CN113769075B - In-situ vaccine and preparation method thereof - Google Patents
In-situ vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN113769075B CN113769075B CN202111119339.XA CN202111119339A CN113769075B CN 113769075 B CN113769075 B CN 113769075B CN 202111119339 A CN202111119339 A CN 202111119339A CN 113769075 B CN113769075 B CN 113769075B
- Authority
- CN
- China
- Prior art keywords
- mal
- nano material
- solution
- adjuvant
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 44
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 239000002086 nanomaterial Substances 0.000 claims abstract description 60
- 102000036639 antigens Human genes 0.000 claims abstract description 53
- 239000000427 antigen Substances 0.000 claims abstract description 48
- 108091007433 antigens Proteins 0.000 claims abstract description 48
- 210000004881 tumor cell Anatomy 0.000 claims abstract description 47
- 239000000243 solution Substances 0.000 claims abstract description 46
- 239000002671 adjuvant Substances 0.000 claims abstract description 38
- 239000002245 particle Substances 0.000 claims abstract description 24
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 22
- 239000011550 stock solution Substances 0.000 claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 230000000415 inactivating effect Effects 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 41
- 230000002779 inactivation Effects 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 32
- 239000006228 supernatant Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 16
- 239000002244 precipitate Substances 0.000 claims description 16
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 claims description 12
- 238000000108 ultra-filtration Methods 0.000 claims description 12
- 239000003446 ligand Substances 0.000 claims description 11
- 239000011259 mixed solution Substances 0.000 claims description 9
- 238000001556 precipitation Methods 0.000 claims description 8
- 230000001079 digestive effect Effects 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 7
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 108010019160 Pancreatin Proteins 0.000 claims description 4
- 230000003698 anagen phase Effects 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 229940055695 pancreatin Drugs 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 230000006862 enzymatic digestion Effects 0.000 claims description 3
- 238000000265 homogenisation Methods 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 238000010257 thawing Methods 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims description 2
- 239000008346 aqueous phase Substances 0.000 claims 1
- 239000012071 phase Substances 0.000 claims 1
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 4
- 238000002619 cancer immunotherapy Methods 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 abstract description 3
- 206010057249 Phagocytosis Diseases 0.000 abstract description 3
- 210000000612 antigen-presenting cell Anatomy 0.000 abstract description 3
- 230000008782 phagocytosis Effects 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 125000003396 thiol group Chemical group [H]S* 0.000 abstract description 3
- 239000002105 nanoparticle Substances 0.000 description 24
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 16
- 238000000338 in vitro Methods 0.000 description 15
- 206010006187 Breast cancer Diseases 0.000 description 10
- 208000026310 Breast neoplasm Diseases 0.000 description 10
- 210000004443 dendritic cell Anatomy 0.000 description 10
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 239000007853 buffer solution Substances 0.000 description 6
- 238000009169 immunotherapy Methods 0.000 description 5
- 239000003960 organic solvent Substances 0.000 description 5
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 4
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000036571 hydration Effects 0.000 description 4
- 238000006703 hydration reaction Methods 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 230000000139 costimulatory effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 229910018072 Al 2 O 3 Inorganic materials 0.000 description 2
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 2
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 description 2
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 description 2
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229910001566 austenite Inorganic materials 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000011467 adoptive cell therapy Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 229940030156 cell vaccine Drugs 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 238000002595 magnetic resonance imaging Methods 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229940023094 protein/peptide vaccine Drugs 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Crystallography & Structural Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Condensed Matter Physics & Semiconductors (AREA)
- Manufacturing & Machinery (AREA)
- Biophysics (AREA)
- Composite Materials (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Materials Engineering (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Medical Informatics (AREA)
- Molecular Biology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to an in-situ vaccine and a preparation method thereof, wherein the preparation method comprises the steps of culturing and inactivating tumor cell antigen stock solution, preparing nano material@Mal solution, adding an adjuvant to synthesize nano material@Mal-adjuvant particles, and then mixing and incubating the tumor cell antigen stock solution and the nano material@Mal-adjuvant particles according to the volume ratio of 5:1-5:3 to obtain the nano material@Mal-adjuvant@antigen. The preparation method of the in-situ vaccine is green, simple and easy to operate. The preparation method of the in-situ vaccine is suitable for various tumors and has universality. The maleimide on the surface of the nano material @ Mal can form a stable thioether bond with the sulfhydryl group on the protein so as to capture tumor-related antigens, improve the utilization rate of the antigens, and realize the co-delivery of the adjuvant and the antigens. The nano material @ Mal itself can be used as an adjuvant to activate antigen presenting cells and enhance phagocytosis thereof, and can further activate T cells to improve the safety and curative effect of cancer immunotherapy.
Description
Technical Field
The invention relates to an in-situ vaccine and a preparation method thereof, belonging to the technical field of biology.
Background
In recent years, with the development of immune checkpoint inhibitors and CAR-T, immunotherapy has shown tremendous promise in the field of tumor therapy. New strategies for tumor immunotherapy have evolved rapidly and have become the most attractive area for tumor therapy.
In order to achieve optimal anti-tumor immune effects, it is common to employ "passive" immunotherapy such as adoptive cell therapy, genetically engineered T cells, etc. to directly attack tumor cells, or "active" immunotherapy such as cytokines, tumor vaccines, immune checkpoint inhibitors, etc. to modulate and activate the immune system. Tumor vaccines, an active tumor immunotherapy strategy, have grown in popularity with the identification of more and more tumor neoantigens. Common tumor vaccine types include protein/peptide vaccines, cellular vaccines (tumor cells and immune cells) and genetic vaccines (DNA and RNA). While these vaccines have produced acceptable therapeutic results in some clinical trials, their overall clinical efficacy is not satisfactory. The main reasons include: (1) Patients with "cold" tumors are vaccinated, resulting in patients with a lower or ineffective specific immune response to vaccination. (2) The use of an undesirable vaccination system reduces the immunogenicity and effectiveness of the vaccine. (3) The vaccine has immune tolerance to tumor endogenous autoantigens.
Disclosure of Invention
The invention aims to provide an in-situ vaccine and a preparation method thereof, and the prepared in-situ vaccine has good biocompatibility and high antigen utilization rate, can enhance immunogenicity, and can improve the safety and curative effect of cancer immunotherapy.
In order to achieve the above purpose, the present invention provides the following technical solutions: an in situ vaccine preparation method comprising the steps of:
s1, culturing: resuscitating and culturing tumor cells in the culture solution to reach logarithmic growth phase, collecting cells by using digestive juice and cleaning to obtain tumor cell liquid;
s2, inactivating: performing inactivation treatment on the tumor cell liquid collected in the step S1, incubating the tumor cell liquid after the inactivation treatment, and centrifuging the incubated tumor cell liquid to obtain supernatant fluid to obtain an inactivated antigen stock solution;
s3, preparing a nano material@Mal solution: mixing nano materials and PEG-Mal ligand according to the mass ratio of 1:10-1:15, dissolving to form mixed solution, adding cyclohexane with the volume of 10-15 times of the mixed solution to precipitate the mixed solution, centrifuging to remove supernatant fluid to obtain precipitate, drying the precipitate, adding ultrapure water to dissolve the precipitate, centrifuging and passing through a membrane, ultrafiltering to obtain nano material@Mal solution, and preserving at 2-6 ℃ for later use;
s4, synthesizing nano material @ Mal-adjuvant particles: adding an adjuvant solution into the nano material@Mal solution prepared in the step S3, stirring at room temperature, performing ultrafiltration to obtain synthesized nano material@Mal-adjuvant particles, and preserving at 2-6 ℃ for later use;
s5, preparing a vaccine: mixing the antigen stock solution prepared in the step S2 with the nano material@Mal-adjuvant particles synthesized in the step S4 according to the volume ratio of 5:1-5:3, incubating and ultrafiltering to obtain the nano material@Mal adjuvant@antigen.
Further, the digestive juice in the step S1 is a pancreatin solution with a mass fraction of 0.2%.
Further, the inactivation treatment mode in the step S2 is at least one of a microwave cell inactivation method, an electromagnetic wave cell inactivation method, a laser cell inactivation method, a high-temperature cell inactivation method, an ultrasonic disruption cell inactivation method, a repeated cell freezing and thawing method, a homogenization method, a centrifugation method, a precipitation cell inactivation method and an enzymatic digestion cell inactivation method.
Further, the inactivation treatment in the step S2 is performed by using X-ray irradiation.
Further, the nanomaterial in step S3 is an oil phase nanomaterial, and the nanomaterial @ Mal solution is a water phase.
Further, in the step S3, the nanomaterial is Fe 3 O 4 、γ-Fe 2 O 3 、CuS、MnO 2 And Al 2 O 3 Any one of them.
Further, the ultrafiltration in the step S3 uses an ultrafiltration tube with a molecular weight cut-off of 30k at a rotational speed of 3000-5000rpm.
Further, the adjuvant in the step S4 is any one of STING or TLR.
Further, the incubation time in the step S5 is 2-6h.
The invention also provides an in situ vaccine for medical diagnostic applications prepared by any of the in situ vaccine preparation methods described above.
The invention has the beneficial effects that: the preparation method of the in-situ vaccine is green, simple and easy to operate. The preparation method of the in-situ vaccine is suitable for various tumors and has universality. The maleimide on the surface of the nano material @ Mal can form a stable thioether bond with the sulfhydryl group on the protein so as to capture tumor-related antigens, so that the antigen utilization rate is improved, and the co-delivery of the adjuvant and the antigen can be realized. The nano material @ Mal itself can be used as an adjuvant to activate antigen presenting cells and enhance phagocytosis thereof, and can further activate T cells to improve the safety and curative effect of cancer immunotherapy. Nanomaterial @ Mal is also applicable to medical diagnostics.
The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 shows the preparation of synthetic Fe according to example one 3 O 4 Electron microscopy of the @ Mal-CpG nanoparticles.
FIG. 2 shows the preparation of synthetic Fe according to example one 3 O 4 @Mal and Fe 3 O 4 The hydration particle size map and Zeta potential map of @ Mal CpG, wherein FIG. 2 (a) is Fe 3 O 4 @Mal and Fe 3 O 4 Hydration particle size map of @ Mal CpG, FIG. 2 (b) is Fe 3 O 4 @Mal and Fe 3 O 4 Potential map characterization of @ Mal CpG.
Fig. 3 is an ultraviolet absorption spectrum of a nano material synthesized in example one.
FIG. 4 is a quantitative in vitro capture antigen graph of a nanoparticle synthesized according to example one, wherein FIG. 4 (a) is Fe 3 O 4 @Mal and Fe 3 O 4 Capturing a 4T1 antigen quantitative graph in vitro by using @ Mal-CpG; FIG. 4 (b) is Fe 3 O 4 @Mal and Fe 3 O 4 An in vitro capture 803 antigen quantification map of Mal-CpG; FIG. 4 (c) is Fe 3 O 4 @Mal and Fe 3 O 4 An in vitro capture B16F10 antigen quantification map of Mal-CpG; FIG. 4 (d) is Fe 3 O 4 @Mal and Fe 3 O 4 Capturing MC38 antigen quantitative images in vitro by using @ Mal-CpG; FIG. 4 (e) is Fe 3 O 4 @Mal and Fe 3 O 4 Capturing an L1210 antigen quantitative graph in vitro by using @ Mal-CpG; FIG. 4 (f) is Fe 3 O 4 @Mal and Fe 3 O 4 In vitro capture of B16F10-OVA antigen quantification map with Mal-CpG.
FIG. 5 is a particle size and potential diagram representation of the synthesized nanoparticle prepared in example 1 before and after capturing breast cancer tumor antigen, wherein FIG. 5 (a) is Fe 3 O 4 @Mal and Fe 3 O 4 Particle size diagram of 4T1 antigen before and after capturing 4T1 antigen in vitro at Mal-CpG, FIG. 5 (b) is Fe 3 O 4 @Mal and Fe 3 O 4 Zeta potential map of 4T1 antigen and 4T1 antigen captured in vitro by Mal-CpG.
FIG. 6 is a representation of the maturation of Dendritic Cells (DCs) stimulated in vitro by the preparation of a synthetic in situ vaccine according to example I, wherein FIG. 6 (a) shows the results of CD80 expression of costimulatory molecules on the surface of DCs and FIG. 6 (b) shows the results of CD86 expression of costimulatory molecules on the surface of DCs.
FIG. 7 is a graph showing in vitro capture of antigen by nanoparticles prepared in example one and example three.
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
The invention provides an in-situ vaccine preparation method, which comprises the following steps:
s1, culturing: resuscitating and culturing tumor cells in the culture solution to reach logarithmic growth phase, collecting cells by using digestive juice and cleaning to obtain tumor cell liquid;
s2, inactivating: inactivating the tumor cell liquid collected in the step S1, incubating the tumor cell liquid after the inactivation treatment, and centrifuging the incubated tumor cell liquid to obtain supernatant fluid to obtain an inactivated antigen stock solution;
s3, preparing a nano material@Mal solution: mixing nano material and PEG-Mal ligand according to the mass ratio of 1:10-1:15, dissolving to form mixed liquor, adding cyclohexane with the volume of 10-15 times of the mixed liquor to precipitate the mixed liquor, centrifuging to remove supernatant fluid to obtain precipitate, drying the precipitate, adding ultrapure water to dissolve the precipitate, centrifuging and passing through a membrane, ultrafiltering to obtain nano material@Mal solution, and preserving at 2-6 ℃ for later use;
s4, synthesizing nano material @ Mal-adjuvant particles: adding an adjuvant solution into the nano material@Mal solution prepared in the step S3, stirring at room temperature, performing ultrafiltration to obtain synthesized nano material@Mal-adjuvant particles, and preserving at 2-6 ℃ for later use;
s5, preparing a vaccine: mixing the antigen stock solution prepared in the step S2 with the nano material@Mal-adjuvant particles synthesized in the step S4 according to the volume ratio of 5:1-5:3, incubating and ultrafiltering to obtain the nano material@Mal adjuvant@antigen.
The tumor cells may be isolated from any tumor tissue, for example, the tumor cells may be one or more of ovarian cancer, breast cancer, lung cancer, gastric cancer, colon cancer, liver cancer or melanoma cells, but not limited thereto, and may be other cancer cells. When the tumor cells are cultured to the logarithmic phase, and after the tumor cells are observed by a microscope to be fully paved on the bottom of the culture dish, the non-adherent tumor cells are washed by using a PBS buffer solution, then the adherent tumor cells are changed into a suspension state by using a digestive solution, centrifugally collected and washed by using the PBS buffer solution for a plurality of times, the components of the complete culture medium in the culture dish are removed, so that the tumor cells are suspended in the PBS buffer solution to form a tumor cell solution, and the tumor cell solution is counted. The digestive juice used here is a pancreatin solution with a mass fraction of 0.2%; the washing solution is not limited to PBS, but may be sterile physiological saline, but is not limited to these solvents, and various solvents having a buffer capacity that does not cause allergic reactions in the body may be used.
In the step S2, the tumor cell fluid is subjected to inactivation treatment, wherein the inactivation treatment mode is at least one of a microwave cell inactivation method, an electromagnetic wave cell inactivation method, a laser cell inactivation method, a high-temperature cell inactivation method, an ultrasonic disruption cell inactivation method, a repeated cell freezing and thawing method, a homogenization method, a centrifugation method and a precipitation cell inactivation method, and an enzymatic digestion cell inactivation method. Preferably, the inactivation treatment is performed by X-ray irradiation, so that the tumor cells die and break. And (3) placing the tumor cell liquid subjected to X-ray irradiation into a cell incubator for incubation for 0.5-1h, so that substances in cells are completely exposed in the tumor cell liquid, centrifuging at 3000-5000rpm for 10min, removing cells and cell fragments in the tumor cell liquid, collecting supernatant, centrifuging again, and further removing cell fragments in the solution to obtain the inactivated antigen stock solution.
Weighing the nano material of the oil phase in a centrifuge tube, adding an acetone solution into the centrifuge tube to react with the nano material of the oil phase, so that the nano material wrapped in the oil phase is precipitated, centrifuging at a centrifugal force rotating speed of 5000g for 7-10min to remove supernatant, and adding an organic solvent into the centrifuge tube to completely dissolve the precipitate. The oil phase nanomaterial is mixed with cyclohexane. Weighing a PEG-Mal ligand with proper mass, wherein the mass ratio of the oil phase nano material to the PEG-Mal ligand is 1:10-1:15, and dripping an organic solvent into the PEG-Mal ligand to completely dissolve the PEG-Mal ligand, wherein the nano material and the PEG-Mal are dissolved in the same organic solvent. Adding the nano material dissolved in the organic solvent into PEG-Mal dissolved in the same organic solvent to form mixed solution, and carrying out ultrasonic treatment for 1-2h to completely mix the mixed solution. Cooling to room temperature, adding 10-15 times volume of cyclohexane into the mixed solution for precipitation, centrifuging at 3000-5000rpm for 10min, and removing supernatant to obtain precipitate. After the precipitate was dried, ultrapure water was added to dissolve the precipitate completely, and the precipitate was centrifuged and passed through a membrane. Centrifuging with 30K ultrafiltration centrifuge tube at 3000-5000rpm for 5-7min, ultrafiltering for 3-4 times, removing excessive cyclohexane to obtain water phase nanometer material @ Mal solution, and storing at 2-6deg.C. The nano material is used as a carrier, and can simultaneously load the adjuvant and the antigen, so that the co-delivery of the adjuvant and the antigen is realized. The nanomaterial is preferably a magnetic nanomaterial, and the magnetism facilitates in vivo magnetic resonance imaging and is used for detecting tumor volume change. The nano material in the step S3 is Fe 3 O 4 、γ-Fe 2 O 3 、CuS、MnO 2 And Al 2 O 3 Any of these may be other magnetic nanomaterials. Using Fe 3 O 4 When nano particles and the like are used as magnetic nano materials, fe 3 O 4 The nanoparticles themselves may also be used as adjuvants.
The adjuvant in the step S4 is any one of STING or TLR, and other adjuvants can be used to obtain nano material@Mal-adjuvant particles. The stirring may be magnetic stirring, but not limited to this, other stirring methods may be used, and are not limited to this
And S5, adding the antigen stock solution obtained in the step S2 into the nano material@Mal-adjuvant particles synthesized in the step S4, incubating for 2-6h, performing ultrafiltration for a plurality of times to remove the antigen which is not combined with the nano material@Mal-adjuvant particles, and finally collecting the solution to obtain the nano material@Mal-adjuvant@antigen.
The invention also provides an in situ vaccine for medical diagnostic applications prepared by any of the above methods of in situ vaccine preparation.
The following is a detailed description of specific embodiments:
example 1
An in situ vaccine was prepared as follows:
(a) Resuscitating and culturing breast cancer tumor cells (4T 1) to reach the logarithmic growth phase, observing the condition that the 4T1 cells are fully paved at the bottom of a culture dish through a microscope, washing non-adherent tumor cells by using a PBS buffer solution, then digesting the adherent tumor cells into a suspension state by using a pancreatin solution with the mass fraction of 0.2%, centrifugally collecting, washing for 4 times by using the PBS buffer solution, removing complete culture medium components in the culture dish, suspending the tumor cells in the PBS buffer solution to form tumor cell liquid, counting, and adjusting the cell quantity to be about 4M;
(b) Performing X-ray irradiation on the collected cells, then placing the cells into a cell culture box for incubation for 0.5h, centrifuging to remove the cells and cell fragments, collecting supernatant, centrifuging again to remove residual cell fragments for later use;
(c) Firstly, 1mL of oil phase Fe with the concentration of 8.2mg/mL is absorbed 3 O 4 Oil phase Fe in a 15mL centrifuge tube 3 O 4 With cyclohexane, 10mL of acetone solution was added to the centrifuge tube to complete precipitation, and then the supernatant was removed by centrifugation at 5000g for 5min, and then 1mL of tetrahydrofuran solution was added to complete dissolution of the precipitate. 82mg of PEG-Mal ligand was then weighed out, at which time the oil phase Fe 3 O 4 And PEG-Mal ligand in a mass ratio of =1:10,and tetrahydrofuran solution was added dropwise thereto and shaken to dissolve it completely. Fe to be dissolved in tetrahydrofuran 3 O 4 Added to PEG-Mal, also dissolved in tetrahydrofuran, and sonicated for 1h. Cooling to room temperature, adding 10 times of cyclohexane, centrifuging to remove supernatant, adding an appropriate amount of ultrapure water after precipitation and drying to dissolve completely, centrifuging and performing membrane passing. Finally, the mixture was centrifuged with a 30K ultrafiltration tube at 4000rpm for 5min and ultrafiltered 3 times to remove excess cyclohexane. Finally obtain water phase Fe 3 O 4 The @ Mal solution is preserved at 4 ℃ for standby;
(d) 150. Mu.L of CpG solution with a molar concentration of 100. Mu.M was added to 35mM Fe with a volume of 430. Mu.L 3 O 4 Stirring at room temperature in the @ Mal solution (c). Subsequently, the collected solution was subjected to ultrafiltration three times. Finally preparing Fe 3 O 4 The @ Mal-CpG nanoparticles are stored at 4 ℃ for standby;
(e) Adding the collected supernatant (b) to the synthesized Fe 3 O 4 In the @ Mal CpG nanomaterial (d), the supernatant (b) and Fe 3 O 4 Incubating for 2 hours with the volume ratio of @ Mal-CpG nano material being 5:1, ultrafiltering for three times to remove antigen which is not combined with the nano material, and finally collecting the solution to obtain Fe 3 O 4 The @ Mal CpG @4T1 vaccine.
Example two
An in situ vaccine was prepared as follows:
(a) The same as in the first embodiment;
(b) The same as in the first embodiment;
(c) Firstly, sucking 2mL of oil phase Fe with the concentration of 6.6mg/mL 3 O 4 Oil phase Fe in a 15mL centrifuge tube 3 O 4 Containing cyclohexane, 15mL of acetone solution was added to the centrifuge tube to complete precipitation, and then the supernatant was removed by centrifugation at 5000g for 5min, and then 2mL of tetrahydrofuran solution was added to complete dissolution of the precipitate. Then 158mg of PEG-Mal ligand was weighed out, at which time the oil phase Fe 3 O 4 And PEG-Mal ligand in a mass ratio of =1:12, and a tetrahydrofuran solution was added dropwise thereto and shaken to completely dissolve it. Fe to be dissolved in tetrahydrofuran 3 O 4 Added toAlso dissolved in PEG-Mal in tetrahydrofuran, sonicated for 1h. Cooling to room temperature, adding 10 times of cyclohexane, centrifuging to remove supernatant, adding an appropriate amount of ultrapure water after precipitation and drying to dissolve completely, centrifuging and performing membrane passing. Finally, the mixture was centrifuged with a 30K ultrafiltration tube at 4000rpm for 5min and ultrafiltered 3 times to remove excess cyclohexane. Finally obtain water phase Fe 3 O 4 The @ Mal solution is preserved at 4 ℃ for standby;
(d) The same as in the first embodiment;
(e) As in the first embodiment.
Example III
An in situ vaccine was prepared as follows:
(a) The same as in the first embodiment;
(b) The same as in the first embodiment;
(c) The same as in the first embodiment;
(d) The same as in the first embodiment;
(e) Adding the collected supernatant (b) to the synthesized Fe 3 O 4 In the @ Mal CpG nanomaterial (d), the supernatant (b) and Fe 3 O 4 Incubating for 2 hours with the volume ratio of @ Mal-CpG nano material being 5:2, ultrafiltering for three times to remove antigen which is not combined with the nano material, and finally collecting the solution to obtain Fe 3 O 4 The @ Mal CpG @4T1 vaccine.
Fe prepared in examples one to three 3 O 4 The size of the @ Mal CpG nanoparticle is small. FIG. 1 shows the preparation of synthetic Fe according to example 1 3 O 4 The electron microscope image of the@Mal-CpG nanoparticle has the size of about 3.9+/-0.6 nm, uniform size, high stability and high biocompatibility.
FIG. 2 shows the preparation of synthetic Fe according to example 1 3 O 4 Nano material @ Mal and Fe 3 O 4 The hydration particle size diagram and the Zeta potential diagram of the @ Mal-CpG nanoparticle are characterized. Looking at FIG. 2 (a), fe 3 O 4 Nano material @ Mal and Fe 3 O 4 The hydration particle size of the @ Mal CpG nano particles is similar, and the particle size distribution of different sizes is similar, namely, fe is caused by coupling with CpG materials 3 O 4 No major change in particle size @ MalAnd (5) melting. FIG. 2 (b) is a view of Fe 3 O 4 @Mal and Fe 3 O 4 Zeta potential diagram of @ Mal CpG, fe 3 O 4 Zeta potential of @ Mal is about-1 mV, fe 3 O 4 The Zeta potential of @ Mal CpG was approximately-7 mA. From the point of view of stability, the larger the Zeta potential absolute value, the more stable the solution, i.e. Fe 3 O 4 Synthesis of Fe by Mal 3 O 4 After @ Mal-CpG, the solution was more stable.
Please refer to fig. 3, which shows Fe 3 O 4 Nano material @ Mal, cpG material marked with Cy5.5 fluorescent dye and Fe 3 O 4 Cy5.5 fluorescent dye Fe synthesized by CpG material marked with Cy5.5 fluorescent dye and coated with Mal nano material 3 O 4 Ultraviolet absorption spectrum of Mal-CpG nano particle, detecting ultraviolet absorption spectrum in 350-800nm, observing and finding Fe 3 O 4 @Mal-CpG Cy5 . 5 The absorption peak of the nanoparticle contains Fe at the same time 3 O 4 The absorption peak characteristics of @ Mal and CpG labeled with Cy5.5 fluorescent dye prove that CpG Cy5 . 5 Successfully modify to Fe 3 O 4 On the @ Mal nanomaterial, thereby indirectly proving the successful synthesis of Fe 3 O 4 @Mal-CpG nanoparticles.
Please refer to fig. 4 (a) to fig. 4 (f), which are in vitro capture antigen quantification diagrams of the nanoparticles prepared in example one. The observation diagram shows that the prepared nano particles are respectively used for incubating with 4T1, 803, B16F10, MC38, L1210 and B16F10-OVA tumor cells, and the nano particles can capture antigens, can capture different tumor cells and have universality on the tumor cells.
Referring to fig. 5, the particle size and Zeta potential profile of the nanoparticles prepared in example one before and after capturing breast cancer tumor cells are shown. Looking at FIG. 5 (a), fe 3 O 4 @Mal and Fe 3 O 4 After capturing breast cancer tumor cell antigens, the particle size of the@Mal-CpG nanoparticles is increased. Looking at FIG. 5 (b), fe 3 O 4 After the@Mal nanoparticle captures breast cancer tumor cell antigen, the absolute value of the potential is larger than that of Fe 3 O 4 The nano-particles @ Mal were prepared,less than the absolute value of the electric potential of breast cancer tumor cells; fe (Fe) 3 O 4 After capturing breast cancer tumor cells by the@Mal-CpG nano particles, the absolute value of the potential is smaller than that of Fe 3 O 4 Absolute value of the potentials of the @ Mal-CpG nanoparticles and breast cancer tumor cells.
Please refer to fig. 6, which is a representation of the in situ vaccine prepared in example one, stimulating Dendritic Cell (DCs) maturation in vitro. As can be seen from FIGS. 6 (a) and 6 (b), ctrl is used as a comparison, and the nanoparticle Fe prepared in the examples 3 O 4 @Mal and Fe 3 O 4 The @ Mal-CpG can raise the expression of the costimulatory molecules CD80 and CD86 on the surface of the DCs, and can further up-regulate the expression of the CD80 and CD86 after in-situ vaccine is formed with breast cancer tumor antigen in vitro, so that the DCs are activated to mature. Namely Fe 3 O 4 @Mal@4T1、Fe 3 O 4 Both @ Mal-CpG @4T1 further up-regulates the expression of CD80 and CD86, activating maturation of DCs cells. At the same time prove that Fe 3 O 4 The antigen can be used as a carrier to co-deliver antigen and an adjuvant, and can also be used as an adjuvant to stimulate DCs to mature and improve the utilization rate of the antigen.
Referring to fig. 7, a quantitative comparison chart of in vitro capture antigens of nanoparticles prepared in example one and example three is shown. Example three Fe in step (e) 3 O 4 The addition amount of the @ Mal CpG nanomaterial is Fe in the first embodiment 3 O 4 Double the addition of the @ Mal CpG nanomaterial, FIG. 7 is seen as Fe 3 O 4 An increase in the @ Mal-CpG nanomaterial, which captures an increased antigen.
The invention also provides an in-situ vaccine for medical diagnosis application, which is prepared by the in-situ vaccine preparation method.
In conclusion, the preparation method of the in-situ vaccine is green, simple and easy to operate. The preparation method of the in-situ vaccine is suitable for various tumors and has universality. The maleimide on the surface of the nano material @ Mal can form a stable thioether bond with the sulfhydryl group on the protein so as to capture tumor-related antigens, so that the antigen utilization rate is improved, and the co-delivery of the adjuvant and the antigen can be realized. The nano material @ Mal itself can be used as an adjuvant to activate antigen presenting cells and enhance phagocytosis thereof, and can further activate T cells to improve the safety and curative effect of cancer immunotherapy. Nanomaterial @ Mal is also applicable to medical diagnostics.
The technical features and the detection items of the above-described embodiments may be arbitrarily combined, and for brevity of description, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, they should be regarded as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (7)
1. An in situ vaccine preparation method, which is characterized by comprising the following steps:
s1, culturing: resuscitating and culturing tumor cells in the culture solution to reach logarithmic growth phase, collecting cells by using digestive juice and cleaning to obtain tumor cell liquid;
s2, inactivating: performing inactivation treatment on the tumor cell liquid collected in the step S1, incubating the tumor cell liquid after the inactivation treatment, and centrifuging the incubated tumor cell liquid to obtain supernatant fluid to obtain an inactivated antigen stock solution;
s3, preparing a nano material@Mal solution: mixing nano materials and PEG-Mal ligand according to the mass ratio of 1:10-1:15, dissolving to form mixed solution, adding cyclohexane with the volume of 10-15 times of the mixed solution to precipitate the mixed solution, centrifuging to remove supernatant fluid to obtain precipitate, drying the precipitate, adding ultrapure water to dissolve the precipitate, centrifuging and passing through a membrane, ultrafiltering to obtain nano material@Mal solution, and preserving at 2-6 ℃ for later use;
s4, synthesizing nano material @ Mal-adjuvant particles: adding an adjuvant solution into the nano material@Mal solution prepared in the step S3, stirring at room temperature, performing ultrafiltration to obtain synthesized nano material@Mal-adjuvant particles, and preserving at 2-6 ℃ for later use;
s5, preparing a vaccine: mixing the antigen stock solution prepared in the step S2 with the nano material@Mal-adjuvant particles synthesized in the step S4 according to the volume ratio of 5:1-5:3, incubating and ultrafiltering to obtain the nano material@Mal adjuvant@antigen;
the ultrafiltration in the step S3 uses an ultrafiltration tube with the molecular weight cut-off of 30k, and the rotating speed is 3000-5000rpm;
the nano material is Fe 3 O 4 The adjuvant is CPG.
2. The method of claim 1, wherein the digestive juice in step S1 is a pancreatin solution with a mass fraction of 0.2%.
3. The method for preparing an in-situ vaccine according to claim 1, wherein the inactivation treatment in the step S2 is at least one of a microwave cell inactivation method, an electromagnetic wave cell inactivation method, a laser cell inactivation method, a high temperature cell inactivation method, an ultrasonic disruption cell inactivation method, a repeated cell freeze thawing, a homogenization, centrifugation, a precipitation cell inactivation method, and an enzymatic digestion cell inactivation method.
4. The method for preparing an in-situ vaccine according to claim 3, wherein the inactivation treatment in the step S2 is performed by using X-ray irradiation.
5. The method for preparing an in-situ vaccine according to claim 1, wherein the nanomaterial in step S3 is an oil phase nanomaterial and the nanomaterial @ Mal solution is an aqueous phase.
6. The method of claim 1, wherein the incubation period in step S5 is 2-6h.
7. An in situ vaccine for tumor therapeutic applications prepared by the in situ vaccine preparation method of any one of claims 1 to 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111119339.XA CN113769075B (en) | 2021-09-24 | 2021-09-24 | In-situ vaccine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111119339.XA CN113769075B (en) | 2021-09-24 | 2021-09-24 | In-situ vaccine and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113769075A CN113769075A (en) | 2021-12-10 |
| CN113769075B true CN113769075B (en) | 2023-11-03 |
Family
ID=78852953
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111119339.XA Active CN113769075B (en) | 2021-09-24 | 2021-09-24 | In-situ vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113769075B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116421738B (en) * | 2023-02-28 | 2024-03-19 | 中国医科大学附属第一医院 | Oncolytic virus for capturing antigen and pharmaceutical composition, preparation and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110898215A (en) * | 2019-12-06 | 2020-03-24 | 郑州大学 | Preparation method and application of anti-tumor vaccine based on cell microvesicles |
| WO2020250153A1 (en) * | 2019-06-11 | 2020-12-17 | Pharmaexceed S.R.L. | Nanoparticles for use in the redirection against the tumour of a non-tumour-specific immune response, based on a pre-existing immunity |
-
2021
- 2021-09-24 CN CN202111119339.XA patent/CN113769075B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020250153A1 (en) * | 2019-06-11 | 2020-12-17 | Pharmaexceed S.R.L. | Nanoparticles for use in the redirection against the tumour of a non-tumour-specific immune response, based on a pre-existing immunity |
| CN110898215A (en) * | 2019-12-06 | 2020-03-24 | 郑州大学 | Preparation method and application of anti-tumor vaccine based on cell microvesicles |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113769075A (en) | 2021-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111658767B (en) | Hydrophilic antigen and/or hydrophobic antigen vaccine delivery system and preparation method thereof | |
| CN109666695B (en) | Targeted integrin alphavbeta 3 exosome vector and preparation method and application thereof | |
| CN111346236B (en) | Tumor antigen-loaded polydopamine nanoparticle and preparation method and application thereof | |
| US11357843B2 (en) | Bacteriologically-modified whole-cell tumor vaccine and method of making same | |
| CN107456575A (en) | A kind of manganese dioxide nano adjuvant and preparation method thereof, application | |
| CN104257631A (en) | Magnetic carrier micro-encapsulated stem cell and preparation method thereof | |
| CN113769075B (en) | In-situ vaccine and preparation method thereof | |
| CN113493223A (en) | Preparation method and application of hollow manganese dioxide nanospheres | |
| CN110613844A (en) | Mini combined adjuvant nanoparticle and preparation method and application thereof | |
| CN113663062A (en) | Tumor vaccine based on injectable hydrogel and preparation method and application thereof | |
| CN110755607B (en) | Zinc oxide and antigen co-drug-loaded nano vaccine, and preparation method and application thereof | |
| CN110101853B (en) | Dandelion type heterogeneous nano vesicle and application thereof | |
| CN112807289B (en) | Method for modifying surface of living cell based on nano-particles and nano-particles used by method | |
| CN115252645A (en) | Application of arsenical protein nano preparation in aspects related to tumor immune synergistic treatment | |
| CN114225029A (en) | Sound-sensitive response nanoparticle and application thereof | |
| CN108531514B (en) | Endogenous hyperbranched polyspermine cationic gene vector and preparation method and application thereof | |
| CN1800408B (en) | Nucleic acid transferring vector and transferring system and uses | |
| CN111617054B (en) | Nano acoustic genetics sensitive microsphere and preparation method and application thereof | |
| CN111407897B (en) | Avidin nanoparticle based on co-combination of fluorescent dye and Coomassie brilliant blue, preparation method thereof and application thereof in construction of tumor vaccine | |
| CN113995852B (en) | Arg-liposome microcapsule, microencapsulated protamine-siRNA complex, and preparation method and application thereof | |
| CN110522906B (en) | Preparation method of multidimensional, multivalent and prostate cancer specific tumor antigen assembly | |
| CN117503731B (en) | Nanocapsule method of umbilical blood mesenchymal stem cell source exosome | |
| CN114053433B (en) | Stimulus response type nano material and application thereof in preparation of in-situ tumor vaccine | |
| CN114177283A (en) | Magnetic drive nanometer motor Fe3O4/Ca@MnCO3Preparation of (A) and its use in DC vaccines | |
| CN114288408B (en) | Double-adjuvant self-carrier in-situ nano vaccine and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |