Ultrasound-assisted immunostimulatory tumor vaccine and preparation method and application thereof

Abstract

The application provides an ultrasonic-assisted immune-activated tumor vaccine and a preparation method and application thereof, and belongs to the field of tumor biological immunotherapy. The tumor vaccine provided by the application is obtained by co-loading tumor cell membrane proteins, total tumor cell RNA and a sound sensitizer chlorine 6 into a polylactic acid-glycolic acid copolymer (PLGA) carrier, and the total tumor cell RNA is protected by polymerization with poly-L-lysine (PLL). The application promotes the escape of the tumor vaccine in lysosomes, improves the MHC class I antigen presentation level, and the vaccine can promote the maturation of dendritic cells and enhance T cell immune response by generating ROS. The vaccine has high biological safety, and the nanoparticle size level is more easily phagocytosed by cells and enters lymph nodes, and can be used for preventing the recurrence and metastasis of various tumors after surgery.

Description

Technical Field

[0001] This invention belongs to the field of tumor biological immunotherapy, specifically relating to an ultrasound-assisted immune-activated tumor vaccine, its preparation method, and its application. Background Technology

[0002] Cancer is a global public health problem that threatens human life and health. Although cancer treatment has been around for over a century, the application of various methods such as surgery, drug therapy, and radiotherapy has greatly reduced the overall mortality rate of cancer. However, a considerable number of refractory cancers are still insensitive to multiple treatment methods. In particular, postoperative recurrence and metastasis often lead to the death of patients. Therefore, it is necessary to continuously develop new cancer treatment methods to meet new challenges.

[0003] Tumor vaccines are a type of tumor immunotherapy. They are prepared by combining tumor antigens with immune adjuvants to create a vaccine formulation that activates a specific immune response against the tumor antigen in the human body. This response utilizes the immune system to recognize and kill T cells, thus treating the tumor. For a tumor vaccine to be effective, the antigen must be presented to T cells, and simultaneously expressed on tumor cells. However, currently, tumor vaccine antigens are mainly derived from tumor tissue removed from patients. These pre-existing antigens are outdated and cannot address new antigens generated by continuous tumor mutations. Furthermore, the availability of adjuvants that can activate cellular immunity is limited, and biosafety issues are significant. Current tumor vaccines are primarily used to treat advanced tumors; however, in the later stages of tumor development, immune function declines, antigen-presenting cells gradually lose their function, and vaccine efficacy is poor. These problems restrict the research and application of tumor vaccines. Therefore, improvements should be made to tumor vaccines in terms of antigens, immune adjuvants, carriers, and the timing of application.

[0004] Antigens are a crucial component of tumor vaccines, determining whether the activated immune system can accurately recognize and kill tumor cells. Currently, various types of tumor vaccines are developed, broadly categorized by antigen type into protein antigen-based, nucleic acid antigen-based, and whole-cell-based vaccines. It is believed that three main antigen presentation processes exist within dendritic cells: primary presentation to activate cellular immunity, secondary presentation to activate humoral immunity, and cross-presentation. Protein vaccines primarily activate cellular immunity through cross-presentation, while nucleic acid vaccines activate immunity through primary presentation. Tumor vaccines exert their effects mainly through antigen-presenting cells, specifically dendritic cells, which present antigens to cytotoxic T cells. This process is the activation of cellular immunity. Protein antigen vaccines primarily utilize cross-presentation to activate cellular immunity; nucleic acid vaccines synthesize corresponding proteins in the cytoplasm, undergo degradation by the proteasome, and present them through a primary presentation pathway; whole-cell vaccines primarily involve activating dendritic cells in vitro and then reinfusing them into the body, but their manufacturing process is far more complex than simple antigen vaccines, thus limiting their clinical application.

[0005] Immune adjuvants can stimulate dendritic cell maturation and improve antigen presentation efficiency. Providing antigens without effectively stimulating dendritic cell maturation can easily lead to immune tolerance; therefore, modern vaccine formulations all contain immune adjuvant components. The development of immune adjuvants has spanned approximately one hundred years. Because most vaccines utilize activated humoral immunity to produce antibodies for preventative effects, adjuvants have primarily been developed to target humoral immunity. Six adjuvants have been clinically approved, all for activating humoral immunity, with relatively poor activation effects on cellular immunity against tumors. Adjuvants used in research experiments include CpG and poly(I:C), which, while effectively activating cellular immunity in animals, have controversial effects in human trials, and their safety profile, including systemic inflammation and autoimmune reactions, warrants attention. Given the current lack of cellular immune adjuvants, continued efforts are needed to find safe and effective cellular immune adjuvants to support the development of tumor vaccines.

[0006] Modern vaccines utilize various vectors, including viruses, bacteria, cells, lipid nanoparticles, and polymers. Selecting the appropriate vector based on the antigen and immune response type plays a crucial role in enhancing the level of immune response and improving the immunogenicity of the antigen. Taking mRNA as an example, lipid nanoparticles (LNPs) are used as delivery vectors. Transporting the antigen via a vector can increase protection against degradation of the mRNA and convert the antigen into a particulate form, making it easier for dendritic cells to recognize and induce an immune response. Although LNP technology is widely used in mRNA vaccines, its limited stability, requiring storage at around -80°C, restricts its application. Furthermore, LNPs cannot encapsulate protein components, making it difficult to load multiple antigen types. While viral vectors can promote immune activation, their safety, production complexity, and reduced potency after repeated use limit their application. Viral vectors also struggle to load multiple antigen types. Polymers such as polylactic-co-glycolic acid copolymer (PLGA) are biosafe, in vivo sustained-release materials with advantages such as high drug loading capacity, the ability to simultaneously load hydrophobic and hydrophilic drugs, and simple preparation processes, making them a highly promising type of vaccine vector. As for the carrier, multiple parameters such as sustained release time, vaccine particle size, and preparation process need to be considered to select a reasonable PLGA type.

[0007] Most current tumor vaccines under research or clinical trials are therapeutic vaccines, used after tumor development as therapeutic drugs to enhance the body's anti-tumor immune response. While this approach may play a role in inhibiting tumor progression, it lacks the preventative properties that vaccines should inherently possess. The mechanisms involved in tumor progression and escape are highly complex, and the disruption of the immune system and the formation of a local suppressive immune microenvironment are among the reasons for immunotherapy failure. Taking immune checkpoint blockade therapy as an example, relevant guidelines are gradually advancing the timing of administration because in the later stages of tumor development, due to systemic immunosuppression, even if immune checkpoints are blocked, T cells are unlikely to exert their cytotoxic effects. The timing of tumor vaccine application must also be considered. If the vaccine is administered late after surgical removal of the tumor, not only will systemic immunosuppression affect its effectiveness, but the local suppressive immune microenvironment following tumor focal formation will also prevent T cell infiltration and cytotoxic effects. Therefore, tumor vaccines should be administered as early as possible, provided that appropriate antigens are available, to achieve a preventative effect. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide an ultrasound-assisted immune activation tumor vaccine with high presentation efficiency and high biosafety, which can be used to prevent recurrence and metastasis of various tumors after surgery.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides an ultrasound-assisted immune activation tumor vaccine, wherein the vaccine is obtained by loading tumor cell membrane proteins, total tumor cell RNA and chlorin e6 onto PLGA as a carrier; the total tumor cell RNA is polymerized and protected using PLL.

[0011] This invention also provides a method for preparing the above-mentioned tumor vaccine, comprising the following steps: extracting total RNA and cell membrane from tumor cells respectively; mixing the RNA solution with PLL solution thoroughly and then adding cell membrane suspension as the aqueous phase; mixing PLGA with chlorine 6 and then adding dichloromethane as the oil phase; after the aqueous phase undergoes a first homogenization treatment, adding the oil phase for a second homogenization treatment; pouring the homogenized system into a first PVA aqueous solution for a third homogenization treatment; adding the treated system dropwise to a second PVA aqueous solution under stirring, and continuing stirring; centrifuging the stirred suspension, and obtaining the precipitate as the tumor vaccine.

[0012] Preferably, tumor cells are treated with the small molecule compound PladienolideB before total RNA or cell membrane extraction from tumor cells.

[0013] Preferably, in the aqueous phase, the concentration of the PLL solution is 0.1–100 μg / μl, the concentration of the RNA solution is 0.1–10 μg / μl, and the mass ratio of RNA to PLL is 1:0.0162–6.12; in the cell membrane suspension, the concentration of protein is 0.1–10 μg / μl, and the mass ratio of RNA to cell membrane is 1:300–300:1.

[0014] Preferably, in the oil phase, the PLGA concentration is 50–200 mg / mL and the chlorin e6 concentration is 0.1–10 mg / mL.

[0015] Preferably, the volume ratio of the aqueous phase to the oil phase is 1:100 to 1:3.

[0016] Preferably, the homogenization is ultrasonic homogenization, the homogenization time is 10-100s, and the parameters are set to amplitude 1%-80%, 5s on / 5s off.

[0017] Preferably, the concentration of the first PVA aqueous solution is 15-25 mg / mL; and the concentration of the second PVA aqueous solution is 2-8 mg / mL.

[0018] The present invention also provides the application of the above-mentioned tumor vaccine or the above-mentioned preparation method in the preparation of drugs for preventing postoperative recurrence or metastasis of tumors.

[0019] The present invention also provides a lyophilized tumor vaccine powder with ultrasound-assisted immune activation. The precipitate obtained by the above-mentioned tumor vaccine or the above-mentioned preparation method is resuspended in trehalose aqueous solution and lyophilized to obtain the lyophilized tumor vaccine powder.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention uses total membrane proteins and total RNA from tumor cells as antigens. Compared to current mainstream tumor vaccine technologies that load only a few predetermined proteins or nucleic acids as antigens, this invention broadens the antigen spectrum of the vaccine and induces the immune system to recognize unknown antigens. Compared to single-peptide or nucleic acid vaccines, it can comprehensively utilize both cross-presentation of antigens from dendritic cells and classic MHC class 1 presentation methods, improving the efficiency of MHC class 1 antigen presentation. The proteins and RNA used are derived from the tumor cells themselves, making them autogenous antigens for the individual receiving the vaccine, and neither poses a risk of genomic integration, ensuring safe use.

[0022] This invention uses the sonosensitive agent chlorin e6 as an immunoadjuvant. Compared with commonly used adjuvants such as CpG and poly(I:C), chlorin e6 generates ROS under ultrasound, promoting the escape of tumor vaccines from endosomes / lysosomes. By releasing antigens into the cytoplasm, it promotes the presentation of MHC class I antigens. Furthermore, moderate intracellular oxidative stress can induce the production of heat shock proteins, thereby increasing the efficiency of intracellular protein degradation in the proteasome, generating more antigenic epitopes, and increasing the protein degradation rate. Simultaneously, oxidative stress can also stimulate dendritic cells to mature, ultimately activating antigen-specific T-cell immune responses.

[0023] This invention utilizes biodegradable polylactic-co-glycolic acid copolymer (PLGA) to load proteins, RNA, and chlorin e6 to prepare a tumor vaccine. It exhibits high biosafety, and its nanoparticle size facilitates phagocytosis by cells and entry into lymph nodes. The tumor vaccine described in this invention allows for pre-immunization before tumor recurrence and metastasis, leveraging a relatively intact immune system to maximize its early effectiveness.

[0024] The tumor vaccine prepared according to this invention is relatively simple and can be used for various types of tumors. Future clinical translation could involve rapidly preparing the vaccine from surgically removed tumor tissue after processing, for the prevention of postoperative tumor recurrence and metastasis, showing broad application prospects. Attached Figure Description

[0025] Figure 1 Extraction and protein identification of tumor cell membranes;

[0026] Figure 2 Schematic diagram of tumor vaccine preparation;

[0027] Figure 3 Agarose gel electrophoresis results with different N / P ratios;

[0028] Figure 4 Transmission electron microscopy (left) and scanning electron microscopy (right) images of tumor vaccines;

[0029] Figure 5 Results of particle size distribution (left) and zeta potential analysis (right) of tumor vaccines;

[0030] Figure 6 UV-Vis absorbance curves of tumor vaccine lysates loaded with different components;

[0031] Figure 7 Co-localization of tumor vaccines with DC2.4 cells;

[0032] Figure 8 Confocal fluorescence images showing the distribution of tumor vaccines within lymph nodes;

[0033] Figure 9In vivo fluorescence imaging detection of tumor vaccine distribution in lymph nodes;

[0034] Figure 10 CCK-8 assay was used to detect cell viability after applying different treatment conditions.

[0035] Figure 11 Tumor vaccines co-localize with lysosomes and ultrasound irradiation promote endosome escape;

[0036] Figure 12 Confocal image showing how ultrasound irradiation of a tumor vaccine promotes intracellular ROS production in DC2.4 cells;

[0037] Figure 13 Western blot analysis of HSP70 protein expression in DC2.4 cells;

[0038] Figure 14 Flow cytometry was used to detect the presentation effect of the model antigen SIINFEKL under ultrasound-assisted conditions.

[0039] Figure 15 : Growth curve of tumors in mice after pre-immunization with tumor vaccines;

[0040] Figure 16 qPCR was used to detect alternative splicing changes in representative genes, and RNA-seq was used to detect the ratio of introns to exons in the total transcriptome.

[0041] Figure 17 CCK-8 assay to detect the effect of Pladienolide B treatment on 4T1 cell viability;

[0042] Figure 18 Inhibitory effect of tumor vaccines loaded with alternative splicing interference neoantigens on mouse 4T1 breast cancer model. Detailed Implementation

[0043] This invention provides an ultrasound-assisted immune activation tumor vaccine, wherein the tumor vaccine is obtained by co-loading tumor cell membrane proteins, total tumor cell RNA and the sonosensitive agent chlorin e6 with polylactic-co-glycolic acid copolymer (PLGA) as a carrier, and the total tumor cell RNA is polymerized and protected with poly-L-Lysine (PLL).

[0044] This invention employs a double emulsion method to prepare a tumor vaccine, the preparation method comprising the following steps:

[0045] Total RNA and cell membranes of tumor cells were extracted separately. The RNA solution was thoroughly mixed with PLL solution and then added to the cell membrane suspension as the aqueous phase. PLGA and chlorin e6 were mixed and then dichloromethane was added as the oil phase. The aqueous phase was homogenized for the first time and then added to the oil phase for the second homogenization. The homogenized system was poured into the first PVA aqueous solution for the third homogenization. The treated system was then added dropwise to the second PVA aqueous solution while stirring. The mixture was then centrifuged, and the precipitate was the tumor vaccine.

[0046] In this invention, the type of tumor cell selected for total RNA and cell membrane extraction corresponds to the type of tumor vaccine being prepared. Prior to the extraction of total RNA and cell membrane from the tumor cells, the tumor cells are treated with the small molecule compound Pladienolide B.

[0047] This invention uses DMSO to prepare a Pladienolide B solution, which is then added to a culture medium to culture tumor cells. The preferred volume ratio of Pladienolide B solution to culture medium is 1:5000. Before total RNA extraction from the tumor cells, tumor cells are cultured in a 1-1000 nM Pladienolide B solution for 0.5-24 h, with the preferred concentration being 100-200 nM and the culture time being 3-4 h. Before tumor cell membrane extraction, tumor cells are cultured in a 1-1000 nM Pladienolide B solution for 1-48 h, with the preferred concentration being 2.5-3 nM and the culture time being 22-24 h.

[0048] Alternative splicing dysregulation occurs during tumor recurrence and metastasis, producing proteins with splicing abnormalities, which are one source of tumor neoantigens. This invention uses the small molecule compound Pladienolide B, which inhibits spliceosome function, to treat tumor cells, inducing tumor splicing dysregulation in vitro. This induces the production of proteins and RNA with abnormal splicing, which can then be used as antigens to construct tumor vaccines. This allows the immune system to recognize mutated antigens before they appear in the tumor. Once mutated antigens are generated during tumor recurrence or metastasis, immune cells can recognize and kill the tumor cells immediately. This invention also considers unknown antigens that may arise during future tumor progression, aiming for better efficacy in tumor treatment and prevention.

[0049] In this invention, the total RNA solution of extracted tumor cells is thoroughly mixed with PLL solution, and then cell membrane suspension is added to obtain an aqueous phase system, which is placed on ice for later use.

[0050] In this invention, the total RNA extracted from tumor cells is polymerized and protected using PLL (the amount of RNA mixed with PLL is calculated based on the ratio of the amount of ammonium groups to phosphate groups carried by the PLL, i.e., the N / P ratio is 0.1 to 10).

[0051] In the aqueous phase system of this invention: the PLL solution is preferably prepared using ultrapure water, and the concentration of the solution is 0.1–100 μg / μl, preferably 10–20 μg / μl; the RNA solution is preferably prepared by dissolving in ultrapure water, and the concentration of the RNA solution is 0.1–10 μg / μl; the mass ratio of RNA to PLL is 1:0.0162–6.12, preferably 1:1.53; the protein concentration in the cell membrane suspension is 0.1–10 μg / μl; the mass ratio of RNA to cell membrane is 1:300–300:1, preferably 1:1.

[0052] In this invention, PLGA and chlorin e6 are mixed, and then dichloromethane (analytical grade) is added to prepare a dichloromethane solution containing PLGA and chlorin e6, which serves as the oil phase.

[0053] In the oil phase system of this invention, the final concentration of PLGA is 50-200 mg / mL, preferably 100 mg / mL; the final concentration of chlorin e6 is 0.1-10 mg / mL, preferably 4 mg / mL.

[0054] In this invention, the aqueous phase system is placed on ice and homogenized for the first time before being added to the oil phase system. The volume ratio of the aqueous phase to the oil phase is 1:100 to 1:3, preferably 3:10. The resulting mixture is then homogenized a second time, and the homogenized system is poured into a first PVA aqueous solution for a third homogenization. The volume of the first PVA aqueous solution is 2 to 3 times that of the mixture (total volume of aqueous and oil phases), preferably 2.5 times. The mixture is then added dropwise to a second PVA aqueous solution under stirring, and stirring is continued for 4 hours. The stirring is preferably carried out in the dark, at room temperature, using magnetic stirring, and the stirring speed is preferably 800 rpm. The resulting suspension is centrifuged to obtain a precipitate, which is the tumor vaccine.

[0055] In this invention, the homogenization is ultrasonic homogenization, performed on ice; the homogenization time is 10–100 s, and the parameters are set to amplitude 1%–80%, preferably 40%, with a 5 s on / 5 s off cycle. The first homogenization treatment is preferably 20–25 s; the second homogenization treatment is preferably 30–90 s; and the third homogenization treatment is preferably 30–90 s.

[0056] In this invention, the polyvinyl alcohol (PVA) aqueous solution is prepared by dissolving in ultrapure water and then subjecting it to ultrasonic treatment in a water bath. The concentration of the first PVA aqueous solution is 15–25 mg / mL, preferably 20 mg / mL; the concentration of the second PVA aqueous solution is 2–8 mg / mL, preferably 5 mg / mL.

[0057] In this invention, the centrifugation conditions are preferably 4°C, centrifuged at 12000g for 15 min, and the precipitate obtained by centrifugation is preferably resuspended and washed 2-3 times with ultrapure water.

[0058] This invention also provides the application of the above-mentioned tumor vaccine or the above-mentioned preparation method in the preparation of drugs for preventing postoperative recurrence or metastasis of tumors, which can pre-immunize before tumor recurrence or metastasis, and is beneficial to utilize the relatively intact immune system to exert the effect of tumor vaccine in the early stage.

[0059] This invention also provides a lyophilized tumor vaccine powder with ultrasound-assisted immune activation, obtained by resuspending the aforementioned tumor vaccine (or the prepared centrifuged precipitate) in a trehalose aqueous solution and then lyophilizing it. As an optional embodiment, this invention uses ultrapure water to prepare a 4% w / v trehalose aqueous solution, transfers it to a centrifuge tube, and freezes it overnight at -80°C. Then, the tumor vaccine pre-frozen at -80°C is transferred to a vacuum freeze dryer for lyophilization to obtain the lyophilized tumor vaccine powder.

[0060] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0061] Unless otherwise specified, the following embodiments are all conventional methods.

[0062] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0063] Example 1

[0064] Methods for preparing tumor vaccines loaded with tumor cell membrane proteins, total RNA, and chlorin E6:

[0065] 1. Total RNA and cell membrane extraction from 4T1 mouse breast cancer cells:

[0066] Total RNA extraction: using Tripe TM Total RNA was extracted using the RNA extraction buffer according to the instructions, dissolved in ultrapure water, quantified by UV absorbance, and the concentration was adjusted to 6 μg / μl.

[0067] Cell membrane extraction:

[0068] a. Prepare cell hypotonic buffer: 20 mM Tris-HCl, pH 7.5; 10 mM KCl; 2 mM MgCl2; add 1 tablet of cOpletemini EDTA-free protease inhibitor (Roche, 4693159001) per 10 mL of hypotonic buffer. Store at 4°C.

[0069] b. Digest with trypsin and collect 1×10 8 4T1 cells were centrifuged three times at room temperature using 200g of 1×PBS buffer, and the residual PBS buffer was discarded. 20mL (about 10 times the volume) of cell hypotonic buffer from step a was added, and the cells were placed on ice for 30min.

[0070] c. Assemble the Dounce homogenizer (Kimble, 3110), add 2 mL of cell hypotonic buffer, and rinse the homogenizer using a type B homogenizer. Add 2 mL of the cell suspension to the homogenizer each time, homogenize 40 times on ice, and then transfer the cell suspension to a new centrifuge tube.

[0071] d. Centrifuge the ground cell suspension at 4℃ using differential speed. Centrifuge at 700g for 10 min, discard the precipitate, and collect the supernatant into a new centrifuge tube; centrifuge the supernatant at 7000g for 30 min, discard the precipitate, and collect the supernatant; centrifuge the supernatant at 100000g for 2 h, discard the supernatant, and resuspend the precipitate in ultrapure water. This precipitate contains protein-containing cell membrane components. Quantify the protein using the BCA method and adjust the concentration to 6 μg / μl. Store at -80℃. The results of cell membrane extraction and protein identification are shown below. Figure 1 . Figure 1 In the table, A represents the cell membrane extraction process; B to C represent the protein identification results of the cell membrane, where Celllysate is the cell lysis buffer group and Cellmembrane is the cell membrane lysis buffer group.

[0072] 2. Vaccine preparation (see details of the preparation process) Figure 2 )

[0073] (1) A tumor vaccine was prepared using PLGA as a carrier and co-loaded with cell membrane proteins, total RNA, and chlorin e6 using the double emulsion method. To avoid RNA damage during preparation, poly-L-Lysine (PLL) was used to polymerize and protect the RNA beforehand.

[0074] (2) The N / P ratio was calculated based on the ratio of PLL residues to nucleotide phosphate groups. Different ratio gradients were set, and the optimal ratio was analyzed using agarose gel electrophoresis. The final N / P ratio was selected as 2.5, which was converted to reagent concentration for tumor vaccine preparation (see the results of agarose gel electrophoresis for different N / P ratios). Figure 3 ).

[0075] The specific method is as follows: a. Prepare PLL (Sigma-Aldrich, P7890) stock solution with ultrapure water at a concentration of 10 μg / μl; b. Add 100 μl of 6 μg / μl RNA to a centrifuge tube, place it on ice, then add 92 μl of PLL stock solution, gently shake to mix the RNA and PLL thoroughly, and place on ice for 30 min.

[0076] (3) Add 100 μl of cell membrane suspension with a protein quantification of 6 μg / μl to the PLL-protected RNA suspension, and continue to add 8 μl of ultrapure water to make the total volume of the system 300 μl. This part of the system is used as the aqueous phase and placed on ice for the next step.

[0077] (4) Prepare a PLGA dichloromethane solution containing chlorin e6 in the dark: Accurately weigh 100 mg of PLGA (RESOMER@RG502H, Mw = 7000-17000, Sigma-Aldrich 719897) into an ampoule, weigh 4 mg of chlorin e6 (Cayman, No. 21684), and mix with PLGA. Add dichloromethane (analytical grade) to the PLGA and chlorin e6 mixture and dilute to 1 mL to prepare a dichloromethane solution containing 100 mg / mL PLGA and 4 mg / mL chlorin e6, which will serve as the oil phase.

[0078] (5) Preparation of polyvinyl alcohol (PVA) aqueous solution: Weigh 1g of PVA (Mw = 9000-10000, Sigma-Aldrich 360627) into a centrifuge tube, add ultrapure water to make up to 50mL, and use a water bath to sonicate to dissolve the PVA, so as to obtain a PVA aqueous solution with a concentration of 20mg / mL. This will be used as the external aqueous phase.

[0079] (6) Preparation of tumor vaccine by double breast method: a. Place the system obtained in step (3) on ice and homogenize it for 20s using a probe-type ultrasonic homogenizer (ColeParmer, Ultrasonic Homogenizer 4710 series) with parameters set to amplitude 40%, 5s on / 5s off.

[0080] (7) Add 1 mL of the oil phase system prepared in step (4) to the system in step (6) in the dark, and sonicate on ice for 60 s with parameters of amplitude 40% and 5 s on / 5 s off.

[0081] (8) In the dark, pour the homogenized system from step (7) into 3.25 mL of 20 mg / mL PVA aqueous solution (the PVA aqueous solution is 2.5 times the total volume of the inner aqueous phase and oil phase), and sonicate on ice for 60 s with parameters of amplitude 40% and 5 s on / 5 s off.

[0082] (9) Prepare 40 mL of 5 mg / mL PVA aqueous solution using 20 mg / mL PVA aqueous solution, and stir with a magnetic stirrer at 800 rpm at room temperature. Add the system from step (8) dropwise to the stirring PVA aqueous solution, and stir in the dark for 4 h.

[0083] (10) Transfer the stirred suspension to a centrifuge tube, centrifuge at 12000g for 15 minutes at 4°C, discard the supernatant, add 50mL of ultrapure water to resuspend and wash the precipitate, repeat the above centrifugation, wash 3 times, discard the supernatant, and the precipitate is the tumor vaccine.

[0084] (11) Prepare a 4% w / v trehalose (Beyotime, ST1245) aqueous solution using ultrapure water. Resuspend the (10) step precipitate in 10 mL of trehalose aqueous solution, transfer it to a 5 mL centrifuge tube, and freeze it overnight at -80°C.

[0085] (12) The tumor vaccine pre-frozen at -80°C in step (11) was transferred to a vacuum freeze dryer (SCIENTZ, N series) and freeze-dried for 48 hours to obtain the freeze-dried tumor vaccine powder for subsequent validation.

[0086] Example 2

[0087] Characterization and identification of tumor vaccines (lyophilized powder form)

[0088] 1. Transmission electron microscopy morphological identification: (1) Weigh 2 mg of lyophilized tumor vaccine powder, add 1 mL of ultrapure water to dissolve it again, centrifuge at 12000 g at 4℃, and wash 3 times; (2) Add the tumor vaccine suspension to a copper grid, let it stand for half an hour, add an appropriate amount of 2% phosphotungstic acid staining solution for negative staining for 15 min. Dry overnight at 4℃; (3) Observe the morphology and size of the tumor vaccine using a transmission electron microscope. It can be seen that the tumor vaccine is a spherical particle with a diameter of 150-200 nm. See the results below. Figure 4 .

[0089] 2. Scanning electron microscopy morphology and surface identification: (1) The tumor vaccine, after being washed with ultrapure water, was dropped onto the surface of tin foil and dried overnight; (2) the surface was treated with gold sputtering; (3) the surface morphology of the tumor vaccine was observed using a scanning electron microscope. It was found that the tumor vaccine consisted of smooth spherical particles with a diameter consistent with that observed under a transmission electron microscope. The results are shown in […]. Figure 4 .

[0090] 3. Particle size and Zeta potential analysis: An appropriate volume of the tumor vaccine was weighed, the precipitate was resuspended in 1 mL of PBS, centrifuged at 12000 g, washed three times, and resuspended in an appropriate volume of PBS. The particle size distribution was analyzed using NanoPlus, and the results showed that the particle size was mainly concentrated around 150–200 nm. An appropriate volume of the tumor vaccine was weighed, the precipitate was resuspended in 1 mL of PBS, centrifuged at 12000 g, washed three times, and resuspended in an appropriate volume of PBS. The Zeta potential was analyzed using NanoPlus, and the results showed a Zeta potential of -26.32 mV, indicating good dispersibility. The results of the tumor vaccine particle size distribution and Zeta potential analysis are shown below. Figure 5 .

[0091] 4. Validation of Chlorin e6 loading and calculation of encapsulation efficiency: (1) Weigh 1 mg of tumor vaccine, dissolve it in 30 μl DMSO for 30 minutes, add 170 μl of ultrapure water and mix well, centrifuge at 12000g for 5 minutes; (2) Prepare a mixed solution of 30 μl DMSO and 170 μl ultrapure water as a calibration solution; (3) Analyze the absorbance of the solution at 404 nm using a UV-Vis analyzer, and calculate the encapsulation efficiency of chlorin e6 in the CM-RNA@Ce6 / PLGA group, which is 15.2%.

[0092] 5. Prepare nanoparticles loaded with different components (method as in step 2), and measure the UV-Vis absorbance curves of each nanoparticle, see [see figure]. Figure 6 ( Figure 6 Blank@PLGA represents nanoparticles containing only PLGA components; CM@Ce6 / PLGA represents PLGA nanoparticles loaded with cell membrane and chlorin e6; RNA@Ce6 / PLGA represents PLGA nanoparticles loaded with RNA and chlorin e6; CM-RNA@Ce6 / PLGA represents PLGA nanoparticles loaded with cell membrane, RNA, and chlorin e6 (as described in this invention's tumor vaccine). Figure 6 The UV-Vis absorbance curves from bottom to top are Blank@PLGA, CM@Ce6 / PLGA, RNA@Ce6 / PLGA, and CM-RNA@Ce6 / PLGA. The PLGA nanoparticles loaded with cell membrane, RNA, and chlorin e6 have the strongest absorbance.

[0093] 6. Protein quantification: (1) Preparation of hydrolysate: Prepared with ultrapure water, containing 0.05% NaOH and 0.1% Tween 20 (range: NaOH 0.001-1%, Tween 20 0.001%-1%); (2) Weigh 1 mg of tumor vaccine, add 100 μl of hydrolysate and mix thoroughly, 90℃ for 1 h, shake at 37℃ for 18 h; (3) Centrifuge at 12000g for 15 min, take the supernatant and add an equal volume of 0.05M HCl to adjust the pH, use the BCA method to quantify the protein, and calculate the protein encapsulation rate as 68.3%;

[0094] 7. RNA detection: (1) Weigh 1 mg of tumor vaccine, add 30 μl of DMSO and let stand at room temperature for 30 min to dissolve the tumor vaccine; (2) Add Tripe TM (3) Mix 500 μl of RNA extract with vaccine lysate; (4) Add 100 μl of chloroform and mix well, centrifuge at 12000 g for 15 min at 4 °C; (5) Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, mix well, add 1 μl of glycogen, and let stand at -20 °C for 2 hours; (6) Centrifuge at 12000 g for 15 min at 4 °C to extract the RNA precipitate, dissolve in 20 μl of DEPC water, and perform UV quantitative analysis of the RNA amount. The encapsulation rate is calculated to be 31.5%.

[0095] Example 3

[0096] Validation of tumor vaccine delivery efficacy

[0097] 1. The ability of cells to endocytose tumor vaccines: (1) The tumor vaccine was co-cultured with DC2.4 cells for 12 h; (2) Hoechst33342 and phalloidin-FITC were used to label the cell nucleus and cytoskeleton, respectively. The colocalization of the tumor vaccine and cells was detected by confocal microscopy to assess the entry of the tumor vaccine into the cells. Figure 7 The co-localization of the tumor vaccine with DC2.4 cells indicates that the tumor vaccine can be effectively internalized by the cells.

[0098] 2. In vivo delivery of tumor vaccine: 2 mg of tumor vaccine was weighed and a suspension was prepared using 100 μl PBS. The vaccine was subcutaneously injected into mice. Twelve hours later, lymph nodes near the injection site were collected and frozen sections were prepared. Cell nuclei were labeled using Hoechst 33342, and the distribution of the tumor vaccine within the lymph nodes was detected using confocal microscopy. During the tumor vaccine preparation process, the fluorescent dye DiR was added to the oil phase to prepare DiR-labeled tumor vaccine. 2 mg of DiR-labeled tumor vaccine was weighed and a suspension was prepared using 100 μl PBS. The vaccine was subcutaneously injected. Twelve hours later, lymph nodes and fat cells near the injection site were collected, and the fluorescence expression in the lymph nodes and fat cells was detected using an in vivo fluorescence imaging system. Figure 8 Confocal fluorescence of the distribution of tumor vaccines within lymph nodes; Figure 9This study used in vivo fluorescence imaging to detect the distribution of tumor vaccines in lymph nodes. The results demonstrated that subcutaneously injected tumor vaccines can rapidly accumulate in lymph nodes.

[0099] Example 4

[0100] Validation of the effect of ultrasound irradiation on promoting antigen presentation

[0101] 1. Determination of ultrasound and Ce6 parameters: (1) DC2.4 cells were cultured in 96-well plates to a density of 60%, and the cells were treated with ultrasound of different intensities. Cell viability was detected by CCK-8 assay; (2) Determination of 1.0 W / cm 2 The experiment was conducted using high-intensity ultrasound. Cells were co-incubated with different concentrations of Ce6. Cell viability was detected by CCK-8 assay after ultrasound treatment. The optimal Ce6 concentration was determined to be 2 μg / ml. (3) Cells were treated with Ce6 at a concentration of 2 μg / ml. Different ultrasound parameters were applied, and cell viability was detected by CCK-8 assay again. Figure 10 To detect cell viability under different treatment conditions using CCK-8 assays, a represents the viability of DC2.4 cells at different ultrasound intensities; b represents the viability of DC2.4 cells at a Ce6 concentration of 2 μg / ml at different ultrasound intensities; and c represents the viability of DC2.4 cells at an ultrasound intensity of 1.0 W / cm². 2 The activity of DC2.4 cells at different Ce6 concentrations. Figure 10 The results showed that sonication alone did not significantly affect cell viability, but cell viability decreased significantly with increasing Ce6 concentration. Using appropriate Ce6 concentrations and sonication parameters can effectively reduce cytotoxicity.

[0102] 2. Detection of intracellular localization of tumor vaccine and ultrasound-induced endosome escape: DC2.4 cells were cultured in confocal culture dishes, and the tumor vaccine was co-cultured with the cells for 6 hours. After 6 hours, the culture medium was replaced with fresh culture medium without the tumor vaccine, and the cells were cultured for another 6 hours. Lysosomes were labeled with Lysotracker-FITC lysosomal fluorescent probes at different time points, and the results were observed using a confocal microscope. The ultrasound-treated group was treated with 1 W / cm² ultrasound after 6 hours of culture. 2 The tumor vaccine was irradiated with ultrasound of high intensity for 1 minute, and its intracellular distribution was observed using confocal microscopy 24 hours later. Figure 11 This is a confocal microscopy image showing the co-localization of tumor vaccines with lysosomes. The results indicate that the co-localization of tumor vaccines with lysosomes gradually increases with time, suggesting that the tumor vaccine gradually enters the lysosomes after entering the cell. In the ultrasound irradiation group, scattered tumor vaccine fluorescence appeared in the cytoplasm, indicating that ultrasound treatment promoted the escape of the tumor vaccine from the endosomes.

[0103] 3. Detection of the ability of tumor vaccine to promote ROS production in cells under ultrasound: (1) Co-culture 0.5 mg / mL of tumor vaccine with DC2.4 dendritic cells for 12 h, change the medium and then apply ultrasound treatment with parameters of 1 W / cm 2 (1) Time 1 min; (2) Use DCFH-DA to detect intracellular ROS and confocal microscopy to detect changes in intracellular ROS levels. Figure 12 This is a confocal image of DC2.4 cells. The results indicate that the tumor vaccine generated reactive oxygen species in the cells after ultrasound treatment.

[0104] 4. HSP70 expression detection in cells: DC2.4 cells were cultured in six-well plates and co-cultured with a tumor vaccine at 0.5 mg / mL for 12 h. After medium replacement, a 1.0 W / cm² dose was applied. 2 Cells were subjected to high-intensity sonication for 1 min and 1 h later, and the expression of HSP70 protein in the cells was detected by Western blot. Figure 13 Western blot analysis of HSP70 protein expression in DC2.4 cells showed that ultrasound-assisted tumor vaccines increased the expression of heat shock proteins in cells. Nanoparticles loaded with different antigenic components also promoted HSP70 protein production in cells, while empty PLGA vectors without any loaded components failed to increase HSP70 expression.

[0105] 5. Validation of antigen presentation effect: (1) In order to study the effect of ultrasound irradiation on antigen presentation, ovalbumin (OVA) was used as the model protein. After processing in the cell, OVA protein can present the SIINFEKL epitope to the cell surface to detect the antigen presentation efficiency. First, a tumor vaccine containing OVA antigen was constructed: OVA expression vector was transfected into HEK293T cells to make the cells express OVA protein, and cell membrane and total RNA were collected as antigen components; (2) A tumor vaccine containing OVA antigen was constructed using the method described in step 2; (3) 0.5 mg / mL of this vaccine was co-cultured with primary bone marrow dendritic cells of C57 mice for 6 h, and the ultrasound treatment group used 1 W / cm 2 (3) The cells were subjected to intense sonication for 1 min and cultured for another 48 h. (4) Flow cytometry was used to detect the proportion of dendritic cells presenting the SIINFEKL epitope. The results are shown in the figure. Figure 14 The results showed that after co-culturing dendritic cells with the OVA antigen tumor vaccine, the cells could present the SIINFEKL epitope. Increased ultrasound irradiation significantly increased the proportion of cells presenting the epitope, indicating that ultrasound irradiation can improve antigen presentation efficiency.

[0106] 6. Verification of the tumor growth inhibition effect of tumor vaccine: Female BALB / c mice aged 6-8 weeks were selected as experimental animals and immunized subcutaneously with 100 μl of lyophilized tumor vaccine resuspended in PBS (containing 2 mg of lyophilized powder). Twelve hours later, 1 W / cm² of the vaccine was administered. 2 Ultrasound was applied to the adjacent lymph nodes for 1 minute. Immunization was performed every 5 days, followed by ultrasound irradiation after each immunization. A total of 3 immunizations were administered. After immunization, 4T1 tumor-bearing cells (5 × 10⁶ cells) were implanted in situ into the mouse mammary pads. 5 (cells). Continuous measurement of tumor volume changes over time, see […]. Figure 15 The results showed that tumor vaccines co-loaded with membrane proteins and RNA had the best tumor growth inhibition effect compared with nanoparticles loaded with a single membrane protein or RNA.

[0107] Example 5

[0108] Validation of the efficacy of tumor vaccines loaded with alternative splicing interference neoantigens

[0109] 1. Effects of PladienolideB on alternative splicing: (1) 4T1 cells were treated with different concentrations of PladienolideB small molecule compound for 4 h, and RNA was extracted from the cells; (2) The expression of introns in the Dnajb1 and Cdkn1b genes was detected by qPCR; (3) 4T1 cells were treated with 100 nM PladienolideB for 4 h, and the ratio of introns to exons in total mRNA was detected by second-generation transcriptome sequencing. Figure 16 qPCR was used to detect intron expression levels, and RNA-seq was used to detect the ratio of introns to exons in the total transcriptome. The results showed that the expression levels of representative gene introns increased with increasing pladienolide B concentration, indicating that pladienolide B can affect alternative splicing, and the effect increases with increasing concentration. Cell sequencing results further confirmed the alteration of the intron-to-exon ratio in the overall transcriptome caused by pladienolide B treatment.

[0110] 2. Using a mouse 4T1 breast cancer model, investigate the inhibitory effect of tumor vaccines loaded with neoantigens on tumor growth:

[0111] A tumor vaccine containing a novel antigen of alternative splicing interference was prepared. Since high doses of Pladienolide B are cytotoxic, the appropriate concentration of Pladienolide B needed to be determined first. The effect of different Pladienolide B treatments for 24 h on the viability of 4T1 cells was detected using the CCK-8 assay. Figure 17The results showed that when the concentration of Pladienolide B exceeded 2.5 nM, the survival rate of most cells was low. Therefore, to obtain sufficient cell membrane proteins, 4T1 cells were treated with a concentration of 2.5 nM for 24 h, and the cell membranes were extracted. To obtain a greater amount of alternative splicing interference RNA, 4T1 cells were treated with a concentration of 100 nM Pladienolide B for 4 h, and the cells were collected to extract RNA. The extracted cell membranes and RNA were used as antigens.

[0112] Treatment of 4T1 mouse breast cancer cells with the small molecule compound PladienolideB followed by RNA and cell membrane extraction:

[0113] (1) Prepare stock solutions of Pladienolide B (TOCRIS, Cat. No. 6070) at different concentrations using DMSO.

[0114] (2) Using 100 nM Pladienolide B stock solution, the stock solution was mixed with 1640 medium at a volume ratio of 1:5000, and 4T1 cells were cultured for 4 h. The expression levels of introns of the Dnajb1 and Cdkn1b genes were detected by qPCR using Triplet. TM Total RNA was extracted using the RNA extraction buffer according to the instructions, dissolved in ultrapure water, quantified by UV absorbance, and the concentration was adjusted to 6 μg / μl.

[0115] (3) Use 2.5 nM Pladienolide B stock solution to mix with 1640 medium at a volume ratio of 1:5000, culture 4T1 cells for 24 h, use CCK8 kit to detect cell viability, collect cells, extract cell membranes, and the cell membrane extraction method is the same as step 1 in Example 1.

[0116] The tumor vaccine prepared using the method described in step 2 of Example 1 was named "PlaB-Vaccine". Meanwhile, the tumor vaccine prepared from 4T1 without PladienolideB treatment in Example 1 was named "4T1-Vaccine".

[0117] Female BALB / c mice aged 6–8 weeks were subcutaneously injected with 100 μl of lyophilized tumor vaccine resuspended in PBS (containing 2 mg of lyophilized powder). Twelve hours later, 1.0 W / cm² of the vaccine was administered to the lymph nodes adjacent to the injection site. 2 Intensity sonication for 1 minute. Three vaccinations were administered, each 5 days apart. After the immunization process, 4T1 cells (5×10⁻⁶) were used. 5 (Number of tumors were implanted in situ in the breast pads of mice, and tumor growth was monitored. Flow cytometry was used to detect the number of infiltrating T cells in the tumor tissue, and lung tissue was stained to observe the number of metastatic lesions. Results are shown below.) Figure 18A shows the growth status of 4T1 cells bearing tumors, B shows the number of metastatic lesions stained in lung tissue, and C shows the number of infiltrating T cells in tumor tissue. The results indicate that PlaB-Vaccine, compared to the tumor vaccine 4T1-Vaccine loaded with ordinary 4T1 tumor cell antigen, has a better tumor-suppressive effect, improves the tumor microenvironment, and effectively reduces distant metastasis of tumors in the lungs.

[0118] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A tumor vaccine activated by ultrasound-assisted immunity, characterized in that, The vaccine is prepared by loading tumor cell membrane proteins, total tumor cell RNA and chlorin e6 onto a PLGA vector; the total tumor cell RNA is polymerized and protected using PLL. The method for preparing the tumor vaccine includes the following steps: Total RNA and cell membranes of tumor cells were extracted separately. The RNA solution was thoroughly mixed with PLL solution and then added to the cell membrane suspension as the aqueous phase. PLGA and chlorin e6 were mixed in the dark and then added to dichloromethane as the oil phase. After the aqueous phase was homogenized for the first time, the oil phase was added in the dark for a second homogenization. The homogenized system was then poured into the first PVA aqueous solution for a third homogenization. The homogenized system was then added dropwise to the second PVA aqueous solution while stirring in the dark. The mixture was then centrifuged, and the precipitate was the tumor vaccine.

2. The tumor vaccine according to claim 1, characterized in that, Before total RNA or cell membrane extraction from tumor cells, the tumor cells were treated with the small molecule compound Pladienolide B.

3. The tumor vaccine according to claim 1, characterized in that, In the aqueous phase, the concentration of PLL solution is 0.1–100 μg / μl, the concentration of RNA solution is 0.1–10 μg / μl, and the mass ratio of RNA to PLL is 1:0.0162–6.12; in the cell membrane suspension, the concentration of protein is 0.1–10 μg / μl, and the mass ratio of RNA to cell membrane is 1:300–300:

1.

4. The tumor vaccine according to claim 1, characterized in that, In the oil phase, the concentration of PLGA is 50–200 mg / mL, and the concentration of chlorin e6 is 0.1–10 mg / mL.

5. The tumor vaccine according to claim 1, characterized in that, The volume ratio of the aqueous phase to the oil phase is 1:100 to 1:

3.

6. The tumor vaccine according to claim 1, characterized in that, The homogenization is ultrasonic homogenization, with a homogenization time of 10–100 s and parameters set to amplitude 1%–80%, 5 s on / 5 s off.

7. The tumor vaccine according to claim 1, characterized in that, The concentration of the first PVA aqueous solution is 15–25 mg / mL; the concentration of the second PVA aqueous solution is 2–8 mg / mL.

8. The use of the tumor vaccine according to claim 1 in the preparation of a drug combined with ultrasound for preventing postoperative recurrence or metastasis of breast tumors.

9. A lyophilized powder of a tumor vaccine activated by ultrasound-assisted immunity, characterized in that, The tumor vaccine according to any one of claims 1 to 7 is obtained by resuspending in trehalose aqueous solution and freeze-drying.

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