Cell inactivation composition based on metal polyphenol, inactivation method and inactivated cell

Abstract

The invention discloses a cell inactivation composition based on metal polyphenol, an inactivation method and inactivated cells. The inactivation method comprises the following steps: mixing a plant polyphenol solution and a metal ion solution to obtain a first solution; adding a first buffer solution into the first solution to obtain a metal polyphenol particle suspension; adding the metal polyphenol particle suspension into a cell suspension of cells to be inactivated to obtain a second solution; adding a second buffer solution into the second solution, and inactivating the cells to be inactivated in the second solution; the first buffer solution is an acidic buffer solution or a neutral buffer solution, the second buffer solution is a neutral buffer solution or an alkaline buffer solution, and the pH value of the first buffer solution is smaller than that of the second buffer solution. According to the invention, stable metal polyphenol nanoparticles are formed by plant polyphenol and metal ions, and the metal polyphenol nanoparticles are tightly adhered to the surfaces of cells to be inactivated to form a compact nano coating, so that proliferation and metabolic pathways of the cells are physically blocked, and the purpose of efficient inactivation is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical research and applications, and particularly to a cell inactivation composition based on metal polyphenols and an inactivation method, as well as inactivated cells inactivated by the inactivation method or the inactivation composition. Background Art

[0002] In biomedical research and applications, inactivated cells play a crucial role. For example, in the development of viral vaccines, mammalian cells are inactivated to retain antigenicity and stimulate the body's immune response to resist live viruses, such as influenza vaccines; another example is that in tumor immunotherapy and vaccine research and development, inactivated tumor cells can serve as an antigen source for the immune system to recognize tumors and induce an immune response to inhibit tumor growth.

[0003] Existing cell inactivation methods include radiation, chemical drug treatment, physical freezing, high temperature, etc. Radiation inactivation destroys the DNA or RNA of tumor cells through ionizing radiation, making them lose their reproductive ability, but this method may cause changes in cell antigenicity and affect the immune effect. Physical freezing and high temperature treatment can inactivate cells quickly, but may cause damage to cell structure and affect immunogenicity; high temperature or strong chemical treatment cannot balance inactivation efficiency and antigen integrity, and residual reagents may cause systemic inflammatory reactions. Chemical drug treatment usually uses chemotherapy drugs, such as cisplatin, doxorubicin, etc. Although these drugs can effectively kill tumor cells, they may also have certain toxicity to normal cells, and the operation process is complex.

[0004] In recent years, some new tumor cell inactivation technologies have gradually attracted attention, such as lactate-induced tumor cell inactivation, using specific cell surface anchoring molecules, etc. Patent CN109908334A discloses a method for enhancing the immunogenicity of tumor cells by treating them with lactic acid, but the inactivation efficiency and mildness of this method still need to be improved. Patent CN116669767A mentions various methods for tumor cell inactivation and immune enhancement, including using specific antibody-binding molecules and immune activity enhancers, but these methods often require complex preparation processes and the cooperation of multiple components. Summary of the Invention

[0005] The object of the present invention is to provide a cell inactivation method based on plant polyphenols, which forms stable metal polyphenol nanoparticles by the combination of plant polyphenols and metal ions. The metal polyphenol nanoparticles tightly adhere to the surface of the cells to be inactivated, forming a dense nano-coating, physically blocking the proliferation and metabolic pathways of the cells to achieve the purpose of efficient inactivation. At the same time, this cell inactivation method is easy to operate, has mild conditions, can effectively improve the test efficiency in the process of biomedical research, and enhance the safety of the test.

[0006] The present invention is achieved by the following technical solutions:

[0007] A method for inactivating cells based on metal polyphenols, comprising the following steps:

[0008] Mix a plant polyphenol solution and a metal ion solution to obtain a first solution;

[0009] Add a first buffer solution to the first solution to obtain a metal polyphenol particle suspension;

[0010] Add the metal polyphenol particle suspension to a cell suspension of cells to be inactivated to obtain a second solution;

[0011] Add a second buffer solution to the second solution to inactivate the cells to be inactivated in the second solution;

[0012] Wherein, the first buffer solution is an acidic buffer solution or a neutral buffer solution, the second buffer solution is a neutral buffer solution or an alkaline buffer solution, and the pH value of the first buffer solution is less than the pH value of the second buffer solution.

[0013] In this technical solution, first, a plant polyphenol solution and a metal ion solution are mixed to obtain a first solution. In some preferred embodiments, the plant polyphenol solution is first dispersed in physiological saline, and then the metal ion solution is added. After mixing evenly, the first solution is obtained. In one or more embodiments, the plant polyphenol solution is obtained by dissolving plant polyphenols in a solvent, and the metal ion solution is obtained by dissolving metal salts in a solvent. Wherein, the solvent is preferably deionized water.

[0014] In this technical solution, after obtaining a uniformly mixed first solution, the pH value of the first solution is adjusted by adding a first buffer solution to obtain a metal polyphenol particle suspension. Among them, the first buffer solution is an acidic buffer solution or a neutral buffer solution. In some preferred embodiments, the pH value of the first buffer solution is 4-6.5. In one or more embodiments, the first buffer solution can be at least one of acetate buffer solution, citrate buffer solution, phosphate buffer solution, and succinate buffer solution. Further preferably, the first buffer solution uses a phosphate buffer solution with a pH value of 5.0-6.0. In this technical solution, by utilizing the abundant phenolic hydroxyl groups in plant polyphenols, metal polyphenol nanoparticles can be formed through coordination with metal ions. At the same time, by regulating the pH value of the solution with the first buffer solution, the size of the nanoparticles formed by metal polyphenol complexation can be only 5-50 nanometers.

[0015] In this technical solution, a metal polyphenol particle suspension is added to the cell suspension of the cells to be inactivated to obtain a second solution, and then a second buffer solution is added to the second solution to inactivate the cells to be inactivated in the second solution. Among them, the second buffer solution is a neutral buffer solution or an alkaline buffer solution. In some preferred embodiments, the pH value of the second buffer solution is 7-9. In one or more embodiments, the second buffer solution may be at least one of a phosphate buffer solution, a Tris buffer solution, and a borate buffer solution. Further preferably, the second buffer solution is a phosphate buffer solution with a pH value of 8.0-9.0. In this technical solution, after the fine metal polyphenol nanoparticles adhere to the cell surface, the pH value of the second solution is increased by a buffer solution system with a higher pH value, so that the fine nanoparticles are tightly linked to form a dense network, thereby physically blocking the cell proliferation and metabolism pathways and finally inactivating the cells in the second solution.

[0016] In this technical solution, the pH value is adjusted twice. During the first adjustment, the lower solution pH value results in incomplete complexation of plant polyphenols and metal ions. At this time, the formed metal polyphenol nanoparticles are very small in size, and the connection between the metal polyphenol nanoparticles is still weak. When the small-sized metal polyphenol nanoparticles bind to the bacterial surface, the second adjustment increases the solution pH value, promoting closer connection between the fine metal polyphenol nanoparticles and between the particles and the cells, and then forming a dense network with very small gaps on the cell surface. The dense network with smaller gaps can effectively block the cell proliferation and metabolism pathways, achieving the purpose of inactivating the cells.

[0017] Moreover, in some embodiments, a stabilizer, such as a hyaluronic acid solution, can also be added to the first solution to improve the stability of the metal polyphenol nanoparticles and form a denser nano-coating on the cell surface. This can not only play a role in inactivation but also effectively prevent the leakage of cell contents, maintain the integrity of the cell structure, and reduce the risk brought by the leakage of contents. For example, after encapsulating stem cells with the nano-coating, it can avoid the leakage of key contents of stem cells, such as TGF-β and IGF-1, during in vitro operation and transplantation, and maintain the therapeutic potential of stem cells.

[0018] Furthermore, the molar ratio of the metal ions in the metal ion solution to the plant polyphenols in the plant polyphenol solution is 4:1 to 1:3.

[0019] In this technical solution, the content of metal ions affects the size and stability of metal polyphenol nanoparticles. If the content of metal ions is too low, it is not easy to form metal polyphenol nanoparticles, and the formed metal polyphenol nanoparticles have poor stability. The metal polyphenol nanoparticles cannot be stably connected to cells to form a nano-coating, resulting in a decrease in the inactivation effect. On the contrary, if the content of metal ions is too high, the stability of the metal polyphenol nanoparticles is improved, but the particle size is larger, resulting in an increase in the gap of the nano-coating, also reducing the inactivation effect. Therefore, in this technical solution, the molar ratio of the preferred metal ions to plant polyphenols is determined to be 4:1 to 1:3.

[0020] Furthermore, the concentration of the plant polyphenol solution is 100 - 400 mg / mL, and the concentration of the metal ion solution is 40 - 250 mg / L.

[0021] In some embodiments, the concentration of the plant polyphenol solution is preferably 150 - 250 mg / mL. In some embodiments, the concentration of the metal ion solution is preferably 40 - 150 mg / L.

[0022] Furthermore, the cells to be inactivated are resuspended in physiological saline, and after centrifugal concentration, the centrifuged cells to be inactivated are resuspended in physiological saline to obtain the cell suspension.

[0023] In this technical solution, the pretreated tumor cells are resuspended in physiological saline (0.9% sodium chloride solution, 1 mL) to make the cell concentration reach 1×10 5 ~1×10^7 cells / mL. The cell suspension is centrifugally concentrated (200 - 800 rcf, 3 - 5 min), and the centrifuged cells are resuspended in 50 - 400 μL of physiological saline.

[0024] Furthermore, the plant polyphenol in the plant polyphenol solution is at least one of proanthocyanidin (PC), tara tannin (Tara), bayberry tannin (BT), black wattle tannin (BWT), larch tannin (LT), tannic acid (TA), ellagic acid (EA), epigallocatechin gallate (EGCG), catechin gallate (CG), or catechin.

[0025] Furthermore, the metal ions in the metal ion solution can be at least one of zinc ions, manganese ions, iron ions, and aluminum ions.

[0026] The present invention also provides an inactivated cell, which is obtained by inactivating a cell to be inactivated by any one of the foregoing metal polyphenol-based cell inactivation methods, and the surface of the inactivated cell is coated with a metal polyphenol nanolayer composed of metal polyphenol particles.

[0027] In this technical solution, the inactivated cells can be various types of cells. For example, the inactivated cells can be tumor cells or bacterial cells, providing highly efficient and mild inactivated cells for tumor immunotherapy and the research and development of influenza vaccines. The inactivated cells can also be stem cells, retaining their key contents after inactivation and maintaining the therapeutic potential of the stem cells. The inactivated cells can also be virus cells, reducing the leakage of endotoxin through the nano-coating on the surface and improving the safety of use. In summary, the inactivation method provided by the present invention has wide applicability and generality, and can meet the needs of cell inactivation in different fields.

[0028] In addition, this cell surface modification strategy based on metal polyphenol nanostructures has dynamic and modular characteristics, making metal polyphenol nanoparticles have significant advantages in applications that require adaptability. It can be used as a cell-independent and modular cell surface functionalization method, providing new ideas and methods for the field of cell engineering.

[0029] The present invention also provides a metal polyphenol-based cell inactivation composition, including a metal ion solution, a plant polyphenol solution, a first buffer solution, and a second buffer solution. Among them, the metal ion solution and the plant polyphenol solution are used to form a first solution after mixing, the first buffer solution is used to adjust the pH value of the first solution to acidic or neutral to form a metal polyphenol particle suspension, and the second buffer solution is used to adjust the pH value of the cell suspension added with the metal polyphenol particle suspension to neutral or alkaline, forming a metal polyphenol nanolayer on the surface of the cells to be inactivated in the cell suspension to inactivate the cells to be inactivated.

[0030] In this technical solution, the metal ion solution, the plant polyphenol solution, the first buffer solution, and the second buffer solution can be used to inactivate cells by any one of the foregoing inactivation methods. In some embodiments, the metal ion solution, the plant polyphenol solution, the first buffer solution, and the second buffer solution are preferably four reagents packaged independently. In one or more embodiments, the metal ion solution and the plant polyphenol solution can be mixed and packaged together. In one or more embodiments, the metal ion solution, the plant polyphenol solution, and the first buffer solution can also be mixed and packaged together.

[0031] Furthermore, the pH value of the first buffer solution is 4-6.5, and the pH value of the second buffer solution is 7-9.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] 1. The present invention forms stable metal polyphenol nanoparticles by the combination of plant polyphenols and metal ions. The metal polyphenol nanoparticles adhere tightly to the surface of the cells to be inactivated, forming a dense nano-coating, physically blocking the proliferation and metabolic pathways of the cells, and achieving the purpose of highly efficient inactivation.

[0034] 2. The inactivation process of the present invention does not require complex equipment and cumbersome steps. It can be completed only through simple operations such as preparing solutions, mixing cells with nanoparticle suspensions, etc., greatly simplifying the operation process, reducing the operation difficulty and cost, and at the same time reducing the risks and errors that may be brought by complex operations, and improving the safety.

[0035] 3. Through two pH value adjustments in the present invention, the solution with a lower pH value makes the complexation of plant polyphenols and metal ions incomplete, forming metal polyphenol nanoparticles with small sizes and loose connections. When the small-sized metal polyphenol nanoparticles bind to the bacterial surface, the solution with a higher pH value promotes the closer connection between the small metal polyphenol nanoparticles and between the particles and the cells, and then wraps around the cell surface to form a dense network with very small gaps. The dense network with smaller gaps can effectively block the proliferation and metabolic pathways of the cells, achieving the purpose of inactivating the cells.

[0036] 4. The inactivation method of the present invention can not only play an inactivation role, but also effectively prevent the leakage of cell contents, maintain the integrity of the cell structure, and reduce the risks brought by the leakage of contents.

[0037] 5. By reasonably setting the molar ratio of metal ions to plant polyphenols in the present invention, more stable and smaller-sized metal polyphenol nanoparticles can be formed on the cell surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, form a part of this application, and do not constitute a limitation on the embodiments of the present invention. In the drawings:

[0039] Figure 1 is a flowchart of the cell inactivation method in a specific embodiment of the present invention;

[0040] Figure 2 shows the surface of untreated tumor cells and the surface of inactivated tumor cells C1 characterized by a transmission electron microscope in a specific embodiment of the present invention;

[0041] Figure 3 shows the polyphenols on the cell surface of fluorescein-bovine serum albumin-labeled tumor cells C1 in a specific embodiment of the present invention;

[0042] Figure 4Shows the surface of untreated bacterial cells and the surface of inactivated bacterial cell C6 characterized by transmission electron microscopy in specific embodiments of the present invention;

[0043] Figure 5 Shows the polyphenols on the cell surface of fluorescein-bovine serum albumin-labeled bacterial cell C6 in specific embodiments of the present invention;

[0044] Figure 6 Shows the comparison diagram of tumor cells before and after inactivation treated by Calcein / PI live-dead cell staining method in specific embodiments of the present invention;

[0045] Figure 7 Shows the cell viability statistics of untreated tumor cells and tumor cell C1 at different times in specific embodiments of the present invention;

[0046] Figure 8 Shows the cell survival rates of tumor cells C1 - C5 in specific embodiments of the present invention;

[0047] Figure 9 Shows the potential characterization of the cell surfaces of tumor cells C1 - C5 in specific embodiments of the present invention;

[0048] Figure 10 Shows the survival rates of bacterial cell C6 before and after inactivation in specific embodiments of the present invention;

[0049] Figure 11 Shows the electrophoresis results of the precipitates after rupture of untreated stem cells and stem cell C7 in specific embodiments of the present invention;

[0050] Figure 12 Shows the protein content and RNA content collected after rupture of stem cell C7 in specific embodiments of the present invention. Detailed Embodiments

[0051] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with embodiments and drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and do not limit the present invention.

[0052] For all raw materials of the present invention, there are no special restrictions on their sources, and they can be purchased on the market or prepared according to the conventional methods well-known to those skilled in the art. For all raw materials of the present invention, there are no special restrictions on their purities. The present invention preferably adopts analytical pure or the conventional purity requirements in the field of cell inactivation. For all raw materials of the present invention, their trademarks and abbreviations are all conventional trademarks and abbreviations in the field. Each trademark and abbreviation is clear and definite in the field of its relevant uses. Those skilled in the art can purchase them from the market or prepare them by conventional methods according to the trademark, abbreviation and corresponding uses.

[0053] In the present invention, terms such as "first" and "second" (e.g., first solution, second solution, first phosphate buffer solution, second phosphate buffer solution, etc.) are only used to distinguish the corresponding components for clarity, and are not intended to limit any order or emphasize importance, etc. In addition, the term "connection" used herein, without special explanation, can be directly connected or indirectly connected via other groups.

[0054] I. Preparation of inactivated cells

[0055] At the present stage, metal polyphenol particles are mainly used for cell delivery, such as probiotic delivery, which requires maintaining the activity and function of cells during the delivery process. Therefore, when preparing metal polyphenol particles, it is necessary to first connect polyphenols with cells, and then combine metal ions with polyphenols to form a nano-coating. The gap of this nano-coating is very large, and cells can also achieve their functions through this large gap.

[0056] In the inactivation method of the present invention, in order to form a dense metal polyphenol particle coating on the cell surface, first, loose and fine metal polyphenol nanoparticles are formed based on metal polyphenols in a low pH environment. After connecting them with cells, the pH is then adjusted to a high value to form a dense network on the cell surface, physically blocking the proliferation and metabolic pathways of cells, so as to achieve the purpose of inactivating the cells in the second solution.

[0057] The cell inactivation method based on metal polyphenols adopted in this embodiment includes the following steps:

[0058] Mix a plant polyphenol solution and a metal ion solution to obtain a first solution;

[0059] Add a first buffer solution to the first solution to obtain a metal polyphenol particle suspension;

[0060] Add the metal polyphenol particle suspension to the cell suspension of the cells to be inactivated to obtain a second solution;

[0061] Add a second buffer solution to the second solution to inactivate the cells to be inactivated in the second solution;

[0062] Wherein, the first buffer solution is an acidic buffer solution or a neutral buffer solution, the second buffer solution is a neutral buffer solution or an alkaline buffer solution, and the pH value of the first buffer solution is less than the pH value of the second buffer solution.

[0063] Specifically, in Examples 1 to 7, preferred examples of inactivating cells by forming a nano-coating on the cell surface using plant polyphenols and metal ions are shown.

[0064]

Example 1

[0065] In this example, inactivated tumor cells encapsulated with a PC-Zn nanocoating were prepared:

[0066] S1: Disperse the procyanidin PC solution (200 μL, 150 mg / mL) in 1 mL of physiological saline, and then add zinc chloride ZnCl2 solution (40 μL, 150 mg / mL). Shake well in a shaker (100 rpm, 5 min) to obtain the first solution;

[0067] S2: Add the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL) to the first solution, and shake well in a shaker (100 rpm, 5 min) to obtain a PC-Zn nanoparticle suspension;

[0068] S3: Resuspend the pretreated B16F10 tumor cells (2×10 5 cells) in physiological saline (0.9% sodium chloride solution, 1 mL). Add 400 μL of the PC-Zn nanoparticle suspension to the cell suspension, and shake well in a shaker (200 rpm, 5 min) to obtain the second solution;

[0069] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the second solution, and shake well in a shaker (200 rpm, 5 min) to obtain inactivated tumor cells C1 encapsulated with a PC-Zn nanocoating.

[0070]

Example 2

[0071] In this example, inactivated tumor cells encapsulated with an EGCG-Mn nanocoating were prepared:

[0072] S1: Disperse the epigallocatechin gallate EGCG solution (400 μL, 200 mg / mL) in 1 mL of physiological saline, and then add manganese chloride MnCl2 solution (80 μL, 100 mg / mL). Shake well in a shaker (100 rpm, 5 min) to obtain the first solution;

[0073] S2: Add the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL) to the first solution, and shake well in a shaker (100 rpm, 5 min) to obtain an EGCG-Mn nanoparticle suspension;

[0074] S3: Resuspend the pretreated B16F10 tumor cells (2×10 5 cells) in physiological saline (0.9% sodium chloride solution, 1 mL). Add 400 μL of the EGCG-Mn nanoparticle suspension to the cell suspension, and shake well in a shaker (200 rpm, 5 min) to obtain the second solution;

[0075] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the second solution, and shake it well on a shaker (200 rpm, 5 min) to obtain inactivated tumor cells C2 wrapped with EGCG-Mn nanocoating.

[0076]

Example 3

[0077] This example prepares inactivated tumor cells wrapped with TA-Fe nanocoating:

[0078] S1: Disperse the tannic acid TA solution (100 μL, 250 mg / mL) in 1 mL of physiological saline, then add the ferric chloride FeCl3 solution (50 μL, 100 mg / mL), and shake it well on a shaker (100 rpm, 5 min) to obtain the first solution;

[0079] S2: Add the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL) to the first solution, and shake it well on a shaker (100 rpm, 5 min) to obtain a TA-Fe nanoparticle suspension;

[0080] S3: Resuspend the pretreated B16F10 tumor cells (2×10 5 cells) in physiological saline (0.9% sodium chloride solution, 1 mL), add 400 μL of the TA-Fe nanoparticle suspension to the cell suspension, and shake it well on a shaker (200 rpm, 5 min) to obtain the second solution;

[0081] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the second solution, and shake it well on a shaker (200 rpm, 5 min) to obtain inactivated tumor cells C3 wrapped with TA-Fe nanocoating.

[0082]

Example 4

[0083] This example prepares inactivated tumor cells only inactivated by the plant polyphenol tara tannin:

[0084] S1: Disperse the tara tannin Tara solution (300 μL, 250 mg / mL) in 1 mL of physiological saline, then add the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL), and shake it well on a shaker (100 rpm, 5 min) to obtain a Tara nanoparticle suspension;

[0085] S2: Resuspend the pretreated B16F10 tumor cells (2×10 5 cells) in physiological saline (0.9% sodium chloride solution, 1 mL), add 400 μL of the Tara nanoparticle suspension to the cell suspension, and shake it well on a shaker (200 rpm, 5 min);

[0086] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the cell suspension, and shake it well on a shaker (200 rpm, 5 min) to obtain inactivated tumor cells C4 wrapped with Tara nanocoating.

[0087]

Example 5

[0088] This example prepares tumor cells modified only with manganese ions (Mn 2+ ):

[0089] S1: Disperse the manganese chloride MnCl2 solution (300 μL, 200 mg / mL) in 1 mL of physiological saline, then add the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL), and shake it well on a shaker (100 rpm, 5 min) to obtain a Mn 2+ / Mn(OH)2 nanoparticle suspension;

[0090] S2: Resuspend the pretreated B16F10 tumor cells (2 × 10 5 cells) in physiological saline (0.9% sodium chloride solution, 1 mL), add 400 μL of Mn 2+ / Mn(OH)2 nanoparticle suspension to the cell suspension, and shake it well on a shaker (200 rpm, 5 min);

[0091] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the cell suspension, and shake it well on a shaker (200 rpm, 5 min) to obtain tumor cells C5 treated with Mn 2+ / Mn(OH)2 nanoparticles.

[0092]

Example 6

[0093] This example prepares inactivated bacterial cells wrapped with PC-Zn nanocoating:

[0094] S1: Disperse the procyanidin PC solution (200 μL, 150 mg / mL) in 1 mL of physiological saline, then add the zinc chloride ZnCl2 solution (40 μL, 150 mg / mL), and shake it well on a shaker (100 rpm, 5 min) to obtain the first solution;

[0095] S2: Add the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL) to the first solution, and shake it well on a shaker (100 rpm, 5 min) to obtain a PC-Zn nanoparticle suspension;

[0096] S3: Resuspend the pretreated bacterial cells (Staphylococcus aureus S.aureus, 1 × 10 7 cells / mL, OD 600About 0.1) was resuspended in physiological saline (0.9% sodium chloride solution, 1 mL), and 400 μL of PC-Zn nanoparticle suspension was added to the cell suspension, and the mixture was shaken well on a shaker (200 rpm, 5 min) to obtain a second solution;

[0097] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the second solution, and shake well on a shaker (200 rpm, 5 min) to obtain inactivated bacterial cells C6 wrapped with a PC-Zn nanocoating.

[0098]

Example 7

[0099] In this example, inactivated stem cells wrapped with an EGCG-Mn nanocoating were prepared.

[0100] S1: Disperse the epigallocatechin gallate EGCG solution (200 μL, 150 mg / mL) in 1 mL of physiological saline, and then add manganese chloride MnCl2 solution (40 μL, 150 mg / mL), and shake well on a shaker (100 rpm, 5 min) to obtain a first solution;

[0101] S2: Add hyaluronic acid solution (1 mg / mL) and the first phosphate buffer solution (pH = 5.0, 20 mM, 1 mL) to the first solution, and shake well on a shaker (100 rpm, 5 min) to obtain an EGCG-Mn nanoparticle suspension;

[0102] S3: Resuspend the pretreated stem cells (2×10 5 cells) in physiological saline (0.9% sodium chloride solution, 1 mL), add 400 μL of EGCG-Mn nanoparticle suspension to the cell suspension, and shake well on a shaker (200 rpm, 5 min) to obtain a second solution;

[0103] S4: Add the second phosphate buffer solution (pH = 8, 20 mM, 500 μL) to the second solution, and shake well on a shaker (200 rpm, 5 min) to obtain stem cells C7 wrapped with a coating composed of HA-embedded EGCG-Mn nanoparticles.

[0104] In some embodiments, the concentration range of plant polyphenol solutions such as procyanidin PC solution and tara tannin Tara solution can be 100-400 mg / mL, preferably 150-250 mg / mL. The plant polyphenol solution is obtained by dissolving polyphenols in a solvent. Preferably, the solvent is deionized water.

[0105] In some embodiments, the concentration range of metal ion solutions can be 40-250 mg / L, preferably 40-150 mg / L. The metal ion solution is obtained by dissolving a metal salt in a solvent. Preferably, the solvent is deionized water.

[0106] In one or more embodiments, the method for collecting and preprocessing allogeneic tumor cells is to collect tumor cells in the logarithmic growth phase, remove the serum in the culture medium by centrifugation (800 - 1000 rpm, 3 - 5 min), and wash the cells 1 - 2 times with serum-free medium or PBS to ensure the cell surface is clean for subsequent processing.

[0107] In one or more embodiments, the method for collecting and preprocessing autologous tumor cells is to isolate autologous tumor cells, and use enzymatic digestion, mechanical shearing, or manual tissue grinding to process the tumor tissue into isolated autologous tumor cells. Wash the cells 1 - 2 times with serum-free medium or PBS to ensure the cell surface is clean for subsequent processing.

[0108] In one or more embodiments, the method for collecting and preprocessing bacterial cells is to collect bacterial cells and suspend them in physiological saline or phosphate buffer for subsequent processing.

[0109] In some embodiments, after cell inactivation, for the collection of mammalian cells, the main steps are as follows: after the reaction, collect the inactivated cells by centrifugation (200 - 800 rcf, 3 - 5 min), and wash them 3 - 5 times with phosphate buffer (pH = 7 - 9, 5 - 100 mM, 600 - 1500 μL). Resuspend the cell suspension in 1 mL of physiological saline. Keep the cells static at 4 - 30 °C for 8 - 24 hours to achieve efficient and gentle inactivation of the cells. In one or more embodiments, the mammalian cells involved include tumor cells, immune cells, red blood cells, etc.

[0110] In some embodiments, after cell inactivation, for bacterial cells, after the reaction, collect the inactivated bacterial cells by centrifugation (2000 - 4000 rcf, 3 - 5 min), and wash them 3 - 5 times with phosphate buffer (pH = 7 - 9, 5 - 100 mM, 600 - 1500 μL). Resuspend the bacterial cell suspension in 1 mL of physiological saline. Keep the cells static at 4 - 30 °C for 8 - 24 hours to achieve efficient and gentle inactivation of the cells. In one or more embodiments, the bacteria involved include Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Streptococcus pyogenes (S. pyogenes), Pseudomonas aeruginosa (S. aeruginosa), etc.

[0111] In some embodiments, resuspend the inactivated cells in an appropriate buffer, and they can be used for subsequent vaccine research, preparation, or other immunotherapy applications.

[0112] II. Characterization and performance testing of inactivated cells

[0113]

Example 8

[0114] In this example, the inactivated tumor cells C1 encapsulated with the PC-Zn nanocoating were characterized.

[0115] Specifically, the cell surface was characterized using a transmission electron microscope. Figure 2 The TEM images of the tumor cells C1 and the cross-section of the original tumor cells are shown. It can be seen from the figure that a dense nanocoating was formed on the surface of the tumor cells C1 encapsulated with the PC-Zn nanocoating, while a smooth cell membrane could be seen on the original tumor cells. It can be seen that the tumor cells C1 were completely encapsulated by the PC-Zn nanocoating formed by using PC and Zn ions.

[0116] Furthermore, after modifying the cells with PC-Zn, the fluorescence probe fluorescein-bovine serum albumin (FITC-BSA) was added. The test results are as Figure 3 shown. The polyphenols labeled with fluorescent protein demonstrated the loading of PC-Zn on the cell surface, indicating the presence of the PC-Zn nanocoating on the cell surface.

[0117]

Example 9

[0118] In this example, the inactivated bacterial cells C6 encapsulated with the PC-Zn nanocoating were characterized.

[0119] The surface of the bacterial cells (S. aureus) was characterized using a transmission electron microscope. Figure 4 The TEM images of the bacterial cells C6 and the original bacterial cells are shown. It can be seen from the figure that a dense nanocoating was formed on the surface of the bacterial cells treated with PC-Zn nanoparticles, while a smooth cell membrane could be seen on the original bacterial cells. Thus, it can be seen that the bacterial cells were completely encapsulated by the PC-Zn nanocoating formed by using PC and Zn.

[0120] Furthermore, after modifying the bacterial cells with PC-Zn, the fluorescence probe fluorescein-bovine serum albumin (FITC-BSA) was added. As Figure 5 shown, the confocal microscopy results showed fluorescence around the cells, indicating the presence of PC-Zn nanoparticles on the cell surface.

[0121]

Example 10

[0122] In this example, the Calcein / PI cell viability staining method was used to detect the viability of cells inactivated by the procyanidin PC-Zn ion nano-coating. Specifically, tumor cells C1 were incubated in DMEM medium (10% serum, 1% double antibody) in a cell incubator (37 °C, 5% CO2) for varying lengths of time up to 12 h. Then the cells were collected, washed with PBS, and 195 μL of Annexin V-FITC and 5 μL of PI (per 5×10 4 cells) were added. Incubate in the dark at room temperature for 20 min and detect with a laser confocal microscope. The test results are as shown in Figure 6 . The tumor cells C1 treated by the inactivation method of the present invention completely lost their proliferative ability within 12 hours, and the cell viability was close to zero, indicating that the inactivation effect was significant.

[0123] In this example, the CCK-8 assay was also used to measure the viability of tumor cells C1 after inactivation treatment. As shown in Figure 7 , within 12 hours, the tumor cells C1 treated by this inactivation method had completely lost their proliferative ability and the cell viability was almost zero, which also proved that the inactivation effect was very significant.

[0124]

Example 11

[0125] In this example, the CCK8 method was used to detect the viability of tumor cells C1-C5. Specifically, tumor cells C1-C5 were incubated in an opaque white 96-well plate for 12 h. Subsequently, 10 μL of CCK8 solution was added to each well, and after incubation in a cell incubator for 1 h, the absorbance at 450 nm was measured using a microplate reader (microplate reader, 200PRO, Tecan, Switzerland). Add CCK-8 reagent to each well, then incubate for 60 minutes, and measure the absorbance. At the same time, the original B16F10 tumor cells were used as a control group, and all experiments were independently repeated three times.

[0126] The test results are as shown in Figure 8 . The cell survival rates of tumor cells C1, C2, and C3 inactivated by the metal polyphenol nano-coating were almost zero, while the inactivation effects of tumor cells C4 and C5 treated with only polyphenols or metal-modified surfaces were much less effective than those of the metal polyphenol nano-coating, indicating that the synergistic effect of plant polyphenols and metal ions can form a denser nano-coating, thereby physically blocking the proliferation and metabolic pathways of cells and achieving an efficient inactivation effect.

[0127]

Example 12

[0128] In this example, the Zeta potential of tumor cells C1-C5 was measured. The test results are as shown in Figure 9As shown, after the surface charge of the original tumor cells is wrapped by the nano - coating formed by metal - polyphenol, the Zeta potential value moves towards a more negative direction. This is caused by the negative charge of plant polyphenols, indicating that a nano - coating composed of metal and polyphenols is formed on the cell surface. The cell surfaces of tumor cells C1 to C3 show different potential values, which are mainly related to the selected polyphenols, metal ions and their composition. At the same time, the potential values on the cell surfaces of tumor cells C4 and C5 do not change significantly, indicating that neither the single metal nor polyphenol nano - coating can stably wrap the cells.

[0129]

Example 13

[0130] In this example, the activity of inactivated bacterial cells was determined by the plate - counting method. The experiment shows that after treatment with the metal - polyphenol nanomaterial for 12 h, the activity of bacterial cell C6 is zero, proving that this method is also very effective in inactivating bacteria.

[0131]

Example 14

[0132] In this example, the changes in the content of the lysed contents of stem cells without metal - polyphenol nanoparticles on the surface were compared with those of stem cell C7 with EGCG - Mn.

[0133] Specifically, the same amount of untreated stem cells and stem cell C7 treated with EGCG - Mn were lysed by hypotonic treatment, centrifuged at 300 g for 3 min, and then the supernatant and precipitate were separated.

[0134] SDS - PAGE was used to analyze the protein content in the precipitate. As Figure 11 shown, lane 1 is the proteome of the control stem cells, lane 2 is the proteome collected from the untreated stem cells; lane 3 is the proteome collected after inactivating stem cell C7 with an EGCG - Mn coating.

[0135] It can be seen from the figure that the protein content collected from the stem cells (mesenchymal stem cells) inactivated by the method of the present invention is basically the same as that of the original cells, indicating that the nano - coating formed by metal - polyphenol nanoparticles can effectively retain the cell contents and avoid the leakage of key contents of stem cells, such as TGF - β and IGF - 1, during in vitro operation and transplantation, maintaining the therapeutic potential of stem cells. For example, in the treatment of myocardial infarction, retaining the vascular growth factor VEGF secreted by stem cells can significantly enhance the angiogenesis effect and improve the treatment success rate.

[0136] Furthermore, proteins were extracted from cells using RIPA lysis and extraction buffer containing protease inhibitors (Beyotime, China). According to the manufacturer's instruction manual, a BCA protein assay kit was used to determine the protein concentration. For RNA concentration testing, Triquick reagent (a Trizol alternative, Solarbio Science & Technology Co., Ltd., China) was used to extract cellular RNA. The RNA content was quantified using NanoDrop2000 (Thermo Fisher Scientific, USA).

[0137] Figure 12 The protein content and RNA content of stem cells C7 with EGCG-Mn coating in the precipitate and the control stem cells are shown. It can be seen that the nanocoating effectively prevents the leakage of stem cell contents, retaining a large amount of RNA and almost all proteins in the stem cells.

[0138] The specific embodiments described above further elaborate on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for cell inactivation based on metal polyphenols, characterized in that, Comprising the following steps: Mixing a plant polyphenol solution and a metal ion solution to obtain a first solution; Adding a first buffer solution to the first solution to obtain a metal polyphenol particle suspension; Adding the metal polyphenol particle suspension to a cell suspension of cells to be inactivated to obtain a second solution; Adding a second buffer solution to the second solution to inactivate the cells to be inactivated in the second solution; Wherein, the first buffer solution is an acidic buffer solution or a neutral buffer solution, the second buffer solution is a neutral buffer solution or an alkaline buffer solution, and the pH value of the first buffer solution is less than the pH value of the second buffer solution.

2. The method for inactivating cells based on metal polyphenols according to claim 1, wherein The pH value of the first buffer solution is 4 to 6.5, and the pH value of the second buffer solution is 7 to 9.

3. The method for inactivating cells based on metal polyphenols according to claim 1, wherein, The molar ratio of the metal ions in the metal ion solution to the plant polyphenols in the plant polyphenol solution is 4:1 to 1:

3.

4. The method for inactivating cells based on metal polyphenols according to claim 1, wherein, The concentration of the plant polyphenol solution is 100 to 400 mg / mL, and the concentration of the metal ion solution is 40 to 250 mg / L.

5. The method for inactivating cells based on metal polyphenols according to claim 1, wherein The cells to be inactivated are resuspended in physiological saline, and after centrifugal concentration, the centrifuged cells to be inactivated are resuspended in physiological saline to obtain the cell suspension.

6. The method for inactivating cells based on metal polyphenols according to any one of claims 1 to 5, characterized in that, The plant polyphenols in the plant polyphenol solution are at least one of procyanidin, tara tannin, myricetin tannin, black wattle bark tannin, larch tannin, tannic acid, ellagic acid, epigallocatechin gallate, catechin gallate, or catechin.

7. The method for inactivating cells based on metal polyphenols according to any one of claims 1 to 5, characterized in that, The metal ions in the metal ion solution can be at least one of zinc ions, manganese ions, iron ions, and aluminum ions.

8. An inactivated cell, characterized in that, After inactivating the cells to be inactivated by using the metal polyphenol-based cell inactivation method according to any one of claims 1 to 7, the inactivated cells are obtained, and the surface of the inactivated cells is coated with a metal polyphenol nanolayer composed of metal polyphenol particles.

9. A metal polyphenol-based cell inactivation composition, characterized in that, Comprising a metal ion solution, a plant polyphenol solution, a first buffer solution, and a second buffer solution, wherein the metal ion solution and the plant polyphenol solution are used for mixing to form a first solution, the first buffer solution is used for adjusting the pH value of the first solution to acidic or neutral to form a metal polyphenol particle suspension, and the second buffer solution is used for adjusting the pH value of the cell suspension added with the metal polyphenol particle suspension to neutral or alkaline to form a metal polyphenol nanolayer on the surface of the cells to be inactivated in the cell suspension to inactivate the cells to be inactivated.

10. A cell-inactivating composition based on metal polyphenols according to claim 9, characterized in that, The pH value of the first buffer solution is 4 to 6.5, and the pH value of the second buffer solution is 7 to 9.

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