WO2023087441A1 - 一种预防或治疗癌症的疫苗系统及其应用 - Google Patents

一种预防或治疗癌症的疫苗系统及其应用 Download PDF

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WO2023087441A1
WO2023087441A1 PCT/CN2021/137431 CN2021137431W WO2023087441A1 WO 2023087441 A1 WO2023087441 A1 WO 2023087441A1 CN 2021137431 W CN2021137431 W CN 2021137431W WO 2023087441 A1 WO2023087441 A1 WO 2023087441A1
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water
vaccine
cancer
components
cells
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French (fr)
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刘密
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苏州尔生生物医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the technical field of immunotherapy, in particular to a vaccine system for preventing or treating cancer and its application.
  • Immunity refers to the function of the body's immune system to recognize self and foreign substances, and eliminate antigenic foreign substances through immune response, so as to maintain the body's physiological balance. Immunotechnology has developed rapidly in recent years, especially in the field of cancer immunotherapy. Cancer vaccines are one of the important approaches in cancer immunotherapy and prevention. The basis for developing cancer vaccines is to select appropriate cancer antigens to activate the human immune system to recognize abnormally mutated cancer cells, and cancer cells or cancer tumor tissues themselves are the best source of cancer antigens. Such as Wang Dandan et al. (Research progress of MUC-1 cancer vaccine in the treatment of non-small cell lung cancer[J].
  • MUC-1 mucin-1
  • NSCLC non-small cell lung cancer
  • the methods used are to extract and analyze the differences between cancer cells and normal cells from a small part of cancer cells in tumor tissue, and then look for different polypeptides, but the cancer cells in different parts of the tumor tissue and the heterogeneity of each cancer cell are very large, so this type of method and technology can only find a limited number of antigenic polypeptides, which greatly limits the application of this type of method.
  • the present invention provides a micro or nano vaccine system loaded with water-soluble components or water-insoluble components in the whole cell components of one or more cancer cells and/or tumor tissues, simplifying The preparation process of cancer vaccine is improved, and it still has better therapeutic effect.
  • a vaccine system for preventing or treating cancer of the present invention comprising delivery particles and cell components loaded therein, the delivery particles are nanoparticles or microparticles, and the cell components are derived from one or more cancer cells Water-soluble components or a mixture of water-soluble components in the whole cell components of cells and/or tumor tissues, or non- A mixture of water soluble or water insoluble components.
  • the water-soluble components or a mixture of water-soluble components in the whole cell components derived from one or more cancer cells and/or tumor tissues are loaded inside or on the surface of the delivery particles modified with target heads , or a mixture of water-insoluble components or water-insoluble components in whole cell components derived from one or more cancer cells and/or tumor tissues.
  • the present invention uses cancer cells or cancer tissue
  • the water-soluble components or water-insoluble components of the whole cell components are very promising methods as the source of vaccines for the prevention and treatment of cancer.
  • the vaccine system composed of delivery particles containing target heads on the surface and loaded with water-soluble components is used to prevent or treat cancer
  • the effect achieved is better than that of loading water-soluble components and water-insoluble components.
  • the delivery particles greatly optimize the therapeutic effect of water-soluble components, and are more suitable for clinical application. Compared with loading water-soluble and non-water-soluble components at the same time, the pain during treatment is significantly reduced, and the operation is more convenient.
  • the target head is a small molecular compound, antibody, polypeptide, carbohydrate, lipid, or nucleic acid that can target specific cells.
  • the vaccine system composed of mannose and DEC205 antibody is listed as the target head. In practical application, it can also be used including but not limited to CD32 antibody, CD11c antibody, CD103 antibody, CD44 antibody, and CD40 antibody.
  • the target head targets dendritic cells, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils or basophils, which are mostly present in lymph nodes , thymus, spleen or bone marrow.
  • freeze the cells or tissues containing cancer-related antigens at -20°C to -273°C add water or an aqueous solution without a solubilizer, and freeze and thaw the cells repeatedly.
  • the supernatant is a water-soluble component
  • the precipitate is The soluble part after solubilization is the water-insoluble component.
  • solubilizer used for solubilization is selected from urea, guanidine hydrochloride, sodium deoxycholate, sodium dodecyl sulfate (SDS), glycerin, protein degrading enzymes, albumin, lecithin, 0.1-2000mg/mL inorganic Salt, Triton, Tween, acetic acid, cholesterol, amino acid, glycoside, choline, Brij TM -35, octaethylene glycol monododecyl ether, 3-[3-(cholamidopropyl) dimethyl Amino]propanesulfonic acid inner salt (CHAPS), digitonin (Digitonin), lauryldimethylamine oxide (lauryldimethylamine oxide), One of CA-630, dimethyl sulfoxide (DMSO), acetonitrile, ethanol, methanol, N,N-dimethylformamide (DMF), isopropanol, methylene chloride, propyl
  • the interior or surface of the delivery particle is loaded with immunopotentiators, including but not limited to immunopotentiators derived from microorganisms, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, etc.
  • immunopotentiators including but not limited to immunopotentiators derived from microorganisms, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, etc.
  • the immune enhancing adjuvants include but not limited to pattern recognition receptor agonists, Bacillus Calmette-Guerin (BCG), BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl di Peptides, Mycobacterium phlei, Polyantisin A, Mineral oil, Virus-like particles, Reconstructed influenza virions for immune enhancement, Cholera enterotoxin, Saponins and their derivatives, Resiquimod, Thymosin, Neonatal bovine liver active peptide, Rice Quimod, polysaccharide, curcumin, immune adjuvant CpG, immune adjuvant poly(I:C), mRNA, immune adjuvant poly ICLC, Corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, poly Cytidylic acid, interleukin, interferon, polyinosinic acid, polyadenylic acid
  • the water-soluble component or a mixture of water-soluble components, the water-insoluble component or a mixture of water-insoluble components are loaded on the interior and/or surface of the delivery particle.
  • the delivery particles are prepared from organic synthetic polymer materials, natural polymer materials or inorganic materials.
  • Organic synthetic polymer materials are polylactic acid-glycolic acid copolymer PLGA, polylactic acid PLA, polyglycolic acid PGA, polyethylene glycol PEG, polycaprolactone PCL, Poloxamer, polyvinyl alcohol PVA, polyvinyl pyrrolidone PVP, polyetherimide PEI, polytrimethylene carbonate PTMC, polyanhydride, polydioxanone PDON, polydioxanone PPDO, polymethyl methacrylate PMMA, polyamino acid, synthetic Polypeptides or synthetic lipids; natural polymer materials are lecithin, cholesterol, sodium alginate, albumin, collagen, gelatin, cell membranes (including whole cell membrane components or partial cell membrane components), exosomes, polypeptides, starch or Sugars; inorganic materials are materials without obvious biological toxicity such as ferric oxide, ferric oxide, calcium carbonate or calcium
  • the delivery particles are spherical, ellipsoidal, barrel-shaped, polygonal, rod-shaped, sheet-shaped, linear, worm-shaped, square, triangular, butterfly-shaped or disc-shaped.
  • the particle diameter of the nanoscale particles is 1 nm-1000 nm, and the particle diameter of the micron-scale particles is 1 ⁇ m-1000 ⁇ m.
  • This particle size range ensures that the vaccine can be phagocytized by antigen-presenting cells, and in order to improve the phagocytosis efficiency, the particle size should be within an appropriate range.
  • the nano-scale particle size is 30nm-1000nm, most preferably 100nm-600nm ;
  • the particle size of the micron-sized particles is 1 ⁇ m-10 ⁇ m, most preferably 1 ⁇ m-5 ⁇ m.
  • the delivery particles can be uncharged, negatively charged or positively charged.
  • the delivery particles can be prepared according to the developed preparation methods, including but not limited to solvent evaporation method, dialysis method, extrusion method and hot melt method.
  • the delivery particles are prepared by the double emulsion method in the solvent evaporation method, and the specific steps are as follows:
  • the aqueous phase solution is a solution containing cancer cell or tumor cell lysate ( containing or not containing immunopotentiator), the lysate contains water-soluble components or the original water-insoluble components dissolved in the solubilizer;
  • the organic phase solution is organic synthetic polymer materials, natural polymer materials or inorganic materials dissolved in Solutions obtained in organic solvents;
  • step (2) the mixed solution obtained in step (1) is nanosized or micronized;
  • step (3) adding the mixture obtained in step (2) into a third predetermined volume of an aqueous solution containing an emulsifier with a third predetermined concentration and performing nanometerization or micronization;
  • step (3) (4) adding the liquid obtained in step (3) into a fourth predetermined volume of an emulsifier aqueous solution of a fourth predetermined concentration, and stirring until a predetermined stirring condition is met;
  • step (4) After centrifuging or ultrafiltering the mixed solution obtained in step (4), take the precipitate or ultrafiltration product and mix it with the water-soluble component or the original water-insoluble component dissolved in the solubilizer to obtain the nano-vaccine or micron vaccines.
  • step (5) After centrifugation in step (5), resuspend the precipitate in an aqueous solution containing a lyoprotectant of a fifth predetermined concentration or in PBS (or physiological saline);
  • the first predetermined concentration requires that the protein polypeptide concentration be greater than 0.01ng/mL, preferably 1mg/mL-100mg/mL, because when the protein polypeptide concentration is greater than 0.01ng/mL, enough cancer antigens can be loaded to activate the relevant immune response.
  • concentration of the immune adjuvant in the initial aqueous phase is greater than 0.001 pg/mL, preferably 0.01 mg/mL-20 mg/mL.
  • the organic solvent can be DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate, etc., preferably dichloromethane.
  • the second predetermined concentration is 0.5 mg/mL-5000 mg/mL, preferably 100 mg/mL.
  • the organic synthetic polymer material is preferably PLGA, which has a certain immune regulation function and is suitable as an auxiliary material for vaccine preparation.
  • step (1) the ratio of the first predetermined volume to the second predetermined volume is 1:1.1-5000, preferably 1:10.
  • nanonization or micronization is carried out by ultrasonication, stirring, homogeneous treatment or microfluidic control, and the time, speed and pressure can control the size of the prepared delivery particles.
  • the ultrasonic power is 50W-500W, and the time is greater than 0.1 seconds, such as 2-200 seconds; when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, and the stirring time is greater than or equal to 1 minute, such as 50rpm-500rpm , the mixing time is 60-6000 seconds; use a high-pressure/ultra-high-pressure homogenizer or a high-shear homogenizer for homogenization, the pressure is greater than 20psi, such as 20psi-100psi, and the speed is greater than 100rpm when using a high-shear homogenizer. For example, 1000rpm-5000rpm; the microfluidic flow rate is greater than 0.01mL/min, such as 0.1mL/min-100
  • the emulsifier aqueous solution is polyvinyl alcohol (PVA) aqueous solution
  • the third predetermined concentration is 10-50 mg/mL, preferably 20 mg/mL.
  • the fourth predetermined concentration is 1-20 mg/mL, preferably 5 mg/mL.
  • the third predetermined volume is adjusted according to its ratio to the second predetermined volume. In the present invention, the ratio of the second predetermined volume to the third predetermined volume is 1:1.1-1000, preferably 2:5.
  • the ratio of the third predetermined volume to the fourth predetermined volume is 1:1.5-2000, preferably 1:10.
  • the predetermined stirring condition is until the organic solvent volatilization is completed.
  • the lyoprotectant is preferably trehalose.
  • the fifth predetermined concentration is 1-15% by mass, in order not to affect the freeze-drying effect in subsequent freeze-drying.
  • the present invention also claims the application of the above-mentioned vaccine system in the preparation of medicines for preventing or treating cancer.
  • At least one cancer cell or tumor tissue in the vaccine system is the same as the type of disease that the drug prevents or treats.
  • the vaccine described in the present invention can be used before or after cancer occurs. It is administered multiple times after surgery or surgical removal of tumor tissue to activate the immune system to slow the progression of cancer, treat cancer, or prevent cancer from recurring.
  • the present invention has at least the following advantages:
  • the invention provides a vaccine system for delivering cell water-soluble components or non-water-soluble components by using nanometer or micrometer particles, as well as its application in preparing vaccines for preventing and treating cancer.
  • Figures 1-9 are the experimental results of using nano-vaccine or micro-vaccine to prevent or treat cancer in Examples 1-9; wherein, a, the experimental results of tumor growth rate when nano-vaccine or micro-vaccine is preventing or treating cancer (n ⁇ 8) ; b, the experimental results of the survival period of mice when nano-vaccine or micro-vaccine prevents or treats other cancers (n ⁇ 8), each data point is the mean ⁇ standard error (mean ⁇ SEM); tumor growth inhibition experiment in the figure a
  • the significant difference in the figure is analyzed by ANOVA method, and the significant difference in b is analyzed by Kaplan-Meier and log-rank test; ** indicates that there is a significant difference at p ⁇ 0.005 between the vaccine group and the PBS blank control group; ## represents Compared with the blank nanoparticle + free lysate component control group, there is a significant difference at p ⁇ 0.005 between the vaccine group and the PBS blank control group; *** indicates that there is a significant difference at p ⁇ 0.0005 between the
  • Example 1 The water-soluble component in the whole cell fraction of melanoma tumor tissue is loaded inside and on the surface of nanoparticles for the prevention of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a nano-vaccine loaded with water-soluble components in the whole cell fraction of melanoma tumor tissue, and apply the vaccine to prevent melanoma.
  • B16F10 mouse melanoma was used as a cancer model.
  • B16F10 melanoma tumor tissues were first lysed and water-soluble fractions were collected.
  • the organic polymer material PLGA was used as the nanoparticle framework material
  • Polyinosinic-polycytidylic acid (poly(I:C)) was used as the immune adjuvant to prepare nanovaccine loaded with water-soluble components of tumor tissue by solvent evaporation method. The nanovaccine was then used to prevent melanoma.
  • B16-F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse, and the mice were sacrificed when the tumors grew to a volume of about 1000 mm 3 , and the tumor tissues were harvested. After the tumor tissue was cut into pieces, it was ground, and an appropriate amount of pure water was added through a cell strainer, followed by repeated freezing and thawing 5 times, accompanied by ultrasound to destroy and lyse the cells. After the cells are lysed, the lysate is centrifuged at a speed greater than 1000g for 5 minutes, and the supernatant is taken as the water-soluble component soluble in pure water.
  • this embodiment adopts the water-soluble polypeptide B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD) and B16-M27 (REGVELCPGNKYEMRRHGTTHSLVIHD) loaded in equal proportions.
  • the nano-vaccine, the blank nano-particles used as the control, and the nanoparticles loaded with various polypeptides are prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nano-particle preparation material PLGA used is 24KDa-38KDa.
  • the adjuvant is poly(I:C) and poly(I:C) is only distributed inside the nanoparticles.
  • the preparation method is as described above.
  • the average particle size of the nano-vaccine loaded with water-soluble components in the whole cell component is about 310nm, and the surface potential of the nano-vaccine is about -6mV; each 1mg of PLGA nanoparticles is loaded with about 170 ⁇ g of protein or polypeptide components, and each 1mg of PLGA nanoparticles is used inside and outside
  • the total poly(I:C) immune adjuvant is about 0.02mg.
  • the particle size of the blank nanoparticles is about 250nm. When preparing the blank nanoparticles, pure water or 8M urea containing the same amount of poly(I:C) was used to replace the corresponding water-soluble components.
  • the preparation method of polypeptide-loaded nanoparticles is the same as the preparation method of preparing loaded whole cell components, and the amount of poly(I:C) used is also the same.
  • the particle size of polypeptide nanoparticles is about 305nm, and each 1mg of PLGA nanoparticles loads about 160 ⁇ g polypeptide components.
  • control groups in this study were the PBS group and the blank nanoparticles + free water-soluble group.
  • the administration regimen of the nanovaccine group was as follows: 400 ⁇ L of 4 mg PLGA nanovaccine loaded with water-soluble components were subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the protocol of the PBS control group was as follows: 400 ⁇ L of PBS was injected subcutaneously on the 49th, 42nd, 35th, 28th and 14th day before melanoma inoculation. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right back of each mouse. Blank nanoparticles + cell lysate control group: subcutaneously inject 400 ⁇ L of blank nanoparticles and the same amount of free water-soluble components as the vaccine load on days 49, 42, 35, 28, and 14 before inoculation of melanoma . Blank nanoparticles and free water-soluble fractions were injected at different sites. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the dosage regimen of the polypeptide nanoparticle group was as follows: 200 ⁇ L of 2 mg PLGA nanoparticles loaded with water-soluble polypeptide components on the inside and on the surface were subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. particle. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the size of the mouse tumor volume was recorded every 3 days from the 3rd day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • the nano-vaccine loaded with water-soluble components of various tumor tissues according to the present invention has a good preventive effect on melanoma.
  • Example 2 Water-soluble components of melanoma and colon cancer cancer cells loaded inside and on the surface of microparticles for the prevention of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a micron vaccine loaded with water-soluble components of melanoma and colon cancer cells, and apply the vaccine to prevent melanoma.
  • B16F10 melanoma and MC38 colon cancer cells were lysed to prepare water-soluble components.
  • the organic macromolecule material PLGA is used as the framework material of the micron particle
  • the CpG is used as the immune adjuvant to prepare the micron vaccine loaded with the water-soluble component of the whole cell by solvent evaporation method.
  • the micron vaccine was then used to prevent melanoma.
  • Collect a certain amount of B16F10 cells or MC38 cells remove the medium and freeze at -20°C to -273°C, add a certain amount of ultrapure water and freeze-thaw more than 3 times repeatedly, and can be accompanied by ultrasound to destroy and lyse the cells.
  • the lysate was centrifuged at a speed of 3000g for 5 minutes to obtain the supernatant, which was the water-soluble fraction in the LLC lung cancer cells that could be dissolved in pure water.
  • the above-mentioned water-soluble components derived from two kinds of cancer cell lysates are mixed at a ratio of 1:1, which is the antigen source for preparing micron vaccines.
  • the preparation of micron vaccines and blank micron particles as a control adopts the double emulsion method in the solvent volatilization method.
  • the micron particle preparation material used is an organic polymer material PLGA with a molecular weight of 38KDa-54KDa, and the immune adjuvant used is CPG and CPG are both distributed inside the microparticles and loaded on the surface of the microparticles.
  • the preparation method is as described above.
  • the particle size of the micron vaccine obtained after loading the cell component and the immune adjuvant on the surface of the micron particle is about 1.80 ⁇ m, and the average surface potential of the micron particle is about -5mV.
  • Each 1mg of PLGA microparticles is loaded with 220 ⁇ g of protein or polypeptide components, and the CPG immune adjuvant used inside and outside of each 1mg of PLGA microparticles is 0.02mg, and the inside and outside are divided in half.
  • the particle size of the blank microparticles is about 1.70 ⁇ m, and the corresponding water-soluble components are replaced by pure water containing the same amount of CPG when the blank microparticles are prepared.
  • the micron vaccine group’s protocol was as follows: 400 ⁇ L of 4 mg PLGA micron vaccine loaded with cancer water-soluble components were subcutaneously injected on the 28th day, the 21st day, the 14th day and the 7th day before melanoma inoculation. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the protocol of the PBS blank control group was as follows: 400 ⁇ L of PBS was subcutaneously injected on the 28th day, the 21st day, the 14th day and the 7th day before melanoma inoculation.
  • the size of the mouse tumor volume was recorded every 3 days from the 3rd day.
  • the tumor volume growth rate of the mice in the micron vaccine administration group was significantly slower and the survival period of the mice was significantly prolonged .
  • some tumors in mice in the micron vaccine administration group completely disappeared after inoculation. It can be seen that the micro-vaccine of the present invention loaded with water-soluble components of melanoma and colon cancer cells has a preventive effect on melanoma.
  • Example 3 The non-water-soluble component in the whole cell fraction of melanoma tumor tissue is loaded inside and on the surface of nanoparticles for the prevention of melanoma
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a nano-vaccine loaded with water-insoluble components in the whole cell fraction of melanoma tumor tissue, and apply the vaccine to prevent melanoma.
  • B16F10 mouse melanoma cells were used as the cancer model.
  • B16F10 melanoma tumor tissue was first lysed and the original water-insoluble fraction was solubilized with 8M urea.
  • the nano-vaccine loaded with the water-insoluble component of the tumor tissue was prepared by using the organic polymer material PLGA as the nanoparticle framework material and poly(I:C) as the immune adjuvant. The nanovaccine was then employed to prevent melanoma.
  • B16-F10 cells were subcutaneously inoculated on the back of each C57BL/6 mouse, and the mice were sacrificed when the tumors grew to a volume of about 1000 mm 3 , and the tumor tissues were harvested. After the tumor tissue was cut into pieces, it was ground, and an appropriate amount of pure water was added through a cell strainer, followed by repeated freezing and thawing 5 times, accompanied by ultrasound to destroy and lyse the cells. After the cells are lysed, centrifuge the lysate at a speed of 1000g for 5 minutes, remove the supernatant and add 8M urea to the precipitated part to dissolve the precipitated part. Soluble in aqueous solution. It is the source of raw materials for the preparation of vaccines.
  • this embodiment uses the water-insoluble polypeptide B16-M05 (Eef2, FVVKAYLPVNESFAFTADLRSNTGGQA), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDDADQ) loaded with 8M urea solubilized, and TRP2:180-188 (SVYDFFVWL).
  • the nano-vaccine, the blank nano-particles used as the control, and the nanoparticles loaded with various polypeptides are prepared by the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nano-particle preparation material PLGA used is 24KDa-38KDa.
  • the adjuvant is poly(I:C), and poly(I:C) is distributed inside the nanoparticles and loaded on the surface of the nanoparticles.
  • the preparation method is as described above.
  • the average particle size of the nano-vaccine loaded with the non-water-soluble component of the whole cell component is about 310nm, and the surface potential of the nano-vaccine is about -6mV; each 1mg PLGA nanoparticle is loaded with about 170 ⁇ g protein or polypeptide component, and each 1mg PLGA nanoparticle contains
  • the poly(I:C) immune adjuvant used is about 0.02mg in total and half inside and outside.
  • the particle size of the blank nanoparticles is about 260nm, and 8M urea containing the same amount of poly(I:C) is used to replace the corresponding non-water-soluble components during the preparation of the blank nanoparticles.
  • the preparation method of the nanoparticle loaded with polypeptide is the same as the preparation method of preparing the loaded non-water-soluble component, and the amount of poly(I:C) used is also the same.
  • the particle size of the polypeptide nanoparticle is about 320nm, and each 1mg of PLGA nanoparticle is about 160 ⁇ g loaded. Polypeptide components.
  • control groups in this study were the PBS group and the blank nanoparticles + free non-water-soluble component group.
  • the administration regimen of the nanovaccine group was as follows: 4 mg PLGA nanovaccine loaded with 400 ⁇ L of the non-water-soluble components in the lysate was subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before melanoma inoculation. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the protocol of the PBS control group was as follows: 400 ⁇ L of PBS was injected subcutaneously on the 49th, 42nd, 35th, 28th and 14th day before melanoma inoculation. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse. Blank nanoparticles + free non-water-soluble component control group: 400 ⁇ L of blank nanoparticles and the same amount of free non-water-soluble components as the vaccine load were subcutaneously injected on the 49th, 42th, 35th, 28th and 14th days before melanoma inoculation. water soluble components. Blank nanoparticles and free water-insoluble fraction were injected at different sites. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the dosage regimen of the polypeptide nanoparticle group was as follows: 400 ⁇ L of 400 ⁇ L of both the interior and the surface were subcutaneously injected on the 49th day, the 42nd day, the 35th day, the 28th day and the 14th day before inoculation of the melanoma, and the original non-water-soluble protein in 8M urea was injected. Composition of 4mg PLGA nanoparticles. On day 0, 1.5 ⁇ 105 B16F10 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the size of the mouse tumor volume was recorded every 3 days from the 3rd day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • the nano-vaccine loaded with water-insoluble components of tumor tissue according to the present invention has a good preventive effect on melanoma.
  • Example 4 Lung cancer tumor tissue and liver cancer tumor tissue lysate components are non-water-soluble loaded in the interior and surface of micro-particles for the prevention of liver cancer
  • mice the non-water-soluble components of mouse liver cancer and lung cancer tumor tissue lysates were loaded on the inside and surface of nanoparticles at a ratio of 1:2 to prepare micron vaccines.
  • mouse lung cancer and liver cancer tumor tissues were obtained and lysed to prepare the original water-insoluble fraction of the tumor mass tissue dissolved in 6M guanidine hydrochloride.
  • PLGA as the microparticle framework material
  • poly (I:C) as immune adjuvant to prepare micron vaccine.
  • the micron vaccine was then used to prevent tumors in Hepa 1-6 liver tumor-bearing mice.
  • Method is with embodiment 2. However, in this example, a 1:2 mixture of water-insoluble fractions in liver and lung cancer tumor tissue lysates was loaded.
  • Blank microparticles + free non-water-soluble component control group 400 ⁇ L of blank microparticles and vaccine-loaded microparticles were subcutaneously injected on the 49th day, 42nd day, 35th day, 28th day and 14th day before inoculation of liver cancer cells. The same amount of free non-water-soluble components; blank microparticles and free cell lysates were injected at different sites; on day 0, 2 ⁇ 10 6 Hepa 1-6 liver cancer cells were subcutaneously inoculated into the right axilla of each mouse. In the experiment, the size of the mouse tumor volume was recorded every 3 days from the 3rd day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • liver cancer tumors in mice in the PBS control group and the control group of blank microparticles + free non-water-soluble components was faster. 40% of the tumors disappeared in mice in the micron vaccine administration group after tumor inoculation. Moreover, the therapeutic effect of adjuvanted micron vaccines is better than that of non-adjuvanted micron vaccines. It can be seen that the micro-vaccine loaded with non-water-soluble components in the lysate of lung cancer tumor tissue and liver cancer tumor tissue according to the present invention has a preventive effect on liver cancer.
  • Example 5 The water-soluble component in the lysed fraction of pancreatic cancer tumor tissue is loaded inside and on the surface of nanoparticles for the treatment of pancreatic cancer
  • This example uses mouse pancreatic cancer as a cancer model to illustrate how to prepare a nano-vaccine loaded with water-soluble components in pancreatic cancer tumor tissue lysate, and use the vaccine to treat pancreatic cancer.
  • the water-soluble components in mouse Pan02 pancreatic cancer tumor tissue were loaded on the interior and surface of nanoparticles to prepare nanovaccine.
  • mouse pancreatic cancer tumor tissue was obtained and lysed to prepare water-soluble fractions.
  • nanovaccine was prepared by solvent evaporation method. The nanovaccine was then used to treat tumors in Pan02 pancreatic cancer-bearing mice.
  • Each C57BL/6 mouse was subcutaneously inoculated with 1 ⁇ 10 6 Pan02 pancreatic cancer cells in the armpit.
  • the inoculated tumors in each mouse grew to a volume of about 1000 mm 3 , the mice were sacrificed and the tumor tissues were harvested.
  • the lysing method of cancer cells and the collection method of each component are the same as above.
  • the preparation method of the nano-vaccine is the same as above.
  • Pan02 cells were subcutaneously inoculated into the lower right lower back of each mouse.
  • the vaccine group received subcutaneous injections of 400 ⁇ L of 4 mg PLGA nanovaccine on days 4, 7, 10, 15 and 20, respectively.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L of PBS on the 4th day, 7th day, 10th day, 15th day and 20th day.
  • Blank nanoparticles + water-soluble component control group were subcutaneously injected with 400 ⁇ L of blank nanoparticles and the same amount of free water-soluble components loaded with the vaccine on the 4th day, 7th day, 10th day, 15th day and 20th day. point.
  • the size of the tumor volume of the mice was recorded every 3 days from the 3rd day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • the tumor growth rate of the nano-vaccine group was significantly slower and the survival period of the mice was significantly prolonged. Moreover, tumors in some mice disappeared after inoculation. It can be seen that the nano-vaccine loaded with water-soluble components in the pancreatic cancer tumor tissue lysate of the present invention has a therapeutic effect on pancreatic cancer.
  • Example 6 Water-soluble components of lung cancer tumor tissue loaded inside mannose-modified microparticles for the prevention of lung cancer
  • This example uses mouse lung cancer as a cancer model to illustrate how to prepare a micron vaccine loaded with water-soluble components in the whole cell fraction of lung cancer tumor tissue, and apply the vaccine to prevent lung cancer.
  • the specific dosage form, adjuvant, administration time, administration frequency, and administration regimen can be adjusted according to the situation.
  • the tumor tissue of mouse lung cancer was first obtained and lysed to prepare the water-soluble fraction. Then, using PLGA and mannose-modified PLGA as the microparticle framework material, and CpG as the immune adjuvant, the microvaccine was prepared by the solvent evaporation method.
  • the micron vaccine has the ability to target dendritic cells.
  • Each C57BL/6 mouse was subcutaneously inoculated with 2 ⁇ 10 6 LLC lung cancer cells in the armpit, and the mice were sacrificed when the inoculated tumor grew to 1000 mm 3 and the tumor tissue was removed.
  • Other processing methods are the same as above.
  • the micronized vaccine and the empty micronized particles used as the control adopt the double emulsion method in the solvent volatilization method.
  • (50:50) molecular weight is 38KDa-54KDa.
  • the mass ratio of unmodified PLGA to mannose-modified PLGA is 8:2.
  • the immune adjuvant used is CpG and CpG is distributed inside the microparticles.
  • the preparation method is as mentioned above.
  • the average particle size of the micron particles is about 1.90 ⁇ m, and the average surface potential is about -8mV.
  • Each 1 mg PLGA nanoparticle is loaded with 75 ⁇ g of protein or polypeptide components, and the CpG immune adjuvant used in each 1 mg PLGA microparticle is 0.06 mg.
  • the particle size of the blank microparticles is about 1.85 ⁇ m, and the corresponding water-soluble components are replaced by pure water containing the same amount of CpG when the blank microparticles are prepared.
  • the vaccine group was subcutaneously injected with 400 ⁇ L of 4 mg nano-vaccine loaded with water-soluble components on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L PBS on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • Blank nanoparticles + free water-soluble component control group were subcutaneously injected with 400 ⁇ L of blank nanoparticles and the same amount of vaccine-loaded Free water soluble components.
  • Example 7 The water-soluble components in the whole cell fraction of liver cancer tumor tissue are loaded on the inside and surface of nanoparticles and the nano-vaccine with Bacillus Calmette-Guerin (BCG) as an immune adjuvant is used for the prevention of liver cancer
  • BCG Bacillus Calmette-Guerin
  • This example uses mouse liver cancer as a cancer model and uses water-soluble components in BCG as an immune adjuvant to illustrate how to prepare a nano-vaccine loaded with water-soluble components in the whole cell fraction of liver cancer tumors and apply the vaccine to prevent liver cancer.
  • the liver cancer tumor tissue was firstly lysed and the water-soluble components were collected. Then, the nano-vaccine was prepared by solvent evaporation using PLGA as the nanoparticle framework material and BCG as the immune adjuvant.
  • Lysis of tumor tissue and collection of lysates in this embodiment are the same as above.
  • the method for lysing BCG and collecting and solubilizing the lysate in this embodiment is the same as the method for lysing cancer cells in Example 1, except that the cancer cells are replaced with BCG.
  • Example 1 of the preparation method of the nano-vaccine in this example was replaced by the water-soluble component of BCG from poly(I:C).
  • the vaccine group was subcutaneously injected with 400 ⁇ L of 4 mg PLGA nanovaccine loaded with water-soluble components on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L PBS on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • Blank nanoparticles + free water-soluble component control group were subcutaneously injected with 400 ⁇ L of blank nanoparticles and the same amount of vaccine-loaded Free water soluble components.
  • the tumor growth rate of the nanovaccine administration group with BCG as adjuvant was significantly slower and the survival period of mice was significantly prolonged. It can be seen that the nano-vaccine loaded with water-soluble components in the whole cell components of liver cancer tumor tissue according to the present invention can prevent liver cancer.
  • Example 8 Water-soluble components of lung cancer cancer cells loaded inside DEC205 antibody-modified nanoparticles for the prevention of lung cancer
  • This example uses mouse lung cancer as a cancer model to illustrate how to prepare a nano-vaccine loaded with water-soluble components in the whole cell components of lung cancer cells, and apply the vaccine to prevent lung cancer.
  • the specific dosage form, adjuvant, administration time, administration frequency, and administration regimen can be adjusted according to the situation.
  • mouse LLC lung cancer cells were first lysed to prepare water-soluble fractions. Then, PLGA and DEC205 antibody-modified PLGA were used as nanoparticle framework materials, and CpG was used as immune adjuvant to prepare nanovaccine by solvent evaporation method.
  • the nanovaccine has the ability to target dendritic cells.
  • the methods for collecting, lysing and collecting water-soluble components of LLC lung cancer cells are the same as above.
  • the nano-vaccine and the empty nanoparticles used as a control are prepared by the double emulsion method in the solvent evaporation method, and the molecular weight of the nano-particle preparation material PLGA (50:50) used is 24KDa-38KDa, and the PLGA modified by the DEC205 antibody is used. (50:50) molecular weight is 24KDa-38KDa.
  • the mass ratio of unmodified PLGA to DEC205 antibody-modified PLGA was 8:2.
  • the immune adjuvant used is MnCl 2 and the MnCl 2 is distributed inside the nanoparticles.
  • the preparation method is as described above.
  • the average particle size of the nanoparticles is about 300nm.
  • Each 1mg PLGA nanoparticle is loaded with 55 ⁇ g protein or polypeptide components, and the MnCl 2 immune adjuvant used in each 1mg PLGA nanoparticle is 0.06mg.
  • the particle size of the blank nanoparticles is about 280nm, and the corresponding water-soluble components are replaced by pure water containing the same amount of MnCl 2 when preparing the blank nanoparticles.
  • the vaccine group was subcutaneously injected with 400 ⁇ L of 4 mg nano-vaccine loaded with water-soluble components on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L PBS on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • Blank nanoparticles + free water-soluble component control group were subcutaneously injected with 400 ⁇ L of blank nanoparticles and the same amount of vaccine-loaded Free water soluble components.
  • the tumor growth rate of the mice in the nanovaccine group was significantly slower and the survival period of the mice was significantly prolonged. This shows that the active targeting nano-vaccine loaded with water-soluble components in lung cancer cancer cells of the present invention has a preventive effect on lung cancer.
  • Example 9 Water-soluble components of colon cancer tumor tissue and cancer cells loaded inside DEC205 antibody-modified nanoparticles for the prevention of colon cancer
  • This example uses mouse colon cancer as a cancer model to illustrate how to prepare a nano-vaccine loaded with water-soluble components in colon cancer tumor tissue and whole cell components of cancer cells, and apply the vaccine to prevent colon cancer.
  • the specific dosage form, adjuvant, administration time, administration frequency, and administration regimen can be adjusted according to the situation.
  • the tumor tissue of mouse colon cancer and the cultured cancer cells were first obtained and lysed to prepare a water-soluble component mixture and a water-insoluble component mixture. Then, PLGA and DEC205 antibody-modified PLGA were used as nanoparticle framework materials, and CpG was used as immune adjuvant to prepare nanovaccine by solvent evaporation method.
  • the nanovaccine has the ability to target dendritic cells.
  • the mice are sacrificed and the tumor tissue is removed.
  • the method of lysing the tumor tissue is the same as above. After cracking with ultrapure water, centrifuge at 5000 g for 5 minutes, collect the supernatant to be the water-soluble component, and use 8M urea to dissolve the precipitated part, which is the original water-insoluble component.
  • the method of collecting MC38 cells obtained from cell culture is to discard the medium supernatant after centrifugation, wash and centrifuge twice with PBS, add ultrapure water to the cells collected by centrifugation, and freeze and thaw repeatedly at -20°C for more than 5 times to lyse the cancer cells. After water splitting, centrifuge at 5000g for 5 minutes, collect the supernatant, which is the water-soluble component, and use 8M urea to dissolve the precipitated part, which is the original water-insoluble component.
  • the water-soluble components of tumor tissue and the water-soluble components of cancer cells are mixed at a mass ratio of 1:1 to be the water-soluble component mixture for preparing nano vaccines; the water-insoluble components of tumor tissue and cancer cells are mixed The water-insoluble components are mixed in a mass ratio of 1:1 to be the water-insoluble component mixture for preparing nano vaccines.
  • the nanoparticle preparation material PLGA (50:50) used in the experiment has a molecular weight of 24KDa-38KDa
  • the DEC205 antibody-modified PLGA (50:50) has a molecular weight of 24KDa-38KDa.
  • the mass ratio of unmodified PLGA to DEC205 antibody-modified PLGA was 8:2.
  • the targeted nano-vaccine is only loaded with a mixture of water-soluble components of tumor tissue and cancer cells; the immune adjuvant used is CpG and CpG is distributed inside the nanoparticles; the average particle size of the nano-vaccine is about 290nm; each 1mg of PLGA nanoparticles is loaded with 50 ⁇ g Protein or polypeptide components; the CpG immune adjuvant used in every 1mg of PLGA nanoparticles is 0.04mg.
  • the nano-vaccine without a target head is loaded with a mixture of tumor tissue and cancer cell water-soluble components or a mixture of water-insoluble components; the immune adjuvant used is CpG and CpG is distributed inside the nanoparticles; the average particle size of the nano-vaccine is 290nm About; 50 ⁇ g of protein or polypeptide components are loaded per 1 mg of PLGA nanoparticles; the CpG immune adjuvant used in each 1 mg of PLGA nanoparticles is 0.04 mg.
  • the particle size of the blank nanoparticles is about 270nm, and the solutions containing the same amount of CpG are used to replace the corresponding components when preparing the blank nanoparticles.
  • the nanovaccine group was subcutaneously injected with 400 ⁇ L of 4 mg nanovaccine loaded with water-soluble components on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • the PBS blank control group was subcutaneously injected with 400 ⁇ L PBS on the 35th day, 28th day, 21st day, 14th day and 7th day before tumor inoculation.
  • Blank nanoparticles + free water-soluble component control group were subcutaneously injected with 400 ⁇ L of blank nanoparticles and the same amount of vaccine-loaded Free water soluble components.
  • the control nanovaccine group was subcutaneously injected with 200 ⁇ L of 2 mg nanovaccine loaded with water-soluble components and 200 ⁇ L of 2 mg nanovaccine loaded with non-water-soluble components on the 35th, 28th, 21st, 14th and 7th days before tumor inoculation, respectively. nano vaccine.
  • 2 ⁇ 10 6 MC38 colon cancer cells were subcutaneously inoculated into the lower right back of each mouse.
  • the size of the tumor volume of the mice was recorded every 3 days from the 3rd day.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • the tumor growth rate of the mice in the control nanovaccine group and the DEC205 antibody-targeted modified nanovaccine group was significantly slower and the mice Survival period was significantly prolonged.
  • the survival period of mice in the DEC205 antibody-targeted modified nano-vaccine group was better than that in the control nano-vaccine group.

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Abstract

一种预防或治疗癌症的疫苗系统，包括递送粒子及其负载的细胞组分，递送粒子为纳米粒子或微米粒子，细胞组分为来源于一种或一种以上癌细胞和/或肿瘤组织的全细胞组分中的水溶性组分或水溶性组分形成的混合物，或来源于一种或一种以上癌细胞和/或肿瘤组织的全细胞组分中的非水溶性组分或非水溶性组分形成的混合物，疫苗系统可用于癌症的预防和治疗。

Description

一种预防或治疗癌症的疫苗系统及其应用 技术领域
本发明涉及免疫治疗技术领域，尤其涉及一种预防或治疗癌症的疫苗系统及其应用。
背景技术
免疫是指机体免疫系统识别自身与异己物质，并通过免疫应答排除抗原性异物，以维持机体生理平衡的功能。近些年来免疫技术发展很快，尤其是癌症的免疫治疗领域。癌症疫苗在癌症免疫治疗和预防的重要方法之一。开发癌症疫苗的基础是选择合适的癌症抗原来激活人体免疫系统对异常突变的癌细胞的识别，而癌症细胞或者癌症肿瘤组织本身是最好的癌症抗原的来源。如王丹丹等(MUC-1癌症疫苗治疗非小细胞肺癌的研究进展[J].癌症进展,2016,14(5):419-422.DOI:10.11877/j.issn.1672-1535.2016.14.05.06.)对于靶向黏蛋白-1(MUC-1)抗原表位的疫苗进行临床研究，采用该疫苗治疗非小细胞肺癌(NSCLC)。还有科学家曾采用新技术从癌症病人的肿瘤细胞分析鉴别癌症特异性的或癌症相关的抗原多肽，然后体外人工合成以制备癌症疫苗用于癌症的治疗。该技术在癌症病人的临床试验中表现出了一定的疗效，但是该类方法费时费力，花费巨大。而且所采用的方法都是从肿瘤组织中的一小部分癌细胞中提取分析癌细胞与正常细胞的差异进而寻找有差异的多肽，但是肿瘤组织中不同部位的癌细胞和各个癌细胞异质性都很大，因而该类方法和技术只能找到有限的几种的抗原多肽，从而极大的限制了该类方法的应用。
发明内容
为解决上述技术问题，本发明提供了一种负载一种或多种癌症细胞和/ 或肿瘤组织的全细胞组分中的水溶性组分或非水溶性组分的微米或纳米疫苗系统，简化了癌症疫苗的制备过程，且仍具有较优的治疗效果。
本发明的一种预防或治疗癌症的疫苗系统，包括递送粒子及其负载的细胞组分，所述递送粒子为纳米粒子或微米粒子，所述细胞组分为来源于一种或一种以上癌细胞和/或肿瘤组织的全细胞组分中的水溶性组分或水溶性组分形成的混合物，或来源于一种或一种以上癌细胞和/或肿瘤组织的全细胞组分中的非水溶性组分或非水溶性组分形成的混合物。优选地，在表面修饰有靶头的递送粒子内部或表面负载来源于一种或一种以上癌细胞和/或肿瘤组织的全细胞组分中的水溶性组分或水溶性组分形成的混合物，或来源于一种或一种以上癌细胞和/或肿瘤组织的全细胞组分中的非水溶性组分或非水溶性组分形成的混合物。
目前已有方法将癌细胞全细胞组分作为疫苗用于预防和治疗癌症，但同时负载水溶性组分和非水溶性组分的工作量巨大、步骤繁琐，因此本发明将癌细胞或癌症组织的全细胞组分中的水溶性组分或者非水溶性组分作为疫苗用于预防和治疗癌症的疫苗的来源是很有前景的方法。更重要的是，本发明中，表面含有靶头的递送粒子负载水溶性组分所组成的疫苗系统用于预防或治疗癌症时，达到的效果优于负载水溶性组分和非水溶性组分的递送粒子，大大优化了水溶性组分的治疗效果，同时更适合于临床施用，与同时负载水溶性和非水溶性组分相比，治疗时疼痛感明显减少，且操作更加便捷。
进一步地，靶头为可靶向特定细胞的小分子化合物、抗体、多肽、糖类、脂类、核酸。本发明的实施例中列举了甘露糖和DEC205抗体作为靶头构成的疫苗系统，在实际应用中还可以采用包括但不限于CD32抗体、CD11c抗体、CD103抗体、CD44抗体、CD40抗体。
进一步地，靶头靶向树突状细胞、巨噬细胞、B细胞、T细胞、NK细胞、NKT细胞、中性粒细胞、嗜酸性粒细胞或嗜碱性粒细胞，这些细胞大多存在于淋巴结、胸腺、脾脏或骨髓中。
进一步地，将含有与癌症相关抗原的细胞或组织在-20℃～-273℃下冷 冻，加水或不含增溶剂的水溶液后反复冻融裂解细胞，上清液为水溶性组分，沉淀中经增溶后可溶的部分为非水溶性组分。
进一步地，增溶所用的增溶剂选自尿素、盐酸胍、脱氧胆酸钠、十二烷基硫酸钠(SDS)、甘油、蛋白质降解酶、白蛋白、卵磷脂、0.1-2000mg/mL的无机盐、Triton、吐温、醋酸、胆固醇、氨基酸、糖苷、胆碱、Brij TM-35、八乙二醇单月桂醚(Octaethylene glycol monododecyl ether)、3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐(CHAPS)、洋地黄皂苷(Digitonin)、月桂基二甲胺氧化物(lauryldimethylamine oxide)、
Figure PCTCN2021137431-appb-000001
CA-630、二甲基亚砜(DMSO)、乙腈、乙醇、甲醇、N,N-二甲基甲酰胺(DMF)、异丙醇、二氯甲烷、丙醇和乙酸乙酯中的一种或多种。当然，本领域技术人员可以采用其他增溶剂或其他手段使非水溶性组分变为可溶。
进一步地，递送粒子的内部或表面负载免疫增强剂，包括但不限于微生物来源的免疫增强剂、人或动物免疫系统的产物、固有免疫激动剂、适应性免疫激动剂、化学合成药物、真菌多糖类、中药及其他类中的至少一类；所述免疫增强佐剂包括但不限于模式识别受体激动剂、卡介苗(BCG)、卡介苗细胞壁骨架、卡介苗甲醇提取残余物、卡介苗胞壁酰二肽、草分枝杆菌、多抗甲素、矿物油、病毒样颗粒、免疫增强的再造流感病毒小体、霍乱肠毒素、皂苷及其衍生物、Resiquimod、胸腺素、新生牛肝活性肽、米喹莫特、多糖、姜黄素、免疫佐剂CpG、免疫佐剂poly(I:C)、mRNA、免疫佐剂poly ICLC、短小棒状杆菌苗、溶血性链球菌制剂、辅酶Q10、左旋咪唑、聚胞苷酸、白细胞介素、干扰素、聚肌苷酸、聚腺苷酸、明矾、锰佐剂、锌佐剂、钙佐剂、硅佐剂、羊毛脂、植物油、内毒素、脂质体佐剂、GM-CSF、MF59、双链RNA、双链DNA、铝佐剂、CAF01、人参、黄芪的有效成分中的至少一种。
进一步地，水溶性组分或水溶性组分形成的混合物、非水溶性组分或非水溶性组分形成的混合物负载于递送粒子的内部和/或表面。
进一步地，递送粒子由有机合成高分子材料、天然高分子材料或无机材 料制备得到。有机合成高分子材料为聚乳酸-羟基乙酸共聚物PLGA、聚乳酸PLA、聚乙醇酸PGA、聚乙二醇PEG、聚己内酯PCL、泊洛沙姆Poloxamer、聚乙烯醇PVA、聚乙烯吡咯烷酮PVP、聚醚酰亚胺PEI、聚三亚甲基碳酸酯PTMC、聚酸酐、聚对二氧六环酮PDON、聚对二氧环己酮PPDO、聚甲基丙烯酸甲酯PMMA、聚氨基酸、合成多肽或合成脂质；天然高分子材料为卵磷脂、胆固醇、海藻酸钠、白蛋白、胶原蛋白、明胶、细胞膜(包括全细胞膜组分或部分细胞膜组分)、外泌体、多肽、淀粉或糖类；无机材料为三氧化二铁、四氧化三铁、碳酸钙或磷酸钙等无明显生物毒性的材料。
进一步地，递送粒子为球形、椭球形、桶形、多角形、棒状、片状、线形、蠕虫形、方形、三角形、蝶形或圆盘形。
进一步地，纳米级粒子的粒径为1nm-1000nm，微米级粒子的粒径为1μm-1000μm。此粒径范围保证疫苗能被抗原提呈细胞吞噬，而为了提高吞噬效率，粒径大小要在适宜的范围内，优选地，纳米级粒子粒径大小为30nm-1000nm，最优选为100nm-600nm；优选地，微米级粒子粒径大小为1μm-10μm，最优选为1μm-5μm。
进一步地，递送粒子可不带电、带负电或带正电。
进一步地，递送粒子可按照已开发的制备方法制备，包括但不仅限于溶剂挥发法、透析法、挤出法和热熔法。在本发明的一些实施例中，递送粒子采用溶剂挥发法中的复乳法制备得到，具体步骤如下：
(1)将第一预定体积的含有第一预定浓度的水相溶液加入第二预定体积的含有第二预定浓度的有机相溶液中；水相溶液为含有癌细胞或肿瘤细胞裂解液的溶液(含有或不含有免疫增强剂)，裂解液中含有水溶性组分或溶于增溶剂中的原非水溶性组分；有机相溶液为有机合成高分子材料、天然高分子材料或无机材料溶解于有机溶剂中得到的溶液；
(2)将步骤(1)得到的混合液进行纳米化或微米化；
(3)将步骤(2)得到的混合物加入第三预定体积的含有第三预定浓度乳化剂水溶液中并进行纳米化或微米化；
(4)将步骤(3)得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中，并进行搅拌直至满足预定搅拌条件；
(5)将步骤(4)得到的混合液离心或超滤后，取沉淀物或超滤产物并与水溶性组分或者溶于增溶剂中的原非水溶性组分混合后即得纳米疫苗或微米疫苗。
或在步骤(1)-(4)之后进行以下步骤S1-S3：
S1、在步骤(5)离心后，将沉淀物重悬于第五预定浓度的含有冻干保护剂的水溶液中或者PBS(或生理盐水)中；
S2、将S1得到的混悬液进行冷冻干燥处理后，将冻干物质备用；
S3、将S1中得到的混悬液或S2得到的冻干物质，与水溶性组分或者溶于增溶剂的原非水溶性组分混合后即得纳米疫苗或微米疫苗。
其中，
在步骤(1)中，第一预定浓度要求蛋白质多肽浓度大于0.01ng/mL，优选1mg/mL-100mg/mL，因为当蛋白质多肽浓度大于0.01ng/mL时，才能负载足够癌症抗原以激活相关免疫反应。免疫佐剂在初始水相中的浓度为大于0.001pg/mL，优选0.01mg/mL-20mg/mL。
在步骤(1)中，有机溶剂可选用DMSO、乙腈、乙醇、氯仿、甲醇、DMF、异丙醇、二氯甲烷、丙醇、乙酸乙酯等，优选二氯甲烷。
在步骤(1)中，第二预定浓度为0.5mg/mL-5000mg/mL，优选为100mg/mL。有机合成高分子材料优选为PLGA，其具有一定的免疫调节功能，适合作为疫苗制备时的辅料。
在步骤(1)中，第一预定体积和第二预定体积之比为1:1.1-5000，优选为1:10。
在步骤(2)或(3)中，通过超声、搅拌、均质处理或者微流控进行纳米化或微米化，时间、速度、压力能控制制备的递送粒子大小。超声处理时，超声功率为50W～500W，时间大于0.1秒，比如2～200秒；搅拌为机械搅 拌或者磁力搅拌时，搅拌速度大于50rpm，搅拌时间大于等于1分钟，比如搅拌速度为50rpm～500rpm，搅拌时间为60～6000秒；均质处理时使用高压/超高压均质机或高剪切均质机，压力大于20psi，比如20psi～100psi，使用高剪切均质机时转速大于100rpm，比如1000rpm～5000rpm；微流控流速大于0.01mL/min，比如0.1mL/min-100mL/min。
在步骤(3)或(4)中，乳化剂水溶液为聚乙烯醇(PVA)水溶液，第三预定浓度为10-50mg/mL，优选为20mg/mL。第四预定浓度为1-20mg/mL，优选为5mg/mL。第三预定体积根据其与第二预定体积的比例进行调整。在本发明中，第二预定体积与第三预定体积之比为1:1.1-1000，优选为2:5。第三预定体积与第四预定体积之比为1:1.5-2000，优选为1:10。预定搅拌条件为直至有机溶剂挥发完成。
在步骤S1中，冻干保护剂优选为海藻糖。第五预定浓度为质量百分比1-15％，为了在后续进行冷冻干燥中不影响冻干效果。
本发明还要求保护上述疫苗系统在制备预防或治疗癌症的药物中的应用。
进一步地，疫苗系统中至少有一种癌细胞或肿瘤组织与药物预防或治疗的疾病类型相同，在用作癌症疫苗进行预防和治疗癌症时，本发明所述的疫苗可以在癌症发生前或癌症发生后或手术切除肿瘤组织后多次给药以激活免疫系统，从而延缓癌症的进展、治疗癌症或者预防癌症的复发。
借由上述方案，本发明至少具有以下优点：
本发明提供了一种利用纳米级或微米级的粒子递送细胞水溶性组分或非水溶性组分的疫苗系统，以及用于制备预防和治疗癌症的疫苗中的应用。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明。
图1-9为实施例1-9中采用纳米疫苗或微米疫苗预防或治疗癌症的实验结果；其中，a,纳米疫苗或微米疫苗预防或治疗癌症时的肿瘤生长速度实验结果(n≥8)；b,纳米疫苗或微米疫苗预防或治疗其他癌症时的小鼠生存期实验结果(n≥8)，每个数据点为平均值±标准误差(mean±SEM)；a图中肿瘤生长抑制实验的显著性差异采用ANOVA法分析，b图中显著性差异采用Kaplan-Meier和log-rank test分析；**表示疫苗组与PBS空白对照组相比p＜0.005，有显著性差异；##代表疫苗组与空白纳米粒+游离裂解物组分对照组相比p＜0.005，有显著性差异；***表示疫苗组与PBS空白对照组相比p＜0.0005，有显著性差异；###代表疫苗组与空白纳米粒+细胞裂解物对照组相比p＜0.0005，有显著性差异。￥￥￥表示疫苗组与多肽纳米粒组相比p＜0.0005，有显著性差异；&代表疫苗组与无佐剂疫苗组相比p＜0.05，有显著性差异；★代表靶向疫苗(只负载水溶性组分)组与无靶头疫苗(负载水溶性和非水溶性组分)组相比p＜0.05，有显著性差异。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1 黑色素瘤肿瘤组织全细胞组分中的水溶性组分负载于纳米粒子内部和表面用于黑色素瘤的预防
本实施例以小鼠黑色素瘤为癌症模型来说明如何制备负载有黑色素瘤肿瘤组织全细胞组分中水溶性组分的纳米疫苗，并应用该疫苗预防黑色素瘤。
本实施例中，以B16F10小鼠黑色素瘤为癌症模型。首先裂解B16F10黑色素瘤肿瘤组织并收集水溶性组分。然后，以有机高分子材料PLGA为纳米粒骨架材料，以Polyinosinic-polycytidylic acid(poly(I:C))为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织的水溶性组分的纳米疫苗。然后采用该纳米疫 苗来预防黑色素瘤。
(1)肿瘤组织的裂解及水溶性组分的收集
在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16-F10细胞，在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨，通过细胞过滤网加入适量纯水并反复冻融5次，并可伴有超声以破坏裂解细胞。待细胞裂解后，将裂解物以大于1000g的转速离心5分钟并取上清液即为可溶于纯水的水溶性组分。在对照纳米疫苗中，本实施例采用负载等比例水溶性多肽B16-M20(Tubb3,FRRKAFLHWYTGEAMDEMEFTEAESNM)，B16-M24(Dag1,TAVITPPTTTTKKARVSTPKPATPSTD)和B16-M27(REGVELCPGNKYEMRRHGTTHSLVIHD)。
(2)纳米疫苗的制备
本实施例中纳米疫苗及作为对照的空白纳米粒以及负载多种多肽的纳米粒采用溶剂挥发法中的复乳法制备，所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的免疫佐剂为poly(I:C)且poly(I:C)只分布于纳米粒子内部。制备方法如前所述。负载全细胞组分中水溶性组分的纳米疫苗平均粒径为310nm左右，纳米疫苗表面电位为-6mV左右；每1mg PLGA纳米粒子约负载170μg蛋白质或多肽组分，每1mgPLGA纳米粒内外所使用的poly(I:C)免疫佐剂共约为0.02mg。空白纳米粒粒径为250nm左右，空白纳米粒制备时分别采用含有等量poly(I:C)的纯水或8M尿素代替相对应的水溶性组分。负载多肽的纳米粒制备方法同制备负载全细胞组分的制备方法，所使用的poly(I:C)的量也相同，多肽纳米粒粒径约为305nm,每1mg PLGA纳米粒子约负载160μg多肽组分。
(3)纳米疫苗用于癌症的预防
本研究对照组分别是PBS组和空白纳米粒+游离水溶性组分组。选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。
纳米疫苗组给药方案如下：在接种黑色素瘤之前第49天、第42天、第35天、第28天和第14天分别皮下注射400μL负载水溶性成分的4mg PLGA纳米 疫苗。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
PBS对照组方案如下：在接种黑色素瘤之前第49天、42天、35天、28天和14天分别皮下注射400μL PBS。在第0天给每只小鼠背部右下方皮下接种1.5×105个B16F10细胞。空白纳米粒+细胞裂解物对照组：在接种黑色素瘤之前第49天、42天、35天、28天和14天分别皮下注射400μL空白纳米粒和与疫苗负载的等量的游离水溶性组分。空白纳米粒和游离水溶性组分注射在不同部位。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
多肽纳米粒组给药方案如下：在接种黑色素瘤之前第49天、第42天、第35天、第28天和第14天分别皮下注射200μL内部和表面都负载水溶性多肽成分的2mg PLGA纳米粒子。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
在实验中，从第3天开始每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图1所示，负载B16F10肿瘤组织全细胞组分中水溶性组分的疫苗处理组小鼠的肿瘤在接种后50％消失；而作为对照的，负载水溶性多肽的纳米粒子组小鼠的肿瘤在接种后只有13％左右消失。而PBS对照组和空白纳米粒对照组小鼠的肿瘤都长大。这说明与几种水溶性多肽抗原相比，肿瘤组织水溶性组分中多样性的抗原有利于刺激产生免疫反应。综上所述，本发明所述的负载多种肿瘤组织的水溶性组分的纳米疫苗对黑色素瘤具有良好的预防效果。
实施例2 黑色素瘤和结肠癌癌细胞水溶性组分负载于微米粒子内部和表面用于黑色素瘤的预防
本实施例以小鼠黑色素瘤为癌症模型来说明如何制备负载有黑色素瘤和结肠癌细胞组分中水溶性部分的微米疫苗，并应用该疫苗预防黑色素瘤。
本实施例中，首先裂解B16F10黑色素瘤和MC38结肠癌细胞以制备水溶性组分组分。然后，以有机高分子材料PLGA为微米粒子骨架材料，以CpG为免疫佐剂采用溶剂挥发法制备负载有全细胞的水溶性组分的微米疫苗。然后采用该微米疫苗来预防黑色素瘤。
(1)癌细胞的裂解及各组分的收集
收集一定量的B16F10细胞或MC38细胞，去除培养基后采用-20℃到-273℃冷冻，加一定量超纯水后反复冻融3次以上，并可伴有超声以破坏裂解细胞。待细胞裂解后，将裂解物以3000g的转速离心5min取上清液即为LLC肺癌细胞中可溶于纯水的水溶性组分。上述所得来源于两种癌细胞裂解物的水溶性组分按1:1混合即为制备微米疫苗的抗原来源。
(2)微米疫苗的制备
本实施例中制备微米疫苗及作为对照的空白微米粒采用溶剂挥发法中的复乳法，所采用的微米粒子制备材料为有机高分子材料PLGA分子量为38KDa-54KDa，所采用的免疫佐剂为CPG且CPG既分布于微米粒子内部也负载于微米粒子表面。制备方法如前所述。在微米粒子表面负载细胞组分和免疫佐剂后所得微米疫苗粒径为1.80μm左右，微米粒子平均表面电位为-5mV左右。每1mg PLGA微米粒子负载220μg蛋白质或多肽组分，每1mg PLGA微米粒内外所使用的CPG免疫佐剂为0.02mg且内外各半。空白微米粒粒径为1.70μm左右，空白微米粒制备时分别采用含有等量CPG的纯水代替相对应的水溶性组分。
(3)微米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6制备黑色素瘤荷瘤小鼠。微米疫苗组方案如下：在接种黑色素瘤之前第28天、第21天、第14天和第7天分别皮下注射400μL负载癌水溶性成分的4mg PLGA微米疫苗。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。PBS空白对照组方案如下：在接种黑色素瘤之前第28天、第21天、第14天和第7天分别皮下注射400μL PBS。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。空白微米粒+细胞裂解物 对照组：在接种黑色素瘤之前第28天、第21天、第14天和第7天分别皮下注射400μL空白微米粒子和与疫苗中等量的水溶性组分。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
在实验中，从第3天开始每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。由于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图2所示，与PBS空白对照组相比，空白微米粒+水溶性组分对照组相比，微米疫苗给药组中小鼠肿瘤体积生长速度均明显变慢且小鼠生存期均明显延长。而且，微米疫苗给药组中小鼠有部分小鼠肿瘤接种后完全消失。由此可见，本发明所述的负载黑色素瘤和结肠癌细胞水溶性组分的微米疫苗对黑色素瘤具有预防效果。
实施例3 黑色素瘤肿瘤组织全细胞组分中的非水溶性组分负载于纳米粒子内部和表面用于黑色素瘤的预防
本实施例以小鼠黑色素瘤为癌症模型来说明如何制备负载有黑色素瘤肿瘤组织全细胞组分中非水溶性组分的纳米疫苗，并应用该疫苗预防黑色素瘤。
本实施例中，以B16F10小鼠黑色素瘤细胞为癌症模型。首先裂解B16F10黑色素瘤肿瘤组织并以8M尿素增溶原非水溶性组分。然后，以有机高分子材料PLGA为纳米粒骨架材料，以poly(I:C)为免疫佐剂制备负载有肿瘤组织的非水溶性组分的纳米疫苗。然后采用该纳米疫苗预防黑色素瘤。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠背部皮下接种1.5×10 5个B16-F10细胞，在肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨，通过细胞过滤网加入适量纯水并反复冻融5次，并可伴有超声以破坏裂解细 胞。待细胞裂解后，将裂解物以1000g的转速离心5分钟，将上清液去掉并在沉淀部分中加入8M尿素溶解沉淀部分即可将不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。即为制备疫苗的原料来源。在对照纳米疫苗中，本实施例采用负载8M尿素增溶的水不溶性多肽B16-M05(Eef2,FVVKAYLPVNESFAFTADLRSNTGGQA),B16-M46(Actn4,NHSGLVTFQAFIDVMSRETTDTDTADQ),和TRP2:180-188(SVYDFFVWL)。
(2)纳米疫苗的制备
本实施例中纳米疫苗及作为对照的空白纳米粒以及负载多种多肽的纳米粒采用溶剂挥发法中的复乳法制备，所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的免疫佐剂为poly(I:C)且poly(I:C)既分布于纳米粒子内部也负载于纳米粒子表面。制备方法如前所述。负载全细胞组分中非水溶性组分的纳米疫苗平均粒径为310nm左右，纳米疫苗表面电位为-6mV左右；每1mg PLGA纳米粒子约负载170μg蛋白质或多肽组分，每1mgPLGA纳米粒内外所使用的poly(I:C)免疫佐剂共约为0.02mg且内外各半。空白纳米粒粒径为260nm左右，空白纳米粒制备时采用含有等量poly(I:C)的8M尿素代替相对应非水溶性组分。负载多肽的纳米粒制备方法同制备负载非水溶性组分的制备方法，所使用的poly(I:C)的量也相同，多肽纳米粒粒径约为320nm,每1mg PLGA纳米粒子约负载160μg多肽组分。
(3)纳米疫苗用于癌症的预防
本研究对照组分别是PBS组和空白纳米粒+游离非水溶性组分组。选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。
纳米疫苗组给药方案如下：在接种黑色素瘤之前第49天、第42天、第35天、第28天和第14天分别皮下注射400μL负载裂解物中非水溶性成分的4mg PLGA纳米疫苗。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
PBS对照组方案如下：在接种黑色素瘤之前第49天、42天、35天、28天和14天分别皮下注射400μL PBS。在第0天给每只小鼠背部右下方皮下接种 1.5×10 5个B16F10细胞。空白纳米粒+游离非水溶性组分对照组：在接种黑色素瘤之前第49天、42天、35天、28天和14天分别皮下注射400μL空白纳米粒和与疫苗负载的等量的游离非水溶性组分。空白纳米粒和游离非水溶性组分注射在不同部位。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
多肽纳米粒组给药方案如下：在接种黑色素瘤之前第49天、第42天、第35天、第28天和第14天分别皮下注射400μL内部和表面都负载溶于8M尿素中原非水溶性成分的4mg PLGA纳米粒子。在第0天给每只小鼠背部右下方皮下接种1.5×10 5个B16F10细胞。
在实验中，从第3天开始每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图3所示，负载B16F10肿瘤组织全细胞组分中非水溶性组分的疫苗处理组小鼠的肿瘤在接种后40％消失；作为对照的，负载非水溶性多肽的纳米粒子组小鼠的肿瘤在接种后只有13％左右消失。而PBS对照组和空白纳米粒对照组小鼠的肿瘤都长大。综上所述，本发明所述的负载肿瘤组织的非水溶性组分的纳米疫苗对黑色素瘤具有良好的预防效果。
实施例4 肺癌肿瘤组织和肝癌肿瘤组织裂解组分中非水溶性负载于微米粒子内部和表面用于肝癌的预防
本实施例以如何制备负载有肝癌和肺癌肿瘤组织裂解物组分中非水溶性组分的微米疫苗，并应用该疫苗预防肝癌。
本实施例中，将小鼠肝癌和肺癌肿瘤组织裂解物中分非水溶性组分按1:2的比例负载于纳米粒子内部和表面以制备微米疫苗。首先取得小鼠肺癌和肝癌肿瘤组织并将其裂解以制备瘤块组织的溶于6M盐酸胍中的原非水溶性组分。然后，以PLGA为微米粒骨架材料，以poly(I:C)为免疫佐剂制备微 米疫苗。然后采用该微米疫苗来预防Hepa 1-6肝癌荷瘤小鼠体内的肿瘤。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠腋下皮下接种2×10 6个Hepa 1-6细胞或者2×10 6个LLC肺癌细胞，在各只小鼠所接种肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。后续处理方法同实施例3，只不过采用6M盐酸胍溶解非水溶性组分。
(2)微米疫苗的制备
方法同实施例2。不过，在本实施例中负载的是肝癌和肺癌肿瘤组织裂解物中分非水溶性组分1:2的混合物。
(3)微米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6制备Hepa 1-6肝癌荷瘤小鼠。
在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射400μL负载非水溶性成分的4mg PLGA微米疫苗；在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。PBS空白对照组方案如下：在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射400μL PBS；在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。空白微米粒+游离非水溶性组分对照组：在接种肝癌细胞之前第49天、第42天、第35天、第28天和第14天分别皮下注射400μL空白微米粒和与疫苗所负载的等量的游离非水溶性组分；空白微米粒和游离细胞裂解物注射在不同部位；在第0天给每只小鼠右腋下皮下接种2×10 6个Hepa 1-6肝癌细胞。在实验中，从第3天开始每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图4所示，PBS对照组以及空白微米粒+游离非水溶性组分对照组小 鼠的肝癌肿瘤生长均较快。微米疫苗给药组小鼠在接种肿瘤后40％肿瘤消失。而且，含有佐剂的微米疫苗治疗效果好于无佐剂的微米疫苗。由此可见，本发明所述的负载肺癌肿瘤组织和肝癌肿瘤组织裂解物中非水溶性组分的微米疫苗对肝癌具有预防效果。
实施例5 胰腺癌肿瘤组织裂解组分中的水溶性组分负载于纳米粒子内部和表面用于胰腺癌的治疗
本实施例以小鼠胰腺癌为癌症模型来说明如何制备负载有胰腺癌肿瘤组织裂解物中水溶性组分的纳米疫苗，并应用该疫苗治疗胰腺癌。
本实施例中，将小鼠Pan02胰腺癌肿瘤组织中的水溶性组分负载于纳米粒子内部和表面以制备纳米疫苗。首先取得小鼠胰腺癌肿瘤组织并将其裂解以制备水溶性组分。以PLGA为纳米粒子骨架材料，以poly(I:C)为免疫佐剂采用溶剂挥发法制备纳米疫苗。然后采用该纳米疫苗来治疗Pan02胰腺癌荷瘤小鼠体内的肿瘤。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠腋下皮下接种接种1×10 6个Pan02胰腺癌细胞，在各只小鼠所接种肿瘤长到体积分别为约1000mm 3时处死小鼠并摘取肿瘤组织。癌细胞的裂解方法及各组分的收集方法同上。
(2)纳米疫苗的制备
本实施例中制备纳米疫苗制备方法同上。
(3)纳米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6制备胰腺癌瘤小鼠。在第0天给每只小鼠背部右下方皮下接种1×10 6个个Pan02细胞。疫苗组在第4天、第7天、第10天、第15天和第20天分别皮下注射400μL的4mg PLGA纳米疫苗。PBS空白对照组在第4天、第7天、第10天、第15天和第20天分别皮下注射400μL PBS。空白纳米粒+水溶性组分对照组在第4天、第7天、第10天、第15天和第20天分别皮下注射400μL空白纳米粒和与疫苗所负载的等量的游离水溶性组分。在实 验中，从第3天起每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图5所示，与对照组相比，纳米疫苗组肿瘤生长速度明显变慢且小鼠生存期明显延长。而且，有部分小鼠肿瘤接种后消失。由此可见，本发明所述的负载胰腺癌肿瘤组织裂解物中水溶性组分的纳米疫苗对胰腺癌具有治疗效果。
实施例6 肺癌肿瘤组织水溶性组分负载于甘露糖修饰的微米粒子内部用于肺癌的预防
本实施例以小鼠肺癌为癌症模型来说明如何制备负载有肺癌肿瘤组织全细胞组分中水溶性组分的微米疫苗，并应用该疫苗预防肺癌。在实际应用时具体剂型，佐剂，给药时间、给药次数、给药方案可根据情况调整。
本实施例中，首先取得小鼠肺癌的肿瘤组织并将其裂解以制备水溶性组分。然后，以PLGA和甘露糖修饰的PLGA为微米粒骨架材料，以CpG为免疫佐剂采用溶剂挥发法制备微米疫苗。该微米疫苗具有靶向树突状细胞的能力。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠腋下皮下接种2×10 6个LLC肺癌细胞，在小鼠所接种肿瘤长到1000mm 3时处死小鼠并摘取肿瘤组织。其他处理方法同上。
(2)微米疫苗的制备
本实施例中制微米疫苗及作为对照的空微米粒采用溶剂挥发法中的复乳法，所采用微米粒子制备材料PLGA(50:50)分子量为38KDa-54KDa，所采用的甘露糖修饰的PLGA(50:50)分子量为38KDa-54KDa。未修饰PLGA，甘露糖修饰的PLGA的质量比为8:2。所采用的免疫佐剂为CpG且CpG分布于 微米粒子内部。制备方法如前所述微米粒子平均粒径为1.90μm左右，平均表面电位为-8mV左右。每1mg PLGA纳米粒子负载75μg蛋白质或多肽组分,每1mg PLGA微米粒内所使用的CpG免疫佐剂为0.06mg。空白微米粒粒径为1.85μm左右，空白微米粒制备时分别采用含有等量CpG的纯水代替相对应的水溶性组分。
(3)靶向树突状细胞的微米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备肺癌荷瘤小鼠。疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL负载水溶性成分的4mg纳米疫苗。PBS空白对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL PBS。空白纳米粒+游离水溶性组分对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL空白纳米粒和与疫苗所负载的等量的游离水溶性组分。在第0天给每只小鼠背部右下方皮下接种2×10 6个个LLC肺癌细胞。在实验中，从第3天起每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图6所示，与PBS对照组和空白微米粒+游离水溶性组分对照组相比，微米疫苗组小鼠肿瘤生长速度明显变慢且小鼠生存期明显延长。这说明本发明所述的负载肺癌肿瘤组织中水溶性组分主动靶向微米疫苗对肺癌具有预防效果。
实施例7 肝癌肿瘤组织全细胞组分中水溶性组分负载于纳米粒子内部和表面并以卡介苗(BCG)为免疫佐剂的纳米疫苗用于肝癌的预防
本实施例以小鼠肝癌为癌症模型并以BCG中的水溶性组分为免疫佐剂来说明如何制备负载有肝癌肿瘤全细胞组分中水溶性组分的纳米疫苗并应用该疫苗预防肝癌。
本实施例中，首先裂解肝癌肿瘤组织并收集水溶性组分然后，以PLGA为纳米粒子骨架材料，以BCG为免疫佐剂采用溶剂挥发法制备纳米疫苗。
(1)肿瘤组织的裂解及各组分的收集
该实施例中肿瘤组织的裂解及裂解物收集同上。
(2)BCG的裂解及各组分的收集
该实施例中BCG的裂解及裂解物收集和增溶方法同实施例1中癌细胞的裂解方法，只是将癌细胞换成BCG。
(3)纳米疫苗的制备
本实施例中纳米疫苗的制备方法实施例1。但是在该实施例中，纳米疫苗负载的免疫佐剂由poly(I:C)换成了BCG中的水溶性组分。
(4)纳米疫苗用于肝癌的预防
选取雌性C57BL/6为模型小鼠制备肝癌荷瘤小鼠。疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL负载水溶性成分的4mg PLGA纳米疫苗。PBS空白对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL PBS。空白纳米粒+游离水溶性组分对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL空白纳米粒和与疫苗所负载的等量的游离水溶性组分。在第0天给每只小鼠背部右下方皮下接种2×10 6个Hepa1-6肝癌细胞。在实验中，从第3天起每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图7所示，与对照组相比，以BCG为佐剂的纳米疫苗给药组肿瘤生长速度明显变慢且小鼠生存期明显延长。由此可见，本发明所述负载肝癌肿瘤组织全细胞组分中水溶性组分的纳米疫苗可以预防肝癌。
实施例8 肺癌癌细胞水溶性组分负载于DEC205抗体修饰的纳米粒子内部用于肺癌的预防
本实施例以小鼠肺癌为癌症模型来说明如何制备负载有肺癌癌细胞全细胞组分中水溶性组分的纳米疫苗，并应用该疫苗预防肺癌。在实际应用时具体剂型，佐剂，给药时间、给药次数、给药方案可根据情况调整。
本实施例中，首先将小鼠LLC肺癌细胞裂解以制备水溶性组分。然后，以PLGA和DEC205抗体修饰的PLGA为纳米粒骨架材料，以CpG为免疫佐剂采用溶剂挥发法制备纳米疫苗。该纳米疫苗具有靶向树突状细胞的能力。
(1)肿瘤细胞的裂解及各组分的收集
LLC肺癌细胞的收集、裂解和水溶性组分的收集等处理方法同上。
(2)纳米疫苗的制备
本实施例中纳米疫苗及作为对照的空纳米粒采用溶剂挥发法中的复乳法制备，所采用纳米粒子制备材料PLGA(50:50)分子量为24KDa-38KDa，所采用的DEC205抗体修饰的PLGA(50:50)分子量为24KDa-38KDa。未修饰PLGA与DEC205抗体修饰的PLGA的质量比为8:2。所采用的免疫佐剂为MnCl 2且MnCl 2分布于纳米粒子内部。制备方法如前所述纳米粒子平均粒径为300nm左右。每1mg PLGA纳米粒子负载55μg蛋白质或多肽组分,每1mg PLGA纳米粒内所使用的MnCl 2免疫佐剂为0.06mg。空白纳米粒粒径为280nm左右，空白纳米粒制备时分别采用含有等量MnCl 2的纯水代替相对应的水溶性组分。
(3)靶向树突状细胞的纳米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备肺癌荷瘤小鼠。疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL负载水溶性成分的4mg纳米疫苗。PBS空白对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL PBS。空白纳米粒+游离水溶性组分对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮 下注射400μL空白纳米粒和与疫苗所负载的等量的游离水溶性组分。在第0天给每只小鼠背部右下方皮下接种2×10 6个个LLC肺癌细胞。在实验中，从第3天起每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图8所示，与PBS对照组和空白纳米粒+游离水溶性组分对照组相比，纳米疫苗组小鼠肿瘤生长速度明显变慢且小鼠生存期明显延长。这说明本发明所述的负载肺癌癌细胞中水溶性组分主动靶向纳米疫苗对肺癌具有预防效果。
实施例9 结肠癌肿瘤组织和癌细胞水溶性组分负载于DEC205抗体修饰的纳米粒子内部用于结肠癌的预防
本实施例以小鼠结肠癌为癌症模型来说明如何制备负载有结肠癌肿瘤组织和癌细胞全细胞组分中水溶性组分的纳米疫苗，并应用该疫苗预防结肠癌。在实际应用时具体剂型，佐剂，给药时间、给药次数、给药方案可根据情况调整。
本实施例中，首先取得小鼠结肠癌的肿瘤组织和培养的癌细胞并将其裂解以制备水溶性组分混合物和非水溶性组分混合物。然后，以PLGA和DEC205抗体修饰的PLGA为纳米粒骨架材料，以CpG为免疫佐剂采用溶剂挥发法制备纳米疫苗。该纳米疫苗具有靶向树突状细胞的能力。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠腋下皮下接种2×10 6个MC38结肠癌细胞，在小鼠所接种肿瘤长到1000mm 3时处死小鼠并摘取肿瘤组织，肿瘤组织裂解方法同上，肿瘤组织采用超纯水裂解后在5000g离心5分钟，收集上清液即为水溶性组分，采用8M尿素溶解沉淀部分即为原非水溶性组分。细胞培养得到的MC38细胞收集方法为离心后弃去培养基上清，并用PBS洗涤离心两 遍，离心收集的细胞加入超纯水后在-20℃反复冻融5遍以上裂解癌细胞，超纯水裂解后在5000g离心5分钟，收集上清液即为水溶性组分，采用8M尿素溶解沉淀部分即为原非水溶性组分。将肿瘤组织的水溶性组分和癌细胞的水溶性组分按1:1的质量比混合即为用于制备纳米疫苗的水溶性组分混合物；将肿瘤组织的非水溶性组分和癌细胞的非水溶性组分按1:1的质量比混合即为用于制备纳米疫苗的非水溶性组分混合物。
(2)纳米疫苗的制备
本实施例中的纳米疫苗及作为对照的空纳米粒和对照纳米疫苗采用溶剂挥发法中的复乳法，各种纳米粒制备方法如前所述。实验中所采用纳米粒子制备材料PLGA(50:50)分子量为24KDa-38KDa，所采用的DEC205抗体修饰的PLGA(50:50)分子量为24KDa-38KDa。未修饰PLGA与DEC205抗体修饰的PLGA的质量比为8:2。靶向纳米疫苗内部只负载肿瘤组织和癌细胞水溶性组分混合物；所采用的免疫佐剂为CpG且CpG分布于纳米粒子内部；纳米疫苗平均粒径为290nm左右；每1mg PLGA纳米粒子负载50μg蛋白质或多肽组分；每1mg PLGA纳米粒内所使用的CpG免疫佐剂为0.04mg。无靶头的纳米疫苗内部负载肿瘤组织和癌细胞水溶性组分混合物或非水溶性组分的混合物；所采用的免疫佐剂为CpG且CpG分布于纳米粒子内部；纳米疫苗平均粒径为290nm左右；每1mg PLGA纳米粒子负载50μg蛋白质或多肽组分；每1mg PLGA纳米粒内所使用的CpG免疫佐剂为0.04mg。空白纳米粒粒径为270nm左右，空白纳米粒制备时分别采用含有等量CpG的溶液代替相对应的组分。
(3)靶向树突状细胞的纳米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6为模型小鼠制备结肠癌荷瘤小鼠。纳米疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL内部负载水溶性成分的4mg纳米疫苗。PBS空白对照组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射400μL PBS。空白纳米粒+游离水溶性组分对照组在肿瘤接种前第35天、第28 天、第21天、第14天和第7天分别皮下注射400μL空白纳米粒和与疫苗所负载的等量的游离水溶性组分。对照纳米疫苗组在肿瘤接种前第35天、第28天、第21天、第14天和第7天分别皮下注射200μL内部负载水溶性成分的2mg纳米疫苗和200μL内部负载非水溶性成分的2mg纳米疫苗。在第0天给每只小鼠背部右下方皮下接种2×10 6个MC38结肠癌细胞。在实验中，从第3天起每3天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52×a×b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm 3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图所示，与PBS对照组和空白纳米粒+游离水溶性组分对照组相比，对照纳米疫苗组小鼠和DEC205抗体靶向修饰纳米疫苗组小鼠肿瘤生长速度明显变慢且小鼠生存期明显延长。而且，DEC205抗体靶向修饰纳米疫苗组小鼠的生存期比对照纳米疫苗组要好。这说明本发明所述的负载结肠癌肿瘤组织和癌细胞中水溶性组分的主动靶向纳米疫苗对结肠癌具有良好的预防效果。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种预防或治疗癌症的疫苗系统，其特征在于：所述疫苗系统包括递送粒子及其负载的细胞组分，所述递送粒子为纳米粒子或微米粒子，所述细胞组分为来源于一种或一种以上癌细胞和/或肿瘤组织的全细胞组分中的水溶性组分或水溶性组分形成的混合物，或来源于一种或一种以上癌细胞和/或肿瘤组织的全细胞组分中的非水溶性组分或非水溶性组分形成的混合物。
  2. 根据权利要求1所述的疫苗系统，其特征在于：疫苗系统包括表面修饰有靶头的递送粒子及其负载的细胞组分，所述递送粒子为纳米粒子或微米粒子，所述细胞组分为来源于一种或一种以上癌细胞和/或肿瘤组织的全细胞组分中的水溶性组分或水溶性组分形成的混合物，或来源于一种或一种以上癌细胞和/或肿瘤组织的全细胞组分中的非水溶性组分或非水溶性组分形成的混合物。
  3. 根据权利要求2所述的疫苗系统，其特征在于：所述靶头为可靶向特定细胞的小分子化合物、抗体、多肽、糖类、脂类或核酸。
  4. 根据权利要求2所述的疫苗系统，其特征在于：所述靶头为甘露糖DEC205抗体、CD32抗体、CD11c抗体、CD103抗体、CD44抗体或CD40抗体。
  5. 根据权利要求2所述的疫苗系统，其特征在于：所述靶头靶向树突状细胞、巨噬细胞、B细胞、T细胞、NK细胞、NKT细胞、中性粒细胞、嗜酸性粒细胞或嗜碱性粒细胞。
  6. 根据权利要求1所述的疫苗系统，其特征在于：将癌细胞或肿瘤组织在-20℃～-273℃下冷冻，加水或不含增溶剂的水溶液后反复冻融裂解细胞，上清液为所述水溶性组分，沉淀中经增溶后可溶的部分为所述非水溶性组分。
  7. 根据权利要求6所述的疫苗系统，其特征在于：增溶所用的增溶剂选自尿素、盐酸胍、脱氧胆酸钠、十二烷基硫酸钠、甘油、蛋白质降解酶、白蛋白、卵磷脂、0.1-2000mg/mL的无机盐、Triton、吐温、醋酸、胆固醇、 氨基酸、糖苷、胆碱、Brij TM-35、八乙二醇单月桂醚、3-[3-(胆酰胺丙基)二甲氨基]丙磺酸内盐、洋地黄皂苷、月桂基二甲胺氧化物、
    Figure PCTCN2021137431-appb-100001
    CA-630、二甲基亚砜、乙腈、乙醇、甲醇、N,N-二甲基甲酰胺、异丙醇、二氯甲烷、丙醇和乙酸乙酯中的一种或多种。
  8. 根据权利要求1所述的疫苗系统，其特征在于：所述水溶性组分或水溶性组分形成的混合物、所述非水溶性组分或非水溶性组分形成的混合物负载于递送粒子的内部和/或表面。
  9. 权利要求1-8所述的疫苗系统在制备预防或治疗癌症的药物中的应用。
  10. 根据权利要求9所述的应用，其特征在于：所述药物用于癌症发生前、癌症发生后或切除肿瘤组织后。
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