CN114887049A - Immunologic adjuvant composition, cancer vaccine based on composition and application of immunologic adjuvant composition - Google Patents
Immunologic adjuvant composition, cancer vaccine based on composition and application of immunologic adjuvant composition Download PDFInfo
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Abstract
The invention relates to an immune adjuvant composition, a cancer vaccine based on the composition and application thereof, wherein the immune adjuvant composition at least comprises the combination of (1) and (2) in the following components, (1) Poly (I: C) or Poly (ICLC), (2) CpG-ODN, wherein the CpG-ODN is at least two of A-class CpG-ODN, B-class CpG-ODN and C-class CpG-ODN, at least one of the A-class CpG-ODN, the B-class CpG-ODN and the C-class CpG-ODN, and (3) amino acid, polypeptide, lipid, carbohydrate, protein or inorganic salt. Based on the immune adjuvant composition, a cancer vaccine is provided, which comprises nano particles or micro particles, and an antigen component and an immune adjuvant composition loaded on the nano particles or the micro particles. The immune adjuvant composition provided by the invention can fully play a role in enhancing the response of the adjuvant to activate the cancer specific T cells, and better assists the vaccine to play a role.
Description
Technical Field
The invention relates to the field of immunotherapy, in particular to an immunoadjuvant composition, a cancer vaccine based on the composition and application of the composition.
Background
Cancer vaccines are one of the important approaches for cancer immunotherapy and prevention. The two most important parts of cancer vaccines are antigens and immunological adjuvants, wherein the antigens can provide identification labels, and the immunological adjuvants can effectively enhance the identification of antigens by the immune system of a body. Cancer vaccines function primarily by virtue of antigen-specific activation of cancer-specific T cells, i.e., cellular immunity. Toll-like receptor agonists are substances that can activate the innate immune response and are potential vaccine adjuvants. There are many types of Toll-like receptors, such as Toll-like receptor 3(TLR3), Toll-like receptor 4, Toll-like receptor 7, Toll-like receptor 8 and Toll-like receptor 9(TLR 9).
Poly (I: C) or Poly (ICLC) is an agonist of Toll-like receptor 3, while CpG oligodeoxynucleotides (CpG-ODN) are agonists of Toll-like receptor 9. Poly (I: C) (polyinosinic acid cytidylic acid) is a synthetic double-stranded RNA (dsRNA) analog, a molecular pattern associated with viral infection. Poly (I: C) is recognized by TLR3, inducing activation of NF-kB and cytokine production. Poly (ICLC) is a TLR3 agonist modified by Poly (I: C) with appropriate stabilization, and functions similarly to Poly (I: C). CpG-ODN are artificially synthesized Oligodeoxynucleotides (ODN) containing unmethylated cytosine-guanine dinucleotides (CpG) that mimic bacterial DNA in stimulating immune cells in a variety of mammals including humans. The structural features and immune effects of different types of CpG-ODN vary according to their chemical structures and biological properties, and are generally classified into A, B, C categories. The A class CpG-ODN takes a palindromic sequence containing CpG dinucleotide as a core, two ends of the palindromic sequence are poly G tails, a phosphodiester bond skeleton is partially sulfo-modified, and a high-level structure is formed by the palindromic sequence and the poly G, so that a large amount of I-type interferon can be induced by activating plasmacytoid dendritic cells, and the activity of the A class CpG-ODN on B cells is weak. The B class CpG-ODN is a full-sulfo modified linear CpG ODN, has strong immune stimulation activity on B cells, but can not activate plasmacytoid dendritic cells. The C-class CpG-ODN is a class of full-sulfo modified CpG ODN, can form a dimer through a palindromic sequence, has the activity of both A-type and B-type CpG-ODN, and can activate both plasmacytoid dendritic cells and B cells.
In the process of antigen activation of cancer-specific T cells, in addition to the presentation of the epitope to the surface of the antigen-presenting cell, secondary and tertiary signals are required to assist in activation of the cancer-specific T cells, otherwise the antigen is not effective in activating the cancer-specific T cells. The adjuvant can assist in activating cancer specific T cells, and the function of the vaccine is better exerted. However, the function of the immunoadjuvant still needs to be further developed so as to fully exert the activation effect on the cancer-specific T cells.
Disclosure of Invention
In order to solve the technical problems, the invention provides an immunologic adjuvant composition and a vaccine system for preventing or treating cancers by co-loading Poly (I: C)/Poly (ICLC) and CpG-ODN (and amino acid, polypeptide, lipid, carbohydrate, protein or inorganic salt) and antigen components. The immune adjuvant composition can better enhance the efficacy of activating cancer specific T cell reaction and better assist the vaccine to play a role.
The first object of the present invention is to provide an immunoadjuvant composition which is a combination comprising at least (1) and (2) of the following components: (1) poly (I: C) or Poly (ICLC), (2) CpG-ODN, wherein CpG-ODN is at least two of A class CpG-ODN, B class CpG-ODN and C class CpG-ODN, at least one of them is B class CpG-ODN or C class CpG-ODN, and (3) amino acid, polypeptide, lipid, carbohydrate, protein or inorganic salt.
Further, the A class CpG-ODN includes, but is not limited to, CpG-ODN2216, CpG-ODN 1585, CpG-ODN 2336, etc.
Further, B class CpG-ODN include, but are not limited to, CpG-ODN 1018, CpG-ODN2006, CpG-ODN1826, CpG-ODN 1668, CpG-ODN2007, CpG-ODN BW006, CpG-ODN SL01, etc.
Further, the C-class CpG-ODN includes, but is not limited to, CpG-ODN 2395, CpG-ODN SL03, CpG-ODN M362, etc.
Further, in the immunoadjuvant composition, the mass ratio of the total mass of CpG-ODN, the mass of Poly (I: C) or Poly (ICLC), and the mass of amino acids, polypeptides, lipids, saccharides, proteins or inorganic salts is 0.05-25:1: 0.25-50.
Further, the amino acid is a positively charged amino acid, preferably a combination comprising at least two positively charged amino acids, such as: arginine + histidine, arginine + lysine + histidine.
Further, the inorganic salt is releasable H + Or an inorganic salt of an acidic substance to form a proton sponge effect, preferably an ammonium salt, a carbonate, a bicarbonate, a phosphate.
The second purpose of the invention is to provide a cancer vaccine loaded with the immune adjuvant composition, which comprises nano-particles or micro-particles, and an antigen component and an immune adjuvant composition loaded on the nano-particles or micro-particles, wherein the antigen component is at least one polypeptide with the same type as the cancer, preferably a whole cell component antigen derived from cancer cells and/or tumor tissues.
Further, the preparation method of the whole cell component antigen comprises the following steps: lysing cancer cells or tumor tissue with water or a solution containing no lytic agent, and collecting a soluble fraction as a water-soluble fraction; dissolving the insoluble part with a dissolving agent, and converting into a soluble part which is a water-insoluble component, wherein the water-soluble component and the water-insoluble component are whole cell component antigens; wherein the dissolving agent is selected from urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate (such as sodium dodecyl sulfate, SDS), glycerol, protein degrading enzyme, albumin, lecithin, 0.1-2000mg/mL inorganic salt, Triton, Tween, DMSO (dimethyl sulfoxide), acetonitrile, ethanol, methanol, DMF (N, N-dimethylformamide), propanol, isopropanol, acetic acid, cholesterol, amino acid, glycoside, choline, Brij, and their mixture TM -35, octaethyleneglycol monodecyl ether (octaethyleneglycol monododecylether), CHAPS (3- [3- (cholamidopropyl) dimethylamino)]Propanesulfonic acid inner salt), Digitonin (Digitonin), lauryldimethyimine oxide (N, N-dimethyl alkyl-C10-16-amine-N-oxide),
CA-630 nonionic surfactant;
or cancer cells or tumor tissues are lysed by a lytic agent, and then the lysate is lysed to obtain the whole cell component antigen.
The water soluble and water insoluble portions of the cellular fraction encompass the components and constituents of the whole cell. Wherein the unmutated proteins, polypeptides and genes that are identical to normal cellular components do not elicit an immune response due to immune tolerance produced during development of the autoimmune system; the mutation of genes, proteins and polypeptides due to cancer is immunogenic and can activate the body's immune response against cancer cells because there is no immune tolerance generated during the development of the autoimmune system. The use of these substances with cancer cell-specific immunogenicity due to disease mutations in the whole cell fraction can be used for the prevention and treatment of cancer. The invention improves the comprehensiveness and immunogenicity of antigens loaded by cancer vaccines by converting components insoluble in pure water or aqueous solutions without a lytic agent in cells into components soluble in a specific lysis solution and can be used for preparing cancer vaccines by using the lysis agent-containing solution.
Further, the lysis of tumor tissue comprises the steps of: cutting tumor tissue into pieces, grinding, filtering with cell filter screen, adding appropriate amount of water or solvent-free solution, repeatedly freezing and thawing, and optionally ultrasonic to destroy lysed cells, centrifuging lysate after cell lysis, and collecting supernatant as water soluble component; adding a dissolving agent into the precipitate, wherein the soluble part converted in the precipitate is a water-insoluble component, which is the source of the antigen raw material for preparing the cancer vaccine.
Further, the lysis of cancer cells comprises the steps of: culturing cancer cell lines, centrifuging, removing supernatant, re-suspending cells with pure water or solvent-free solution, repeatedly freezing and thawing, and optionally ultrasonic to destroy lysed cells, centrifuging lysate after cell lysis, and collecting supernatant as water-soluble component; adding a dissolving agent into the precipitate, wherein the soluble part converted in the precipitate is a water-insoluble component, which is the source of the antigen raw material for preparing the cancer vaccine.
Further, the cancer vaccine is connected with a target head with an active targeting function, such as mannose, mannan, a CD32 antibody, a CD11c antibody, a CD103 antibody, a CD44 antibody and the like. The targeting head can lead the cancer vaccine to target specific tissues or cells, such as dendritic cells, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils, basophils, lymph nodes, thymus, spleen, bone marrow and the like.
Further, the antigen component and the immunoadjuvant are entrapped inside the nanoparticle or microparticle and/or on the surface of the nanoparticle or microparticle. Wherein, Poly (I: C) or Poly (ICLC) and multiple CpG-ODNs can be loaded in the interior and/or on the surface of the nano-particle or the micro-particle respectively.
Further, the particle size of the nano particles is 1nm-1000 nm; the particle size of the micron particles is 1-1000 μm; the surface of the nano vaccine or the micro vaccine prepared by the nano particles or the micro particles is neutral, negatively charged or positively charged.
Further, the cancer vaccine can be prepared according to the preparation method developed by nano-sized particles and micro-sized particles, including but not limited to common solvent evaporation method, dialysis method, extrusion method, hot melt method. In some embodiments of the invention, the cancer vaccine is prepared by a multiple emulsion method in a solvent evaporation method.
Further, the preparation material of the nano-particles or micro-particles includes, but is not limited to, organic synthetic polymer materials, natural polymer materials or inorganic materials. Wherein, the organic synthetic polymer material is biocompatible or degradable polymer material, including but not limited to PLGA (polylactic acid-glycolic acid copolymer), PLA (polylactic acid), PGA (polyglycolic acid), PEG (polyethylene glycol), PCL (polycaprolactone), Poloxamer, PVA (polyvinyl alcohol), PVP (polyvinylpyrrolidone), PEI (polyethyleneimine), PTMC (polytrimethylene carbonate), polyanhydride, PDON (poly-p-dioxanone), PPDO (poly-p-dioxanone), PMMA (polymethyl methacrylate), PLGA-PEG, PLA-PEG, PGA-PEG, polyamino acid, synthetic polypeptide, synthetic lipid, etc.; the natural polymer material is biocompatible or degradable, and includes but is not limited to lecithin, cholesterol, alginate, albumin, collagen, gelatin, cell membrane, starch, saccharide, polypeptide, etc.; inorganic materials are materials without significant biological toxicity, including but not limited to ferric oxide, ferroferric oxide, calcium carbonate, calcium phosphate, and the like.
The third purpose of the invention is to provide the application of the immune adjuvant composition or the cancer vaccine in preparing a cancer treatment medicament or a prevention medicament.
The fourth purpose of the invention is to provide the application of the immune adjuvant composition in preparing a cell immune activator. The B class CpG-ODN has strong immunostimulation activity on B cells but can not activate plasmacytoid dendritic cells, and the C class CpG-ODN can activate the plasmacytoid dendritic cells and B cells but mainly activates the B cells. Therefore, theoretically, the B-class and C-class CpG-ODN have better humoral immunity activation effect and poorer assistance effect in the aspect of cellular immunity activation; the A-class CpG-ODN can activate plasmacytoid dendritic cells to induce a large amount of I-type interferon, the activity on B cells is weak, the A-class activated cell has better immune effect, and the capability of activating humoral immunity is poorer. However, the invention unexpectedly finds that when the CpG-ODN mixed adjuvant containing the B-class CpG-ODN or the C-class CpG-ODN is combined with PolyIC or Poly ICLC, unexpected excellent effect can be obtained, and the effect is obviously superior to that when A-class CpG-ODN is used.
Further, at least one antigen in the cancer vaccine corresponds to a disease that is treated or prevented by the drug.
By the scheme, the invention at least has the following advantages:
according to the invention, the target points which can be activated by Poly (I: C) and different CpG-ODNs are different, amino acids, polypeptides, lipids, saccharides, proteins, inorganic salts and the like can assist and increase lysosome escape, and when the antigen components are co-loaded in a nano vaccine or a micro vaccine, the effect is obviously better than that of the mixture of Poly (I: C) and one CpG or the use of a plurality of CpGs. Therefore, the immune adjuvant composition can effectively enhance the ability of the loaded adjuvant to assist the antigen to activate specific immune response, greatly improves the prevention and treatment effects on diseases, and can be used for preparing medicaments for preventing and treating cancers.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.
FIG. 1 is a schematic diagram of the preparation and application of a vaccine in which the antigen component of the present invention is a whole cell component; a, respectively collecting water-soluble components and water-insoluble components and preparing a schematic diagram of a nano vaccine or a micro vaccine; dissolving the whole cell component by using a dissolving solution containing a dissolving agent and preparing a schematic diagram of the nano vaccine or the micro vaccine;
FIGS. 2 to 17 are schematic structural views of nano-sized or micro-sized particles loaded with water-soluble components and water-insoluble components, and when loaded with a polypeptide, the polypeptide may be divided into two parts of a water-soluble polypeptide and a water-insoluble polypeptide and loaded with reference to a whole cell component; wherein 1, water soluble components of cell or tissue components; 2. water insoluble components in cellular or tissue components; 3. an immunological adjuvant; 4. nanoparticles or microparticles; 5. an inner core portion in the nanoparticle;
FIGS. 18-28 are schematic structural diagrams of nanoparticles or microparticles loaded with water-soluble and water-insoluble components actively targeted to target modification, wherein when loading polypeptide, the polypeptide can also be divided into two parts of water-soluble polypeptide and water-insoluble polypeptide and loaded with reference to whole cell components; wherein 1, water soluble components of cell or tissue components; 2. water insoluble components in cellular or tissue components; 3. an immunological adjuvant; 4. nanoparticles or microparticles; 5. an inner core portion in the nanoparticle; 6.a target that can target a particular cell or tissue;
FIGS. 29-51 are experimental results of mouse tumor growth rate and survival when the CpG-ODN and Poly (I: C) or Poly (ICLC) mixed adjuvant and cancer cells or tumor tissue lysate were co-loaded into nano-or micro-vaccine for preventing or treating cancer in examples 1-23, respectively; wherein, a, the nano vaccine or the micro vaccine prevents or treats the tumor growth speed in cancerExperimental results (n is more than or equal to 8); b. the survival period experimental result 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), and each data point is the mean value plus or minus standard error (mean plus or minus SEM); the significant differences in the tumor growth inhibition experiments in panel a were analyzed by ANOVA, and in panel b by Kaplan-Meier and log-rank test; indicates a significant difference in p < 0.0005 compared to PBS blank control; # represents that p is less than 0.005 and has significant difference compared with the control group of blank nanoparticles and cell lysate; the # indicates that p is less than 0.0005 compared with the control group of blank nanoparticles and cell lysate, and has significant difference.&Or $ represents a significant difference with p < 0.05 compared to a specific ratio loaded with CpG and Poly (I: C) or Poly ICLC, or to a vaccine group loaded with only class 1 CpG and Poly (I: C) or Poly ICLC;&&or $ represents that there is a significant difference with p < 0.01 compared to the specific ratio of CpG and Poly (I: C) or Poly ICLC loaded, or compared to the vaccine group loaded with only 1 class of CpG and Poly (I: C) or Poly ICLC;
shows that compared with a vaccine group which is loaded with CpG and Poly (I: C) or Poly ICLC mixed with adjuvant in a specific ratio and only uses one CpG, the p is less than 0.01 and has significant difference; tau is significantly different from the control vaccine group loaded with lysate fraction alone without any immunoadjuvant by p < 0.005; ρ represents a significant difference compared to the vaccine group using CpG and Poly ic or Poly iclc mixed adjuvant and CpG: Poly ═ 1:1, p < 0.05; lambda represents a significant difference compared to the vaccine group using only the CpG immunoadjuvant and lysate components, p < 0.01; epsilon represents a significant difference compared to the vaccine group using only PolyIC adjuvant and lysate fractions with p < 0.01; δ δ δ represents a significant difference compared to using a particle group loaded with only two a-class CpG and one C-class CpG immunoadjuvants with p < 0.005; beta represents a significant difference compared to the vaccine group loaded with lysate fraction and arginine without any adjuvant, p < 0.005; phi represents a significant difference compared to the vaccine group using two CpG and PolyIC or PolyICLC mixed adjuvants with glycine addition of p < 0.05; phi stands for a mixture with only one CpG and PolyIC or PolyICLCCompared with a vaccine group added with adjuvant and a substance for increasing lysosome escape, the vaccine group has the significant difference that p is less than 0.05; phi represents a significant difference compared to the vaccine group using only one CpG and PolyIC or PolyICLC mixed adjuvant and adding lysosome escape increasing substances, p < 0.01; major differences were noted in: < 0.05 for: > p ≦ v, compared to vaccine groups using CpG and PolyIC or poly iclc mixed adjuvants but without any lysosomal escape increasing material; pi represents a significant difference compared with the vaccine group using two A-class CpG and PolyIC or PolyICLC as mixed adjuvant, p is less than 0.05; pi represents a significant difference compared to the vaccine group using two A-class CpG and PolyIC or PolyICLC as mixed adjuvants, with p < 0.01; psi represents a significant difference compared to the vaccine + CD4 deletion group with p < 0.05; ξ ξ ξ represents that p is less than 0.005 compared with the vaccine + CD8 replication group, and has significant difference; kappa represents that p is less than 0.005 and has significant difference compared with the vaccine + CD8 replication + CD4 replication group.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
The nano-vaccine and/or micro-vaccine system can be used for preparing a vaccine for preventing and/or treating cancer, and the preparation process and the application field thereof are shown in figure 1. During preparation, the cells or tissues can be cracked, and then the water-soluble component and the water-insoluble component are respectively collected and the nano vaccine or the micro vaccine is respectively prepared; or directly using a dissolving solution containing a dissolving agent to directly crack cells or tissues and dissolve whole cell components to prepare the nano vaccine or the micro vaccine.
The whole cell component can be inactivated or denatured and then prepared into the nano vaccine or the micro vaccine before (and) cracking, or can be directly prepared into the nano vaccine or the micro vaccine without any inactivation, enzyme treatment or (and) denaturation. In some embodiments of the present invention, the tumor tissue cells are inactivated or (and) denatured before being lysed, and may be inactivated, enzymatically treated, or (and) denatured after being lysed during the actual use process, or both before and after being lysed; in some embodiments of the present invention, the inactivation or denaturation treatment method is ultraviolet irradiation and high temperature heating, and in the actual use process, radiation irradiation, high pressure, freeze drying, formaldehyde and other inactivation or denaturation treatment methods can also be adopted.
The vaccine system of the present invention is schematically shown in fig. 2-28. In FIGS. 2-5, the surface and the interior of the nano-particle or the micro-particle contain immune adjuvant; in FIGS. 6-9, the immunoadjuvant is distributed only inside the nanoparticle or microparticle; in FIGS. 10-13, the immunoadjuvant is only on the outer surface of the nanoparticle or microparticle; figures 14-17 are devoid of immunopotentiating adjuvants on both the interior and exterior surfaces of the nanoparticle or microparticle; the water soluble or non-water soluble components of the cellular or tissue components of figures 2, 6, 10 and 14 are distributed within the nanoparticle or microparticle without forming a distinct inner core; the water-soluble or water-insoluble components of the cell or tissue components of fig. 3, 7, 11 and 15, when distributed within the nanoparticle or microparticle, form a core portion, which may be formed during the manufacturing process or by using polymers or inorganic salts, etc.; the water-soluble or water-insoluble components of the cell or tissue components of fig. 4, 8, 12 and 16, when distributed within the nanoparticle or microparticle, form a plurality of inner core portions, which may be formed during the manufacturing process or by using polymers or inorganic salts, etc.; the water soluble or non-water soluble components of the cellular or tissue components of figures 5, 9, 13 and 17 are located in the outer layer of the inner core formed when distributed within the interior of the nanoparticle or microparticle; a: both the inside load and the surface load of the nano particles or the micro particles are water-soluble components in cell or tissue components; b: both the inside and the surface of the nano-particle or the micro-particle are loaded with water-insoluble components in cell or tissue components; c: the nano particles or the micro particles are internally loaded with water-insoluble components in cell or tissue components, and the surface of the nano particles or the micro particles is loaded with water-soluble components in the cell or tissue components; d: the nano particles or the micro particles are internally loaded with water-soluble components in cell or tissue components, and the surface of the nano particles or the micro particles is loaded with water-insoluble components in the cell or tissue components; e: the water-soluble component and the water-insoluble component in the cell or tissue component are simultaneously loaded in the nano-particle or the micro-particle, and the water-soluble component and the water-insoluble component in the cell or tissue component are simultaneously loaded on the surface of the nano-particle or the micro-particle; the water-soluble component and the water-insoluble component in the cell or tissue component are simultaneously encapsulated in the nano-particle or the micro-particle, and the water-soluble component in the cell or tissue component is only loaded on the surface of the nano-particle or the micro-particle; the water-soluble component and the water-insoluble component in the cell or tissue component are simultaneously encapsulated in the nano-particle or the micro-particle, and the water-insoluble component in the cell or tissue component is only loaded on the surface of the nano-particle or the micro-particle; h: the inside of the nano particle or the micro particle only loads the water-insoluble component in the cell or tissue component, and the surface of the nano particle or the micro particle simultaneously loads the water-soluble component and the water-insoluble component in the cell or tissue component; i, only loading the water-soluble components in the cell or tissue components inside the nano-particle or the micro-particle, and simultaneously loading the water-soluble components and the water-insoluble components in the cell or tissue components on the surface of the nano-particle or the micro-particle;
in FIGS. 18-19, the surface and interior of the nanoparticle or microparticle contains an immunoadjuvant; in FIGS. 20-21, the immunoadjuvant is distributed only inside the nanoparticle or microparticle; in FIGS. 22-23 the nanoparticles or microparticles contain immunoadjuvants only on the outer surface; FIGS. 24-25 show no immunological adjuvant on the inner and outer surface of the nanoparticle or microparticle; FIG. 26 cellular components and/or immunoadjuvants are distributed only inside nanoparticles or microparticles; FIG. 27 the cellular components and/or immunoadjuvants are distributed only on the outside of the nanoparticle or microparticle; figure 28 cellular components and immunoadjuvants are distributed inside or outside nanoparticles or microparticles, respectively. In FIGS. 18-25, the water-soluble or water-insoluble component of the cell or tissue component carried by the nanoparticles or microparticles of FIGS. 2.a-2.i in FIG. 18, 6.a-6.i in FIG. 20, 10.a-10.i in FIG. 22, and 14.a-14.i in FIG. 24 does not form a distinct inner core when distributed within the nanoparticle or microparticle; 3.a-3.i in fig. 19, 7.a-7.i in fig. 20, 11.a-11.i in fig. 22 and 15.a-15.i in fig. 24, the water-soluble or water-insoluble component of the nanoparticle or microparticle-loaded cell or tissue component is distributed within an inner core portion of the nanoparticle or microparticle; the water soluble or non-water soluble component of the cellular or tissue component carried by the nanoparticles or microparticles of 4.a-4.i in fig. 19, 8.a-8.i in fig. 21, 12.a-12.i in fig. 23 and 16.a-16.i in fig. 25 is distributed over a plurality of core segments inside the nanoparticles or microparticles; the water-soluble or water-insoluble component of the cell or tissue fraction encapsulated by the nanoparticles or microparticles of 5.a-5.i in FIG. 19, 9.a-9.i in FIG. 21, 13.a-13.i in FIG. 23, and 17.a-17.i in FIG. 25 is distributed in the outer layer of the inner core formed within the nanoparticles or microparticles; a: both the inside load and the surface load of the nano particles or the micro particles are water-soluble components in cell or tissue components; b: both the inside and the surface of the nano-particle or the micro-particle are loaded with water-insoluble components in cell or tissue components; c: the nano particles or the micro particles are internally loaded with water-insoluble components in cell or tissue components, and the surface of the nano particles or the micro particles is loaded with water-soluble components in the cell or tissue components; d: the nano particles or the micro particles are internally loaded with water-soluble components in cell or tissue components, and the surface of the nano particles or the micro particles is loaded with water-insoluble components in the cell or tissue components; e: the water-soluble component and the water-insoluble component in the cell or tissue component are simultaneously loaded in the nano-particle or the micro-particle, and the water-soluble component and the water-insoluble component in the cell or tissue component are simultaneously loaded on the surface of the nano-particle or the micro-particle; the water-soluble component and the water-insoluble component in the cell or tissue component are simultaneously encapsulated in the nano-particle or the micro-particle, and the water-soluble component in the cell or tissue component is only loaded on the surface of the nano-particle or the micro-particle; the water-soluble component and the water-insoluble component in the cell or tissue component are simultaneously encapsulated in the nano-particle or the micro-particle, and the water-insoluble component in the cell or tissue component is only loaded on the surface of the nano-particle or the micro-particle; h: the inside of the nano particle or the micro particle only loads the water-insoluble component in the cell or tissue component, and the surface of the nano particle or the micro particle simultaneously loads the water-soluble component and the water-insoluble component in the cell or tissue component; i, only loading the water-soluble components in the cell or tissue components inside the nano-particle or the micro-particle, and simultaneously loading the water-soluble components and the water-insoluble components in the cell or tissue components on the surface of the nano-particle or the micro-particle. In FIGS. 26-28, the water-soluble or water-insoluble components of the cellular or tissue components carried by the nanoparticles or microparticles in a, b, and c are distributed within the nanoparticle or microparticle without forming a distinct inner core; d, in e and f, the water-soluble component or the water-insoluble component in the cell or tissue component loaded by the nano-particle or the micro-particle is distributed in an inner core part inside the nano-particle or the micro-particle; g, h and i, wherein the water-soluble component or the water-insoluble component in the cell or tissue component loaded by the nano-particle or the micro-particle is distributed in a plurality of inner core parts inside the nano-particle or the micro-particle; the water-soluble ingredients or the water-insoluble ingredients in the cell or tissue components encapsulated by the nano-particles or the micro-particles in j, k and l are distributed in the outer layer of the inner core formed in the nano-particles or the micro-particles; the nano particles or micro particles in a, d, g and j are loaded with water-soluble components in cell or tissue components; b, e, h and k are loaded with water-insoluble components in cell or tissue components by nano-particles or micro-particles; the nanoparticles or microparticles of c, f, i and l simultaneously carry water soluble and water insoluble components of a cellular or tissue component.
In the actual use process, only one kind of nano-particles and/or micro-particles with a specific structure can be used, or one or more kinds of nano-particles and/or micro-particles with different structures can be used simultaneously.
In some embodiments, the specific preparation method of the multiple emulsion process employed in the present invention is as follows:
In some embodiments, the aqueous phase solution may contain components of the cancer cell lysate and an immunoadjuvant; the components of the cancer cell lysate are water-soluble components or original water-insoluble components dissolved in a dissolving agent such as 8M urea or 6M guanidine hydrochloride. The aqueous solution contains a concentration of water soluble components from the cancer cells or a concentration of primary water insoluble components from the cancer cells dissolved in a lytic agent, i.e., the first predetermined concentration requires a protein polypeptide concentration greater than 1ng/mL that is loaded with sufficient cancer antigen to activate an associated immune response. The concentration of the immunopotentiating adjuvant in the initial aqueous phase is greater than 0.01 ng/mL.
The second predetermined concentration of the medical polymer material is in the range of 0.5mg/mL to 5000mg/mL, preferably 100 mg/mL.
In practice, the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the aqueous phase, in the present invention the ratio between the first predetermined volume of the aqueous phase and the second predetermined volume of the organic phase ranges from 1:1.1 to 1:5000, preferably 1: 10.
Preferably, when the aqueous phase solution is a lysate component solution, the concentration of the protein and the polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL; when the aqueous phase solution is lysate component/immune adjuvant solution, the concentration of protein and polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL, and the concentration of immune adjuvant is more than 0.01ng/mL, preferably 0.01 mg/mL-20 mg/mL. In the organic phase solution of the high molecular material, 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 0.5mg/mL to 5000mg/mL, preferably 100 mg/mL.
And 2, carrying out ultrasonic treatment, stirring, homogenizing treatment or micro-fluidic treatment 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 50rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is more than 5W, and the time is more than 0.1 second, such as 2-200 seconds; the homogenizing treatment is carried out by using a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer, wherein the pressure is more than 5psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 100rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used. The nano-crystallization and/or the micron crystallization are carried out by ultrasonic treatment, stirring treatment, homogenizing treatment or micro-fluidic treatment, the size of the prepared nano-particles and/or micron particles can be controlled by the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time, and the particle size can be changed when the particles are too large or too small.
And 3, adding the mixture obtained after the treatment in the step 2 into a third preset volume of aqueous solution containing a third emulsifier with a 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. Adding the mixture obtained in the step 2 into an emulsifier aqueous solution, and continuing to carry out ultrasonic treatment or stirring for nano-crystallization or micro-crystallization. The step is to carry out nano-crystallization or micro-crystallization, the size of the prepared nano-particles or micro-particles can be controlled by the length of the ultrasonic time or the stirring speed and time, and the change of the particle size can be brought by overlong or overlong ultrasonic time, so that the proper ultrasonic time needs to be selected. In the invention, the ultrasonic time is more than 0.1 second, such as 2-200 seconds, the stirring speed is more than 50rpm, such as 50-500 rpm, and the stirring time is more than 1 minute, such as 60-6000 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 50rpm to 1500rpm, and the stirring time is 0.5 hour to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W-500W, and the time is more than 0.1 second, such as 2-200 seconds; the homogenizing treatment is carried out by using a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer, wherein the pressure is more than 20psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 1000rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates of greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used.
In the present invention, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) solution, the third predetermined volume is 5mL, 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 and the third predetermined volume is set from 1:1.1 to 1:1000, preferably 2: 5. The ratio of the second predetermined volume to the third predetermined volume may be adjusted during the implementation in order to control the size of the nanoparticles or microparticles. Similarly, the ultrasonic time or stirring time, the volume of the emulsifier aqueous solution and the concentration of the emulsifier aqueous solution are all taken according to the values to obtain the nano-particles or micro-particles with proper size.
And 4, adding the liquid obtained after the treatment in the step 3 into a fourth preset volume of emulsifier aqueous solution with a fourth preset concentration, and stirring until preset stirring conditions are met.
In this step, the emulsifier aqueous solution is still PVA.
The fourth predetermined concentration is 5mg/mL, and the fourth predetermined concentration is selected based on obtaining nanoparticles or microparticles of a suitable 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 to 1:2000, preferably 1: 10. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted during implementation to control the size of the nanoparticles or microparticles.
In the present invention, the predetermined stirring condition in this step is until the volatilization of the organic solvent is completed, that is, the volatilization of dichloromethane in step 1 is completed.
And 5, centrifuging the mixed solution processed in the step 4 and meeting the preset stirring condition at the rotating speed of more than 100rpm for more than 1 minute, removing the supernatant, and resuspending the remaining precipitate in a fifth preset volume of aqueous solution with a fifth preset concentration of the lyoprotectant or a sixth preset volume of PBS (or physiological saline).
In some embodiments of the present invention, the pellet obtained in step 5 is resuspended in PBS (or physiological saline) of a sixth predetermined volume without lyophilization, and the subsequent experiments related to the adsorption of cancer cell lysate on the surface of nanoparticles or microparticles can be directly performed or directly used as nano-vaccine or micro-vaccine.
In some embodiments of the present invention, the precipitate obtained in step 5 is re-suspended in an aqueous solution containing a lyoprotectant, and is subjected to freeze-drying, and then subsequent experiments related to the adsorption of cancer cell lysate on the surface of nanoparticles or microparticles are performed after freeze-drying, or is directly used as a nano-vaccine or a micro-vaccine.
In the invention, the freeze-drying protective agent is Trehalose (Trehalose).
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 keeping the freeze-dried substance for later use.
And 7, mixing the nanoparticle-containing suspension obtained in the sixth preset volume in the step 5 or the freeze-dried substance containing the nanoparticles or the microparticles and the freeze-drying protective agent obtained in the step 6 by adopting the sixth preset volume of PBS (or normal saline) to re-suspend, with the seventh preset volume of water-soluble component or the original non-water-soluble component dissolved in a solvent such as 8M urea and the like, so as to obtain the nano vaccine or the micron 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 9mL, and the volume of the cancer cell lysate or the water-soluble component of the tumor tissue lysate or the original water-insoluble component dissolved in the lytic agent is 1 mL. The volume and the proportion of the two can be adjusted according to the needs when in actual use.
The nano-sized vaccine or the micro-sized vaccine has a particle size of nano-sized or micro-sized, which ensures that the vaccine is phagocytosed by antigen presenting cells, and the particle size is within a proper range in order to improve phagocytosis efficiency. The nano vaccine has a particle size of 1nm-1000nm, more preferably, a particle size of 30nm-1000nm, and most preferably, a particle size of 100nm-600 nm; the particle size of the micro-vaccine is 1 μm to 1000 μm, more preferably 1 μm to 100 μm, more preferably 1 μm to 10 μm, most preferably 1 μm to 5 μm. In the embodiment, the particle size of the nanoparticle vaccine is 100nm-600nm, and the particle size of the micrometer vaccine is 1 μm-5 μm.
In other embodiments, the specific method for preparing the nano-or micro-vaccine by using the multiple emulsion method is as follows:
In some embodiments, the aqueous solution may contain components of the cancer cell lysate and immune enhancing adjuvants Poly (I: C) and CpG-ODN, or Poly ICLC and CpG-ODN; the aqueous solution contains a concentration of water soluble components from the cancer cells or a concentration of primary water insoluble components from the cancer cells dissolved in a lytic agent, i.e., the first predetermined concentration requires a protein polypeptide concentration greater than 0.01ng/mL to be loaded with sufficient cancer antigen to activate an 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 second predetermined concentration of the medical material ranges from 0.5mg/mL to 5000mg/mL, preferably 100 mg/mL.
In practice, the second predetermined volume of the organic phase is set according to the ratio thereof 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 first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the nanoparticles or microparticles produced during the implementation process.
Preferably, when the aqueous phase solution is a lysate component solution, the concentration of the protein and the polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL; when the aqueous phase solution is lysate component/immunoadjuvant solution, the concentration of protein and polypeptide is more than 1ng/mL, preferably 1 mg/mL-100 mg/mL, and the concentration of immunoadjuvant is more than 0.01ng/mL, preferably 0.01 mg/mL-20 mg/mL. In the organic phase solution of the high molecular material, 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 0.5mg/mL to 5000mg/mL, preferably 100 mg/mL.
And 2, carrying out ultrasonic treatment, stirring, homogenizing treatment or micro-fluidic treatment 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 50rpm, the stirring time is greater than 1 minute, for example, the stirring speed is 50rpm to 1500rpm, and the stirring time is 0.1 hour to 24 hours; during ultrasonic treatment, the ultrasonic power is more than 5W, and the time is more than 0.1 second, such as 2-200 seconds; the homogenizing treatment is carried out by using a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer, wherein the pressure is more than 5psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 100rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates of greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used. The nano-grade and/or micron-grade particles are subjected to ultrasonic treatment, stirring treatment, homogenizing treatment or microfluidic treatment, the size of the prepared micro-nano particles can be controlled by the ultrasonic time or the stirring speed or the homogenizing treatment pressure and time, and the particle size can be changed when the particles are too large or too small.
And 3, adding the mixture obtained after the treatment in the step 2 into a third preset volume of aqueous solution containing a third preset concentration of emulsifier, and performing ultrasonic treatment, stirring, homogenizing treatment or microfluidic treatment. Adding the mixture obtained in the step 2 into an emulsifier aqueous solution, and continuing to carry out ultrasonic treatment or stirring for nano-crystallization or micro-crystallization. The step is to carry out nano-crystallization or micro-crystallization, the ultrasonic time is long or short, or the stirring speed and the stirring time can control the size of the prepared nano-particles or micro-particles, and the change of the particle size can be caused by overlong or overlong ultrasonic time, so that the proper ultrasonic time is required to be selected. In the invention, the ultrasonic time is more than 0.1 second, such as 2-200 seconds, the stirring speed is more than 50rpm, such as 50-500 rpm, and the stirring time is more than 1 minute, such as 60-6000 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 50rpm to 1500rpm, and the stirring time is 0.5 hour to 5 hours; during ultrasonic treatment, the ultrasonic power is 50W-500W, and the time is more than 0.1 second, such as 2-200 seconds; the homogenizing treatment is carried out by using a high pressure/ultrahigh pressure homogenizer or a high shear homogenizer, wherein the pressure is more than 20psi, such as 20 psi-100 psi, when the high pressure/ultrahigh pressure homogenizer is used, and the rotating speed is more than 1000rpm, such as 1000 rpm-5000 rpm, when the high shear homogenizer is used; microfluidic processing flow rates of greater than 0.01mL/min, such as 0.1mL/min to 100mL/min, are used.
In the present invention, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) solution, the third predetermined volume is 5mL, 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 and the third predetermined volume is set in the range of 1:1.1 to 1:1000, preferably 2: 5. The ratio of the second predetermined volume to the third predetermined volume may be adjusted during the implementation in order to control the size of the nanoparticles or microparticles. Similarly, the ultrasonic time or stirring time, the volume of the emulsifier aqueous solution and the concentration of the emulsifier aqueous solution are all taken according to the values to obtain the nano-particles or micro-particles with proper size.
And 4, adding the liquid obtained after the treatment in the step 3 into a fourth predetermined volume of emulsifier aqueous solution with a fourth predetermined concentration, and stirring until a predetermined stirring condition is met or directly carrying out subsequent treatment without stirring.
In this step, the emulsifier aqueous solution is still PVA.
The fourth predetermined concentration is 5mg/mL, and the fourth predetermined concentration is selected based on obtaining nanoparticles or microparticles of a suitable 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 to 1:2000, preferably 1: 10. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted during the implementation to control the size of the nanoparticles or microparticles.
In the present invention, the predetermined stirring condition in this step is that the volatilization of the organic solvent is completed, that is, the volatilization of dichloromethane in step 1 is completed.
And 5, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition at the rotating speed of more than 100RPM for more than 1 minute, removing the supernatant, and re-suspending the remaining precipitate in a fifth preset volume of solution containing the water-soluble and/or non-water-soluble components in the whole-cell components at a fifth preset concentration, or re-suspending the remaining precipitate in a fifth preset volume of solution containing the water-soluble and/or non-water-soluble components and the adjuvant in the whole-cell components at a fifth preset concentration.
And 6, centrifuging the mixed solution which is processed in the step 5 and meets the preset stirring condition for more than 1 minute at the rotating speed of more than 100RPM, removing the supernatant, resuspending the remaining precipitate in a sixth preset volume of solidification treatment reagent or mineralization treatment reagent, performing centrifugal washing after a certain time of action, and adding a seventh preset substance containing positively or negatively charged substances and performing a certain time of action.
In some embodiments of the present invention, the pellet obtained in step 6 may be resuspended in the seventh predetermined volume of the charged substance without lyophilization, and may be directly subjected to subsequent experiments related to the loading of cancer cell/tissue lysate on the surface of nanoparticles or microparticles, or directly used as nano-vaccine or micro-vaccine.
In some embodiments of the present invention, the precipitate obtained in step 6 is re-suspended in an aqueous solution containing a dry protective agent, and then is subjected to vacuum drying at room temperature or freeze vacuum drying, and then is subjected to subsequent experiments related to the adsorption of cancer cell lysate on the surface of the nano-particle or micro-particle after drying, or is directly used as a nano-vaccine or a micro-vaccine.
In the invention, the freeze-drying protective agent is Trehalose (Trehalose) or a mixed solution of mannitol and sucrose. In the present invention, the concentration of the drying protective agent in this step is set so as not to affect the drying effect in the subsequent drying, because the concentration is 4% by mass.
And 7, drying the suspension containing the drying protective agent obtained in the step 6, and then keeping the dried substance for later use.
8, resuspending the suspension containing the nanoparticles in PBS (or physiological saline) obtained in the step 6 with an eighth predetermined volume or resuspending the dried substance containing the nanoparticles or microparticles and the dry protective agent obtained in the step 7 with PBS (or physiological saline) with an eighth predetermined volume for direct use; or mixed with a ninth predetermined volume of a water-soluble component or a water-insoluble component.
In the present invention, the modification and antigen loading steps of steps 5-8 can be repeated multiple times to increase the antigen loading. When a substance having a positive or negative charge is added, a substance having the same charge may be added a plurality of times or a substance having different charges may be added alternately.
In some embodiments, the volume of the resuspended nanoparticle suspension is 9mL, and the volume of the cancer cell lysate or the tumor tissue lysate containing water-soluble components or the original water-insoluble components is 0.1-100 mL. The volume and the proportion of the two can be adjusted according to the needs when in actual use.
Example 1 adjuvant and melanoma tumor tissue antigen Loading inside nanoparticles for prevention of melanoma
In this example, a mouse melanoma is used as a cancer model to illustrate how to prepare a nano vaccine co-loaded with an immune adjuvant and a melanoma tumor tissue whole cell component, and to prevent melanoma using the nano vaccine. In this example, B16F10 mouse melanoma cells were used as a cancer model. B16F10 melanoma tumor tissue was first lysed and water soluble and water insoluble fractions of the tumor tissue were prepared. Then, the organic polymer material PLGA is used as a nanoparticle framework material, CpG-ODN 1018(B class), CpG-ODN 2395(C class) and poly (I: C) are used as immune adjuvants, and a solvent evaporation method is adopted to prepare the nano vaccine loaded with water-soluble components and water-insoluble components of tumor tissues. The nano-vaccine is then used to prevent melanoma.
(1) Lysis of tumor tissue and Collection of fractions
Subcutaneous inoculation of 1 in the dorsal part of each C57BL/6 mouse.5×10 5 B16-F10 cells, which grow to a volume of about 1000mm in tumor 3 Mice were sacrificed and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, and a proper amount of pure water is added through a cell filter screen and repeated freeze thawing is carried out for 5 times, and ultrasonic waves are accompanied to destroy the cells of the lysed tissue. After the tissue cells are cracked, centrifuging the lysate for 3 minutes at the rotating speed of 5000g, and taking supernatant fluid as a water-soluble component soluble in pure water; the addition of 8M urea to the resulting precipitate portion dissolves the precipitate portion, converting the water-insoluble components that are insoluble in pure water to soluble in an 8M aqueous urea solution.
(2) Preparation of nano-vaccine
The nano vaccine and the blank nano particle used as a reference in the embodiment are prepared by a solvent volatilization method, the molecular weight of PLGA used as a nano particle preparation material is 7KDa-17KDa, and the nano vaccine loaded with the water-soluble component and the nano vaccine loaded with the water-insoluble component are respectively prepared and used together when in use. The adopted immune adjuvants are CpG-ODN 1018, CpG-ODN 2395 and poly (I: C), and the mass ratio of the CpG-ODN 2395 to the poly (I: C) is 0.5:1: 1. preparation method as described above, PLGA nanoparticles entrap an immunoadjuvant and a whole cell component in a nano-vaccine. The average particle size of the nano vaccine loaded with the whole cell component is about 280nm, and the surface potential of the nano vaccine is about-5 mV; about 80 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, the total mass of the adjuvant used on each 1mg of PLGA nano particle is 0.05mg, wherein the CpG-ODN 1018 is 0.01mg, the CpG-ODN 2395 is 0.02mg, and the poly (I: C) is 0.02 mg. The blank nanoparticle only loaded with 0.05mg of immunologic adjuvant (CpG-ODN 1018 is 0.01mg, CpG-ODN 2395 is 0.02mg, poly (I: C) is 0.02mg) has a particle size of about 270nm, and pure water or 8M urea containing the same amount of adjuvant is respectively adopted to replace corresponding water-soluble components and non-water-soluble components during preparation of the blank nanoparticle. The vaccine only loading the B16F10 tumor tissue whole cell component is prepared by taking the water-soluble component from the B16F10 tumor tissue and the original water-insoluble component dissolved in 8M urea as raw material sources, each 1mg of PLGA nano particle is loaded with about 80 mu g of protein or polypeptide component, and the surface potential of the nano vaccine is about-5 mV. The particle size of the control nano vaccine is 280nm, about 80 mu g of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, the total mass of the adjuvant used on each 1mg of PLGA nano particle is 0.05mg, wherein the CpG-ODN 1018 is 0.0125mg, the CpG-ODN 2395 is 0.0125mg, the poly (I: C) is 0.025mg, and the surface potential of the nano vaccine is about-5 mV.
(3) Nano-vaccine for cancer prevention
The study control groups were PBS group and blank nanoparticle + free tissue lysate group, respectively. Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice.
The nano vaccine group administration scheme is as follows: subcutaneously injecting 100. mu.L of 1mg PLGA nano-vaccine loaded with a water-soluble component and 100. mu.L of 1mg PLGA nano-vaccine loaded with an original water-insoluble component on days 42, 35, 28, 21 and 7, respectively, before the melanoma inoculation; each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 And B16F10 cells.
The PBS control protocol was as follows: subcutaneous injections of 200 μ L PBS were given on day 42, 35, 28, 21, and 7, respectively, before melanoma inoculation; each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 And B16F10 cells.
Blank nanoparticle + free lysate control group: subcutaneous injection of 200 μ L of blank nanoparticles and equivalent amounts of free lysate was administered on days 42, 35, 28, 21 and 7, respectively, before melanoma inoculation; injecting blank nanoparticles and free lysate at different parts; each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 And B16F10 cells.
In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 3. Tumor volume is calculated by the formula v ═ 0.52 × a × b 2 Calculation, where v is tumor volume, a is tumor length, and b is tumor width. From animal experiment ethics, when the tumor volume of the mouse exceeds 2000mm in the life test of the mouse 3 I.e. the mice were considered dead and were euthanized.
(4) Results of the experiment
As shown in fig. 29, the tumors of both PBS control and blank nanoparticle control mice grew and were produced at a fast rate. The curative effect of the nano vaccine group is better than that of a PBS control group and a blank nano particle control group, and most of tumors of a mouse in the nano vaccine treatment group loaded with a mixed adjuvant and a tumor tissue whole cell component in a ratio of 1.5:1 (CpG-ODN: poly (I: C)) disappear after inoculation, which is better than that of a vaccine loaded with a mixed adjuvant and a tumor tissue whole cell component in a ratio of 1:1 (CpG-ODN: poly (I: C)). In conclusion, the nano vaccine loaded with a plurality of CpG-ODN and Poly (I: C) and antigen components in a specific ratio has a good prevention effect on melanoma.
Example 2 adjuvant and polypeptide Loading inside nanoparticles for prevention of melanoma
In this example, mouse melanoma is used as a cancer model to illustrate how to prepare a nano vaccine co-loaded with immune adjuvant and polypeptide, and to prevent melanoma using the vaccine. In this example, B16F10 mouse melanoma cells were used as a cancer model. The organic high molecular material PLGA is used as a nanoparticle framework material, poly (I: C), CpG-ODN2006 (B class) and CpG-ODN2216(A class) are used as immune adjuvants, and a solvent evaporation method is adopted to prepare the nano vaccine loaded with the adjuvant and the polypeptide antigen. The nano-vaccine is then used to prevent melanoma.
(1) Polypeptide antigens
The present example uses water-insoluble polypeptide antigens B16-M05(Eef2, FVVKAYLPVNESFAFTADLRSNTGGQA), B16-M46(Actn4, NHSGLVTFQAFIDVMSRETTDTDTADQ) and TRP2:180-188(SVYDFFVWL) solubilized by water-soluble polypeptide antigens B16-M20(Tubb3, FRRKAFLHWYTGEAMDEMEFTEAESNM), B16-M24(Dag1, TAVITPPTTTTKKARVSTPKPATPSTD), B16-M27(REGVELCPGNKYEMRRHGTTHSLVIHD) and 8M aqueous urea solution (containing 500mM sodium chloride). The respective polypeptide antigens contained in the water-insoluble polypeptide antigen in which the water-soluble polypeptide antigen and the 8M aqueous urea solution (containing 500mM sodium chloride) were dissolved were mixed in equal amounts by mass ratio and used as an antigen component.
(2) Preparation of nano-vaccine
The nano-vaccine, the blank nano-particle used as a reference and the nano-particle loaded with a plurality of polypeptides in the embodiment are prepared by a multiple emulsion method in a solvent volatilization method, the molecular weight of PLGA used as a nano-particle preparation material is 24KDa-38KDa, and the adopted immune adjuvants are CpG-ODN2006 (B class), CpG-ODN2216(A class) and poly (I: C), and the mass ratio of the CpG-ODN2006 to the poly (A class) to the poly (I: C) is 1:1: 0.5. The preparation method is as described above. The average particle size of the polypeptide-loaded nano vaccine is about 270nm, and the surface potential of the nano vaccine is about-5 mV; about 70 micrograms of polypeptide components are loaded on each 1mg of PLGA nano-particle, and the total amount of immunologic adjuvants used inside and outside each 1mg of PLGA nano-particle is 0.05mg, wherein the CpG-ODN2006 is 0.02mg, the CpG-ODN2216 is 0.02mg, and the poly (I: C) is 0.01 mg. The particle size of the blank nanoparticle only containing the immunologic adjuvant is about 250nm, and pure water or 8M urea aqueous solution (containing 500mM sodium chloride) containing the same amount of adjuvant is respectively adopted to replace corresponding polypeptide when the blank nanoparticle is prepared. The particle size of the contrast polypeptide nano vaccine is about 270nm, about 70 mug of polypeptide component is loaded on each 1mg of PLGA nano particle, and the total amount of immunologic adjuvants used inside and outside each 1mg of PLGA nano particle is 0.05mg, wherein the CpG-ODN2006 is 0.025mg, the CpG-ODN2216 is 0.024mg, and the poly (I: C) is 0.001 mg.
(3) Nano-vaccine for cancer prevention
The study control groups are respectively a PBS group and a blank nanoparticle + free polypeptide group. Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice.
The nano vaccine group administration scheme is as follows: subcutaneously injecting 200 μ L of 2mg PLGA nano-vaccine at 49, 42, 35, 28 and 14 days before melanoma inoculation; each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 And B16F10 cells.
The PBS control protocol was as follows: subcutaneous injections of 200 μ L PBS were given on days 49, 42, 35, 28, and 14, respectively, before melanoma inoculation; each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 And B16F10 cells.
Blank nanoparticle + free polypeptide control group: subcutaneous injection of 200 μ L of blank nanoparticles and an equivalent amount of free lysate to the vaccine load on days 49, 42, 35, 28 and 14, respectively, prior to melanoma vaccination; injecting blank nanoparticles and free polypeptide at different parts; each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 And B16F10 cells.
In the experiment, the tumor volume of the mice was recorded every 3 days from day 3Is small. Tumor volume is calculated by the formula v ═ 0.52 × a × b 2 Calculation, where v is tumor volume, a is tumor length, and b is tumor width. From animal experiment ethics, when the tumor volume of the mouse exceeds 2000mm in the life test of the mouse 3 I.e. the mice were considered dead and were euthanized.
(4) Results of the experiment
As shown in fig. 30, the tumors of both PBS control and blank nanoparticle control mice grew and were produced at a fast rate. The curative effect of the nano vaccine group is better than that of the PBS control group and the blank nano particle control group, and the tumor of the mouse of the nano vaccine treatment group loaded with the mixed adjuvant and the polypeptide antigen in the ratio of 4:1 (CpG-ODN: poly (I: C)) is mostly disappeared after inoculation, which is better than that of the vaccine loaded with the mixed adjuvant and the polypeptide antigen in the ratio of 49:1 (CpG-ODN: poly (I: C)). In conclusion, the nano vaccine loaded with a plurality of CpG and Poly (I: C) and antigen components in a specific ratio has a good prevention effect on melanoma.
Example 3 adjuvant and melanoma cancer cells Loading inside nanoparticles for treatment of melanoma
In this example, a mouse melanoma is used as a cancer model to illustrate how to prepare a nano vaccine co-loaded with an immune adjuvant and a melanoma cancer cell whole cell component, and to treat melanoma using the nano vaccine. In this example, B16F10 mouse melanoma cells were used as a cancer model. B16F10 melanoma cancer cells were first lysed and water soluble and water insoluble fractions were prepared. Then, the organic polymer material PLGA is used as a nanoparticle framework material, and poly (I: C), CpG-ODN 1018(B class), CpG-ODN1826(B class) and CpG-ODN 2336(A class) are used as immunologic adjuvants to prepare the nano vaccine loaded with water-soluble components and water-insoluble components of cancer cells by a solvent evaporation method. The nano-vaccine is then used to treat melanoma.
(1) Lysis of cells and Collection of fractions
Removing the culture medium from the cultured B16F10 cancer cell line, centrifuging for 5 minutes at 400g, resuspending and washing twice by using PBS, then resuspending the cancer cells by using ultrapure water, repeatedly freezing and thawing for five times, cracking the cancer cells by using ultrasound assistance in the freezing and thawing process, centrifuging for 5 minutes at 10000g, and collecting the supernatant, namely the water-soluble component. Dissolving the precipitate with 8M urea to obtain the original water-insoluble component. Mixing the water-soluble component from the B16F10 cancer cell and the original water-insoluble component dissolved in 8M urea according to the mass ratio of 1:1 to obtain the raw material source for preparing the vaccine.
(2) Preparation of nano-vaccine
The nano-vaccine in the embodiment is prepared by a multiple emulsion method in a solvent volatilization method, the molecular weight of PLGA used as a nano-particle preparation material is 7KDa-17KDa, an adjuvant and an antigen component are loaded in the nano-vaccine, and the adopted immune adjuvants are poly (I: C), CpG-ODN 1018, CpG-ODN1826 and CpG-ODN 2336. The preparation method is as described above. The average grain diameter of the nano vaccine loaded with the whole cell component is about 320 nm; the surface potential of the nano vaccine is about-5 mV; about 90 μ g of protein or polypeptide component is loaded per 1mg of PLGA nanoparticle, and 0.05mg of adjuvant is used per 1mg of PLGA nanoparticle, wherein the amount of poly (I: C) is 0.01mg, the amount of CpG-ODN 1018 is 0.01mg, the amount of CpG-ODN1826 is 0.01mg, and the amount of CpG-ODN 2336 is 0.02 mg. The preparation method of the contrast nano vaccine is the same, the particle size is 320nm, and the surface potential of the vaccine is about-5 mV; every 1mg PLGA nano particle is loaded with about 90 mug of protein or polypeptide component; the adjuvant is 0.05mg, the poly (I: C) is 0.049mg, the CpG-ODN 1018 is 0.0003mg, the CpG-ODN1826 is 0.0003mg, and the CpG-ODN 2336 is 0.0004mg, per 1mg of PLGA nano-particle.
(3) Nano-vaccine for cancer treatment
Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice.
The nano vaccine group administration scheme is as follows: each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 B16F10 cells; 100 μ L of 2mg PLGA nano-vaccine was subcutaneously injected on days 4, 7, 10, 15 and 20 after melanoma inoculation, respectively.
The PBS control protocol was as follows: each mouse was subcutaneously inoculated on day 0 at the lower right back with 1.5X 10 5 B16F10 cells; 200 μ L of PBS were injected subcutaneously on days 4, 7, 10, 15, and 20, respectively, after melanoma inoculation.
In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 3. Tumor volume is calculated by the formula v ═ 0.52 × a × b 2 Calculation, where v is tumor volume, a is tumor length, and b is tumor width. From animal experiment ethics, when the tumor volume of the mouse exceeds 2000mm in the life test of the mouse 3 I.e. the mice were considered dead and were euthanized.
(4) Results of the experiment
As shown in FIG. 31, the tumors of the PBS control mice all grew and the production rate was very fast. The efficacy of the nano vaccine group is better than that of the PBS control group, and the tumor of the nano vaccine treatment group mouse loaded with 0.5:0.5:1:0.5(CpG-ODN 1018: CpG-ODN 1826: CpG-ODN 2336: poly (I: C)) proportion mixed adjuvant and tumor tissue whole cell component is mostly disappeared after inoculation, which is better than that of the vaccine loaded with 3:3:4:490(CpG-ODN 1018: CpG-ODN 1826: CpG-ODN 2336: poly (I: C)) proportion mixed adjuvant and tumor tissue whole cell component. In conclusion, the nano vaccine loaded with a plurality of CpG and Poly (I: C) and antigen components in a specific ratio has a good treatment effect on melanoma.
Example 4 adjuvant and melanoma tumor tissue Loading inside nanoparticles for treatment of melanoma
In this example, mouse melanoma is used as a cancer model to illustrate how to prepare a nano vaccine co-loaded with an immunoadjuvant and a melanoma tumor tissue whole cell component, and to treat melanoma using the vaccine. In this example, B16F10 mouse melanoma cells were used as a cancer model. B16F10 melanoma tumor tissue was first lysed and water soluble and water insoluble fractions were prepared. Then, the organic polymer material PLGA is used as the nano-particle framework material, poly (I: C), CpG-ODN 2336(A class) and CpG-ODN 1018(B class) are used as the immunologic adjuvant, and the nano-vaccine loaded with the water-soluble component and the water-insoluble component of the cancer cells is prepared by a solvent evaporation method. The nano-vaccine is then used to treat melanoma.
(1) Lysis of tumor tissue and Collection of fractions
The same as in example 1. Mixing the water-soluble component and the original water-insoluble component dissolved in 5% sodium deoxycholate according to the mass ratio of 1:1 to obtain the raw material source for preparing the vaccine.
(2) Preparation of nano-vaccine
The nano-vaccine in the embodiment is prepared by a multiple emulsion method, the molecular weight of PLGA used as a nano-particle preparation material is 24KDa-38KDa, an adjuvant and an antigen component are loaded in the nano-vaccine, and the adopted immune adjuvants are poly (I: C), CpG-ODN 2336 and CpG-ODN 1018. The preparation method is as described above. The average grain diameter of the nano vaccine loaded with the whole cell component is about 350 nm; the surface potential of the nano vaccine is about-5 mV; about 90 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano-particle, and the adjuvant used for each 1mg of PLGA nano-particle is 0.03mg, wherein the poly (I: C) is 0.01mg, the CpG-ODN 2336 is 0.01mg, and the CpG-ODN 1018 is 0.01 mg. The preparation method of the contrast nano vaccine 1 is the same, the particle size is 340nm, and the surface potential of the vaccine is about-5 mV; loading about 90 μ g of protein or polypeptide component per 1mg of PLGA nanoparticle; 0.03mg of adjuvant is used per 1mg of PLGA nanoparticle, wherein 0.01mg of poly (I: C), 0.01mg of CpG-ODN 2336 and 0.01mg of CpG-ODN 2216. The preparation method of the contrast nano vaccine 2 is the same, the particle size is 340nm, and the surface potential of the vaccine is about-5 mV; loading about 90 μ g of protein or polypeptide component per 1mg of PLGA nanoparticle; 0.03mg of adjuvant is used for every 1mg of PLGA nano-particles, wherein 0.01mg of CpG-ODN 1018, 0.01mg of CpG-ODN 2336 and 0.01mg of CpG-ODN2216 are used;
(3) nano-vaccine for cancer treatment
The same as in example 3.
(4) Results of the experiment
As shown in fig. 32, the efficacy of the nano-vaccine group was better than that of the PBS control group, and CpG-ODN (class B) was loaded: CpG-ODN (class A): the tumor of the nano vaccine treatment group mouse mixed with adjuvant and tumor tissue whole cell component in the ratio of poly (I: C): 1:1:1 mostly disappeared after inoculation, better than the loaded CpG-ODN (class A): CpG-ODN (class A): control nano-vaccine 1 treatment group mice, mixed with adjuvant and tumor tissue whole cell component in a poly (I: C) ═ 1:1:1 ratio, were also better than CpG-ODN (class a): CpG-ODN (class A): control nano-vaccine 2 treatment group mice mixed with adjuvant and tumor tissue whole cell fraction at CpG-ODN (class B) ═ 1:1:1 ratio. In conclusion, the nano vaccine loaded with a plurality of CpG and Poly (I: C) and antigen components in a specific ratio has a good treatment effect on melanoma.
Example 5 melanoma and Lung cancer cell Water soluble cell fraction Loading inside and on microparticles for prevention of melanoma
This example uses mouse melanoma as a cancer model to illustrate how to prepare a micro vaccine loaded with only the water soluble fraction of melanoma and lung cancer cell fractions and to use this vaccine to prevent melanoma. In this example, B16F10 melanoma and LLC lung cancer cells were first lysed to prepare a water soluble fraction and a water insoluble fraction. Then, using organic polymer material PLGA (24KDa-38KDa) as a microparticle framework material, using poly (I: C) and two CpG-ODNs as immune adjuvants to prepare the micro vaccine loaded with the water-soluble components of the whole cells, and using the micro vaccine 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-20 deg.C, adding a certain amount of ultrapure water, heating, irradiating with ultraviolet, freeze thawing for more than 3 times, and lysing cells with ultrasonic disruption. After cell lysis, the lysate is centrifuged at 8000g for 5min, and the supernatant is the water-soluble component which can be dissolved in pure water in B16F10 melanoma or LLC lung cancer cells. The obtained water-soluble components from the two cancer cell lysates are mixed according to the mass ratio of 1:1 to obtain the antigen source for preparing the micron vaccine.
(2) Preparation of micron vaccine
In the embodiment, the micro-vaccine and the blank micro-particles used as the reference adopt a multiple emulsion method in a solvent volatilization method, the adopted micro-particle preparation material is PLGA, and the adopted immunologic adjuvant comprises poly (IC), CpG-ODN2006 (B class) and CpG-ODN2216(A class), and the mass ratio of the poly (IC), the CpG-ODN2006 (B class) and the CpG-ODN2216(A class) is 2:2: 1. The antigen component is loaded in the micro-vaccine and adsorbed on the surface of the micro-particles, and the adjuvant is loaded only in the micro-vaccine. The preparation method is as described above. The particle diameter of the obtained micro vaccine is 2.10 mu m after the micro particles are loaded with cell components and immunologic adjuvants, the surface potential is about-5 mV, 150 mu g of protein or polypeptide components are loaded on each 1mg of PLGA micro particles, and 0.05mg of adjuvant is used, wherein 0.02mg of Poly (I: C), 0.78 mg of CpG-ODN 20060.02 mg and 60.01mg of CpG-ODN22160. Control Microvaccine 1 used only one CpG and poly (I: C) as a mixed adjuvant with a particle size of 2.10 μm, a surface potential of-5 mV, 150 μ g protein or polypeptide fraction per 1mg PLGA microparticle, and 0.05mg adjuvant, where Poly (IC)0.02mg, CpG-ODN 20060.03mg. The particle size of the control mini-vaccine 2 was 2.10 μm, the surface potential was-5 mV, and 150 μ g of protein or polypeptide fraction was loaded per 1mg PLGA microparticles, using 0.05mg of adjuvant, among which Poly (IC)0.025mg, CpG-ODN 20060.02 mg, CpG-ODN 22160.005 mg.
(3) Micron vaccine for preventing cancer
Female C57BL/6 at 6-8 weeks was selected to prepare melanoma-bearing mice.
The scheme of the micron vaccine is as follows: subcutaneously injecting 200 μ L of 2mg PLGA micro-vaccine loaded with water-soluble components in cancer cell lysate on days 35, 28, 21, and 14, respectively, before melanoma inoculation; each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 And B16F10 cells. The PBS blank control protocol was as follows: subcutaneous injections of 200 μ L PBS were given on days 35, 28, 21, and 14, respectively, before melanoma inoculation; each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 And B16F10 cells. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 3. Tumor volume is calculated by the formula v ═ 0.52 × a × b 2 Calculation, where v is tumor volume, a is tumor length, and b is tumor width. Due to animal experiment ethics, when the tumor volume of the mouse exceeds 2000mm in the life test of the mouse 3 I.e. the mice were considered dead and were euthanized.
(4) Results of the experiment
As shown in fig. 33, the growth rate of tumor volume of mice in the group administered with the mu-m vaccine was significantly reduced and the survival time of mice was significantly prolonged, compared to the PBS blank control group. Furthermore, using CpG-ODN: the micro-vaccine using Poly (I: C) ═ 1.5:1 as adjuvant is better than the micro-vaccine group using CpG-ODN: Poly (I: C) ratio of 1:1 as adjuvant, and the effect of using two CpG is better than that of using only one CpG.
Example 6 liver cancer tumor tissue lysis component loaded inside and on the surface of microparticles for prevention of liver cancer
In this example, a micro vaccine loaded with the liver cancer tumor tissue lysate fraction was prepared and applied to prevent liver cancer. In this example, the mouse liver cancer tumor tissue lysis component was loaded inside and on the surface of the micrometer particles to prepare a micrometer vaccine. First, mouse liver cancer tumor tissue was obtained, and 8M urea was used to lyse tumor tissue and dissolve the tumor tissue lysate fraction. Then, PLGA (38KDa-54KDa) is used as a micron particle framework material, Poly ICLC, CpG-ODN M362(C type) and CpG-ODN2216(A type) are used as immune adjuvants to prepare the micron vaccine, and the micron vaccine is applied to prevent tumors in Hepa 1-6 liver cancer bearing mice.
(1) Lysis of tumor tissue and Collection of fractions
Each C57BL/6 mouse was inoculated subcutaneously under the axilla of 2X 10 mice 6 A Hepa 1-6 cell or 2X 10 6 LLC lung cancer cells, each mouse inoculated with tumor growing to a volume of about 1000mm 3 Mice were sacrificed and tumor tissue was harvested. Tumor tissue was minced through a cell mesh, and then lysed using 8M urea.
(2) Preparation of micron vaccine
In the embodiment, the micro-vaccine and the blank micro-particles used as the reference adopt a multiple emulsion method in a solvent volatilization method, the adopted micro-particle preparation material is PLGA, and the adopted immunologic adjuvants are Poly ICLC, CpG-ODN M362 and CpG-ODN2216, and the mass ratio of the three is 1:3: 1. The antigen component is loaded in the micro-vaccine and adsorbed on the surface of the micro-particles, and the adjuvant is loaded only in the micro-vaccine. The preparation method comprises the steps of firstly adopting a dissolution and volatilization method to wrap antigen components and adjuvants inside the microparticles, then centrifuging for 15min at 8000g, collecting precipitates, re-suspending 100mg PLGA microparticles by using a 4% trehalose solution, then freeze-drying for 48 hours, and refrigerating for later use. Before injection, 10mg PLGA microparticles were resuspended in 0.9mL PBS or physiological saline, and then mixed with 0.1mL of a cell lysate containing 8M urea (60mg/mL) and an immunoadjuvant (2mg/mL, wherein the mass ratio of Poly ICLC: CpG-ODN M362: CpG-ODN 2216: 1:3:1) at room temperature for 10min, and then the injection was performed. The obtained micrometer vaccine has particle diameter of 2.10 μm, surface potential of-5 mV, 150 μ g protein or polypeptide component loaded on each 1mg PLGA micrometer particle, and adjuvant 0.05mg, wherein PolyICLC 0.01mg, CpG-ODN M3620.03mg, CpG-ODN 22160.01 mg. The particle size of the control micron vaccine is 2.10 μ M, the surface potential is-5 mV, 150 μ g of protein or polypeptide component is loaded per 1mg PLGA micron particle, and Poly ICLC, CpG-ODN M362 and CpG-ODN2216 are used as adjuvants, wherein Poly ICLC is 0.025mg, CpG-ODN M3620.0125 mg and CpG-ODN 22160.0125 mg are used. The particle size of the blank microparticle loaded with only the immunological adjuvant was 2.00. mu.m, and 8M urea containing equal amounts of Poly ICLC, CpG-ODN M362 and CpG-ODN2216 was used instead of the corresponding lysate components when the blank microparticle was prepared.
(3) Use of micro-vaccine for cancer prevention
Selecting female C57BL/6 for 6-8 weeks to prepare a Hepa 1-6 hepatoma tumor-bearing mouse.
200 μ L of 2mg PLGA micro-vaccine were subcutaneously injected on days 49, 42, 35, 28 and 14, respectively, before the inoculation of hepatoma cells. Each mouse was inoculated subcutaneously to the right underarm at day 0 at 2X 10 6 And each Hepa 1-6 liver cancer cell.
The PBS blank control protocol was as follows: 200 μ L of PBS was injected subcutaneously on days 49, 42, 35, 28, and 14, respectively, before the inoculation of hepatoma cells. Each mouse was inoculated subcutaneously at day 0 in the right underarm at 2X 10 6 And each Hepa 1-6 liver cancer cell.
Blank microparticles + free lysate control group: injecting subcutaneously 200 μ L of empty microparticles and an equivalent amount of free lysate to the vaccine load on days 49, 42, 35, 28 and 14, respectively, prior to inoculation with hepatoma cells; the empty microparticles and free cell lysate were injected at different sites. Each mouse was inoculated subcutaneously to the right underarm at day 0 at 2X 10 6 And each Hepa 1-6 liver cancer cell. In the experiment, the method for monitoring the growth of the mouse tumor is the same as the method.
(4) Results of the experiment
As shown in fig. 34, the growth of liver cancer tumors was faster in both the PBS control group and the blank microparticle + free lysate control group of mice. The majority of mice in the group administered with the minivaccine had lost their tumor after tumor inoculation. Therefore, the micrometer vaccine loaded with the liver cancer tumor tissue lysate has a prevention effect on liver cancer. Furthermore, the total content of various CpG-ODNs in the adjuvant: the effect of the Poly ICLC ═ 4:1 micron vaccine is better than the total content of multiple CpG-ODN in the adjuvant: poly ICLC ═ 1:1 micron vaccine.
Example 7 Whole cell fraction of lung cancer and liver cancer tumor tissue loaded inside nanoparticles for prevention of liver cancer
In this example, mouse liver cancer is used as a cancer model to illustrate how to prepare a nano vaccine loaded with whole cell components of lung cancer and liver cancer tumor tissues, and to prevent liver cancer by using the nano vaccine. First, lung cancer and liver cancer tumor tissues were lysed to prepare water-soluble components and water-insoluble components of whole cell components, and the water-soluble components were mixed in a mass ratio of 1:1, and the water-insoluble components were also mixed in a mass ratio of 1:1. Then, PLGA is used as a nanoparticle framework material, Poly (I: C), CpG-ODN1826(B type) and CpG-ODN 1018(B type) are mixed according to the mass ratio of 1:1:1 and used as an immunologic adjuvant to prepare the nano vaccine loaded with the water-soluble component or the water-insoluble component, and when the nano vaccine is used, the nano vaccine loaded with the water-soluble component and the nano vaccine loaded with the water-insoluble component are mixed to prevent the liver cancer.
(1) Lysis of cancer cells and collection of fractions
Each C57BL/6 mouse was inoculated subcutaneously into the back of 1.0X 10 6 LLC cells or 1.0X 10 6 The growth to volume of each Hepa 1-6 cell in the tumor is about 1000mm 3 Mice were sacrificed and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, and a proper amount of pure water is added through a cell filter screen and is repeatedly frozen and thawed for 5 times, and ultrasonic waves are carried out to destroy the tissue cells. After the tissue cells are cracked, degrading nucleic acid in the whole cell components by using nuclease, then heating at 95 ℃ for 5 minutes to inactivate the nuclease, then centrifuging the lysate for 5 minutes at the rotating speed of 1000g, and taking supernatant fluid which is a water-soluble component soluble in pure water; in the obtaining ofThe insoluble water-insoluble fraction insoluble in pure water can be converted to soluble in a 10% aqueous solution of octyl glucoside by adding 10% octyl glucoside to the precipitated fraction to dissolve the precipitated fraction. After collecting the water-soluble component and the water-insoluble component, mixing the water-soluble component in a mass ratio of 1:1, and mixing the water-insoluble component in a mass ratio of 1:1. The obtained water-soluble component mixture or water-insoluble component mixture is the antigen raw material for preparing the nano vaccine.
(2) Preparation of nano-vaccine
In the embodiment, a multiple emulsion method in a solvent volatilization method is adopted for preparing the nano vaccine, the molecular weight of PLGA used as a nano particle preparation material is 24-38 KDa, and Poly (I: C), CpG-ODN1826 and CpG-ODN 1018 are used as mixed adjuvants according to the mass ratio of 1:1: 1. When the vaccine is prepared, the water-soluble component mixture and the water-insoluble component mixture are respectively prepared into the nano vaccine and then used together, and the antigen component and the adjuvant are loaded on the nano vaccine only in a nanoparticle entrapment mode. The preparation method is the same as above. The nano vaccine has the particle size of about 280nm and the surface potential of about-6 mV, about 100 mu g of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, and 0.03mg of adjuvant is used on each 1mg of PLGA nano particle. The particle size of the blank nanoparticle is about 250nm, and the corresponding water-soluble components and water-insoluble components are replaced by solutions containing the same amount of adjuvant when the blank nanoparticle is prepared. The particle size of the control nano-vaccine is about 280nm, the surface potential is about-6 mV, about 100 mu g of protein or polypeptide component is loaded in each 1mg of PLGA nano-particle, and 0.03mg of adjuvant Poly (I: C) or 0.03mg of adjuvant CpG-ODN1826 + CpG-ODN 1018 (mass ratio of 1:1) is used in each 1mg of PLGA nano-particle.
(3) Nano-vaccine for cancer prevention
Selecting female C57BL/6 for 6-8 weeks to prepare a Hepa 1-6 hepatoma tumor-bearing mouse. 100 μ L of 1mg PLGA water-soluble component nano-vaccine and 100 μ L of 1mg PLGA water-insoluble component nano-vaccine were subcutaneously injected on days 49, 42, 35, 28 and 14, respectively, before the inoculation of hepatoma cells. Each mouse was inoculated subcutaneously to the right underarm at day 0 at 2X 10 6 And each Hepa 1-6 liver cancer cell. The PBS blank control protocol was as follows: 4 th before inoculation of hepatoma cells200 μ L of PBS was injected subcutaneously on days 9, 42, 35, 28, and 14, respectively. Each mouse was inoculated subcutaneously to the right underarm at day 0 at 2X 10 6 And each Hepa 1-6 liver cancer cell. Blank nanoparticle + free lysate control group: injecting 200 μ L of blank nanoparticles and an equivalent amount of free lysate loaded with vaccine subcutaneously on day 49, 42, 35, 28 and 14, respectively, prior to inoculation with hepatoma cells; blank nanoparticles and free cell lysate were injected at different sites. Each mouse was inoculated subcutaneously to the right underarm at day 0 at 2X 10 6 And each Hepa 1-6 liver cancer cell. In the experiment, the method for monitoring the growth of the mouse tumor is the same as the method.
(4) Results of the experiment
As shown in fig. 35, both tumor growth rate and survival time of mice were significantly different in the vaccine-protected group compared to the control group. Furthermore, the vaccine group mice had partially disappeared after tumor inoculation. Moreover, the nano-vaccine using both CpG-ODN and Poly (I: C) (the mass ratio of total CpG-ODN to Poly (I: C) is 2:1) as adjuvant has better prevention effect than the nano-vaccine using only Poly (I: C) or only CpG-ODN.
Example 8 Loading of pancreatic cancer tumor tissue lysis component inside nanoparticles for treatment of pancreatic cancer
In this example, a mouse pancreatic cancer is used as a cancer model to illustrate how to prepare a nano vaccine loaded with a pancreatic cancer tumor tissue lysate component and apply the vaccine to treat pancreatic cancer. Mouse pancreatic cancer tumor tissue was first taken and lysed to prepare a water-soluble fraction and a primary water-insoluble fraction dissolved in 6M guanidine hydrochloride. PLGA (molecular weight 7-17 KDa) is used as a nano particle framework material, and Poly (I: C), CpG-ODN 2395 and CpG-ODN2216 are used as immune adjuvants to prepare the nano vaccine.
(1) Lysis of tumor tissue and Collection of fractions
C57BL/6 mice were each inoculated subcutaneously in the axilla at 1X 10 6 A Pan02 pancreatic cancer cell was inoculated to a tumor growth volume of about 1000mm in each mouse 3 Mice were sacrificed and tumor tissue was harvested. The tumor tissue lysis and fractions were collected as above except that 6M guanidine hydrochloride was used instead of 8M urea.
(2) Preparation of nano-vaccine
In this example, the multiple emulsion method of the solvent evaporation method was used to prepare the nano-vaccine, the molecular weight of the nanoparticle preparation material PLGA was 24kDa-38kDa, and the ratio of Poly (I: C): CpG-ODN 2395: CpG-ODN2216 is used as a mixed adjuvant according to the mass ratio of 1:0.6: 0.6. When the vaccine is prepared, the water-soluble component mixture and the water-insoluble component mixture are respectively prepared into the nano vaccine and then used together, and the antigen component and the adjuvant are loaded on the nano vaccine only in a nanoparticle entrapment mode. The preparation method is the same as above. The nano vaccine has the particle size of about 270nm and the surface potential of about-5 mV, about 80 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, 0.044mg of adjuvant is used on each 1mg of PLGA nano particle, wherein Poly (I: C) is 0.02mg, CpG-ODN 2395 is 0.012 mg: CpG-ODN2216 is 0.012 mg. The particle diameter of the control nano vaccine 1 is about 280nm, the surface potential is about-5 mV, about 80 mug protein or polypeptide component is loaded on each 1mg PLGA nano particle, 0.044mg of adjuvant is used on each 1mg PLGA nano particle, wherein Poly (I: C) is 0.02mg, CpG-ODN 1585 is 0.012 mg: CpG-ODN2216 is 0.012 mg. The particle size of the control nano vaccine 2 is about 280nm, the surface potential is about-6 mV, about 80 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, 0.044mg of adjuvant is used on each 1mg of PLGA nano particle, wherein CpG1585 is 0.02mg, CpG-ODN 2395 is 0.012 mg: CpG-ODN2216 is 0.012 mg.
(3) Nano-vaccine for cancer treatment
Female C57BL/6 at 6-8 weeks was selected to prepare pancreatic carcinoma mice. Each mouse was inoculated subcutaneously at day 0 at the lower right back at 1X 10 6 Individual Pan02 cells. The vaccine group was subcutaneously injected with 100. mu.L of 1mg PLGA nano vaccine loaded with a water-soluble component and 100. mu.L of 1mg PLGA nano vaccine loaded with a water-insoluble component on days 4, 7, 10, 15 and 20, respectively. The PBS blank control group was injected subcutaneously with 200 μ L PBS on days 4, 7, 10, 15, and 20, respectively. In the experiment, the mouse tumor monitoring and volume calculation methods are the same as above.
(4) Results of the experiment
As shown in fig. 36, both tumor growth rate and survival of mice were significantly different in the vaccine-treated group compared to the control group. Furthermore, the vaccine group mice had partially disappeared after tumor inoculation. And CpG-ODN (class C) loaded: CpG-ODN (class A): the tumor of the nano vaccine treatment group mice mixed with adjuvant and tumor tissue whole cell component in the ratio of poly (I: C) ═ 0.6:0.6:1, mostly disappeared after inoculation, better than the loading of CpG-ODN (class a): CpG-ODN (class A): control nano-vaccine 1 treated group mice mixed with adjuvant and tumor tissue whole cell fraction at a ratio of poly (I: C) ═ 0.6:0.6: 1. Also better than CpG-ODN loaded (class A): CpG-ODN (class C): CpG-ODN (class A): control nano vaccine 2 treatment group mice mixed with adjuvant and tumor tissue whole cell fraction at a ratio of 0.6:0.6: 1; in conclusion, the nano vaccine loaded with a plurality of CpG and Poly (I: C) and antigen components in a specific ratio has a good treatment effect on cancer.
Example 9 Whole cell fraction Loading inside mannose modified microparticles for prevention of Breast cancer
In this example, a mouse breast cancer is used as a cancer model to illustrate how to prepare a micro vaccine loaded with a whole cell component of breast cancer tumor tissue and cancer cells, and to prevent breast cancer by using the micro vaccine. In practical application, the particle size, administration time, administration frequency and administration scheme can be adjusted according to the situation.
In this example, cancer cells of mouse breast and lung cancers were first taken and lysed to produce a water-soluble fraction and a primary water-insoluble fraction dissolved in 8M urea. Then, PLGA and mannose modified PLGA are used as a micron-particle framework material, Poly (I: C), CpG-ODN 1018(B class) and CpG-ODN1826(B class) are used as immunologic adjuvants, and a solvent evaporation method is adopted to prepare the micron vaccine. The micro-vaccine has the ability to target dendritic cells.
(1) Lysis of tumor tissue and Collection of fractions
Axilla inoculation of each BALB/c mouse 2X 10 6 4T1 Breast cancer cells, inoculated with tumors growing to 1000mm in mice 3 Mice were sacrificed and tumor tissue was harvested. The method for collecting and treating lung cancer cells is the same as that for melanoma cancer cells. Tumor tissue and cancer cell lysis and fractions collection were as above. Water solubility in cancer cell lysates of breast and lung cancerMixing the components according to the mass ratio of 4:1, and mixing the water-insoluble components in the cancer cell lysate of the breast cancer and the lung cancer according to the mass ratio of 4: 1; and then mixing the water-soluble component mixture and the water-insoluble component mixture according to the mass ratio of 1:1 to obtain the antigen component for preparing the micron vaccine.
(2) Preparation of micron vaccine
In this example, the micron vaccine and the control hollow micron particles were prepared by multiple emulsion method. The molecular weight of the microparticle preparation material PLGA (50:50) is 24-38 KDa, and the molecular weight of the adopted mannose modified PLGA (50:50) is 24-38 KDa. The mass ratio of unmodified PLGA to mannose-modified PLGA was 8: 2. Preparation method as described above, the antigen component and the adjuvant are co-loaded in the micro-vaccine. The preparation method is as described above. The average particle diameter of the micro-vaccine is about 1.50 mu m, the average surface potential is about-7 mV, each 1mg PLGA micro-particle is loaded with 85 mu g protein or polypeptide component, each 1mg PLGA micro-vaccine uses 0.05mg adjuvant, wherein, the CpG-ODN 1018 is 0.025mg, the CpG-ODN1826 is 0.023mg, and the Poly (I: C) is 0.002 mg. The particle size of the blank micron particle is about 1.45 mu m, and the blank micron particle is prepared by loading the same amount of adjuvant but not loading antigen components. The average particle size of the control micro-vaccine is about 1.50 μm, the average surface potential is about-7 mV, each 1mg PLGA micro-particle is loaded with 85 μ g protein or polypeptide component, each 1mg PLGA micro-vaccine is loaded with 0.05mg adjuvant, wherein the CpG-ODN 1018 is 0.025mg, the CpG-ODN1826 is 0.024mg, and the Poly (I: C) is 0.001 mg.
(3) Dendritic cell-targeted micro-vaccines for cancer prevention
Female BALB/c mice of 6-8 weeks were selected to prepare breast cancer-bearing mice. Vaccine groups 200 μ L of 2mg PLGA micro-vaccine was injected subcutaneously on days 35, 28, 21, 14 and 7 prior to tumor vaccination. The PBS blank control group was injected subcutaneously with 200 μ L PBS on days 35, 28, 21, 14 and 7, respectively, prior to tumor inoculation. The empty microwell + lysate control group was injected subcutaneously with 200 μ L of empty microwell and an equivalent amount of free lysate to the vaccine load on days 35, 28, 21, 14, and 7, respectively, prior to tumor inoculation. Each mouse was inoculated subcutaneously 4X 10 in the lower right back on day 0 5 4T1 breast cancer cells. In the experiment, the method for monitoring the growth of the mouse tumor is the same as the method.
(4) Results of the experiment
As shown in fig. 37, both tumor growth rate and survival time of mice were significantly different in the vaccine-protected group compared to the control group. Furthermore, the vaccine group mice had partially disappeared after tumor inoculation. Moreover, the prevention effect of the micro-vaccine taking the total CpG-ODN and Poly (I: C) with the mass ratio of 25:1 as the adjuvant is better than that of the micro-vaccine taking the total CpG-ODN and Poly (I: C) with the mass ratio of 49:1 as the adjuvant.
Example 10 melanoma tumor tissue and cancer cell Whole cell fractions Loading on Nanoprotein for treatment of melanoma
In this example, a mouse melanoma is used as a cancer model and Poly (I: C), CpG-ODN 1018 and CpG-ODN2006 are used as immunological adjuvants to illustrate how to prepare a nano vaccine loaded with melanoma tumor tissues and cancer cell whole cell components and to treat melanoma by using the nano vaccine.
In this example, the water soluble and water insoluble components of liver cancer and melanoma tumor tissues were first lysed and mixed at a ratio of 3:1, respectively. Then, PLGA is used as the nanometer particle framework material to prepare the nanometer vaccine by a solvent volatilization method.
(1) Lysis of tumor tissue and Collection of fractions
In this example, tumor tissue and cancer cells were lysed and the lysate collected as above. The water insoluble fraction was dissolved using 5% SDS. Mixing water-soluble components in the lysate of the melanoma tissue and the cancer cells according to a mass ratio of 3:1, and mixing water-insoluble components of the melanoma tissue and the cancer cells according to a mass ratio of 3: 1; and then mixing the water-soluble component mixture and the water-insoluble component mixture according to the mass ratio of 2:1 to obtain the antigen component for preparing the nano vaccine.
(3) Preparation of nano-vaccine
In this example, the nano vaccine and the blank nanoparticles used as a control were prepared by multiple emulsion method. The molecular weight of the nano particle preparation material PLGA (50:50) is 24KDa-38KDa, the molecular weight of the adopted mannose modified PLGA (50:50) is 24KDa-38KDa, and the mass ratio of the unmodified PLGA to the mannose modified PLGA is 9: 1. The preparation method is as described above, and the antigen component and the adjuvant are co-loaded in the nano vaccine. The average particle size of the nano-vaccine is about 280nm, the average surface potential is about-4 mV, 95 mug protein or polypeptide component is loaded on each 1mg PLGA nano-vaccine, 0.05mg of adjuvant is used for each 1mg PLGA nano-vaccine, wherein the CpG-ODN 1018 is 0.025mg, the CpG-ODN2006 is 0.02mg, and the Poly (I: C) is 0.005 mg. The average particle size of the control nano vaccine 1 is about 280nm, the average surface potential is about-5 mV, 95 mu g of protein or polypeptide component is loaded on each 1mg of PLGA nano vaccine, and 0.05mg of adjuvant is used for each 1mg of PLGA nano vaccine, wherein the CpG-ODN 1018 is 0.025mg, the CpG-ODN2006 is 0.024mg, and the Poly (I: C) is 0.001mg (the mass ratio is 25:24: 1). The average particle size of the control nano vaccine 2 is about 280nm, the average surface potential is about-5 mV, 95 mu g of protein or polypeptide component is loaded on each 1mg of PLGA nano vaccine, and 0.05mg of adjuvant is used on each 1mg of PLGA nano vaccine, wherein the CpG-ODN 1018 is 0.02mg, the CpG-ODN2006 is 0.005mg, and the Poly (I: C) is 0.025mg (mass ratio is 4:1: 5).
(4) Nano vaccine for treating melanoma
Female C57BL/6 was selected as a model mouse to prepare tumor-bearing mice. Each group was inoculated subcutaneously at day 0 to the lower right of the back of each mouse at 1.5X 10 5 And (3) B16F10 melanoma cells. Vaccine groups 200 μ L of 2mg PLGA nano-vaccine was subcutaneously injected on days 4, 7, 10, 15 and 20 after tumor vaccination, respectively. The PBS blank control group was injected subcutaneously with 200 μ L PBS on days 4, 7, 10, 15, and 20, respectively, after tumor inoculation. Mouse tumor growth was monitored as above.
(4) Results of the experiment
As shown in fig. 38, there were significant differences in both tumor growth rate and survival time of mice in the vaccine-treated group compared to the control group. Furthermore, the vaccine group mice had partially disappeared after tumor inoculation. Moreover, the prevention effect of the nano-vaccine taking the total CpG-ODN and Poly (I: C) in the mass ratio of 9:1 as the adjuvant is better than that of the nano-vaccine taking the total CpG-ODN and Poly (I: C) in the mass ratio of 49:1 and 1:1 as the adjuvant.
Example 118M Urea solubilization of Breast cancer tumor tissue Components and Loading of nanoparticles for treatment of Breast cancer
This example illustrates how to lyse whole cell fractions using 8M urea and prepare whole cell fraction loaded nano-vaccines for the treatment of breast cancer. In this example, 4T1 mouse triple negative breast cancer was used as a cancer model. Breast cancer tumor tissue cells were first inactivated and denatured and tumor tissue lysed with 8M urea and whole cell fractions lysed. Then, PLGA is used as a nano-particle framework material, Poly (I: C), CpG-ODN 1018(B class) and CpG-ODN M362(C class) are used as immunologic adjuvants, and a solvent evaporation method is adopted to prepare the nano-vaccine loaded with the tumor tissue whole cell component.
(1) Lysis of tumor tissue and Collection of fractions
(2) Preparation of nano-vaccine
In this example, the nano vaccine and the control blank nanoparticles were prepared by multiple emulsion method. The molecular weight of the nano particle preparation material PLGA (50:50) is 7KDa-17 KDa. The preparation method is as described above, and the antigen component and the adjuvant are co-loaded in the nano vaccine. The average particle size of the nano-vaccine is about 250nm, the average surface potential is about-4 mV, 90 mug protein or polypeptide component is loaded on each 1mg PLGA nano-vaccine, 0.048mg of adjuvant is used on each 1mg PLGA nano-vaccine, wherein the CpG-ODN 1018 is 0.0056mg, the CpG-ODN M362 is 0.04mg, and the Poly (I: C) is 0.0024 mg. The average particle diameter of the control nano vaccine 1 is about 250nm, the average surface potential is about-5 mV, 95 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano vaccine, 0.048mg of adjuvant is used on each 1mg of PLGA nano vaccine, wherein the CpG-ODN M362 is 0.047mg, and the Poly (I: C) is 0.001mg (the mass ratio is 47: 1). The average particle size of the control nano vaccine 2 is about 250nm, the average surface potential is about-5 mV, and each 1mg of PLGA nano vaccine is loaded with 90 mug of protein or polypeptide component and does not contain any adjuvant.
(3) Nano-vaccine for cancer treatment
Female BALB/c from 6-8 weeks was selected to prepare 4T1 tumor-bearing mice. Each mouse was inoculated subcutaneously 4X 10 in the lower right back on day 0 5 4T1 cells.
The PBS blank control group was injected subcutaneously with 200 μ L of PBS on days 4, 7, 10, 15, and 20, respectively.
The control group of blank nanoparticles + free lysate were injected subcutaneously with equal amounts of tumor tissue lysate and 2mg PLGA blank nanoparticles on days 4, 7, 10, 15 and 20, respectively. In the experiment, the mouse tumor volume monitoring and calculation method is the same as the above.
(4) Results of the experiment
As shown in fig. 39, there were significant differences in both tumor growth rate and survival time of mice in the vaccine-treated group compared to the control group. Furthermore, the vaccine group mice had partially disappeared after tumor inoculation. Moreover, the prevention effect of the nano-vaccine taking the total CpG-ODN and Poly (I: C) in the mass ratio of 19:1 as the adjuvant is better than that of the nano-vaccine taking the total CpG-ODN and Poly (I: C) in the mass ratio of 47:1 as the adjuvant and the vaccine without any adjuvant.
Example 12 Whole cell fractions of melanoma tumor tissue and lung cancer cells loaded into and on a Nanoprotein vaccine for treatment of Lung cancer
This example illustrates how to prepare a nano-vaccine loaded with a whole-cell fraction of melanoma tumor tissue and lung cancer cells and use the vaccine to treat lung cancer. In this example, B16F10 melanoma tumor tissue and LLC cancer cells were first lysed to prepare the corresponding water soluble fraction and the water insoluble fraction dissolved in 8M aqueous urea (containing 500mM sodium chloride). Then, mixing the water-soluble component from the tumor tissue and the water-soluble component from the cancer cells according to the mass ratio of 1:1, mixing to obtain water-soluble components used in the experiment; mixing a water-insoluble component from a tumor tissue and a water-insoluble component from a cancer cell in a mass ratio of 1:1, mixing to obtain a water-insoluble component used in the experiment; then, PLGA is used as a framework material, and Poly (I: C), CpG-ODN 1018(B class) and CpG-ODN1826(B class) are used as immune adjuvants to prepare the nano-vaccine.
(1) Lysis of tumor tissue and cancer cells and collection of fractions
B16F10 melanoma tumor tissue and LLC lung cancer cell lysis method is the same as above, after melanoma tumor tissue or LLC cancer cell lysis, centrifuging lysate for 5 minutes at 8000g rotation speed and taking supernatant as water soluble component soluble in pure water; the water-insoluble component insoluble in pure water was converted to be soluble in an 8M aqueous urea solution (containing 500mM sodium chloride) by dissolving the precipitated fraction with the 8M aqueous urea solution (containing 500mM sodium chloride). The water-soluble component and the water-insoluble component are respectively mixed according to the mass ratio of 1:1 to obtain a water-soluble component mixture and a water-insoluble component mixture, namely a raw material source for preparing the vaccine.
(2) Preparation of nano-vaccine
In the embodiment, the nano-vaccine and the blank nano-particle are prepared by adopting a properly improved multiple emulsion method, the water-soluble component mixture is loaded inside the nano-particle, the non-water-soluble component mixture is loaded on the surface of the nano-vaccine, the PLGA (polylactic-co-glycolic acid) used as a nano-particle preparation material has the molecular weight of 7KDa-17KDa, and the adopted immunologic adjuvant is formed by distributing Poly (I: C), CpG-ODN 1018 and CpG-ODN1826 inside and on the surface of the nano-particle. In the preparation process of the nano particles, two modification methods of low-temperature silicification and charged substance addition are adopted to improve the antigen loading. The preparation method is as described above, in the preparation process, the water-soluble component and the adjuvant are loaded in the nano particles by a multiple emulsion method, after loading antigen and adjuvant inside, 100mg nanoparticles were centrifuged at 10000g for 20min, then the nanoparticles were resuspended using 7mL of PBS and mixed with 3mL of PBS solution containing a mixture of water-insoluble components (60mg/mL), followed by centrifugation at 10000g for 20 minutes, then resuspended in 10mL of silicate solution (containing 150mM NaCl, 80mM tetramethyl orthosilicate and 1.0mM HCl, pH 3.0) and fixed at room temperature for 10min, then fixed at-80 ℃ for 24h, washed centrifugally with ultrapure water, then resuspended in 3mL of PBS containing protamine (5mg/mL) and polylysine (10mg/mL) and allowed to act for 10min, then 10000g of the mixture is centrifuged for 20min for washing, and the mixture is frozen and dried for 48h after being resuspended by 10mL of ultrapure water containing 4 percent of trehalose; the particles were resuspended in 9mL PBS before use and then 1mL of water insoluble fraction (protein concentration 50mg/mL) containing immunoadjuvant (1mg/mL) was added for 10min at room temperature to give a siliconized and cationic species-added modified nanoparticle system loaded with both the lysate fraction on the inside and outside. The average particle diameter of the nano particles is about 350nm, and the surface potential of the nano particles is about-3 mV; about 250 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano-particle, 0.05mg of immunologic adjuvant is used on each 1mg of PLGA nano-particle, wherein Poly (I: C) is 0.01mg, CpG-ODN 1018(B class) is 0.03mg, and CpG-ODN1826(B class) is 0.01 mg.
The preparation method of the contrast nano vaccine is the same as that of the contrast nano vaccine, the average particle size of the nano particles is about 350nm, and the surface potential of the nano particles is about-4 mV; about 250 micrograms of protein or polypeptide components are loaded per 1mg of PLGA nanoparticles, 0.05mg of immunologic adjuvant is used per 1mg of PLGA nanoparticles, wherein 0.01mg of Poly (I: C), 0.03mg of CpG-ODN 1585 (class A) and 0.01mg of CpG-ODN2216 (class A) are used.
The blank nanoparticles had a particle size of about 320nm and contained equal amounts of Poly (I: C), CpG-ODN 1018 and CpG-ODN1826, but no lysate fraction.
(3) Nano vaccine for treating lung cancer
Female C57BL/6 mice of 6-8 weeks were selected to prepare lung cancer-bearing mice. Each mouse was inoculated subcutaneously at day 0 in the lower right-hand side of the back at 1X 10 6 LLC lung cancer cells.
The PBS blank control group was injected subcutaneously with 200 μ L of PBS on days 4, 7, 10, 15, and 20, respectively.
The control group of blank nanoparticles + free lysate were injected subcutaneously with equal amounts of tumor tissue lysate and 2mg PLGA blank nanoparticles on days 4, 7, 10, 15 and 20, respectively. In the experiment, the mouse tumor volume monitoring and calculation method is the same as the above.
(4) Results of the experiment
As shown in fig. 40, both tumor growth rate and survival time of mice were significantly different in the vaccine-treated group compared to the control group. Furthermore, the vaccine group mice had partially disappeared after tumor inoculation. Moreover, the treatment effect of the nano-vaccine using the mixture of the B-class CpG-ODN and the Poly (I: C) (the mass ratio of 4:1) as the adjuvant is better than that of the nano-vaccine using the A-class CpG-ODN and the Poly (I: C) (the mass ratio of 4:1) as the adjuvant.
Example 13 Whole cell fraction-loaded Nanoprotein of Colon cancer tumor tissue for treatment of Colon cancer
This example illustrates how to prepare a nano-vaccine loaded with a whole cell fraction of a colon cancer tumor tissue and use the vaccine to treat colon cancer. In this example, MC38 colon cancer tumor tissue was first lysed to prepare the corresponding water soluble fraction and the water insoluble fraction dissolved in 8M urea. Mixing water-soluble components and water-insoluble components from tumor tissues according to a mass ratio of 1:1 mixing to obtain a lysate component used in an experiment; then, PLGA was used as a scaffold material, Poly (I: C), CpG-ODN 2395(C class) and CpG-ODN SL03(C class) were used as immunoadjuvants, NH was used as a scaffold material, and 4 HCO 3 in order to increase the lysosome escaping substances, nano-vaccines are prepared.
(1) Lysis of tumor tissue and cancer cells and collection of fractions
Female C57BL/6 mice were selected for 6-8 weeks and inoculated on the back with 2X 10 6 MC38 colon cancer cells, mice were sacrificed when tumors grew to a volume of approximately 1000mm3, respectively, and tumor tissue was harvested. Tumor tissue is cut into pieces and ground, a proper amount of pure water is added through a cell filter screen, ultraviolet irradiation and heating treatment at 45 ℃ are used, freezing and thawing are repeated for 5 times, and ultrasonic is accompanied to destroy the lysed tissue cells. After the tissue cells are cracked, the lysate is centrifuged for 5 minutes at the rotating speed of 8000g, and the supernatant is taken as a water-soluble component which can be dissolved in pure water; the precipitation fraction was dissolved by adding 8M urea to the resulting precipitation fraction to convert the water-insoluble fraction insoluble in pure water into soluble in an 8M aqueous urea solution. Mixing water soluble component and water insoluble component at a mass ratio of 1:1 to obtain water soluble component mixture and water insoluble component mixture, which is used as raw material for preparing vaccineAnd (4) material source.
(2) Preparation of nano-vaccine
In the embodiment, the nano-vaccine and the blank nano-particle are prepared by adopting a properly improved multiple emulsion method, the water-soluble component mixture is loaded inside the nano-particle, the non-water-soluble component mixture is loaded on the surface of the nano-vaccine, the PLGA (polylactic-co-glycolic acid) material used for preparing the nano-particle has the molecular weight of 7-17 KDa, the adopted immunologic adjuvant is that Poly (I: C), CpG-ODN 2395 and CpG-ODN SL03 are distributed inside and on the surface of the nano-particle, and NH (NH-ODN) SL03 is distributed on the inside and on the surface of the nano-particle 4 HCO 3 Is wrapped in the nano vaccine. The preparation method is as described above, in the preparation process, firstly, the lysate component and the adjuvant are loaded inside the nanoparticles by a multiple emulsion method, after the antigen and the adjuvant are loaded inside, 100mg of the nanoparticles are centrifuged at 10000g for 20 minutes, and after being resuspended by 10mL of ultrapure water containing 4% trehalose, the nanoparticles are frozen and dried for 48 hours for later use. The particles were resuspended in 9mL of PBS before use and then treated with 1mL of water-insoluble fraction (protein concentration 50mg/mL) containing an immunoadjuvant (1mg/mL) at room temperature for 10 min. The average particle diameter of the nano particles is about 250nm, and the surface potential of the nano particles is about-4 mV; about 140 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano-particle, 0.04mg of immunologic adjuvant is used on each 1mg of PLGA nano-particle, wherein Poly (I: C) is 0.01mg, CpG-ODN 2395(C class) is 0.02mg, CpG-ODN SL03(C class) is 0.01mg, NH is loaded 4 HCO 3 0.05mg。
The preparation method of the contrast nano vaccine is the same as that of the contrast nano vaccine, the average particle size of the nano particles is about 250nm, and the surface potential of the nano particles is about-4 mV; about 140 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, 0.04mg of immunologic adjuvant is used on each 1mg of PLGA nano particle, wherein Poly (I: C) is 0.01mg, CpG-ODN 1585(A class) is 0.02mg, CpG-ODN 2336(A class) is 0.01mg, NH is loaded 4 HCO 3 0.05mg。
The blank nanoparticles have a particle size of about 240nm, and contain equal amounts of Poly (I: C), CpG-ODN 2395(C class), CpG-ODN SL03(C class) and NH 4 HCO 3 But no lysate component.
(3) Nanometer vaccine for treating colon cancer
Selecting for 6-8 weeksFemale C57BL/6 to prepare colon cancer tumor-bearing mice. Each mouse was inoculated subcutaneously at day 0 in the lower right-hand side of the back at 1X 10 6 And MC38 colon cancer cell.
The PBS blank control group was injected subcutaneously with 200 μ L of PBS on days 4, 7, 10, 15, and 20, respectively.
The control group of blank nanoparticles + free lysate were injected subcutaneously with equal amounts of tumor tissue lysate and 2mg PLGA blank nanoparticles on days 4, 7, 10, 15 and 20, respectively. In the experiment, the mouse tumor volume monitoring and calculation method is the same as the above.
(4) Results of the experiment
As shown in fig. 41, both tumor growth rate and survival time of mice were significantly different in the vaccine-treated group compared to the control group. Furthermore, the vaccine group mice had partially disappeared after tumor inoculation. Moreover, the treatment effect of the nano-vaccine using the mixture of the C-class CpG-ODN and the Poly (I: C) (the mass ratio of 3:1) as the adjuvant is better than that of the nano-vaccine using the A-class CpG-ODN and the Poly (I: C) (the mass ratio of 3:1) as the adjuvant.
Example 14 Loading of Water-soluble cellular Components inside and on the surface of microparticles for prevention of melanoma
This example uses mouse melanoma as a cancer model to illustrate how to prepare a micro vaccine loaded with only the water soluble fraction of melanoma and lung cancer cell fractions and to use this vaccine to prevent melanoma. In this example, B16F10 melanoma and LLC lung cancer cells were first lysed to prepare a water soluble fraction and a water insoluble fraction. Then, PLGA (24KDa-38KDa) is used as a microparticle framework material, poly (I: C) and two CpG-ODNs are used as immune adjuvants to prepare the whole-cell loaded micro vaccine with water-soluble components, and the micro vaccine is applied 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-20 deg.C, adding a certain amount of ultrapure water, heating, irradiating with ultraviolet radiation, freeze thawing for more than 3 times, and lysing cells with ultrasonic destruction. After cell lysis, the lysate is centrifuged at 8000g for 5min, and the supernatant is the water-soluble component which can be dissolved in pure water in B16F10 melanoma or LLC lung cancer cells. The obtained water-soluble components from the two cancer cell lysates are mixed according to the mass ratio of 1:1 to obtain the antigen source for preparing the micron vaccine.
(2) Preparation of micron vaccine
In the embodiment, the micro-vaccine and the blank micro-particles used as the reference adopt a multiple emulsion method in a solvent volatilization method, the adopted micro-particle preparation material is PLGA, and the adopted immunologic adjuvants are poly (I: C), CpG-ODN2006 (B class) and CpG-ODN2216(A class), and the mass ratio of the poly (I: C), the CpG-ODN2006 (B class) and the CpG-ODN2216(A class) is 4:0.5: 0.5. The antigen component is loaded in the micro-vaccine and adsorbed on the surface of the micro-particles, and the adjuvant is loaded only in the micro-vaccine. The preparation method is as described above. The particle diameter of the obtained micro vaccine 1 is 2.10 μm after loading cell components and immune adjuvants on the surface of the micro particles, the surface potential is about-5 mV, 150 μ g of protein or polypeptide components are loaded on each 1mg of PLGA micro particles, and 0.05mg of adjuvants are used, wherein 0.04mg of Poly (I: C), 0.78 mg of CpG-ODN 20060.005 mg and 22160.005 mg of CpG-ODN. The control mini-vaccine 1 has a particle size of 2.10 μm, a surface potential of-5 mV, and 150 μ g of protein or polypeptide fraction is loaded per 1mg PLGA mini-particle, using poly (I: C) and CpG-ODN2006 as adjuvants, where poly (I: C) is 0.04mg and CpG-ODN 20060.01 mg. The particle size of the control mini-vaccine 2 was 2.10 μm, the surface potential was-5 mV, and 150 μ g of protein or polypeptide fraction was loaded per 1mg PLGA microparticles, and poly (I: C), CpG-ODN2006 and CpG-ODN2216 were used as adjuvants, wherein poly (IC)0.025mg, CpG-ODN 20060.0125 mg and CpG-ODN 22160.0125 mg.
(3) Micron vaccine for preventing cancer
Female C57BL/6 at 6-8 weeks was selected to prepare melanoma-bearing mice. The scheme of the micron vaccine is as follows: subcutaneously injecting 200 μ L of 2mg PLGA micrometer vaccine at 35, 28, 21 and 14 days before melanoma inoculation; each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 And B16F10 cells. The PBS blank control protocol was as follows: in inoculation of melanomaSubcutaneous injections of 200 μ L PBS were given on days 35, 28, 21, and 14, respectively, before; each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 And B16F10 cells. In the experiment, the size of the tumor volume of the mice was recorded every 3 days from day 3. Tumor volume is calculated by the formula v ═ 0.52 × a × b 2 Calculation, where v is tumor volume, a is tumor length, and b is tumor width. Due to animal experiment ethics, when the tumor volume of the mouse exceeds 2000mm in the life test of the mouse 3 I.e. the mice were considered dead and were euthanized.
(4) Results of the experiment
As shown in fig. 42, the growth rate of tumor volume of mice in the group administered with the mu-vaccine was significantly slowed and the survival time of mice was significantly prolonged compared to the PBS blank control group. Moreover, the micro-vaccine using Poly (I: C) and two CpG-ODNs as mixed adjuvants is better than the micro-vaccine using Poly (I: C) and one CpG-ODN only as mixed adjuvants; the micro-vaccine mixed as an adjuvant at the mass ratio of total CpG-ODN: Poly (I: C) ═ 0.25:1 is better than the micro-vaccine mixed as an adjuvant at the mass ratio of CpG-ODN: Poly (I: C) ═ 1:1.
Example 15 adjuvant and melanoma cancer cells Loading inside nanoparticles for treatment of melanoma
In this example, a mouse melanoma is used as a cancer model to illustrate how to prepare a nano vaccine co-loaded with an immune adjuvant and a melanoma cancer cell whole cell component, and to treat melanoma using the nano vaccine. In this example, B16F10 mouse melanoma cells were used as a cancer model. B16F10 melanoma cancer cells were first lysed and water soluble and water insoluble fractions were prepared. Then, the organic polymer material PLGA is used as the nano-particle framework material, and poly (I: C), CpG-ODN 1018, CpG-ODN 2336 and arginine are used as immunologic adjuvants to prepare the nano-vaccine loaded with the water-soluble component and the water-insoluble component of the cancer cells by a solvent evaporation method. The nano-vaccine is then used to treat melanoma.
(1) Lysis of cells and Collection of fractions
Removing the culture medium from the cultured B16F10 cancer cell line, centrifuging for 5 minutes at 400g, resuspending and washing twice by using PBS, then resuspending the cancer cells by using ultrapure water, repeatedly freezing and thawing for five times, cracking the cancer cells by using ultrasound assistance in the freezing and thawing process, centrifuging for 5 minutes at 10000g, and collecting the supernatant, namely the water-soluble component. Dissolving the precipitate with 8M urea to obtain the original water-insoluble component. Mixing the water-soluble component from the B16F10 cancer cell and the original water-insoluble component dissolved in 8M urea according to the mass ratio of 1:1 to obtain the raw material source for preparing the vaccine.
(2) Preparation of nano-vaccine
The nano-vaccine in the embodiment is prepared by a multiple emulsion method in a solvent volatilization method, the molecular weight of PLGA used as a nano-particle preparation material is 7KDa-17KDa, an adjuvant and an antigen component are loaded in the nano-vaccine, and the adopted immune adjuvants are poly (I: C), CpG-ODN 1018, CpG-ODN 2336 and arginine. The preparation method is as described above. The average grain diameter of the nano vaccine loaded with the whole cell component is about 280 nm; the surface potential of the nano vaccine is about-5 mV; about 90 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano-particle, 0.05mg of adjuvant is used on each 1mg of PLGA nano-particle, wherein the amount of poly (I: C) is 0.02mg, the amount of CpG-ODN 1018 is 0.01mg, the amount of CpG-ODN 2336 is 0.02mg, and the amount of arginine used is 0.5 mg. The preparation method of the contrast nano vaccine 1 is the same, the particle size is 280nm, and the surface potential of the vaccine is about-5 mV; every 1mg PLGA nano particle is loaded with about 90 mug of protein or polypeptide component; the adjuvant is 0.05mg, the poly (I: C) is 0.02mg, the CpG-ODN 1018 is 0.015mg and the CpG-ODN 2336 is 0.015mg for each 1mg of PLGA nano-particle. The preparation method of the contrast nano vaccine 2 is the same, the particle size is 280nm, and the surface potential of the vaccine is about-5 mV; loading about 90 μ g of protein or polypeptide component per 1mg of PLGA nanoparticle; arginine was used as an immunoadjuvant at 0.5mg per 1mgPLGA nanoparticle.
(3) Nano-vaccine for cancer treatment
Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. The nano vaccine group administration scheme is as follows: each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 B16F10 cells; subcutaneously injecting 100 μ L of the water-soluble ingredient-loaded composition on 4 th, 7 th, 10 th, 15 th and 20 th days after melanoma inoculation2mg PLGA nano vaccine. The PBS control protocol was as follows: each mouse was inoculated subcutaneously on day 0, at the lower right back, at 1.5X 10 5 B16F10 cells; 200 μ L of PBS was injected subcutaneously on days 4, 7, 10, 15 and 20 after melanoma inoculation, respectively. Mouse tumor monitoring protocol was as above.
(4) Results of the experiment
As shown in FIG. 43, the tumors of the PBS control mice all grew and the production rate was very fast. The curative effect of the nano vaccine group is better than that of a PBS control group, and the nano vaccine using more than two CpG + Poly (I: C) + arginine as a mixed adjuvant is better than the nano vaccine using more than two CpG + Poly (I: C) as the mixed adjuvant and is also better than the nano vaccine using only arginine as the adjuvant. The nano vaccine loaded with a plurality of CpG and Poly (I: C) and antigen components in a specific ratio has a good treatment effect on melanoma.
Example 16 adjuvant and Breast cancer cell Loading inside nanoparticles for treatment of Breast cancer
In this example, a mouse triple negative breast cancer is used as a cancer model to illustrate how to prepare a nano vaccine which is loaded with an immune adjuvant, a substance for increasing lysosome escape and a triple negative breast cancer cell whole cell component, and to treat triple negative breast cancer by using the nano vaccine. In this example, 4T1 breast cancer cells were first lysed and a water-soluble fraction and a water-insoluble fraction were prepared. Then, PLGA is used as a nanoparticle framework material, Poly (I: C), CpG-ODN 1018 and CpG-ODN 2336 are used as immunologic adjuvants, lysine is used as a substance for increasing escape of lysosomes, and a solvent evaporation method is adopted to prepare the nano-vaccine loaded with water-soluble components and water-insoluble components of cancer cells. Then the nano vaccine is adopted to treat breast cancer.
(1) Lysis of cells and Collection of fractions
Removing a culture medium from a cultured 4T1 cancer cell line, centrifuging for 5 minutes at 400g, resuspending and washing twice by using PBS, then resuspending the cancer cells by using ultrapure water, repeatedly freezing and thawing for five times, cracking the cancer cells by using ultrasound assistance in the freezing and thawing process, adding nuclease for 10 minutes to degrade all nucleic acids, heating for 5 minutes at 95 ℃ to inactivate the nuclease, centrifuging for 5 minutes at 10000g, and collecting a supernatant, namely a water-soluble component. Dissolving the precipitate with 8M urea to obtain the original water-insoluble component. Mixing water-soluble components from cancer cells and original water-insoluble components dissolved in 8M urea according to the mass ratio of 2:1 to obtain a raw material source for preparing the vaccine.
(2) Preparation of nano-vaccine
The nano-vaccine in the embodiment is prepared by a multiple emulsion method in a solvent volatilization method, the molecular weight of PLGA used as a nano-particle preparation material is 7KDa-17KDa, an adjuvant and an antigen component are loaded in the nano-vaccine, and the adopted immune adjuvants are poly (I: C), CpG-ODN 1018, CpG-ODN 2336 and lysine. The preparation method is as described above. The average grain diameter of the nano vaccine loaded with the whole cell component is about 280 nm; the surface potential of the nano vaccine is about-5 mV; about 90 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, 0.05mg of adjuvant is used on each 1mg of PLGA nano particle, wherein the amount of poly (I: C) is 0.02mg, the amount of CpG-ODN 1018 is 0.015mg, the amount of CpG-ODN2216 is 0.015mg, and the amount of lysine is 0.005 mg. The preparation method of the contrast nano vaccine 1 is the same, the particle size is 280nm, and the surface potential of the vaccine is about-5 mV; every 1mg PLGA nano particle is loaded with about 90 mug of protein or polypeptide component; the adjuvant used per 1mg PLGA nanoparticle is 0.05mg, wherein the amount of poly (I: C) is 0.02mg, the amount of CpG-ODN 1018 is 0.015mg, the amount of CpG-ODN2216 is 0.015mg, and the amount of glycine is 0.005 mg. The preparation method of the contrast nano vaccine 2 is the same, the particle size is 280nm, and the surface potential of the vaccine is about-5 mV; loading about 90 μ g of protein or polypeptide component per 1mg of PLGA nanoparticle; the adjuvant is 0.05mg per 1mg PLGA nanoparticle, wherein the amount of poly (I: C) is 0.02mg, the amount of CpG-ODN 1585 is 0.015mg, the amount of CpG-ODN2216 is 0.015mg, and the amount of lysine is 0.005 mg.
(3) Nano-vaccine for cancer treatment
Female BALB/c of 6-8 weeks is selected as a model mouse to prepare a breast cancer tumor-bearing mouse. The nano vaccine group administration scheme is as follows: each mouse was inoculated subcutaneously 4X 10 in the lower right back on day 0 5 4T1 cells; 100 mu L of 2mg PLGA nano-vaccine loaded with water-soluble components was subcutaneously injected on days 4, 7, 10, 15 and 20 after breast cancer inoculation. PBS controlThe formula is as follows: each mouse was inoculated subcutaneously 4X 10 in the lower right back on day 0 5 4T1 cells; 200 μ L of PBS was injected subcutaneously on days 4, 7, 10, 15 and 20 after breast cancer inoculation. Mouse tumor monitoring protocol was as above.
(4) Results of the experiment
As shown in FIG. 44, the tumors of the PBS control mice all grew and the production rate was very fast. The nano vaccine group has better curative effect than a PBS control group, and the nano vaccine using more than two CpG-ODN (A class + B class) + Poly (I: C) + lysine as a mixed adjuvant is better than the nano vaccine using more than two CpG-ODN (A class + B class) + Poly (I: C) as a mixed adjuvant, and is also better than the nano vaccine using more than two CpG-ODN (A class + A class) + Poly (I: C) + lysine as an adjuvant. The nano vaccine loaded with a plurality of CpG and Poly (I: C) and antigen components in a specific ratio has a good treatment effect on breast cancer.
Example 17 Whole cell fraction Loading inside mannan-modified microparticles for Breast cancer prevention
In this example, a mouse breast cancer is used as a cancer model to illustrate how to prepare a micro vaccine loaded with a whole cell component of breast cancer tumor tissue and cancer cells, and to prevent breast cancer by using the micro vaccine. In practical application, the particle size, administration time, administration frequency and administration scheme can be adjusted according to the situation. In this example, tumor tissue and cancer cells of mouse breast cancer were first taken and lysed to prepare a water-soluble fraction and a primary water-insoluble fraction dissolved in 8M urea. Then, PLGA modified by PLGA and mannan is used as a microparticle framework material, Poly ICLC, CpG-ODN 1018(B class) and CpG-ODN 2395(C class) are used as immune adjuvants, TAT polypeptide (YGRKKRRQRRR) is used for increasing substances escaping from immunity, and a solvent evaporation method is adopted to prepare the micro vaccine. TAT polypeptides contain positively charged lysines and arginines. The micro-vaccine has the ability to target dendritic cells.
(1) Lysis of tumor tissue and Collection of fractions
(2) Preparation of micron vaccine
In this example, the micron vaccine and the control hollow micron particles were prepared by multiple emulsion method. The molecular weight of the micro-particle preparation material PLGA is 24KDa-38KDa, and the molecular weight of the mannan-modified PLGA is 24KDa-38 KDa; the mass ratio of unmodified PLGA to mannan-modified PLGA was 8: 2. Preparation method as described above, the lysate fraction and adjuvant are co-loaded into the mini-vaccine. The average particle diameter of the micro vaccine is about 1.50 mu m, the average surface potential is about-7 mV, each 1mg PLGA micro particle is loaded with 85 mu g protein or polypeptide component, each 1mg PLGA micro vaccine uses 0.05mg adjuvant, wherein, 0.01mg Poly ICLC, 0.02mg CpG-ODN 1018(B class), 0.02mg CpG-ODN 2395(C class) and 0.1mg TAT polypeptide. The polymer material and the preparation method for preparing the blank micron particles are the same as those of the blank micron particles, the particle size is about 1.45 mu m, and the blank micron particles are loaded with the same amount of adjuvant and TAT polypeptide but are not loaded with antigen components. The polymer material and the preparation method used for preparing the control micron vaccine are the same, the average particle size is about 1.50 microns, the average surface potential is about-7 mV, each 1mg PLGA micron particle is loaded with 85 μ g protein or polypeptide component, each 1mg PLGA micron vaccine uses 0.05mg adjuvant, wherein the Poly ICLC is 0.01mg, the CpG-ODN 1585 (class A) is 0.01mg, the CpG-ODN 2336 (class A) is 0.01mg, and the TAT polypeptide is 0.1 mg.
(3) Dendritic cell-targeted micro-vaccines for cancer prevention
Female BALB/c mice of 6-8 weeks were selected to prepare breast cancer-bearing mice. Vaccine groups 200 μ L of 2mg PLGA micro-vaccine was injected subcutaneously on days 35, 28, 21, 14 and 7 prior to tumor vaccination. PBS blank control group on day 35, 28, day 35 before tumor inoculationDay 21, day 14 and day 7 were injected subcutaneously with 200 μ L PBS, respectively. The empty microwell + lysate control group was injected subcutaneously with 200 μ L of empty microwell and an equivalent amount of free lysate to the vaccine load on days 35, 28, 21, 14, and 7, respectively, prior to tumor inoculation. Each mouse was inoculated subcutaneously 4X 10 in the lower right back on day 0 5 4T1 breast cancer cells. In the experiment, the method for monitoring the growth of the tumor in the mouse is the same as that described above.
(4) Results of the experiment
As shown in fig. 45, the tumor growth rate and survival time of the mice were significantly different in the vaccine-protected group compared to the control group and the blank microparticle + free lysate group. Furthermore, the micro-vaccine prepared in this example was better than the control micro-vaccine group, which indicates that the use of the mixture of C-class CpG-ODN and B-class CpG-ODN as an adjuvant is superior to the mixture of two A-class CpG-ODN.
Example 18 prevention of liver cancer with lysis fraction of liver cancer tumor tissue loaded on microparticles
In this example, a micro vaccine loaded with components of liver cancer and lung cancer tumor tissue lysates was prepared and applied to prevent liver cancer. In this example, the mouse liver cancer tumor tissue lysis fraction was loaded on a micron vaccine. First, tumor tissues of liver cancer and lung cancer of mice were obtained, and 8M urea was used to lyse the tumor tissues and to dissolve the tumor tissue lysate components. Then, PLA (30KDa) is used as a micron-sized framework material, Poly ICLC, CpG-ODN2007 (B class) and CpG-ODN2216(A class) are used as immune adjuvants, cR8(cyclo (CRRRRRRRRRRC)) polypeptide is used as an immune escape increasing substance, a micron vaccine is prepared, and the micron vaccine is applied to prevent liver cancer. cR8 is a cyclic polypeptide containing arginine.
(1) Lysis of tumor tissue and Collection of fractions
Each C57BL/6 mouse was inoculated subcutaneously under the axilla of 2X 10 mice 6 A Hepa 1-6 cell or 2X 10 6 LLC lung cancer cells, each mouse inoculated with tumor growing to a volume of about 1000mm 3 Mice were sacrificed and tumor tissue was harvested. Cutting the tumor tissue into pieces, sieving with a cell sieve, and dissolving the pieces after the tumor tissue is cracked by 8M urea; the lysate of liver cancer tumor tissue and the lysate of lung cancer tumor tissue are mixed according to the formulaMixing the components in a mass ratio of 1:1 to obtain the antigen components used for preparing the vaccine.
(2) Preparation of micron vaccine
In the embodiment, the micron vaccine is prepared by a solvent volatilization method, the adopted preparation material is PLA, and the adopted immunologic adjuvant is Poly ICLC, CpG-ODN2007 and CpG-ODN2216, wherein the mass ratio of the PLA to the immunologic adjuvant is 1:1: 1; the substance used for increasing lysosome escape is cR8 polypeptide, one fifth of the cR8 polypeptide is located on the surface of the particle through chemical bond modification PLA, and four fifths of the PLA is loaded in the particle. The antigen component is loaded inside the micron vaccine and adsorbed on the surface of the micron particle, and the adjuvant and the lysosome escape increasing matter are loaded inside the micron vaccine only. The preparation method comprises the steps of firstly adopting a dissolving and volatilizing method to wrap antigen components and adjuvants inside the micrometer particles, then centrifuging for 15min at 8000g, collecting precipitates, re-suspending 100mg of PLA micrometer particles by using a 4% trehalose solution, then freeze-drying for 48 hours, and refrigerating for later use. Before injection, 10mg of PLA microparticles are resuspended in 0.9mL of PBS or physiological saline, and then mixed with 0.1mL of a sample containing 8M cell lysate in urea (60mg/mL) and an immunoadjuvant (3mg/mL, four mass ratios 1:1:1) at room temperature for 10min, so that the injection can be performed. The obtained micron vaccine has the particle size of 3.10 mu m, each 1mg PLA micron particle is loaded with 150 mu g of protein or polypeptide component, and 0.045mg of adjuvant is used, wherein 0.015mg of Poly ICLC, 0.015mg of CpG-ODN20070, and 22160.015 mg of CpG-ODN are used; 0.02mg of cR8 polypeptide was used, wherein 0.004mg of the cR8 polypeptide was attached to PLA by chemical modification and located on the surface of the particles, and 0.016mg of the cR8 polypeptide was entrapped inside the particles. The particle size of the control micro-vaccine 1 is 3.10 μm, 150 μ g of protein or polypeptide component is loaded per 1mg PLA microparticles, 0.015mg each of Poly ICLC, CpG-ODN 1585 and CpG-ODN2216 is used; 0.02mg of cR8 polypeptide was used, wherein 0.004mg of the cR8 polypeptide was attached to PLA by chemical modification and located on the surface of the particles, and 0.016mg of the cR8 polypeptide was entrapped inside the particles. The control micrometer vaccine 2 was loaded with immune adjuvant, the particle size of the blank micrometer particles was 3.00 μm, each 1mg PLA micrometer particles was loaded with 150 μ g protein or polypeptide component, 0.015mg Poly ICLC, and 20070.03 mg CpG-ODN was used; 0.02mg of cR8 polypeptide was used, wherein 0.004mg of the cR8 polypeptide was attached to PLA by chemical modification and located on the surface of the particles, and 0.016mg of the cR8 polypeptide was entrapped inside the particles.
(3) Micron vaccine for preventing cancer
Selecting female C57BL/6 for 6-8 weeks to prepare a Hepa 1-6 hepatoma tumor-bearing mouse. 200 μ L of 2mg PLA micron vaccine was injected subcutaneously on days 49, 42, 35, 28, and 14, respectively, before the inoculation of hepatoma cells. Each mouse was inoculated subcutaneously to the right underarm at day 0 at 2X 10 6 And each Hepa 1-6 liver cancer cell. The PBS blank control protocol was as follows: 200 μ L of PBS was injected subcutaneously on days 49, 42, 35, 28, and 14, respectively, before the inoculation of hepatoma cells. Each mouse was inoculated subcutaneously to the right underarm at day 0 at 2X 10 6 And each Hepa 1-6 liver cancer cell. In the experiment, the method for monitoring the growth of the tumor in the mouse is the same as that described above.
(4) Results of the experiment
As shown in fig. 46, the liver cancer tumor growth of the PBS control group mice was faster, and the tumor growth of the mice and the survival time of the mice could be significantly inhibited by the micrometer vaccine administration group. Furthermore, the vaccines used in this example were more effective than control vaccine 1 and control vaccine 2. Therefore, the use of both B-class CpG-ODN and A-class CpG-ODN in combination with Poly ICLC as the mixed adjuvant is better than the use of only B-class CpG-ODN and Poly ICLC in combination with the use of both A-class CpG-ODN and Poly ICLC as the mixed adjuvant.
Example 19 tumor tissue lysis component loaded inside nanoparticles for treatment of pancreatic cancer
In this example, a mouse pancreatic cancer is used as a cancer model to illustrate how to prepare a nano vaccine loaded with a pancreatic cancer tumor tissue lysate component and apply the vaccine to treat pancreatic cancer. Mouse pancreatic cancer tumor tissue was first taken and lysed to prepare a water-soluble fraction and a primary water-insoluble fraction dissolved in 6M guanidine hydrochloride. PLGA (molecular weight 7-17 KDa) is used as a particle framework material, Poly (I: C), CpG-ODN M362 and CpG-ODN 2336 are used as immunological adjuvants, HER2 polypeptide (YDLKEPEEH) is used as a lysosome escape increasing substance, and the nano vaccine is prepared. HER2 polypeptide contains histidine and arginine.
(1) Lysis of tumor tissue and Collection of fractions
C57BL/6 mice were each inoculated subcutaneously in the axilla at 1X 10 6 Pan02 pancreatic cancer cells in each mouseThe inoculated tumors grew to a volume of about 1000mm each 3 Mice were sacrificed and tumor tissue was harvested. Tumor tissue was lysed as in example 18, and after collecting the water-soluble fraction, the water-insoluble fraction was dissolved using 6M guanidine hydrochloride.
(2) Preparation of nano-vaccine
In the embodiment, a solvent evaporation method is adopted for preparing the nano vaccine, the molecular weight of PLGA is 24-38 KDa, Poly (I: C), CpG-ODN M362 and CpG-ODN 2336 are used as mixed adjuvants according to the mass ratio of 1:0.8:0.8, and HER2 polypeptide is used as a lysosome escape increasing substance. When the vaccine is prepared, the water-soluble component mixture and the water-insoluble component mixture are respectively prepared into the nano vaccine and then are used together, and the antigen component and the adjuvant are only loaded in the nano vaccine. The preparation method is the same as above. The nano-vaccine has the particle size of about 270nm and the surface potential of about-7 mV, about 80 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano-particle, 0.026mg of adjuvant is used on each 1mg of PLGA nano-particle, wherein Poly (I: C) is 0.01mg, CpG-ODN M362 is 0.008 mg: CpG-ODN 2336 at 0.008 mg; HER2 polypeptide 0.026mg was used. The particle size of the control nano vaccine 1 is about 280nm, the surface potential is about-8 mV, about 80 mug protein or polypeptide component is loaded on each 1mg PLGA nano particle, 0.026mg adjuvant is used on each 1mg PLGA nano particle, wherein Poly (I: C) is 0.01mg, CpG-ODN M362 is 0.08 mg: CpG-ODN 2336 was 0.08 mg. The particle size of the control nano vaccine 2 is about 280nm, the surface potential is about-8 mV, about 80 mug protein or polypeptide component is loaded on each 1mg PLGA nano particle, 0.026mg adjuvant is used on each 1mg PLGA nano particle, 0.01mg Poly (I: C) is used, wherein CpG1585 is 0.08mg, CpG-ODN2216 is 0.08mg, and HER2 polypeptide is 0.026 mg.
(3) Nano-vaccine for cancer treatment
Female C57BL/6 at 6-8 weeks was selected to prepare pancreatic carcinoma mice. Each mouse was inoculated subcutaneously at day 0 at the lower right back at 1X 10 6 Individual Pan02 cells. The vaccine group was injected subcutaneously with 100. mu.L of 1mg PLGA nano-vaccine loaded with a water-soluble component and 100. mu.L of 1mg PLGA nano-vaccine loaded with an original water-insoluble component on days 4, 7, 10, 15 and 20, respectively. PBS blank control group was injected subcutaneously on days 4, 7, 10, 15 and 20, respectively00 μ L PBS. In the experiment, the mouse tumor monitoring and volume calculation methods are the same as above.
(4) Results of the experiment
As shown in fig. 47, there were significant differences in both tumor growth rate and survival time of mice in the vaccine-treated group compared to the control group. Furthermore, the vaccine group mice had partially disappeared after tumor inoculation. The vaccine described in this example was better than control nano-vaccine 1 and control nano-vaccine 2. In conclusion, the nano vaccine loaded with a plurality of CpG and Poly (I: C) mixed adjuvants and antigen components in a specific ratio has a good treatment effect on cancer, the vaccine effect can be improved by adding the polypeptide containing positively charged amino acid, and the mixed effect of one C class CpG-ODN and one A class CpG-ODN is better than that of two A class CpG-ODNs.
Example 20 Nanoprotein of Nano-size exerts cancer therapeutic efficacy through T cell immunity in cellular immunity
This example uses mouse melanoma as a cancer model to demonstrate that the nano-vaccine is mainly effective through T cell immunity in cellular immunity. In this example, B16F10 melanoma cancer cells were first lysed and a water soluble fraction and a water insoluble fraction were prepared. Then, PLGA is used as a nanoparticle framework material, poly (I: C), CpG-ODN 1018(B class) and CpG-ODN 2336(A class) are used as immunological adjuvants, arginine and polyhistidine are used as substances for increasing lysosome escape, and a solvent evaporation method is adopted to prepare the nano vaccine loaded with water-soluble components and water-insoluble components of cancer cells. The nano-vaccine is then used to treat melanoma.
(1) Lysis of cells and Collection of fractions
Removing the culture medium from the cultured B16F10 cancer cell line, centrifuging for 5 minutes at 400g, resuspending and washing twice by using PBS, then resuspending the cancer cells by using ultrapure water, repeatedly freezing and thawing for five times, cracking the cancer cells by using ultrasound assistance in the freezing and thawing process, centrifuging for 5 minutes at 10000g, and collecting the supernatant, namely the water-soluble component. Dissolving the precipitate with 8M urea to obtain the original water-insoluble component. Mixing the water-soluble component from the B16F10 cancer cell and the original water-insoluble component dissolved in 8M urea according to the mass ratio of 1:1 to obtain the raw material source for preparing the vaccine.
(2) Preparation of nano-vaccine
The nano-vaccine is prepared by a solvent volatilization method, the molecular weight of PLGA is 7KDa-17KDa, the adjuvant, the cracking component, arginine and poly-histidine are encapsulated in the nano-vaccine, the adopted immunologic adjuvant is poly (I: C), CpG-ODN 1018 and CpG-ODN 2336, and the substance for increasing immunologic escape is arginine and poly-histidine. The preparation method is as described above. The average grain diameter of the nano vaccine loaded with the whole cell component is about 280 nm; the surface potential of the nano vaccine is about-8 mV; about 90 mug of protein or polypeptide component is loaded on each 1mg of PLGA nano particle, 0.05mg of adjuvant is used on each 1mg of PLGA nano particle, wherein the amount of poly (I: C) is 0.02mg, the amount of CpG-ODN 1018 is 0.015mg, the amount of CpG-ODN 2336 is 0.015mg, the amount of arginine loaded is 0.005mg, and the amount of polyhistidine loaded is 0.005 mg.
(3) Nano-vaccine for cancer treatment
Female C57BL/6 at 6-8 weeks was selected as a model mouse to prepare melanoma-bearing mice. The nano vaccine group administration scheme is as follows: each mouse was subcutaneously inoculated on day 0 at the lower right back with 1.5X 10 5 B16F10 cells; subcutaneously injecting 100 μ L of 2mg PLGA nano-vaccine on 4 th, 7 th, 10 th, 15 th and 20 th days after melanoma inoculation, respectively; CD4 + T cell antagonistic group in addition to the administration of the vaccine, 10mg/kg of the alpha CD4 antibody was intraperitoneally injected every day starting two days before the 0 th day of cancer cell inoculation to eliminate CD4 in mice + T cells, administered antibody continuously up to day 50; CD8 + T cell antagonistic group in addition to the administration of the vaccine, mice were cleared of CD8 by intraperitoneal injection of 10mg/kg of the antibody to alpha CD8 every day starting on the first two days of day 0 after the inoculation of cancer cells + T cells, antibody was given continuously until day 50. The PBS control protocol was as follows: each mouse was subcutaneously inoculated on day 0 at the lower right back with 1.5X 10 5 B16F10 cells; 200 μ L of PBS were injected subcutaneously on days 4, 7, 10, 15, and 20, respectively, after melanoma inoculation. Mouse tumor monitoring protocol was as above.
(4) Results of the experiment
As shown in FIG. 48, the tumors of the PBS control mice were allThe growth and the production speed are fast, and the growth speed and the survival time of the mouse tumor of the vaccine group are obviously better than those of the PBS control group. Depletion of CD4 in mice Using the alpha CD4 antibody + After T cells, the cancer vaccine still has strong therapeutic effect on cancer; depletion of CD8 in mice Using the alpha CD8 antibody + After T cells, the loss of most efficacy of cancer vaccines has only a weak therapeutic effect on cancer. This demonstrates that the vaccine of the present invention is mainly effective by T cell immunity in cellular immunity.
Example 21 micron vaccine exerts cancer prevention efficacy through T cell immunity in cellular immunity
This example uses mouse breast cancer as a cancer model to demonstrate that the mini-vaccine acts primarily through T cells. In this example, a mouse breast cancer tumor tissue was first obtained and lysed with 8M urea and then dissolved in 8M urea. Then, PLGA and mannose modified PLGA are used as a micron-particle framework material, Poly (I: C), CpG-ODN 1018(B class) and CpG-ODN2216(A class) are used as immunologic adjuvants, and a solvent evaporation method is adopted to prepare the micron vaccine.
(1) Lysis of tumor tissue and Collection of fractions
(2) Preparation of micron vaccine
In the present example, the micron vaccine and the empty micron particles used as the control were prepared by multiple emulsion method. The molecular weight of the micro-particle preparation material PLGA is 38KDa-54KDa, and the molecular weight of the mannose modified PLGA is 38KDa-54 KDa. The mass ratio of unmodified PLGA to mannose-modified PLGA was 8: 2. Preparation method as described above, the lysate fraction and adjuvant are co-loaded into the mini-vaccine. The preparation method is as described above. The average particle size of the micro-vaccine is about 2.50 mu m, the average surface potential is about-9 mV, each 1mg PLGA micro-particle is loaded with 85 mu g protein or polypeptide component, each 1mg PLGA micro-vaccine uses 0.05mg adjuvant, wherein, the CpG-ODN 1018 is 0.02mg, the CpG-ODN2216 is 0.01mg, and the Poly (I: C) is 0.002 mg.
(3) Dendritic cell-targeted micro-vaccines for cancer prevention
Female BALB/c mice of 6-8 weeks were selected to prepare breast cancer-bearing mice. Vaccine groups 200 μ L of 2mg PLGA micro vaccine was injected subcutaneously on days 35, 28, 21, 14 and 7 prior to tumor vaccination; each mouse was inoculated subcutaneously 4X 10 in the lower right back on day 0 5 4T1 breast cancer cells; CD4 + T cells and CD8 + T cell Simultaneous antagonism group in addition to the administration of the vaccine, mice were cleared of CD4 by intraperitoneal injection of 10mg/kg of the alpha CD4 antibody and 10mg/kg of the alpha CD8 antibody every day starting on the first two days of day 0 after the cancer cell inoculation + T cells and CD8 + T cells, administered antibody continuously up to day 50; CD8 + T cell antagonistic group in addition to the administration of the vaccine, 10mg/kg of the alpha CD8 antibody was intraperitoneally injected every day starting two days before the 0 th day of cancer cell inoculation to eliminate CD8 in mice + T cells, antibody was given continuously until day 50. PBS blank control group was injected subcutaneously with 200 μ L PBS on days 35, 28, 21, 14 and 7, respectively, prior to tumor inoculation; each mouse was inoculated subcutaneously 4X 10 in the lower right back on day 0 5 4T1 breast cancer cells. In the experiment, the method for monitoring the growth of the mouse tumor is the same as the method.
(4) Results of the experiment
As shown in fig. 49, both tumor growth rate and survival time of mice were significantly different in the vaccine-protected group compared to the control group. Depletion of CD4 in mice Using the alpha CD4 antibody + After T cells, the cancer vaccine still has strong prevention effect on cancer; depletion of CD8 in mice Using the alpha CD8 antibody + After T cells, the loss of most efficacy of cancer vaccines has only a weak preventive effect on cancer. This demonstrates that the vaccine of the present invention is mainly effective by T cell immunity in cellular immunity.
Example 22 Whole cell fraction Loading mannose modified microparticles for Breast cancer treatment
In this example, mouse breast cancer is used as a cancer model to illustrate how to prepare a micro vaccine loaded with whole cell components of breast cancer tumor tissues and use the vaccine to treat breast cancer. In practical application, the particle size, administration time, administration frequency and administration scheme can be adjusted according to the situation. In this example, a micro vaccine was prepared using PLGA and mannose-modified PLGA as a micro-particulate scaffold material, Poly ICLC, CpG-ODN 1018 (class B) and CpG-ODN 2395 (class C) as immunoadjuvants, and a mixture of arginine and lysine as an immune escape increasing substance. The micro-vaccine has the ability to target dendritic cells.
(1) Lysis of tumor tissue and Collection of fractions
(2) Preparation of a micro-vaccine
In this example, the micron vaccine and the control hollow micron particles were prepared by multiple emulsion method. The molecular weight of the micro-particle preparation material PLGA is 24KDa-38KDa, and the molecular weight of the mannose-modified PLGA is 24KDa-38 KDa; the mass ratio of unmodified PLGA to mannose-modified PLGA was 7: 3. Preparation method as described above, the lysate fraction and adjuvant are co-loaded into the mini-vaccine. The average particle diameter of the micro vaccine is about 1.50 mu m, the average surface potential is about-7 mV, each 1mg PLGA micro particle is loaded with 85 mu g protein or polypeptide component, each 1mg PLGA micro vaccine uses 0.05mg adjuvant, wherein 0.01mg Poly ICLC, 0.02mg CpG-ODN 1018(B class), 0.02mg CpG-ODN 2395(C class), 0.05mg arginine and 0.05mg lysine are used. The particle size of the control micrometer vaccine 1 is about 1.50 micrometers, the average surface potential is about-7 mV, and the control micrometer vaccine is loaded with 0.1mg arginine by equal amounts of adjuvant and cell lysate components. The average particle diameter of the control micro-vaccine 2 is about 1.50 mu m, the average surface potential is about-7 mV, each 1mg PLGA micro-particle is loaded with 85 mu g protein or polypeptide component, each 1mg PLGA micro-vaccine is used with 0.05mg adjuvant, wherein the Poly ICLC is 0.01mg, the CpG-ODN 1018(B class) is 0.02mg, the CpG-ODN 2395(C class) is 0.02mg, and the loading lysine is 0.1 mg.
(3) Dendritic cell-targeted micro-vaccines for the treatment of cancer
Female BALB/c mice of 6-8 weeks were selected to prepare breast cancer bearing tumor mice. Each mouse was inoculated subcutaneously at day 0 in the lower right back of 4X 10 cells 5 4T1 breast cancer cells. Vaccine groups 200 μ L of 2mg PLGA micro vaccine was injected subcutaneously on days 4, 7, 10, 14, 19 and 24 post tumor vaccination. The PBS blank control group was injected subcutaneously with 200 μ L PBS on days 4, 7, 10, 14, 19 and 24, respectively, after tumor inoculation. In the experiment, the mouse tumor growth monitoring method is the same as above.
(4) Results of the experiment
As shown in fig. 50, both tumor growth rate and survival time of mice were significantly different in the vaccine-treated group compared to the control group. Furthermore, the micro-vaccine prepared in this example was better than the control micro-vaccine group, indicating that the use of mixed amino acids was more effective than the use of single amino acids.
Example 23 liver cancer tumor tissue lysis fraction loaded on microparticles for treating liver cancer
In this example, a micro vaccine loaded with the liver cancer tumor tissue lysate fraction was prepared and applied to prevent liver cancer. In this example, PLGA (38KDa-54KDa) was used as a microparticle scaffold material, Poly ICLC, CpG-ODN2007 (class B) and CpG-ODN2216 (class a) were used as immunoadjuvants, and histidine was used as a substance that promotes lysosomal escape, to prepare a micro vaccine, and to treat liver cancer using the micro vaccine.
(1) Lysis of tumor tissue and Collection of fractions
Each C57BL/6 mouse was inoculated subcutaneously under the axilla of 2X 10 mice 6 The tumor inoculated on each mouse of the Hepa 1-6 liver cancer cells grows to the volume of about 1000mm 3 Mice were sacrificed and tumor tissue was harvested. Cutting tumor tissue, sieving with cell sieve, cracking tumor tissue with 6M guanidine hydrochloride, and dissolving the cracked components to obtain vaccineThe antigenic component used.
(2) Preparation of micron vaccine
In the embodiment, the micro-vaccine is prepared by a solvent evaporation method, the adopted preparation material is PLGA, and the adopted immunologic adjuvant is Poly ICLC, CpG-ODN2007 and CpG-ODN2216, wherein the mass ratio of the Poly ICLC, the CpG-ODN2007 and the CpG-ODN2216 is 1:1: 1; the substance used to increase lysosomal escape was histidine. An antigenic component, an adjuvant, and a substance that increases lysosomal escape are entrapped inside the micro-vaccine. The preparation method comprises the steps of firstly adopting a dissolving and volatilizing method to wrap antigen components and adjuvants inside the micrometer particles, then centrifuging for 15min at 8000g, collecting precipitates, re-suspending 100mg of PLA micrometer particles by using a 4% trehalose solution, then freeze-drying for 48 hours, and refrigerating for later use. The obtained micrometer vaccine has particle diameter of 2.60 μm, and each 1mg PLGA micrometer particle is loaded with 100 μ g protein or polypeptide component, and adjuvant 0.045mg, wherein Poly ICLC 0.015mg, CpG-ODN 20070.015mg, CpG-ODN 22160.015 mg; histidine 0.02mg was used. The particle size of the control micro vaccine 1 is 2.60 μm, 100 μ g of protein or polypeptide component is loaded per 1mg of PLGA micro particle, 0.015mg each of Poly ICLC, CpG-ODN2007 and CpG-ODN2216 is used; glutamic acid 0.02mg was used. The particle size of the control micro-vaccine 2 is 2.60 μm, 100 μ g of protein or polypeptide component is loaded on each 1mg PLGA micro-particle, and 0.015mg of Poly ICLC, 20070.015mg of CpG-ODN, 22160.015 mg of CpG-ODN are used; histidine-containing 8 peptide (LHQACVPGL) was used at 0.02 mg.
(3) Micron vaccine for treating cancer
Selecting female C57BL/6 for 6-8 weeks to prepare a Hepa 1-6 hepatoma tumor-bearing mouse. Each mouse was inoculated subcutaneously to the right underarm at day 0 at 2X 10 6 And each Hepa 1-6 liver cancer cell. 200 μ L of 2mg PLA micron vaccine was injected subcutaneously on days 4, 7, 10, 15, 20 and 25 after the inoculation of hepatoma cells, respectively. In the experiment, the method for monitoring the growth of the mouse tumor is the same as the method.
(4) Results of the experiment
As shown in fig. 51, the liver cancer tumor growth of the PBS control group mice was faster, and the tumor growth of the mice and the survival time of the mice could be significantly inhibited by the micrometer vaccine administration group. Furthermore, the vaccines used in this example were more effective than control vaccine 1 and control vaccine 2. It can be seen that the use of positively charged amino acids is more effective than negatively charged amino acids, and that pure histidine is more effective than polypeptides containing partial histidine.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Claims (14)
1. An immunoadjuvant composition, characterized in that it comprises at least a combination of (1) and (2) of the following components:
(1) poly (I: C) or Poly (ICLC),
(2) CpG-ODN, wherein the CpG-ODN is at least two of A class CpG-ODN, B class CpG-ODN and C class CpG-ODN, and at least one of them is B class CpG-ODN or C class CpG-ODN;
(3) amino acids, polypeptides, lipids, carbohydrates, proteins or inorganic salts.
2. The immunoadjuvant composition according to claim 1, characterized in that: the A class CpG-ODN is selected from CpG-ODN2216, CpG-ODN 1585 or CpG-ODN 2336.
3. The immunoadjuvant composition according to claim 1, characterized in that: the B class CpG-ODN is selected from CpG-ODN 1018, CpG-ODN2006, CpG-ODN1826, CpG-ODN 1668, CpG-ODN2007, CpG-ODN BW006 or CpG-ODN SL 01.
4. The immunoadjuvant composition according to claim 1, characterized in that: the C class CpG-ODN is selected from CpG-ODN 2395, CpG-ODN SL03 or CpG-ODN M362.
5. The immunoadjuvant composition according to claim 1, characterized in that: the amino acid is a positively charged amino acid.
6. The immunoadjuvant composition according to claim 1, characterized in that: the amino acid is a combination comprising at least two positively charged amino acids.
7. The immunoadjuvant composition according to claim 1, characterized in that: the inorganic salt is releasable H + Or an acidic substance to form a proton sponge effect.
8. Use of an immunoadjuvant composition according to any one of claims 1 to 7 for the preparation of a cellular immune activator.
9.A cellular immune activator characterized by: the cellular immune activator comprises the immune adjuvant composition of any one of claims 1-7.
10.A cancer vaccine loaded with the immunoadjuvant composition of any one of claims 1 to 7, characterized in that: the cancer vaccine comprises nanoparticles or microparticles, and an antigen component and an immunoadjuvant composition loaded on the nanoparticles or microparticles.
11.A cancer vaccine as claimed in claim 10, wherein: the antigen component is a whole cell component antigen derived from cancer cells and/or tumor tissue.
12. The cancer vaccine of claim 11, wherein the whole cell component antigen is prepared by a method comprising the steps of:
cracking cancer cells or tumor tissues by using water or a solution without a dissolving agent, collecting a soluble part as a water-soluble component, dissolving an insoluble part by using the dissolving agent, and then converting the insoluble part into a soluble part as a water-insoluble component, wherein the water-soluble component and the water-insoluble component are whole cell component antigens;
or cancer cells or tumor tissues are cracked by a lytic agent, and then the cracked substance is dissolved to obtain the whole cell component antigen;
wherein the dissolving agent is selected from urea, guanidine hydrochloride, deoxycholate, dodecyl sulfate, glycerol, protein degrading enzyme, albumin, lecithin, 0.1-2000mg/mL inorganic salt, Triton, Tween, dimethyl sulfoxide, acetonitrile, ethanol, methanol, N-dimethylformamide, propanol, isopropanol, acetic acid, cholesterol, amino acid, glycoside, choline, Brij TM -35, octaethyleneglycol monodecyl ether, CHAPS, Digitonin, lauryldimethyimine oxide and
at least one of CA-630 nonionic surfactants.
13.A cancer vaccine as claimed in claim 10, wherein: the cancer vaccine is connected with a target head with an active targeting function.
14. Use of an immunoadjuvant composition according to any one of claims 1 to 7 or a cancer vaccine according to any one of claims 10 to 13 for the manufacture of a medicament for the treatment or prevention of cancer.
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| PCT/CN2022/092201 WO2023201786A1 (en) | 2022-04-18 | 2022-05-11 | Immunological adjuvant composition and cancer vaccine based on composition and application thereof |
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