Metal-polyphenol nano-coating-wrapped tumor whole cell, preparation method therefor, and use thereof

Abstract

Disclosed are a metal-polyphenol nano-coating-wrapped tumor whole cell, a preparation method therefor, and use thereof. A plant polyphenol in the present invention and manganese ions can be rapidly assembled at room temperature to form a dense coating on the membrane of a tumor cell. The nano-coating wrapping inactivates the tumor cell, ensuring that the vaccine is safe. The nano-coating can prevent any potential tumor antigen from being lost under physiological conditions. The nano-coating is further modified with a lipopolysaccharide, which can promote the endocytosis of the formed whole-cell vaccine by antigen-presenting cells. While forming the structural coating, ions of the metal manganese can stimulate the STING pathway to enhance the anti-tumor effect.

Description

Tumor whole cells wrapped in metal-polyphenol nanocoating, preparation method and application Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a whole tumor cell wrapped in a metal-polyphenol nanocoating, a preparation method and an application thereof. Background Art

[0002] Immunotherapy is a new type of tumor treatment. It activates the body's own immunity, allowing immune cells to actively exert their effectiveness, and causes tumor cells to undergo apoptosis through direct or indirect contact with tumor cells, thereby delaying or inhibiting the progression or metastasis of the tumor.

[0003] Tumor vaccines are an important immunotherapy strategy that stimulates tumor-specific immunity and hold enormous potential for cancer treatment. Currently, most tumor vaccines target individual tumor-associated antigens. While modern sequencing technologies have significantly advanced the development of personalized neoantigen vaccines, they still lack comprehensive coverage of all individual tumor cell antigens. Furthermore, due to the lack of immune recognition mechanisms, their clinical effectiveness is extremely limited, hindering widespread adoption.

[0004] Because whole-cell vaccines retain a complete set of tumor-associated antigens, they can overcome the limitations of a broad range of antigens across different tumor types and the immune escape caused by insufficient expression of single or specific antigens, thereby achieving tumor treatment or prevention. The main approaches to constructing whole-cell vaccines include recombinant tumor cell lysates and modification of intact tumor cells, including genetic modification, cryogenic engineering, microneedle patch delivery, and macroporous cryogels. Although these strategies show promise, they fail to retain all potential tumor antigens and achieve robust anti-tumor immune responses, and the preparation process is time-consuming, limiting their clinical application.

[0005] The current method for constructing whole-cell tumor vaccines, such as application number 201910810918.5, uses gene editing to knock out specific genes in melanoma cells, causing them to mutate during replication, thereby expressing non-self antigens that are easily recognized by immune cells, thereby increasing the expression of more co-stimulatory proteins by dendritic cells (DCs) in co-culture conditions, and significantly improving the immune response in mice. The risk of gene editing lies in unknown mutations. For example, application number 202011095900.0, the constructed vaccine modifies tumor cells by covalent bonding, coupling tumor cells and small molecule agonists to enhance the recognition of tumor cells by immune cells. However, this method introduces chemical modification, which has high requirements for the safety and reactivity of the coupling agent molecules, and separation and purification also leads to high manufacturing costs. In addition, small molecule agonists, as the main recognition antigens, may be tolerated. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a tumor whole cell wrapped with a metal-polyphenol nano-coating, a preparation method and an application thereof.

[0007] The technical solution adopted by the present invention is: a method for preparing whole tumor cells wrapped with a metal-polyphenol nanocoating, comprising the following steps:

[0008] Step 1: Thoroughly mix the plant polyphenol solution and tumor cell suspension;

[0009] Step 2: adding a manganese ion compound solution to the mixed solution obtained in step 1 and mixing thoroughly; wherein the mass ratio of the plant polyphenols to the metal ion compound is 1:2-8;

[0010] Step 3: Add buffer to the solution obtained in step 2, mix thoroughly, wash, and centrifuge to obtain coated tumor cells (MnTA cells).

[0011] Furthermore, the plant polyphenol is one of tannic acid (TA), tara tannin (Tara tanninn, Tara) and tea polyphenol (EGCG).

[0012] Furthermore, the metal ion compound in step 2 is a manganese ion (Mn 2+ ) compounds, zinc ions (Zn 2+ ) compounds, containing ferrous ions (Fe 2+ ) compounds, wherein the manganese ion-containing compound is manganese sulfate monohydrate.

[0013] Furthermore, the step 3 may further include the following steps: the coated tumor whole cells (MnTA cells) obtained in step 3 are fully mixed with the lipopolysaccharide solution, washed, and centrifuged to obtain the desired tumor whole cells (LMP cells, or LMP vaccine).

[0014] Furthermore, the steps 1, 2 and 3 are all mixed by vortexing.

[0015] Furthermore, the MnTA cells are first mixed with a binder and then mixed with lipopolysaccharide; the mass ratio of the binder to the plant polyphenols is 1 to 10:1.

[0016] A tumor whole cell wrapped in a polyphenol-nano coating, wherein the surface of the coated tumor whole cell is constructed with a plant polyphenol-metallic manganese ion coating; while forming a structural coating, the metal manganese ion can stimulate the STING pathway to enhance the anti-tumor effect.

[0017] The invention discloses an application of a whole tumor cell wrapped with a metal-polyphenol nano coating, wherein the whole tumor cell is one of a melanoma cell, a DC2.4 cell, and a Raw264.7 cell.

[0018] Furthermore, the coated tumor whole cells are used to prepare whole cell vaccines.

[0019] Furthermore, the coated tumor whole cells and / or whole cell vaccines are used in the preparation of anti-tumor drugs; the coated tumor whole cells and / or whole cell vaccines are combined with pharmaceutically acceptable adjuvants or other ingredients to obtain pharmaceutically acceptable preparations.

[0020] The beneficial effects of the present invention are:

[0021] (1) The plant polyphenols and manganese ions in the present invention can be rapidly assembled at room temperature to form a dense nanocoating on the tumor cell membrane. The nanocoating inactivates the tumor cells, ensuring the safety of the vaccine;

[0022] (2) The metal-polyphenol nanocoating of the present invention can prevent the loss of any potential tumor antigens under physiological conditions, solving the problem that existing tumor vaccines are limited to single or specific tumor antigens and insufficient expression of tumor antigens;

[0023] (3) The manganese ions in the present invention can activate the STING pathway of immune cells while forming a coating, thereby enhancing the expression of type I interferon and enhancing the anti-tumor effect;

[0024] (4) The present invention further modifies the surface of the nanocoating with lipopolysaccharide, which can promote the internalization of the formed LMP whole-cell vaccine by antigen-presenting cells. The obtained LMP whole-cell vaccine has excellent anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG1 shows the potential measurement results of cells obtained at various stages in Example 1 of the present invention.

[0026] FIG2 shows the potential measurement results of tumor cells obtained in Examples 2 and 3 of the present invention.

[0027] FIG3 is a SEM image of LMP cells and original tumor cells obtained in Example 1 of the present invention.

[0028] FIG4 is a SEM image of the LMP cells obtained in Example 2 and Example 3 of the present invention.

[0029] FIG5 is an inductively coupled plasma-optical emission spectrum of the LMP cells obtained in Example 1 of the present invention.

[0030] FIG6 is a TEM image of LMP cells and primary tumor cells obtained in Example 1 of the present invention.

[0031] FIG7 is a fluorescence micrograph of the LMP cells and primary tumor cells obtained in Example 1 of the present invention after being cultured in DMEM for 12 days and stained with Calcein / PI.

[0032] FIG8 is a statistical result of cell viability of LMP cells obtained in Example 1 of the present invention after being cultured in DMEM medium for 12 hours.

[0033] FIG9 shows the protein concentration determination results of LMP cells obtained in Example 1 of the present invention.

[0034] FIG10 is a schematic diagram showing a comparison of protein concentrations of LMP cells and original tumor cells obtained in Example 1 of the present invention.

[0035] FIG11 is a graph showing the results of DC phagocytosis analysis by flow cytometry of the LMP cells obtained in Example 1 of the present invention and the cells obtained in the comparative example.

[0036] FIG12 is a laser confocal micrograph of DC phagocytosis of LMP cells obtained in Example 1 of the present invention.

[0037] FIG13 is a SEM image of LPS-encapsulated tumor cells.

[0038] FIG14 is a schematic diagram showing the expression of p65, TBK1 and IRF3 in LMP cells obtained in Example 1 of the present invention and control cells.

[0039] FIG15 is a comparison of tumor sizes in animal models after treatment with LMP cells obtained in Example 1 of the present invention and cells of a comparative example.

[0040] FIG16 is a schematic diagram of staining of the main organs of an animal model after the animal model was treated with LMP cells obtained in Example 1 of the present invention and cells of a control example.

[0041] FIG17 is a serum test result of the animal model after the animal model was treated with the LMP cells obtained in Example 1 of the present invention and the control cells. DETAILED DESCRIPTION

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0043] A method for preparing whole tumor cells coated with a metal-polyphenol nanocoating comprises the following steps:

[0044] Step 1: Vortex the plant polyphenol solution and the tumor cell suspension; the plant polyphenol is one of tannic acid, tara tannin and tea polyphenol.

[0045] Step 2: Add a manganese ion compound solution to the mixed solution obtained in step 1 and vortex mix; wherein the mass ratio of the plant polyphenol to the metal ion compound is 1:2-8; the metal ion compound is one of a manganese ion compound, a zinc ion compound, and a ferrous ion compound, and the manganese ion compound is manganese sulfate monohydrate.

[0046] Step 3: Add buffer to the solution obtained in step 2, vortex mix, wash, and centrifuge to obtain coated tumor cells (MnTA cells).

[0047] The coated whole tumor cells obtained in step 3 are vortex-mixed with the lipopolysaccharide solution, washed, and centrifuged to obtain the desired LMP tumor cells. The coated whole tumor cells are first mixed with a binder and then with lipopolysaccharide (the amount and concentration of lipopolysaccharide are not limited here and are selected based on actual conditions). The mass ratio of the binder to the plant polyphenol is 1 to 10:1.

[0048] The surface of the coated tumor cells is coated with a plant polyphenol-metallic manganese ion coating. If lipopolysaccharide is added, the plant polyphenol-metallic manganese ion coating is coated with a layer of lipopolysaccharide. Adhesives such as polyethyleneimine and lysine can be added to achieve a tighter bond.

[0049] The tumor cells are melanoma cells, DC2.4 cells, and one of Raw264.7 cells.

[0050] The coated tumor whole cells are used to prepare whole-cell vaccines. The coated tumor whole cells and / or whole-cell vaccines are used in the preparation of anti-tumor drugs. The coated tumor whole cells and / or whole-cell vaccines are combined with pharmaceutically acceptable adjuvants or other ingredients to obtain pharmaceutically acceptable preparations.

[0051] Example 1

[0052] Prepare whole tumor cells coated with metal-polyphenol nanoparticles by the following steps:

[0053] Step 1: Collect B16F10 cells and obtain a single cell suspension (2×10 6 cells / mL); tannic acid solution (10 μL, 20 mg / mL) was added to 200 μL of the cell suspension and vortexed for 10 seconds to obtain a tannic acid-modified cell TA cell solution.

[0054] Step 2: Add manganese sulfate monohydrate (MnSO4·H2O) solution (50 μL, 10 mg / mL) to the solution obtained in step 1 and vortex mix for 10 seconds.

[0055] Step 3: Add 500 μL of phosphate buffered saline (PBS, 100 mM, pH = 7.4) to the solution obtained in step 2. Vortex mix for 10 seconds and centrifuge (500 × g, 5 minutes) to separate the cells. Subsequently, wash the separated cells by adding PBS solution (1 mL, 100 mM, pH = 7.4) to evenly disperse the cells. Centrifuge (500 × g, 5 minutes) and remove the supernatant. Repeat the washing process three times to remove excess TA and Mn. 2+ ions. After washing, centrifugation was performed to obtain Mn 2+ and TA nanocoating-modified B16F10 melanoma cells MnTA cells.

[0056] Step 4: The MnTA cells obtained in Step 3 were suspended in 1 mL of adhesive (polyethyleneimine solution, 0.1 mg / mL) and vortexed for 10 seconds to obtain PEI-MnTA cells. The lipopolysaccharide solution was then added and vortexed for 15 seconds. Subsequently, the cells were washed with PBS (100 mM, 7.4°C) and centrifuged to obtain LMP cells (hereinafter referred to as LMP whole-cell vaccine for convenience).

[0057] Example 2

[0058] Prepare the metal-polyphenol nanoparticle-coated encapsulated whole tumor cells according to the following steps:

[0059] Step 1: Collect B16F10 cells and obtain a single cell suspension (2×10 6 cells / mL); Tara tannin solution (10 μL, 20 mg / mL) was added to 200 μL of the cell suspension and vortexed for 10 seconds to obtain a tannic acid-modified Tara cell solution.

[0060] Step 2: Add manganese sulfate monohydrate (MnSO4·H2O) solution (50 μL, 10 mg / mL) to the solution obtained in step 1 and vortex mix for 10 seconds.

[0061] Step 3: Add 500 μL of phosphate buffered saline (PBS, 100 mM, pH = 7.4) to the solution obtained in step 2. Vortex mix for 10 seconds and centrifuge (500 × g, 5 minutes) to separate the cells. Subsequently, wash the separated cells by adding PBS solution (1 mL, 100 mM, pH = 7.4) to evenly disperse the cells. Centrifuge (500 × g, 5 minutes) and remove the supernatant. Repeat the washing process three times to remove excess taratannin and Mn. 2+ ions. After washing, centrifugation was performed to obtain Mn 2+ and Tara tannin nanocoating-modified B16F10 melanoma cells Mn-Tara cells.

[0062] Step 4: The MnTA cells obtained in Step 3 were suspended in 1 mL of polyethyleneimine solution (PEI, 0.1 mg / mL) and vortexed for 10 seconds. The lipopolysaccharide solution was then added and vortexed for 15 seconds. The cells were then washed with PBS (100 mM, 7.4°C) and centrifuged to obtain LMP-Tara cells (hereinafter referred to as LMP-Tara whole-cell vaccine for convenience).

[0063] Example 3

[0064] Prepare whole tumor cells coated with metal-polyphenol nanoparticles by the following steps:

[0065] Step 1: Collect B16F10 cells and obtain a single cell suspension (2×10 6 cells / mL); epicatechin gallate EGCG solution (10 μL, 20 mg / mL) was added to 200 μL of the cell suspension and vortexed for 10 seconds to obtain a tannic acid-modified cell EGCG solution.

[0066] Step 2: Add manganese sulfate monohydrate (MnSO4·H2O) solution (50 μL, 10 mg / mL) to the solution obtained in step 1 and vortex mix for 10 seconds.

[0067] Step 3: Add 500 μL of phosphate buffered saline (PBS, 100 mM, pH = 7.4) to the solution obtained in step 2. Vortex mix for 10 seconds and centrifuge (500 × g, 5 minutes) to separate the cells. Subsequently, wash the separated cells by adding PBS solution (1 mL, 100 mM, pH = 7.4) to evenly disperse the cells. Centrifuge (500 × g, 5 minutes) and remove the supernatant. Repeat the washing process three times to remove excess taratannin and Mn. 2+ ions. After washing, centrifugation was performed to obtain Mn 2+ and EGCG nanocoating-modified B16F10 melanoma cells Mn-EGCG cells.

[0068] Step 4: The MnTA cells obtained in step 3 were suspended in 1 mL of polyethyleneimine solution (PEI, 0.1 mg / mL) and vortexed for 10 seconds. The lipopolysaccharide solution was then added and vortexed for 15 seconds. The cells were then washed with PBS (100 mM, 7.4) and centrifuged to obtain LMP-EGCG cells (hereinafter referred to as LMP-EGCG whole-cell vaccine for convenience).

[0069] Example 4

[0070] Prepare whole tumor cells encapsulated with metal-polyphenol nanoparticles by the following steps:

[0071] Step 1: Collect DC 2.4 cells and obtain a single cell suspension (2×10 6 cells / mL); tannic acid solution (10 μL, 20 mg / mL) was added to 200 μL of the cell suspension and vortexed for 10 seconds to obtain a tannic acid-modified cell TA cell solution.

[0072] Step 2: Add manganese sulfate monohydrate (MnSO4·H2O) solution (50 μL, 10 mg / mL) to the solution obtained in step 1 and vortex mix for 10 seconds.

[0073] Step 3: Add 500 μL of phosphate buffered saline (PBS, 100 mM, pH = 7.4) to the solution obtained in step 2. Vortex mix for 10 seconds and centrifuge (500 × g, 5 minutes) to separate the cells. Subsequently, wash the separated cells by adding PBS solution (1 mL, 100 mM, pH = 7.4) to evenly disperse the cells. Centrifuge (500 × g, 5 minutes) and remove the supernatant. Repeat the washing process three times to remove excess TA and Mn. 2+ ions. After washing, centrifugation was performed to obtain Mn 2+ and TA nanocoating-modified B16F10 melanoma cells MnTA cells.

[0074] Step 4: The MnTA cells obtained in step 3 were suspended in 1 mL of polyethyleneimine solution (PEI, 0.1 mg / mL), vortexed for 10 seconds, and then lipopolysaccharide solution was added and vortexed for 15 seconds. Subsequently, the cells were washed with PBS (100 mM, 7.4) and centrifuged to obtain the coated cells.

[0075] Example 5

[0076] Prepare whole tumor cells encapsulated with metal-polyphenol nanoparticles by the following steps:

[0077] Step 1: Collect Raw264.7 cells and obtain single cell suspension (2×10 6 cells / mL); tannic acid solution (10 μL, 20 mg / mL) was added to 200 μL of the cell suspension and vortexed for 10 seconds to obtain a tannic acid-modified cell TA cell solution.

[0078] Step 2: Add manganese sulfate monohydrate (MnSO4·H2O) solution (50 μL, 10 mg / mL) to the solution obtained in step 1 and vortex mix for 10 seconds.

[0079] Step 3: Add 500 μL of phosphate buffered saline (PBS, 100 mM, pH = 7.4) to the solution obtained in step 2. Vortex mix for 10 seconds and centrifuge (500 × g, 5 minutes) to separate the cells. Subsequently, wash the separated cells by adding PBS solution (1 mL, 100 mM, pH = 7.4) to evenly disperse the cells. Centrifuge (500 × g, 5 minutes) and remove the supernatant. Repeat the washing process three times to remove excess TA and Mn. 2+ ions. After washing, centrifugation was performed to obtain Mn 2+ and TA nanocoating-modified B16F10 melanoma cells MnTA cells.

[0080] Step 4: The MnTA cells obtained in step 3 were suspended in 1 mL of polyethyleneimine solution (PEI, 0.1 mg / mL), vortexed for 10 seconds, and then lipopolysaccharide solution was added and vortexed for 15 seconds. Subsequently, the cells were washed with PBS (100 mM, 7.4) and centrifuged to obtain the coated cells.

[0081] Example 6

[0082] Prepare whole tumor cells coated with metal-polyphenol nanoparticles by the following steps:

[0083] Step 1: Collect B16F10 cells and obtain a single cell suspension (2×10 6 cells / mL); tannic acid solution (10 μL, 20 mg / mL) was added to 200 μL of the cell suspension and vortexed for 10 seconds to obtain a tannic acid-modified cell TA cell solution.

[0084] Step 2: Add manganese sulfate monohydrate (MnSO4·H2O) solution (40 μL, 10 mg / mL) to the solution obtained in step 1 and vortex mix for 10 seconds.

[0085] Step 3: Add 500 μL of phosphate buffered saline (PBS, 100 mM, pH = 7.4) to the solution obtained in step 2. Vortex mix for 10 seconds and centrifuge (500 × g, 5 minutes) to separate the cells. Subsequently, wash the separated cells by adding PBS solution (1 mL, 100 mM, pH = 7.4) to evenly disperse the cells. Centrifuge (500 × g, 5 minutes) and remove the supernatant. Repeat the washing process three times to remove excess TA and Mn. 2+ ions. After washing, centrifugation was performed to obtain Mn 2+ and TA nanocoating-modified B16F10 melanoma cells MnTA cells.

[0086] Step 4: The MnTA cells obtained in Step 3 were suspended in 1 mL of polyethyleneimine solution (PEI, 0.02 mg / mL) and vortexed for 10 seconds. The lipopolysaccharide solution was then added and vortexed for 15 seconds. The cells were then washed with PBS (100 mM, 7.4°C) and centrifuged to obtain LMP cells (hereinafter referred to as LMP whole-cell vaccine for convenience).

[0087] Example 7

[0088] Prepare whole tumor cells coated with metal-polyphenol nanoparticles by the following steps:

[0089] Step 1: Collect B16F10 cells and obtain a single cell suspension (2×10 6 cells / mL); tannic acid solution (10 μL, 20 mg / mL) was added to 200 μL of the cell suspension and vortexed for 10 seconds to obtain a tannic acid-modified cell TA cell solution.

[0090] Step 2: Add manganese sulfate monohydrate (MnSO4·H2O) solution (160 μL, 10 mg / mL) to the solution obtained in step 1 and vortex mix for 10 seconds.

[0091] Step 3: Add 500 μL of phosphate buffered saline (PBS, 100 mM, pH = 7.4) to the solution obtained in step 2. Vortex mix for 10 seconds and centrifuge (500 × g, 5 minutes) to separate the cells. Subsequently, wash the separated cells by adding PBS solution (1 mL, 100 mM, pH = 7.4) to evenly disperse the cells. Centrifuge (500 × g, 5 minutes) and remove the supernatant. Repeat the washing process three times to remove excess TA and Mn. 2+ ions. After washing, centrifugation was performed to obtain Mn 2+ and TA nanocoating-modified B16F10 melanoma cells MnTA cells.

[0092] Step 4: The MnTA cells obtained in Step 3 were suspended in 1 mL of polyethyleneimine solution (PEI, 0.2 mg / mL) and vortexed for 10 seconds. The lipopolysaccharide solution was then added and vortexed for 15 seconds. The cells were then washed with PBS (100 mM, 7.4°C) and centrifuged to obtain LMP cells (hereinafter referred to as LMP whole-cell vaccine for convenience).

[0093] In the following tests and experiments, TA cells are B16F10 melanoma cells modified with tannic acid TA, and MnTA cells are Mn 2+ The B16F10 melanoma cells modified with TA nanocoating and the LMP whole cell vaccine were MnTA cells modified with lipopolysaccharide LPS.

[0094] The cell culture conditions in the above examples are as follows: B16F10 cells, DC 2.4 cells, TA cells, MnTA cells, and LMP whole-cell vaccines were incubated in DMEM medium (Sigma, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin at 37°C in an environment with a CO concentration of 5%. Solutions in all examples were freshly prepared and filtered through a 0.2 μm filter membrane for immediate use.

[0095] Figure 1 shows the Zeta potential measurement results of cells obtained in each step of Example 1 of the present invention. It can be seen from the figure that the surface charge of the original tumor cells is 2+ After the nano-coating formed with TA was wrapped into MnTA cells, its Zeta potential value moved to a more negative direction, which was caused by the negative charge of plant polyphenols TA, indicating that Mn 2+ The nanocoating formed with TA on the surface of tumor cells. After PEI modification, the whole-cell LMP vaccine was prepared, and its Zeta potential shifted slightly to a positive level (1.30 eV), indicating the successful preparation of the LMP vaccine.

[0096] FIG2 shows the Zeta potential measurement results of the cells obtained in each step of Example 2 and Example 3. It can be seen from the figure that the results are the same as those in Example 1.

[0097] Figure 3 is a SEM image of the LMP cells and original tumor cells obtained in Example 1. Figure 4 is a SEM image of the cells obtained in Examples 2 and 3. As can be seen from Figure 3, the LMP vaccine has the same size as the original tumor cells, but the surface is relatively rough. As can be seen from Figure 4, the use of Tara and EGCG also makes the surface of the encapsulated cells rough (Figure 4), indicating that Tara and EGCG can also achieve the same effect as Mn 2+ Formation of metal polyphenol networks to modify individual cells.

[0098] FIG5 is the inductively coupled plasma-optical emission spectroscopy ICP-OEC result of the LMP cells obtained in Example 1 of the present invention, as can be seen from the figure.

[0099] Figure 6 is a TEM image of the cross section of the LMP cells and the original tumor cells obtained in Example 1 of the present invention. As can be seen from the figure, a continuous nanocoating is formed on the cell membrane of the LMP cell vaccine, while a smooth cell membrane can be seen on the original tumor cells. It can be seen that by using Mn 2+ The nanocoating formed with TA completely encapsulated the tumor cells.

[0100] When constructing a whole-cell vaccine, the following three aspects require important consideration:

[0101] (1) Whether tumor cells can be effectively inactivated. Vaccines constructed based on whole tumor cells retain all tumor-associated antigens and may induce new tumors in the host after injection. Therefore, the effective inactivation of whole cell vaccines is the first step in determining whether whole cell vaccines can be used clinically.

[0102] (2) Whether all tumor cell antigens can be effectively retained. Only by retaining as many tumor cell antigens as possible can we overcome the defects of immune escape caused by insufficient expression of a single antigen or a specific antigen and achieve effective tumor treatment.

[0103] (3) Equipped with adjuvants such as antigen-presenting cell activators (such as interferon). Whole-cell vaccines activate the immune system and directly rely on the continuous activation of antigen-presenting cells (such as DC cells) associated with innate immune stimulation. In addition to the direct cytotoxic effect on tumor cells, activators can also promote the maturation and antigen presentation of dendritic cells, thereby linking the innate immune response with the adaptive immune response; this problem does not exist in the whole-cell vaccine of the present invention.

[0104] Safety assessment of LMP whole-cell vaccine:

[0105] 1. Detection of cell activity by staining: Example 1 B16F10 cells modified with MnTA nanoparticles were incubated with DMEM medium (10% serum, 1% double antibody) in a cell culture incubator (37°C, 5% CO2) for 12 hours. The cells were then collected, washed with PBS, and 195 μL Annexin V-FITC, 5 μL PI (per 5×10 4 The cells were incubated at room temperature in the dark for 20 min and detected by flow cytometry.

[0106] 2. CCK8 method: Simply incubate cells (B16F10 cells, LMP cells) at 1.0×10 5 The cells were incubated in an opaque white 96-well plate at a density of 10 cells / mL for 24 hours. 10 μL of CCK8 solution was then added to each well and incubated in a cell culture incubator for 1 hour. The cells were then analyzed using a microplate reader (microwell reader, The absorbance at 450 nm was detected using a 200PRO (Tecan, Switzerland) CCK-8 reagent was added to each well, followed by incubation for 60 minutes, and the absorbance was measured.

[0107] Original B16F10 cells were used as a control group, and all experiments were repeated three times independently.

[0108] For the survival of the LMP whole-cell vaccine, necrosis-dependent cell death was confirmed by staining with an apoptosis kit (Annexin V-FITC / PI). Figure 7 is a fluorescent micrograph of the LMP cells obtained in Example 1 and the original tumor cells stained with Calcein / PI after being cultured in DMEM for 12 hours. It can be seen from the figure that almost all of the LMP whole-cell vaccines were positive, indicating that the cells had died. Figure 8 is the statistical results of cell viability of the LMP cells obtained in Example 1 after being cultured in DMEM culture medium for 12 hours. It can be seen from the figure that compared with the original tumor cells, the LMP whole-cell vaccine cells did not show proliferation activity in the cell counting kit-8 (CCK8) test. After incubation in the culture medium for less than 12 hours, the LMP whole-cell vaccine cells completely lost their ability to proliferate. It can be seen from the test results that the tumor cell-based LMP whole-cell vaccine is non-tumorigenic, eliminating the possibility of unexpected secondary tumors.

[0109] Figure 9 shows the protein concentration measurement results of LMP cells obtained in Example 1 of the present invention. Equal amounts of LMP whole-cell vaccine and primary tumor cells were suspended in PBS. Protein concentration was determined using a Bradford protein assay kit according to the manufacturer's instructions. Protein concentration was calculated based on the standard curve and sample volume, and the results are shown in Figures 9 and 10. As can be seen from the figures, the protein concentration of the primary tumor cells was 591.4 ± 24 μg mL -1 (1182.8 ± 4.8 pg per cell), while the protein concentration of LMP vaccine was 589.2 ± 85 μg mL -1 (1178.4±17.8pg per vaccine). In addition, there was no significant difference in RNA concentration between the original tumor cells and the LMP whole cell vaccine. More than 99% of the potential tumor antigens were trapped by the MnTA nanocamouflage, indicating that Mn 2+ The nanocoating formed with TA can retain all tumor antigens and is beneficial for immune stimulation.

[0110] Testing the effect of LPS coating on DC uptake and activation

[0111] Assessment of the extent of antigen internalization by antigen-presenting cells (DCs), such as dendritic cells (DCs), is an important indicator of vaccine efficacy. After phagocytosis of tumor antigens, immature DCs further mature by secreting proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Mature DCs have higher expression of CD80 and CD86. Bone marrow-derived rabbit-like cells (BMDCs) were co-incubated with a whole-cell LMP vaccine, which was first stained with a cell membrane far-infrared fluorescent probe (DiD). The phagocytic (uptake) ability of dendritic cells to phagocytose (whole-cell) vaccines was observed using confocal laser scanning microscopy (CLSM, Leica TCS SP8). B16F10 cells were labeled with DiD (denoted as DiD-B16F10) by diluting DiD to 10 μM, mixing it with B16F10 cells, and staining for 20 minutes. DiD-labeled B16F10 cells were prepared into LMP vaccine according to the method of Example 1 to prepare DiD-labeled LMP whole-cell vaccine (DiD-LMP vaccine).

[0112] The BMDCs were extracted as follows: mice were killed by cervical dislocation and soaked in 75% alcohol for 2 minutes. The mice were removed and the humerus and tibia were cut. After the remaining tissue was removed, the humerus and tibia were first soaked in 75% alcohol for 2 minutes and then placed in PBS (pH = 7.4). The ends of the humerus and tibia were then cut, and sterile PBS was drawn up with a sterile syringe to flush out bone marrow cells from both ends of the humerus and tibia. The bone marrow cell suspension was collected and centrifuged (1500 rpm, 6 minutes) to remove the PBS and collect the cells. The bone marrow cells were cultured in six-well plates in RPMI-1640 medium (containing 10% fetal bovine serum) containing granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 ng / mL) and interleukin-4 (IL-4, 5 ng / mL) to induce bone marrow cell differentiation. Finally, the bone marrow cells were cultured in an incubator (37°C, 5% CO2). The extraction time was recorded as day 0. The culture medium was replaced on the third day and culture was continued. On the fifth day, the suspended cells were collected as immature BMDCs.

[0113] After DC2.4 cells were incubated in a confocal microscopy dish for 24 hours, the DiD-LMP vaccine was added and incubated with DC2.4 cells for an additional 6 hours. The cells were then washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and incubated at 4°C for 12 hours. The cells were washed with PBS, permeabilized with 0.1% Triton-X in PBS for 15 minutes, blocked with 1% BSA for 20 minutes, and then labeled with phalloidin-FITC in 1% BSA for 1 hour. After a final wash in PBS, DCs were stained with DAPI and observed using a laser confocal microscope. Flow cytometry: The DiD-LMP vaccine was co-cultured with DC2.4 cells for 6 hours. The cells were then harvested and analyzed directly by flow cytometry for fluorescence.

[0114] Activation of dendritic cells by the LMP vaccine: Immature BMDCs were incubated overnight. DiD-B16F10, DiD-MnTA-modified B16F10 cells, DiD-LMP vaccine, and DiD-TA-modified B16F10 cells were co-cultured with BMDCs for 24 hours (37°C, 5% CO2). The cells were then incubated with FITC-CD11c for 15 minutes, and fluorescence intensity was analyzed by flow cytometry. DiD-B16F10, DiD-MnTA-modified B16F10 cells, and DiD-TA-modified B16F10 cells were used as control samples. To demonstrate the synergistic effect of LPS with the metal-polyphenol coating, LPS was encapsulated with tumor cells to form LPS cells. This LPS cell was obtained by vortexing the tumor cells and LPS solution for 10 seconds. The LPS cell was also used as a control sample.

[0115] Figure 11 shows flow cytometric analysis of DC phagocytosis of LMP cells obtained in Example 1 of the present invention and cells obtained in a comparative example. Figure 12 shows a confocal laser scanning microscopy image of DC phagocytosis of LMP cells obtained in Example 1 of the present invention. Figure 13 shows an SEM image of LPS-encapsulated tumor cells. As shown in Figure 11, cellular internalization of the LMP vaccine was enhanced twofold compared to MnTA cells, indicating that LPS functionalization plays a key role in the LMP vaccine. Compared to cells treated with LPS alone, cellular internalization of the LMP vaccine was enhanced threefold, demonstrating that the combination of LPS and TA-Mn synergistically enhances DC endocytosis of the LMP vaccine. Figure 12 shows that the DiD-labeled LMP vaccine was co-incubated with BMDCs, and confocal laser scanning microscopy (CLSM) revealed that the LMP vaccine (red) was internalized by DCs. This suggests that the increased uptake of the LMP whole-cell vaccine is attributed to LPS. Tumor cells coated with pure LPS exhibited low fluorescence intensity, indicating that LPS was unable to directly adhere to the cell surface for encapsulation, resulting in suboptimal internalization. As can be seen from Figure 13 , the surface of cells treated with LPS remains smooth, which is due to the presence of a small amount of LPS, thus resulting in low DC cell internalization efficiency.

[0116] In vitro experiments on activation of the STING pathway

[0117] The experimental process is as follows:

[0118] BMDCs were cultured at 1×10 per well. 6 Cells were seeded at a density of 100 μg / ml in 6-well plates and cultured overnight. The LMP vaccine was then incubated with BMDCs for 24 hours. A portion of the cells was trypsinized, harvested, and placed in PBS. Protein samples were extracted from the BMDCs, dispersed in PBS, and protein concentration was determined using the BCA assay. The extracted protein was then mixed with protein loading buffer and denatured in a boiling water bath for 5 minutes. The denatured protein and marker were then loaded into the gel wells for electrophoresis, followed by staining and analysis of TBK1, p-TBK1, p65, p-p65, IRF3, and pIRF3 expression. Another portion of the cells was harvested and total RNA was extracted using TriQuick extraction reagent. RNA (500 ng) was then quantified using a NanoDrop 2000 and reverse transcribed into cDNA. Real-time quantitative PCR was performed using SYBR Green dye on a Quant Studio 3 (Applied Biosystems). Primer sequences are shown in Table 1. PCR experimental parameters were: initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s, and primer annealing at 60°C for 30 s. Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an endogenous control, and the 2-ΔΔCt method was used for comparative quantification.

[0119] Table 1. Primer sequences for RT-qPCR

[0120] First, proteins potentially involved in the STING pathway were evaluated in BMDCs. After treatment, BMDCs were incubated with primary tumor cells, TA-coated primary tumor cells (TA cells, prepared as described in Example 1), and the LMP whole-cell vaccine. The LMP whole-cell vaccine had the most significant effect on the STING pathway-related phosphorylation of p65, TBK1, and IRF3 (phos-p65, phos-TBK1, and phos-IRF3). As shown in Figure 14, the left figure shows the gel electrophoresis analysis results of p65, TBK1, and IRF3; the right figure shows the expression results of phos-p65, phos-TBK1, and phos-IRF3.

[0121] The LMP whole-cell vaccine obtained in the example was used for in vivo treatment research, and the process was as follows:

[0122] Using subcutaneous melanoma mice as a model, the establishment process of the melanoma mouse model is as follows (the animal model in the accompanying figure description is this mouse model):

[0123] B16F10 cells with good growth status were collected and resuspended in PBS to a cell density of 6×10 6 A subcutaneous melanoma model was established by injecting 100 μL into the dorsal region above the right leg of four-week-old C57BL / 6 mice, designated as day 0. Tumor size was measured using a vernier caliper for subsequent experiments after inoculation.

[0124] Cell therapy using LMP vaccine and immunotherapy (loaded with PD-L1) for synergistic treatment:

[0125] Thirty-two tumor-bearing C57BL / 6 mice were randomly divided into four groups (n=8): PBS group, LMP whole-cell vaccine group, TA vaccine group, and LMP+anti-PD-L1 group. Anti-PD-L1 (anti-PD-L1: 7.5 mg / kg) was administered via tail vein injection on days 3, 6, 9, and 14. The LMP whole-cell vaccine group and the TA vaccine group were injected with LMP whole-cell vaccine (3×10 5 The LMP+anti-PD-L1 group was given anti-PD-L1 (7.5 mg / kg) by intravenous injection every three days starting from the third day, and the LMP whole cell vaccine (3×10 5 The mice were weighed every two days during treatment, and the size of their tumors was measured with a vernier caliper until day 24. The tumor volume was calculated according to the following formula:

[0126] V(mm 3 )=L×W 2 / 2

[0127] V is the tumor volume, L is the tumor length, and W is the tumor width.

[0128] The experimental results are as follows:

[0129] As can be seen from Figure 15, compared with the PBS group, the LMP vaccine significantly inhibited tumor growth, while the TA cell group showed a moderate growth inhibitory effect, indicating the importance of MnTA nanocamouflage in activating potent anti-tumor immunotherapy. After adding anti-PD-L1 antibodies to the treatment model, further improved anti-tumor effects were achieved. It should be noted that all mice in the synergistic group had only the smallest tumor volume, which was significantly reduced after treatment. The data results show that the combined use of the whole-cell vaccine and immunotherapy proposed by the present invention helps to inhibit tumor growth. Its application in the treatment of tumors has been demonstrated.

[0130] After the above in vivo experiments were completed, the mice were sacrificed, the main organs (heart, liver, spleen, lung, and kidney) were separated, the tumors were collected, fixed in 4% paraformaldehyde, and pathological examination was performed. The results of pathological analysis (hemoglobin and eosinophil staining, H&E staining) of the histological sections of the main organs (heart, liver, spleen, and kidney) are shown in Figure 16. As can be seen from the figure, there were no significant differences between the mice after different treatments. The collected serum was used to perform a comprehensive toxicity assessment of key blood biochemical analysis indicators including aspartate aminotransferase (AST), albumin (ALB), alanine aminotransferase (ALT), creatine kinase (CK), creatinine (CREA), and urea (UREA). The results showed that all parameters were within the reference range, as shown in Figure 17. It can be seen that the toxicity of the LMP whole-cell vaccine meets the requirements, and the LMP vaccine is safe and biocompatible.

[0131] In the present invention, plant polyphenols and metal ions form a dense nanocoating on the tumor surface in the form of non-covalent bonds (complex bonds) at room temperature. As described in the embodiments and claims, vortexing for about 10 seconds is sufficient, which is very short, indicating that the formation speed is relatively fast. Tumor cells are physically modified into single cells, and a dense single-cell coating is formed to achieve rapid cell inactivation. Existing tumor cell vaccines mainly involve cell lysis and protein recombination, or inactivation by quick freezing of cells, or inactivation by incubation in pure water. The present invention forms a coating by polyphenols and metal ions, which is inactivated at room temperature.

[0132] Metal ions, which have both structural and functional roles, complex with plant polyphenols to form a nanocoating. Furthermore, they act as immune adjuvants. After being internalized by antigen-presenting cells, the metal ions are released into the intracellular acidic environment, acting as immune adjuvants to activate the immune system's STING signaling pathway and release type I interferon. The strong interaction between polyphenols and proteins preserves all tumor cell antigens, resolving the limitations of existing tumor vaccines, which are limited to single or specific tumor antigens and insufficient expression of tumor antigens.

[0133] The multiple interactions of plant polyphenols facilitate the introduction of functional molecules, such as lipopolysaccharide coatings and anti-PD-L1. Further modification of the lipopolysaccharide nanocoating can promote the internalization of LMP whole-cell vaccines by antigen-presenting cells (such as DCs). This further promotes DC maturation and elicits potent anti-tumor immunity. Due to the adhesive properties of TA, PD-L1 can be easily loaded onto the cell surface via non-covalent bonds, enabling the integration of cell therapy with checkpoint blockade immunotherapy.

[0134] Therefore, the present invention prepares tumor whole-cell vaccines through a new method, which is rapidly inactivated under mild conditions, retains complete tumor antigens, and can be loaded with activators to enhance its anti-tumor activity.

Claims

1. A method for preparing tumor whole cells encapsulated with a metal-polyphenol nanocoating, characterized in that, It includes the following steps: Step 1: Thoroughly mix the plant polyphenol solution and the tumor cell suspension; Step 2: Add a manganese ion-containing compound solution to the mixed solution obtained in Step 1 and mix thoroughly; the mass ratio of plant polyphenol to the metal ion-containing compound is 1:2 to 8; Step 3: Add a buffer solution to the solution obtained in Step 2, mix thoroughly, wash, and centrifuge to obtain tumor cells coated with a coating.

2. The preparation method of tumor whole cells encapsulated with a metal-polyphenol nanocoating according to claim 1, wherein, The plant polyphenol is one of tannic acid, tara tannin, and tea polyphenol.

3. The preparation method of tumor whole cells encapsulated with a metal-polyphenol nanocoating according to claim 1, wherein, The metal ion compound in Step 2 is one of a manganese ion-containing compound, a zinc ion-containing compound, and an iron(II) ion-containing compound, and the manganese ion-containing compound is manganese sulfate monohydrate.

4. The preparation method of tumor whole cells encapsulated with a metal-polyphenol nanocoating according to claim 1, wherein After Step 3, the following steps are further included: Thoroughly mix the tumor whole cells coated with a coating obtained in Step 3 with a lipopolysaccharide solution, wash, and centrifuge to obtain the required tumor whole cells.

5. The preparation method of tumor whole cells encapsulated by a metal-polyphenol nanocoating according to claim 4, wherein In Step 1, Step 2, and Step 3, the vortex method is used for mixing.

6. The preparation method of tumor whole cells encapsulated with a metal-polyphenol nanocoating according to claim 4, wherein The MnTA cells are first mixed with a binder and then with lipopolysaccharide; the mass ratio of the binder to the plant polyphenol is 1 to 10:

1.

7. The tumor whole cells encapsulated by the metal polyphenol-nanocoating obtained by the preparation method according to any one of claims 1 to 6, characterized in that, A plant polyphenol-metal manganese ion coating is constructed on the surface of the tumor whole cells coated with a coating.

8. The application of a tumor whole cell encapsulated with a metal-polyphenol nanocoating as described in claim 7, wherein, The tumor cells are one of melanoma cells, DC2.4 cells, and Raw264.7 cells.

9. Use of a tumor whole cell encapsulated with a metal-polyphenol nanocoating according to claim 8, characterized in that, The tumor whole cells coated with a coating are used for preparing a whole cell vaccine.

10. Use of a tumor whole cell encapsulated with a metal-polyphenol nanocoating according to claim 9, characterized in that, The application of the tumor whole cells coated with a coating and / or the whole cell vaccine in preparing an anti-tumor drug; the tumor whole cells coated with a coating and / or the whole cell vaccine are combined with a pharmaceutically acceptable adjuvant or other components to obtain a pharmaceutically acceptable preparation.

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