatible matrix.

3. The formulation according to claim 2, characterized in that, The gene that inhibits macrophage phagocytosis is CD47, and the gene that promotes macrophage phagocytosis is CRT; among them, the inducer that induces downregulation of CD47 gene expression is catechin; and the inducer that induces upregulation of CRT gene expression is butamin.

4. The formulation according to claim 1, characterized in that, The negative hydrogen ion carrier has a core-shell structure; wherein the shell part is a polylactic acid-glycolic acid copolymer, and the core part includes the following components by weight: 30-50 parts of cyclodextrin, 15-25 parts of hyaluronic acid, 20-40 parts of American plum blossom extract, and 5-15 parts of negative hydrogen ion powder.

5. The formulation as described in claim 1, characterized in that, The volume ratio of the engineered stem cell dispersion to the pharmaceutically acceptable carrier is 1:(1-5), the volume-to-mass ratio of the engineered stem cell dispersion to the negative hydrogen ion carrier is 1mL:(0.2-1)mg, and the volume-to-mass ratio of the pharmaceutically acceptable carrier to the negative hydrogen ion carrier is (1-10)mL:1mg; the dosage form of the preparation is an injection, and the injection method is intravenous injection.

6. A method for preparing an anti-tumor stem cell-based negative hydrogen ion preparation, used to prepare the preparation according to any one of claims 1-5, characterized in that, Specifically, the following steps are included: S1. Mesenchymal stem cells cultured to the third generation were seeded into 96-well plates and cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2 for 12 hours. After culturing, they were cultured in DMEM medium containing catechin and then in DMEM medium containing butamin for 24-48 hours to obtain stem cells that promote macrophage phagocytosis. After both cultures, the stem cells were washed out for later use. Linkage of S2 and PAR1 proteins: N-hydroxysuccinimide palmitate was dissolved in dimethyl sulfoxide and added to phosphate buffer containing PAR1 protein. The mixture was reacted at room temperature for 1-4 hours, dialyzed with deionized water for 24 hours, and then lyophilized to obtain PA-PAR1. Stem cells that promote macrophage phagocytosis are cultured in DMEM medium containing PA-PAR1 for 5-15 min; the culture conditions are 37℃ and 5% CO2 atmosphere, and engineered stem cells are obtained after elution; the engineered stem cell dispersion is obtained after dispersion with physiological saline. S3. Preparation of negative hydrogen ion carriers: Cyclodextrin, negative hydrogen ion powder, hyaluronic acid, and Western mountain plum extract were mixed in a certain proportion, then soaked in a polylactic acid-hydroxyacetic acid copolymer solution for encapsulation, the solvent was removed and freeze-dried to obtain a negative hydrogen ion carrier. S4. Mix and disperse the engineered stem cell dispersion, negative hydrogen ion carrier and pharmaceutically acceptable carrier in proportion, and stir at room temperature at 50-100 rpm to obtain an anti-tumor stem cell-based negative hydrogen ion preparation.

7. The preparation method according to claim 6, characterized in that, In step S1, the concentrations of catechin and buteavorin in the DMEM medium are 30-70 μM and 5-10 μM, respectively.

8. The preparation method according to claim 6, characterized in that, The concentration of N-hydroxysuccinimide palmitate dissolved in dimethyl sulfoxide in step S2 is 3-10 mg / mL, the concentration of PAR1 protein in phosphate buffer is 0.1 mg / mL, and the concentration of PA-PAR1 in DMEM medium containing PA-PAR1 is 1-20 μg / mL.

9. The preparation method according to claim 6, characterized in that, Step S3 specifically includes the following steps: Cyclodextrin, negative hydrogen ion powder, American plum blossom extract and hyaluronic acid were mixed in proportion and then soaked in an ethyl acetate solution of polylactic acid-hydroxyacetic acid copolymer with a final concentration of 1-5 mg / mL. After being mixed evenly at room temperature, the mixture was vacuum rotary evaporated in a water bath at 30-40℃ to solidify the copolymer and uniformly coat the surface of the pre-coated body. The negative hydrogen ion carrier was obtained by freeze drying. The preparation of the extract from the Western Mountain plum blossom includes the following steps: Fresh plum blossoms from Xiyang Mountain are broken into small pieces of 1-2 cm, washed with clean water, and then mixed with deionized water at a mass-to-volume ratio of 1 g: (5-10) mL. The pH is adjusted to 4.0-6.0, and 1-2% of a compound hydrolytic enzyme is added to the mixture. The mixture is then hydrolyzed at 40-50℃ for 4-8 hours. After hydrolysis, the temperature is raised to 90-95℃ to inactivate the enzyme, yielding the hydrolysate. The compound hydrolytic enzyme is a mixture of cellulase and ligninase at a mass ratio of 4:

1. The enzymatic hydrolysate was microfiltered through a filter membrane with a pore size of 0.1-0.2 μm to remove macromolecular fibers, and the permeate was collected. The permeate was concentrated and then spray-dried to obtain the extract of Prunus cerasifera flower.

10. The application of an anti-tumor stem cell combined with a negative hydrogen ion preparation in the preparation of a drug for treating lung cancer.