WO2023010776A1 - 具有肿瘤免疫微环境调节功能的纳米药物及其制备方法与应用 - Google Patents

具有肿瘤免疫微环境调节功能的纳米药物及其制备方法与应用 Download PDF

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WO2023010776A1
WO2023010776A1 PCT/CN2021/141283 CN2021141283W WO2023010776A1 WO 2023010776 A1 WO2023010776 A1 WO 2023010776A1 CN 2021141283 W CN2021141283 W CN 2021141283W WO 2023010776 A1 WO2023010776 A1 WO 2023010776A1
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nano
tumor
phospholipids
medicine
glycero
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刘庄
冯良珠
王春杰
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Suzhou University
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the invention relates to a nano-medicine with the function of regulating the tumor immune microenvironment, its preparation method and application, and belongs to the technical field of nano-medicine for tumor treatment.
  • indoleamine 2,3-dioxygenase IDO1
  • extracellular- 5'-nucleotidase CD73
  • IDO1 indoleamine 2,3-dioxygenase
  • Trp tryptophan
  • Kyn kynurenine
  • Kyn kynurenine
  • IDO1 indoleamine 2,3-dioxygenase
  • these small molecule inhibitors have encountered many difficulties in the actual clinical transformation application, such as poor water solubility of molecules, easy drug resistance, lack of tumor selectivity and targeting, etc.
  • the development of a drug carrier with a high loading rate is expected to achieve tumor-targeted delivery of these small molecule inhibitors, thereby reshaping the immunosuppressive metabolic tumor microenvironment and effectively improving the efficacy of tumor therapy.
  • radiotherapy is a treatment method that uses high-energy rays to generate reactive oxygen species (ROS) to kill cancer cells, and is currently the most used treatment method in clinical tumor treatment.
  • ROS reactive oxygen species
  • the present invention provides a simple preparation, which can be efficiently enriched into the tumor site, quickly neutralizes protons, and the released immune regulatory small molecules can effectively regulate the tumor microenvironment and reverse the immune response caused by tumor cell metabolism.
  • Nanomedicines for inhibitory microenvironments Combined with radiotherapy, it can efficiently inhibit tumor growth, and at the same time, it can inhibit tumor metastasis and recurrence by stimulating the body's immune response.
  • the first object of the present invention is to provide a nano-medicine with the function of regulating the tumor immune microenvironment, said nano-medicine comprising: nano-medicine particles formed by calcium carbonate nanoparticles and complexes coated on the surface of calcium carbonate nanoparticles , and, the liposome carrier used to load the nano-medicine particles; wherein, the complex is formed by coordination of immune regulation small molecules and metal ions.
  • the particle size of the calcium carbonate nanoparticles is 60-300nm. Preferably it is 80-160 nm.
  • the immunoregulatory small molecules are indoleamine 2,3-dioxygenase inhibitors, transforming growth factor- ⁇ (TGF- ⁇ ) inhibitors, interleukin receptor inhibitors, oxidative phosphorylation inhibitors, One or more of PD-1/PD-L1 interaction inhibitors.
  • the indoleamine 2,3-dioxygenase inhibitor is 4-phenylimidazole (4PI) or navomod (NLG919).
  • TGF- ⁇ transforming growth factor- ⁇
  • SB431542 LY364947, SB505124 or SB525334.
  • the interleukin receptor inhibitor is dexamethasone sodium phosphate.
  • the oxidative phosphorylation inhibitor is 4-methyl-2-oxopentanoic acid.
  • the PD-1/PD-L1 interaction inhibitor is BMS-1001, BMS-1166 or BMS-1.
  • the metal ions are one or more of zinc ions, manganese ions, iron ions, nickel ions, and copper ions.
  • the second object of the present invention is to provide a kind of preparation method of described nano drug, comprising the following steps:
  • the mass ratio of the calcium carbonate nano-particles, immunoregulatory small molecules and metal ions is 1:(1-8):(0.1-4).
  • the preferred mass ratio is 1:(2-2.5):(0.1-1.2).
  • the mass ratio of the nano drug particles, phosphatidic acid series phospholipids, cholesterol, choline series phospholipids, and phosphoethanolamine series phospholipids is (5-20): 1:1: (1-4): (2-8 ).
  • the preferred mass ratio is (10-12):1:1:(2-3):(4-6).
  • the phosphatidic acid series phospholipids mainly include 1,2-dioleoyl-sn-glyceryl-3-phosphate (sodium salt) (DOPA), 1,2-distearoyl-sn-glyceryl- 3-Phosphate (sodium salt) (DSPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA) or 1,2-dimyristoyl-sn-glycero-3 - one or more of phosphoric acid (sodium salt) (DMPA).
  • DOPA 1,2-dioleoyl-sn-glyceryl-3-phosphate
  • DSPA 1,2-distearoyl-sn-glyceryl- 3-Phosphate
  • DPPA 1,2-dipalmitoyl-sn-glycero-3-phosphate
  • DMPA 1,2-dimyristoyl-sn-glycero-3 - one or more of phosphoric
  • the choline series phospholipids mainly include 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine alkali (DSPC), 1-octadecanoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SOPC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine ( DMPC) or one or more of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine alkali
  • SOPC 1-octadecanoyl-2-palmitoyl-sn-glycero-3-phosphocholine
  • SOPC 1-
  • the phosphoethanolamine series phospholipids mainly include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxyl (polyethylene glycol) (DSPE-PEG), 1, 2-Didecanoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol) (DDPE-PEG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate Ethanolamine-N-(methoxyl (polyethylene glycol) (POPE-PEG), 1-stearyl-2-linoleic acid-sn-glycerol-3-phosphoethanolamine-N-(methoxyl (polyethylene glycol) diol), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine-N-(methoxy(polyethylene glycol) (DPPE-PEG), 1,2-dimyristoyl-sn-glycerol
  • the ultrasonic treatment time is 15-25 minutes.
  • nano-medicine can be ultrasonically hydrated with water to obtain a water-soluble nano-medicine solution.
  • the third object of the present invention is to provide the application of the nanomedicine with tumor immune microenvironment regulation function in the preparation of drugs for enhancing tumor radiotherapy.
  • the present invention provides a preparation of nano-medicine with the function of regulating the microenvironment of tumor metabolism, and its synthesis method is simple and easy to operate. After intravenous injection, it can be efficiently enriched into the tumor site, quickly neutralize the protons in the tumor, and alleviate the relief of tumor microacidity; at the same time, the released immune regulatory small molecules can effectively regulate the tumor microenvironment and reverse the immune suppression of tumors. Microenvironment.
  • the invention also discloses the use of the nanometer drug for enhancing tumor radiotherapy.
  • Intravenous injection of nanomedicine combined with radiotherapy can effectively regulate the tumor immunosuppressive microenvironment and reduce the content of immunosuppressive cells in tumors, such as regulatory T cells (regulatory T cells, Tregs), M2 macrophages, myeloid-derived Suppressor cells (myeloid-derived suppressor cells, MDSCs); increase the content of immune cells capable of killing tumors, such as M1 macrophages, natural killer cells (natural killer cells, NKs), CD8 + T lymphocytes.
  • regulatory T cells regulatory T cells
  • Tregs regulatory T cells
  • M2 macrophages myeloid-derived Suppressor cells
  • MDSCs myeloid-derived suppressor cells
  • immune cells capable of killing tumors such as M1 macrophages, natural killer cells (natural killer cells, NKs), CD8 + T lymphocytes.
  • NKs natural killer cells
  • Fig. 1 is the nano-medicine ultraviolet absorption spectrogram and transmission electron micrograph that contain 4PI and zinc ion;
  • Fig. 2 is the ultraviolet absorption spectrogram and the transmission electron microscope figure of the nano-medicine containing 4PI and iron ion;
  • Fig. 3 is the ultraviolet absorption spectrogram and the transmission electron micrograph of the nano-medicine containing NLG919 and manganese ions;
  • Fig. 4 is the ultraviolet absorption spectrogram and the transmission electron micrograph of the nano-medicine containing NLG919 and iron ions;
  • Fig. 5 is the test result of nano medicine property
  • Fig. 6 is the determination that nano-medicine inhibits intracellular IDO activity
  • Figure 7 is the determination of the ability of nanomedicine combined with radiation to cause DNA damage in cells
  • Figure 8 shows the enrichment of nanomedicine in the tumor site after intravenous injection, and its blood circulation time
  • Figure 9 is the result of regulating the tumor immune microenvironment after intravenous injection of nanomedicine.
  • Figure 10 is the tumor growth curves of colon cancer subcutaneous tumors and breast cancer subcutaneous tumors in different groups of mice after treatment;
  • Figure 11 is a schematic diagram of the nanomedicine used to treat bilateral colon cancer models in mice, the growth curves of the right tumors of the colon cancer in mice in different groups after intravenous injection of nanomedicine, and the growth curves of the left tumors of the colon cancer in mice;
  • Figure 12 is the tumor growth curves of mice in different groups after the second inoculation of mouse colon cancer tumors.
  • Embodiment 1 the preparation method of nano medicine
  • Embodiment 1.1 Preparation method of nano-medicine containing 4PI and zinc ions:
  • Figure 1a is the ultraviolet absorption curve of the nanomedicine containing 4PI and zinc ions
  • Figure 1b is its transmission electron microscope image. It can be seen from the ultraviolet absorption curve that the obtained nanomedicine has the characteristic absorption peak of 4PI, and it can be seen from the transmission electron microscope that the calcium carbonate particles indicate that a very thin layer of complex is wrapped. These data indicate that 4PI is successfully coated on the surface of calcium carbonate nanoparticles.
  • Embodiment 1.2 the preparation method of the nano drug containing 4PI and iron ion:
  • Figure 2a is the ultraviolet absorption curve of the nanomedicine containing 4PI and iron ions
  • Figure 2b is its transmission electron microscope image. It can be seen from the ultraviolet absorption curve that the obtained nanomedicine has the characteristic absorption peak of 4PI, and it can be seen from the transmission electron microscope that the calcium carbonate particles indicate that a very thin layer of complex is wrapped. These data indicate that 4PI is successfully coated on the surface of calcium carbonate nanoparticles.
  • Example 1.3 Preparation method of nanomedicine containing NLG919 and manganese ions:
  • Figure 3 is the ultraviolet absorption curve of the nanomedicine containing NLG919 and manganese ions
  • Figure 3b is its transmission electron microscope image. It can be seen from the ultraviolet absorption curve that the obtained nanomedicine has the characteristic absorption peak of NLG919, and from the transmission electron microscope, it can be seen that the calcium carbonate particles are wrapped with a very thin layer of complex, these data indicate that NLG919 was successfully coated on the surface of calcium carbonate nanoparticles.
  • Example 1.4 Preparation method of nanomedicine containing NLG919 and iron ions:
  • Figure 4a is the ultraviolet absorption curve of the nanomedicine containing NLG919 and iron ions
  • Figure 4b is its transmission electron microscope image. It can be seen from the ultraviolet absorption curve that the obtained nanomedicine has the characteristic absorption peak of NLG919, and from the transmission electron microscope, it can be seen that the calcium carbonate particles are wrapped with a very thin layer of complex, these data indicate that NLG919 was successfully coated on the surface of calcium carbonate nanoparticles.
  • the above-prepared nano-medicines are modified. Dissolve nano-drugs and DOPA in ethanol and chloroform respectively according to a certain mass ratio, then mix them, sonicate for 20 minutes, and centrifuge to obtain nano-drug complexes coated with DOPA on the surface; weigh a certain mass of cholesterol, DPPC, DSPE- PEG and DOPA-coated nano-drugs were dispersed into chloroform, mixed and stirred overnight, chloroform was removed by rotary evaporation, water was added for ultrasonic hydration, and finally a modified nano-drug solution dispersed in the water phase was obtained.
  • nano-medicines are based on nano-medicines containing 4PI and zinc ions.
  • Embodiment 2 the detection of nano drug properties
  • the nanomedicine prepared in Example 1 was qualitatively detected, and the transmission electron microscope detection, stability detection, proton neutralization ability detection, and acid-responsive drug release ability detection were respectively carried out.
  • the ethanol-phase nanomedicine prepared in Example 1 was detected by transmission electron microscopy, as shown in Figure 5a, the results show that the nanomedicine prepared in Example 1 has uniform particle size and uniform shape.
  • the modified nano-medicine obtained in Example 1 was tested for stability, and dispersed in different solvents (H 2 O, PBS, 0.9% NaCl, RPMI 1640), by detecting the particle size change of the nano-medicine in different solutions to judge its stability. As shown in Figure 5b, the modified nanomedicine prepared in Example 1 maintains a stable particle size in different solutions, and there is no significant particle size change within 24h, indicating that the modified nanomedicine prepared in Example 1 Has good stability.
  • solvents H 2 O, PBS, 0.9% NaCl, RPMI 1640
  • the proton neutralization ability of the modified nanomedicine prepared in Example 1 was tested, as shown in Figure 5c, the results show that the modified nanomedicine prepared in Example 1 has a proton neutralization ability similar to that of calcium carbonate, and can quickly neutralize and protons in acidic solution.
  • the acid-responsive drug release ability of the modified nano-drug prepared in Example 1 was tested. Transfer the nanomedicine to a dialysis bag and immerse it in different pH buffer solutions (i.e. pH 7.4 solution, pH 6.5 solution, pH 5.5 solution) at room temperature. At predetermined time points, the external solution was collected and the concentration of 4PI was measured with a UV-Vis spectrophotometer. The test results are shown in Figure 5d, and the results show that the modified nanomedicine prepared in Example 1 shows better drug release ability under acidic conditions.
  • pH buffer solutions i.e. pH 7.4 solution, pH 6.5 solution, pH 5.5 solution
  • Embodiment 3 The IDO inhibitory property detection of 4PI in nanomedicine
  • IDO1 inhibitor 4PI Due to the presence of indoleamine 2,3-dioxygenase (IDO1) inhibitor 4PI in the modified nano-medicine obtained in Example 1, which can inhibit the activity of IDO1 enzymes, the modified nano-medicine obtained in Example 1 was detected. IDO1 inhibitory effects of drugs. Add 4PI and the modified nanomedicine prepared in Example 1 to colon cancer cells at the same time as IFN- ⁇ and incubate for 48 hours, then add 30% trichloroacetic acid, and incubate at 50°C for 6 hours to hydrolyze formylkynurenine For kynurenine. Finally, add Ehrlich reagent, develop color at room temperature for 10 min, and measure absorption at 490 nm wavelength.
  • IDO1 inhibitory effects of drugs Add 4PI and the modified nanomedicine prepared in Example 1 to colon cancer cells at the same time as IFN- ⁇ and incubate for 48 hours, then add 30% trichloroacetic acid, and incubate at 50°C for 6 hours to hydro
  • Embodiment 4 Detection of cellular DNA damage ability caused by nanomedicine combined with radiation
  • Example 1 Since the modified nanomedicine prepared in Example 1 has good proton neutralization ability, we tested the ability of the nanomedicine to reverse acidic radiation resistance. We cultured the cells in pH 6.5 and pH 7.4 media, then treated the cells with nanomedicine, exposed them to X-rays after a period of time, and detected the X - ray exposure-induced DNA double-strand break.
  • the test results are shown in Figure 7.
  • the results showed that the intracellular ⁇ -H 2 AX signal of cells treated with nanomedicine, pH 6.5 medium and X-ray irradiation was significantly different from that of cells treated only with pH 7.4 medium and X-ray irradiation. Comparable, but much higher than cells treated only with pH 6.5 medium and X-ray irradiation. Therefore, the prepared nanomedicine treatment can induce more DNA double-strand breaks after exposure to X-rays, which can reverse the effect of acidic radiation resistance.
  • Example 5 In vivo behavior of nanomedicine in mice after tail vein injection
  • the nanomedicine prepared in Example 1 in mice after intravenous injection, we labeled the nanomedicine with small fluorescent molecules, and used in vivo fluorescence imaging to understand its behavior in mice.
  • the nano-drug complex labeled with fluorescent small molecule DiR was injected intravenously into mice with colon cancer tumors, and at different time points, real-time pictures were collected using a small animal in vivo imaging system. Observe the enrichment of nano-medicine in the tumor site.
  • the test results are shown in Figure 8a. The results show that the fluorescent signal of the tumor site gradually becomes stronger with time, and still maintains a high fluorescent signal at the time point of 48 hours, indicating that the nanomedicine has good tumor accumulation behavior. .
  • nano-drugs labeled with DiR fluorescent molecules were injected intravenously into mice, blood was collected at specific time points, and the concentration of drugs in the blood was determined by cleavage. Fluorescence signal value, and then calculate the blood circulation half-life of the nanomedicine.
  • the test results are shown in Figure 8b, and the results show that the nano drug complex has a rather long blood circulation time.
  • Embodiment 6 Nanomedicine regulates tumor immunosuppressive microenvironment ability detection
  • the nanomedicine was intravenously injected into mice with colon cancer tumors.
  • pre-injection (0 h) and post-injection (24 h) microelectrode probes were used to monitor the changes in intratumoral pH in mice before and after intravenous injection of the material.
  • the test results are shown in Figure 9a, and the results showed that the pH value in the mouse tumors showed a significant increase 24 h after intravenous injection of the material, indicating that the nanomedicine can serve as an effective tumor acidity regulator.
  • nanomedicine was injected intravenously into mice with colon cancer tumors. After 24 hours, the tumor was removed, lysed by homogenate, and the contents of tryptophan (Trp) and kynurenine (Kyn) in the tumor were determined by HPLC. The test results are shown in Figure 9b. The results showed that the ratio of Kyn/Trp in the tumor treated with the nanomedicine showed a significant decrease, indicating that the activity of IDO1 was significantly inhibited.
  • Embodiment 7 the treatment of different groups of mouse colon cancer subcutaneous tumors and mouse subcutaneous breast cancer tumors
  • the present invention also explores the treatment of different tumors by intravenous injection of nanomedicine combined with radiotherapy.
  • mice with subcutaneous tumor models of colon cancer were divided into six groups, including: the first group, the control group (injection of normal saline only); the second group, the radiotherapy treatment group; the third group, the intravenous injection of calcium carbonate combined with radiotherapy The treatment group; the fourth group, the intravenous injection of 4PI-Zn combined with radiotherapy treatment group; the fifth group, the intravenous injection of nano-medicine treatment group; the sixth group, the intravenous injection of nano-medicine combined with radiotherapy treatment group. Twenty-four hours after the intravenous injection of the material, the tumor site of the mice was irradiated with radiation at a dose of 5Gy, and then the growth of the tumor was measured. The results are shown in FIG. 10 .
  • Figure 10b is the tumor growth curves of mice in different treatment groups
  • Figure 10c is the survival curves of different treatment groups. The results showed that compared with the first, second, third, fourth, and fifth groups, the tumor growth in the sixth group was effectively inhibited, indicating that nanomedicine combined with radiotherapy can achieve efficient treatment of tumors.
  • mice with subcutaneous tumor models of breast cancer were divided into six groups, including: the first group, the control group (only injected with normal saline); the second group , the radiotherapy treatment group; the third group, the intravenous injection of calcium carbonate combined with radiotherapy treatment group; the fourth group, the intravenous injection of 4PI-Zn combined with radiotherapy treatment group; the fifth group, the intravenous injection of nano drug treatment group; the sixth group, the intravenous injection of nano Drug combined with radiotherapy treatment group.
  • the tumor site of the mice was irradiated with radiation at a dose of 5Gy, and then the growth of the tumor was measured.
  • FIG. 10 The results are shown in FIG. 10 .
  • Figure 10e is the growth curves of tumors in different treatment groups
  • Figure 10f is the survival curves of different treatment groups.
  • the results showed that compared with the first, second, third, fourth, and fifth groups, the tumor growth of the sixth group was slightly inhibited, indicating that nanomedicine combined with radiotherapy can not only achieve high-efficiency treatment of immunogenic tumors, but also Treatment of poorly immunogenic tumors can also be achieved.
  • Example 8 Nanomedicine combined with radiotherapy for the treatment of bilateral tumors of colon cancer in mice
  • mice with bilateral colon cancer subcutaneous tumors were randomly divided into four groups, including: the first group, the control group (only injected with saline); the second group, the radiotherapy treatment group; the third group, the intravenous injection of nano-medicine treatment group; The fourth group, intravenous injection of nano-medicine combined with radiotherapy treatment group.
  • the tumor growth was measured, and the results are shown in FIG. 11 .
  • Figures 11a and 11b are the growth curves of primary tumors and distant tumors in mice in different treatment groups, respectively. The results showed that, compared with the control group, the left tumor growth of the mice treated with intravenous injection of nanomedicine combined with radiotherapy was effectively inhibited, indicating that the nanomedicine compound combined with radiotherapy can inhibit distant metastases.
  • Example 9 Immunological memory of mouse subcutaneous tumor models of colon cancer in different groups after treatment
  • mice cured from the subcutaneous tumor model of colon cancer in Example 7 (Group 6, group treated with nanomedicine combined with radiotherapy) were inoculated again with the same number of colon cancer cells to construct tumors.
  • the growth of the tumor was measured, and the results are shown in FIG. 12 .
  • Figure 12 shows the growth curves of mice in different treatment groups. Compared with the control group, the tumor growth of cured mice in the sixth group was significantly inhibited. It shows that nanomedicine combined with radiotherapy can stimulate strong immune memory and inhibit tumor recurrence.
  • the immune memory cells in the cured group were at a higher level, and TNF- ⁇ and IFN- ⁇ were stimulated by reinoculation of tumors, compared with the control group.
  • a higher level can achieve the killing effect on recurrent tumors.

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Abstract

一种具有肿瘤免疫微环境调节功能的纳米药物，包括：由碳酸钙纳米颗粒和包覆在碳酸钙纳米颗粒表面的复合物形成的纳米药物颗粒，以及，用于负载纳米药物颗粒的脂质体载体；其中，复合物由免疫调控小分子和金属离子通过配位作用形成。一种具有肿瘤免疫微环境调节功能的纳米药物的制备方法及其在制备增强肿瘤放射治疗的药物中的应用。通过静脉注射，纳米药物在肿瘤部位具有高效的富集行为及相当长的血液循环时间，可实现快速的质子中和，调节肿瘤的微酸环境，释放的免疫调控小分子可有效地调节肿瘤微环境，逆转免疫抑制肿瘤微环境。通过联合放射治疗，增强了放射治疗的疗效。

Description

具有肿瘤免疫微环境调节功能的纳米药物及其制备方法与应用 技术领域
本发明涉及一种具有肿瘤免疫微环境调节功能的纳米药物及其制备方法与应用，属于肿瘤治疗纳米药物技术领域。
背景技术
截至目前，肿瘤仍然是威胁人类健康首要的疾病。随着人们对肿瘤生物学及其代谢的深入了解，研究者发现代谢重编程是肿瘤的典型特征之一，也是作为研究肿瘤治疗的重要潜在靶点。癌细胞代谢通路的改变被认为是肿瘤发生、转移以及治疗失败的主要原因之一。越来越多的研究表明，癌细胞通过低效的糖酵解方式来快速产生ATP和其它代谢中间体，以此来维持肿瘤细胞的快速增殖。乳酸，作为糖酵解代谢的一种副产物，其在肿瘤内的积聚导致了肿瘤微环境的酸化，使得免疫细胞的功能障碍，促进了肿瘤的不断恶化。除此之外，为了适应肿瘤的快速生长，肿瘤微环境内的不同细胞也表达了高水平的营养消耗相关外酶，比如吲哚胺2,3-双加氧酶(IDO1)、胞外-5’-核苷酸酶(CD73)。其中吲哚胺2,3-双加氧酶(IDO1)是色氨酸(Trp)沿犬尿氨酸(Kyn)途径代谢的关键酶，其催化生成的犬尿氨酸抑制了T细胞的活化。肿瘤微环境的酸化和犬尿氨酸的生成导致了免疫抑制微环境的产生，从而使得肿瘤细胞逃避了免疫细胞的监测，促使了肿瘤的增殖。虽然目前有多种小分子抑制剂，比如吲哚胺2,3-双加氧酶(IDO1)抑制剂，被用来抑制肿瘤细胞内相关酶的活性以逆转免疫抑制性微环境，从而协同增效传统的肿瘤治疗，但是这些小分子抑制剂在实际的临床转化应用中遇到了重重困难，例如，分子水溶性较差、容易产生耐药性、缺乏肿瘤选择性和靶向性等。鉴于纳米医学的快速发展，开发一种具有高装载率的药物载体有望实现这些小分子抑制剂的肿瘤靶向递送，进而重塑免疫抑制代谢肿瘤微环境，有效提高肿瘤治疗的疗效。
此外，放射治疗(radiotherapy，RT)是一种利用高能量射线产生活性氧(reactive oxygen species，ROS)从而杀死癌细胞的治疗手段，是目前临床肿瘤治疗中使用最多的治疗方式。但是，大量的研究表明，放射治疗的疗效在很大程度上受到了肿瘤微环 境的影响，其临床治疗结果远远不尽人意。鉴于氧气是放疗过程中产生活性氧的来源，研究者们设计了各种策略来试图改善肿瘤部位的乏氧，并且获得了积极的疗效。有研究发现肿瘤细胞的微酸环境会使得其对射线更加耐受，且碱性条件下的细胞对射线的耐受程度会降低。尽管相关的pH依赖性放疗耐受机制没有详细的解释，但是已经有相关研究表明，中和肿瘤微环境内的微酸将会增强射线对肿瘤细胞的杀伤能力。因此，开发一种能够快速中和质子和实现肿瘤微环境的有效调节，协同增效肿瘤放射治疗的纳米药物是非常有必要的。
发明内容
为解决上述技术问题，本发明提供一种制备简单，并且能够高效富集到肿瘤部位，快速中和质子，且释放的免疫调控小分子能够有效地调节肿瘤微环境，逆转肿瘤细胞代谢导致的免疫抑制性微环境的纳米药物。其与放疗联用，能够高效地抑制肿瘤的生长，同时通过激起机体的免疫反应，能够抑制肿瘤的转移和复发。
本发明的第一个目的是提供一种具有肿瘤免疫微环境调节功能的纳米药物，所述纳米药物包括：由碳酸钙纳米颗粒和包覆在碳酸钙纳米颗粒表面的复合物形成的纳米药物颗粒，以及，用于负载所述纳米药物颗粒的脂质体载体；其中，所述的复合物由免疫调控小分子和金属离子通过配位作用形成。
进一步地，所述的碳酸钙纳米颗粒得粒径为60～300nm。优选80～160nm。
进一步地，所述的免疫调控小分子为吲哚胺2,3-双加氧酶抑制剂、转化生长因子-β(TGF-β)抑制剂、白介素受体抑制剂、氧化磷酸化抑制剂、PD-1/PD-L1相互作用抑制剂中的一种或多种。
进一步地，所述的吲哚胺2,3-双加氧酶抑制剂为4-苯基咪唑(4PI)或那伏莫德(NLG919)。
进一步地，所述的转化生长因子-β(TGF-β)抑制剂为SB431542、LY364947、SB505124或SB525334。
进一步地，所述的白介素受体抑制剂为地塞米松磷酸钠。
进一步地，所述的氧化磷酸化抑制剂为4-甲基-2-氧代戊酸。
进一步地，所述的PD-1/PD-L1相互作用抑制剂为BMS-1001、BMS-1166或BMS-1。
进一步地，所述的金属离子为锌离子、锰离子、铁离子、镍离子、铜离子中的一种或多种。
本发明的第二个目的是提供一种所述的纳米药物的制备方法，包括如下步骤：
S1、将碳酸钙纳米颗粒、免疫调控小分子和金属离子溶于无水乙醇中，搅拌反应，离心洗涤后，得到纳米药物颗粒；
S2、将纳米药物颗粒溶于无水乙醇中，与磷脂酸系列磷脂的氯仿溶液混合，超声处理后离心，得到包裹有磷脂酸系列磷脂的纳米药物颗粒；
S3、将包裹有磷脂酸系列磷脂的纳米药物颗粒溶于氯仿，与胆固醇、胆碱系列磷脂和磷酸乙醇胺系列磷脂混合搅拌处理10～30小时，去除氯仿后得到所述的纳米药物。
进一步地，所述的碳酸钙纳米颗粒、免疫调控小分子和金属离子的质量比为1:(1～8):(0.1～4)。优选质量比为1:(2～2.5):(0.1～1.2)。
进一步地，所述的纳米药物颗粒、磷脂酸系列磷脂、胆固醇、胆碱系列磷脂、磷酸乙醇胺系列磷脂的质量比为(5～20)：1：1：(1～4)：(2～8)。优选质量比为(10～12):1:1:(2～3):(4～6)。
进一步地，所述的磷脂酸系列磷脂主要包括1,2-二油酰-sn-甘油基-3-磷酸(钠盐)(DOPA)、1,2-二硬脂酰-sn-甘油基-3-磷酸(钠盐)(DSPA)、1,2-二棕榈酰-sn-甘油基-3-磷酸(钠盐)(DPPA)或1,2-二肉豆蔻酰-sn-甘油基-3-磷酸(钠盐)(DMPA)中的一种或多种。
进一步地，所述的胆碱系列磷脂主要包括1,2-二棕榈酰-sn-甘油-3-磷酸胆碱(DPPC)、1,2-二硬脂酰-sn-甘油-3-磷酸胆碱(DSPC)、1-十八烷酰-2-棕榈酰-sn-甘油-3-磷酸胆碱(SOPC)、1-豆蔻酰-2-棕榈酰-sn-甘油-3-磷酸胆碱(DMPC)或1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)中的一种或多种。
进一步地，所述的磷酸乙醇胺系列磷脂主要包括1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)(DSPE-PEG)、1,2-二癸酰基-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)(DDPE-PEG)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)(POPE-PEG)、1-硬脂酰-2-亚油酸-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)(DPPE-PEG)、1,2-二豆蔻酰基-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)(DMPE-PEG)或1,2-二油酰基-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)(DOPE-PEG)中的一种或多种。
进一步地，在S2步骤中，超声处理的时间为15～25分钟。
进一步地，所述的纳米药物可加水超声水化处理，得到溶于水的纳米药物溶液。
本发明的第三个目的是提供所述的具有肿瘤免疫微环境调节功能的纳米药物在制备增强肿瘤放射治疗的药物中的应用。
本发明的有益效果是：
本发明提供了一种具有肿瘤代谢微环境调节功能的纳米药物制备，其合成方法简单易操 作。在静脉注射后，其能够高效地富集到肿瘤部位，快速地中和肿瘤中的质子，缓解肿瘤微酸缓解；同时释放的免疫调控小分子，可以有效调节肿瘤微环境，逆转了免疫抑制肿瘤微环境。
本发明还公开了纳米药物用于增强肿瘤放疗的用途。静脉注射纳米药物，同时联合放疗，能够有效调控肿瘤免疫抑制微环境，减少肿瘤中免疫抑制性细胞的含量，如调节性T细胞(regulatory T cells,Tregs)，M2巨噬细胞，髓系来源的抑制性细胞(myeloid-derived suppressor cells，MDSCs)；增加能够杀伤肿瘤的免疫细胞的含量，如M1巨噬细胞，自然杀伤细胞(natural killer cells,NKs)，CD8 +T淋巴细胞。有效扭转肿瘤免疫抑制微环境，激活机体的免疫反应，抑制肿瘤的转移和复发。
附图说明：
图1为含有4PI和锌离子的纳米药物紫外吸收谱图和透射电镜图；
图2为含有4PI和铁离子的纳米药物紫外吸收谱图和透射电镜图；
图3为含有NLG919和锰离子的纳米药物紫外吸收谱图和透射电镜图；
图4为含有NLG919和铁离子的纳米药物紫外吸收谱图和透射电镜图；
图5为纳米药物性质的测试结果；
图6为纳米药物抑制细胞内IDO活性的测定；
图7为纳米药物联合射线引起细胞内DNA damage的能力测定；
图8为静脉注射纳米药物后在肿瘤部位的富集情况，以及其血液循环时间；
图9为静脉注射纳米药物后调节肿瘤免疫微环境的结果；
图10为不同分组的小鼠结肠癌皮下肿瘤和乳腺癌皮下肿瘤在治疗后的肿瘤生长曲线；
图11为纳米药物用于治疗小鼠双边结肠癌模型示意图、不同分组的小鼠结肠癌右侧肿瘤在静脉注射纳米药物治疗后的生长曲线、小鼠结肠癌左侧肿瘤的生长曲线；
图12为不同分组的小鼠在第二次接种小鼠结肠癌肿瘤后肿瘤的生长曲线。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：纳米药物的制备方法
实施例1.1：含有4PI和锌离子的纳米药物制备方法：
S1：称取12mg的4PI溶于1mL的乙醇中，然后将其加入到2mL的纳米碳酸钙乙醇溶液中(3mg mL -1)，常温搅拌5min；
S2：称取一定质量的硝酸锌(Zn：6mg)，溶于1mL的乙醇中，然后将其加入到含有 纳米碳酸钙和4PI的混合溶液中，继续搅拌反应4h；
S3：14800rpm离心，用无水乙醇洗涤三次，重悬于1mL的乙醇中备用。
获得的纳米药物的相关表征如图1所示，其中图1a为含有4PI和锌离子纳米药物的紫外吸收曲线，图1b为其透射电镜图。从紫外吸收曲线可以看出，获得的纳米药物具有4PI的特征吸收峰，且从透射电镜图可以看出，碳酸钙颗粒表明包裹了很薄的一层复合物，这些数据表明4PI被成功包覆在碳酸钙纳米颗粒表面。
实施例1.2：含有4PI和铁离子的纳米药物制备方法：
S1：称取12mg的4PI溶于1mL的乙醇中，然后将其加入到2mL的纳米碳酸钙乙醇溶液中(3mg mL -1)，常温搅拌5min；
S2：称取一定质量的六水合氯化铁(Fe：0.6mg)，溶于1mL的乙醇中，然后将其加入到含有纳米碳酸钙和4PI的混合溶液中，继续搅拌反应4h；
S3：14800rpm离心，用无水乙醇洗涤三次，重悬于1mL的乙醇中备用。
获得的纳米药物的相关表征如图2所示，其中图2a为含有4PI和铁离子纳米药物的紫外吸收曲线，图2b为其透射电镜图。从紫外吸收曲线可以看出，获得的纳米药物具有4PI的特征吸收峰，且从透射电镜图可以看出，碳酸钙颗粒表明包裹了很薄的一层复合物，这些数据表明4PI被成功包覆在碳酸钙纳米颗粒表面。
实施例1.3：含有NLG919和锰离子的纳米药物制备方法：
S1：称取12mg的NLG919溶于1mL的乙醇中，然后将其加入到2mL的纳米碳酸钙乙醇溶液中(3mg mL -1)，常温搅拌5min；
S2：称取一定质量的氯化锰(Mn：3mg)，溶于1mL的乙醇中，然后将其加入到含有纳米碳酸钙和NLG919的混合溶液中，继续搅拌反应4h；
S3：14800rpm离心，用无水乙醇洗涤三次，重悬于1mL的乙醇中备用。
获得的纳米药物的相关表征如图3所示，其中图3a为含有NLG919和锰离子纳米药物的紫外吸收曲线，图3b为其透射电镜图。从紫外吸收曲线可以看出，获得的纳米药物具有NLG919的特征吸收峰，且从透射电镜图可以看出，碳酸钙颗粒表明包裹了很薄的一层复合物，这些数据表明NLG919被成功包覆在碳酸钙纳米颗粒表面。
实施例1.4：含有NLG919和铁离子的纳米药物制备方法：
S1：称取12mg的NLG919溶于1mL的乙醇中，然后将其加入到2mL的纳米碳酸钙乙醇溶液中(3mg mL -1)，常温搅拌5min；
S2：称取一定质量的硝酸锌(Fe：1.2mg)，溶于1mL的乙醇中，然后将其加入到含有纳米碳酸钙和NLG919的混合溶液中，继续搅拌反应4h；
S3：14800rpm离心，用无水乙醇洗涤三次，重悬于1mL的乙醇中备用。
获得的纳米药物的相关表征如图4所示，其中图4a为含有NLG919和铁离子纳米药物的紫外吸收曲线，图4b为其透射电镜图。从紫外吸收曲线可以看出，获得的纳米药物具有NLG919的特征吸收峰，且从透射电镜图可以看出，碳酸钙颗粒表明包裹了很薄的一层复合物，这些数据表明NLG919被成功包覆在碳酸钙纳米颗粒表面。
对以上制备得到的纳米药物进行修饰。将纳米药物和DOPA按照一定的质量比分别溶于乙醇和氯仿中，然后将其混合，超声20min，离心，得到表面包裹有DOPA的纳米药物复合物；称取一定质量的胆固醇、DPPC、DSPE-PEG，和包裹有DOPA的纳米药物一起分散到氯仿中，混合搅拌过夜，旋蒸去除氯仿，加水超声水化，最终得到修饰后分散在水相中的纳米药物溶液。
以下关于纳米药物的实施例均是基于含有4PI和锌离子的纳米药物进行的实施例。
实施例2：纳米药物性质的检测
对实施例1制得的纳米药物进行定性检测，分别进行透射电镜检测、稳定性检测、质子中和能力检测、酸响应药物释放能力检测。
对实施例1制得的乙醇相纳米药物进行透射电镜检测，如图5a，结果显示，实施例1制得的纳米药物的粒径均一、形貌均一。
对实施例1制得修饰后的纳米药物进行稳定性检测，将其分散于不同溶剂中(H 2O，PBS，0.9％NaCl，RPMI 1640)，通过检测纳米药物在不同溶液中的粒径变化来判断其稳定性。如图5b所示，实施例1制得修饰后的纳米药物在不同的溶液中均保持稳定的粒径，且24h之内没有显著的粒径变化，表明实施例1制得修饰后的纳米药物具有良好的稳定性。
对实施例1制得修饰后的纳米药物进行质子中和能力检测，如图5c，结果显示，实施例1制得的修饰后纳米药物具有和碳酸钙相似的质子中和能力，能够快速地中和酸性溶液中的质子。
对实施例1制得修饰后的纳米药物进行酸响应药物释放能力检测。将纳米药物转移到透析袋中，室温下浸入到不同pH缓冲溶液中(即pH 7.4溶液、pH 6.5溶液、pH 5.5溶液)。在预定的时间点，收集外部溶液，用紫外-可见分光光度计测定4PI浓度。测试结果如图5d所示，结果显示，实施例1制得修饰后的纳米药物在酸性条件下显示出更好的药物释放能力。
实施例3：纳米药物中4PI的IDO抑制性质检测
由于实施例1制得修饰后的纳米药物中存在吲哚胺2,3-双加氧酶(IDO1)抑制剂4PI，其能够抑制IDO1酶的活性，因此检测实施例1制得修饰后的纳米药物的IDO1抑制效果。将4PI与实施例1制得修饰后的纳米药物分别与IFN-γ同时加入结肠癌细胞中孵育48h，再 加入30％三氯乙酸，在50℃下孵育6h，将甲酰犬尿氨酸水解为犬尿氨酸。最后加入埃利希试剂，室温显色10min，测490nm波长处吸收。
测试结果如图6所示，结果显示，实施例1制得修饰后的纳米药物具有与单独4PI相似的IDO1抑制性能。
实施例4：纳米药物联合射线引起的细胞DNA damage能力检测
由于实施例1制得修饰后的纳米药物具有良好的质子中和能力，因此我们检测了纳米药物对逆转酸性辐射耐药的能力。我们通过将细胞在pH 6.5和pH 7.4的培养基中培养，然后对细胞进行纳米药物的处理，一段时间后，暴露于X射线，之后通过γ-H 2AX免疫荧光染色检测X射线暴露诱导的DNA双链断裂。
测试结果如图7所示，结果显示，经纳米药物、pH 6.5培养基和X射线照射处理的细胞，其胞内γ-H 2AX信号与仅受pH 7.4培养基和X射线照射处理的细胞相当，但远高于仅受pH6.5培养基和X射线照射处理的细胞。因此，经所制备得的纳米药物处理，在暴露于X射线后可以诱导更多的DNA双链断裂，可以逆转酸性辐射耐药的影响。
实施例5：纳米药物尾静脉注射后在小鼠的体内行为
为了了解实施例1制得修饰后的纳米药物通过静脉注射后在小鼠体内的行为，我们将纳米药物标记上荧光小分子，通过活体荧光成像来了解其在小鼠体内的行为。简单来说，将标记有荧光小分子DiR的纳米药物复合物通过静脉注射到带有结肠癌肿瘤的小鼠体内，在不同的时间点，用小动物活体成像系统对小鼠进行实时图片采集，观察纳米药物在肿瘤部位的富集量。测试结果如图8a所示，结果显示，随着时间的迁移，肿瘤部位的荧光信号逐渐变强，在48h时间点仍然保持较高的荧光信号，表明该纳米药物具有很好的肿瘤富集行为。
除此之外，为了了解纳米药物在小鼠体内的血液循环行为，将标记有DiR荧光分子的纳米药物通过静脉注射到小鼠体内，在特定的时间点取血，通过裂解测定血液中药物的荧光信号值，然后计算出纳米药物的血液循环半衰期。测试结果如图8b所示，结果显示，该纳米药物复合物具有相当长的血液循环时间。
实施例6:纳米药物调节肿瘤免疫抑制性微环境能力检测
为了检测实施例1制得修饰后的纳米药物调节肿瘤内酸性微环境的能力，将纳米药物通过静脉注射到带有结肠癌肿瘤的小鼠体内。在注射前(0h)和注射后(24h)这两个时间点，使用微电极探针监测小鼠肿瘤内pH值在静脉注射材料前后的变化。测试结果如图9a所示，结果显示，小鼠肿瘤内的pH值在静脉注射材料后24h表现出明显的增加，表明该纳米药物可以作为有效的肿瘤酸性调节剂。
同时，为了检测纳米药物抑制肿瘤内IDO1活性的能力，将纳米药物通过静脉注射到带 有结肠癌肿瘤的小鼠体内。24小时之后，将肿瘤取下，通过匀浆裂解，使用HPLC测定肿瘤内的色氨酸(Trp)和犬尿氨酸(Kyn)的含量。测试结果如图9b所示，结果显示，经该纳米药物处理后的肿瘤内Kyn/Trp的比值表现出明显的下降，说明IDO1的活性受到了显著性的抑制。
实施例7：不同分组的小鼠结肠癌皮下肿瘤、小鼠皮下乳腺癌肿瘤的治疗
在以上结果的基础上，本发明还探究了静脉注射纳米药物联合放疗对于不同肿瘤的治疗情况。
将带有结肠癌皮下肿瘤模型的小鼠分为六组，其中包括：第一组，对照组(仅注射生理盐水)；第二组，放疗治疗组；第三组，静脉注射碳酸钙联合放疗治疗组；第四组，静脉注射4PI-Zn联合放疗治疗组；第五组，静脉注射纳米药物治疗组；第六组，静脉注射纳米药物联合放疗治疗组。静脉注射材料后24小时，对小鼠的肿瘤部位照射线，射线的剂量为5Gy，之后测量肿瘤的生长，结果见图10。图10b为不同治疗组小鼠肿瘤的生长曲线，图10c为不同治疗组的生存曲线。结果表明，相比较于第一、二、三、四、五组，第六组的肿瘤生长得到了有效的抑制，表明纳米药物联合放疗治疗能够实现对肿瘤的高效治疗。
为了验证上述治疗能对免疫原性弱的肿瘤也有效果，将带有乳腺癌皮下肿瘤模型的小鼠分为六组，其中包括：第一组，对照组(仅注射生理盐水)；第二组，放疗治疗组；第三组，静脉注射碳酸钙联合放疗治疗组；第四组，静脉注射4PI-Zn联合放疗治疗组；第五组，静脉注射纳米药物治疗组；第六组，静脉注射纳米药物联合放疗治疗组。静脉注射材料后24小时，对小鼠的肿瘤部位照射线，射线的剂量为5Gy，之后测量肿瘤的生长，结果见图10。图10e为不同治疗组小鼠肿瘤的生长曲线，图10f为不同治疗组的生存曲线。结果表明，相比较于第一、二、三、四、五组，第六组的肿瘤生长得到了轻微的抑制，表明纳米药物联合放疗治疗不仅能够实现对免疫原性强的肿瘤的高效治疗，还能够实现免疫原性弱的肿瘤的治疗。
实施例8：纳米药物联合放疗用于小鼠结肠癌双边瘤的治疗
将双边结肠癌皮下肿瘤的小鼠随机分为四组，其中包括：第一组，对照组(仅注射生理盐水)；第二组，放疗治疗组；第三组，静脉注射纳米药物治疗组；第四组，静脉注射纳米药物联合放疗治疗组。对小鼠进行相应的治疗后，测量其肿瘤的生长，结果见图11。图11a、11b分别为不同治疗组小鼠原发肿瘤、远端肿瘤的生长曲线。结果表明，与对照组相比，静脉注射纳米药物联合放疗治疗组小鼠左侧肿瘤生长被有效的抑制，表明纳米药物复合物联合放疗能够实现对远端转移瘤的抑制。
实施例9：不同分组的小鼠结肠癌皮下肿瘤模型在治疗后免疫记忆的情况
将实施例7中结肠癌皮下肿瘤模型的小鼠治愈的小鼠(第六组，纳米药物联合放疗治疗组)，再次接种同样数量的结肠癌细胞构建肿瘤。测量其肿瘤的生长，结果见于图12。图12为不同治疗组小鼠的生长曲线，相比对照组，第六组治愈的小鼠肿瘤的生长得到了明显的抑制。表明纳米药物联合放疗能够激起很强的免疫记忆抑制肿瘤的复发。同时对血液中免疫细胞的检测我们发现，相比于对照组，治愈组的免疫记忆细胞处于较高水平，且TNF-α与IFN-γ在再次接种肿瘤的刺激下，相比于对照组处于较高的水平，实现对复发肿瘤的杀伤作用。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (10)

  1. 一种具有肿瘤免疫微环境调节功能的纳米药物，其特征在于，所述纳米药物包括：由碳酸钙纳米颗粒和包覆在碳酸钙纳米颗粒表面的复合物形成的纳米药物颗粒，以及，用于负载所述纳米药物颗粒的脂质体载体；其中，所述的复合物由免疫调控小分子和金属离子通过配位作用形成。
  2. 根据权利要求1所述的纳米药物，其特征在于，所述的碳酸钙纳米颗粒得粒径为60～300nm。
  3. 根据权利要求1所述的纳米药物，其特征在于，所述的免疫调控小分子为吲哚胺2,3-双加氧酶抑制剂、转化生长因子-β抑制剂、白介素受体抑制剂、氧化磷酸化抑制剂、PD-1/PD-L1相互作用抑制剂中的一种或多种。
  4. 根据权利要求1所述的纳米药物，其特征在于，所述的金属离子为锌离子、锰离子、铁离子、镍离子、铜离子中的一种或多种。
  5. 一种权利要求1～4任一项所述的具有肿瘤免疫微环境调节功能的纳米药物的制备方法，其特征在于，包括如下步骤：
    S1、将碳酸钙纳米颗粒、免疫调控小分子和金属离子溶于无水乙醇中，搅拌反应，离心洗涤后，得到纳米药物颗粒；
    S2、将纳米药物颗粒溶于无水乙醇中，与磷脂酸系列磷脂的氯仿溶液混合，超声处理后离心，得到包裹有磷脂酸系列磷脂的纳米药物颗粒；
    S3、将包裹有磷脂酸系列磷脂的纳米药物颗粒溶于氯仿，与胆固醇、胆碱系列磷脂和磷酸乙醇胺系列磷脂混合搅拌处理10～30小时，去除氯仿后得到所述的纳米药物。
  6. 根据权利要求5所述的方法，其特征在于，所述的碳酸钙纳米颗粒、免疫调控小分子和金属离子的质量比为1:(1～8):(0.1～4)。
  7. 根据权利要求5所述的方法，其特征在于，所述的纳米药物颗粒、磷脂酸系列磷脂、胆固醇、胆碱系列磷脂、磷酸乙醇胺系列磷脂的质量比为(5～20)：1：1：(1～4)：(2～8)。
  8. 根据权利要求5所述的方法，其特征在于，所述的磷脂酸系列磷脂主要包括1,2-二油酰-sn-甘油基-3-磷酸、1,2-二硬脂酰-sn-甘油基-3-磷酸、1,2-二棕榈酰-sn-甘油基-3-磷酸或1,2-二肉豆蔻酰-sn-甘油基-3-磷酸中的一种或多种；所述的胆碱系列磷脂主要包括1,2-二棕榈酰-sn-甘油-3-磷酸胆碱、1,2-二硬脂酰-sn-甘油-3-磷酸胆碱、1-十八烷酰-2-棕榈酰-sn-甘油-3-磷酸胆碱、1-豆蔻酰-2-棕榈酰-sn-甘油-3-磷酸胆碱或1,2-二油酰基 -sn-甘油-3-磷酸胆碱中的一种或多种；所述的磷酸乙醇胺系列磷脂主要包括1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)、1,2-二癸酰基-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)、1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)、1-硬脂酰-2-亚油酸-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)、1,2-二豆蔻酰基-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)或1,2-二油酰基-sn-甘油-3-磷酸乙醇胺-N-(甲氧基(聚乙二醇)中的一种或多种。
  9. 根据权利要求5所述的方法，其特征在于，所述的纳米药物可加水超声水化处理，得到溶于水的纳米药物溶液。
  10. 权利要求1～4任一项所述的具有肿瘤免疫微环境调节功能的纳米药物在制备增强肿瘤放射治疗的药物中的应用。
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