Bladder cancer nano vaccine

Abstract

The invention discloses a bladder cancer nano vaccine, which is characterized in that nano erdarinib is entrapped in platelets to complete the construction of the bladder cancer nano vaccine in a resting state, bacillus subtilis is adopted for in-vitro activation before use, and the bladder cancer nano vaccine is administered through intravesical perfusion. The nanocrystallized Erdatinib is prepared by dissolving Erdatinib in dimethyl sulfoxide and modifying soybean phospholipid and BSA (Bovine Serum Albumin) on the surface. According to the invention, platelets are used as a drug carrier and bacillus subtilis is used as an activator of platelets, so that the platelets are rapidly activated, and rapid recognition and targeted drug delivery of bladder cancer are realized; the nanocrystallized Erdatinib causes pyroptosis by activating a Caspase-3 / GSDME pathway of bladder cancer cells, so that the aim of removing tumor cells is fulfilled.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a bladder cancer nano vaccine. Background Art

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system. Tumor vaccines are a new technology for cancer immunotherapy. Cancer can trigger tumor-specific immune responses and improve efficacy. Tumor vaccines can improve immunotherapy, have the characteristics of universality and high efficiency in killing cancer, and are an effective strategy to achieve complete tumor elimination. The principle of tumor vaccines is to improve the effect of tumor immunotherapy by inducing and activating cytotoxic CD8+T lymphocytes (CTL). Specifically, exogenous antigens are presented to CD8+T lymphocytes through MHC-Ⅰ antigen presentation, activating CTL immune responses, thereby initiating adaptive immune responses and improving immune efficacy. As a result, tumor therapeutic vaccines can induce cancer-specific immune responses in the body, minimize adverse autoimmune reactions, help relieve immunosuppression, and form synergistic effects with other immunotherapies. However, because the exogenous drugs are insufficient, it is impossible to effectively stimulate the production of sufficient mature immune cells, thereby greatly reducing the effectiveness of tumor vaccines. Therefore, improving the antigen presentation of tumor cells is an effective method to improve the effect of bladder cancer vaccines and achieve immunotherapy.

[0003] Pyroptosis is a recently reported rapid burst of programmed death that is different from apoptosis. It is characterized by a small number of cells expressing high levels of cysteine ​​proteases (caspases) under the action of drugs, cleaving gasdermin family proteins (such as GSDME) to activate them, causing the cells to explode and die, and causing other related cells to undergo similar rapid burst of programmed death. It is an effective and rapid method to activate the body's immune T cells. Summary of the invention

[0004] The purpose of the present invention is to solve the defects in the prior art and provide a nano vaccine that can effectively improve the effect of tumor immunotherapy.

[0005] In order to achieve the above object, the present invention provides a bladder cancer nano vaccine, which is prepared by the following method:

[0006] S1. Preparation of nano-erdafitinib solution: dissolve erdafitinib (BASA) in dimethyl sulfoxide (DMSO) to obtain oil phase-DMSO; take soybean lecithin in pure water, heat and stir, then drop the oil phase-DMSO, after the dropwise addition, add BSA (bovine serum albumin), stir well, filter with a 0.22 micron membrane, and obtain nano-erdafitinib nano-BASA solution;

[0007] S2. Platelet preparation: fresh blood was collected and platelet fluid was obtained by gradient centrifugation;

[0008] S3, ultrasonic encapsulation: the nano-BASA@PLT solution prepared in step S1 and the platelet solution prepared in step S2 are fully ultrasonically mixed and then ultrasonically encapsulated to obtain a nano-BASA@PLT solution;

[0009] S4. In vitro activation of Bacillus subtilis: The nano-BASA@PLT solution obtained in step S3 was uniformly mixed with the Bacillus subtilis bacterial solution in vitro at 37° C. for 30 min to obtain the activated bladder cancer nanovaccine.

[0010] The present invention achieves efficient targeted drug delivery by nano-modifying Erdafitinib and encapsulating it with platelets under the action of in vitro activation of Bacillus subtilis, and can perform bladder instillation with low toxic and side effects.

[0011] More specifically, the bladder cancer nanovaccine of the present invention is prepared by the following method:

[0012] S1. Preparation of nano-sized Erdafitinib solution: dissolve Erdafitinib in dimethyl sulfoxide to obtain oil phase-DMSO; take soybean lecithin in pure water, heat and control the temperature at 60-70°C, and stir continuously under a magnetic stirrer at a speed of 2000 r·min -1 Then, add the oil phase-DMSO dropwise, add BSA after the addition is complete, stir thoroughly, filter through a 0.22 μm membrane to obtain a nano-BASA solution of Erdafitinib, and dilute with PBS buffer to an effective concentration of Erdafitinib of 25 μg / ml;

[0013] S2. Platelet preparation: fresh blood was collected and platelet fluid was obtained by gradient centrifugation;

[0014] S3, ultrasonic loading: the nano-BASA@PLT solution prepared in step S1 and the platelet solution prepared in step S2 were mixed in a ratio of 1:2, and ultrasonic mixing was performed using a 100 W bath type ultrasonicator at room temperature for 10 min, and after the ultrasonication was completed, the solution was incubated at 37° C. for 30 min to obtain a nano-BASA@PLT solution;

[0015] S4. In vitro activation of Bacillus subtilis: The nano-BASA@PLT solution obtained in step S3 was mixed with a Bacillus subtilis bacterial solution (the bacterial solution concentration was 5×10 5 The activated bladder cancer nano-vaccine is obtained by uniformly mixing the nanoparticles / ml in a volume ratio of 1:2 to 2:1 (preferably 1:1) in vitro at 37° C. for 30 min.

[0016] Furthermore, for the convenience of storage, after the S3 ultrasonic encapsulation, the nano-BASA@PLT solution is centrifuged, and the precipitate is the nano-BASA@PLT nanovaccine. Before the bladder instillation, the nano-BASA@PLT nanovaccine and Bacillus subtilis are activated in vitro by the method of step S4.

[0017] The present invention also provides the use of the bladder cancer nano vaccine in preparing a vaccine for bladder instillation.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. The present invention nano-modifies Erdafitinib and encapsulates the nano-drug through platelets. Under the action of in vitro activation of Bacillus subtilis, efficient targeted drug delivery is achieved, thereby regulating bladder cancer cell pyroptosis, improving tumor antigen presentation, and realizing rapid tumor immune T cell transformation, thereby improving the effect of tumor immunotherapy;

[0020] 2. The bladder cancer nanovaccine of the present invention can be administered by bladder instillation, with low toxic and side effects;

[0021] 3. The nano-vaccine in the present invention acts on tumor cells by targeting them, activating the Caspase-3 / GSDME pathway, inducing pyroptosis in tumor cells, and releasing a large number of pro-inflammatory factors. This provides a rapid and efficient immunotherapy approach for tumor immunotherapy, and also provides basic data for the clinical application of nano-vaccine therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of the method for preparing the nano vaccine of the present invention;

[0023] Figure 2 The particle size changes during the construction of the nanovaccine of the present invention;

[0024] Figure 3 The change of Zeta potential during the construction of the nanovaccine of the present invention;

[0025] Figure 4 The SEM image of the nano vaccine of the present invention is compared with the SEM image of normal platelets;

[0026] Figure 5 This is a confocal microscope image of the nanovaccine of the present invention;

[0027] Figure 6 This is a SEM image of the activation process of the nano vaccine of the present invention after being stimulated by Bacillus subtilis;

[0028] Figure 7 Screening for the ratio of Bacillus subtilis to activate this patent;

[0029] Figure 8 The expression changes of P-selectin before and after the nano vaccine of the present invention is activated by Bacillus subtilis;

[0030] Fig. 9 The results of the nano vaccine of the present invention targeting bladder cancer cells after being activated by Bacillus subtilis;

[0031] Fig.10 The results of the nano vaccine of the present invention on normal bladder epithelial cells after being activated by Bacillus subtilis;

[0032] Fig.11 This is a fluorescence image of the nanovaccine of the present invention targeting and penetrating into 3D bladder cancer cell spheres;

[0033] Fig.12 The nano vaccine of the present invention causes pyroptosis of bladder cancer cells;

[0034] Fig.13 The expression results of Caspase-3, a key molecule in the pyroptosis pathway of tumor cells activated by the nanovaccine of the present invention;

[0035] Fig.14 This is the expression result of GSDME, a key molecule in the pyroptosis pathway of tumor cells activated by the nanovaccine of the present invention. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to specific embodiments and accompanying drawings.

[0037] Example 1

[0038] Example of preparation of bladder cancer nanovaccine

[0039] like Figure 1 As shown, the preparation process of the bladder cancer nanovaccine of the present invention is as follows:

[0040] S1. Preparation of nano-sized Erdafitinib solution: 10 mg of Erdafitinib was dissolved in dimethyl sulfoxide to obtain an oil phase-DMSO; 300 mg of soybean lecithin was added into 10 mL of pure water, heated and the temperature was controlled at 60-70°C, and stirred continuously under a magnetic stirrer at a speed of 2000 r·min. -1 Then, the oil phase-DMSO was added dropwise within 10 minutes, followed by the addition of 100 mg BSA. After stirring for 5 minutes, the mixture was filtered through a 0.22 μm membrane to obtain the nano-BASA solution of Erdafitinib. The mixed solution was diluted to an effective concentration of Erdafitinib of 25 μg / ml.

[0041] S2. Platelet preparation: 20 ml of fresh SD rat blood was collected, platelet precipitate was obtained by gradient centrifugation, and then the platelet precipitate was resuspended and mixed with 1 ml PBS to obtain platelet solution;

[0042] S3, ultrasonic loading: the nano-BASA@PLT solution prepared in step S1 and the platelet solution prepared in step S2 were mixed in a ratio of 1:2, and ultrasonic mixing was performed using a 100 W bath type ultrasonicator at room temperature for 10 min, and after the ultrasonication was completed, the solution was incubated at 37° C. for 30 min to obtain a nano-BASA@PLT solution;

[0043] S4, centrifugal collection:

[0044] The solution after ultrasonic encapsulation is centrifuged to obtain the precipitate, which is the nanovaccine (nano-BASA@PLT), which is convenient for storage.

[0045] S5. In vitro activation of Bacillus subtilis: The nano-BASA@PLT solution obtained in step S3 was mixed with a Bacillus subtilis bacterial solution (purchased from Beijing Huayueyang Biotechnology Co., Ltd., with a bacterial solution concentration of 5×10 5 / ml. ) are uniformly mixed in vitro at a volume ratio of 1:2 to 2:1 (in this embodiment, the volume ratio is 1:1) at 37°C for 30 minutes to obtain the activated bladder cancer nano vaccine.

[0046] like Figure 2 Shown is a comparison of the particle sizes of nano-BASA, platelet PLT and the nanovaccine nano-BASA@PLT of the present invention (prepared in step S4): the particle size of the constructed bladder cancer nanovaccine is close to that of normal platelets, indicating that the bladder cancer nanovaccine we constructed is in a dormant state before being activated by Bacillus subtilis.

[0047] like Figure 3 As shown, after the nano-BASA@PLT of the present invention is constructed, its potential is close to that of normal platelet PLT, indicating that the surface of the bladder cancer nano-vaccine is the platelet membrane, proving that we have successfully completed the platelet-encapsulated nano-erdatinib.

[0048] like Figure 4 As shown, the original platelet PLT is flat disc-shaped, and after being coated with drugs, it presents a three-dimensional spherical shape with an abrupt and uneven surface, similar to normal platelets, indicating that the vaccine nano-BASA@PLT constructed in this example is in a resting state.

[0049] like Figure 5 As shown, the fluorescence labeling results of the nanovaccine nano-BASA@PLT of the present invention showed that most of Erdafitinib was encapsulated in platelets.

[0050] like Figure 6As shown, tracking step S5 is the activation process of the nanovaccine of the present invention after stimulation by Bacillus subtilis. Bacillus subtilis can rapidly activate platelets in vitro and release internal drugs. After activation, platelets are spindle-shaped and extend multiple tentacles. After activation, platelets will aggregate and release drugs. After releasing the drugs, the platelets will break into fragments and the drugs will be released.

[0051] Example 2

[0052] Comparison of activation effects of Bacillus subtilis

[0053] The nano-vaccine nano-BASA@PLT prepared in steps S1 to S4 of Example 1 was activated with Bacillus subtilis liquid at different ratios, such as Figure 7 As shown, under the activation effect of different ratios of Bacillus subtilis / bladder cancer nanovaccine, the nanovaccine was most obviously activated after adding it to bladder cancer cells at a volume ratio of 1:2 to 2:1; then as the proportion of Bacillus subtilis increased, the activated bladder cancer nanovaccine would adsorb on the Bacillus subtilis to form aggregates, which was not conducive to later treatment.

[0054] Example 3

[0055] Targeted test of the nano vaccine of the present invention

[0056] like Figure 8 As shown, immunofluorescence experiments were used to detect the changes in the expression of P-selectin (CD62p) of PLT before and after activation with Bacillus subtilis (as shown in the figure, the Bacillus subtilis activation group corresponds to the activated bladder cancer nanovaccine obtained in step S5 of Example 1, and the group without Bacillus subtilis activation corresponds to the nanovaccine nano-BASA@PLT obtained in step S4 of Example 1), confirming that the targeting of PLT is enhanced after Bacillus subtilis activation.

[0057] like Fig. 9 As shown, using the Transwell system, Erdafitinib in the nanovaccine is fluorescently labeled with rhodamine (red), and the bladder cancer cell nuclei are fluorescently labeled with hoechst33342 (blue), separated by a 0.4 micron pore membrane in the chamber, MB49 (bladder cancer cells) are cultured in the lower chamber, and the bladder cancer nanovaccine is added to the upper chamber. After 24 hours of culture, PBS is rinsed, and it can be seen that the bladder cancer nanovaccine activated by Bacillus subtilis (i.e., the activated bladder cancer nanovaccine obtained in step S5) passes through the middle channel of the Transwell, reaches the lower chamber, and attaches to the bladder cancer cells.

[0058] However, the bladder cancer nanovaccine that was not activated by Bacillus subtilis (i.e., the nanovaccine nano-BASA@PLT obtained in step S4) did not pass through the middle channel of the Transwell, and no bladder cancer nanovaccine was found in the lower chamber.

[0059] like Fig.10 As shown, using the Transwell system, MBEC (bladder urothelial cells, normal tissue cells) were cultured in the lower chamber, and the bladder cancer nanovaccine was added to the upper chamber. After 24 hours of culture, PBS was rinsed, and it can be seen that neither the bladder cancer nanovaccine activated by Bacillus subtilis nor the bladder cancer nanovaccine not activated by Bacillus subtilis targeted cells.

[0060] This is mainly because the activated bladder cancer nanovaccine has no targeted recognition effect on normal tissue cells.

[0061] like Fig.11 As shown, erdafitinib was labeled with rhodamine, PLT was labeled with FITC, and bladder cancer nanovaccine was constructed. The bladder cancer nanovaccine activated by Bacillus subtilis (i.e. Fig.11 The nano-BASA@PLT group shown in the figure was added to the 3D cell tumor spheres. The results showed that the activated bladder cancer nanovaccine was able to target tumor cells and penetrate into the tumor.

[0062] While free administration of nanoerdatinib (i.e. Fig.11 The nano-BASA free administration group shown in the figure was unable to penetrate into the tumor. The possible reason is that the freely administered nano-erdafitinib was evenly dispersed in the solution, resulting in a lower concentration in the tumor.

[0063] Example 4

[0064] Immune Effect

[0065] Fig.12 In the figure, after the activated bladder cancer nanovaccine obtained in step S5 of Example 1 was used to act on bladder cancer cells in vitro (the action conditions were: effective erdafitinib concentration of 10 μM; action time of 24 h; action temperature of 37°C), typical morphological characteristics of cell pyroptosis, such as cell swelling, rupture, release of contents, and vacuolation, were observed in bladder cancer cells under the microscope (red arrows).

[0066] Fig.13 and 14 In the present study, immunofluorescence experiments were used to detect that after the activated bladder cancer nanovaccine was used to act on bladder cancer cells in vitro (the action conditions were: effective erdafitinib concentration of 10 μM; action time of 24 h; action temperature of 37°C), the expression of caspase3 and GSDME in bladder cancer cells increased significantly, which preliminarily verified the pyroptosis effect of the nanovaccine on tumor cells.

Claims

1. A bladder cancer nanovaccine, characterized in that: The bladder cancer nanovaccine is prepared by the following method: S1. Preparation of nano-sized Erdafitinib solution: dissolve Erdafitinib in dimethyl sulfoxide to obtain oil phase-DMSO; take soybean lecithin in pure water, heat and stir, then drop the oil phase-DMSO, after the dropwise addition is complete, add BSA, stir well, and filter through a 0.22 micron membrane to obtain the nano-sized Erdafitinib solution; S2. Platelet preparation: fresh blood was collected and platelet fluid was obtained by gradient centrifugation; S3, ultrasonic encapsulation: the nano-BASA@PLT solution prepared in step S1 and the platelet solution prepared in step S2 are fully ultrasonically mixed and then ultrasonically encapsulated to obtain a nano-BASA@PLT solution; S4. In vitro activation of Bacillus subtilis: The nano-BASA@PLT solution obtained in step S3 was uniformly mixed with the Bacillus subtilis bacterial solution in vitro at 37° C. for 30 min to obtain the activated bladder cancer nanovaccine.

2. The bladder cancer nanovaccine according to claim 1, characterized in that In the step S1, the addition ratio of erdafitinib, soybean lecithin and BSA is 10 mg:300 mg:100 mg, and the mixed solution is diluted with PBS buffer to an effective concentration of erdafitinib of 25 μg / ml.

3. The bladder cancer nanovaccine according to claim 2, characterized in that In step S3, the volume ratio of the nano-sized Erdafitinib solution to the platelet solution is 1:

2.

4. The bladder cancer nanovaccine according to claim 3, characterized in that In step S4, the concentration of the Bacillus subtilis liquid is: 5×10 5 / ml, and the volume ratio of nano-BASA solution to Bacillus subtilis solution was 1:2 to 2:

1.

5. The bladder cancer nanovaccine according to claim 4, characterized in that In the step S4, the volume ratio of the nano-BASA solution to the Bacillus subtilis solution is 1:

1.

6. The bladder cancer nanovaccine according to claim 5, characterized in that The conditions for ultrasonic loading in step S3 are: ultrasonic mixing using a bath-type ultrasonicator with a power of 100 W, at room temperature, for 10 minutes, and after the ultrasonication is completed, the solution is placed and incubated at 37° C. for 30 minutes.

7. Use of the bladder cancer nanovaccine according to any one of claims 1 to 6 in the preparation of a vaccine for bladder instillation.

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