WO2022082869A1 - 一种负载全细胞组分的靶向输送系统及其应用 - Google Patents

一种负载全细胞组分的靶向输送系统及其应用 Download PDF

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WO2022082869A1
WO2022082869A1 PCT/CN2020/126655 CN2020126655W WO2022082869A1 WO 2022082869 A1 WO2022082869 A1 WO 2022082869A1 CN 2020126655 W CN2020126655 W CN 2020126655W WO 2022082869 A1 WO2022082869 A1 WO 2022082869A1
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water
cells
components
cancer
delivery system
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PCT/CN2020/126655
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English (en)
French (fr)
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刘密
马琳
刁璐
李柏松
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苏州大学
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Priority to KR1020237010736A priority Critical patent/KR20230093248A/ko
Priority to AU2020473977A priority patent/AU2020473977A1/en
Priority to JP2023521074A priority patent/JP2023547789A/ja
Priority to EP20958449.9A priority patent/EP4233891A1/en
Priority to US18/028,084 priority patent/US20230330199A1/en
Priority to CA3196495A priority patent/CA3196495A1/en
Publication of WO2022082869A1 publication Critical patent/WO2022082869A1/zh

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Definitions

  • the invention relates to the technical field of immunotherapy, in particular to a targeted delivery system loaded with whole cell components and its application.
  • Immunity is a physiological function of the human body.
  • the human body relies on this function to identify "self” and “non-self” components, thereby destroying and rejecting antigenic substances (such as viruses and bacteria) that enter the human body, or damaged cells produced by the human body itself. and tumor cells to maintain human health.
  • antigenic substances such as viruses and bacteria
  • the development of immunotechnology has been extremely rapid, especially in the field of immunotherapy of cancer. With the continuous improvement of cancer awareness, it has been found that the body's immune system and various immune cells play a key role in inhibiting the occurrence and development of cancer.
  • cancer immunotherapy has developed rapidly in recent years, and cancer vaccines are one of the important methods for cancer immunotherapy and prevention.
  • Effective cancer vaccines need to be loaded with cancer-specific antigens, and these loaded antigens can be efficiently delivered to antigen-presenting cells to activate the body's immune system to recognize and attack cancer cells.
  • scientists identify cancer-specific or cancer-related antigenic polypeptides from tumor cell analysis of cancer patients, and then artificially synthesize them in vitro to prepare cancer vaccines for cancer treatment.
  • the method has shown certain efficacy in clinical trials of cancer patients.
  • the number of cancer antigens that can be found by this type of method is limited, and it is time-consuming, labor-intensive, and costly, and the obtained polypeptide vaccine has no ability to target antigen-presenting cells.
  • antigens need to be delivered to antigen-presenting cells first, and then antigen-presenting cells process cancer antigens and present them to the surface of antigen-presenting cells and interact with T cells to activate T cells against cancer.
  • T cells Once T cells can recognize specific cancer antigens, they will recognize and kill cancer cells that contain those cancer antigens.
  • cancer cell antigens are different even within each patient of the same cancer. Therefore, it is difficult to prepare cancer vaccines that can be used by most people from a limited number of cancer cell antigens.
  • Cancer cells and tumor tissues from patients contain each individual's unique cancer antigens and mutations, so they belong to personalized antigens, and the prepared cancer vaccines also belong to personalized vaccines.
  • Tumor tissue contains various cancer antigens. If tumor tissue or whole cell components of cancer cells can be delivered to antigen-presenting cells as a vaccine, the vaccine will have good preventive and therapeutic effects on cancer.
  • researchers due to the limitations of current technical means, researchers have focused their research on water-soluble components, and cannot effectively apply water-insoluble components, which is the current difficulty in the application of whole-cell components.
  • antigens need to be delivered and presented to antigen-presenting cells first, and then antigen-presenting cells process cancer antigens and present them on the surface of antigen-presenting cells and interact with T cells to activate T cells. Immune recognition of cancer cells. Once T cells can recognize specific cancer antigens, they will recognize and kill cancer cells that contain those cancer antigens.
  • T cells can recognize specific cancer antigens, they will recognize and kill cancer cells that contain those cancer antigens.
  • Currently targeting antigen-presenting cells by particle size is called passive targeting.
  • passive targeting alone cannot efficiently present to the surface of antigen-presenting cells and interact with T cells. Taking 300-nanometer nanoparticles as an example, they can be phagocytosed by antigen-presenting cells such as dendritic cells and B cells. , but may also be phagocytosed by other cells such as fibroblasts.
  • the object of the present invention is to provide a targeted delivery system loaded with whole cell components, so that the targeted delivery system can actively target the water-soluble and water-insoluble components of cancer cells or tissues.
  • the whole-cell component of the component is presented to antigen-presenting cells, and can improve the effect of preventing and treating tumors;
  • Another object of the present invention is to provide the application of the above-mentioned targeted delivery system in the preparation of vaccines for preventing and/or treating cancer.
  • the present invention provides the following technical solutions:
  • a whole-cell component-loaded targeted delivery system which is a nanoscale or micron-sized particle with a target on the surface, and the particle is loaded with the whole cell component of cancer cells or cancer tissues, and the non-
  • the water-soluble components are dissolved by the solubilizer; the whole cell components are water-soluble components and water-insoluble components of the whole cells in the cell or tissue; the target head is combined with molecules on the surface of specific cells or tissues to help the particles enter cells or tissues.
  • the water-soluble components that are soluble in pure water or an aqueous solution without a solubilizer are first obtained, and then the water-insoluble components are dissolved in a solubilizer by using a solubilizing aqueous solution containing a specific solubilizer.
  • all cellular components can be converted into components that can be dissolved in aqueous solution and then loaded inside and outside nanoparticles or microparticles to prepare a targeted delivery system, thus ensuring that most antigenic substances are loaded into the prepared targeted delivery system.
  • cells or tissues can also be lysed directly using a solubilizing aqueous solution containing a solubilizer to dissolve whole cell components without collecting water-soluble and non-water-soluble components separately.
  • Cell component preparation targeted delivery system are examples of cells or tissues that are soluble in pure water or an aqueous solution without a solubilizer.
  • the water-soluble and water-insoluble fractions of cellular components in cancer cells or tumor tissue encompass the constituents and components of the entire cell.
  • the unmutated proteins, polypeptides and genes that are the same as the normal cell components will not cause an immune response because of the immune tolerance generated during the development of the autoimmune system; while the mutations of the genes, proteins and polypeptides produced by cancer do not cause an autoimmune system.
  • the immune tolerance developed during development is thus immunogenic and activates the body's immune response against cancer cells.
  • the use of these cancer cell-specific immunogenic substances produced by disease mutations in whole cell fractions can be used for cancer prevention and treatment.
  • the whole cell component can be divided into two parts according to the solubility in pure water or an aqueous solution without a solubilizer: a water-soluble component and a water-insoluble component.
  • the water-soluble component is the original water-soluble part that is soluble in pure water or an aqueous solution without a solubilizer
  • the water-insoluble component is the original water-insoluble part that is insoluble in pure water.
  • the fraction that is insoluble in water or the aqueous solution without the solubilizer becomes soluble in the aqueous solution containing the solubilizer.
  • Both the water-soluble portion and the water-insoluble portion of the whole cell fraction can be solubilized by a solubilizing aqueous solution containing a solubilizing agent.
  • the solubilizer includes, but is not limited to, urea, guanidine hydrochloride, sodium deoxycholate, SDS, glycerol, alkaline solution with pH greater than 7, acidic solution with pH less than 7, each Protein degrading enzymes, albumin, lecithin, high concentration of inorganic salts, Triton, Tween, DMSO, acetonitrile, ethanol, methanol, DMF, propanol, isopropanol, acetic acid, cholesterol, amino acids, glycosides, choline, Brij TM -35, Octaethylene glycol monododecyl ether, CHAPS, Digitonin, lauryldimethylamine oxide, One or more of CA-630, DMSO, acetonitrile, ethanol, methanol, DMF, isopropanol, propanol, dichloromethane and ethyl acetate can be selected.
  • the water-insoluble component can also be changed from insoluble to soluble in pure water by other methods that can solubilize protein and polypeptide fragments. But in the specific implementation process of the present invention, the effect of urea, guanidine hydrochloride, SDS, sodium deoxycholate or glycerol is obviously better than other solubilizers such as PEG.
  • the present invention directly uses a solubilizing solution containing urea or guanidine hydrochloride to directly lyse cells or tissues and directly dissolve whole cell components.
  • 8M urea and 6M guanidine hydrochloride aqueous solution are used to dissolve the water-insoluble components in tumor tissue or cancer cells. In practical applications, any other components that can solubilize whole cells such as SDS and glycerol can also be used. Solubilizer for water-insoluble components.
  • Microparticles or nanoparticles have an optimal particle size for targeting antigen-presenting cells, but the optimal size of nanoparticles or microparticles prepared from different materials may vary. For example, it is currently believed that the optimal size of cationic liposomes is around 50-150 nanometers, and the nanoparticles prepared in PLGA may be around 200-500 nanometers. And micron particles theoretically believe that 1.5-5 microns is the most suitable. This optimum size varies depending on the particle material. Targeting antigen-presenting cells by particle size is called passive targeting.
  • the present invention also uses an active targeting strategy, that is, a target molecule that can target specific cells is connected outside the nanoparticles or microparticles.
  • an active targeting strategy that is, a target molecule that can target specific cells is connected outside the nanoparticles or microparticles.
  • nanoparticles or microparticles can be directly targeted to the surface of specific cells or tissues, and the particles can enter cells or tissues through ligand-receptor binding.
  • These cells include, but are not limited to, dendritic cells in leukocytes, macrophages, B cells, T cells, NK cells, NKT cells, neutrophils, eosinophils, basophils
  • the target Tissues that can be targeted include, but are not limited to, lymph nodes, thymus, spleen, bone marrow.
  • 300-nanometer nanoparticles can be phagocytosed by antigen-presenting cells such as dendritic cells and B cells, but may also be phagocytosed by other cells such as fibroblasts. If an active targeting strategy is used, it can be directed to be phagocytosed only by the most critical antigen-presenting cells, dendritic cells.
  • the mannose (ie, target) modified nanoparticles or microparticles target dendritic cells. In practical applications, any target or target that can target dendritic cells can also be used. Other types of specific cells or tissues.
  • the particle size of nano-vaccine or micro-vaccine is nano-scale or micro-scale, which can ensure that the vaccine is phagocytosed by antigen-presenting cells, and in order to improve the phagocytosis efficiency, the particle size should be within an appropriate range.
  • the particle size of the nano-sized particles is 1 nm-1000 nm.
  • the nano-sized particles have a particle size of 30 nm to 800 nm.
  • the particle size of the nano-sized particles is 50 nm-600 nm.
  • the particle size of the micron-sized particles is 1 ⁇ m-1000 ⁇ m. In some embodiments, the micron-sized particles have a particle size of 1 ⁇ m to 100 ⁇ m. In some embodiments, the micron-sized particles have a particle size of 1 ⁇ m to 10 ⁇ m. Further, in some embodiments, the particle size of the micron-sized particles is 1 ⁇ m-5 ⁇ m.
  • the surfaces of the nano-sized particles or micro-sized particles can be electrically neutral, negatively charged or positively charged.
  • immune adjuvants with immunomodulatory functions can also be added to it, such as pattern recognition receptor agonists, BCG cell wall skeleton, BCG methanol extraction residue, BCG muramyl Dipeptide, Mycobacterium phlei, Polyantibiotic A, Mineral Oil, Virus-Like Particles, Reconstituted Influenza Virosomes with Immune Enhancement, Cholera Enterotoxins, Saponins and Their Derivatives, Resiquimod, Thymosin, Neonatal Bovine Liver Active Peptides, Miquimod, polysaccharide, curcumin, immune adjuvant poly ICLC, corynebacterium brevis vaccine, hemolytic streptococcus preparation, coenzyme Q10, levamisole, polycytidylic acid, interleukin, interferon, polyinosinic acid, Polyadenylic acid, alum, aluminum phosphate, lanolin, vegetable oil, endo
  • the method of adding the immunoadjuvant of the present invention includes loading in nanoparticles or microparticles, or loading on the surface of nanoparticles or microparticles, or loading both in nanoparticles or microparticles and on the surface of nanoparticles or microparticles .
  • the immune adjuvant is added to the whole cell fraction.
  • Polyinosinic-polycytidylic acid poly(I:C)
  • Bacillus Calmette-Guerin BCG
  • CpG Bacillus Calmette-Guerin
  • concentration of poly(I:C), BCG or CpG is preferably greater than 1 ng/mL to separate and dissolve the original water-insoluble components dissolved in the solubilizer.
  • the present invention also includes adding a PEG protective film outside the particles.
  • the preparation materials of the nano-sized or micro-sized particles are one or more of organic synthetic polymer materials, natural polymer materials, inorganic materials, bacteria or viruses.
  • the organic synthetic polymer material is a biocompatible or degradable polymer material, including but not limited to PLGA, PLA, PGA, Poloxamer, PEG, PCL, PEI, PVA, PVP, PTMC, polyanhydride, PDON, PPDO , PMMA, polyamino acids, synthetic peptides.
  • the natural polymer materials are biocompatible or degradable polymer materials, including but not limited to phospholipids, cholesterol, starch, carbohydrates, polypeptides, sodium alginate, albumin, collagen, gelatin, and cell membrane components.
  • the inorganic material is a material without obvious biological toxicity, including but not limited to ferric oxide, ferric tetroxide, calcium carbonate, and calcium phosphate.
  • the shape of the targeted delivery system of the present invention is any common shape, including but not limited to spherical, ellipsoid, barrel, polygonal, rod, sheet, line, worm, square, triangle, butterfly or circle disc shape.
  • the loading method is that the water-soluble components and the water-insoluble components of the whole cells are separately or simultaneously loaded inside the particle, and/or separately or simultaneously loaded on the particle surface.
  • the water-soluble components are simultaneously loaded in the particles and on the surface of the particles
  • the water-insoluble components are simultaneously loaded in the particles and loaded on the surface of the particles
  • the water-soluble components are loaded in the particles and the non-water-soluble components are loaded on the surface of the particles
  • the water-insoluble component is loaded in the particle and the water-soluble component is loaded on the particle surface
  • the water-soluble component and the water-insoluble component are loaded in the particle and only the water-insoluble component is loaded on the particle surface
  • the water-soluble component and the water-insoluble component are loaded on the particle surface.
  • the water-soluble component is loaded in the particle, and the water-soluble component and the water-insoluble component are simultaneously supported on the surface of the particle, the water-insoluble component is loaded in the particle, and the water-soluble component and the water-insoluble component are loaded.
  • the components are simultaneously supported on the particle surface, the water-soluble component and the water-insoluble component are simultaneously supported in the particle, and the water-soluble component and the water-insoluble component are simultaneously supported on the particle surface.
  • FIG. 2 to 17 Schematic diagrams of the structure of the delivery system of the whole cell component of the present invention are shown in Figures 2 to 17 .
  • only one nanoparticle or microparticle with a specific structure may be used, or two or more nanoparticles or microparticles with different structures may be used at the same time.
  • the targeted delivery system of the present invention can be prepared according to any preparation method that has been found for nano-sized particles and micro-sized particles, including but not limited to common solvent evaporation methods, dialysis methods, extrusion methods, and hot melt methods.
  • the delivery system is prepared by a double emulsion method in a solvent evaporation method.
  • the targeted delivery system of the whole cell component of the present invention can deliver the loaded whole cell component to the relevant immune cells, and activate and enhance the killing effect of the autoimmune system on cancer cells through the immunogenicity of the loaded component. Therefore, the present invention also provides the application of the whole-cell component targeted delivery system in the preparation of vaccines for preventing and/or treating cancer.
  • the cancers are solid tumors or hematological tumors, including but not limited to endocrine system tumors, nervous system tumors, reproductive system tumors, digestive system tumors, urinary system tumors, immune system tumors, circulatory system tumors, respiratory system tumors, blood Systemic tumors, skin system tumors.
  • the targeted delivery system of the present invention can be administered multiple times before or after the occurrence of cancer to activate the body's immune system, thereby delaying the progression of cancer, treating cancer or preventing cancer Cancer recurrence.
  • the present invention converts the components in the cells that are insoluble in pure water or the aqueous solution without the solubilizer into soluble in the specific solubilizer and can be used to prepare nanoparticles by using an aqueous solution containing a specific solubilizer. and microparticles, thereby improving the comprehensiveness and immunogenicity of antigenic substances or components carried by nanoparticles or microparticles.
  • a target head that can be targeted to antigen-presenting cells is added to improve the phagocytosis efficiency of antigen-presenting cells in an active targeting manner, thereby improving the effect of preventing or treating cancer.
  • Fig. 1 shows the preparation process and application field schematic diagram of vaccine of the present invention
  • a water-soluble component and water-insoluble component collect and prepare the schematic diagram of nano-vaccine or micro-vaccine respectively
  • b adopt the solubilizing agent containing solubilizer Schematic diagram of solution dissolving whole cell components and preparing nano-vaccine or micro-vaccine;
  • Figures 2-12 are schematic diagrams showing the structure of nanoparticles or microparticles loaded with water-soluble and water-insoluble cellular components, wherein 1: water-soluble components in cells or tissue components; 2: cells or tissue components 3: immune adjuvant; 4: nanoparticle or microparticle; 5: inner core part of nanoparticle; 6: target that can target specific cells or tissues.
  • 1 water-soluble components in cells or tissue components
  • 2 cells or tissue components
  • 3 immune adjuvant
  • 4 nanoparticle or microparticle
  • 5 inner core part of nanoparticle
  • 6 target that can target specific cells or tissues.
  • the surface and interior of the nanoparticles or micro-particles contain immune adjuvants
  • Fig. 4-Fig. 5 the immune adjuvant is only distributed inside the nanoparticles or micro-particles
  • the nanoparticles or micro-particles contains immune adjuvant only on the outer surface;
  • Figure 8- Figure 9 has no immune adjuvant inside and outside the nanoparticle or microparticle;
  • Figure 10 Cell components and/or immune adjuvant are only distributed inside the nanoparticle or microparticle;
  • Figure 11 Cell components and/or immune adjuvants are only distributed outside the nanoparticles or microparticles;
  • Figure 12 Cell components and immune adjuvants are distributed inside or outside the nanoparticles or microparticles, respectively;
  • FIG. 3 5.a-5.i in Fig. 5, 9.a-9.i in Fig. 5, 13.a-13.i in Fig. 7 and 17.a-17.i in Fig.
  • the water-soluble or non-water-soluble components in the cell or tissue components are distributed in the outer layer of the inner core formed by the nanoparticles or microparticles; a: The inner and surface loading of the nanoparticles or microparticles are both cells or tissue groups water-soluble components in the components; b: the nano-particles or micro-particles are encapsulated inside and surface-loaded are non-water-soluble components in the cell or tissue components; c: the nano-particles or micro-particles are encapsulated inside the cells or tissues
  • the water-insoluble components in the components and the surface-loaded components are all water-soluble components in the cells or tissue components; d: the water-soluble components in the cells or tissue components are contained within the nanoparticles or microparticles and the surface-loaded components are are all water-insoluble components in cells or tissue components; e: water-soluble components and water-insoluble components in cells or tissue components simultaneously encapsulated inside nanoparticles
  • the water-soluble or water-insoluble components in the cells or tissue components carried by the nanoparticles or microparticles do not form a distinct core when distributed inside the nanoparticles or microparticles ;
  • the water-soluble or water-insoluble components in the cells or tissue components carried by the nanoparticles or microparticles are distributed in a core part inside the nanoparticles or microparticles; in g, h and i, the nanoparticle
  • the water-soluble or water-insoluble components in the cells or tissue components carried by the particles or microparticles are distributed in multiple inner core parts inside the nanoparticles or microparticles; in j, k and l, the nanoparticles or microparticles are carried
  • the water-soluble or water-insoluble components in the cell or tissue components are distributed in the outer layer of the inner core formed by the nanoparticles or microparticles; in a, d, g and j,
  • Water-soluble components in tissue components are loaded with nanoparticles or microparticles that are non-water-soluble components in cells or tissue components;
  • c, f, i, and l include nanoparticles or microparticles Particles load both water-soluble and water-insoluble components in cellular or tissue components;
  • Figures 13-19 show the results for cancer treatment and prevention when tumor tissue or cancer cell whole cell components are loaded on targeted nanoparticles or microparticles in Example 1-7; a, tumor tissue or cancer cell whole cell components are loaded Targeted nanoparticle or microparticle structure of water-soluble components/or simultaneously water-insoluble components in cellular components; b, targeted nanoparticle or water-insoluble components loaded with tumor tissue or cancer cell whole cell components Microparticle structure; c, tumor growth inhibition test results of targeted nano-vaccine or micro-vaccine; d, mouse survival test results.
  • the embodiment of the present invention discloses a targeted delivery system loaded with whole-cell components and its application, and those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications apparent to those skilled in the art are deemed to be included in the present invention.
  • the targeted delivery system and its applications of the present invention have been described with reference to the preferred embodiments, and it is obvious that modifications to the targeted delivery systems and their applications described herein can be made without departing from the content, spirit and scope of the present invention. Or appropriate changes and combinations to implement and apply the technology of the present invention.
  • the whole-cell component delivery system of the present invention can be used to prepare vaccines for preventing and/or treating cancer, and the preparation process and application fields thereof are shown in FIG. 1 .
  • cells or tissues can be lysed and then water-soluble components and water-insoluble components can be collected separately to prepare nano-vaccine or micro-vaccine; or a solubilizing solution containing a solubilizing agent can be used to directly lyse cells or tissues and dissolve whole cells. components and prepare nano-vaccine or micro-vaccine.
  • the method for preparing a targeted delivery system (also referred to as a nano-vaccine or a micro-vaccine) described in the present invention is a common preparation method.
  • the preparation of the nano-vaccine adopts the double emulsion method in the solvent evaporation method
  • the nano-particle preparation materials used are organic polymer polylactic acid-glycolic acid copolymer (PLGA) and polylactic acid (PLA).
  • the immunoadjuvant employed was poly(I:C), Bacille Calmette-Guerin (BCG) or CpG.
  • the specific preparation method of the double emulsion method adopted in the present invention is as follows:
  • Step 1 adding a first predetermined volume of an aqueous phase solution containing a first predetermined concentration to a second predetermined volume of an organic phase containing a medical polymer material of a second predetermined concentration.
  • the aqueous solution contains each component in the cancer cell lysate and the immunopotentiating adjuvant poly(I:C), BCG or CpG; each component in the cancer cell lysate is water-soluble when prepared The active component or the original water-insoluble component dissolved in 8M urea.
  • the concentration of water-soluble components from cancer cells contained in the aqueous phase solution or the concentration of the original water-insoluble components dissolved in 8M urea from cancer cells, that is, the first predetermined concentration requires that the protein polypeptide concentration content is greater than 1ng/ mL, the reason for choosing such a concentration is that the inventors have found through a large number of experiments that the larger the concentration of the aqueous solution of cell lysate used, the more various cancer antigens are contained in the prepared nanoparticles.
  • the prepared protein and polypeptide concentration is greater than 1 ng/mL, that is, when the first predetermined concentration is greater than 1 ng/mL, enough cancer antigens can be loaded to activate the relevant immune response.
  • the concentration of the immune adjuvant in the initial aqueous phase was greater than 0.01 ng/mL.
  • the first predetermined volume is 600 ⁇ L.
  • the aqueous solution contains each component in the tumor tissue lysate and the immunopotentiating adjuvant poly(I:C), BCG or CpG; each component in the tumor tissue lysate is water-soluble during preparation The active component or the original water-insoluble component dissolved in 8M urea.
  • the concentration of the water-soluble component from the tumor tissue contained in the aqueous phase solution or the concentration of the original water-insoluble component from the tumor tissue dissolved in 8M urea, that is, the first predetermined concentration requires that the protein polypeptide concentration content is greater than 1ng
  • the reason for choosing such a concentration is that the inventors have found through a large number of experiments that the higher the concentration of the aqueous solution of tumor tissue lysate generally used, the more various cancer antigens are encapsulated in the prepared nanoparticles. Only when the concentration is greater than 1 ng/mL, that is, the first predetermined concentration is greater than 1 ng/mL, can enough cancer antigens be loaded to activate the relevant immune response.
  • the concentration of the immune adjuvant in the initial aqueous phase was greater than 0.01 ng/mL.
  • the first predetermined volume is 600 ⁇ L.
  • the medical polymer material is dissolved in the organic solvent to obtain a second predetermined volume of the organic phase containing the medical polymer material of the second predetermined concentration.
  • the medical polymer material is target-modified PLGA, a mixture of PLGA and PLA, and dichloromethane is selected as the organic solvent, and the volume of the obtained organic phase, that is, the second predetermined volume, is 2 mL.
  • the range of the second predetermined concentration of the medical polymer material is 0.5 mg/mL-5000 mg/mL, preferably 100 mg/mL.
  • PLGA and target-modified PLGA are selected because the material is a biodegradable material and has been approved by the FDA as a drug dressing, and any other materials that can be used to prepare nano-sized materials can also be used in actual use.
  • a mixture of particle or micron-sized particle material and target modification material are selected because the material is a biodegradable material and has been approved by the FDA as a drug dressing, and any other materials that can be used to prepare nano-sized materials can also be used in actual use.
  • a mixture of particle or micron-sized particle material and target modification material are examples of target modification material.
  • the second predetermined volume of the organic phase is set according to its ratio to the first predetermined volume of the water phase.
  • the range of the ratio of the first predetermined volume of the water phase to the second predetermined volume of the organic phase is 1:1.1-1:5000, 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 required to adjust the size of the prepared nanoparticles.
  • step 2 the mixed solution obtained in step 1 is subjected to ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute.
  • the purpose of this step is to carry out nanometerization.
  • the length of the ultrasonic time or the stirring speed and time can control the size of the prepared nanoparticles. Too long or too short will bring about changes in the particle size. For this reason, an appropriate ultrasonic time needs to be selected.
  • the ultrasonic time is greater than 2 seconds
  • the stirring speed is greater than 50 rpm
  • the stirring time is greater than 1 minute.
  • step 3 the mixture obtained after the treatment in step 2 is added to a third predetermined volume of an aqueous solution containing a third predetermined concentration of emulsifier and subjected to ultrasonic treatment for more than 2 seconds or stirring for more than 1 minute.
  • step 2 the mixture obtained in step 2 is added to the aqueous emulsifier solution to continue ultrasonication or stirring for nanometerization.
  • the emulsifier aqueous solution is a polyvinyl alcohol (PVA) aqueous solution
  • the third predetermined volume is 5 mL
  • the third predetermined concentration is 20 mg/mL.
  • the third predetermined volume is adjusted according to its ratio to the second predetermined volume.
  • the range between the second predetermined volume and the third predetermined volume is set to be 1:1.1-1:1000, preferably 2:5.
  • the ratio of the second predetermined volume to the third predetermined volume can be adjusted.
  • the ultrasonic time or stirring time in this step the volume and concentration of the emulsifier aqueous solution are based on the values to obtain nanoparticles of suitable size.
  • step 4 the liquid obtained after the treatment in step 3 is added to a fourth predetermined volume of the emulsifier aqueous solution of a fourth predetermined concentration, and stirred until the predetermined stirring condition is satisfied.
  • the emulsifier aqueous solution is still PVA, and the fourth predetermined volume is greater than 50mL,
  • the fourth predetermined concentration is 5 mg/mL, and the selection of the fourth predetermined concentration is based on obtaining nanoparticles of suitable size.
  • the selection of the fourth predetermined volume is determined according to the ratio of the third predetermined volume to the fourth predetermined volume.
  • the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:1.5-1:2000, preferably 1:10.
  • the ratio of the third predetermined volume to the fourth predetermined volume may be adjusted in order to control the size of the nanoparticles.
  • the predetermined stirring condition in this step is until the volatilization of the organic solvent is completed, that is, the volatilization of the dichloromethane in step 1 is completed.
  • Step 5 After centrifuging the mixture that meets the predetermined stirring conditions in step 4 at a speed of more than 100 RPM for more than 1 minute, remove the supernatant, and resuspend the remaining precipitate in the fifth predetermined volume of the first.
  • the pellet obtained in step 5 does not need to be lyophilized when resuspended in a sixth predetermined volume of PBS (or physiological saline), and subsequent experiments related to the adsorption of cancer cell lysates on the surface of nanoparticles can be performed directly.
  • the precipitate obtained in step 5 needs to be freeze-dried when resuspended in an aqueous solution containing a freeze-drying protective agent, and then the subsequent experiments related to the adsorption of cancer cell lysates on the surface of nanoparticles are carried out after freeze-drying.
  • the freeze-drying protective agent is selected from Trehalose.
  • the fifth predetermined volume of the freeze-drying protective agent in this step is 20 mL, and the fifth predetermined concentration is 4% by mass.
  • the reason for this setting is to not affect the freeze-drying effect in the subsequent freeze-drying.
  • Step 6 after subjecting the suspension containing the freeze-drying protective agent obtained in step 5 to freeze-drying treatment, the freeze-dried substance is used for later use.
  • Step 7 resuspend the nanoparticle-containing suspension obtained in step 5 of the sixth predetermined volume in PBS (or physiological saline) or use the sixth predetermined volume of PBS (or physiological saline) to resuspend the suspension obtained in step 6
  • the freeze-dried lyophilized substance containing nanoparticle and freeze-drying protective agent after mixing with the water-soluble component of the seventh predetermined volume or the original water-insoluble component dissolved in 8M urea is allowed to stand for more than 0.1 minutes to obtain Nano-vaccine or micro-vaccine.
  • the volume ratio of the sixth predetermined volume to the seventh predetermined volume is 1:10000 to 10000:1, the preferred volume ratio is 1:100 to 100:1, and the optimal volume ratio is 1:30 to 30:1 .
  • the resuspended nanoparticle suspension has a volume of 10 mL and contains cancer cell lysate or contains water-soluble components in tumor tissue lysates or original water-insoluble components dissolved in 8M urea The volume and 1mL.
  • the present invention firstly encapsulates the water-soluble part or (and) the water-insoluble part soluble in pure water in the cell component by a solubilizer and then encapsulates it on nanoparticles or micrometers.
  • the immune enhancer is simultaneously loaded; then, the water-soluble part or (and) the water-insoluble part of the cellular component is adsorbed on the surface of the nanoparticle, and the immune enhancer is simultaneously adsorbed. In this way, the loading capacity of the water-soluble or water-insoluble components of the cells in the nanoparticles or microparticles can be maximized.
  • a solubilizing solution containing a solubilizer (such as 8M urea aqueous solution or 6M guanidine hydrochloride aqueous solution) can also be used to directly lyse cells or tissues and directly dissolve whole cell components, and then prepare nano-vaccine or micro-vaccine.
  • a solubilizer such as 8M urea aqueous solution or 6M guanidine hydrochloride aqueous solution
  • Example 1 Whole cell components of melanoma tumor tissue loaded inside mannose-modified nanoparticles for cancer treatment
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a nano-vaccine loaded with tumor tissue whole cell components, and use the vaccine to treat melanoma.
  • the specific dosage form, adjuvant, administration time, administration frequency, and administration schedule can be adjusted according to the situation.
  • B16-F10 mouse melanoma was used as a cancer model.
  • the lysed components of mouse melanoma tumor tissue were loaded inside and on the surface of nanoparticles to prepare nano-vaccine.
  • Mouse melanoma tumor mass was first obtained and lysed to prepare the water-soluble fraction of tumor mass tissue and the original water-insoluble fraction dissolved in 8M urea.
  • PLGA 50:50
  • PLA nanoparticle framework materials
  • CpG immune adjuvant
  • a nanovaccine loaded with water-soluble and water-insoluble components of tumor tissue lysate was prepared by solvent evaporation method. The nanovaccine was then used to treat tumors in melanoma tumor-bearing mice.
  • B16F10 melanoma cells were subcutaneously inoculated on the back of each C57BL/6 mouse, and the mice were sacrificed when the inoculated tumors of each mouse grew to a volume of 400 mm3 to 1500 mm3 and the tumor tissue was excised.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of pure water was added through a cell strainer, followed by repeated freezing and thawing and ultrasonication at least 5 times.
  • the cell lysate of the tumor tissue was centrifuged at a speed of more than 12000 RPM for 5 min, and the supernatant was obtained as the water-soluble components in the tumor tissue that were soluble in pure water;
  • the 8M urea aqueous solution dissolves the precipitation part to convert the original water-insoluble components that are insoluble in pure water into 8M urea aqueous solution soluble.
  • the water-soluble components of the tumor tissue lysate obtained above and the original water-insoluble components dissolved in 8M urea are the raw material sources for preparing the nano-vaccine for treating cancer cells.
  • the preparation of nano-vaccine and blank nanoparticles as control adopts the double emulsion method in the solvent evaporation method.
  • the molecular weight of the nano-particle preparation material PLGA (50:50) is 24KDa-38KDa.
  • the molecular weight of PLGA (50:50) is 24KDa-38KDa, and the molecular weight of PLA used is 10KDa.
  • the mass ratio of unmodified PLGA, mannose-modified PLGA and PLA was 8:1:2.
  • the immunoadjuvant employed was CpG and CpG was distributed inside the nanoparticles.
  • the preparation method is as described above.
  • the average particle size of the nanoparticles is about 280nm, and the average surface potential Zeta potential of the nanoparticles is about -8mV.
  • Each 1 mg of PLGA nanoparticles was loaded with 60 ⁇ g of protein or polypeptide components, and the amount of CpG immune adjuvant used inside and outside of each 1 mg of PLGA nanoparticles was 0.01 mg, half inside and outside.
  • the particle size of the blank nanoparticles was about 240 nm, and pure water containing the same amount of CpG or 8M urea was used to replace the corresponding water-soluble and water-insoluble components when the blank nanoparticles were prepared.
  • the experimental groups used in this study were set as follows: nanovaccine group; PBS control group, blank nanoparticle + cell lysate group.
  • the dosing schedule of the nanovaccine group was as follows: 150,000 B16-F10 cells were subcutaneously inoculated on the lower right back of each mouse on day 0, and on days 4, 7, 10, 15, and 20, respectively. 200 ⁇ L of 2 mg PLGA nanoparticles loaded with water-soluble components in cancer cell lysate both internally and on the surface and 200 ⁇ L of 2 mg PLGA nanoparticles loaded both internally and on the surface with the original water-insoluble components in 8M urea were injected subcutaneously.
  • the PBS blank control group protocol was as follows: on the 0th day, 150,000 B16-F10 cells were subcutaneously inoculated on the lower right side of the back of each mouse, and on the 4th day, the 7th day, the 10th day, the 15th day and the 20th day respectively. Inject 400 ⁇ L of PBS.
  • Blank nanoparticles + tissue lysate control group 150,000 B16-F10 cells were inoculated subcutaneously in the lower right back of each mouse on day 0, and on days 4, 7, 10, 15 and 20
  • the water-soluble components in the same amount of tumor tissue lysates, the original water-insoluble components in the same amount of lysates dissolved in 8M urea, and the 4 mg PLGA blank nanoparticles loaded with the same amount of CpG without any lysate components were subcutaneously injected on the same day. Note that the above three should be administered separately and injected at different sites to avoid adsorption of free lysate on the surface of blank nanoparticles.
  • mice tumor volume was recorded every three days starting from day 6.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • the tumor growth rate of the mice in the nanovaccine group was significantly slower (p ⁇ 0.05) and the survival time of the mice was significantly prolonged (p ⁇ 0.05). 0.05). This shows that the nanovaccine loaded with water-soluble and water-insoluble components of cancer cells according to the present invention has a therapeutic effect on melanoma and can partially cure melanoma.
  • Example 2 Whole cell components of melanoma tumor tissue loaded inside and on the surface of mannose-modified microparticles for cancer treatment
  • This example uses B16F-10 mouse melanoma as a cancer model to illustrate how to use the micro-vaccine to treat melanoma.
  • the specific dosage form can be adjusted according to the situation.
  • mice all the lysed components of mouse melanoma tumor tissue were simultaneously loaded inside and on the surface of the microparticles to prepare a micron vaccine.
  • Mouse melanoma tumor mass was first obtained and lysed to prepare the water-soluble fraction of tumor mass tissue and the original water-insoluble fraction dissolved in 8M urea.
  • PLGA 50:50
  • poly(I:C) as immune adjuvant
  • a solvent-evaporation method was used to prepare a composite containing both water-soluble and water-insoluble components of tumor tissue lysate.
  • Micron vaccine The micro-vaccine was then used to treat tumors in melanoma tumor-bearing mice.
  • the treatment method is the same as that of Example 1.
  • the preparation of micron vaccine and blank micron particles as control adopts the double emulsion method in the solvent evaporation method.
  • the molecular weight of the micron particle preparation material PLGA is 24KDa-38KDa
  • the mannose-modified PLGA (50:50 ) molecular weight is 24KDa-38KDa.
  • the mass ratio of unmodified PLGA and mannose-modified PLGA (50:50) was 10:1.
  • the immunoadjuvant used is poly(I:C) and the poly(I:C) is distributed inside the microparticles.
  • the preparation method is as described above, and the water-soluble component and the water-insoluble component are simultaneously supported in the same microparticle.
  • whole cell components were loaded both inside and on the surface of the microparticles.
  • the microparticles that have been loaded with the whole cell component and the cell component are mixed at a volume ratio of 10:1 and then allowed to stand for 15 minutes.
  • the average particle size of microparticles is about 2.0 ⁇ m, and the average surface potential Zeta potential of nanoparticles is about -17mV.
  • Each 1 mg of PLGA microparticles was loaded with 70 ⁇ g of protein or polypeptide components, and the amount of poly(I:C) immunoadjuvant used inside and outside of each 1 mg of PLGA microparticles was 0.01 mg, with half and half.
  • the particle size of the blank microparticles is about 1.8 ⁇ m, and pure water containing the same amount of poly(I:C) or 8M urea was used to replace the corresponding water-soluble and water-insoluble components when the blank microparticles were prepared.
  • mice The experimental groups in this study were set as follows: micron vaccine group; PBS control group, blank microparticles + cell lysate group. Select 6-8 week old female C57BL/6 as model mice to prepare melanoma tumor-bearing mice.
  • the dosage regimen of the micron vaccine group was as follows: 150,000 B16-F10 cells were inoculated subcutaneously in the lower right back of each mouse on day 0, and on day 4, day 7, day 10, day 15, and day 20, respectively A subcutaneous injection of 400 ⁇ L of 4 mg PLGA micro-vaccine loaded with both water-soluble and water-insoluble components in cancer cell lysates both internally and on the surface.
  • the PBS blank control group protocol was as follows: on the 0th day, 150,000 B16-F10 cells were subcutaneously inoculated on the lower right side of the back of each mouse, and on the 4th day, the 7th day, the 10th day, the 15th day and the 20th day respectively. Inject 400 ⁇ L of PBS.
  • Blank microparticles + cell lysate control group 150,000 B16-F10 cells were inoculated subcutaneously in the lower right back of each mouse on day 0, on days 4, 7, 10, 15 and 20 The same amount of water-soluble component + water-insoluble component in cancer cell lysate and 4 mg of PLGA blank nanoparticles loaded with equal amount of poly(I:C) without any cell lysate component were injected subcutaneously on each day. Note that the above two should be administered separately and injected at different sites to prevent free cell lysates from adsorbing on the surface of blank microparticles.
  • the size of mouse tumor volume was recorded every three days starting from day 6.
  • the tumor growth rate of the mice in the microvaccine group was significantly slower (p ⁇ 0.05) and the survival time of the mice was significantly prolonged (p ⁇ 0.05). 0.05). This shows that the micro-vaccine loaded with water-soluble and water-insoluble components of cancer cells according to the present invention has a therapeutic effect on melanoma and can partially cure melanoma.
  • Example 3 Whole cell components of melanoma tumor tissue loaded inside and on the surface of mannose-modified nanoparticles for cancer prevention
  • This example uses mouse melanoma as a cancer model to illustrate how to prepare a nano-vaccine loaded with tumor tissue whole cell components, and use the vaccine to treat melanoma.
  • the specific dosage form, adjuvant, administration time, administration frequency, and administration schedule can be adjusted according to the situation.
  • B16-F10 mouse melanoma was used as a cancer model.
  • the lysed components of mouse melanoma tumor tissue were loaded inside and on the surface of nanoparticles to prepare nano-vaccine.
  • Mouse melanoma tumor mass was first obtained and lysed to prepare the water-soluble fraction of tumor mass tissue and the original water-insoluble fraction dissolved in 8M urea.
  • the nanoparticle loaded with the water-soluble and water-insoluble components of the tumor tissue lysate was prepared by solvent evaporation method. vaccine.
  • the nanovaccine was then used to treat tumors in melanoma tumor-bearing mice.
  • the preparation of the nano-vaccine and the blank nano-particles used as a control adopts the double emulsion method in the solvent evaporation method. 38KDa.
  • the mass ratio of unmodified PLGA and mannose-modified PLGA was 10:1.
  • the immunoadjuvant used was poly(I:C) and poly(I:C) was distributed inside the nanoparticles.
  • the preparation method is as described above, the difference is that in this example, whole cell components are loaded on the inside and on the surface of the nanoparticles at the same time.
  • the nanoparticle that has been loaded with the whole cell component and the cell component are mixed at a volume ratio of 10:1, and then left to stand for 15 minutes.
  • the average particle size of the nanoparticles is about 300nm, and the average surface potential Zeta potential of the nanoparticles is about -8mV.
  • Each 1 mg of PLGA nanoparticles was loaded with 60 ⁇ g of protein or polypeptide components, and the amount of poly(I:C) immunoadjuvant used inside and outside of each 1 mg of PLGA nanoparticles was 0.01 mg, half inside and outside.
  • the particle size of the blank nanoparticles was about 260 nm, and pure water containing the same amount of poly(I:C) or 8M urea was used to replace the corresponding water-soluble and water-insoluble components.
  • the PBS blank control regimen was as follows: 400 ⁇ L of PBS was subcutaneously injected on the 42nd day before, the 35th day before, the 28th day before, the 21st day before and the 14th day before inoculation of B16F10 cancer cells, respectively. On day 0, 150,000 B16-F10 cells were inoculated subcutaneously in the lower right back of each mouse.
  • the size of mouse tumor volume was recorded every three days starting from day 6.
  • the tumor growth rate of the nanovaccine prevention group was significantly slower (p ⁇ 0.05) and the survival time of the mice was significantly prolonged (p ⁇ 0.05). This shows that the nanovaccine with dendritic cell targeting ability loaded with cancer cell water-soluble components and water-insoluble components has a preventive effect on melanoma.
  • Example 4 Whole cell components of lung cancer tumor tissue are loaded into mannose-modified nanoparticles for the prevention of lung cancer
  • This example uses mouse lung cancer as a cancer model to illustrate how to prepare a nano-vaccine loaded with tumor tissue whole cell components, and use the vaccine to prevent lung cancer.
  • the specific dosage form, adjuvant, administration time, administration frequency, and administration schedule can be adjusted according to the situation.
  • LLC mouse lung cancer was used as a cancer model.
  • the lysed components of mouse lung cancer tumor tissue were loaded on the inside and on the surface of nanoparticles to prepare a nanovaccine.
  • mouse lung cancer tumor mass was obtained and lysed to prepare the water-soluble component of tumor mass tissue and the original water-insoluble component dissolved in 8M urea.
  • PLGA 50:50
  • nanoparticle framework material and (poly(I:C)) as immune adjuvant
  • the solvent evaporation method was used to prepare the water-soluble and water-insoluble components loaded with tumor tissue lysate.
  • Nanovaccine The nanovaccine was then used to treat tumors in lung cancer-bearing mice.
  • 1,000,000 LLC melanoma cells were subcutaneously inoculated into the flank of each C57BL/6 mouse, and the mice were sacrificed when the inoculated tumors of each mouse grew to a volume of 400 mm3 to 1500 mm3 and the tumor tissue was excised.
  • the tumor tissue was cut into pieces and ground, and an appropriate amount of pure water was added through a cell strainer, followed by repeated freezing and thawing and ultrasonication at least 5 times.
  • the cell lysate of the tumor tissue was centrifuged at a speed of more than 12000 RPM for 5 min, and the supernatant was obtained as the water-soluble components in the tumor tissue that were soluble in pure water;
  • the 8M urea aqueous solution dissolves the precipitation part to convert the original water-insoluble components that are insoluble in pure water into 8M urea aqueous solution soluble.
  • the water-soluble components of the tumor tissue lysate obtained above and the original water-insoluble components dissolved in 8M urea are the raw material sources for preparing the nano-vaccine for treating cancer cells.
  • the preparation of the nano-vaccine and the blank nano-particles used as a control adopts the double emulsion method in the solvent evaporation method. 38KDa.
  • the mass ratio of unmodified PLGA and mannose-modified PLGA was 10:1.
  • the immunoadjuvant used is poly(I:C) and the poly(I:C) is distributed inside the nanoparticles.
  • the preparation method is as described above.
  • the average particle size of the nanoparticles is about 300nm, and the average surface potential Zeta potential of the nanoparticles is about -8mV.
  • Each 1 mg of PLGA nanoparticles was loaded with 60 ⁇ g of protein or polypeptide components, and the amount of poly(I:C) immunoadjuvant used inside and outside of each 1 mg of PLGA nanoparticles was 0.01 mg, half inside and outside.
  • the particle size of the blank nanoparticles was about 260 nm, and pure water containing the same amount of poly(I:C) or 8M urea was used to replace the corresponding water-soluble and water-insoluble components.
  • the 6-8 week old female C57BL/6 was selected as model mice to prepare lung cancer tumor-bearing mice.
  • the dosing schedule for the nanovaccine group was as follows: 200 ⁇ L of both internal and surface-loaded cancer cell lysis was injected subcutaneously on days -49, -42, -35, -28, and -14, respectively, before tumor cell inoculation.
  • the 2 mg PLGA nanoparticles of the water-soluble component in the medium and 200 ⁇ L of the 2 mg PLGA nanoparticles dissolved in the original water-insoluble component of 8M urea were loaded inside and on the surface.
  • the PBS control group was injected with 400-200 ⁇ L PBS on the corresponding days. On day 0, 1,000,000 LLC lung cancer cells were inoculated subcutaneously in the lower right back of each mouse.
  • the size of mouse tumor volume was recorded every three days starting from day 6.
  • the tumor growth rate of the mice in the nanovaccine group was significantly slower (p ⁇ 0.05) and the survival time of the mice was significantly prolonged (p ⁇ 0.05). This shows that the nanovaccine loaded with the water-soluble and water-insoluble components of cancer cells according to the present invention has a preventive effect on lung cancer.
  • Example 5 Whole cell components of breast cancer cells are loaded into mannose-modified nanoparticles for cancer treatment
  • This example uses the treatment of breast cancer in mice to illustrate how to prepare a nano-vaccine loaded with whole cell components and use the vaccine to treat breast cancer.
  • 4T1 mouse triple-negative breast cancer cells were used as cancer cell models.
  • 4T1 cells were first lysed to prepare water-soluble and water-insoluble components of 4T1 cells. Then, using PLGA and mannose-modified PLGA (50:50) as nanoparticle framework materials, a nanovaccine loaded with water-soluble and water-insoluble components of 4T1 cells was prepared by solvent evaporation method.
  • Bacillus Calmette-Guérin (BCG) adjuvant was used as an immune adjuvant, and the nano-vaccine was used to treat tumors in 4T1 breast cancer-bearing mice.
  • BCG Bacillus Calmette-Guérin
  • Collect a certain amount of 4T1 cells remove the medium and freeze at -20°C to -273°C, add a certain amount of ultrapure water, freeze and thaw for more than 3 times, and sonicate to destroy the lysed cells.
  • the lysate is centrifuged at a speed of more than 100g for more than 1 minute, and the supernatant is taken as the water-soluble component soluble in pure water in 4T1; 8M urea is added to the obtained precipitation part to dissolve the precipitation part.
  • the water-insoluble components in 4T1 that were insoluble in pure water were converted to be soluble in 8M urea aqueous solution.
  • the obtained water-soluble components derived from cancer cell lysates and the original water-insoluble components dissolved in 8M urea are the raw material sources for preparing nano-vaccine for cancer treatment.
  • the preparation method and materials used for the nano-vaccine in this example are basically the same as those in Example 4, except that LLC cells are replaced with 4T1 cells, and the adjuvant is replaced with BCG adjuvant.
  • mice 6-8 week old female BALB/c mice were selected to prepare 4T1 tumor-bearing mice.
  • the dosing schedule of the nanovaccine group was as follows: 400,000 4T1 cells were inoculated subcutaneously on the lower right side of the back of each mouse on day 0, and were injected subcutaneously on days 4, 7, 10, 15, and 20, respectively. 200 ⁇ L of 2mg PLGA nanoparticles + 0.5mg BCG adjuvant loaded with water-soluble components in cancer cell lysate both internally and on the surface and 200 ⁇ L of 2mg PLGA nanoparticles + 0.5mg BCG loaded with the original water-insoluble components in 8M urea both internally and on the surface adjuvant.
  • the PBS blank control group protocol was as follows: on the 0th day, 400,000 4T1 cells were subcutaneously inoculated on the lower right side of the back of each mouse, and 400 ⁇ L were subcutaneously injected on the 4th, 7th, 10th, 15th and 20th days respectively. PBS.
  • Blank nanoparticles + cell lysate control group 400,000 4T1 cells were inoculated subcutaneously in the lower right back of each mouse on day 0, and on day 4, day 7, day 10, day 15, and day 20, respectively Subcutaneous injection of water-soluble components in cancer cell lysate, original water-insoluble components dissolved in 8M urea, equal amount of BCG and 4mg PLGA blank nanoparticles in cancer cell lysate. Note that the above three should be administered separately and injected into different subcutaneous sites to prevent free cell lysates from adsorbing on the surface of blank nanoparticles.
  • the size of mouse tumor volume was recorded every three days from day 6.
  • v was the tumor volume
  • a was the tumor length
  • b was the tumor width.
  • the tumor growth rate in the nanovaccine administration group was significantly slower (p ⁇ 0.05) and the survival time of mice was significantly prolonged (p ⁇ 0.05). 0.05). It can be seen that the nanovaccine loaded with the water-soluble and water-insoluble components of cancer cells according to the present invention has a therapeutic effect on breast cancer.
  • Example 6 Whole cell components of breast cancer cells are loaded into mannose-modified nanoparticles for cancer treatment
  • This example uses the treatment of breast cancer in mice to illustrate how to prepare a nano-vaccine loaded with whole cell components and use the vaccine to treat breast cancer.
  • 4T1 mouse triple-negative breast cancer cells were used as cancer cell models.
  • 4T1 cells were first lysed to prepare water-soluble and water-insoluble components of 4T1 cells. Then, using PLGA and mannose-modified PLGA (50:50) as nanoparticle framework materials, a nanovaccine loaded with water-soluble and water-insoluble components of 4T1 cells was prepared by solvent evaporation method.
  • Bacillus Calmette-Guérin (BCG) adjuvant was used as an immune adjuvant, and the nano-vaccine was used to treat tumors in 4T1 breast cancer-bearing mice.
  • BCG Bacillus Calmette-Guérin
  • the preparation method is the same as in Example 5.
  • the preparation method and materials used for the nano-vaccine in this example are basically the same as those in Example 5, and the whole cell components are only loaded inside the nanoparticles.
  • the vaccine group containing PEG protective film 3.5% PEG-PLGA (the molecular weight of the PEG part was 5000 and the molecular weight of the PLAG part was 25000) was added to the PLGA organic phase during preparation.
  • mice 6-8 week old female BALB/c mice were selected to prepare 4T1 tumor-bearing mice.
  • the dosage regimen of the nanovaccine group with PEG protective film and the nanovaccine group without PEG protective film was as follows: on the 0th day, 400,000 4T1 cells were subcutaneously inoculated on the lower right side of the back of each mouse, and on the 4th and 7th days On the 10th, 15th and 20th days, 200 ⁇ L of 2mg PLGA nanoparticles loaded with water-soluble components + 0.5mg BCG adjuvant and 200 ⁇ L of 2mg PLGA nanoparticles + 0.5mg BCG adjuvant loaded with original water-insoluble components were injected subcutaneously, respectively. .
  • the PBS blank control group protocol was as follows: on the 0th day, 400,000 4T1 cells were subcutaneously inoculated on the lower right side of the back of each mouse, and 400 ⁇ L were subcutaneously injected on the 4th, 7th, 10th, 15th and 20th days respectively. PBS.
  • the size of mouse tumor volume was recorded every three days from day 6.
  • the tumor growth rate of the nanovaccine group containing the PEG protective film was significantly slower (p ⁇ 0.05) and the survival time of the mice was significantly prolonged. (p ⁇ 0.05). It can be seen that the PEG protective film can protect the nanoparticles from being phagocytosed by cells other than dendritic cells and prolong the circulation time of the nanoparticles in the body, thereby improving the targeting and efficacy of the nanovaccine.
  • Example 7 Solubility of different solubilizers for water-insoluble components
  • This example uses the treatment of breast cancer in mice to illustrate how to prepare a nano-vaccine loaded with whole cell components and use the vaccine to treat breast cancer.
  • 4T1 mouse triple-negative breast cancer cells were used as cancer cell models.
  • 4T1 cells were first lysed to prepare water-soluble and water-insoluble components of 4T1 cells.
  • the water-insoluble components were dissolved with 6M guanidine hydrochloride and PEG (5000KD), respectively.
  • nanovaccine was prepared by using PLG(50:50)A and mannose-modified PLGA(50:50) as nanoparticle framework materials.
  • Bacillus Calmette-Guérin (BCG) adjuvant was used as an immune adjuvant, and the nano-vaccine was used to treat tumors in 4T1 breast cancer-bearing mice.
  • BCG Bacillus Calmette-Guérin
  • the preparation method is the same as in Example 5. The difference is that in this example, 6M guanidine hydrochloride and PEG (5000KD) are respectively used to dissolve the water-insoluble components in the lysed cancer cells.
  • 6M guanidine hydrochloride and PEG (5000KD) are respectively used to dissolve the water-insoluble components in the lysed cancer cells.
  • the ability of 6M guanidine hydrochloride to solubilize water-insoluble components is significantly stronger than that of PEG (5000KD): the concentration of 6M guanidine hydrochloride solubilization of water-insoluble components can reach 80 mg/mL; PEG (5000KD) can only solubilize the water-insoluble group at the highest Fraction to 1 mg/mL.
  • the preparation method and materials used for the nano-vaccine in this example are basically the same as those in Example 5, and the whole cell components are only loaded inside the nanoparticles. Moreover, when preparing the nanovaccine loaded with water-insoluble components, the concentration of the original water-insoluble component solubilized with 6M guanidine hydrochloride was 60 mg/mL, while the concentration of the original water-insoluble component solubilized with PEG was 60 mg/mL. is 1 mg/mL.
  • the prepared nanovaccine was solubilized with 6M guanidine hydrochloride, and the nanoparticles loaded with water-insoluble components were loaded with 70 ⁇ g of protein/polypeptide components per 1 mg of PLGA nanoparticles; the nanoparticles loaded with non-water-soluble components after solubilization with PEG 1mg PLGA nanoparticles are loaded with 2 ⁇ g protein/peptide fraction.
  • mice 6-8 week old female BALB/c mice were selected to prepare 4T1 tumor-bearing mice.
  • the dosing schedule for the nanovaccine group was as follows: 200 ⁇ L of both internal and surface-loaded cancer cell lysis was injected subcutaneously on days -49, -42, -35, -28, and -14, respectively, before tumor cell inoculation. 2 mg of PLGA nanoparticles (0.5 mg BCG adjuvant) of the water-soluble component in the medium and 200 ⁇ L of 2 mg PLGA nanoparticles (0.5 mg BCG adjuvant) of the original water-insoluble component dissolved in 8 M urea were loaded both internally and on the surface. The PBS control group was injected with 400 ⁇ L of PBS on the corresponding days. On day 0, 400,000 4T1 cells were inoculated subcutaneously in the lower right back of each mouse. In the experiment, the size of mouse tumor volume was recorded every three days from day 6. The tumor volume was calculated in the same manner as in Example 1.
  • the tumor growth rate of the nanovaccine group prepared by using 6M guanidine hydrochloride solubilization of the water-insoluble components was significantly slower (p ⁇ 0.05) and the survival time of mice was significantly prolonged (p ⁇ 0.05). It can be seen that the solubilization solution and method are important for improving the effect of nanovaccine.

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Abstract

一种负载全细胞组分的靶向输送系统，其涉及免疫治疗技术领域。该靶向输送系统为表面有靶头的纳米级尺寸或微米级尺寸的粒子，且所述粒子负载有癌细胞或癌组织的全细胞组分；所述全细胞组分为细胞或组织中全细胞的水溶性成分和非水溶性成分，所述非水溶性成分通过增溶剂溶解；所述靶头与特定细胞或组织表面的分子结合进而帮助所述粒子进入细胞或组织。该靶向输送系统采用特定增溶剂增溶非水溶性部分，使其可以在水溶液中溶解，从而可以综合癌症细胞或组织中水溶性成分和非水溶性组分的的全细胞抗原来制备癌症疫苗，同时附加可靶向于抗原提呈细胞的靶头，以主动靶向的方式提高抗原提呈细胞吞噬效率，进而提高预防或治疗癌症的效果。

Description

一种负载全细胞组分的靶向输送系统及其应用
本申请要求于2020年10月23日提交中国专利局、申请号为202011146241.9、发明名称为“一种负载全细胞组分的靶向输送系统及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及免疫治疗技术领域，具体的说是涉及一种负载全细胞组分的靶向输送系统及其应用。
背景技术
免疫是人体的一种生理功能，人体依靠这种功能识别“自己”和“非己”成分，从而破坏和排斥进入人体的抗原物质(如病毒和细菌等)，或人体本身所产生的损伤细胞和肿瘤细胞等，以维持人体的健康。近些年来免疫技术的发展极其迅速，尤其是癌症的免疫治疗领域。随着对癌症认识的不断提高，人们发现人体的免疫系统和各类免疫细胞在抑制癌症发生、发展的过程中扮演着关键角色。
近些年以后癌症免疫治疗发展迅速，癌症疫苗是癌症免疫治疗和预防的重要方法之一。而有效的癌症疫苗需要负载癌症特异性的抗原，并能把这些负载的抗原高效的输送到抗原提呈细胞以激活机体免疫系统对癌症细胞的识别和攻击。目前，科学家从癌症病人的肿瘤细胞分析鉴别癌症特异性的或癌症相关的抗原多肽，然后体外人工合成以制备癌症疫苗用于癌症的治疗。该方法在癌症病人的临床试验中表现出了一定的疗效。但是该类方法所能找到的癌症抗原数量有限，而且费时费力，花费巨大，而且，所得多肽疫苗无靶向抗原提呈细胞的能力。
为了发挥疫苗的功能，需要先把抗原递送提呈给抗原提呈细胞，然后抗原提呈细胞处理癌症抗原后将其提呈到抗原提呈细胞表面并和T细胞相互作用进而激活T细胞对癌细胞的免疫识别能力。一旦T细胞能识别特意的癌症抗原了，就会去识别和杀死含有这些癌症抗原的癌细胞。
而且，目前技术所使用的只是部分有限数量的抗原，无法覆盖病人体内绝大部分的癌症抗原。由于癌细胞抗原的多样性和癌症突变的多样性，即使是同一种癌症中每个患者体内的癌细胞抗原也各不相同。因而就很难通过有限数量的癌细胞抗原来制备多数人可以使用的癌症疫苗。而癌细胞和来自病人的肿瘤组织含有每个人独的癌症抗原和突变，因而属于个性化 抗原，所制备的癌症疫苗也属于个性化疫苗。而肿瘤组织中含有各类癌症抗原，如果能将肿瘤组织或者癌细胞全细胞组分作为疫苗地送给抗原提呈细胞，那么疫苗将对癌症具有良好的预防和治疗效果。但是，由于目前技术手段的限制，研究者将研究重点都放在了水溶性组分，对于非水溶性组分无法有效地的应用，这是当下全细胞组分的应用难点。
此外，为了发挥疫苗的功能，需要先把抗原递送提呈给抗原提呈细胞，然后抗原提呈细胞处理癌症抗原后将其提呈到抗原提呈细胞表面并和T细胞相互作用进而激活T细胞对癌细胞的免疫识别能力。一旦T细胞能识别特意的癌症抗原了，就会去识别和杀死含有这些癌症抗原的癌细胞。目前通过粒子大小靶向抗原提呈细胞称之为被动靶向。然而，单纯依靠被动靶向无法高效的提呈到抗原提呈细胞表面并和T细胞相互作用，以300纳米的纳米粒子为例，其可以被树突状细胞，B细胞等抗原提呈细胞吞噬，但是同时也可能被成纤维细胞等其他细胞吞噬。
发明内容
有鉴于此，本发明的目的在于提供一种负载全细胞组分的靶向输送系统，使得所述靶向输送系统能够以主动靶向的方式将包含癌症细胞或组织的水溶性和非水溶性组分的全细胞组分提呈到抗原提呈细胞，且能够提高预防和治疗肿瘤的效果；
本发明的另外一个目的在于提供上述靶向输送系统在制备预防和/或治疗癌症的疫苗中的应用。
为了实现上述目的，本发明提供如下技术方案：
一种负载全细胞组分的靶向输送系统，其为表面有靶头的纳米级尺寸或微米级尺寸的粒子，且所述粒子负载有癌细胞或癌组织的全细胞组分，所述非水溶性成分通过增溶剂溶解；所述全细胞组分为细胞或组织中全细胞的水溶性成分和非水溶性成分；所述靶头与特定细胞或组织表面的分子结合进而帮助所述粒子进入细胞或组织。
本发明在将癌细胞或组织裂解后首先获取在纯水或不含增溶剂的水溶液中可溶的水溶性组分，而后采用含有特定增溶剂的增溶水溶液将水不溶性的组分溶解于增溶液中，这样就可将所有的细胞组分都转变为可在水溶液中溶解的组分并进而将其负载于纳米粒子或微米粒子内外以制备靶向输送系统，从而保证了绝大部分抗原物质被负载于所制备的靶向输送系 统中。在实际应用中也可将细胞或组织裂解后直接采用含有增溶剂的增溶水溶液溶解全细胞组分而不分别收集水溶性组分和非水溶性组分，并采用增溶水溶液溶解后的全细胞组分制备靶向输送系统。
癌细胞或肿瘤组织中的细胞组分中水溶性部分和非水溶性部分囊括了整个细胞的成分和组分。其中与正常细胞成分相同未突变的蛋白质、多肽和基因因为自身免疫系统发育过程中所产生的免疫耐受不会引起免疫反应；而因为癌症产生的基因、蛋白质和多肽的突变因为没有自身免疫系统发育过程中所产生的免疫耐受因而具有免疫原性且可激活机体针对癌细胞的免疫反应。利用全细胞组分中这些因为疾病突变而产生的具有癌细胞特异性免疫原性的物质即可用于癌症的预防和治疗。
在本发明所述的靶向输送系统中，所述全细胞组分按照在纯水或不含增溶剂的水溶液中的溶解性可分为两部分：水溶性成分和非水溶性成分。所述水溶性成分为可溶于纯水或不含增溶剂的水溶液的原水溶性部分，所述非水溶性成分为在纯水中不溶的原非水溶性部分，采用适当增溶方法由在纯水或不含增溶剂的水溶液中不溶变为在含增溶剂的水溶液中可溶的部分。所述全细胞组分中的水溶性部分和非水溶性部分都可以被含增溶剂的增溶水溶液溶解。
在本发明所述的靶向输送系统中，所述增溶剂包括但不限于尿素、盐酸胍、脱氧胆酸钠、SDS、甘油、pH大于7的碱性溶液、pH小于7的酸性溶液、各类蛋白质降解酶、白蛋白、卵磷脂、高浓度无机盐、Triton、吐温、DMSO、乙腈、乙醇、甲醇、DMF、丙醇、异丙醇、醋酸、胆固醇、氨基酸、糖苷、胆碱、Brij TM-35、Octaethylene glycol monododecyl ether、CHAPS、Digitonin、lauryldimethylamine oxide、
Figure PCTCN2020126655-appb-000001
CA-630、DMSO、乙腈、乙醇、甲醇、DMF、异丙醇、丙醇、二氯甲烷和乙酸乙酯，可以选择其中的一种和两种以上。
所述非水溶性成分也可采用其他可使蛋白质和多肽片段增溶的方法由在纯水中不溶变为可溶。但在本发明具体实施过程中，尿素、盐酸胍、SDS、脱氧胆酸纳或甘油的效果要明显好于PEG等其他增溶剂。作为优选，本发明直接采用含有尿素或盐酸胍的增溶液，直接裂解细胞或组织并直接溶解全细胞组分。在本发明所列实施例中采用8M尿素和6M盐酸胍水溶液溶解肿瘤组织或癌细胞中的非水溶性组分，在实际应用中也可以使用SDS和甘油等其他任何可以增溶全细胞组分中非水溶性组分的增溶液。
微米粒子或者纳米粒子在靶向抗原提呈细胞方面具有最适的粒子大小，但是不同材料制备的纳米粒子或微米粒子最适大小可能有所不同。比 如，目前认为阳离子脂质体最适大小在50-150纳米左右，在PLGA制备的纳米粒子可能在200-500纳米左右。而微米粒子理论上认为1.5-5微米最适。这个最适大小因粒子材料不同而异。通过粒子大小靶向抗原提呈细胞称之为被动靶向。
本发明除了采用被动靶向还使用了主动靶向的策略，即在纳米粒子或者微米粒子外部再连接有可以靶向特定细胞的靶头分子。这样就可以直接将纳米粒子或微米粒子靶向到特定细胞或组织表面，通过配体受体结合方式帮助所述粒子进入细胞或组织。这些细胞包括但不限于白细胞中的树突状细胞，巨噬细胞，B细胞，T细胞，NK细胞，NKT细胞，中性粒细胞，嗜酸性粒细胞，嗜碱性粒细胞，所述靶头可以靶向的组织包括但不限于淋巴结，胸腺，脾脏，骨髓。
举例来说，单纯依靠被动靶向，300纳米的纳米粒子可以被树突状细胞，B细胞等抗原提呈细胞吞噬，但是同时也可能被成纤维细胞等其他细胞吞噬。而如果使用主动靶向策略的话，就可以定向只被最关键的抗原提呈细胞树突状细胞吞噬。在本发明所列实施例中甘露糖(即靶头)修饰的纳米粒子或微米粒子靶向树突状细胞，在实际应用中也可以使用任何可以靶向树突状细胞的靶头或者靶向其他类型的特定细胞或组织。
纳米疫苗或微米疫苗的粒径大小为纳米级或微米级，这样能保证疫苗被抗原提呈细胞吞噬，而为了提高吞噬效率，粒径大小要在适宜的范围内。本发明所述的靶向输送系统中，所述纳米级尺寸的粒子的粒径为1nm-1000nm。在一些实施方案中，所述纳米尺寸粒子的粒径为30nm-800nm。进一步的，在一些实施方案中，所述纳米尺寸粒子的粒径为50nm-600nm。
本发明所述的靶向输送系统中，所述微米级尺寸的粒子的粒径为1μm-1000μm。在一些实施方案中，所述微米尺寸粒子的粒径为1μm-100μm。在一些实施方案中，所述微米尺寸粒子的粒径为1μm-10μm。进一步的，在一些实施方案中，所述微米尺寸粒子的粒径为1μm-5μm。
在本发明所述的输送系统中，所述的纳米尺寸粒子或微米尺寸粒子表面可为电中性，带负电或者带正电。
为了增强靶向输送系统的免疫原性和效果也可在其中加入一定的具有免疫调节功能的免疫佐剂，如模式识别受体激动剂、卡介苗细胞壁骨架、卡介苗甲醇提取残余物、卡介苗胞壁酰二肽、草分枝杆菌、多抗甲素、矿物油、病毒样颗粒、免疫增强的再造流感病毒小体、霍乱肠毒素、皂苷及 其衍生物、Resiquimod、胸腺素、新生牛肝活性肽、米喹莫特、多糖、姜黄素、免疫佐剂poly ICLC、短小棒状杆菌苗、溶血性链球菌制剂、辅酶Q10、左旋咪唑、聚胞苷酸、白细胞介素、干扰素、聚肌苷酸、聚腺苷酸、明矾、磷酸铝、羊毛脂、植物油、内毒素、脂质体佐剂、GM-CSF、MF59、双链RNA、双链DNA、氢氧化铝、CAF01、人参、黄芪等中药有效成分，可以选择其中的一种或两种以上。本发明所述的免疫佐剂的添加方式包括装载于纳米粒子或微米粒子内，或者负载于纳米粒子或微米粒子表面，或者同时装载于纳米粒子或微米粒子内及负载于纳米粒子或微米粒子表面。作为优选，免疫佐剂添加在全细胞组分中。
在本发明具体实施方式中，采用Polyinosinic-polycytidylic acid(poly(I:C))、卡介苗(BCG)或CpG为免疫佐剂，添加到含有癌细胞裂解物或含有肿瘤组织裂解物中水溶性组分和溶于增溶剂的原非水溶性组分，poly(I:C)、BCG或CpG的浓度优选为大于1ng/mL。
为了提高提高靶向输送系统的靶向性和效果，本发明还包括在粒子外部添加PEG保护膜。
本发明所述的靶向输送系统，所述纳米级尺寸或微米级尺寸的粒子的制备材料为有机合成高分子材料、天然高分子材料、无机材料、细菌或者病毒中的一种或多种。
其中所述有机合成高分子材料为生物相容或可降解的高分子材料，包括但不限于PLGA、PLA、PGA、Poloxamer、PEG、PCL、PEI、PVA、PVP、PTMC、聚酸酐、PDON、PPDO、PMMA、聚氨基酸、合成多肽。
所述的天然高分子材料为生物相容或可降解的高分子材料，包括但不限于磷脂、胆固醇、淀粉、糖类、多肽、海藻酸钠、白蛋白、胶原蛋白、明胶、细胞膜成分。
所述的无机材料为无明显生物毒性的材料，包括但不限于三氧化二铁、四氧化三铁、碳酸钙、磷酸钙。
本发明所述的靶向输送系统的形状为常见的任意形状，包括但不限于球形、椭球形、桶形、多角形、棒状、片状、线形、蠕虫形、方形、三角形、蝶形或圆盘形。
在本发明所述的靶向输送系统中，所述负载方式为全细胞的水溶性成分和非水溶性成分分别或同时被包载于粒子内部，和/或分别或同时负载于粒子表面。包括但不仅限于水溶性成分同时装载于粒子中和负载于粒子表面，非水溶性成分同时装载于粒子中和负载于粒子表面，水溶性成分装载于粒子中而非水溶性成分负载于粒子表面，非水溶性成分装载于粒子中 而水溶性成分负载于粒子表面，水溶性成分和非水溶性成分装载于粒子中而只有非水溶性成分负载于粒子表面，水溶性成分和非水溶性成分装载于粒子中而只有水溶性成分负载于粒子表面，水溶性成分装载于粒子中而水溶性成分和非水溶性成分同时负载于粒子表面，非水溶性成分装载于粒子中而水溶性成分和非水溶性成分同时负载于粒子表面，水溶性成分和非水溶性成分同时装载于粒子中而且水溶性成分和非水溶性成分同时负载于粒子表面。
本发明所述全细胞组分的输送系统其结构示意图如图2-图17所示。在实际使用过程中可以为只使用其中的某一种特定结构的纳米粒子或微米粒子，或者是同时使用两种或两种以上的不同结构的纳米粒子或微米粒子。
本发明所述的靶向输送系统的可以按照纳米尺寸粒子和微米尺寸粒子已发现的任何制备方法制备得到，包括但不仅限于常见的溶剂挥发法、透析法、挤出法、热熔法。在一些实施方案中，所述的输送系统采用溶剂挥发法中的复乳法制备得到。
本发明所述全细胞组分的靶向输送系统可将装载的全细胞组分递送给相关免疫细胞，通过所装载成分的免疫原性而激活和增强自身免疫系统对癌细胞的杀伤作用。因此本发明还提供了所述全细胞组分的靶向输送系统在制备预防和/或治疗癌症的疫苗中的应用。
其中，所述癌症为实体瘤或血液系统肿瘤，包括但不限于内分泌系统肿瘤、神经系统肿瘤、生殖系统肿瘤、消化系统肿瘤、泌尿系统肿瘤、免疫系统肿瘤、循环系统肿瘤、呼吸系统肿瘤、血液系统肿瘤、皮肤系统肿瘤。
在用作癌症疫苗以预防和治疗癌症时，本发明所述的靶向输送系统可以在癌症发生前或癌症发生后多次给药以激活机体免疫系统，从而延缓癌症的进展、治疗癌症或者预防癌症的复发。
由以上技术方案可知，本发明通过采用含有特定增溶剂的水溶液将细胞中不溶于纯水或不含增溶剂水溶液的组分转化为在特定增溶溶液中可溶并可被用于制备纳米粒子和微米粒子，从而提高了纳米粒子或微米粒子所负载的抗原物质或成分的全面性和免疫原性。同时附加可靶向于抗原提呈细胞的靶头，以主动靶向的方式提高抗原提呈细胞吞噬效率，进而提高预防或治疗癌症的效果。
附图说明
图1所示为本发明所述疫苗的制备过程及应用领域示意图；a:水溶性组分和非水溶性组分分别收集和制备纳米疫苗或微米疫苗的示意图；b:采用含有增溶剂的增溶液溶解全细胞组分和制备纳米疫苗或微米疫苗的示意图；
图2-图12所示为载有水溶性和非水溶性细胞组分的纳米粒子或微米粒子的结构示意图，其中1：细胞或组织组分中的水溶性成分；2：细胞或组织组分中的非水溶性成分；3：免疫佐剂；4：纳米粒子或微米粒子；5：纳米粒子中的内核部分；6：可以靶向特定细胞或者组织的靶头。图2-图3中纳米粒子或微米粒子表面和内部均含有免疫佐剂；图4-图5中免疫佐剂只分布于纳米粒子或微米粒子的内部；图6-图7中纳米粒子或微米粒子只在外表面含有免疫佐剂；图8-图9纳米粒子或微米粒子内部和外表面均无免疫佐剂；图10细胞组分和/或免疫佐剂只分布于纳米粒子或微米粒子内部；图11细胞组分和/或免疫佐剂只分布于纳米粒子或微米粒子外部；图12细胞组分和免疫佐剂分别分布于纳米粒子或微米粒子内部或外部；
在图2-9中，图2中2.a-2.i，图4中6.a-6.i，图6中10.a-10.i和图8中14.a-14.i纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部时未形成明显的内核；图2中3.a-3.i，图4中7.a-7.i，图6中11.a-11.i和图8中15.a-15.i中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的一个内核部分；图3中4.a-4.i，图5中8.a-8.i，图7中12.a-12.i和图9中16.a-16.i纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的多个内核部分；图3中5.a-5.i，图5中9.a-9.i，图7中13.a-13.i和图9中17.a-17.i纳米粒子或微米粒子所包载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部所形成内核的外层；a：纳米粒子或微米粒子内部包载和表面负载的均为细胞或组织组分中的水溶性成分；b：纳米粒子或微米粒子内部包载和表面负载的均为细胞或组织组分中的非水溶性成分；c：纳米粒子或微米粒子内部包载的为细胞或组织组分中的非水溶性成分而表面负载的均为细胞或组织组分中的水溶性成分；d：纳米粒子或微米粒子内部包载的为细胞或组织组分中的水溶性成分而表面负载的均为细胞或组织组分中的非水溶性成分；e：纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水 溶性成分和非水溶性成分，而纳米粒子或微米粒子表面也同时负载细胞或组织组分中的水溶性成分和非水溶性成分；f:纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面只负载细胞或组织组分中的水溶性成分；g:纳米粒子或微米粒子内部同时包载的细胞或组织组分中的水溶性成分和非水溶性成分，而纳米粒子或微米粒子表面只负载细胞或组织组分中的非水溶性成分；h：纳米粒子或微米粒子内部只包载的细胞或组织组分中的非水溶性成分，而纳米粒子或微米粒子表面同时负载细胞或组织组分中的水溶性成分和非水溶性成分；i:纳米粒子或微米粒子内部只包载的细胞或组织组分中的水溶性成分，而纳米粒子或微米粒子表面同时负载细胞或组织组分中的水溶性成分和非水溶性成分；
在图10-12中，a,b和c中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部时未形成明显的内核；d,e和f中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的一个内核部分；g，h和i中纳米粒子或微米粒子所负载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部的多个内核部分；j，k和l中纳米粒子或微米粒子所包载的细胞或组织组分中的水溶性成分或非水溶性成分分布于纳米粒子或微米粒子内部所形成内核的外层；a,d,g和j中纳米粒子或微米粒子负载的均为细胞或组织组分中的水溶性成分；b，e，h和k中纳米粒子或微米粒子负载的均为细胞或组织组分中的非水溶性成分；c，f，i和l中纳米粒子或微米粒子同时负载细胞或组织组分中的水溶性成分和非水溶性成分；
图13-19所示为实施例1-7中肿瘤组织或癌细胞全细胞组分负载于靶向纳米粒子或微米粒子时用于癌症治疗和预防的结果；a,负载肿瘤组织或癌细胞全细胞组分中水溶性组分/或同时非水溶性组分的靶向纳米粒子或微米粒子结构；b,负载肿瘤组织或癌细胞全细胞组分中非水溶性组分的靶向纳米粒子或微米粒子结构；c,靶向纳米疫苗或微米疫苗对肿瘤生长抑制实验结果；d,小鼠生存期实验结果。实验结果中肿瘤生长抑制实验图中每个数据点为平均值±标准误差(mean±SEM)；肿瘤生长抑制实验和小鼠生存期实验中n＝10。图c中肿瘤生长抑制实验的显著性差异采用ANOVA法分析；图d中显著性差异采用Kaplan-Meier和log-rank  test分析；*表示该组与PBS对照组相比P＜0.05，有显著性差异；☆表示该组与空白纳米粒子或空白微米粒子对照组相比P＜0.05，有显著性差异；
Figure PCTCN2020126655-appb-000002
表示该组与不含PEG保护层的纳米疫苗相比P＜0.05，有显著性差异；★表示该组与PEG增溶非水溶性组分制备的纳米疫苗相比P＜0.05，有显著性差异。
具体实施方式
本发明实施例公开了一种负载全细胞组分的靶向输送系统及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明内。本发明所述靶向输送系统及其应用已通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的靶向输送系统及其应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明所述全细胞组分的输送系统可用于制备预防和/或治疗癌症的疫苗，其制备过程及应用领域如图1所示。在制备时可裂解细胞或组织后先分别收集水溶性组分和水不溶性组分并分别制备纳米疫苗或微米疫苗；或者也可以直接采用含有增溶剂的增溶液直接裂解细胞或组织并溶解全细胞组分并制备纳米疫苗或微米疫苗。
在本发明所述制备靶向输送系统(也可称为纳米疫苗或微米疫苗)的方法为常用制备方法。在一些实施方案中，制备纳米疫苗采用溶剂挥发法中的复乳法，所采用的纳米粒子制备材料为有机高分子聚乳酸-羟基乙酸共聚物(PLGA)和聚乳酸(PLA)。所采用的免疫佐剂为poly(I:C)、卡介苗(BCG)或CpG。本领域技术人员可以理解，在实际应用过程中技术人员可根据具体情况对制备方法、制备过程、所采用的纳米粒子制备材料、免疫佐剂的种类和浓度等进行适当调整。
在一些实施方案中，本发明所采用的复乳法的具体制备方法如下：
步骤1，将第一预定体积的含有第一预定浓度的水相溶液加入第二预定体积的含有第二预定浓度医用高分子材料的有机相中。
在一些实施例中，水相溶液含有癌细胞裂解物中的各组分以及免疫增强佐剂poly(I:C)、BCG或CpG；癌细胞裂解物中的各组分在制备时分别为水溶性组分或者是溶于8M尿素中的原非水溶性组分。水相溶液所含有来自癌细胞的水溶性组分的浓度或者是来自癌细胞的溶于8M尿素中的 原非水溶性组分的浓度，也即第一预定浓度要求蛋白质多肽浓度含量大于1ng/mL，之所以选择这样的浓度，是发明人经大量试验发现，一般所用细胞裂解物水溶液浓度越大，制备的纳米粒中包载的各类癌症抗原越多，当制备得到的蛋白质多肽浓度大于1ng/mL，也即第一预定浓度大于1ng/mL时，才能负载足够癌症抗原以激活相关免疫反应。免疫佐剂在初始水相中的浓度为大于0.01ng/mL。另外，第一预定体积为600μL。
在一些实施例中，水相溶液含有肿瘤组织裂解物中的各组分以及免疫增强佐剂poly(I:C)，BCG或CpG；肿瘤组织裂解物中的各组分在制备时分别为水溶性组分或者是溶于8M尿素中的原非水溶性组分。水相溶液所含有得来自肿瘤组织的水溶性组分的浓度或者是来自肿瘤组织的溶于8M尿素中的原非水溶性组分的浓度，也即第一预定浓度要求蛋白质多肽浓度含量大于1ng/mL，之所以选择这样的浓度，是发明人经大量试验发现，一般所用肿瘤组织裂解物水溶液浓度越大，制备的纳米粒中包载的各类癌症抗原越多，当制备得到的蛋白质多肽浓度大于1ng/mL，也即第一预定浓度大于1ng/mL时，才能负载足够癌症抗原以激活相关免疫反应。免疫佐剂在初始水相中的浓度为大于0.01ng/mL。另外，第一预定体积为600μL。
在本发明中，将医用高分子材料溶解于有机溶剂中，得到第二预定体积的含有第二预定浓度医用高分子材料的有机相。在一些实施例中，医用高分子材料为靶头修饰的PLGA、PLGA和PLA的混合物，有机溶剂选用二氯甲烷，得到的有机相的体积，也即第二预定体积为2mL。另外，在一些实施例中，医用高分子材料的第二预定浓度的范围为0.5mg/mL-5000mg/mL，优选为100mg/mL。
在本发明中，之所以选择PLGA和靶头修饰的PLGA，是由于该材料为生物可降解材料且已被FDA批准用作药物敷料，在实际使用时也可以选用任何其他可以用于制备纳米尺寸粒子或微米尺寸粒子的材料和靶头修饰材料的混合物。
实际中，有机相的第二预定体积根据其和水相的第一预定体积的比例进行设定，在本发明中，水相的第一预定体积和有机相的第二预定体积之比的范围为1:1.1-1:5000，优先地为1:10。在具体实施过程中可根据需要对第一预定体积、第二预定体积和第一预定体积与第二预定体积之比进行调整以调整制备的纳米粒的尺寸大小。
步骤2，将步骤1得到的混合液进行大于2秒的超声处理或大于1分钟的搅拌。
该步骤是为了进行纳米化，超声时间长短或搅拌速度及时间能控制制备的纳米粒子大小，过长或过短都会带来粒径大小的变化，为此，需要选择合适的超声时间。在本发明中，超声时间大于2秒，搅拌速度大于50rpm，搅拌时间大于1分钟。
步骤3，将步骤2处理后得到的混合物加入第三预定体积的含有第三预定浓度乳化剂的水溶液中并进行大于2秒的超声处理或大于1分钟的搅拌。
该步骤将步骤2得到的混合物加入到乳化剂水溶液中继续超声或搅拌纳米化。
在本发明中，乳化剂水溶液为聚乙烯醇(PVA)水溶液，第三预定体积为5mL，第三预定浓度为20mg/mL。第三预定体积根据其与第二预定体积的比例进行调整。在本发明中，第二预定体积与第三预定体积之的范围为1:1.1-1:1000进行设定，优先地可以为2:5。在具体实施过程中为了控制纳米粒子的尺寸，可以对第二预定体积和第三预定体积之比进行调整。
同样地，本步骤的超声时间或搅拌时间、乳化剂水溶液的体积以及浓度的取值根据，均为了得到尺寸大小合适的纳米粒。
步骤4，将步骤3处理后得到的液体加入第四预定体积的第四预定浓度的乳化剂水溶液中，并进行搅拌直至满足预定搅拌条件。
本步骤中，乳化剂水溶液依然为PVA，第四预定体积为大于50mL，
第四预定浓度为5mg/mL，第四预定浓度的选择，以得到尺寸大小合适的纳米粒为依据。第四预定体积的选择依据第三预定体积与第四预定体积之比决定。在本发明中，第三预定体积与第三预定体积之比为范围为1:1.5-1:2000，优先地为1:10。在具体实施过程中为了控制纳米粒子的尺寸可以对第三预定体积和第四预定体积之比进行调整。
在本发明中，本步骤的预定搅拌条件为直至有机溶剂挥发完成，也即步骤1中的二氯甲烷挥发完成。
步骤5，将步骤4处理满足预定搅拌条件的混合液在以大于100RPM的转速进行大于1分钟的离心后，去除上清液，并将剩下的沉淀物重新混悬于第五预定体积的第五预定浓度的含有冻干保护剂的水溶液中或者第六预定体积的PBS(或生理盐水)中。
在本发明一些实施方案中，步骤5所得沉淀重新混悬于第六预定体积的PBS(或生理盐水)中时不需要冻干，可直接进行后续纳米粒子表面吸附癌细胞裂解物的相关实验。
在本发明一些实施方案中，步骤5所得沉淀重新混悬于含有冻干保护剂的水溶液中时需进行冷冻干燥，再冷冻干燥以后再进行后续纳米粒子表面吸附癌细胞裂解物的相关实验。
在本发明中，所述冻干保护剂选用海藻糖(Trehalose)。
在本发明中，该步骤的冻干保护剂的第五预定体积为20mL，第五预定浓度为质量百分比4％，之所以如此设定，是为了在后续进行冷冻干燥中不影响冻干效果。
步骤6，将步骤5得到的含有冻干保护剂的混悬液进行冷冻干燥处理后，将冻干物质备用。
步骤7，将第六预定体积的步骤5中得到的重悬于PBS(或生理盐水)中的含纳米粒的混悬液或者采用第六预定体积的PBS(或生理盐水)重悬步骤6得到的冷冻干燥后的含有纳米粒和冻干保护剂的冻干物质，与第七预定体积的水溶性组分或者溶于8M尿素中的原非水溶性组分混合后静置大于0.1分钟即得纳米疫苗或微米疫苗。
在本发明中，第六预定体积与第七预定体积的体积比为1:10000到10000:1，优先体积比为1:100到100:1，最优体积比为1:30到30:1。
在一些实施例中，所述重悬的纳米粒子混悬液体积为10mL，含有癌细胞裂解物或含有肿瘤组织裂解物中的水溶性组分或者溶于8M尿素中的原非水溶性组分的体积与为1mL。
在一些实施例中，如图2，本发明先将细胞组分中的可溶于纯水的水溶性部分或(和)非水溶性部分经增溶剂进行增溶后包载于纳米粒子或微米粒子内，同时包载免疫增强剂；然后，将细胞组分中的水溶性部分或(和)非水溶性部分吸附于纳米粒子表面，同时吸附有免疫增强剂。这样就使得纳米粒子或微米粒子中细胞的水溶性组分或非水溶性组分的负载能力可以达到最大。在实际应用中，也可以直接采用含有增溶剂的增溶液(如8M尿素水溶液或6M盐酸胍水溶液)直接裂解细胞或组织并直接溶解全细胞组分，而后以此制备纳米疫苗或微米疫苗。
以下就本发明所提供的一种负载全细胞组分的靶向输送系统及其应用做进一步说明。
实施例1：黑色素瘤肿瘤组织全细胞组分负载于甘露糖修饰的纳米粒子内部用于癌症的治疗
本实施例以小鼠黑色素瘤为癌症模型来说明如何制备负载有肿瘤组织全细胞组分的纳米疫苗，并应用该疫苗治疗黑色素瘤。在实际应用时具体剂型，佐剂，给药时间、给药次数、给药方案可根据情况调整。
本实施例中，以B16-F10小鼠黑色素瘤为癌症模型。本实施例中，将小鼠黑色素瘤肿瘤组织裂解组分负载于纳米粒子内部和表面以制备纳米疫苗。首先取得小鼠黑色素瘤肿瘤瘤块并将其裂解以制备瘤块组织的水溶性组分和溶于8M尿素中的原非水溶性组分。然后，以PLGA(50:50)和PLA为纳米粒骨架材料，以CpG为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织裂解物的水溶性组分和非水溶性组分的纳米疫苗。然后采用该纳米疫苗来治疗黑色素瘤荷瘤小鼠体内的肿瘤。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠背部皮下接种150000个B16F10黑色素瘤细胞，在各只小鼠所接种肿瘤长到体积分别为400mm3到1500mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨，通过细胞过滤网加入适量纯水并反复冻融和超声处理至少5次。待肿瘤组织部位细胞裂解后，将瘤块组织的细胞裂解物以大于12000RPM的转速离心5min取上清液即为瘤块组织中可溶于纯水的水溶性组分；在所得沉淀部分中加入8M尿素水溶液溶解沉淀部分即可将不溶于纯水的原非水溶性组分转化为在8M尿素水溶液中可溶。上述所得肿瘤组织裂解物的水溶性组分和溶解于8M尿素中的原非水溶性组分即为制备用于治疗癌细胞的纳米疫苗的原料来源。
(2)纳米疫苗的制备
本实施例中制备纳米疫苗及作为对照的空白纳米粒采用溶剂挥发法中的复乳法，所采用的纳米粒子制备材料PLGA(50:50)分子量为24KDa-38KDa，所采用的甘露糖修饰的PLGA(50:50)分子量为24KDa-38KDa，所采用的的PLA分子量为10KDa。未修饰PLGA，甘露糖修饰的PLGA和PLA的质量比为8:1:2。所采用的免疫佐剂为CpG且CpG分布于纳米粒子内部。制备方法如前所述。纳米粒子平均粒径为280nm左右，纳米粒子平均表面电位Zeta potential为-8mV左右。每1mg PLGA纳米粒子负载60μg蛋白质或多肽组分,每1mg PLGA纳米粒内外所使用的CpG免疫佐剂为0.01mg且内外各半。空白纳米粒粒径为240nm左右，空白纳米粒制备时分别采用含有等量CpG的纯水或8M尿素代替相对应的水溶性组分和非水溶性组分。
(3)纳米疫苗用于癌症的治疗
本研究分所采用的实验组设置如下：纳米疫苗组；PBS对照组，空白纳米粒+细胞裂解物组。
选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。
纳米疫苗组给药方案如下：在第0天给每只小鼠背部右下方皮下接种150000个B16-F10细胞，在第4天、第7天、第10天、第15天和第20天分别皮下注射200μL内部和表面都负载癌细胞裂解物中水溶性成分的2mg PLGA纳米粒子和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA纳米粒子。
PBS空白对照组方案如下：在第0天给每只小鼠背部右下方皮下接种150000个B16-F10细胞，在第4天、第7天、第10天、第15天和第20天分别皮下注射400μL PBS。
空白纳米粒+组织裂解物对照组：在第0天给每只小鼠背部右下方皮下接种150000个B16-F10细胞，在第4天、第7天、第10天、第15天和第20天分别皮下注射等量肿瘤组织裂解物中水溶性成分，等量裂解物中溶于8M尿素中的原非水溶性成分以及负载等量CpG而不负载任何裂解物成分的4mg PLGA空白纳米粒。注意以上三者需分开给药并注射在不同部位，以免游离的裂解物吸附于空白纳米粒表面。
在实验中，从第6天开始每三天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52*a*b2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。出于动物实验伦理，在小鼠生存期试验中当小鼠肿瘤体积超过2000mm3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图13所示，与PBS空白对照组和空白纳米粒+细胞裂解物对照组相比，纳米疫苗组小鼠肿瘤生长速度明显变慢(p<0.05)且小鼠生存期明显延长(p<0.05)。这说明本发明所述的负载癌细胞水溶性组分和非水溶性组分的纳米疫苗对黑色素瘤具有治疗效果且可部分治愈黑色素瘤。
实施例2：黑色素瘤肿瘤组织全细胞组分负载于甘露糖修饰的微米粒子内部和表面用于癌症的治疗
本实施例以B16F-10小鼠黑色素瘤为癌症模型来说明如何使用微米疫苗治疗黑色素瘤。在实际应用时具体剂型可根据情况调整。
本实施例中，将小鼠黑色素瘤肿瘤组织所有裂解组分同时负载于微米粒子内部和表面以制备微米疫苗。首先取得小鼠黑色素瘤肿瘤瘤块并将其裂解以制备瘤块组织的水溶性组分和溶于8M尿素中的原非水溶性组分。 然后，以PLGA(50:50)为纳米粒骨架材料，以poly(I:C)为免疫佐剂采用溶剂挥发法制备同时负载有肿瘤组织裂解物的水溶性组分和非水溶性组分的微米疫苗。然后采用该微米疫苗来治疗黑色素瘤荷瘤小鼠体内的肿瘤。
(1)肿瘤组织的裂解及各组分的收集
处理方法同实施例1。
(2)微米疫苗的制备
本实施例中制备微米疫苗及作为对照的空白微米粒采用溶剂挥发法中的复乳法，所采用的微米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的甘露糖修饰的PLGA(50:50)分子量为24KDa-38KDa。未修饰PLGA和甘露糖修饰的PLGA(50:50)的质量比为10:1。所采用的免疫佐剂为poly(I:C)且poly(I:C)分布于微米粒子内部。制备方法如前所述，水溶性组分和非水溶性组分同时负载于同一微米粒子中。在本实施例中将全细胞组分同时负载于微米粒子内部和表面。在将全细胞组分负载于微米粒子表面时将已经负载有全细胞组分的微米粒子与细胞组分按10:1的体积比混合后静置15分钟即可。微米粒子平均粒径为2.0μm左右，纳米粒子平均表面电位Zeta potential为-17mV左右。每1mg PLGA微米粒子负载70μg蛋白质或多肽组分,每1mg PLGA微米粒子内外所使用的poly(I:C)免疫佐剂为0.01mg且内外各半。空白微米粒粒径为1.8μm左右，空白微米粒子制备时分别采用含有等量poly(I:C)的纯水或8M尿素代替相对应的水溶性组分和非水溶性组分。
(3)微米疫苗用于癌症的治疗
本研究实验组设置如下：微米疫苗组；PBS对照组，空白微米粒+细胞裂解物组。选取6-8周的雌性C57BL/6为模型小鼠制备黑色素瘤荷瘤小鼠。
微米疫苗组给药方案如下：在第0天给每只小鼠背部右下方皮下接种150000个B16-F10细胞，在第4天、第7天、第10天、第15天和第20天分别皮下注射400μL内部和表面都同时负载癌细胞裂解物中水溶性成分和非水溶性成分的4mg PLGA微米疫苗。
PBS空白对照组方案如下：在第0天给每只小鼠背部右下方皮下接种150000个B16-F10细胞，在第4天、第7天、第10天、第15天和第20天分别皮下注射400μL PBS。
空白微米粒+细胞裂解物对照组：在第0天给每只小鼠背部右下方皮下接种150000个B16-F10细胞，在第4天、第7天、第10天、第15天和第20 天分别皮下注射等量癌细胞裂解物中水溶性成分+非水溶性成分以及负载等量poly(I:C)而不负载任何细胞裂解物成分的4mg PLGA空白纳米粒。注意以上二者需分开给药并注射在不同部位，以免游离的细胞裂解物吸附于空白微米粒子表面。
在实验中，从第6天开始每三天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52*a*b2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。
(4)实验结果
如图14所示，与PBS空白对照组和空白微米粒子+细胞裂解物对照组相比，微米疫苗组小鼠肿瘤生长速度明显变慢(p<0.05)且小鼠生存期明显延长(p<0.05)。这说明本发明所述的负载癌细胞水溶性组分和非水溶性组分的微米疫苗对黑色素瘤具有治疗效果且可部分治愈黑色素瘤。
实施例3：黑色素瘤肿瘤组织全细胞组分负载于甘露糖修饰的纳米粒子内部和表面用于癌症的预防
本实施例以小鼠黑色素瘤为癌症模型来说明如何制备负载有肿瘤组织全细胞组分的纳米疫苗，并应用该疫苗治疗黑色素瘤。在实际应用时具体剂型，佐剂，给药时间、给药次数、给药方案可根据情况调整。
本实施例中，以B16-F10小鼠黑色素瘤为癌症模型。本实施例中，将小鼠黑色素瘤肿瘤组织裂解组分负载于纳米粒子内部和表面以制备纳米疫苗。首先取得小鼠黑色素瘤肿瘤瘤块并将其裂解以制备瘤块组织的水溶性组分和溶于8M尿素中的原非水溶性组分。然后，以PLGA(50:50)为纳米粒骨架材料，以poly(I:C)为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织裂解物的水溶性组分和非水溶性组分的纳米疫苗。然后采用该纳米疫苗来治疗黑色素瘤荷瘤小鼠体内的肿瘤。
(1)肿瘤组织的裂解及各组分的收集
方法同实施例1。
(2)纳米疫苗的制备
本实施例中制备纳米疫苗及作为对照的空白纳米粒采用溶剂挥发法中的复乳法，所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的甘露糖修饰的PLGA分子量为24KDa-38KDa。未修饰PLGA和甘露糖修饰的PLGA的质量比为10:1。所采用的免疫佐剂为poly(I:C)且poly(I:C)既布于纳米粒子内部。制备方法如前所述，不同的是本实施例中将全细胞组分同时负载于纳米粒子内部和表面。在将全细胞组分负载于纳 米粒子表面时将已经负载有全细胞组分的纳米粒子与细胞组分按10:1的体积比混合后静置15分钟即可。纳米粒子平均粒径为300nm左右，纳米粒子平均表面电位Zeta potential为-8mV左右。每1mg PLGA纳米粒子负载60μg蛋白质或多肽组分,每1mgPLGA纳米粒内外所使用的poly(I:C)免疫佐剂为0.01mg且内外各半。空白纳米粒粒径为260nm左右，空白纳米粒制备时分别采用含有等量poly(I:C)的纯水或8M尿素代替相对应的水溶性组分和非水溶性组分。
(3)纳米疫苗用于癌症的预防
选取6-8周的雌性C57BL/6制备黑色素瘤荷瘤小鼠。在接种B16F10癌细胞之前第42天、之前第35天、之前第28天、之前第21天和之前第14天分别皮下注射200μL内部和表面都负载癌细胞裂解物中水溶性成分的2mg PLGA纳米疫苗和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA纳米疫苗。最后一针纳米疫苗注射完后14天，在每只小鼠背部右下方皮下接种150000个B16-F10细胞，并将该天设为肿瘤细胞接种的第0天。在本实验中，PBS空白对照组方案如下：在接种B16F10癌细胞之前第42天、之前第35天、之前第28天、之前第21天和之前第14天分别皮下注射400μL PBS。在第0天给每只小鼠背部右下方皮下接种150000个B16-F10细胞。
在实验中，从第6天开始每三天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52*a*b2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。
(4)实验结果
如图15所示，与PBS对照组相比，纳米疫苗预防组肿瘤生长速度明显变慢(p<0.05)且小鼠生存期明显延长(p<0.05)。这说明本发明所述的具有树突状细胞靶向能力的负载癌细胞水溶性组分和非水溶性组分的纳米疫苗对黑色素瘤具有预防效果。
实施例4：肺癌肿瘤组织全细胞组分负载于甘露糖修饰的纳米粒子内部用于肺癌的预防
本实施例以小鼠肺癌为癌症模型来说明如何制备负载有肿瘤组织全细胞组分的纳米疫苗，并应用该疫苗预防肺癌。在实际应用时具体剂型，佐剂，给药时间、给药次数、给药方案可根据情况调整。
本实施例中，以LLC小鼠肺癌为癌症模型。本实施例中，将小鼠肺癌肿瘤组织裂解组分负载于纳米粒子内部和表面以制备纳米疫苗。首先取得 小鼠肺癌肿瘤瘤块并将其裂解以制备瘤块组织的水溶性组分和溶于8M尿素中的原非水溶性组分。然后，以PLGA(50:50)纳米粒骨架材料，以(poly(I:C))为免疫佐剂采用溶剂挥发法制备负载有肿瘤组织裂解物的水溶性组分和非水溶性组分的纳米疫苗。然后采用该纳米疫苗来治疗肺癌荷瘤小鼠体内的肿瘤。
(1)肿瘤组织的裂解及各组分的收集
在每只C57BL/6小鼠侧腹部皮下接种1000000个LLC黑色素瘤细胞，在各只小鼠所接种肿瘤长到体积分别为400mm3到1500mm3时处死小鼠并摘取肿瘤组织。将肿瘤组织切块后研磨，通过细胞过滤网加入适量纯水并反复冻融和超声处理至少5次。待肿瘤组织部位细胞裂解后，将瘤块组织的细胞裂解物以大于12000RPM的转速离心5min取上清液即为瘤块组织中可溶于纯水的水溶性组分；在所得沉淀部分中加入8M尿素水溶液溶解沉淀部分即可将不溶于纯水的原非水溶性组分转化为在8M尿素水溶液中可溶。上述所得肿瘤组织裂解物的水溶性组分和溶解于8M尿素中的原非水溶性组分即为制备用于治疗癌细胞的纳米疫苗的原料来源。
(2)纳米疫苗的制备
本实施例中制备纳米疫苗及作为对照的空白纳米粒采用溶剂挥发法中的复乳法，所采用的纳米粒子制备材料PLGA分子量为24KDa-38KDa，所采用的甘露糖修饰的PLGA分子量为24KDa-38KDa。未修饰PLGA和甘露糖修饰的PLGA的质量比为10:1。所采用的免疫佐剂为poly(I:C)且poly(I:C)布于纳米粒子内部。制备方法如前所述。纳米粒子平均粒径为300nm左右，纳米粒子平均表面电位Zeta potential为-8mV左右。每1mg PLGA纳米粒子负载60μg蛋白质或多肽组分,每1mgPLGA纳米粒内外所使用的poly(I:C)免疫佐剂为0.01mg且内外各半。空白纳米粒粒径为260nm左右，空白纳米粒制备时分别采用含有等量poly(I:C)的纯水或8M尿素代替相对应的水溶性组分和非水溶性组分。
(3)纳米疫苗用于肺癌的预防
选取6-8周的雌性C57BL/6为模型小鼠制备肺癌荷瘤小鼠。
纳米疫苗组给药方案如下：在肿瘤细胞接种前的第-49天、第-42天、第-35天、第-28天和第-14天分别皮下注射200μL内部和表面都负载癌细胞裂解物中水溶性成分的2mg PLGA纳米粒子和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA纳米粒子。PBS对照组在相应天数注射400200μL PBS。第0天给每只小鼠背部右下方皮下接种1000000个LLC肺癌细胞。
在实验中，从第6天开始每三天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52*a*b 2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。
(4)实验结果
如图16所示，与PBS空白对照组相比，纳米疫苗组小鼠肿瘤生长速度明显变慢(p<0.05)且小鼠生存期明显延长(p<0.05)。这说明本发明所述的负载癌细胞水溶性组分和非水溶性组分的纳米疫苗对肺癌具有预防效果。
实施例5：乳腺癌细胞全细胞组分负载于甘露糖修饰的纳米粒子内部用于癌症的治疗
本实施例以小鼠乳腺癌治疗来说明如何制备负载有全细胞组分的纳米疫苗并应用该疫苗治疗乳腺癌。本实施例中，以4T1小鼠三阴性乳腺癌细胞为癌细胞模型。首先裂解4T1细胞以制备4T1细胞的水溶性组分和非水溶性组分。然后，以PLGA和甘露糖修饰的PLGA(50:50)为纳米粒子骨架材料，采用溶剂挥发法制备负载有4T1细胞的水溶性组分和非水溶性组分的纳米疫苗。并以卡介苗(BCG)佐剂为免疫佐剂，采用该纳米疫苗治疗4T1乳腺癌荷瘤小鼠体内的肿瘤。
(1)癌细胞的裂解及各组分的收集
收集一定量的4T1细胞，去除培养基后采用-20℃到-273℃冷冻，加一定量超纯水后反复冻融3次以上，并可伴有超声以破坏裂解细胞。待细胞裂解后，将裂解物以大于100g的转速离心1分钟以上并取上清液即为4T1中可溶于纯水的水溶性组分；在所得沉淀部分中加入8M尿素溶解沉淀部分即可将4T1中不溶于纯水的非水溶性组分转化为在8M尿素水溶液中可溶。上述所得来源于癌细胞裂解物的水溶性组分和溶解于8M尿素中的原非水溶性组分即为制备用于治疗癌症的纳米疫苗的原料来源。
(2)纳米疫苗的制备
本实施例中纳米疫苗的制备方法、所使用的材料等与实施例4基本相同，不同的是将LLC细胞换成4T1细胞，并将佐剂换成了BCG佐剂。
(3)纳米疫苗用于癌症的治疗
选取6-8周的雌性BALB/c小鼠制备4T1荷瘤小鼠。
纳米疫苗组给药方案如下：在第0天给每只小鼠背部右下方皮下接种400000个4T1细胞，在第4天、第7天、第10天、第15天和第20天分别皮下注射200μL内部和表面都负载癌细胞裂解物中水溶性成分的2mg PLGA纳 米粒子+0.5mg BCG佐剂和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA纳米粒子+0.5mg BCG佐剂。
PBS空白对照组方案如下：在第0天给每只小鼠背部右下方皮下接种400000个4T1细胞，在第4天、第7天、第10天、第15天和第20天分别皮下注射400μL PBS。
空白纳米粒+细胞裂解物对照组：在第0天给每只小鼠背部右下方皮下接种400000个4T1细胞，在第4天、第7天、第10天、第15天和第20天分别皮下注射癌细胞裂解物中水溶性成分，癌细胞裂解物中溶于8M尿素中的原非水溶性成分、等量BCG以及4mg PLGA空白纳米粒。注意以上三者需分开给药并注射在不同皮下部位，以免游离的细胞裂解物吸附于空白纳米粒表面。
在实验中，从第6天起每三天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52*a*b2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。在小鼠生存期试验中当小鼠肿瘤体积超过2000mm3即视为小鼠死亡并将小鼠安乐死。
(4)实验结果
如图17所示，与PBS空白对照组和空白纳米粒+细胞裂解物对照组相比，纳米疫苗给药组肿瘤生长速度明显变慢(p<0.05)且小鼠生存期明显延长(p<0.05)。由此可见，本发明所述的负载癌细胞水溶性组分和非水溶性组分的纳米疫苗对乳腺癌具有治疗效果。
实施例6：乳腺癌细胞全细胞组分负载于甘露糖修饰的纳米粒子内部用于癌症的治疗
本实施例以小鼠乳腺癌治疗来说明如何制备负载有全细胞组分的纳米疫苗并应用该疫苗治疗乳腺癌。本实施例中，以4T1小鼠三阴性乳腺癌细胞为癌细胞模型。首先裂解4T1细胞以制备4T1细胞的水溶性组分和非水溶性组分。然后，以PLGA和甘露糖修饰的PLGA(50:50)为纳米粒子骨架材料，采用溶剂挥发法制备负载有4T1细胞的水溶性组分和非水溶性组分的纳米疫苗。并以卡介苗(BCG)佐剂为免疫佐剂，采用该纳米疫苗治疗4T1乳腺癌荷瘤小鼠体内的肿瘤。
(1)癌细胞的裂解及各组分的收集
制备方法同实施例5。
(2)纳米疫苗的制备
本实施例中纳米疫苗的制备方法、所使用的材料等与实施例5基本相同，全细胞组分只负载于纳米粒子内部。此外，在含有PEG保护膜的疫苗组中在制备时在PLGA有机相中加入了3.5％的PEG-PLGA(PEG部分分子量5000，PLAG部分分子量25000)。
(3)纳米疫苗用于癌症的治疗
选取6-8周的雌性BALB/c小鼠制备4T1荷瘤小鼠。
含有PEG保护膜的纳米疫苗组和不含PEG保护膜的纳米疫苗组给药方案如下：在第0天给每只小鼠背部右下方皮下接种400000个4T1细胞，在第4天、第7天、第10天、第15天和第20天分别皮下注射200μL负载水溶性成分的2mg PLGA纳米粒子+0.5mg BCG佐剂和200μL负载原非水溶性成分的2mg PLGA纳米粒子+0.5mg BCG佐剂。
PBS空白对照组方案如下：在第0天给每只小鼠背部右下方皮下接种400000个4T1细胞，在第4天、第7天、第10天、第15天和第20天分别皮下注射400μL PBS。
在实验中，从第6天起每三天记录一次小鼠肿瘤体积的大小。肿瘤体积采用公式v＝0.52*a*b2计算，其中v为肿瘤体积，a为肿瘤长度，b为肿瘤宽度。
(4)实验结果
如图18所示，与PBS空白对照组和不含PGE保护膜的纳米疫苗组相比，含有PEG保护膜的纳米疫苗组肿瘤生长速度明显变慢(p<0.05)且小鼠生存期明显延长(p<0.05)。由此可见，PEG保护膜可以保护纳米粒子不被除树突状细胞以外的细胞吞噬和延长纳米粒子在体内的循环时间，从而提高纳米疫苗的靶向性和效果。
实施例7：不同增溶剂对于非水溶性组分的溶解性
本实施例以小鼠乳腺癌治疗来说明如何制备负载有全细胞组分的纳米疫苗并应用该疫苗治疗乳腺癌。本实施例中，以4T1小鼠三阴性乳腺癌细胞为癌细胞模型。首先裂解4T1细胞以制备4T1细胞的水溶性组分和非水溶性组分。分别采用6M盐酸胍和PEG(5000KD)溶解非水溶性组分。然后，以PLG(50:50)A和甘露糖修饰的PLGA(50:50)为纳米粒子骨架材料制备纳米疫苗。并以卡介苗(BCG)佐剂为免疫佐剂，采用该纳米疫苗治疗4T1乳腺癌荷瘤小鼠体内的肿瘤。
(1)癌细胞的裂解及各组分的收集
制备方法同实施例5。不同的是本实施例分别采用6M盐酸胍和PEG(5000KD)分别溶解所裂解癌细胞中的非水溶性组分。6M盐酸胍增溶非水溶性组分能力明显强于PEG(5000KD)：6M盐酸胍增溶非水溶性组分的浓度可达80mg/mL；PEG(5000KD)最高只能增溶非水溶性组分达到1mg/mL。
(2)纳米疫苗的制备
本实施例中纳米疫苗的制备方法、所使用的材料等与实施例5基本相同，全细胞组分只负载于纳米粒子内部。而且，在制备负载非水溶性组分的纳米疫苗时采用6M盐酸胍增溶的原非水溶性组分的浓度为60mg/mL，而所使用的PEG增溶的原非水溶性组分的浓度为1mg/mL。所以制备所得纳米疫苗以6M盐酸胍增溶后的负载非水溶性组分的纳米粒子每1mg PLGA纳米粒子负载70μg蛋白质/多肽组分；PEG增溶后的负载非水溶性组分的纳米粒子每1mg PLGA纳米粒子负载2μg蛋白质/多肽组分。
(3)纳米疫苗用于癌症的预防
选取6-8周的雌性BALB/c小鼠制备4T1荷瘤小鼠。
纳米疫苗组给药方案如下：在肿瘤细胞接种前的第-49天、第-42天、第-35天、第-28天和第-14天分别皮下注射200μL内部和表面都负载癌细胞裂解物中水溶性成分的2mg PLGA纳米粒子(0.5mg BCG佐剂)和200μL内部和表面都负载溶于8M尿素中原非水溶性成分的2mg PLGA纳米粒子(0.5mg BCG佐剂)。PBS对照组在相应天数注射400μL PBS。第0天给每只小鼠背部右下方皮下接种400000个4T1细胞。在实验中，从第6天起每三天记录一次小鼠肿瘤体积的大小。肿瘤体积计算方式同实施例1。
(4)实验结果
如图19所示，与PBS空白对照组和PEG增溶非水溶性组分制备的纳米疫苗组相比，采用6M盐酸胍增溶非水溶性组分制备的纳米疫苗组肿瘤生长速度明显变慢(p<0.05)且小鼠生存期明显延长(p<0.05)。由此可见，增溶溶液和方法对于提高纳米疫苗的效果很重要。
以上所述只是用于理解本发明的方法及其核心思想，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利的保护范围。

Claims (10)

  1. 一种负载全细胞组分的靶向输送系统，其特征在于，其为表面有靶头的纳米级尺寸或微米级尺寸的粒子，且所述粒子负载有癌细胞或癌组织的全细胞组分，所述非水溶性成分通过增溶剂溶解；所述全细胞组分为细胞或组织中全细胞的水溶性成分和非水溶性成分；所述靶头与特定细胞或组织表面的分子结合进而帮助所述粒子进入细胞或组织。
  2. 根据权利要求1所述靶向输送系统，其特征在于，所述增溶剂为尿素、盐酸胍、脱氧胆酸钠、SDS、甘油、pH大于7的碱性溶液、pH小于7的酸性溶液、各类蛋白质降解酶、白蛋白、卵磷脂、高浓度无机盐、Triton、吐温、DMSO、乙腈、乙醇、甲醇、DMF、丙醇、异丙醇、醋酸、胆固醇、氨基酸、糖苷、胆碱、Brij-35、Octaethylene glycol monododecyl ether、CHAPS、Digitonin、lauryldimethylamine oxide、
    Figure PCTCN2020126655-appb-100001
    Figure PCTCN2020126655-appb-100002
    DMSO、乙腈、乙醇、甲醇、DMF、异丙醇、丙醇、二氯甲烷、乙酸乙酯中的一种或两种以上。
  3. 根据权利要求1所述靶向输送系统，其特征在于，所述靶头为甘露糖。
  4. 根据权利要求1所述靶向输送系统，其特征在于，所述特定细胞或组织为树突状细胞、巨噬细胞、B细胞、T细胞、NK细胞、NKT细胞、中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、淋巴结、胸腺、脾脏、骨髓中的一种或两种以上。
  5. 根据权利要求1所述靶向输送系统，其特征在于，所述纳米级尺寸的粒子的粒径为1nm-1000nm；所述微米级尺寸的粒子的粒径为1μm-1000μm。
  6. 根权利要求1或5所述靶向输送系统，所述纳米级尺寸粒子或微米级尺寸的粒子的制备材料为有机合成高分子材料、天然高分子材料、无机材料、细菌或者病毒中的一种或两种以上。
  7. 根据权利要求1所述靶向输送系统，其特征在于，其形状为球形、椭球形、桶形、多角形、棒状、片状、线形、蠕虫形、方形、三角形、蝶形或圆盘形。
  8. 根据权利要求1所述靶向输送系统，其特征在于，还包括免疫佐剂。
  9. 根据权利要求1-8任意一项所述靶向输送系统，其特征在于，还包括在粒子外部添加PEG保护膜。
  10. 权利要求1-9任意一项所述靶向输送系统在制备预防和/或治疗癌症的疫苗中的应用。
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CN114958739A (zh) * 2022-04-29 2022-08-30 苏州尔生生物医药有限公司 一种细胞系统及其应用、以及激活广谱癌细胞特异性t细胞的方法
WO2023206684A1 (zh) * 2022-04-29 2023-11-02 苏州尔生生物医药有限公司 一种细胞系统及其应用、以及激活广谱癌细胞特异性t细胞的方法
CN115040660A (zh) * 2022-06-28 2022-09-13 南通大学 一种甘露醇修饰的纳米粒及其制备方法和应用
WO2024016688A1 (zh) * 2022-07-19 2024-01-25 苏州尔生生物医药有限公司 基于激活的抗原提呈细胞的核酸递送粒子、核酸递送系统及制备方法

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