CN114288397B - Vaccine system for preventing or treating cancer based on whole cell components of various cancer cells and/or tumor tissues, preparation and application thereof - Google Patents
Vaccine system for preventing or treating cancer based on whole cell components of various cancer cells and/or tumor tissues, preparation and application thereof Download PDFInfo
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Abstract
The application belongs to the field of immunotherapy, and discloses a vaccine system for preventing or treating cancers based on whole cell components of various cancer cells and/or tumor tissues, a preparation method and application thereof, wherein water-soluble components and non-water-soluble components of the whole cell components mixed by various cells are delivered by particles with nanoscale or microscale, and the vaccine is used for preparing vaccines for preventing and treating cancers. Both the water soluble and insoluble portions are loaded in the nanovaccine or the minivaccine, so that variant proteins or polypeptides produced by the cancer in the cellular fraction are loaded in the nanovaccine or the minivaccine. The use of these immunogenic substances in the whole cell fraction, which are produced by disease mutations, can be used for the prophylaxis and treatment of cancer. Therefore, the nanometer vaccine and/or the micrometer vaccine system of whole cell components with different cell sources can be used for preparing medicines for preventing and/or treating cancers.
Description
Technical Field
The invention belongs to the field of immunotherapy, in particular relates to a nano or micron cancer vaccine based on different cancer cells and/or different tumor tissues, and especially relates to a cancer vaccine based on whole cell components of cancer cells and/or tumor tissues of two or more cancers and application thereof in preventing and treating cancers.
Background
Immunization is a physiological function of the human body, by which the human body recognizes "own" and "nonhexose" components, thereby destroying and eliminating abnormal substances (such as viruses, bacteria, etc.) in the human body, or injured cells and tumor cells, etc. generated by the human body itself, to maintain the health of the human body. In recent years, the development of immunological techniques has been rapid, especially in the field of immunotherapy of cancer. Along with the continuous improvement of the knowledge of cancer, people find that the immune system of human body and various immune cells play a key role in the process of inhibiting the occurrence and development of cancer. By regulating the balance of the immune system of the body, it is expected to influence and control the occurrence, development and treatment of cancer.
Cancer vaccines are one of the important approaches in cancer immunotherapy and prophylaxis. The basis for developing cancer vaccines is to select an appropriate cancer antigen to activate recognition of abnormally mutated cancer cells by the human immune system, with the cancer cells or cancer tumor tissue itself being the best source of cancer antigen. Scientists have used new techniques to identify cancer-specific or cancer-associated antigenic polypeptides from tumor cell analysis of cancer patients and then synthesized artificially in vitro to prepare cancer vaccines for the treatment of cancer. The technology shows a certain curative effect in clinical trials of cancer patients, but the method is time-consuming and labor-consuming and has huge cost. The method only extracts and analyzes the difference between the cancer cells and normal cells from the water-soluble components of the cancer cells so as to find the polypeptides with the difference, so that the method and the technology can only find a limited number of antigen polypeptides with good water solubility, thereby greatly limiting the application of the method. Many antigen proteins or polypeptides which are highly immunogenic in the real environment of the human body are insoluble in pure water and are present in the body by binding to proteins, adsorbing to proteins or being located on or on the surface of membranes, so that the insoluble part of the water-insoluble proteins and polypeptides in pure water is very important and critical. The use of whole cell fractions of cancer cells or cancer tissues as a vaccine source for vaccines for the prevention and treatment of cancer is a promising approach. In the prior art, a cancer vaccine system is constructed by adopting a cancer cell or a tumor tissue whole cell, and is used for treating the cancer, and no report on the use of a cancer vaccine prepared by adopting various cancer cells or tumor tissues for better preventing and treating the cancer is yet seen.
Disclosure of Invention
Accordingly, it is an object of the present invention to provide a method for preventing or treating cancer by a micro-or nano-vaccine system loaded with whole cell components of various cancer cells and/or tumor tissues, in view of the problems of the prior art.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a nano-and/or micro-vaccine system for preventing or treating cancer based on a plurality of cancer cells and/or whole cell components of tumor tissue, comprising nano-and/or micro-particles, whole cell component mixture; the whole cell component mixture is a whole cell component of various cancer cells and/or tumor tissues.
The vaccine system for preventing or treating cancers based on the whole cell components of various cancer cells and/or tumor tissues is a nano or micron vaccine system, is called nano vaccine or micron vaccine, can prevent or treat cancers, and consists of a mixture of particles with nano or micron size and the whole cell components loaded by the particles, or consists of a mixture of particles with nano or micron size and the whole cell components loaded by the particles, and an immunoadjuvant; the whole cell fraction is a mixture of water-soluble and/or non-water-soluble components of whole cells in different cancer cells and/or different tumor tissues. The mixture may be, but is not limited to, water-soluble components intermixed, or water-insoluble components intermixed, or all and/or part of the water-soluble components intermixed with all and/or part of the water-soluble components.
The preparation method of the nano-and/or micro-vaccine system for preventing or treating cancers based on the whole cell components of various cancer cells and/or tumor tissues comprises the steps of loading the whole cell components of various cancer cells and/or tumor tissues into nano-and/or micro-particles and/or on the surfaces of the nano-and/or micro-particles to obtain the nano-and/or micro-vaccine system for preventing or treating cancers based on the whole cell components of various cancer cells and/or tumor tissues; or loading various cancer cells and/or tumor tissue whole cell components and an immune adjuvant into and/or on the interior and/or the surface of nano-sized and/or micro-sized particles to obtain the nano-sized and/or micro-sized vaccine system for preventing or treating cancers based on the various cancer cells and/or tumor tissue whole cell components. In particular, the vaccine system for preventing or treating cancer based on the whole cell components of various cancer cells and/or tumor tissues according to the present invention can be prepared according to the preparation methods developed for nano-sized particles and micro-sized particles, including but not limited to the common solvent evaporation method, dialysis method, extrusion method, and hot melting method. In some embodiments, the vaccine system employs a multiple emulsion process in a solvent evaporation process.
The active ingredient whole cell component mixture of the nano-and/or micro-vaccine system for preventing or treating cancers based on the whole cell components of various cancer cells and/or tumor tissues is a water-soluble component mixture and/or a non-water-soluble component mixture of whole cells, and is prepared from two or more cancer cells and/or tumor tissues, can be prepared from two or more cancer cells, can be prepared from two or more tumor tissues, and can also be prepared from one or more cancer cells and tumor tissues. The invention is the inventive point, the prior art adopts a cancer cell or tumor tissue to prepare whole cell component, which is used for constructing cancer vaccine by nanometer and/or micrometer particles, the invention adopts two or more cancer cells and/or tumor tissue to prepare cancer vaccine, and the unexpected technical effect is improved obviously.
The nano-and/or micro-vaccine system for preventing or treating cancers based on whole cell components of various cancer cells and/or tumor tissues is used for preventing or treating cancers and recurrence thereof. In the nano-and/or micro-vaccine system for preventing or treating cancer based on whole cell components of various cancer cells and/or tumor tissues, one of the cancer cells or tumor tissues is the same as the type of cancer used for preventing or treating.
In the nano-and/or micro-vaccine system for preventing or treating cancers based on the whole cell components of various cancer cells and/or tumor tissues, the loading mode is that the water-soluble components and the water-insoluble components of the whole cells are respectively or simultaneously coated inside the particles and/or respectively or simultaneously coated on the surfaces of the particles.
In the nano-and/or micro-vaccine system for preventing or treating cancer based on various cancer cell and/or tumor tissue whole cell components, the whole cell components are loaded in and/or on the interior and/or surface of nano-and/or micro-particles, specifically, the loading mode is that the water-soluble components and the water-insoluble components of the whole cells are respectively or simultaneously loaded in and/or on the surface of the particles, including but not limited to the loading of the water-soluble components in and on the particle at the same time, the loading of the water-insoluble components in and on the particle at the same time, the loading of the water-soluble components in and the water-insoluble components on the particle surface, the water-insoluble component is loaded in the particles and the water-insoluble component is loaded on the particle surfaces, the water-soluble component and the water-insoluble component are loaded in the particles and only the water-soluble component is loaded on the particle surfaces, the water-soluble component is loaded in the particles and the water-soluble component and the water-insoluble component are simultaneously loaded on the particle surfaces, the water-soluble component and the water-insoluble component are simultaneously loaded in the particles and the water-soluble component and the water-insoluble component are simultaneously loaded on the particle surfaces.
In the nano-and/or micro-vaccine system for preventing or treating cancers based on whole cell components of various cancer cells and/or tumor tissues, the inside and/or the surface of the nano-and/or micro-particles further comprise an immune adjuvant. The immune enhancer is added in a mode of loading in nano particles or micro particles, or loading on the surfaces of the nano particles or the micro particles, or loading in the nano particles or the micro particles and loading on the surfaces of the nano particles or the micro particles at the same time. The immunopotentiating adjuvants include, but are not limited to, at least one of microbial-derived immunopotentiators, products of the human or animal immune system, innate immune agonists, adaptive immune agonists, chemically synthesized drugs, fungal polysaccharides, traditional Chinese medicines, and other classes; the immunopotentiating adjuvants include, but are not limited to, at least one of the active ingredients of pattern recognition receptor agonists, BCG cell wall skeleton, BCG methanol extraction residue, BCG cell wall dipeptide, mycobacterium phlei, polyoxin a, mineral oil, virus-like particles, immunopotentiating reconstituted influenza virus minibodies, cholera enterotoxin, saponins and derivatives thereof, resiquimod, thymosin, neobovine liver active peptide, mi Kuimo t, polysaccharide, curcumin, immunoadjuvant CpG, poly (I: C), immunoadjuvant poly ICLC, pony bacterin, lysostreptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukins, interferons, polyminosinic acid, polyadenylic acid, lanolin, vegetable oil, endotoxins, liposome adjuvants, GM-CSF, MF59, double stranded RNA, double stranded DNA, aluminum adjuvants, manganese adjuvants, CAF01, ginseng, astragalus. It will be appreciated by those skilled in the art that other substances that enhance the immune response may be used as the immune enhancing adjuvant.
The surface of the nano-and/or micro-vaccine system for preventing or treating cancers based on the whole cell components of various cancer cells and/or tumor tissues can be not connected with a target head with an active targeting function or connected with the target head with the active targeting function; the target head can direct the delivery system to target a particular cell; the specific cells or tissues are one or more than two of dendritic cells, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils, basophils, lymph nodes, thymus, spleen and bone marrow.
In the nano-and/or micro-vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention, the whole cell components can be divided into two parts according to solubility in pure water or an aqueous solution without a solubilizing agent: a water-soluble component and a water-insoluble component. The water-soluble component is a raw water-soluble portion soluble in pure water or an aqueous solution containing no solubilizing agent, and the water-insoluble component is a raw water-insoluble portion insoluble in pure water, and is changed from insoluble in pure water or an aqueous solution containing no solubilizing agent to soluble in an aqueous solution containing solubilizing agent or an organic solvent by a suitable solubilizing method. Both the water soluble and non-water soluble portions of the whole cell fraction can be solubilized by the solubilizing aqueous solution or organic solvent containing solubilizing agent. The solubilizer is at least one of solubilizers which can increase the solubility of proteins or polypeptides in aqueous solution; the organic solvent is an organic solvent capable of dissolving proteins or polypeptides. The solubilizing agents include, but are not limited to, urea, guanidine hydrochloride, sodium deoxycholate, SDS, glycerol, alkaline solutions with a pH greater than 7, acidic solutions with a pH less than 7, various types of protein degrading enzymes, albumin, lecithin, high concentration inorganic salts, triton, tween, DMSO, acetonitrile, ethanol, methanol, DMF, propanol, isopropanol, acetic acid, cholesterol, amino acids, glycosides, choline BrijTM-35Octaethylene glycol monododecyl etherCHAPSDigitoninlauryldimethylamine oxideIGEPALCA-630., it being understood by those skilled in the art that the water insoluble components may also be rendered soluble by other means of solubilizing the protein and polypeptide fragments from insoluble in pure water. The organic solvents include, but are not limited to, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, dichloromethane, ethyl acetate. It will be appreciated by those skilled in the art that other methods of containing organic solvents that solubilize proteins and polypeptide fragments can be used as the organic solvent.
In the nano-and/or micro-vaccine system for preventing or treating cancer based on the whole cell components of various cancer cells and/or tumor tissues, the shape of nano-and/or micro-particles is any common shape, including but not limited to sphere, ellipsoid, barrel, polygon, rod, sheet, line, worm, square, triangle, butterfly or disc. In the nano-and/or micro-vaccine system for preventing or treating cancers based on the whole cell components of various cancer cells and/or tumor tissues, nano-and/or micro-particles are nano-sized particles and/or micro-sized particles. The particle size of the nanovaccine and nanoscale-sized particles, respectively, is from 1nm to 1000nm, in some embodiments from 50nm to 800nm, and further in some embodiments from 100nm to 600nm. The micrometer vaccine and micrometer sized particles each have a particle size of from 1 m to 1000 m, in some embodiments from 1 m to 100 m, in some embodiments from 1 m to 10 m, and further in some embodiments, from 1 m to 5 m. The surface of the nano-sized particles or the micro-sized particles can be neutral, negatively charged or positively charged.
In the vaccine system for preventing or treating cancers based on the whole cell components of various cancer cells and/or tumor tissues, the preparation materials of nano and/or micro particles comprise organic synthetic high polymer materials, natural high polymer materials or inorganic materials. Wherein the organic synthetic polymer material is biocompatible or biodegradable polymer material including, but not limited to PLGA, PLA, PGA, poloxamer, PEG, PCL, PEI, PVA, PVP, PTMC, polyanhydrides, PDON, PPDO, PMMA, polyamino acids, synthetic polypeptides, synthetic lipids. The natural polymer material is biocompatible or degradable polymer material, including but not limited to lecithin, cholesterol, starch, lipid, saccharide, polypeptide, sodium alginate, albumin, collagen, gelatin, and cell membrane component. The inorganic material is a material without obvious biological toxicity, including but not limited to ferric oxide, calcium carbonate and calcium phosphate.
The nano-and/or micro-vaccine system for preventing or treating cancer based on various cancer cells and/or tumor tissue whole cell components of the present invention can deliver the loaded whole cell components to related immune cells, activate and enhance the killing effect of autoimmune system on cancer cells through immunogenicity of the loaded components. The invention also provides application of the vaccine system for preventing or treating cancers based on various cancer cells and/or tumor tissue whole cell components in preparing vaccines for preventing and/or treating cancers.
The whole cell fraction cancer vaccine system of the present invention may use both water-soluble fraction-only nanoparticles and/or microparticles and non-water-soluble fraction-only nanoparticles and/or microparticles, non-water-soluble fraction-only nanoparticles and/or microparticles, or both water-soluble fraction and non-water-soluble fraction-only nanoparticles and/or microparticles in the prevention or treatment of disease.
According to the technical scheme, the invention provides a nano vaccine and/or micro vaccine system for delivering water-soluble components and non-water-soluble components of cells by using particles with nano-scale or micro-scale dimensions, and application of the nano vaccine and/or micro vaccine system in preparation of vaccines for preventing and treating cancers. Because the whole cell fraction of the relevant cells or tissues is divided into two parts according to solubility in pure water, a water-soluble part soluble in pure water and a water-insoluble part insoluble in pure water, and both the water-soluble part and the water-insoluble part are supported in nano-particles or micro-particles, variant proteins or polypeptides produced in the cell fraction due to cancer are mostly supported in nano-particles or micro-particles. The water soluble fraction and the water insoluble fraction of the cell fraction comprise the components of the whole cell; the water-soluble fraction and the water-insoluble fraction of the cell fraction may also be simultaneously dissolved by an aqueous solution containing a solubilizing agent. Wherein the same unmutated proteins, polypeptides and genes as normal cellular components do not elicit an immune response due to immune tolerance generated during development of the autoimmune system; mutations in genes, proteins and polypeptides produced by cancer and the like are immunogenic and activate immune responses because they are not immune tolerant to development of the autoimmune system. The use of these immunogenic substances in the whole cell fraction, which are generated by disease mutations, can be used for the treatment of cancer.
The whole cell fraction cancer vaccine system of the present invention can be used for preparing a vaccine for preventing and/or treating cancer. In the use as a cancer vaccine to prevent and treat cancer, the vaccine of the present invention may be administered multiple times before or after the occurrence of cancer or after surgical removal of tumor tissue to activate the immune system of the body, thereby delaying the progression of cancer, treating cancer, or preventing recurrence of cancer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram of the preparation process and application field of the vaccine according to the present invention; a, respectively collecting and preparing a nano vaccine or a micro vaccine by the water-soluble component and the water-insoluble component; and b, dissolving the whole cell component by using a solubilizing liquid containing a solubilizing agent and preparing a nano vaccine or a micro vaccine.
Fig. 2-17 are schematic structural diagrams of nano-or micro-sized particles loaded with water-soluble and water-insoluble cellular components, wherein 1: a water-soluble component in the cellular or tissue component; 2, a water insoluble component of the cellular or tissue component; 3, an immunopotentiating adjuvant; 4, nanoparticles or microparticles; 5: a core moiety in the nanoparticle; the surface and the interior of the nano-particle or the micro-particle in fig. 2-5 contain an immune enhancing adjuvant; the immunopotentiator of fig. 6-9 is distributed only inside the nanoparticle or microparticle; the nanoparticle or microparticle of figures 10-13 contains only immunopotentiating adjuvants on the outer surface; FIGS. 14-17, nanoparticle or microparticle having no immunopotentiating adjuvant both inside and outside surfaces; fig. 2, 6, 10 and 14 show that the water-soluble or non-water-soluble components of the cellular or tissue components loaded by the nanoparticles or microparticles do not form a distinct core when distributed within the nanoparticles or microparticles; in fig. 3, fig. 7, fig. 11 and fig. 15, the water-soluble or non-water-soluble components in the cellular or tissue components loaded by the nanoparticles or microparticles form a core part when distributed inside the nanoparticles or microparticles, and the core can be formed during the preparation process or by using polymers or inorganic salts, etc.; fig. 4, 8, 12 and 16 show that the water-soluble or non-water-soluble components of the cellular or tissue components loaded by the nanoparticles or microparticles form a plurality of inner core portions when distributed inside the nanoparticles or microparticles, and the inner core may be formed during the preparation process or by using polymers or inorganic salts, etc.; fig. 5, 9, 13 and 17 show that the water-soluble or non-water-soluble components of the cellular or tissue components entrapped by the nanoparticles or microparticles are located on the outer layer of the formed core when distributed inside the nanoparticles or microparticles; a: both nanoparticle or microparticle internal entrapment and surface loading are water soluble components in the cellular or tissue components; b: both nanoparticle or microparticle internal entrapment and surface loading are water insoluble components in the cellular or tissue components; c: the nano-particle or the micro-particle is internally encapsulated with water-insoluble components in the cell or tissue component, and the surface of the nano-particle or the micro-particle is encapsulated with water-soluble components in the cell or tissue component; d: the nano-particle or the micro-particle is internally encapsulated with water-soluble components in the cell or tissue component, and the surface of the nano-particle or the micro-particle is encapsulated with water-insoluble components in the cell or tissue component; e: the interior of the nanoparticle or the microparticle simultaneously loads water-soluble components and water-insoluble components in the cell or tissue components, and the surface of the nanoparticle or the microparticle simultaneously loads the water-soluble components and the water-insoluble components in the cell or the tissue components; the interior of the nanoparticle or the microparticle is simultaneously coated with water-soluble components and non-water-soluble components in the cell or tissue components, and the surface of the nanoparticle or the microparticle is only coated with the water-soluble components in the cell or the tissue components; the water-soluble components and the water-insoluble components in the cell or tissue components are simultaneously encapsulated in the nano-particle or the micro-particle, and the surface of the nano-particle or the micro-particle only carries the water-insoluble components in the cell or the tissue components; h: the nanometer particle or the micrometer particle only contains water-insoluble components in the cell or tissue components, and the surface of the nanometer particle or the micrometer particle simultaneously loads the water-soluble components and the water-insoluble components in the cell or the tissue components; i, the nano-particle or the micro-particle internally only comprises water-soluble components in the cell or tissue components, and the surface of the nano-particle or the micro-particle simultaneously loads the water-soluble components and the water-insoluble components in the cell or tissue components.
Fig. 18-28 are schematic structural diagrams of active targeting of a targeting modified nanoparticle or microparticle loaded with water-soluble and water-insoluble cellular components, wherein 1: a water-soluble component in the cellular or tissue component; 2: a water insoluble component in the cellular or tissue component; 3: an immunoadjuvant; 4: nanoparticles or microparticles; 5: a core moiety in the nanoparticle; 6: a target head that can target a particular cell or tissue. The nanoparticle or microparticle of FIGS. 18-19 contains an immunoadjuvant both on the surface and inside; the immunoadjuvant of figures 20-21 is distributed only inside the nanoparticle or microparticle; the nanoparticles or microparticles of fig. 22-23 contain an immunoadjuvant only on the outer surface; FIGS. 24-25, nanoparticle or microparticle having no immunoadjuvant both inside and outside surfaces; FIG. 26 the cellular components and/or immunoadjuvant are distributed only within the nanoparticle or microparticle; FIG. 27 the cellular components and/or immunoadjuvant are distributed only outside the nanoparticle or microparticle; figure 28 cellular components and immunoadjuvant are distributed inside or outside the nanoparticle or microparticle, respectively. In fig. 18-25, the water soluble or non-water soluble components of the cellular or tissue components supported by the nanoparticles or microparticles of fig. 18, fig. 2.A-2.I, fig. 20, 6.a-6.I, fig. 22, 10.A-10.I, and fig. 24, 14.A-14.I, do not form a distinct core when distributed within the nanoparticle or microparticle; 1 the water soluble or non-water soluble components of the cellular or tissue components supported by the nanoparticles or microparticles in fig. 19, fig. 20, fig. 7.a-7, fig. 22, fig. 11.A-11, and fig. 24, 15.A-15, are distributed within a core portion of the nanoparticle or microparticle; the water soluble or non-water soluble components of the cellular or tissue components supported by the nanoparticles or microparticles of fig. 19, fig. 4.A-4.I, fig. 21, fig. 8.A-8.I, fig. 23, fig. 12.A-12.I, and fig. 25 are distributed within the multiple core portions of the nanoparticle or microparticle; 1 5.a-5.i of FIG. 19, 9.a-9.i of FIG. 21, 13.A-13.I of FIG. 23 and 17.A-17.I of FIG. 25 the water soluble or non-water soluble components of the cellular or tissue components entrapped by the nanoparticles or microparticles are distributed within the outer layer of the inner core formed by the nanoparticles or microparticles; a: both nanoparticle or microparticle internal entrapment and surface loading are water soluble components in the cellular or tissue components; b: both nanoparticle or microparticle internal entrapment and surface loading are water insoluble components in the cellular or tissue components; c: the nano-particle or the micro-particle is internally encapsulated with water-insoluble components in the cell or tissue component, and the surface of the nano-particle or the micro-particle is encapsulated with water-soluble components in the cell or tissue component; d: the nano-particle or the micro-particle is internally encapsulated with water-soluble components in the cell or tissue component, and the surface of the nano-particle or the micro-particle is encapsulated with water-insoluble components in the cell or tissue component; e: the interior of the nanoparticle or the microparticle simultaneously loads water-soluble components and water-insoluble components in the cell or tissue components, and the surface of the nanoparticle or the microparticle simultaneously loads the water-soluble components and the water-insoluble components in the cell or the tissue components; the interior of the nanoparticle or the microparticle is simultaneously coated with water-soluble components and non-water-soluble components in the cell or tissue components, and the surface of the nanoparticle or the microparticle is only coated with the water-soluble components in the cell or the tissue components; the water-soluble components and the water-insoluble components in the cell or tissue components are simultaneously encapsulated in the nano-particle or the micro-particle, and the surface of the nano-particle or the micro-particle only carries the water-insoluble components in the cell or the tissue components; h: the nanometer particle or the micrometer particle only contains water-insoluble components in the cell or tissue components, and the surface of the nanometer particle or the micrometer particle simultaneously loads the water-soluble components and the water-insoluble components in the cell or the tissue components; i, only the water-soluble components in the cell or tissue components are encapsulated in the nano-particle or the micro-particle, and the water-soluble components and the water-insoluble components in the cell or tissue components are simultaneously loaded on the surface of the nano-particle or the micro-particle; 26-28, the water-soluble or non-water-soluble components of the cellular or tissue components supported by the nanoparticles or microparticles in a, b, and c do not form a distinct core when distributed within the nanoparticles or microparticles; d, e and f, wherein the water-soluble component or the non-water-soluble component in the cell or tissue component loaded by the nanoparticle or the microparticle is distributed in an inner core part inside the nanoparticle or the microparticle; the water-soluble components or the non-water-soluble components in the cell or tissue components loaded by the nano-particles or the micro-particles in g, h and i are distributed in a plurality of inner core parts inside the nano-particles or the micro-particles; the water-soluble components or non-water-soluble components in the cell or tissue components entrapped by the nano-particles or the micro-particles in j, k and l are distributed on the outer layer of the inner core formed inside the nano-particles or the micro-particles; the nano-particle or micro-particle load in a, d, g and j is the water-soluble component in the cell or tissue component; the nano-particle or micro-particle load in b, e, h and k is the water insoluble component in the cell or tissue component; the nanoparticles or microparticles in c, f, i and l simultaneously support water soluble and non-water soluble components in the cell or tissue components.
FIGS. 29-37 are experimental results of the growth rate and survival time of a mouse tumor when the nano-or micro-vaccine prepared with various cancer cells or tumor tissues in examples 1-9 was used for preventing or treating cancer, respectively; a, experimental results of tumor growth speed (n is more than or equal to 8) when the nano vaccine or the micro vaccine is used for preventing or treating cancers; b, the experimental result of the survival time of the mice when the nano vaccine or the micro vaccine is used for preventing or treating other cancers (n is more than or equal to 8), wherein each data point is the average value plus or minus standard error (mean plus or minus SEM); the significant difference of the tumor growth inhibition experiment in the a graph is analyzed by adopting an ANOVA method, and the significant difference in the b graph is analyzed by adopting Kaplan-Meier and log-rank test; * Represents that the vaccine group has a significant difference in p < 0.005 compared to the PBS blank group; # represents a significant difference in p < 0.005 in the vaccine group compared to the blank nanoparticle + cell lysate control group; * P < 0.0005, showing significant differences in vaccine groups compared to PBS blank; # # represents a significant difference in p < 0.0005 in the vaccine group compared to the blank nanoparticle + cell lysate control group. The vaccine group has a p less than 0.0005 compared with the polypeptide nano particle group, and has a significant difference; represents that the vaccine group has a significant difference in p < 0.05 compared with the control vaccine group (containing only B16F10 component or only liver cancer tissue component); represents the vaccine group versus the adjuvant-free vaccine group (p < 0.05 with significant differences).
Detailed Description
The invention discloses a broad-spectrum cancer vaccine system loaded with more than one cancer cell and/or tumor tissue whole cell component in nano-scale or micro-scale and application thereof in preventing or treating cancer. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the method and product of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods described herein without departing from the spirit and scope of the invention.
The present invention firstly obtains water-soluble components which are soluble in pure water or an aqueous solution without a solubilizing agent after the cancer cells and/or tissues are lysed, and then adopts the solubilizing aqueous solution containing the solubilizing agent to dissolve the water-insoluble components in the solubilizing solution, so that all the cell components can be converted into components which can be dissolved in the aqueous solution and then are loaded inside and outside nano particles or micro particles to prepare nano vaccines or micro vaccines for the prevention and treatment of cancers. In practical application, the whole cell component can be directly dissolved by using the solubilizing aqueous solution containing the solubilizing agent after the cell or tissue is lysed without collecting the water-soluble component and the non-water-soluble component respectively, and the whole cell component after the solubilizing aqueous solution is dissolved is used for preparing the nano vaccine or the micro vaccine.
The invention improves the comprehensiveness and immunogenicity of antigen substances or components loaded by nano-particles or micro-particles by adopting the water solution containing the solubilizing agent to convert the components insoluble in pure water or the water solution without the solubilizing agent in cells into the components soluble in specific solubilizing solution and can be used for preparing nano-particles and micro-particles.
The invention divides the whole cell component in cancer cells and/or tumor tissues into a water-soluble part which can be dissolved in pure water or an aqueous solution without a solubilizing agent and a water-insoluble part which can be dissolved in the aqueous solution by a certain solubilizing agent, and the water-soluble part and the water-insoluble part are packed in nano particles or micro particles and loaded on the surfaces thereof, thereby ensuring that most of antigen substances are loaded in the prepared vaccine.
The water soluble fraction and the water insoluble fraction of the cell fraction encompass the constituents and components of the whole cell. Wherein the same unmutated proteins, polypeptides and genes as normal cellular components do not elicit an immune response due to immune tolerance generated during development of the autoimmune system; mutations in genes, proteins and polypeptides produced by cancer are immunogenic and activate the immune response of the body against cancer cells because they are not immune tolerant to the development of the autoimmune system. The use of these substances in the whole cell fraction, which are specific for cancer cells and are immunogenic in the event of a disease mutation, can be used for the prophylaxis and treatment of cancer.
The nano vaccine and/or micro vaccine system can be used for preparing a vaccine for preventing and/or treating cancers, and the preparation process and the application field of the nano vaccine and/or micro vaccine system are shown in figure 1. After cells or tissues can be lysed during preparation, respectively collecting water-soluble components and water-insoluble components, and respectively preparing nano vaccine or micro vaccine; alternatively, the solubilizing liquid containing the solubilizing agent can be directly used for directly lysing cells or tissues and dissolving whole cell components to prepare nano-or micro-vaccines.
The whole cell component can be subjected to inactivation or denaturation treatment before or after the lysis to prepare the nano vaccine or the micro vaccine, or can be subjected to no inactivation, enzyme treatment or denaturation treatment before or after the cell lysis to directly prepare the nano vaccine or the micro vaccine. In some embodiments of the present invention, tumor tissue cells are subjected to inactivation or (and) denaturation before lysis, or may be subjected to inactivation, enzyme treatment, or (and) denaturation after cell lysis in the actual use process, or may be subjected to inactivation, enzyme treatment, or (and) denaturation before and after cell lysis; the inactivation or denaturation treatment method before or after the cell lysis or (and) in some embodiments of the present invention is ultraviolet irradiation and high temperature heating, and in the actual use process, inactivation or denaturation treatment methods such as radiation irradiation, high pressure, freeze drying, formaldehyde and the like may also be used. Those skilled in the art will appreciate that the actual application process can be appropriately adjusted according to the specific circumstances.
The surface of the broad-spectrum cancer vaccine system can be not connected with a target head with an active targeting function or connected with the target head with the active targeting function.
The structural schematic of the vaccine system for preventing or treating cancer based on the whole cell components of various cancer cells and/or tumor tissues is shown in fig. 2-28. In the actual use process, only the nano particles and/or the micro particles with a specific structure are used, or two or more nano particles and/or micro particles with different structures are used simultaneously.
In an embodiment, the immunopotentiator is coated in the nanoparticle or the microparticle and simultaneously supported on the surface of the nanoparticle or the microparticle, and in the actual use process, the immunopotentiator can be coated in the nanoparticle or the microparticle only, or is supported on the surface of the nanoparticle or the microparticle only, or is not added.
In some embodiments, the invention comprises the steps of first solubilising the water-soluble fraction or (and) the non-water-soluble fraction of the cellular fraction, which is soluble in pure water, by a solubiliser and then encapsulating the solubilised fraction in nanoparticles or microparticles, while supporting an immunopotentiator; then, the water-soluble portion or (and) the water-insoluble portion of the cellular component is loaded on the nanoparticle surface while the immunopotentiator is loaded. This allows the loading capacity of the water-soluble or non-water-soluble components of the cells in the nanoparticle or microparticle to be maximized. In practical applications, the solubilizing liquid (such as 8M urea aqueous solution or 6M guanidine hydrochloride aqueous solution) containing the solubilizing agent can also be directly used for directly lysing cells or tissues and directly lysing whole cell components, and then the nano vaccine or the micro vaccine can be prepared.
The method for preparing the nano vaccine and the micro vaccine is a common preparation method. In some embodiments, the nano vaccine is prepared by a multiple emulsion method in a solvent evaporation method, the adopted nano particle preparation material is organic high polymer polylactic acid-glycolic acid copolymer (PLGA) with the molecular weight of 24kDa-38kDa, and the adopted immunological adjuvant is poly (I: C), BCG or CpG. Those skilled in the art will understand that in the practical application process, the preparation method, the preparation process, the nanoparticle preparation material used, the type and concentration of the immunoadjuvant, etc. can be appropriately adjusted according to the specific situation.
In some embodiments, the specific preparation method of the multiple emulsion method adopted by the invention is as follows:
step 1, adding a first preset volume of aqueous phase solution containing a first preset concentration into a second preset volume of organic phase containing a second preset concentration of medical polymer material.
In some embodiments, the aqueous solution may contain components of cancer cell lysate, and immunopotentiating adjuvants poly (I: C), BCG, or CpG; the components in the cancer cell lysate are water-soluble components or raw water-insoluble components dissolved in 8M urea respectively during preparation. The aqueous solution contains the concentration of the water-soluble component from the cancer cells or the concentration of the primary water-insoluble component from the cancer cells dissolved in the solubilizing agent, i.e., the first predetermined concentration requires a concentration of the protein polypeptide of greater than 1 ng/mL, which is sufficient to support the cancer antigen to activate the associated immune response. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01 ng/mL.
In some embodiments, the aqueous solution contains components of tumor tissue lysate and immunopotentiating adjuvant poly (I: C), BCG or CpG; the components in the tumor tissue lysate are respectively water-soluble components or raw water-insoluble components or whole cell components dissolved in 8M urea are respectively dissolved in solubilizers such as 8M urea or 6M guanidine hydrochloride. The aqueous solution contains a concentration of water-soluble components derived from the tumor tissue or a concentration of raw water-insoluble components derived from the tumor tissue dissolved in 8M urea, i.e., the first predetermined concentration requires a protein polypeptide concentration level of greater than 1 ng/mL, which is sufficient to support the cancer antigen to activate the associated immune response. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01 ng/mL.
In the invention, the medical polymer material is dissolved in an organic solvent to obtain a second predetermined volume of an organic phase containing the medical polymer material with a second predetermined concentration. In some embodiments, the medical polymer material is PLGA, and the organic solvent is dichloromethane. Additionally, in some embodiments, the second predetermined concentration of the medical-grade polymeric material ranges from 0.5mg/mL to 5000mg/mL, preferably 100 mg/mL.
In the present invention, PLGA or modified additional PLGA is selected because this material is a biodegradable material and has been approved by the FDA for use as a drug dressing. The study shows that PLGA has certain immunoregulatory function, so it is suitable for use as vaccine preparing supplementary material.
In practice, the second predetermined volume of the organic phase is set according to the ratio of the same to the first predetermined volume of the aqueous phase, in the present invention the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase ranges from 1:1.1 to 1:5000, preferably 1:10. The ratio of the first predetermined volume, the second predetermined volume and the first predetermined volume to the second predetermined volume can be adjusted as needed in the implementation process to adjust the size of the prepared nano-particles or micro-particles.
Preferably, the aqueous solution is a lysate component solution wherein the concentration of protein and polypeptide is greater than 1 ng/mL, preferably 1 mg/mL to 100 mg/mL; when the aqueous phase solution is a lysate component/immunoadjuvant solution, the concentration of the protein and the polypeptide is more than 1 ng/mL, preferably 1 mg/mL-100 mg/mL, and the concentration of the immunoadjuvant is more than 0.01 ng/mL, preferably 0.01 mg/mL-20 mg/mL. In the high polymer material organic phase solution, the solvent is DMSO, acetonitrile, ethanol, chloroform, methanol, DMF, isopropanol, dichloromethane, propanol, ethyl acetate and the like, preferably dichloromethane; the concentration of the polymer material is 0.5 mg/mL-5000 mg/mL, preferably 100 mg/mL. The first emulsifier solution is preferably an aqueous solution of polyvinyl alcohol at a concentration of 10 mg/mL to 50 mg/mL, preferably 20 mg/mL. The second emulsifier solution is preferably an aqueous solution of polyvinyl alcohol at a concentration of 1 mg/mL to 20 mg/mL, preferably 5 mg/mL. The dispersion liquid is PBS buffer solution or normal saline or pure water.
And 2, carrying out ultrasonic treatment for more than 2 seconds or stirring or homogenizing treatment or microfluidic treatment for more than 1 minute on the mixed solution obtained in the step 1. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50 rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm-1500 rpm, and the stirring time is 0.1-24 hours; during ultrasonic treatment, the ultrasonic power is more than 5W, and the time is more than 0.1 seconds, such as 2-200 seconds; the high pressure/ultra-high pressure homogenizer or high shear homogenizer is used for homogenizing treatment, the pressure is more than 5 psi, such as 20 psi-100 psi, and the rotating speed is more than 100 rpm, such as 1000 rpm-5000 rpm; the flow rate is greater than 0.01 mL/min, such as 0.1 mL/min to 100 mL/min, using microfluidic processing. The ultrasonic treatment, stirring treatment, homogenizing treatment or microfluidic treatment is performed for nanocrystallization and/or micronization, the ultrasonic time, stirring speed or homogenizing treatment pressure and time can control the size of the prepared nano and/or micron particles, and the particle size change can be caused by the excessive or excessive small size.
And 3, adding the mixture obtained after the treatment in the step 2 into a third preset volume of aqueous solution containing the emulsifier with the third preset concentration, and performing ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute or performing homogenization treatment or microfluidic treatment. The mixture obtained in the step 2 is added into the aqueous solution of the emulsifier to continue ultrasonic treatment or stirring nanocrystallization or micronization. The step is to carry out nanocrystallization or micrometering, the ultrasonic time or stirring speed and time can control the size of the prepared nano particles or micro particles, and the particle size can be changed due to overlong or too short, so that proper ultrasonic time is required to be selected. In the present invention, the ultrasonic time is more than 0.1 seconds, such as 2 to 200 seconds, the stirring speed is more than 50rpm, such as 50rpm to 500 rpm, and the stirring time is more than 1 minute, such as 60 to 6000 seconds. Preferably, when the stirring is mechanical stirring or magnetic stirring, the stirring speed is greater than 50rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50 rpm-1500 rpm, and the stirring time is 0.5-5 hours; during ultrasonic treatment, the ultrasonic power is 50-500W, and the time is more than 0.1 seconds, such as 2-200 seconds; the high pressure/ultra-high pressure homogenizer or high shear homogenizer is used for homogenizing, the pressure is more than 20psi, such as 20 psi-100 psi, and the rotating speed is more than 1000 rpm, such as 1000 rpm-5000 rpm; the flow rate is greater than 0.01 mL/min, such as 0.1 mL/min to 100 mL/min, using microfluidic processing. The ultrasonic treatment, stirring or homogenizing treatment or micro-fluidic treatment is performed for nanocrystallization or microminiaturization, the ultrasonic time, stirring speed or homogenizing treatment pressure and time can control the size of the prepared nano or micron particles, and the particle size change can be caused by the excessive or excessive small size.
In the present invention, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) solution, the third predetermined volume is 5 mL, and the third predetermined concentration is 20 mg/mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume. In the present invention, the range of the second predetermined volume to the third predetermined volume is 1:1.1-1:1000, preferably may be 2:5. In order to control the size of the nano-or micro-particles during the implementation process, the ratio of the second predetermined volume to the third predetermined volume may be adjusted. Likewise, the ultrasonic time or stirring time, the volume of the aqueous solution of the emulsifier and the concentration of the aqueous solution of the emulsifier in this step are all determined according to the order of obtaining the nano-particles or micro-particles with proper size.
And 4, adding the liquid obtained after the treatment in the step3 into a fourth preset volume of a fourth preset concentration emulsifier water solution, and stirring until a preset stirring condition is met.
In this step, the aqueous emulsifier solution is still PVA.
The fourth predetermined concentration is 5 mg/mL, and the fourth predetermined concentration is selected based on obtaining the nano-particles or the micro-particles with proper size. The fourth predetermined volume is selected based on a ratio of the third predetermined volume to the fourth predetermined volume. In the present invention, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted in order to control the size of the nanoparticles or microparticles in the implementation process.
In the present invention, the predetermined stirring conditions of this step are until the evaporation of the organic solvent is completed, that is, the evaporation of methylene chloride in step 1 is completed.
And 5, centrifuging the mixed solution which meets the preset stirring condition in the step 4 at a speed of more than 100rpm for more than 1 minute, removing supernatant, and re-suspending the remained precipitate in a fifth preset volume of fifth preset-concentration aqueous solution containing a lyoprotectant or a sixth preset volume of PBS (or physiological saline).
In some embodiments of the present invention, the precipitate obtained in step 5 may be resuspended in a sixth predetermined volume of PBS (or physiological saline) without lyophilization, and subsequent experiments involving adsorption of cancer cell lysate onto the nanoparticle or microparticle surface may be performed directly.
In some embodiments of the invention, the precipitate obtained in step 5 is re-suspended in an aqueous solution containing a lyoprotectant, and then subjected to subsequent experiments involving adsorption of cancer cell lysate onto the nanoparticle or microparticle surface after lyophilization.
In the invention, trehalose (Trehalose) is selected as the lyoprotectant.
In the present invention, the fifth predetermined concentration of the lyoprotectant in this step is 4% by mass, and is set so as not to affect the lyophilization effect in the subsequent lyophilization.
And 6, freeze-drying the suspension containing the freeze-drying protective agent obtained in the step 5, and then, reserving the freeze-dried substance.
And 7, re-suspending the nanoparticle-containing suspension obtained in the step 5 in a sixth preset volume or re-suspending the freeze-dried substance containing the nanoparticles or the microparticles and the lyoprotectant obtained in the step 6 in a sixth preset volume of PBS (or normal saline), and mixing the freeze-dried substance with a seventh preset volume of water-soluble component or the original non-water-soluble component dissolved in 8M urea to obtain the nano vaccine or the micro vaccine.
In the present invention, the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferential volume ratio is 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1.
In some embodiments, the volume of the resuspended nanoparticle suspension is 10mL and the sum of the volume of the water soluble component in the cancer cell lysate or tumor tissue lysate or the original water insoluble component dissolved in 8M urea is 1 mL. The volume and the proportion of the two can be adjusted according to the requirement in actual use.
In the present invention, the water-soluble fraction contained in the cancer cell lysate or the tumor tissue lysate or the original water-insoluble fraction dissolved in 8M urea is used to contain poly (I: C), BCG or CpG, and the concentration of poly (I: C), BCG or CpG is more than 1 ng/mL.
The particle size of the nano vaccine or the micro vaccine is nano-scale or micro-scale, so that the vaccine can be guaranteed to be phagocytized by antigen presenting cells, and the particle size is in a proper range for improving the phagocytizing efficiency. The particle size of the nano vaccine is 1nm-1000nm, more preferably 30nm-1000nm, most preferably 100nm-600nm; the micrometer vaccine has a particle size of 1 m to 1000 m, more preferably a particle size of 1 m to 100 m, more preferably a particle size of 1 m to 10 m, and most preferably a particle size of 1 m to 5 m. In this example, the nanoparticle vaccine particle size is 100nm-600nm, and the micrometer vaccine particle size is 1 m-5 m.
In addition, in the present invention, urea and guanidine hydrochloride are used to solubilize the raw water-insoluble components in the cancer cell lysate or tumor tissue lysate, any other solubilizing material that can solubilize the raw water-insoluble components in an aqueous solution in practical use, such as sodium deoxycholate, SDS, alkaline solution with pH of more than 7, acidic solution with pH of less than 7, albumin, lecithin, high concentration inorganic salts, triton, tween, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, acetic acid, cholesterol, amino acids, glycosides, choline BrijTM-35Octaethylene glycol monododecyl etherCHAPSDigitoninlauryldimethylamine oxideIGEPAL CA-630;, or the above solubilizing solution can be used to solubilize both the water-soluble components and the water-insoluble components.
In addition, in the present invention, 8M urea and 6M guanidine hydrochloride aqueous solution are used to solubilize the raw water insoluble components in the cancer cell lysate or tumor tissue lysate, and any other urea concentration or guanidine hydrochloride concentration that can solubilize the raw water insoluble components in the cancer cell lysate or tumor tissue lysate in aqueous solution can be used in practical use; or using an 8M aqueous urea solution to dissolve both the water-soluble and non-water-soluble components.
In addition, in the embodiment of the invention, the nano vaccine and the micro vaccine are prepared by adopting a multiple emulsion method, and any other commonly used nano particle or micro particle preparation method can be adopted in practice.
In addition, in the embodiment of the present invention, the nano vaccine and the micro vaccine are prepared from PLGA, and any other material that can prepare nano particles or micro particles can be used in practice.
In addition, in the embodiment of the invention, the water-soluble component in the cancer cell lysate or the tumor tissue lysate or the original water-insoluble component dissolved in 8M urea is respectively coated inside the nanoparticles and adsorbed on the surfaces of the nanoparticles, and in actual use, the water-soluble component in the cancer cell lysate or the tumor tissue lysate and the original water-insoluble component dissolved in 8M urea can be mixed and then loaded inside the nanoparticles or on the surfaces of the nanoparticles; alternatively, 8M urea may be used to dissolve both the water soluble and non-water soluble components and then entrapped within and/or adsorbed onto the nanoparticle or microparticle surface. The loading means includes chemical bond bonding and/or non-chemical bond adsorption.
In addition, in the present invention, poly (I: C), manganese adjuvant, BCG and CpG are used as immunological adjuvants, and in practice, no immunological adjuvant or any other immunological adjuvant having an immunopotentiating function, such as pattern recognition receptor agonist, BCG cell wall skeleton, BCG methanol extraction residue, BCG cell wall dipeptide, mycobacterium phlei, polyoxin A, mineral oil, virus-like particle, immunopotentiating reconstituted influenza virus minibody, cholera enterotoxin, saponin and its derivatives, resiquimod, thymosin, nascent bovine liver active peptide, mi Kuimo, polysaccharide, curcumin, immunological adjuvant poly ICLC, short coryneform vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyminosinic acid, polyadenylic acid, alum, aluminum adjuvant, lanolin, vegetable oil, endotoxin, liposome adjuvant, GM-CSF, MF59, RNA, double-stranded DNA, CAF01, ginseng, etc. may be used.
In addition, in the present invention, some of the examples used vaccines were nano-vaccines, and some of the examples used micro-vaccines. The person skilled in the art may choose to use nanovaccines and/or microdevices in practice, depending on the circumstances.
In order to further understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise specified, the methods used in the embodiments of the present invention are all conventional methods; the materials, reagents and the like used are all commercially available. The nano-sized particles or micro-sized particle structures, the preparation method, the use strategy during disease treatment, and the like related to the embodiments of the present invention are merely representative methods, and other nano-sized particles or micro-sized particle structures, the preparation method, the use strategy during disease prevention or treatment, and the combination strategy with other drugs may also employ the methods described in the present invention. The examples only list the application of the invention in some cancers, but the invention can also be used in any other type of cancer. With respect to the specific methods or materials used in the embodiments, those skilled in the art may perform conventional alternatives based on the technical idea of the present invention and are not limited to the specific descriptions of the embodiments of the present invention. The specific administration time, the administration times, the administration scheme and the combination condition with other medicines can be adjusted according to the situation in practical application.
EXAMPLE 1 melanoma tumor tissue and Lung cancer tumor tissue Whole cell fraction Supported inside and on nanoparticle surface for prevention of melanoma
In this example, a mouse melanoma is used as a cancer model to demonstrate how to prepare a nanovaccine loaded with whole cell components of melanoma tumor tissue and lung cancer tumor tissue, and to use the vaccine to prevent melanoma.
In this example, B16F10 mouse melanoma cells were used as a cancer model. The B16F10 melanoma tumor tissue and LLC lung cancer tumor tissue were first lysed to prepare water-soluble and water-insoluble components of the tumor tissue. Then, organic polymer material PLGA is used as a nanoparticle framework material, polyinosinic-polycytidylic acid (poly (I: C)) is used as an immunoadjuvant, and a solvent volatilization method is adopted to prepare the nano vaccine loaded with the water-soluble components and the water-insoluble components of the tumor tissue. The nanovaccine is then used to prevent melanoma.
(1) Lysis of tumor tissue and collection of fractions
1.5X10 5 B16-F10 cells or 2X 10 6 LLC lung cancer cells were inoculated subcutaneously in the back of each C57BL/6 mouse, and the mice were sacrificed and tumor tissues were removed when tumors grew to a volume of about 1000 mm 3, respectively. Tumor tissue is diced and ground, and appropriate amount of pure water is added through a cell filter screen and freeze thawing is repeated 5 times, and can be accompanied by ultrasound to destroy the lysed tissue cells. After the tissue cells are lysed, centrifuging the lysate at a rotation speed of 5000g for 3 minutes, and taking supernatant fluid, namely the water-soluble component which is soluble in pure water; the water insoluble component insoluble in pure water can be converted into soluble in 8M urea aqueous solution by adding 8M urea dissolution precipitation section to the obtained precipitation section. The water-soluble components from B16-F10 tumor tissues and LLC lung cancer tumor tissues and the raw water-insoluble components dissolved in 8M urea are respectively mixed according to the ratio of 1:1 to obtain the raw material source for preparing the vaccine. In the control nanovaccine, this example uses a water-insoluble polypeptide solubilized by loading an equal proportion of water-soluble polypeptide B16-M20 (Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM),B16-M24 (Dag1, TAVITPPTTTTKKARVSTPKPATPSTD),B16-M27 (REGVELCPGNKYEMRRHGTTHSLVIHD) and 8M urea B16-M05(Eef2, FVVKAYLPVNESFAFTADLRSNTGGQA), B16-M46 (Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ), and TRP2:180-188(SVYDFFVWL).
(2) Preparation of nanovaccine
In the embodiment, the nano vaccine, blank nano particles serving as a control and nano particles loaded with various polypeptides are prepared by a multiple emulsion method in a solvent volatilization method, the PLGA molecular weight of the adopted nano particle preparation material is 24KDa-38KDa, the adopted immune adjuvant is poly (I: C), and the poly (I: C) is distributed in the nano particles and is loaded on the surfaces of the nano particles. The preparation method is as described above. The average grain diameter of the nanometer vaccine loaded with the whole cell component is about 320nm, and the surface potential of the nanometer vaccine is about-5 mV; about 180. Mu.g of protein or polypeptide component is loaded per 1mg of PLGA nanoparticle, and about 0.01mg of poly (I: C) immunoadjuvant is used per 1mgPLGA of nanoparticle both inside and outside. The particle size of the blank nanoparticle is about 270nm, pure water or 8M urea containing equivalent poly (I: C) is used for replacing corresponding water-soluble components and non-water-soluble components respectively when the blank nanoparticle is prepared, and the poly (I: C) equivalent to the nano vaccine is adsorbed on the outer surface of the blank nanoparticle. The preparation method of the polypeptide-loaded nanoparticle is the same as the preparation method of the whole cell-loaded component, the amount of poly (I: C) used is also the same, the particle size of the polypeptide nanoparticle is about 310nm, and about 150 mug of the polypeptide component is loaded per 1mg of PLGA nanoparticle. The vaccine only loaded with the whole cell component of the B16F10 tumor tissue is prepared by taking the water-soluble component from the B16-F10 tumor tissue and the original water-insoluble component dissolved in 8M urea as raw material sources.
(3) Nanometer vaccine for preventing cancer
The study control groups were PBS and blank nanoparticle + free tissue lysate group, respectively. Female C57BL/6 after 6-8 weeks is selected as a model mouse to prepare melanoma tumor-bearing mice.
The nano vaccine group dosing regimen is as follows: 200. Mu.L of a 2mg PLGA nanovaccine loaded with water-soluble components in the interior and surface and 200. Mu.L of a 2mg PLGA nanovaccine loaded with raw water-insoluble components in the interior and surface, respectively, were subcutaneously injected on the 49 th, 42 th, 35 th, 28 th and 14 th day before melanoma inoculation; each mouse was subcutaneously vaccinated with 1.510 5 B16F10 cells on day 0, lower right back.
The PBS control protocol is as follows: 400 l of PBS was subcutaneously injected 49 days, 42 days, 35 days, 28 days, and 14 days before melanoma inoculation, respectively; each mouse was subcutaneously vaccinated with 1.5x105B 16F10 cells on day 0, lower right back. Blank nanoparticle + free lysate control: 400 l of blank nanoparticles and an equal amount of free lysate to the vaccine load were subcutaneously injected at day 49, 42, 35, 28, and 14, respectively, prior to melanoma inoculation; blank nanoparticles and free lysate were injected at different sites; each mouse was subcutaneously vaccinated with 1.510 5 B16F10 cells on day 0, lower right back.
The dosing regimen for the polypeptide nanoparticle group was as follows: 200. Mu.L of a 2mg PLGA nanovaccine loaded with water-soluble components in the interior and surface and 200. Mu.L of a 2mg PLGA nanovaccine loaded with raw water-insoluble components in the interior and surface, respectively, were subcutaneously injected on the 49 th, 42 th, 35 th, 28 th and 14 th day before melanoma inoculation; each mouse was subcutaneously vaccinated with 1.510 5 B16F10 cells on day 0, lower right back.
In the experiment, the size of the tumor volume of the mice was recorded every 3 days starting on day 3. Tumor volume was calculated using the formula v=0.52ab 2, where v is tumor volume, a is tumor length, and b is tumor width. For ethical animal experiments, mice were considered dead and euthanized when their tumor volume exceeded 2000mm 3 in the mice life cycle test.
(4) Experimental results
As shown in fig. 29, tumors of vaccine-treated mice loaded with whole cell fractions of mixed tumor tissue of B16F10 and LLC all disappeared after inoculation; tumors of about 70% of mice in the vaccine-treated group loaded with B16F10 tumor tissue alone disappeared after inoculation; only about 25% of the tumors in the polypeptide-loaded nanoparticle group disappeared after inoculation. The tumors of the mice in the PBS control group and the blank nanoparticle control group grow and the production speed is high. In conclusion, the nano vaccine loaded with the water-soluble components and the water-insoluble components of various tumor tissues has good prevention effect on melanoma.
Example 2 Water-soluble cellular Components of melanoma and Lung cancer cells Supported inside and on the surface of microparticles for melanoma prevention
This example demonstrates how to prepare a micrometer vaccine loaded with only water soluble portions of melanoma and lung cancer cell fractions using a mouse melanoma as a cancer model, and to use the vaccine to prevent melanoma.
In this example, B16F10 melanoma and LLC lung cancer cells were first lysed to prepare water-soluble components and water-insoluble components. Then, organic polymer material PLGA (38 KDa-54 KDa) is used as a micron particle framework material, poly (I: C) is used as an immunoadjuvant, and a solvent volatilization method is adopted to prepare the micron vaccine loaded with the whole-cell water-soluble component. The micrometer vaccine is then used to prevent melanoma.
(1) Lysis of cancer cells and collection of fractions
Collecting a certain amount of B16F10 cells or LLC cells, removing culture medium, freezing at-20deg.C, adding a certain amount of ultrapure water, and repeatedly freezing and thawing for more than 3 times, which may be accompanied with ultrasound to destroy the lysed cells. After the cells are lysed, centrifuging the lysate at a speed of 3000g for 5min, and taking the supernatant as a water-soluble component soluble in pure water in B16F10 melanoma or LLC lung cancer cells. The water-soluble components derived from the two cancer cell lysates are mixed according to the ratio of 1:1 to obtain the antigen source for preparing the micrometer vaccine.
(2) Preparation of micrometer vaccine
In the embodiment, the preparation of the micrometer vaccine and the blank micro-particles used as the control adopt a multiple emulsion method in a solvent volatilization method, the adopted micrometer particle preparation material is PLGA, the adopted immunological adjuvant is CPG, and the CPG is distributed in the micrometer particles and is loaded on the surfaces of the micrometer particles. The preparation method is as described above. The particle size of the obtained micrometer vaccine is about 1.40 mu m after cell components and immune adjuvants are loaded on the surface of the micrometer particles, and the average surface potential of the micrometer particles is about-5 mV. 210 g of protein or polypeptide component is loaded per 1mg PLGA microns particle, and 0.01mg of CPG immunoadjuvant is used per 1mgPLGA microns inside and outside each half. The particle size of the blank micro-particles is about 1.20 mu m, and pure water containing the same amount of CPG is used for replacing the corresponding water-soluble components during the preparation of the blank micro-particles.
(3) Micron vaccine for prevention of cancer
Female C57BL/6 was selected for 6-8 weeks to prepare melanoma tumor-bearing mice. The micrometer vaccine group protocol is as follows: 400 l of 4mg PLGA micrometer vaccine loaded with water soluble components in cancer cell lysate both internally and topically was subcutaneously injected on day 28, day 21, and day 14, respectively, prior to melanoma inoculation; each mouse was subcutaneously vaccinated with 1.510 5 B16F10 cells on day 0, lower right back. The PBS blank protocol is as follows: 400 l PBS was injected subcutaneously on day 28, day 21, and day 14, respectively, prior to melanoma inoculation; each mouse was subcutaneously vaccinated with 1.510 5 B16F10 cells on day 0, lower right back. Blank microparticles+cell lysate control: 400 l of blank microparticles and an equivalent amount of cancer cell lysate as in the vaccine were subcutaneously injected on day 28, day 21, and day 14, respectively, prior to melanoma inoculation. Each mouse was subcutaneously vaccinated with 1.510 5 B16F10 cells on day 0, lower right back.
In the experiment, the size of the tumor volume of the mice was recorded every 3 days starting on day 3. Tumor volume was calculated using the formula v=0.52ab 2, where v is tumor volume, a is tumor length, and b is tumor width. Due to ethical animal experiments, mice were considered dead and euthanized when their tumor volume exceeded 2000mm 3 in the mice life cycle test.
(4) Experimental results
As shown in fig. 30, the tumor volume growth rate of mice in the micrometer vaccine administration group was significantly slowed and the survival time of mice was significantly prolonged compared to the PBS blank control group, the blank microparticle+cell lysate control group. Furthermore, mice in the micron vaccine dosing group had a complete disappearance of some mice tumor post inoculation. Therefore, the micrometer vaccine loaded with the water-soluble components of the melanoma and lung cancer cells has a preventive effect on the melanoma.
EXAMPLE 3 Lung cancer tumor tissue and liver cancer tumor tissue lysis fraction Supported inside and on the surface of microparticles for prevention of liver cancer
In this example, how to prepare a micrometer vaccine loaded with tumor tissue lysate components of liver cancer and lung cancer, and how to use the vaccine prepared from tumor tissue to prevent liver cancer was described.
In this example, the tumor tissue lysis components of liver cancer and lung cancer in mice were loaded in the ratio of 1:3 into the nanoparticle and on the surface to prepare the micrometer vaccine. First, the lung cancer and liver cancer tumor tissues of the mice are obtained and lysed to prepare water-soluble components of tumor mass tissues and raw water-insoluble components dissolved in 8M urea. Then, PLGA (24 kDa-38 kDa) is used as a microparticle scaffold material, and poly (I: C) is used as an immunoadjuvant to prepare the microparticle vaccine. The micrometer vaccine is then used to prevent tumors in Hepa 1-6 hepatoma tumor-bearing mice.
(1) Lysis of tumor tissue and collection of fractions
Each C57BL/6 mouse was inoculated subcutaneously with 2X 10 6 Hepa 1-6 cells or 2X 10 6 LLC lung cancer cells under the armpit, and when the inoculated tumors grew to a volume of about 1000 mm 3 each, the mice were sacrificed and tumor tissues were removed. The subsequent treatment was the same as in example 1.
(2) Preparation of micrometer vaccine
The procedure is as in example 2.
(3) Micron vaccine for prevention of cancer
Female C57BL/6 after 6-8 weeks is selected to prepare the Hepa 1-6 liver cancer tumor-bearing mice.
200. Mu.L of a 2mg PLGA micrometer vaccine loaded with water-soluble components in the interior and surface and 200. Mu.L of a 2mg PLGA micrometer vaccine loaded with original water-insoluble components in the interior and surface were subcutaneously injected on the 49 th day, 42 th day, 35 th day, 28 th day and 14 th day, respectively, before the inoculation of liver cancer cells. Each mouse was inoculated subcutaneously with 2X 10 6 Hepa 1-6 hepatoma cells on day 0 in the right underarm. The PBS blank protocol is as follows: 400 l PBS was injected subcutaneously on day 49, day 42, day 35, day 28 and day 14, respectively, prior to inoculation of liver cancer cells. Each mouse was inoculated subcutaneously with 2X 10 6 Hepa 1-6 hepatoma cells on day 0 in the right underarm. Blank microparticles+free lysate control: subcutaneous injections of 400 l of blank microparticles and an equivalent amount of free lysate to the vaccine load on day 49, day 42, day 35, day 28, and day 14, respectively, prior to inoculation of hepatoma cells; blank microparticles and free cell lysates were injected at different sites. Each mouse was inoculated subcutaneously with 2X 10 6 Hepa 1-6 hepatoma cells on day 0 in the right underarm. In the experiment, the method for monitoring tumor growth of mice is the same as that described above.
(4) Experimental results
As shown in fig. 31, the liver cancer tumor growth was faster in PBS control and in mice with blank nanoparticle+tissue lysate control. The tumor disappeared after tumor inoculation in the micrometer vaccine administration group mice. Therefore, the micrometer vaccine loaded with the water soluble component and the water insoluble component in lung cancer tumor tissues and liver cancer tumor tissue lysates has a preventive effect on liver cancer.
EXAMPLE 4 prevention of liver cancer by supporting Whole cell fraction of tumor tissue of lung cancer and liver cancer inside nanoparticle
In this example, a mouse liver cancer is used as a cancer model to demonstrate how to prepare a nano vaccine loaded with whole cell components of lung cancer and liver cancer tumor tissues, and to apply the vaccine to prevent liver cancer. First, lung cancer and liver cancer tumor tissues are lysed to prepare water-soluble components and non-water-soluble components of whole cell components and mixed in a ratio of 1:2. Then, PLGA is used as a nanoparticle framework material, poly (I: C) is used as an immunoadjuvant, and a solvent volatilization method is adopted to prepare the nano vaccine loaded with water-soluble components and non-water-soluble components of lung cancer tumor masses and liver cancer tumor masses. The nanometer vaccine is then used in preventing liver cancer.
(1) Lysis of cancer cells and collection of fractions
The procedure is as in example 3.
(2) Preparation of nanovaccine
In the embodiment, the nano vaccine is prepared by a multiple emulsion method in a solvent volatilization method, the molecular weight of the adopted nano particle preparation material PLGA is 24kDa-38kDa, the adopted immune adjuvant is poly (I: C), and the poly (I: C) is distributed in the nano particles and adsorbed on the surfaces of the nano particles. When the vaccine is prepared, the water-soluble component is a mixture of the water-soluble component of lung cancer tumor tissue and the water-soluble component of liver cancer tumor tissue, and is only distributed in the vaccine; the water insoluble component is a mixture of water insoluble component of lung cancer tumor tissue and water insoluble component of liver cancer tumor tissue, and is distributed only in vaccine. The particle size of the nano vaccine obtained after the immune adjuvant is adsorbed on the surface of the nano particle is about 300nm, and the average surface potential Zeta potential of the nano particle is about-6 mV. About 200. Mu.g of protein or polypeptide component is loaded per 1mg PLGA of nanoparticle, and 0.01mg of poly (I: C) immunoadjuvant is used per 1mgPLGA of nanoparticle in each case. The particle size of the blank nanoparticle is about 230nm, pure water or 8M urea containing poly (I: C) is used for replacing the corresponding water-soluble component and non-water-soluble component when the blank nanoparticle is prepared, and the poly (I: C) with the same amount as the nano vaccine is adsorbed on the surface of the blank nanoparticle.
(3) Nanometer vaccine for preventing cancer
The vaccine and PBS control dosing regimen and tumor growth monitoring regimen were as in example 3.
(4) Experimental results
As shown in fig. 32, the vaccine-protected group showed significant differences in tumor growth rate and survival time of mice compared to the control group. Furthermore, the tumors of the mice in the vaccine group disappeared after inoculation. Moreover, the vaccine loaded with lung cancer and liver cancer tumor tissues simultaneously has better preventive effect than the vaccine loaded with liver cancer tumor tissues only. Therefore, the nano vaccine loaded with the water-soluble component and the water-insoluble component in the lung cancer tumor tissue and the liver cancer tumor tissue has a preventive effect on liver cancer.
Example 5 pancreatic cancer tumor tissue and colon cancer tumor tissue lysis fraction supported inside and on the surface of nanoparticles for treatment of pancreatic cancer
In this example, a mouse pancreatic cancer is used as a cancer model to demonstrate how to prepare a nano vaccine loaded with pancreatic cancer tumor tissue and colon cancer tumor tissue lysate components, and to apply the vaccine to treat pancreatic cancer.
In the embodiment, the split components of the pancreatic cancer tumor tissue and the MC38 colon cancer tumor tissue of the mouse Pan02 are loaded in the interior and the surface of the nanoparticle according to the mass ratio of 2:1 to prepare the nano vaccine. The pancreatic and colon cancer tumor tissues of mice were first taken and lysed to prepare a water-soluble fraction and a raw water-insoluble fraction dissolved in 8M urea. In the preparation of the vaccine, the water-soluble component is a mixture of a pancreatic cancer tumor tissue water-soluble component and a colon cancer tumor tissue water-soluble component in a ratio of 2:1; the water insoluble component is a 2:1 mixture of pancreatic cancer tumor tissue water insoluble component and colon cancer tumor tissue water insoluble component. PLGA (molecular weight 7kDa-17 kDa) is used as a nanoparticle skeleton material, poly (I: C) is used as an immunoadjuvant, and a solvent volatilization method is used for preparing the nanometer vaccine. The nano vaccine is then used to treat tumors in Pan02 pancreatic cancer tumor-bearing mice.
(1) Lysis of tumor tissue and collection of fractions
Each C57BL/6 mouse was inoculated subcutaneously with 2 x 10 6 MC38 colon cancer cells or with 1 x 10 6 Pan02 pancreatic cancer cells in the armpit, and when the tumors inoculated in each mouse grew to a volume of about 1000 mm 3, respectively, the mice were sacrificed and tumor tissues were removed. The method for lysing cancer cells and the method for collecting the components are the same as above.
(2) Preparation of nanovaccine
The preparation method of the nano vaccine in the embodiment is the same as that of the nano vaccine.
(3) Nanometer vaccine for treating cancer
Female C57BL/6 for 6-8 weeks was selected for pancreatic carcinoma mice. Each mouse was subcutaneously vaccinated with 1 x 10 6 Pan02 cells on day 0, lower right back. The vaccine group was subcutaneously injected with 400 l of 4mg PLGA nanoparticles internally loaded with water-soluble components and surface-loaded with raw water-insoluble components on day 4, day 7, day 10, day 15, and day 20, respectively. PBS blank was subcutaneously injected with 400 l PBS on day 4, day 7, day 10, day 15, and day 20, respectively. Blank nanoparticle + lysate control groups were subcutaneously injected on days 4, 7, 10, 15 and 20 with 400 l of blank nanoparticle and an equal amount of free lysate to the vaccine load, respectively. In the experiment, the tumor monitoring and volume calculation methods of the mice are the same as those described above.
(4) Experimental results
As shown in fig. 33, the tumor growth rate of the nanovaccine group was significantly slowed and the survival time of mice was significantly prolonged compared to the control group. Furthermore, some mice had tumor disappeared after inoculation. Therefore, the nano vaccine loaded with the whole cell components of pancreatic cancer and colon cancer tumor tissues has a therapeutic effect on pancreatic cancer.
EXAMPLE 6 prevention of lung cancer by supporting Whole cell fraction of breast cancer and Lung cancer tumor tissue inside mannose-modified microparticles
In this example, a mouse lung cancer is used as a cancer model to demonstrate how to prepare a micrometer vaccine loaded with whole cell components of breast cancer and lung cancer tumor tissues, and to apply the vaccine to prevent lung cancer. The specific dosage form, adjuvant, administration time, administration times and administration scheme can be adjusted according to the situation in practical application.
In this example, tumor tissue lysis fractions of breast and lung cancer in mice were loaded at 1:4 into microparticles to prepare a micrometer vaccine. Tumor tissue of breast cancer and lung cancer of mice was first taken and lysed to prepare a water-soluble fraction and a raw water-insoluble fraction dissolved in 8M urea. And then, PLGA and mannose modified PLGA are used as a micron particle framework material, cpG is used as an immunoadjuvant, and a solvent volatilization method is adopted to prepare the micron vaccine. The micrometer vaccine has the capability of targeting dendritic cells.
(1) Lysis of tumor tissue and collection of fractions
2X 10 6 LLC lung cancer cells were inoculated subcutaneously under the armpits of each C57BL/6 mouse or 2X 10 6 4T1 breast cancer cells were inoculated under the armpits of each BALB/C mouse, and the mice were sacrificed and tumor tissues were removed when the tumors inoculated in the mice grew to 1000 mm 3. Other processing methods are the same as above.
(2) Preparation of micrometer vaccine
In the embodiment, the micron vaccine and the empty micron particle serving as the control are prepared by adopting a multiple emulsion method in a solvent volatilization method, the molecular weight of the adopted micron particle preparation material PLGA (50:50) is 38kDa-54kDa, and the molecular weight of the adopted mannose modified PLGA (50:50) is 38kDa-54kDa. The mass ratio of unmodified PLGA to mannose modified PLGA was 8:2. The micron vaccine is internally loaded with water-soluble components in cancer cell lysate, and the vaccine surface is loaded with raw water-insoluble components dissolved in 8M urea, and the adopted immunological adjuvant is CpG and CpG is distributed in the micron particles. The preparation method is that the average grain diameter of the micron particles is about 1.30 mu m, and the average surface potential is about-9 mV. 65 g of protein or polypeptide component is loaded per 1mg PLGA microns particle, and 0.025mg of CpG immunoadjuvant is used per 1mg of PLGA microparticles. The particle size of the blank micro-particles is about 1.20 mu M, and pure water or 8M urea containing the same amount of CpG is respectively adopted to replace the corresponding water-soluble components and non-water-soluble components when the blank micro-particles are prepared.
(3) Dendritic cell-targeted micron vaccine for cancer prevention
Female C57BL/6 after 6-8 weeks is selected as a model mouse to prepare the lung cancer tumor-bearing mouse. Vaccine groups 400 l of 4mg PLGA micron vaccine was injected subcutaneously on day 35, day 28, day 21, day 14 and day 7 before tumor inoculation. PBS blank was subcutaneously injected with 400 l of PBS on day 35, day 28, day 21, day 14, and day 7, respectively, prior to tumor inoculation. Blank microparticles + lysate control groups were subcutaneously injected with 400 l of blank microparticles and an equivalent amount of free lysate to the vaccine load on day 35, day 28, day 21, day 14 and day 7, respectively, prior to tumor inoculation. Each mouse was subcutaneously vaccinated with 2 x 10 6 LLC lung cancer cells on day 0, lower right back. In the experiment, the method for monitoring tumor growth of mice is the same as that described above.
(4) Experimental results
As shown in fig. 34, the tumor growth rate was significantly slower and the survival time was significantly prolonged in the mice in the micro vaccine group compared to the PBS control group and the blank microparticle + lysate control group. This shows that the active targeting micron vaccine loaded with the water soluble component and the water insoluble component in the breast cancer and lung cancer tumor tissue has a preventive effect on lung cancer.
EXAMPLE 7 use of a nanovaccine comprising whole cell fractions of liver cancer and melanoma tumor tissues supported inside and on the surface of nanoparticles and BCG as an immunoadjuvant for liver cancer prevention
In the embodiment, the mouse liver cancer is taken as a cancer model, and BCG is taken as an immune adjuvant to explain how to prepare the nano vaccine loaded with the whole tissue cell components of melanoma and liver cancer tumor and apply the vaccine to prevent liver cancer.
In this example, the water-soluble and non-water-soluble components of liver cancer and melanoma tumor tissues were first lysed and mixed at 3:1, respectively. Then, PLGA is used as a nanoparticle framework material, BCG is used as an immunoadjuvant, and a solvent volatilization method is adopted to prepare the nanometer vaccine.
(1) Lysis of tumor tissue and collection of fractions
In this example tumor tissue was lysed and lysate collected as above.
(2) Cleavage of BCG and collection of fractions
The lysis of BCG and lysate collection and solubilization method in this example are the same as the lysis method of cancer cells in example 1, except that the cancer cells are replaced with BCG.
(3) Preparation of nanovaccine
The preparation method, materials used, etc. of the nanovaccine in this example are the same as those in example 1. In this example, however, the nanovaccine loaded immunoadjuvant is replaced by poly (I: C) with water soluble or non-water soluble components in the BCG lysate.
(4) Nanometer vaccine for preventing liver cancer
Female C57BL/6 is selected as a model mouse to prepare a liver cancer tumor-bearing mouse. Vaccine groups were subcutaneously injected on day 35, day 28, day 21, day 14 and day 7 before tumor inoculation with 200 l of 2mg PLGA nanoparticles loaded with water soluble components in cancer cell lysates both internally and topically and 200 l of 2mg PLGA nanoparticles loaded with raw water insoluble components dissolved in 8M urea both internally and topically, respectively. PBS blank was subcutaneously injected with 400 l of PBS on day 35, day 28, day 21, day 14, and day 7, respectively, prior to tumor inoculation. Blank nanoparticle + lysate control groups were subcutaneously injected with 400 l of blank nanoparticle and an equivalent amount of free lysate to the vaccine load on day 35, day 28, day 21, day 14 and day 7, respectively, prior to tumor inoculation. Each mouse was subcutaneously inoculated with 2X 10 6 Hepa1-6 hepatoma cells on day 0, lower right back. The method for monitoring tumor growth of mice is the same as that described above.
(4) Experimental results
As shown in fig. 35, the tumor growth rate of the nanovaccine-administered group adjuvanted with BCG was significantly slowed and the survival period of mice was significantly prolonged compared to the control group. Therefore, the nano vaccine loaded with the whole cell components of the liver cancer and melanoma tumor tissues can prevent liver cancer.
EXAMPLE 8 6M guanidine hydrochloride solubilizes tumor tissue fractions of breast and colon cancer and is supported within and on the surface of microparticles for treatment of breast cancer
This example illustrates how 6M guanidine hydrochloride can be used to solubilize whole cell fractions and prepare a whole cell fraction loaded micrometer vaccine for the treatment of breast cancer. In this example, triple negative breast cancer in 4T1 mice was used as a cancer model. First, tumor tissue cells of breast cancer and colon cancer are inactivated and denatured, and the tumor tissue is lysed with 6M guanidine hydrochloride and whole cell fractions are lysed. And then, PLGA is used as a micron particle framework material, cpG is used as an immunoadjuvant, and a solvent evaporation method is adopted to prepare the micron vaccine loaded with the tumor tissue whole cell component. The micrometer vaccine was then used to treat tumors in 4T1 breast cancer tumor-bearing mice.
(1) Lysis of tumor tissue and collection of fractions
The BALB/C mice were inoculated subcutaneously with 2X 10 6 4T1 cells in the right armpit or C57BL/6 mice were inoculated subcutaneously with 2X 10 6 MC38 colon cancer cells in the right armpit, and when the tumors had grown to a volume of 1000 mm 3, the mice were sacrificed and tumor tissue was harvested. Cutting tumor tissue, adding collagenase, grinding, filtering by a cell filter screen, and collecting tumor tissue cells obtained by filtering. The obtained tumor tissue cells are respectively subjected to inactivation and denaturation treatment by adopting ultraviolet rays and high-temperature heating, then 6M guanidine hydrochloride is adopted to lyse the tumor tissue cells of breast cancer and colon cancer, and the tissue lysate is dissolved, and the tumor tissue lysate of the breast cancer and the tumor tissue lysate of the colon cancer are mixed according to the proportion of 5:1, so that the vaccine is prepared from the raw materials.
(2) Preparation of micrometer vaccine
The preparation of the micrometer vaccine and blank micrometer particles in this example is as described above using PLGA (50:50) with a molecular weight of 38KD-54 KD. CpG was used as an immunological adjuvant. The average grain diameter of the prepared micrometer vaccine is about 2.6 mu m, and the Zeta potential of the surface of the micrometer particle is-4 mV. The protein and polypeptide components loaded inside and outside the PLGA microparticles are 210 mug, and the total amount of CpG immunoadjuvant used inside and outside the 1mgPLGA nanometer particles is 0.02mg, and the inside and outside are half. The micro vaccine without adjuvant has the same properties as the vaccine with CpG adjuvant, except that it does not contain CpG adjuvant.
(3) Micron vaccine for treatment of cancer
Female BALB/c was selected for 6-8 weeks to prepare 4T1 tumor-bearing mice. Each mouse was subcutaneously vaccinated with 4 x 10 5 4T1 cells on day 0, lower right back. Vaccine treatment groups injected subcutaneously 400 l of 4mg PLGA m vaccine loaded with tumor tissue whole cell fraction both internally and topically on day 4, day 7, day 10, day 15, and day 20. PBS blank was subcutaneously injected with 400 l PBS on day 4, day 7, day 10, day 15, and day 20, respectively. Blank microparticles+free lysate control on days 4, 7, 10, 15 and 20, equal amounts of tumor tissue lysate was subcutaneously injected, respectively, and 4mg PLGA blank microparticles loaded with equal amounts of CpG without any lysate components. In the experiment, the tumor volume monitoring and calculating method of the mice are the same as that of the mice.
(4) Experimental results
As shown in fig. 36, the tumor growth rate was significantly slower and the survival time of mice was significantly prolonged in the micro vaccine administration group compared to the control group. Furthermore, the adjuvant-containing micro vaccine is better in therapeutic effect than the adjuvant-free micro vaccine. Therefore, the micrometer vaccine loaded with the whole cell components of lung cancer and colon cancer tumor tissues has a therapeutic effect on breast cancer.
EXAMPLE 9 melanoma tumor tissue and lung cancer cell Whole cell fraction Supported inside and surface of nanoparticles for prevention of melanoma
This example illustrates how to prepare a nanovaccine loaded with melanoma tumor tissue and whole cell components of lung cancer cells and to apply the vaccine to prevent melanoma. In this example, B16F10 melanoma tumor tissue and LLC cancer cells were first lysed to prepare the corresponding water-soluble fraction and the non-water-soluble fraction dissolved in 8M urea. Then mixing the water-soluble components from the tumor tissue and the water-soluble components from the cancer cells according to the mass ratio of 1:1 to obtain the water-soluble components used in the experiment; the water insoluble component from the tumor tissue and the water insoluble component from the cancer cells are mixed according to the mass ratio of 1:1 to obtain the water insoluble component used in the experiment; then, PLGA is used as a framework material, and colloidal manganese is used as an immune adjuvant to prepare the nano vaccine.
(1) Lysis of tumor tissue and cancer cells and collection of fractions
The method for lysing the melanoma tumor tissue and the LLC lung cancer cells of B16F10 is the same as the method for lysing the melanoma tumor tissue and the LLC lung cancer cells, adding DNA degrading enzyme to act for 20 minutes after the melanoma tumor tissue or the LLC cancer cells are lysed, centrifuging the lysate at 8000g for 5 minutes, and taking supernatant as a water-soluble component which can be dissolved in pure water; the water insoluble components insoluble in pure water can be converted to be soluble in an aqueous 8M urea solution by dissolving the precipitate fraction with 8M urea. The components are respectively mixed according to the mass ratio of 1:1 to obtain the raw material source for preparing the vaccine.
(2) Preparation of nanovaccine
In the embodiment, the nano vaccine and the blank nano particles are prepared by a multiple emulsion method, water-soluble components are loaded in the nano particles, but not water-soluble components are loaded on the surfaces of the nano vaccine, the PLGA molecular weight of the adopted nano particle preparation material is 7KDa-17KDa, and the adopted immune adjuvant is colloidal manganese and the colloidal manganese is distributed in the nano particles. Colloidal manganese was prepared by adding 20. Mu. LMnCl 2 (0.2M) to 180. Mu.L Na 3PO4 (0.028M), then mixed with 300. Mu.L water soluble component (80 mg/mL), then added to 1mL of PLGA in 100mg of methylene chloride and sonicated to prepare colostrum, then the above sample was added to 2.5 mL of 20 mg/mL aqueous polyvinyl alcohol (PVA) solution and sonicated, then 50 mL of 5 mg/mL aqueous PVA solution was added and stirred for 3 hours, then after centrifugation at 12000g 25 min, the nanoparticles were resuspended in 10 mL of 4% aqueous trehalose solution and lyophilized for 48 hours. The sample was used after dissolving in 9mL PBS and mixing the water insoluble components (80 mg/mL) of 1mL in 8M urea and allowing to act at room temperature for 10 min. The average grain diameter of the nanometer vaccine loaded with the whole cell component is about 520nm, and the surface potential of the nanometer vaccine is about-5 mV; about 190 g of protein or polypeptide component is loaded per 1 mg PLGA nanoparticle. The particle size of the blank nanoparticle is about 470nm, and the blank nanoparticle is loaded with the same amount of colloidal manganese.
(3) Nanometer vaccine for preventing cancer
Vaccine group mice were dosed 400 l each time each mouse was dosed with 4 mg PLGA nanovaccine, control group were dosed 400 l each time each mouse was dosed with 400 l PBS or blank nanoparticle + free lysate. Dosing regimen at prophylaxis and mouse tumor vaccination and monitoring regimen example 1.
(4) Experimental results
As shown in fig. 37, tumors of about 90% of mice in the vaccine-treated group disappeared after inoculation; the tumors of the mice in the PBS control group and the blank nanoparticle control group grow and have high growth speed. In conclusion, the nano vaccine loaded with the melanoma tumor tissue and the whole cell component of the lung cancer cells has a preventive effect on melanoma.
Claims (5)
1. Use of a nano-and/or micro-vaccine system for the prevention or treatment of cancer based on cancer cells and/or whole cell fractions of tumour tissue of lung cancer and melanoma in the manufacture of a medicament for the prevention and/or treatment of melanoma, characterized in that the vaccine system comprises nano-and/or micro-particles, a whole cell fraction mixture; the whole cell component mixture is a cancer cell and/or tumor tissue whole cell component of lung cancer and melanoma; the whole cell component is a water-soluble component mixture and/or a non-water-soluble component mixture of whole cells in cancer cells and/or tumor tissues of lung cancer and melanoma, the water-soluble component is a raw water-soluble part of the cells or tissues, which is soluble in pure water or an aqueous solution without a solubilizing agent, and the non-water-soluble component is a part of the cells or tissues, which is originally non-water-soluble, and is insoluble in pure water to be soluble in the aqueous solution with the solubilizing agent or an organic solvent by a solubilizing method.
2. The use according to claim 1, wherein the surface of the vaccine system may not be connected to a target head having an active targeting function or to a target head having an active targeting function.
3. The use according to claim 1, wherein the whole-cell component mixture is entrapped inside and/or supported on the surface of nano-and/or microparticles; the nano-and/or micro-particles also include an immunoadjuvant within and/or on the surface.
4. The use according to claim 1, wherein the nanoparticles are nanoscale-sized particles; the micron particles are micron-sized particles; the preparation materials of the nano and/or micro particles comprise organic synthetic polymer materials, natural polymer materials or inorganic materials; the nano-and/or micro-particles are spherical, ellipsoidal, barrel-shaped, polygonal, rod-shaped, plate-shaped, linear, worm-shaped, square, triangular, butterfly-shaped or disc-shaped.
5. The use according to claim 4, wherein the particle size of the nanovaccine and the nanoparticle is 1nm to 1000nm, respectively; the particle size of the micrometer vaccine and the micrometer particle is 1 m-1000 m respectively; the nano vaccine and/or the micro vaccine are electrically neutral, negatively charged or positively charged on the surface.
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| CN114887049A (en) * | 2022-04-18 | 2022-08-12 | 苏州尔生生物医药有限公司 | Immunologic adjuvant composition, cancer vaccine based on composition and application of immunologic adjuvant composition |
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| CN116942802A (en) * | 2023-06-05 | 2023-10-27 | 苏州尔生生物医药有限公司 | Cancer vaccine based on cancer cell and/or tumor tissue lysate component and synthetic cancer antigen and preparation method thereof |
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| CN110124019A (en) * | 2019-04-22 | 2019-08-16 | 海南医学院 | Bacterisation tumor cell vaccine and preparation method thereof |
| CN112245574A (en) * | 2020-10-23 | 2021-01-22 | 苏州大学 | Targeting conveying system loaded with whole-cell components and application thereof |
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