Tumor whole cell wrapped by metal-polyphenol nano coating as well as preparation method and application of tumor whole cell

Abstract

The invention discloses a tumor whole cell wrapped by a metal-polyphenol nano coating as well as a preparation method and application thereof. The preparation method comprises the following steps: step 1, fully mixing a plant polyphenol solution and a tumor cell suspension; 2, adding a manganese ion-containing compound solution, and fully mixing; 3, adding a buffer solution into the solution obtained in the step 2, fully mixing, washing, and centrifugally separating to obtain tumor cells coated with coatings; the plant polyphenol and the manganese ions can be quickly assembled at room temperature, a compact nano coating is formed on a tumor cell membrane, and tumor cells are inactivated due to the wrapping of the nano coating, so that the safety of the vaccine is ensured; the nano coating can prevent any potential tumor antigen from being lost under physiological conditions; lipopolysaccharide is further modified, so that the formed LMP whole-cell vaccine can be promoted to be endocytosed by antigen presenting cells; the metal manganese ions can stimulate the STING pathway to enhance the anti-tumor effect while forming the structural coating.

Description

Technical Field

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

[0002] Immunotherapy is a new type of tumor treatment method. It activates the body's own immunity, allows 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 one of the important immunotherapy strategies that can stimulate tumor-specific immune stimulation and have great potential for cancer treatment. Currently, most tumor vaccines are based on individual tumor-associated antigens. Although modern sequencing technology has greatly promoted the research of personalized new antigen vaccines, it still cannot fully cover the personalized tumor cell antigens, and due to the lack of immune recognition mechanism, its effectiveness in clinical application is very limited and cannot be widely used.

[0004] Whole-cell vaccines can overcome the limitation of lack of extensive antigens in different types of tumors and the defect of immune escape caused by insufficient expression of single antigens or specific antigens, thus achieving tumor treatment or prevention, as they retain complete tumor-associated antigens. The main methods for constructing whole-cell vaccines include recombination of tumor cell lysates and modification of intact tumor cells, including genetic modification, cryogenic engineering, microneedle patch delivery, and macroporous cryogel. Although these strategies show promise, they fail to achieve retention of all potential tumor antigens and strong anti-tumor immune responses, and the preparation process is time-consuming and limits clinical application.

[0005] At present, the construction method of whole-cell tumor vaccine, 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 (DC) in co-culture conditions, and significantly improving the immune response in mice. The risk of gene editing lies in unknown mutations. For example, the application number is 202011095900.0. The constructed vaccine modifies tumor cells by covalent bonding, and couples 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. And small molecule agonists, as the main recognition antigen, may be tolerated. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a tumor whole cell wrapped by 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 nano-coating, 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-containing compound is 1:2-8;

[0010] Step 3: Add buffer to the solution obtained in step 2, mix thoroughly, wash, and centrifuge to obtain the coated tumor whole 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 further includes 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, in step 1, step 2 and step 3, a vortex method is used for mixing.

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

[0017] An application of a whole tumor cell wrapped by 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 quickly assembled at room temperature to form a dense nano-coating on the tumor cell membrane. The encapsulation of the nano-coating inactivates the tumor cells, thereby 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 metal manganese ions in the present invention can activate the STING pathway of immune cells while forming a coating, enhance the expression of type I interferon, and enhance the anti-tumor effect;

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

[0025] Figure 1 These are the potential measurement results of cells obtained at each stage in Example 1 of the present invention.

[0026] Figure 2 These are the potential measurement results of tumor cells obtained in Examples 2 and 3 of the present invention.

[0027] Figure 3 This is a SEM image of the LMP cells and original tumor cells obtained in Example 1 of the present invention.

[0028] Figure 4 These are SEM images of LMP cells obtained in Examples 2 and 3 of the present invention.

[0029] Figure 5 This is the inductively coupled plasma-optical emission spectrum of the LMP cell obtained in Example 1 of the present invention.

[0030] Figure 6 TEM images of LMP cells and original tumor cells obtained in Example 1 of the present invention.

[0031] Figure 7 This is a fluorescence micrograph of the LMP cells and the original tumor cells obtained in Example 1 of the present invention, which were cultured in DMEM for 12 hours and then stained with Calcein / PI.

[0032] Figure 8 The cell viability statistics of the LMP cells obtained in Example 1 of the present invention after being cultured in DMEM medium for 12 hours.

[0033] Fig. 9 This is the protein concentration measurement result of LMP cells obtained in Example 1 of the present invention.

[0034] Fig.10 This is a schematic diagram comparing the protein concentrations of LMP cells and original tumor cells obtained in Example 1 of the present invention.

[0035] Fig.11 This is a diagram 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] Fig.12 This is a laser confocal image of DC engulfing the LMP cells obtained in Example 1 of the present invention.

[0037] Fig.13 This is the SEM image of LPS-encapsulated tumor cells.

[0038] Fig.14 Schematic diagram of the expression of p65, TBK1 and IRF3 in LMP cells and control cells obtained in Example 1 of the present invention.

[0039] Fig.15 This is a comparison chart of the tumor size 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.

[0040] Fig.16 Schematic diagram of staining of main organs of animal models after the animal models were treated with LMP cells obtained in Example 1 of the present invention and cells of a comparative example.

[0041] Fig.17 Serum test results 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 in conjunction with the accompanying drawings and specific embodiments.

[0043] A method for preparing whole tumor cells wrapped with a metal-polyphenol nano-coating 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 the manganese ion compound solution to the mixed solution obtained in step 1 and vortex mix; wherein the mass ratio of the plant polyphenols to the metal ion-containing compound is 1:2-8; the metal ion compound is one of a manganese ion-containing compound, a zinc ion-containing compound, and a ferrous ion-containing compound, and the manganese ion-containing compound is manganese sulfate monohydrate.

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

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

[0048] The surface of the whole tumor cell coated with the coating is constructed with a plant polyphenol-metallic manganese ion coating; if lipopolysaccharide is added, the surface of the plant polyphenol-metallic manganese ion coating is coated with a layer of lipopolysaccharide. In order to make the adhesion tighter, an adhesive such as polyethyleneimine, lysine, etc. can be added.

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

[0053] Step 1: Collect B16F10 cells and obtain a single cell suspension (2×10 6cells / 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. Then wash, add PBS solution (1 mL, 100 mM, pH = 7.4) to the separated cells to make the cells evenly dispersed, centrifuge (500 × g, 5 minutes), and remove the upper washing solution. Repeat the washing process three times to remove excess TA and Mn 2+ ions. After washing, centrifugal separation 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 PEI, 0.1 mg / mL), vortexed for 10 seconds to obtain PEI-MnTA cells, 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 LMP cells (hereinafter referred to as LMP whole cell vaccine for convenience).

[0057] Example 2

[0058] Prepare the metal-polyphenol nanoparticle-coated encapsulated tumor whole cells as follows:

[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. Then wash, add PBS solution (1 mL, 100 mM, pH = 7.4) to the separated cells to make the cells evenly dispersed, centrifuge (500×g, 5 minutes), and remove the upper washing solution. The washing process is repeated three times to remove excess taratannin and Mn 2+ ions. After washing, centrifugal separation was performed to obtain Mn 2+ and B16F10 melanoma cells Mn-Tara cells modified with Tara tannin nanocoating.

[0062] 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 LMP-Tara cells (hereinafter referred to as LMP-Tara whole cell vaccine for convenience).

[0063] Example 3

[0064] Prepare the whole tumor cells encapsulated 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 cell 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. Then wash, add PBS solution (1 mL, 100 mM, pH = 7.4) to the separated cells to make the cells evenly dispersed, centrifuge (500×g, 5 minutes), and remove the upper washing solution. The washing process is repeated three times to remove excess taratannin and Mn 2+ ions. After washing, centrifugal separation 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), 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 LMP-EGCG cells (hereinafter referred to as LMP-EGCG whole cell vaccine for convenience).

[0069] Example 4

[0070] Prepare the 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. Then wash, add PBS solution (1 mL, 100 mM, pH = 7.4) to the separated cells to make the cells evenly dispersed, centrifuge (500 × g, 5 minutes), and remove the upper washing solution. Repeat the washing process three times to remove excess TA and Mn 2+ ions. After washing, centrifugal separation 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 the 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 6cells / 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. Then wash, add PBS solution (1 mL, 100 mM, pH = 7.4) to the separated cells to make the cells evenly dispersed, centrifuge (500 × g, 5 minutes), and remove the upper washing solution. Repeat the washing process three times to remove excess TA and Mn 2+ ions. After washing, centrifugal separation 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 the whole tumor cells encapsulated 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. Then wash, add PBS solution (1 mL, 100 mM, pH = 7.4) to the separated cells to make the cells evenly dispersed, centrifuge (500 × g, 5 minutes), and remove the upper washing solution. Repeat the washing process three times to remove excess TA and Mn 2+ ions. After washing, centrifugal separation 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), 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 LMP cells (hereinafter referred to as LMP whole cell vaccine for convenience).

[0087] Example 7

[0088] Prepare the whole tumor cells encapsulated 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. Then wash, add PBS solution (1 mL, 100 mM, pH = 7.4) to the separated cells to make the cells evenly dispersed, centrifuge (500 × g, 5 minutes), and remove the upper washing solution. Repeat the washing process three times to remove excess TA and Mn 2+ ions. After washing, centrifugal separation 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), 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 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 nano-coating and the LMP whole cell vaccine were MnTA cells modified with lipopolysaccharide LPS.

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

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

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

[0097] Figure 3 The SEM images of the LMP cells and the original tumor cells obtained in Example 1 are shown. Figure 4 2 and 3 are SEM images of cells obtained in Example 2 and Example 3. Figure 3 It can be seen that the LMP vaccine has the same size as the original tumor cell, but the surface is rougher. Figure 4 As can be seen, the use of Tara and EGCG also made the surface of the encapsulated cells rough ( Figure 4), indicating that Tara and EGCG can also achieve 2+ Formation of metallopolyphenol networks to modify individual cells.

[0098] Figure 5 This 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 TEM images of the cross-sections of LMP cells and original tumor cells obtained in Example 1 of the present invention. It can be seen from the figure that 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] To construct 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-related 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; the whole-cell vaccine of the present invention does not have this problem.

[0104] Evaluation of the safety of LMP whole-cell vaccines:

[0105] 1. Detecting cell activity by staining method: Example 1 B16F10 cells modified with MnTA nanoparticles were incubated 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 at a density of 10 cells / mL in an opaque white 96-well plate for 24 h. Then 10 μL of CCK8 solution was added to each well and incubated in a cell culture incubator for 1 h before using a microplate reader (microwell reader, 200PRO, Tecan, Switzerland) to detect the absorbance at 450 nm. 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 LMP whole cell vaccines, necrosis-dependent cell death was confirmed by staining with an apoptosis kit (Annexin V-FITC / PI). Figure 7 The fluorescence micrographs of the LMP cells and the original tumor cells obtained in Example 1 after being cultured in DMEM for 12 hours and then stained with Calcein / PI are shown. It can be seen from the figure that almost all the LMP whole cell vaccines are positive, indicating that the cells have died. Figure 8 The cell viability statistics of the LMP cells obtained in Example 1 after culturing in DMEM medium for 12 hours. As can be seen from the figure, compared with the original tumor cells, the LMP whole 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 vaccine cells completely lost their proliferation ability. From the test results, it can be seen that the LMP whole cell vaccine based on tumor cells is non-tumorigenic, eliminating the possibility of unexpected secondary tumors.

[0109] Fig. 9 The results of protein concentration determination of LMP cells obtained in Example 1 of the present invention are shown in Table 1. The same number of LMP whole cell vaccines and original tumor cells were suspended in PBS. The protein concentration was determined using a Bradford protein assay kit according to the manufacturer's instructions. The protein concentration was calculated based on the standard curve and sample volume. The results are shown in Table 1. Fig. 9 and Fig.10 As shown in the figure, the protein concentration of the original tumor cells is 591.4±24μg·mL -1 (1182.8±4.8pg per cell), while the protein concentration of LMP vaccine was 589.2±85μg·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 potential tumor antigens were trapped by MnTA nanocamouflage, indicating that Mn 2+The nanocoating formed with TA can retain all tumor antigens and is beneficial for immune stimulation.

[0110] Testing of LPS coating for enhanced uptake and activation of DCs

[0111] Evaluation of the degree of antigen endocytosis by antigen-presenting cells such as DCs is an important indicator for measuring vaccine efficacy. After phagocytosis of tumor antigens, immature DCs further become mature DCs by secreting proinflammatory factors, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Mature DCs have higher expression of CD80 and CD86 factors. Bone marrow-derived lagomorphic BMDCs were co-incubated with LMP whole-cell vaccines, which were first stained with a cell membrane far-infrared fluorescent probe (DiD). The phagocytic (uptake) ability of dendritic cells to whole-cell vaccines was observed using a laser confocal microscope (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. The 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 extraction method of BMDCs is as follows: the mouse is killed by cervical dislocation, soaked in 75% alcohol for 2 minutes, the mouse is taken out, and the humerus and tibia are cut off. After the residual tissue is cleared from the removed humerus and tibia, it is first soaked in 75% alcohol for 2 minutes, and then placed in PBS (pH=7.4). Then the two ends of the humerus and tibia are cut off, and the sterile PBS is drawn from the sterile syringe to flush the bone marrow cells from the two ends of the humerus and tibia in turn. The bone marrow cell suspension is collected, centrifuged (1500rpm, 6min) to remove the PBS and collect the cells, and the bone marrow cells are cultured in a six-well plate, and the culture medium is RPMI-1640 culture medium (containing 10% fetal bovine serum) containing granulocyte-macrophage colony stimulating factor (GM-CSF, 10ng / mL) and interleukin 4 (IL-4, 5ng / mL) to induce differentiation of bone marrow cells. Finally, the bone marrow cells were cultured in an incubator (37° C., 5% CO 2 ), and 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 cells.

[0113] After DC 2.4 was incubated in the confocal dish for 24 h, DiD-LMP vaccine was added and incubated with DC2.4 for another 6 h. The cells were then washed with PBS, fixed with 4% paraformaldehyde for 15 min, and then placed at 4°C for 12 h. The cells were washed with PBS, permeabilized with 0.1% Triton-X in PBS for 15 min, blocked with 1% BSA for 20 min, and then labeled with phalloidin-FITC in 1% BSA for 1 h. After the final wash in PBS, DCs were stained with DAPI. Observed with a laser confocal microscope. Flow cytometry: DiD-LMP vaccine was co-cultured with DC2.4 for 6 h, and the cells were collected and analyzed directly by fluorescence on a flow cytometer.

[0114] Activation of dendritic cells by LMP vaccine: Immature BMDCs were incubated overnight, and DiD-B16F10, DiD-MnTA-modified B16F10 cells, DiD-LMP vaccine, and DiD-TA-modified B16F10 cells were co-cultured with BMDCs for 24h (37°C, 5% CO2). Subsequently, FITC-CD11c was co-incubated with the cells for 15min, and the fluorescence intensity was analyzed by flow cytometry. DiD-B16F10, DiD-MnTA-modified B16F10 cells and DiD-TA-modified B16F10 cells were all used as comparative examples. In order to illustrate whether LPS has a synergistic effect with the metal-polyphenol coating, LPS was encapsulated with tumor cells, namely LPS cells. That is, LPS cells can be obtained by vortexing the tumor cells and the LPS solution for 10s, and LPS cells are also used as comparative examples.

[0115] Fig.11 This is a diagram 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. Fig.12 This is a laser confocal image of DC engulfing the LMP cells obtained in Example 1 of the present invention. Fig.13 This is a SEM image of LPS-encapsulated tumor cells. Fig.11 As can be seen in the figure, compared with MnTA cells, the cellular internalization of LMP vaccine was enhanced by two times. This shows that LPS functionalization plays a key role in LMP vaccine. Compared with cells modified with LPS alone, the cellular internalization of LMP vaccine was enhanced by 3 times, indicating that LPS combined with TA-Mn can synergistically enhance the endocytosis of LMP vaccine by DC cells. Fig.12As can be seen in the figure, the DiD-labeled LMP vaccine was co-incubated with BMDCs, and confocal laser scanning microscopy (CLSM) observed that the LMP vaccine (red) was internalized by DCs. This shows that the increased uptake of the LMP whole-cell vaccine is due to LPS. The fluorescence intensity of tumor cells wrapped with pure LPS is not obvious, indicating that LPS cannot directly adhere to the cell surface for wrapping, so the internalization effect is not ideal. Fig.13 It can be seen that the cell surface treated with LPS remains smooth due to the presence of a small amount of LPS, which leads to low DC cell internalization efficiency.

[0116] In vitro assay to activate the STING pathway

[0117] The experimental process is as follows:

[0118] BMDCs were cultured at 1×10 per well. 6 The cells were inoculated in a 6-well plate at a density of 10 cells and cultured overnight. The LMP vaccine was incubated with BMDCs for 24 hours. Some cells were digested with trypsin, collected and placed in PBS, protein samples were extracted from BMDCs, dispersed in PBS, and the protein concentration was determined by BCA method. The extracted protein was then mixed with protein loading buffer and placed in a boiling water bath for 5 minutes to denature the protein. The denatured protein and marker were then added to the gel wells for electrophoresis, and finally stained to analyze the expression of TBK1, p-TBK1, p65, p-p65, IRF3 and pIRF3. Another part of the cells was collected and total RNA was extracted with Triquick extraction reagent, and then RNA (500ng) was quantified with NanoDrop2000, and then the extracted RNA was reverse transcribed into cDNA. Real-time fluorescence quantitative PCR was performed on quant studio 3 applied biosystems using SYBR green dye. The primer sequences are shown in Table 1. The PCR experimental parameters were as follows: initial denaturation at 95°C for 30 s, denaturation at 95°C for 10 s, 40 cycles, 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 quantitative calculation.

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

[0120]

[0121] First, proteins in BMDCs that may be associated with the STING pathway were evaluated. After treatment, BMDCs were incubated with original tumor cells, TA-coated original tumor cells (TA cells, prepared as in Example 1) and LMP whole-cell vaccines. 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). Fig.14 As shown, 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.

[0122] The LMP whole cell vaccine obtained in the example was used for in vivo treatment research, and the process was as follows:

[0123] 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):

[0124] B16F10 cells with good growth status were collected and resuspended in PBS to a cell density of 6×10 6 100 μL was injected into the back of four-week-old C57BL / 6 mice above the right leg to establish a mouse subcutaneous melanoma model, which was recorded as day 0. After inoculation, the size of the tumor was measured with a vernier caliper for subsequent experiments.

[0125] Cell therapy using LMP vaccine and immunotherapy (loaded with PD-L1) for synergistic treatment:

[0126] Thirty-two tumor-bearing C57BL / 6 mice were randomly divided into four groups (n=8), namely, PBS group, LMP whole cell vaccine group, TA vaccine group, and LMP+anti-PD-L1 group. Anti-PD-L1 was administered via tail vein injection on days 3, 6, 9, and 14 (anti-PD-L1: 7.5 mg / kg); LMP whole cell vaccine group and 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 given 4 times in total. The body weight of the mice was measured every two days during the treatment, and the size of the tumors of the mice was measured with a vernier caliper until the 24th day. The tumor volume was calculated according to the following formula:

[0127] V(mm 3)=L×W 2 / 2

[0128] V is the tumor volume, L is the tumor length, and W is the tumor width.

[0129] The experimental results are as follows:

[0130] from Fig.15 It can be seen that the LMP vaccine significantly inhibited tumor growth compared with the PBS group, while the TA cell group showed a moderate growth inhibition effect, indicating the importance of MnTA nanocamouflage in activating potent anti-tumor immunotherapy. After adding anti-PD-L1 antibodies to the treatment model, a further improved anti-tumor effect was 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 whole cell vaccine and immunotherapy proposed in the present invention are used together to help inhibit tumor growth. Its application in the treatment of tumor drugs has been demonstrated.

[0131] After the above in vivo experiments were completed, the mice were killed, the main organs (heart, liver, spleen, lung, kidney) were separated, the tumors were collected, fixed in 4% paraformaldehyde, and pathological examination was performed. The results of pathological analysis (hemoglobin and eosinophilic staining, H&E staining) of the histological sections of the main organs (heart, liver, spleen and kidney) are shown in Fig.16 As shown in the figure, there is no significant difference between mice after different treatments. The collected serum was used for 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, such as Fig.17 As shown, it can be seen that the toxicity of the LMP whole-cell vaccine meets the requirements, and the LMP vaccine is safe and biocompatible.

[0132] In the present invention, plant polyphenols and metal ions form a dense nano-coating 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 a very short time, 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 quick freezing and inactivation of cells, or incubation in pure water for inactivation. The present invention forms a coating by polyphenols and metal ions, which is inactivated at room temperature.

[0133] Metal ions with dual structural and functional effects, on the one hand, complex with plant polyphenols to form nanocoatings, and on the other hand, are introduced as immune adjuvants. After being internalized into antigen-presenting cells, they are released in the intracellular acidic environment as immune adjuvants, activating the immune system STING signaling pathway and releasing type I interferon. Due to the strong interaction between polyphenols and proteins, all antigens of tumor cells are retained. This solves the problem that existing tumor vaccines are limited to single or specific tumor antigens and insufficient expression of tumor antigens.

[0134] The multiple interactions of plant polyphenols can easily introduce functional molecules, such as lipopolysaccharide coatings, anti-PDL1 and other functional molecules. For example, further modification of lipopolysaccharide on the nanocoating can promote the internalization of LMP whole-cell vaccines by antigen-presenting cells (such as DCs). Further promoting DC maturation triggers strong anti-tumor immunity. Due to the adhesion of TA, PD-L1 can be simply loaded on the cell surface through non-covalent bonds, allowing cell therapy to be combined with checkpoint blockade immunotherapy.

[0135] 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 load activators to enhance its anti-tumor activity.

Claims

1. A method for preparing whole tumor cells wrapped with metal-polyphenol nano-coating, characterized in that: The following steps are involved: Step 1: Thoroughly mix the plant polyphenol solution and tumor cell suspension; Step 2: adding the 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; Step 3: Add buffer to the solution obtained in step 2, mix thoroughly, wash, and centrifuge to obtain coated tumor cells.

2. The method for preparing whole tumor cells wrapped with a metal-polyphenol nano-coating according to claim 1, characterized in that: The plant polyphenol is one of tannic acid, tara tannin and tea polyphenol.

3. The method for preparing whole tumor cells wrapped with a metal-polyphenol nano-coating according to claim 1, characterized in that: The metal ion compound in step 2 is one of a manganese ion-containing compound, a zinc ion-containing compound, and a ferrous ion-containing compound, and the manganese ion-containing compound is manganese sulfate monohydrate.

4. The method for preparing whole tumor cells wrapped with a metal-polyphenol nano-coating according to claim 1, characterized in that: The step 3 also includes the following steps: the whole tumor cells coated with the coating obtained in step 3 are fully mixed with the lipopolysaccharide solution, washed and centrifuged to obtain the desired whole tumor cells.

5. The method for preparing whole tumor cells wrapped with a metal-polyphenol nano-coating according to claim 4, characterized in that: In step 1, step 2 and step 3, a vortex method is used for mixing.

6. The method for preparing whole tumor cells wrapped with a metal-polyphenol nano-coating according to claim 4, characterized in that: 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.

7. The whole tumor cell wrapped by metal polyphenol-nano coating obtained by the preparation method according to any one of claims 1 to 6, characterized in that: The surface of the whole tumor cell coated with the coating is constructed with a plant polyphenol-metal manganese ion coating.

8. The use of a tumor whole cell wrapped by a metal-polyphenol nano coating as claimed in claim 7, characterized in that: The tumor cell is one of a melanoma cell, a DC2.4 cell, and a Raw264.7 cell.

9. The use of a tumor whole cell wrapped by a metal-polyphenol nano coating according to claim 8, characterized in that: The coated tumor whole cells are used to prepare whole cell vaccines.

10. The use of a tumor whole cell wrapped by a metal-polyphenol nano coating according to claim 9, characterized in that: The application of the coated tumor whole cells and / or whole cell vaccines 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.

Images