Plasmid DNA nano-selenium tumor duplex vaccine as well as preparation method and application thereof
By combining plasmid DNA-modified nanoselenium particles with tumor cells and CAF-specific antigens, a plasmid DNA nanoselenium tumor dual vaccine was developed, solving the problem of single targets and poor immunogenicity of existing tumor vaccines, and achieving efficient immune regulation and inhibition of profibroplastic tumors.
Patent Information
- Application Number
- CN202411899781.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-23
- Publication Date
- 2025-03-28
AI Technical Summary
The existing tumor vaccine has a single target, poor immunogenicity, and it is difficult to effectively remove profibroplastic tumor cells and CAF in the early stage of the tumor, resulting in limited efficacy.
The plasmid DNA nanoselenium tumor dual vaccine was developed, and nanoselenium particles modified by plasmid DNA were used as the core and plasmid DNA was used as the shell to bind tumor cell-specific antigens and CAF-specific markers to form a dual antigen system to regulate immune response and inhibit profibroplastic tumors.
This dual vaccine significantly activates macrophages in both in vitro and in vivo experiments, enhances humoral and cellular immune responses, improves the immune memory response to profibroplastic tumors, and significantly inhibits tumor growth.
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Abstract
Description
Technical Field
[0001] This patent relates to the field of medical technology, and in particular to a nano-selenium tumor dual vaccine and a preparation method, as well as its application in inhibiting fibroproliferative tumors. Background Art
[0002] With the continuous advancement of science and technology, the morbidity and mortality of malignant tumors have been alleviated to a certain extent, but they are still major diseases that seriously endanger human health.
[0003] Surgical treatment can effectively remove most tumors, but it cannot remove all tumor cells 100% and may cause some tumor cells to fall off and metastasize. Surgical trauma can cause reactive oxygen free radicals and stress-related hormones. The immune dysfunction and immunosuppressive microenvironment caused by surgical trauma greatly limit the occurrence of the body's anti-tumor immune response and its effective removal of residual or detached tumor cells.
[0004] Chemotherapy and radiotherapy cannot selectively kill tumor cells and have significant toxic side effects.
[0005] Due to the heterogeneity of tumors and their complex microenvironment, patients have low clinical responsiveness to immune checkpoint blockade therapy. The high price of immune checkpoint blockers also greatly limits their widespread use in clinical practice.
[0006] As one of the strategies for tumor immunotherapy, tumor vaccines have unique advantages such as low cost, ability to induce specific anti-tumor immune responses and immune memory effects.
[0007] Current tumor vaccines have a single target: Current tumor vaccines are mainly designed for tumor cell-related / specific antigens (such as tumor neoantigens). However, tumor stromal cells also play a vital role in the occurrence and development of tumors.
[0008] Poor immunogenicity: Although non-viral vector vaccines can avoid the safety issues of viral vector vaccines, their immunogenicity is low and it is difficult to induce efficient antigen-specific immune responses like viral vector vaccines.
[0009] Immune dysfunction leads to limited efficacy: The extremely complex tumor microenvironment, such as weak acidity, hypoxia and immunosuppressive microenvironment, can lead to dysfunction and exhaustion of anti-tumor immune cells (NK / CTL, etc.), greatly limiting the efficient induction of anti-tumor immune response by tumor immunotherapy, including tumor vaccines.
[0010] In the early stage of tumorigenesis, tumor cells activate and induce nearby fibroblasts, endothelial cells or epithelial cells to differentiate into CAFs by secreting a variety of cytokines such as TGF-β and IL-10. Afterwards, CAFs secrete a large amount of collagen and a variety of cell / chemokines such as TGF-β, IL-10, SDF1, CXCL12, etc., and further interact with tumor cells and other stromal cells to promote tumor angiogenesis, improve the tolerance and invasiveness of tumor cells, and the infiltration of immunosuppressive cells-MDSC, Treg, TAM, etc. in the tumor site, inhibit the anti-tumor killing effect of cytotoxic T lymphocytes (CTL) and NK cells, and achieve tumor immune escape.
[0011] Due to the extremely complex microenvironment of the tumor and the limitations of the treatment strategies themselves, the above series of strategies are difficult to eliminate CAFs in a timely and efficient manner in the early stages of tumor development and achieve complete tumor suppression.
[0012] The development of a new dual vaccine targeting tumor cells and CAFs and inducing a dual-antigen-specific immune memory response is expected to achieve selective and rapid clearance of tumor cells and CAFs in the early stages of tumor development, destroying the "seeds and soil" of early tumor development and helping the immune system to strangle the tumor in the "cradle". At the same time, this new dual vaccine can be applied to the vast majority of clinical fibroproliferative tumors, including breast cancer, lung cancer, prostate cancer, melanoma, and squamous cell carcinoma, and has extremely important clinical significance. Summary of the invention
[0013] In view of the deficiencies in the prior art, the primary purpose of the present invention is to provide a plasmid DNA nano-selenium tumor dual vaccine for use in regulating immunity and thereby inhibiting fibroproliferative tumors.
[0014] Another object of the present invention is to provide a plasmid DNA nano-selenium tumor double vaccine and a preparation method thereof.
[0015] Another object of the present invention is to provide the use of plasmid DNA nano-selenium tumor double vaccine in vaccines for inhibiting fibroproliferative tumors.
[0016] The present invention provides a method for preparing a plasmid DNA nano-selenium tumor dual vaccine, comprising the steps of:
[0017] (3) The extracted double plasmid is mixed with Na2SeO3 solution and ascorbic acid for reaction, and dialyzed to obtain the plasmid DNA modified nano-selenium particles.
[0018] Further, the final concentration of the Na2SeO3 solution in step (3) is 1-20 mM; the final concentration of the ascorbic acid solution is 4-80 mM
[0019] Furthermore, in step (3), the amount of plasmid DNA added is a final concentration of 50-800 μg / mL; the vitamin C and Na2SeO3 are mixed in a molar ratio of 2-8:1; and the dialysis is performed using a 6000-100000 dialysis bag for a dialysis time of 10-48 hours.
[0020] Specifically, the binary plasmid includes a combination of pOVA and pFAP, and a combination of pIL15 and pCXCL9.
[0021] Further, step (3) includes: first adding the double plasmid at room temperature or in an ice bath, then dripping Na2SeO3 solution at 50-800rpm, stirring for 5-180min, and then slowly dripping ascorbic acid solution at 50-800rpm, stirring for 1-24h, stirring at room temperature or in an ice bath; dialyzing in ultrapure water with a dialysis bag for 48h to obtain the plasmid DNA nano-selenium tumor double vaccine.
[0022] Furthermore, the method further comprises steps (1) and (2):
[0023] (1) Plasmid transformation
[0024] Add the binary plasmid to the competent E. coli and mix well, place on ice, and then put it in a constant temperature water bath for heat shock, then immediately put it in ice, add LB medium without antibiotics and gently mix well, and finally put the bacterial tube into a constant temperature shaker for culture; collect the bacterial liquid and centrifuge, discard the supernatant and add the culture medium to resuspend the bacteria; use a coating rod to spread the bacteria evenly on the plate containing ampicillin antibiotics that has been poured in advance, leave it at room temperature for a few minutes, and put it into a constant temperature incubator after the bacterial liquid is absorbed by the solid culture medium;
[0025] (2) Plasmid amplification
[0026] Single colonies were selected from the plates and placed in conical flasks containing LB culture medium containing ampicillin antibiotics. The flask mouth was sealed with cotton and gauze, and cultured in a constant temperature shaking incubator. The plasmid was extracted using the Tiangen Plasmid Extraction Endotoxin-Free Kit, and the plasmid concentration was detected using an ELISA instrument for subsequent material synthesis.
[0027] The present invention provides a plasmid DNA nano-selenium tumor double vaccine, which is a plasmid DNA nano-selenium tumor double vaccine made of nano-selenium particles modified with plasmid DNA as core and plasmid DNA as shell.
[0028] The present invention also provides a vaccine for inhibiting fibroproliferative tumors, which is a plasmid DNA nano-selenium tumor double vaccine made of nano-selenium particles modified with plasmid DNA as the core and plasmid DNA as the shell.
[0029] Furthermore, the shell may also contain drugs for regulating immunity and targeting tumors to inhibit fibroproliferative tumors. Of course, the nano-selenium may be used as a carrier or adjuvant to load plasmid DNA.
[0030] The present invention also provides an application of a plasmid DNA nano-selenium tumor double vaccine in regulating immunity and inhibiting desmoplastic tumors, or an application of a plasmid DNA nano-selenium tumor double vaccine in a vaccine for inhibiting desmoplastic tumors. The plasmid DNA nano-selenium tumor double vaccine, which uses the plasmid DNA-modified nano-selenium particles as the core and the plasmid DNA as the shell, is used in regulating immunity and inhibiting desmoplastic tumors and its medicine. The plasmid DNA nano-selenium tumor double vaccine can efficiently deliver antigens and activate immune function to more effectively inhibit the growth of desmoplastic tumors.
[0031] The solution of the present invention has the following advantages and effects compared with the prior art:
[0032] The present invention discloses a plasmid DNA nano-selenium tumor double vaccine as a means of regulating immunity and further inhibiting fibroproliferative tumors. In an in vitro cell experiment, it can effectively inhibit the reactive oxygen species in inflammatory dendritic cells DC2.4, stimulate the maturation of BMDC, and upregulate related inflammatory factors, such as TNF-α and IL-12. The control group SeNPs@pFAP and SeNPs@pOVA have lower maturation promotion and inflammatory factor secretion on BMDC than the double vaccine.
[0033] In in vivo animal experiments, compared with a single nano-selenium vaccine, the plasmid DNA nano-selenium tumor dual vaccine can stimulate the activation of macrophages at a higher level. The nano-selenium tumor dual vaccine can also significantly activate more macrophages, and can also differentiate into more MemoryNK and NKT, while humoral immunity is also enhanced. Such as Tfh, SLPC and GCB. Under the stimulation of antigens, naive B cells will differentiate into short-lived plasma cells and GCB plasma cells, and then differentiate into long-lived plasma cells to produce antigen-specific antibodies to protect the organism from pathogens. Studies have shown that compared with a single antigen, the nano-selenium tumor dual vaccine can significantly increase the proportion of Tfh, GCB, and SLPC, and also induce strong T cell-mediated cellular immunity, which can significantly enhance CD4 + CD8 + T lymphocytes secrete TNF-α, IFN-γ and IL-4 and stimulate T cell proliferation to a certain extent. Therefore, nano-selenium tumor dual vaccine can be developed as a tumor vaccine, which can well regulate immunity and improve the effect of preventing fibroproliferative tumors.
[0034] At present, the existing tumor vaccines have a single target and unsatisfactory immunogenicity. The present invention uses the coding gene pOVA of tumor cell-specific antigen and the coding gene (pFAP) of CAF-specific marker-fibroblast activation protein (FAP) as DNA antigens, and constructs a multifunctional nano-selenium tumor dual nano-vaccine through biomineralization. The nano-selenium tumor dual nano-vaccine plays a role in inhibiting fibroproliferative tumors by targeting CAF ("soil"), tumors ("seeds") and their selenium-regulated immunity.
[0035] The scheme of the present invention provides a method for preparing a nano-selenium tumor dual vaccine. The raw materials of the obtained nano-selenium tumor dual vaccine are relatively easy to obtain, and the synthesis and purification methods are simple and highly operable. By optimizing the process, the synthesis scale can be immediately expanded to realize the commercialization and application of the vaccine, which has broad application prospects for medical vaccines. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 : Water and particle size diagram of nanoparticles of SeNPs@pFAP, SeNPs@pOVA, and SeNPs@pFAP / pOVA nanovaccines in the examples of this application.
[0037] Figure 2 : Zeta potential diagram of the nanoparticles of SeNPs@pFAP, SeNPs@pOVA, and SeNPs@pFAP / pOVA nanovaccines in the examples of this application.
[0038] Figure 3 : Transmission electron microscopy image (SeNPs@pFAP / pOVANanovaccine) in the example of this application.
[0039] Figure 4 : Elemental analysis spectrum of the plasmid DNA modified nano-selenium tumor dual nanovaccine (SeNPs@pFAP / pOVANanovaccine) in the example of this application.
[0040] Figure 5 : XPS analysis diagram of the tumor dual nanovaccine (SeNPs@pFAP / pOVANanovaccine) in the example of this application.
[0041] Figure 6 : This is a picture showing that the plasmid DNA-modified nano-selenium tumor dual nanovaccine (SeNPs@pFAP / pOVANanovaccine) activates BMDC and promotes the cells to secrete TNF-α and IL-12.
[0042] Figure 7: Cellular immune indicators such as macrophage activation ( Figure 7 A) Short-lived plasma cells ( Figure 7 B) GCB( Figure 7 C) Tfh cells ( Figure 7 D), CD4 + T, CD8 + Multifunctionality of T cells Figure 7 E, F).
[0043] Figure 8 :Nano-selenium tumor dual nanovaccine (SeNPs@pFAP / pOVANanovaccine) inhibits tumor volume changes during treatment in tumor-bearing mice. DETAILED DESCRIPTION
[0044] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.
[0045] Example 1 A method for preparing a plasmid DNA-modified nano-selenium tumor dual nano-vaccine (SeNPs@pFAP / pOVANanovaccine), i.e., a plasmid DNA nano-selenium tumor dual vaccine, comprises the following steps:
[0046] (1) Plasmid transformation
[0047] Take 5μL of plasmids pOVA and pFAP, add them to 50μL of competent E. coli and mix them evenly. Place them on ice for 45 minutes, then place them in a constant temperature water bath at 42°C for 45 seconds for heat shock, then immediately place them in ice, add 900μL of LB medium without antibiotics and gently mix them evenly, and finally place the bacterial tube in a 37°C constant temperature shaker at 250rpm for 60 minutes. Collect the bacterial solution and centrifuge it at 4000rpm for 1 minute, discard the supernatant and add 20μL of culture medium to resuspend the bacteria. Use a coating rod to spread the bacteria evenly on the plate containing ampicillin antibiotics that has been poured in advance, place it at room temperature for a few minutes, and after the bacterial solution is absorbed by the solid culture medium, place it in a 37°C constant temperature incubator for 24 hours.
[0048] (2) Plasmid amplification
[0049] Single colonies were selected from the plate and placed in a conical flask containing LB medium containing ampicillin antibiotics. The flask mouth was sealed with cotton and gauze and cultured in a constant temperature shaking incubator at 37°C for 16 hours. The plasmid was extracted using the Tiangen Plasmid Extraction Kit without Endotoxin, and the plasmid concentration was detected using an ELISA instrument for subsequent material synthesis.
[0050] (3) Synthesis of Nano-Selenium Tumor Dual Nano-Vaccine
[0051] At room temperature or in an ice bath, add 1000 μg of the amplified and extracted plasmid to the reaction bottle, stir for 30 minutes, then add Na2SeO3 solution (final concentration 5 mM) and put it into the above solution to mix, stir for 60 minutes, slowly add 800 μL of ascorbic acid, the ascorbic acid and Na2SeO3 solution are mixed in a molar ratio of 4:1, continue to react at room temperature for 2 hours, dialyze in ultrapure water with a dialysis bag for 12 hours, change the water frequently in the middle, and obtain the plasmid DNA modified nano-selenium tumor dual nano vaccine (SeNPs@pFAP / pOVANanovaccine) and store it at 4°C.
[0052] pOVA: plasmid Ovalbumin, the gene encoding chicken ovalbumin, pFAP: plasmidfibroblastactivationprotein, the gene encoding fibroblast activation protein.
[0053] Example 2 A method for preparing a plasmid DNA-modified nano-selenium tumor dual nano-vaccine (SeNPs@pIL15 / pCXCL9Nanovaccine) comprises the following steps:
[0054] (1) Plasmid transformation
[0055] Take 4 μL of plasmids pIL15 and pCXCL9, add them to 45 μL of competent E. coli and mix them evenly. Place them on ice for 30 minutes, then place them in a 42°C constant temperature water bath for 45 seconds for heat shock, then immediately place them in ice, add 900 μL of LB medium without antibiotics and gently mix them evenly, and finally place the bacterial tube in a 37°C constant temperature shaker at 200 rpm for 45 minutes. Collect the bacterial solution and centrifuge it at 4000 rpm for 1 minute, discard the supernatant and add 20 μL of culture medium to resuspend the bacteria. Use a spreading rod to spread the bacteria evenly on the plate containing ampicillin antibiotics that has been poured in advance, place it at room temperature for a few minutes, and after the bacterial solution is absorbed by the solid culture medium, place it in a 37°C constant temperature incubator for 24 hours.
[0056] (2) Plasmid amplification
[0057] Single colonies were selected from the plate and placed in a conical flask containing LB medium containing ampicillin antibiotics. The flask mouth was sealed with cotton and gauze and cultured in a constant temperature shaking incubator at 37°C for 12 hours. The plasmid was extracted using the Tiangen Plasmid Extraction Kit without Endotoxin, and the plasmid concentration was detected using an ELISA instrument for subsequent material synthesis.
[0058] (3) Synthesis of Nano-Selenium Tumor Dual Nano-Vaccine
[0059] At room temperature or in an ice bath, add 100 μg of the amplified and extracted double plasmid into the reaction bottle, stir for 5 minutes, then add Na2SeO3 solution (final concentration 1 mM) and put it into the above solution to mix, stir for 10 minutes, slowly add 700 μL of ascorbic acid, the ascorbic acid and Na2SeO3 solution are mixed in a molar ratio of 8:1, continue to react at room temperature for 10 hours, dialyze in ultrapure water with a dialysis bag for 9 hours, change the water frequently in the middle, and obtain the plasmid DNA modified nano-selenium tumor double nano vaccine (SeNPs@pIL15 / pCXCL9) and store it at 4°C.
[0060] pIL15: plasmid interleukin-15, the encoding gene of interleukin-15, pCXCL9: plasmid C-XC Motif Chemokine Ligand 9, the encoding gene of chemokine CXC ligand 9.
[0061] Example 3 A method for preparing a plasmid DNA-modified nano-selenium tumor dual nano-vaccine (SeNPs@pFAP / pOVANanovaccine) comprises the following steps:
[0062] (1) Plasmid transformation
[0063] Take 4 μL of plasmids pOVA and pFAP, add them to 50 μL of competent E. coli and mix them evenly. Place them on ice for 35 minutes, then place them in a 42°C constant temperature water bath for 50 seconds for heat shock, then immediately place them in ice, add 850 μL of LB medium without antibiotics and gently mix them evenly, and finally place the bacterial tube in a 37°C constant temperature shaker at 220 rpm for 48 minutes. Collect the bacterial solution and centrifuge it at 4000 rpm for 1 minute, discard the supernatant and add 25 μL of culture medium to resuspend the bacteria. Use a coating rod to spread the bacteria evenly on the plate containing ampicillin antibiotics that has been poured in advance, place it at room temperature for a few minutes, and after the bacterial solution is absorbed by the solid culture medium, place it in a 37°C constant temperature incubator for 25 hours.
[0064] (2) Plasmid amplification
[0065] Single colonies were selected from the plate and placed in a conical flask containing LB medium containing ampicillin antibiotics. The flask mouth was sealed with cotton and gauze and cultured in a constant temperature shaking incubator at 37°C for 13 hours. The plasmid was extracted using the Tiangen Plasmid Extraction Kit without Endotoxin, and the plasmid concentration was detected using an ELISA instrument for subsequent material synthesis.
[0066] (3) Synthesis of Nano-Selenium Tumor Dual Nano-Vaccine
[0067] At room temperature or in an ice bath, add 700 μg of the amplified and extracted double plasmid into the reaction bottle, stir for 10 min, then add Na2SeO3 solution (final concentration 10 mM) and put it into the above solution to mix, stir for 20 min, slowly add 750 μL of ascorbic acid, the ascorbic acid and Na2SeO3 solution are mixed in a molar ratio of 6:1, continue to react at room temperature for 20 hours, dialyze in ultrapure water with a dialysis bag for 32 hours, change the water frequently in the middle, and obtain the plasmid DNA modified nano-selenium tumor double nano vaccine (SeNPs@pFAP / pOVA) and store it at 4°C.
[0068] Example 4 A method for preparing a plasmid DNA-modified nano-selenium tumor dual nano-vaccine (SeNPs@pIL15 / pCXCL9), comprising the steps of:
[0069] (1) Plasmid transformation
[0070] Take 5.5μL plasmids pIL15 and pCXCL9, add them to 60μL competent E. coli and mix them evenly. Place them on ice for 42 minutes, then place them in a 42℃ constant temperature water bath for 48 seconds for heat shock, then immediately place them in ice, add 950μL LB medium without antibiotics and gently mix them evenly, and finally place the bacterial tube in a 37℃ constant temperature shaker at 240rpm for 55min. Collect the bacterial solution and centrifuge it at 4000rpm for 1min, discard the supernatant and add 22μL culture medium to resuspend the bacteria. Use a coating rod to spread the bacteria evenly on the plate containing ampicillin antibiotics that has been poured in advance, place it at room temperature for a few minutes, and after the bacterial solution is absorbed by the solid culture medium, place it in a 37℃ constant temperature incubator for 24 hours.
[0071] (2) Plasmid amplification
[0072] A single colony was selected from the plate and placed in a conical flask containing LB medium containing ampicillin antibiotics. The flask was sealed with cotton and gauze and cultured in a constant temperature shaking incubator at 37°C for 15 hours. The plasmid was extracted using the Tiangen Plasmid Extraction Kit without Endotoxin, and the plasmid concentration was detected using an ELISA reader for subsequent material synthesis.
[0073] (3) Synthesis of Nano-Selenium Tumor Dual Nano-Vaccine
[0074] At room temperature or in an ice bath, add 500 μg of the amplified and extracted double plasmid into the reaction bottle, stir for 25 min, then add Na2SeO3 solution (final concentration 8 mM) and put it into the above solution to mix, stir for 50 min, slowly add 800 μL of ascorbic acid, the ascorbic acid and Na2SeO3 solution are mixed in a molar ratio of 4:1, continue to react at room temperature for 24 hours, dialyze in ultrapure water with a dialysis bag for 42 hours, change the water frequently in the middle, and obtain the plasmid DNA modified nano-selenium tumor double nano vaccine (SeNPs@pIL15 / pCXCL9Nanovaccine) and store it at 4°C.
[0075] Example 5 Characterization of DNA-modified nano-selenium tumor dual nano-vaccine (SeNPs@pFAP / pOVA)
[0076] Experimental results: Figure 1 The hydrated particle size of the synthesized SeNPs@pFAP / pOVA nanovaccine was approximately 100 nm. Figure 2 The Zeta potential results also showed that compared with pFAP, pOVA, and SeNPs, the potentials of SeNPs@pFAP / pOVA, SeNPs@pFAP, and SeNPs@pOVA were significantly reduced. Figure 3 The results showed that the synthesized SeNPs@pFAP / pOVA was an irregular spherical structure and the plasmid DNA was obviously wrapped on the surface. Figure 4 The results show that selenium and plasmid DNA are well combined, as can be seen from the overlap of the spectrum of Se element and the spectrum of ligand N and P elements in plasmid DNA. Figure 5 The results showed that SeNPs bind to plasmid DNA through Se-P and Se-O bonds. These results show that plasmid DNA interacts with nano-selenium, and this example successfully prepared a nucleic acid tumor dual vaccine using nano-selenium as an adjuvant or carrier.
[0077] The preparation method of SeNPs@pFAP and SeNPs@pOVA can refer to Examples 1-4, except that the binary plasmids (a combination of pOVA and pFAP, a combination of pIL15 and pCXCL9) are replaced with a single pOVA or pFAP.
[0078] Example 6, study on SeNPs@pFAP / pOVA tumor dual nanovaccine inducing BMDC cell maturation.
[0079] First, bone marrow-derived dendritic cells (BMDCs) were obtained by culturing monodispersed cells from the femoral and tibial bone marrow of female C57BL / 6 mice (6-8 weeks old) in RPMI 1640 medium containing 10% fetal bovine serum, colony stimulating factor (GM-CSF, 20 ng / mL) and β-mercaptoethanol (5 ng / mL) for 6-8 days.
[0080] Then, BMDC cells were treated with SeNPs@pFAP, SeNPs@pOVA, SeNPs@pFAP / pOVA nanovaccine (1 μg / mL pDNA), and lipopolysaccharide (LPS, 1 μg / mL, as a positive control group) for 12 h. Afterwards, the cells were stained with fluorescently labeled antibodies (including CD11c-APC, CD86-APC-Cy7, and CD80-PE) to assess the maturity of BMDCs and analyzed using flow cytometry. Figure 6 As shown, compared with the untreated group (i.e., control), SeNPs@pFAP and SeNPs@pOVA can activate BMDC, but SeNPs@pFAP / pOVA dual nanovaccine can better activate BMDC maturation and activation and secrete more cytokines such as TNF-α.
[0081] Example 7. The present invention provides SeNPs@pFAP / pOVA dual nanovaccine that regulates the immunogenicity of mice using an intradermal injection immunization route.
[0082] (1) Experimental groups
[0083] To evaluate the immunogenicity of the developed SeNPs@pFAP / pOVA nanovaccine in mice, 20 C57BL / 6J mice were equally divided into 4 groups, namely, saline group (control group), SeNPs@pFAP group, SeNPs@pOVA group, and SeNPs@pFAP / pOVA vaccine group. The vaccine dose pDNA was 20 μg per mouse.
[0084] (2) Experimental content
[0085] Immunization was performed once every 2 weeks for a total of three times. The mice were killed on the 7th day after the last immunization, and the spleen, inguinal nodes and serum were collected for subsequent immunological analysis. Flow cytometry was used to measure the macrophage activation in the spleen of SeNPs@pFAP / pOVA nanovaccine mice ( Figure 7 A) Short-lived plasma cells ( Figure 7 B) GCB( Figure 7 C) Tfh cells ( Figure 7 D), multifunctionality of CD4 T and CD8 T cells ( Figure 7E, F). The results showed that the dual vaccine was more effective than a single vaccine.
[0086] Example 8. The present invention provides a SeNPs@pFAP / pOVA dual nanovaccine that inhibits the growth of desmoplastic tumors (b16-OVA melanoma).
[0087] (1) Experimental groups
[0088] To evaluate the immunogenicity of the developed SeNPs@pFAP / pOVA nanovaccine in mice, 20 C57BL / 6J mice were equally divided into 4 groups, namely, saline group (control group), SeNPs@pFAP group, SeNPs@pOVA group, and SeNPs@pFAP / pOVA vaccine group. The vaccine dose pDNA was 20 μg per mouse.
[0089] (2) Experimental content
[0090] The mice were immunized once every 2 weeks for a total of three times. 10W melanoma b16-OVA was subcutaneously inoculated on the 7th day after the last immunization, and then the tumor growth was monitored. Figure 8 The results showed that the dual vaccine can better inhibit the growth of fibroproliferative tumors such as melanoma b16-OVA than single vaccine.
Claims
1. A method for preparing a plasmid DNA nano-selenium tumor dual vaccine, characterized in that: Includes steps: (3) Synthesis of Nano-Selenium Tumor Dual Nano-Vaccine The extracted double plasmid is mixed with Na2SeO3 solution and ascorbic acid for reaction and dialyzation to obtain the plasmid DNA nano-selenium tumor double vaccine.
2. The preparation method according to claim 1, characterized in that: The final concentration of the Na2SeO3 solution is 1-20 mM; the final concentration of the ascorbic acid solution is 4-80 mM.
3. The preparation method according to claim 1, characterized in that: The amount of plasmid DNA added is a final concentration of 10-2000 μg / mL; the vitamin C and Na2SeO3 are mixed in a molar ratio of 1-12:1; the dialysis is performed using a 6000-100000 dialysis bag, and the dialysis time is 10-48 hours.
4. The preparation method according to claim 1, characterized in that: The binary plasmid includes pOVA and pFAP, pIL15 and pCXCL9.
5. The preparation method according to any one of claims 1 to 4, characterized in that: Step (3) comprises: first adding the double plasmid at room temperature or in an ice bath, then dripping Na2SeO3 solution at 50-800rpm, stirring for 5-180min, then slowly dripping ascorbic acid solution at 50-800rpm, stirring for 1-24h, stirring at room temperature or in an ice bath; dialyzing in ultrapure water with a dialysis bag for 48h to obtain the plasmid DNA nano-selenium tumor double vaccine.
6. The preparation method according to claim 5, characterized in that: The method further comprises steps (1) and (2): (1) Plasmid transformation Add the binary plasmid to the competent E. coli and mix well, place on ice, and then put it in a constant temperature water bath for heat shock, then immediately put it in ice, add LB medium without antibiotics and gently mix well, and finally put the bacterial tube into a constant temperature shaker for culture; collect the bacterial liquid and centrifuge, discard the supernatant and add the culture medium to resuspend the bacteria; use a coating rod to spread the bacteria evenly on the plate containing ampicillin antibiotics that has been poured in advance, leave it at room temperature for a few minutes, and put it into a constant temperature incubator after the bacterial liquid is absorbed by the solid culture medium; (2) Plasmid amplification Single colonies were selected from the plates and placed in conical flasks containing LB culture medium containing ampicillin antibiotics. The flask mouth was sealed with cotton and gauze, and cultured in a constant temperature shaking incubator. The plasmid was extracted using the Tiangen Plasmid Extraction Endotoxin-Free Kit, and the plasmid concentration was detected using an ELISA instrument for subsequent material synthesis.
7. A plasmid DNA nano-selenium tumor double vaccine, characterized in that: It is a plasmid DNA nano-selenium tumor double vaccine made of plasmid DNA modified nano-selenium particles as the core and plasmid DNA as the shell.
8. A plasmid DNA nano-selenium tumor double vaccine according to claim 7, characterized in that: The shell contains drugs targeting tumors and tumor-related fibroblasts, which prevent and inhibit the occurrence and growth of fibroproliferative tumors.
9. The plasmid DNA nano-selenium tumor double vaccine according to claim 7, characterized in that: Prepared according to the preparation method according to any one of claims 1 to 4.
10. Use of a plasmid DNA nano-selenium tumor dual vaccine as described in any one of claims 7 to 9 in a vaccine to inhibit fibroproliferative tumors.