Novel titanium functionalized whole-tumor vaccine as well as preparation method and application thereof

Abstract

The invention discloses a novel titanium-functionalized whole-tumor vaccine and a preparation method and application thereof.The preparation method comprises the steps that Lewis lung cancer cells are collected, washed and precipitated, normal saline is added for resuspension at the room temperature, TiBALDH is added at the same time, and the mixture is mixed to be uniform to obtain a mixed solution; oscillating and incubating the obtained mixed solution for a period of time, so that Ti in the mixed solution is in full contact with the tumor cells, and the Ti and the tumor cells are combined to form mineralized particles, thereby realizing biomimetic mineralization to obtain mineralized cells; and centrifuging and washing the obtained mineralized cells to obtain the titanium functionalized whole tumor cell vaccine. And finally adding ultrapure water and carrying out low-temperature resuspension preservation. The titanium-functionalized whole tumor cell vaccine prepared by the invention can enhance immunocompetence, stimulate DC maturation and play an immune activation role, can also retain that a complete tumor antigen can induce immune response without tumorigenic risk, and has good safety, so that the titanium-functionalized whole tumor cell vaccine is expected to be applied to tumor immunotherapy and has a relatively wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a novel titanium-functionalized whole tumor vaccine, a preparation method thereof, and an application thereof. Background Art

[0002] As is well known, malignant tumors have become a major challenge seriously endangering human life and health at present. As an important means of treating malignant tumors, the core purpose of surgery is to directly remove the tumor focus and strive for radical cure. However, even if the surgery is successful, the risk of recurrence of malignant tumors still lurks, because although surgery can effectively remove the visible tumor tissue, it is still difficult to completely avoid the problem of micro-lesions or potential cancer cell spread. This recurrence risk may not only directly threaten the patient's life safety, but also significantly reduce the patient's quality of life. In view of this, preventing postoperative recurrence has become an indispensable key link in the overall treatment plan for malignant tumors.

[0003] With the continuous in-depth research on tumor immunology, tumor treatment strategies are gradually transforming from traditional non-specific treatments such as surgery, radiotherapy, and chemotherapy to more precise and highly targeted immunotherapies. In this process, tumor vaccines, as an important part of immunotherapy, have demonstrated their excellent targeted treatment effects. Tumor vaccines use tumor antigens to induce the body's own immune response to specifically kill tumor cells, thus showing unique advantages in preventing the recurrence of malignant tumors after surgery, and have therefore become an important research topic for in-depth exploration by researchers.

[0004] According to the different antigens contained in the vaccine, tumor vaccines can be classified into multiple categories such as whole cell vaccines, tumor polypeptide vaccines, genetic engineering vaccines, and antibody-mediated tumor vaccines. It is worth noting that whole tumor cell vaccines, as a subclass of whole cell vaccines, have demonstrated great anti-tumor potential by virtue of their comprehensive tumor cell antigen spectrum contained therein. These antigens can induce a wide range of immune responses against a variety of tumor-associated antigens, thus opening up a unique therapeutic path in tumor treatment and indicating broad clinical application prospects.

[0005] The preparation of traditional whole tumor cell vaccines usually includes steps such as collection of tumor cells, inactivation, and preparation of vaccines. However, there are many problems in this preparation process, especially during the inactivation process, antigen leakage or loss may occur, which in turn affects the ability of the vaccine to induce an immune response, resulting in a less intense immune response. The emergence of the method of biomimetic mineralization for preparing whole tumor cells effectively solves this technical problem. During the process of biomimetic mineralization for preparing whole tumor vaccines, intact tumor cells, as antigens, combine with metal ions through specific interactions (such as electrostatic attraction, coordination bonds, etc.) to form mineralized particles.

[0006] The mineralization process usually involves the formation of mineral deposits on the cell surface. This deposition enhances the physical structure of the cell to a certain extent, which not only improves its stability but also reduces the risk of antigen loss. The metal elements used for mineralization each have unique metal immunotherapy effects, which can promote and enhance anti-tumor immune responses to a certain extent. When mineralized particles (foreign substances) enter the body, they are recognized by the immune system and trigger a more comprehensive and strong immune response, thereby achieving the recognition and attack of cancer cells. Therefore, as a new type of therapeutic vaccine, the bionic mineralized whole tumor cell vaccine has shown great clinical significance in preventing the recurrence of malignant tumors after surgery.

[0007] The following are the studies that have been published so far on the preparation of whole tumor cell vaccines through biomimetic mineralization:

[0008] (1) Low-temperature siliconized whole tumor cell vaccine. Guo et al. reported a Si@ovarian cancer cell (BR5-Akt and ID8ova cell lines) whole tumor cell vaccine and achieved good therapeutic effects by decorating the siliconized surface with pathogen-associated molecular patterns (PAMPs) to enhance cell immunogenicity.

[0009] (2) Manganese-mineralized whole tumor cell vaccine. He et al. reported a whole tumor cell vaccine made by modifying tumor cells with a manganese (II) phenol network (MnTA nanonet). 2+ The cGAS-STING pathway can be efficiently activated, so this vaccine can be used in combined immunotherapy.

[0010] (3) Palladium-mineralized whole tumor cell vaccine. Zeng et al. reported a palladium-mineralized whole tumor cell vaccine. 2+ It is a bioorthogonal reaction catalyst that can convert doxorubicin prodrug into doxorubicin for combined cancer treatment.

[0011] In summary, among the many strategies of tumor immunotherapy, the use of metal ion mineralization technology to prepare whole tumor cell vaccines shows great potential, indicating that its application prospects in the field of tumor immunology are broader and provide innovative ideas for tumor treatment. Summary of the invention

[0012] Based on this, the present invention provides a novel titanium-functionalized whole tumor vaccine and its preparation method and application, so that it is expected to utilize the titanium mineralized whole tumor cell vaccine to enhance immune activity, stimulate DC maturation, and exert immune activation effects.

[0013] In order to solve the above technical problems, the present invention is implemented through the following technical solutions:

[0014] A method for preparing a novel titanium functionalized whole tumor vaccine comprises the following steps:

[0015] Step 1) Collect a certain number of Lewis lung cancer cells (LLC-luc).

[0016] Step 2) Wash and precipitate the collected Lewis lung cancer cells.

[0017] Step 3) At room temperature, resuspend the washed Lewis lung cancer cells in normal saline, and at the same time add TiBALDH and mix well according to a certain ratio to obtain a mixed solution.

[0018] Step 4) Incubate the obtained mixed solution with shaking for a period of time to allow the Ti in the mixed solution to fully contact with the tumor cells. Utilize the negative charge on the surface of the tumor cells to adsorb Ti in the solution through the principle of electrostatic adsorption, and the two combine to form mineralized particles, thereby realizing biomimetic mineralization to obtain mineralized cells TiO 2 @Cell;

[0019] Step 5) Centrifuge and wash the obtained mineralized cells TiO 2 @Cell to remove excess TiBALDH and broken cells, thereby obtaining a titanium-functionalized whole tumor cell vaccine.

[0020] Step 6) Finally, add ultrapure water to the titanium-functionalized whole tumor cell vaccine after centrifugation and washing, and resuspend and store it at low temperature.

[0021] Furthermore, in Step 2, the collected Lewis lung cancer cells are washed with normal saline, and the number of washing times is 2 - 3 times.

[0022] Furthermore, in Step 3, the concentration of TiBALDH is 2 mol / L, and it is added to the washed Lewis lung cancer cells at a concentration dilution ratio of 1:20 (the volume ratio of the added TiBALDH to the volume of the final mixed solution in Step 3 is 1:20), that is, the final reaction concentration of TiBALDH is 0.1 mol / L.

[0023] Furthermore, in Step 4, the obtained mixed solution is placed in a shaker and incubated with low-speed shaking at 37 °C for 48 hours.

[0024] Furthermore, in Step 5, the centrifuged mineralized cells TiO 2 @Cell are washed with ultrapure water, and the number of washing times is 2 - 3 times.

[0025] Furthermore, in Step 6, the temperature for resuspension and storage with ultrapure water is 4 °C.

[0026] A novel titanium-functionalized whole tumor vaccine obtained by the above preparation method.

[0027] The potential application of this novel titanium-functionalized whole-tumor vaccine is specifically its application in the preparation of whole-tumor immunotherapy drugs.

[0028] Furthermore, the novel titanium-functionalized whole-tumor vaccine exerts an immune activation effect to synergistically inhibit tumor growth by enhancing immune activity and stimulating the maturation of dendritic cells (DCs).

[0029] The beneficial effects of the present invention are as follows:

[0030] The titanium-functionalized whole-tumor cell vaccine prepared by the present invention enhances immune activity, stimulates the maturation of DCs, exerts an immune activation effect, can also retain intact tumor antigens to induce immune responses but has no tumorigenic risk, and has good safety. Therefore, it is expected to be applied to tumor immunotherapy and the formulation of its strategies, providing innovative ideas for tumor treatment and having a relatively broad application prospect.

[0031] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the invention more clearly and implement it in accordance with the content of the description, the following takes the preferred embodiments of the present invention and describes them in detail in conjunction with the accompanying drawings. The specific implementation manners of the present invention are given in detail by the following embodiments and their accompanying drawings. Brief Description of the Drawings

[0032] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and the schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0033] Figure 1 A shows the in vitro characterization of the TiO 2 @Cell vaccine synthesized in Example 1 at 0 h, 24 h, and 48 h; Figure 1 B shows the SEM characterization of the TiO 2 @Cell vaccine synthesized in Example 1; Figure 1 C shows the synthesis degree curve of the TiO 2 @Cell vaccine synthesized in Example 1.

[0034] Figure 2 A shows the result graph of the viability detection of Live Cell and TiO 2 @Cell vaccine cells in the viability detection of Example 2 of the present invention; Figure 2 B shows the flow cytometry analysis result graph of the TiO 2 @Cell vaccine cells in the viability detection of Example 2 of the present invention; Figure 2 C shows the IVIS monitoring result graph of Live Cell and TiO 2 @Cell vaccine cells on the 1st, 7th, and 11th days in the in vivo tumorigenesis experiment of Example 2 of the present invention.

[0035] Figure 3 A shows the observation of TiO 2 @Cell vaccine under a fluorescence microscope on the 4th, 7th, 8th, and 10th days during the stability detection of the titanium-functionalized whole tumor vaccine in Example 3 of the present invention; Figure 3 B shows the hemolysis phenomenon diagrams of the water group, PBS group, and 1.4M, 0.7M, 0.35M, 0.14M TiO 2 @Cell vaccines in the biosafety detection of the titanium-functionalized whole tumor vaccine in Example 3 of the present invention; Figure 3 C shows the SDS-PAGE analysis result diagrams of TiO 2 @Cell vaccine, Live Cell, and IR@Cell vaccine in the antigen integrity detection of the titanium-functionalized whole tumor vaccine in Example 3 of the present invention; Figure 3 D shows the BCA protein quantification analysis result diagrams of TiO 2 @Cell vaccine, Live Cell, and IR@Cell vaccine in the antigen integrity detection of the titanium-functionalized whole tumor vaccine in Example 3 of the present invention;

[0036] Figure 3 E shows the change diagrams of the DNA content of TiO 2 @Cell vaccine, Live Cell, and IR@Cell vaccine in the antigen integrity detection of the titanium-functionalized whole tumor vaccine in Example 3 of the present invention; Figure 3 F shows the change diagrams of the RNA content of TiO 2 @Cell vaccine, Live Cell, and IR@Cell vaccine in the antigen integrity detection of the titanium-functionalized whole tumor vaccine in Example 3 of the present invention.

[0037] Figure 4 A is the flow cytometry analysis result of the titanium-functionalized whole tumor vaccine in Example 4 of the present invention stimulating DC cells to express CD80 and CD86; Figure 4 B is the flow cytometry analysis result of the titanium-functionalized whole tumor vaccine in Example 4 of the present invention stimulating DC cells to express MHC-II. Detailed implementation manners

[0038] The following will describe the preferred embodiments of the present invention in detail with reference to the accompanying drawings to more clearly understand the purpose, features, and advantages of the invention. It should be understood that the embodiments shown in the drawings are not a limitation on the scope of the present invention, but only to illustrate the essential spirit of the technical solution of the present invention.

[0039] In the following description, certain specific details are set forth in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other instances, well-known devices, structures, and techniques associated with the present application may not be shown or described in detail so as not to unnecessarily obscure the description of the embodiments.

[0040] Unless the context requires otherwise, throughout the specification and claims, the words "comprising" and its variants such as "including" and "having" shall be understood in an open, inclusive sense, i.e., shall be interpreted as "including, but not limited to".

[0041] References to "one embodiment" or "an embodiment" throughout the specification mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Additionally, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0042] As used in this specification and the appended claims, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally used in its "and / or" sense unless the context clearly dictates otherwise.

[0043] Furthermore, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0044] The present invention provides a method for preparing a novel titanium-functionalized whole tumor vaccine, comprising the following steps:

[0045] Step 1) Collect a certain number of Lewis lung cancer cells (LLC-luc).

[0046] Step 2) Wash and precipitate the collected Lewis lung cancer cells.

[0047] Preferably, the collected Lewis lung cancer cells can be washed with physiological saline, and the number of washing times can be 2 - 3 times.

[0048] Step 3) At room temperature, resuspend the washed Lewis lung cancer cells in physiological saline, and at the same time add TiBALDH and mix well in a certain proportion to obtain a mixed solution.

[0049] Preferably, the concentration of TiBALDH can be 2 mol / L, and the mass ratio of Lewis lung cancer cells to TiBALDH is 1:20.

[0050] Step 4) Shake and incubate the obtained mixed solution for a period of time to allow Ti in the mixed solution to fully contact with tumor cells. Utilize the negative charge on the surface of tumor cells to adsorb Ti in the solution through the principle of electrostatic adsorption, and the two combine to form mineralized particles, thereby realizing biomimetic mineralization and obtaining mineralized cells TiO2@Cell.

[0051] Preferably, the obtained mixed solution can be placed in a shaker and incubated with low-speed shaking at 37 °C, and the incubation time can be 48 hours.

[0052] Step 5) Centrifuge and wash the obtained mineralized cells TiO2@Cell to remove excess TiBALDH and broken cells, thereby obtaining a titanium-functionalized whole tumor cell vaccine.

[0053] Preferably, the centrifuged mineralized cells TiO2@Cell can be washed with ultrapure water, and the number of washing times can be 2 - 3 times.

[0054] Step 6) Finally, add ultrapure water to the titanium-functionalized whole tumor cell vaccine after centrifugation and washing, and resuspend and store it at low temperature.

[0055] Preferably, the temperature for resuspension and storage with ultrapure water can be 4 °C.

[0056] Through the above preparation method, a novel titanium-functionalized whole tumor vaccine can be prepared.

[0057] The following details an example of the synthesis and experiment of a titanium-functionalized whole tumor vaccine of the present invention.

[0058] Example 1 Synthesis and characterization of titanium-functionalized whole tumor vaccine;

[0059] 1) Synthesis of TiO 2 @Cell vaccine cells:

[0060] Collect 1×10 6 Lewis lung cancer cells (LLC-luc), add normal saline and wash 2 - 3 times, then add 475 μL normal saline and 25 μL TiBALDH (2 mM) and resuspend in a 1.5 mL EP tube. Place the EP tube in a shaker and incubate with shaking at 37 °C and 390 rpm for 48 h to allow Ti to fully contact with the tumor. Utilize the negative charge on the surface of tumor cells to adsorb Ti in the solution through the principle of electrostatic adsorption, and the two combine to form mineralized particles to achieve biomimetic mineralization. The obtained TiO 2 @Cell is centrifuged to remove the supernatant, washed 2 - 3 times with ultrapure water to remove excess TiBALDH and broken cells, and then added with ultrapure water and resuspended and stored at 4 °C for further experiments.

[0061] 2) TiO 2 @Cell vaccine cell body surface characterization:

[0062] Prepared TiO 2 Add 500 μL of 4% paraformaldehyde tissue fixative to @Cell vaccine and fix for 15 min. Centrifuge to remove the supernatant, then use gradient alcohol to dehydrate the intracellular water (100%, 95%, 85%, 70%, 50%), dehydrating for 15 min at each alcohol concentration. After dehydration, resuspend with 200 μL of ultrapure water, drop it onto a 9.9 mm mica sheet, first observe the cell state and take pictures with a fluorescence microscope, and then place it in a fume hood to dry naturally overnight. After drying, fix the mica sheet to the SEM sample stage with conductive glue, perform sputter coating, and then use SEM for morphology analysis; then take 1 mL of TiO 2 @Cell vaccine is detected for the surface potential of the vaccine using a Malvern zeta potential analyzer. In addition, the surface potentials of normal tumor cells and TiO 2 are also detected, and the differences in surface potentials among the three are compared.

[0063] For the synthesis of titanium-functionalized whole tumor vaccine, in this example, Ti is first biomineralized with LLC-luc cells, and as shown in Figure 1 Figure C, it can be seen that TiO 2 @Cell vaccine is successfully synthesized and in vitro characterization is performed at 0 h, 24 h, and 48 h respectively. As shown in Figure 1 Figure A, it can be seen that the synthesized TiO 2 @Cell has good size uniformity, and the average hydrodynamic diameter is about 13 μm. As shown in the SEM characterization in Figure 1 Figure B, it can be seen that the surface of the mineralized tumor cells is rough and uneven, with obvious material deposition. To further observe the surface structure of the mineralized cells, the surface structure of the cells is further magnified to 500 nm, and it can be seen that the surface of the mineralized cells presents a scaly structure.

[0064] Example 2 Antitumor efficacy of titanium-functionalized whole tumor vaccine;

[0065] 1) TiO 2 @Cell vaccine cell viability detection:

[0066] Respectively take 2×10 2 Live Cell and TiO 6 @Cell cells each, and divide them into the Live Cell group and TiO 2@Cell vaccine group. Dilute both Calcein-AM and Propidium iodide (PI) at a ratio of 1:1000, that is, put 4 mL of ultrapure water into a 10 mL centrifuge tube respectively, and then add 4 μL of Calcein-AM and PI respectively, and shake it 4 times repeatedly to fully disperse the dye. Add the prepared working solution to the Live Cell group and TiO 2 @Cell vaccine group respectively, and incubate in the dark at room temperature for 30 min. Shake it once every 10 min during the staining process to fully stain the cells. After the staining is completed, centrifuge to remove the supernatant, add 1 mL of PBS to wash the cells, and repeat 3 times. After the washing is completed, resuspend the cells in the Live Cell group and TiO 2 @Cell vaccine group with 400 μL of ultrapure water respectively. For the cells observed by confocal microscope, drop them into a confocal dish, shake well, and then detect them on the machine; finally, for the cells used in flow cytometry analysis, directly detect them on the machine with the resuspended cells.

[0067] 2) TiO 2 @Cell vaccine in vivo tumorigenesis experiment:

[0068] To further verify the in vivo tumorigenicity of the vaccine, we injected 2×10 6 normal tumor cells and mineralized TiO 2 @Cell vaccine into 6-8 week old C57 mice to observe whether tumors can form within a certain period of time to detect the biosafety of the vaccine. Depilate and prepare the skin of C57 mice, and randomly divide them into 2 groups, with 3 mice in each group. One group is the Live Cell group, in which mice are inoculated with normal tumor cells, and the other group is the TiO 2 @Cell vaccine group, in which mice are inoculated with mineralized vaccine, to detect the in vivo tumorigenesis risk of the vaccine. Monitor using IVIS on the 1st, 7th, and 11th days after injection. Inject 50 μL of sodium fluorescein intraperitoneally into each mouse before imaging, and then perform imaging on the machine 5 min later.

[0069] See Figure 2 As shown in 2 A, it can be seen that the cell activity of Live Cell is normal (green), and the cell activity of TiO Figure 2 @Cell vaccine is lost (red). At the same time, see 2 B, flow cytometry analysis also proves that the cell activity of TiO 2 @Cell vaccine is lost. In vitro live / dead staining preliminarily verifies the loss of tumorigenicity of TiO Figure 2 @Cell vaccine. As shown in 2There was no fluorescence signal after 11 days of tumor bearing with @Cell vaccine.

[0070] In summary, it can be considered that the TiO prepared by mineralization 2 @Cell vaccine has no tumorigenic risk and has the biological safety as a tumor vaccine.

[0071] Example 3 Stability and integrity of titanium-functionalized whole tumor vaccine;

[0072] 1) Stability of titanium-functionalized whole tumor vaccine:

[0073] The prepared titanium-functionalized whole tumor vaccine TiO 2 @Cell was stored in a 4°C refrigerator. It was resuspended with PBS at the same time period on the 4th, 7th, 8th, and 10th days respectively, and whether the TiO 2 @Cell vaccine was intact was observed under a fluorescence microscope. See Figure 3 As shown in A, it can be seen that within the time period of the 4th, 7th, 8th, and 10th days, the morphological structure of tumor cells was intact, indicating good vaccine stability.

[0074] 2) Biological safety of titanium-functionalized whole tumor vaccine:

[0075] 1.4M, 0.7M, 0.35M, and 0.14M TiO 2 @Cell was suspended in the same volume of PBS. The obtained red blood cell precipitates were respectively mixed with 1.4M, 0.7M, 0.35M, and 0.14M TiO 2 @Cell vaccine, the hemolysis phenomenon was observed, and the hemoglobin concentration of different experimental groups was detected, where water was the positive control group and the PBS group was the negative control group. See Figure 3 As shown in B, it can be seen that the hemolysis phenomenon of the vaccine was not obvious, indicating that the vaccine has high biocompatibility and safety.

[0076] 3) Antigen integrity of titanium-functionalized whole tumor vaccine:

[0077] To verify that compared with the Live Cell and IR@Cell vaccines, the antigen components, DNA, and RNA contents of the TiO 2 @Cell vaccine will not show obvious loss, so as to ensure the complete antigenicity of the vaccine. See Figure 3 As shown in C, the SDS-PAGE analysis results show that compared with Live Cell and IR@Cell, the protein types and contents of the TiO 2 @Cell vaccine are basically the same as theirs.

[0078] To further accurately analyze the protein content changes, the protein contents of the three were analyzed by BCA protein quantification. See Figure 3As shown in D, the results show that TiO 2 @Cell vaccine has slightly less protein than Live Cell and IR@Cell vaccines, presumably due to partial protein loss during the mineralization process.

[0079] Next, the changes in DNA and RNA content were extracted and analyzed by the phenol / chloroform method. As shown in Figure 3 E and 3F, it can be seen that the DNA and RNA content of TiO 2 @Cell vaccine is basically the same as that of Live Cell, but slightly higher than that of the existing IR@Cell vaccine, presumably due to partial DNA and RNA strand damage during the irradiation process.

[0080] All of the above results show that TiO 2 @Cell vaccine has good antigen integrity and thus helps to fully activate the immune system.

[0081] Example 4 Titanium-functionalized whole tumor vaccine stimulates DC maturation;

[0082] Mouse bone marrow cells were extracted from the leg bones of C57 mice. The cells were continuously cultured in a medium containing GM-CSF and IL-2 for 7 days to induce differentiation into BMDCs. Subsequently, 2 mL of fresh medium containing LLC-luc cells treated with different materials (PBS, irradiation-induced, TiBALDH) and TiBALDH alone were added to the BMDCs, as well as TiBALDH alone. The corresponding groups were Ctrl group, IR@Cell group, TiBALDH group, and TiO 2 @Cell group. After co-incubation for 24 h, the BMDCs were washed with PBS, digested with trypsin and collected in 1.5 mL EP tubes, and incubated with flow antibodies MHC-II-AF488 and CD86-FITC for 50 min, washed 3 times with PBS, resuspended with 200 μL PBS, and finally detected by flow cytometry.

[0083] As shown in Figure 4 A-4B, it can be seen from the test results that compared with the other three groups, the percentage of TiO 2 @Cell group stimulating DCs to express CD80 and CD86 is the highest, and the percentage of TiO 2 @Cell group stimulating DCs to express MHC-II is the highest, enabling the function of macrophages to present tumor antigens to T cells and thus playing an immune activation role. The above results show that compared with the Ctrl group, IR@Cell group, and TiBALDH group, TiO 2 @Cell group has the strongest ability to stimulate DC maturation.

[0084] According to the above experimental results, it can be proved that the novel titanium-functionalized whole tumor vaccine prepared by the present invention can enhance immune activity, stimulate the maturation of DCs, and play an immune activation role. Therefore, it has potential application value in tumor immunotherapy, for example, it can be used to prepare whole tumor immunotherapy drugs.

[0085] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a novel titanium functionalized whole tumor vaccine, characterized in that: The following steps are involved: Step 1) collecting a certain number of Lewis lung cancer cells; Step 2) washing and precipitating the collected Lewis lung cancer cells; Step 3) At room temperature, physiological saline is added to the washed Lewis lung cancer cells for re-suspending, and TiBALDH is added in a certain proportion and mixed to obtain a mixed solution; Step 4) The obtained mixed solution is shaken and incubated for a period of time to allow the Ti in the mixed solution to fully contact with the tumor cells, and the negative charge on the surface of the tumor cells is used to adsorb the Ti in the solution through the electrostatic adsorption principle. The two are combined to form mineralized particles, thereby achieving biomimetic mineralization and obtaining mineralized cells TiO2@Cell; Step 5) centrifuging and washing the obtained mineralized cells TiO2@Cell to remove excess TiBALDH and broken cells, thereby obtaining a titanium functionalized whole tumor cell vaccine; Step 6) Finally, ultrapure water is added to the titanium functionalized whole tumor cell vaccine after centrifugation and washing, and the vaccine is resuspended and stored at low temperature.

2. The method for preparing the novel titanium functionalized whole tumor vaccine according to claim 1, characterized in that: In step 2, the collected Lewis lung cancer cells are washed with physiological saline for 2-3 times.

3. The method for preparing the novel titanium functionalized whole tumor vaccine according to claim 1, characterized in that: In step 3, the concentration of TiBALDH is 2 mol / L, and it is added to the washed Lewis lung cancer cells at a concentration dilution ratio of 1:20, that is, the final TiBALDH reaction concentration is 0.1 mol / L.

4. The method for preparing the novel titanium functionalized whole tumor vaccine according to claim 1, characterized in that: In step 4, the obtained mixed solution is placed in an oscillator at 37° C. for low-speed shaking incubation for 48 hours.

5. The method for preparing the novel titanium functionalized whole tumor vaccine according to claim 1, characterized in that: In step 5, the centrifuged mineralized cells TiO2@Cell are washed with ultrapure water for 2-3 times.

6. The method for preparing the novel titanium functionalized whole tumor vaccine according to claim 1, characterized in that: In step 6, the ultrapure water is resuspended and stored at 4°C.

7. A novel titanium functionalized whole tumor vaccine obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the novel titanium functionalized whole tumor vaccine as claimed in claim 7 in the preparation of whole tumor immunotherapy drugs.

9. The use according to claim 8, characterized in that: The novel titanium-functionalized whole tumor vaccine enhances immune activity, stimulates DC maturation, and exerts immune activation to synergistically inhibit tumor growth.

Images