WO2022257401A1 - 黑色素或聚多巴胺纳米颗粒作为免疫检查点Siglec-15抑制剂抗肿瘤的应用 - Google Patents

黑色素或聚多巴胺纳米颗粒作为免疫检查点Siglec-15抑制剂抗肿瘤的应用 Download PDF

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WO2022257401A1
WO2022257401A1 PCT/CN2021/137752 CN2021137752W WO2022257401A1 WO 2022257401 A1 WO2022257401 A1 WO 2022257401A1 CN 2021137752 W CN2021137752 W CN 2021137752W WO 2022257401 A1 WO2022257401 A1 WO 2022257401A1
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cancer
nanoparticles
melanin
polydopamine
tumor
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French (fr)
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李洋
曹国利
张国芳
宋庆乐
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深圳先进技术研究院
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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A61K31/787—Polymers containing nitrogen containing heterocyclic rings having nitrogen as a ring hetero atom
    • 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
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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 the fields of biological science and medical nanometer materials, in particular to the application of melanin nanoparticles or polydopamine nanoparticles.
  • Cancer is currently one of the leading causes of human death.
  • anti-tumor methods mainly include targeted therapy, chemotherapy, radiotherapy, and surgery.
  • New anti-tumor immunotherapy based on immune checkpoints relying on the body's own immune system to kill tumor cells, has received widespread attention.
  • Sites that have an inhibitory immunomodulatory role in the immune response are called immune checkpoints.
  • Activating the body's immune killing against tumors by inhibiting immune checkpoints is immune checkpoint blockade therapy.
  • the first antibody targeting the immune checkpoint CTL4, ipilimumab was approved for marketing, opening a new era of tumor immunotherapy.
  • the 2018 Nobel Prize in Physiology or Medicine goes to James P. Allison and Tasuku Honjo, the discoverers of the CTLA-4 and PD-1 immune checkpoints.
  • Sialic acid-binding immunoglobulin-like lectin-15 (Siglec-15) was newly discovered in 2019 after CTLA-4 and PD-1, and has attracted much attention immune checkpoint.
  • Siglec-15 was originally identified as one of the members of the Siglec gene family, with a characteristic sialic acid-binding immunoglobulin-type lectin structure. The role of Siglec-15 on osteoclast differentiation and bone remodeling has been reported, but less research on its immune function.
  • Professor Chen Lieping built a high-throughput in vitro functional screening system—genome-level T cell activity array (TCAA), and found that Siglec-15 can continuously inhibit T cell activity, and then conducted a series of experiments to verify the role of Siglec-15 in tumor immunity.
  • TCAA gene-level T cell activity array
  • Siglec-15 mRNA is hardly expressed in most normal human tissues and various immune cell subsets, and Siglec-15 mRNA was not detected in bone marrow-derived dendritic cells (BMDCs), but bone marrow-derived Low levels of Siglec-15 mRNA can be detected in macrophages (BMDMs).
  • BMDCs bone marrow-derived dendritic cells
  • M-CSF Macrophage colony-stimulating factor
  • Siglec-15 monoclonal antibody can inhibit the expression of Siglec-15 in macrophages and thus play a role in inhibiting tumor growth.
  • Siglec-15 knockout mice can significantly inhibit tumor growth, and macrophages from Siglec-15 knockout mice can induce T cell proliferation at a higher level compared with wild-type mouse macrophages.
  • B16 -GMCSF granulocyte-macrophage colony-stimulating factor GM-CSF gene-overexpressed B16 mouse melanoma cells
  • B16 -GMCSF granulocyte-macrophage colony-stimulating factor GM-CSF gene-overexpressed B16 mouse melanoma cells
  • Polydopamine is a polymer compound, which is a self-polymer of dopamine.
  • the main component of melanin is polydopamine, which is widely distributed in human hair, skin, liver, spleen, brain and other organs, and has anti-inflammatory and anti-oxidative effects.
  • melanin/polydopamine can protect the ischemic brain from damage induced by ROS and RNS (reactive oxygen species and reactive nitrogen species), LPS stimulates macrophages, and induces macrophage tumor necrosis factor - ⁇ (TNF- ⁇ ) and interleukin-1 ⁇ (IL-1 ⁇ ) expression increased, and melanin/polydopamine could inhibit the expression of TNF- ⁇ and IL-1 ⁇ in macrophages.
  • Polydopamine/polydopamine can be effectively coated on the surface of almost any material, and has the characteristics of metal ion chelation, light-to-heat conversion, high dispersion stability and good biocompatibility.
  • melanin/polydopamine nanoparticles have been reported, which are usually used as carriers of pharmaceutical preparations, and the use of drug carriers can be realized by loading and transporting the active ingredients contained in them, and can also be modified with polydopamine as a drug carrier , to achieve the purpose of long circulation or targeting.
  • melanin/polydopamine due to the photothermal effect of melanin/polydopamine, it has also been reported to be used in photothermal drugs when used in combination with infrared light. Due to the low cytotoxicity of melanin/polydopamine nanoparticles, more applications are yet to be discovered.
  • the purpose of the present invention is to provide an application of melanin nanoparticles or polydopamine nanoparticles as immune checkpoint inhibitory drugs, which can kill tumors by acting on macrophages.
  • One aspect of the present invention provides a use of melanin nanoparticles or polydopamine nanoparticles in the preparation of immune checkpoint inhibitory drugs for treating tumors.
  • Another aspect of the present invention provides a use of melanin nanoparticles or polydopamine nanoparticles in the preparation of Siglec-15 inhibitors.
  • Another aspect of the present invention provides an anti-tumor immune checkpoint inhibitory drug, which uses melanin nanoparticles or polydopamine nanoparticles as the only active ingredient.
  • Another aspect of the present invention provides an inhibitor of Siglec-15, comprising at least one of melanin nanoparticles or polydopamine nanoparticles, preferably, melanin nanoparticles or polydopamine nanoparticles as the only active ingredient.
  • Another aspect of the present invention provides an anti-tumor immunotherapy drug, which includes melanin nanoparticles or polydopamine nanoparticles.
  • Another aspect of the present invention provides an anti-tumor immunization method, comprising the step of administering melanin nanoparticles or polydopamine nanoparticles to a subject.
  • Siglec-15 refers to sialic acid-binding immunoglobulin-type lectin-15.
  • the tumor is selected from melanoma, cervical cancer, pancreatic cancer, colon cancer, gastric cancer, lung cancer, renal cell carcinoma, liver cancer, ovarian cancer, esophageal adenocarcinoma, cholangiocarcinoma, prostate cancer, Multiple osteosarcoma, bowel cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon cancer, ovarian serous cystadenocarcinoma, endometrial cancer, thyroid cancer, head and neck squamous cell carcinoma, multiple Formative glioma, prostate cancer, thymic carcinoma, low-grade glioma of the brain, rectal adenocarcinoma, pheochromocytoma and paraganglioma, clear cell carcinoma of the kidney, adenocarcinoma, urothelial carcinoma of the bladder, renal papilla Stem cell carcinoma, pancreatic cancer, chromopho
  • the melanin nanoparticles or polydopamine nanoparticles do not include the step of irradiating with light during the application process.
  • melanin nanoparticles or polydopamine nanoparticles are not used as phototherapeutic agents.
  • the melanin nanoparticles or the polydopamine nanoparticles do not contain other active substances.
  • melanin nanoparticles or polydopamine nanoparticles are used as the only active ingredient.
  • the melanin nanoparticles are nanoparticles formed from melanin, and the melanin nanoparticles are not loaded with anti-tumor active substances.
  • the melanin nanoparticles are nanoparticles formed by self-assembly of polymers formed by dopamine and basic amino acids. More preferably, the basic amino acid is one or more of histidine, arginine or lysine.
  • polydopamine nanoparticles refer to nanoparticles formed of polydopamine, and the polydopamine nanoparticles are not loaded with anti-tumor active substances.
  • the particle size of the melanin nanoparticles or polydopamine nanoparticles is 1 nm to 1000 nm, preferably in the range of 10-500 nm, 30-300 nm or 30-80 nm.
  • the active ingredient refers to an ingredient that acts on tumor alone and has an anti-tumor effect.
  • the active ingredients can be small molecule compounds, proteins, nucleotides and the like.
  • FIG. 1 is a transmission electron microscope (TEM) image of melanin nanoparticles prepared in Example 1.
  • Fig. 2 is the relative cell activity under different doses of melanin nanoparticles in Example 2 of the present invention, that is, the cytotoxicity test results.
  • Fig. 3A is the result of Example 3 of the present invention aimed at the inhibitory effect of melanin nanoparticles on the transcription level of Siglec-15 mRNA in macrophages.
  • Fig. 3 B is that embodiment of the present invention 3 is aimed at these nanoparticles of melanin nanoparticle to macrophage Siglec-15 protein level
  • Figure 3C is the use of qPCR to detect the effect of M-CSF and melanin nanoparticles on macrophage BMDM Siglec-15 Effects on mRNA expression.
  • Figure 4A is the body weight change curve of the mice in the B16-GM-CSF experimental group and the control group, as shown in the figure, the melanin nanoparticles have no effect on the body weight of the mice, and the toxicity is small;
  • Figure 4B is the change curve of the tumor volume of the mice in the B16-GM-CSF experimental group and the control group, as shown in the figure, the tumor volume of the mice in the melanin nanoparticle group was significantly smaller than that in the control group;
  • Figure 4C is the change curve of the tumor volume of the mice in the B16F10 experimental group and the control group, as shown in the figure, the tumor volume of the mice in the melanin nanoparticle group was significantly smaller than that in the control group;
  • Figure 4D is a graph showing the effect of Siglec-15 transcription levels in tumor tissues of C57BL6 mice in the B16-GM-CSF experimental group and control group;
  • Figure 4E is a graph showing the effect of Melanin on the Siglec-15 protein level in the tumor tissue of C57BL6 mice in the B16-GM-CSF experimental group and the control group;
  • Figure 4F is a graph showing the changes in T cell populations in the tumor tissue of C57BL6 mice treated with Melanin in the B16-GM-CSF experimental group and the control group;
  • Fig. 4G is a statistical diagram of the changes of CD3+T cells and MDSC populations in the tumor tissues of C57BL6 mice treated with Melanin in the B16-GM-CSF experimental group and the control group.
  • nanoparticle refers to particles having a particle size ranging from 1 nm to 1000 nm, where particle size refers to the diameter of an ideal sphere having the same volume as the particle. In specific embodiments, it may be in the range of 10-500 nm, 30-300 nm or 50-100 nm.
  • polydopamine (Polydopamine, PDA) refers to a polymer compound formed by self-polymerization of dopamine or a composition comprising the polymer compound. Specifically, in the present invention, polydopamine is a polymer compound formed by self-polymerization of dopamine.
  • meltanin refers to the polymer formed by dopamine and basic amino acid, and the basic amino acid is one or more of histidine, arginine or lysine. Melanin has self-assembly properties and can self-assemble to form nanoparticles. Melanin has polydopamine in its molecule, so it has properties similar to polypolyamine.
  • the polydopamine nanoparticle refers to a nanoparticle in which polydopamine is a nanoparticle component, and the polydopamine nanoparticle does not contain other active ingredients with antitumor effects.
  • the melanin nanoparticle refers to a nanoparticle with melanin as the nanoparticle component, and the melanin nanoparticle does not contain other active ingredients with anti-tumor effect.
  • Siglec-15 refers to the family of sialic acid-binding immunoglobulin-like lectins (Siglec family) genes that encode a short extracellular domain (ECD). Therefore, as a target for tumor therapy, it can achieve a tumor suppressive effect by inhibiting the transcription or translation of Siglec-15.
  • terapéuticaally effective amount is an amount effective to treat, alleviate, ameliorate, lessen, delay its onset, inhibit its progression, reduce its severity and/or reduce its incidence of one or more symptoms or features of cancer.
  • immune checkpoint inhibitory drugs is a class of drugs that act on the immune system and through the onset of the immune system, can inhibit immune checkpoints, thereby inhibiting tumors. It does not directly kill tumor cells by chemical or physical methods, or directly induce tumor cell apoptosis. Immune checkpoint inhibitory drugs are different from chemotherapy drugs, radiotherapy drugs, and photothermal therapy. Wherein, the method of action of the chemotherapeutic drugs is to cause the death or apoptosis of the tumor cells by acting on the corresponding chemotherapeutic drugs to the tumor cells. However, radiotherapy drugs induce apoptosis of tumor cells by applying physical rays from the outside world, and sometimes radiotherapy sensitizing drugs need to be applied at the same time.
  • Photothermal therapy is physical therapy mediated by near-infrared light, which can generate local high temperature to kill tumor cells without causing loss of normal tissue.
  • Immune checkpoint inhibitory drugs need to act on the immune system to trigger an immune response, such as the activation of the immune system to achieve anti-tumor effects.
  • An embodiment of the present invention provides a use of melanin nanoparticles or polydopamine nanoparticles in the preparation of immune checkpoint inhibitory drugs for treating tumors.
  • Another embodiment of the present invention provides a use of melanin nanoparticles or polydopamine nanoparticles in preparing Siglec-15 inhibitors.
  • melanin nanoparticles or polydopamine nanoparticles in the preparation of Siglec-15 translation, transcription or expression inhibitors.
  • Yet another embodiment of the present invention provides an anti-tumor immune checkpoint inhibitory drug, which contains melanin nanoparticles or polydopamine nanoparticles, preferably, melanin nanoparticles or polydopamine nanoparticles as the only active ingredient.
  • Yet another embodiment of the present invention provides an inhibitor of Siglec-15, which includes at least one of melanin nanoparticles or polydopamine nanoparticles, preferably, melanin nanoparticles or polydopamine nanoparticles as the only active ingredient.
  • Yet another embodiment of the present invention provides a method for treating tumors or delaying tumor progression, which includes the step of administering melanin nanoparticles or polydopamine nanoparticles to a subject. More specifically, it is necessary to administer a therapeutically effective amount of melanin nanoparticles or polydopamine nanoparticles to the subject.
  • a "therapeutically effective amount” is an amount effective in treating, alleviating, ameliorating, alleviating, delaying the onset, inhibiting the progression, reducing the severity and/or reducing the incidence of one or more symptoms or characteristics of a cancer or tumor.
  • Yet another embodiment of the present invention provides the use of melanin nanoparticles or polydopamine nanoparticles as Siglec-15 inhibitors.
  • melanin nanoparticles or polydopamine nanoparticles as inhibitors of translation, transcription or expression of Siglec-15.
  • Siglec-15 refers to sialic acid-binding immunoglobulin-type lectin-15.
  • the tumor is selected from melanoma, cervical cancer, pancreatic cancer, colon cancer, gastric cancer, lung cancer, renal cell carcinoma, liver cancer, ovarian cancer, esophageal adenocarcinoma, cholangiocarcinoma, prostate cancer , multiple osteosarcoma, bowel cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon cancer, ovarian serous cystadenocarcinoma, endometrial cancer, thyroid cancer, head and neck squamous cell carcinoma, Glioblastoma multiforme, prostate cancer, thymic carcinoma, low-grade glioma of the brain, rectal adenocarcinoma, pheochromocytoma and paraganglioma, clear cell carcinoma of the kidney, adenocarcinoma, urothelial carcinoma of the bladder, renal Papillary cell carcinoma, pancreatic cancer, chromophobe renal
  • the melanin nanoparticles or polydopamine nanoparticles do not include the step of irradiating with light during the administration process.
  • melanin nanoparticles or polydopamine nanoparticles are not used as phototherapeutic agents.
  • the melanin nanoparticles or polydopamine nanoparticles do not contain other active substances.
  • melanin nanoparticles or polydopamine nanoparticles are used as the only active ingredient.
  • polydopamine nanoparticles refer to nanoparticles formed from polydopamine, and the polydopamine nanoparticles are not loaded with other anti-tumor active substances.
  • the polydopamine nanoparticles are nanoparticles formed by polymer self-assembly of dopamine.
  • the melanin nanoparticles are nanoparticles formed from melanin, and the melanin nanoparticles are not loaded with anti-tumor active substances.
  • the melanin nanoparticles are nanoparticles formed by self-assembly of polymers formed by dopamine and basic amino acids. More preferably, the basic amino acid is one or more of histidine, arginine or lysine.
  • the particle size of the melanin nanoparticles or polydopamine nanoparticles is in the range of 1 nm to 1000 nm, preferably in the range of 10-500 nm, 30-300 nm or 30-80 nm.
  • polydopamine nanoparticles may also contain long-cycle modifications and/or active targeting modifications, wherein long-cycle modification PEG modifications, active targeting modifications include but are not limited to mediators (folate, flavin mononuclear glycosides, transferrin, etc.), polypeptides (RGD peptide, K237 peptide, etc.), carbohydrates (heparin, hyaluronic acid), and antibodies (single-chain antibody fragments, monoclonal antibody AMG 655).
  • Siglec-15 inhibitors or anti-tumor immune checkpoint inhibitory drugs are injections and lyophilized powder injections.
  • melanin nanoparticles or polydopamine nanoparticles can be obtained through preparation, or can be purchased through commercially available products.
  • the preparation method of melanin nanoparticles or polydopamine nanoparticles includes, but is not limited to, any preparation method disclosed in the prior art.
  • melanin nanoparticles can be obtained by any preparation method disclosed in the prior art, for example, adding melanin into sodium hydroxide solution, ultrasonically oscillating until completely dissolved; slowly adding HCl solution for neutralization, The cells are pulverized, centrifuged, washed with water and freeze-dried to obtain melanin nanoparticles.
  • the melanin nanoparticles are obtained by reacting basic amino acids with dopamine in an aqueous solution to polymerize, and then removing precipitates by centrifugation to obtain a supernatant.
  • the centrifugation method is centrifuging at a speed below 500g to remove the precipitate, and then centrifuging at a speed above 800g to obtain a supernatant.
  • the preparation process of melanin nanoparticles further includes the step of washing the obtained supernatant with alcohol solution and water.
  • the basic amino acid is selected from arginine, histidine or lysine.
  • the mass ratio of basic amino acid to dopamine is 5:1-3, preferably 5:2.
  • the CCK8 method was used to detect the effect of melanin nanoparticles on the survival rate of mouse macrophage RAW264.7 cells.
  • RAW264.7 cells were seeded in a 96-well plate at a density of 1 ⁇ 10 4 cells/well. After 24 h, they were stimulated with a concentration gradient of melanin nanoparticles. The concentration gradient was designed to be 5, 10, 50, 100, 500 ⁇ g/well.
  • CCK8 incubate for 30 min at 37 °C in a 5% CO 2 incubator, then detect the OD value at 450 nm (OD 450 ) with a microplate reader, and calculate the cell survival rate using the following formula.
  • Cell survival rate (%) (experimental well - blank well) / (control well - blank well) ⁇ 100%
  • melanin nanoparticles had no significant effect on the cell viability of mouse macrophage RAW264.7, indicating that melanin nanoparticles had no obvious cytotoxicity.
  • RAW264.7 cells were seeded in 12-well plates at a density of 2 ⁇ 10 5 cells/well. After 24 h, they were stimulated with melanin nanoparticles at concentrations of 0 ⁇ g/mL, 10 ⁇ g/mL and 100 ⁇ g/mL, respectively. After 2 h, the blank control reagent PBS and the stimulation reagent M-CSF (50 ng/mL) were added to the above wells respectively.
  • RAW264.7 was inoculated into a 6-well plate at a density of 1 ⁇ 10 6 cells/wall and cultured overnight, stimulated by melanin nanoparticles at a concentration of 0 ⁇ g/mL and 10 ⁇ g/mL, and cultured at 37°C for 2 h in the above wells Add blank control reagent PBS and stimulation reagent M-CSF (50 ng/mL) respectively.
  • M-CSF can significantly increase Siglec-15 in macrophages
  • the transcript expression level of mRNA, melanin granules can significantly reduce Siglec-15 in macrophages mRNA transcription levels.
  • the degree of reduction is dose-related, the high-dose group 100 ⁇ g/mL is better than the low-dose group 10 ⁇ g/mL.
  • mice melanoma model buy female C57BL/6 mice (five weeks), and divide the mice into four groups randomly, (1) B16-GM-CSF control group; (2) B16-GM-CSF administration group (melanin nanoparticles); (3) B16F10 control group; (4) B16F10 administration group (melanin nanoparticles)
  • the experimental method was as follows: B16F10 cells (1.5 ⁇ 10 5 ) overexpressing GM-CSF gene were implanted on the right side of the back of mice in groups (1) and (2), and implanted on the right side of the back of mice in groups (3) and (4). Into B16F10 cells (1.5 ⁇ 10 5 cells). After the tumor volume reached 20 mm3 , intraperitoneal injection was performed every other day.
  • the experiment was ended when the tumor in the control group reached 2000 mm 3 , and the tumor tissue was taken for backup, and the changes of the mouse tumor CD3+T cell components and tumor MDSC components were detected by flow cytometry, and the B16-GM-CSF tumor tissue collected was The results of the detection of Siglec-15 transcription level and protein level (GAPDH is a housekeeping gene) are shown in Figure 4.
  • Figure 4A is the body weight change curve of the mice in the B16-GM-CSF group and the administration group. As shown in the figure, the melanin nanoparticles have no effect on the body weight of the mice, which shows that the melanin nanoparticles are less toxic;
  • Figure 4B is the B16-GM-CSF The tumor volume change curves of the mice in the administration group and the B16-GM-CSF control group, as shown in the figure, the tumor volume of the mice in the melanin nanoparticle group was significantly smaller than that in the control group.
  • Figure 4C is the change curve of the tumor volume of the mice in the B16F10 administration group and the B16F10 control group.
  • FIG 4D is the results of the effect of melanin nanoparticles (Melanin) on the transcription level of Siglec-15 in the tumor tissue of C57BL6 mice in the administration group and the control group.
  • melanin nanoparticles can reduce Siglec-15 in tumor tissues of mice. 15mRNA transcription level
  • Figure 4E is the result of the effect of melanin nanoparticles on the Siglec-15 protein level in the tumor tissue of C57BL6 mice in the experimental group and the control group.
  • Figure 4F shows that the melanin nanoparticles in the experimental group and the control group significantly increased the composition ratio of CD3+ T cell population in the tumor tissue of C57BL6 mice, and the composition ratio of MDSC was significantly reduced, which proves that the tumor immunity of mice treated with melanin nanoparticles Killing was significantly enhanced;
  • Figure 4G is the statistical graph of Figure 4F.
  • the melanin nanoparticles or polydopamine nanoparticles described in the present invention can inhibit tumor growth.
  • the melanin nanoparticles or polydopamine nanoparticles of the present invention do not affect cell viability, and can inhibit the growth of mouse tumors by inhibiting the expression of the immune checkpoint Siglec-15 in macrophages.
  • melanin nanoparticles or polydopamine have excellent biocompatibility as human biochromes, and have good application prospects in the field of tumor treatment.

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Abstract

黑色素或聚多巴胺纳米颗粒作为免疫检查点Siglec-15抑制剂抗肿瘤的应用，具体公开了一种黑色素纳米颗粒或聚多巴胺纳米颗粒在制备免疫检查点Siglec-15抑制剂中的用途。克服了技术偏见，发现黑色素纳米颗粒或聚多巴胺纳米颗粒可以作为Siglec-15的抑制剂，能够降低其mRNA和蛋白水平。由于Siglec-15可作为一种免疫检查点，调控肿瘤免疫杀伤，进一步发现其单独使用可作为一种免疫检查点抑制剂来进行抗肿瘤治疗。由于这些纳米颗粒并非直接作用于肿瘤，而是通过抑制免疫检查点，激活抗肿瘤免疫应答，达到抗肿瘤效果，因此可以预期这些纳米颗粒具有广谱的抗肿瘤疗效。

Description

黑色素或聚多巴胺纳米颗粒作为免疫检查点Siglec-15抑制剂抗肿瘤的应用 技术领域
本发明涉及生物科学和医用纳米材料领域，尤其涉及一种黑色素纳米颗粒或聚多巴胺纳米颗粒的应用。
 背景技术
癌症是目前导致人类死亡的主要原因之一。当前常用的抗肿瘤方法主要有靶向治疗、化疗、放疗、手术等。基于免疫检查点的新型抗肿瘤免疫疗法，依靠机体自身免疫系统杀伤肿瘤细胞，受到广泛关注。在免疫反应中具有抑制性免疫调节作用的位点，称为免疫检查点。通过抑制免疫检查点活化机体对肿瘤的免疫杀伤即为免疫检查点阻断疗法。2011年第一个靶向免疫检查点CTL4的抗体ipilimumab获批上市，开启了肿瘤免疫治疗的新时代。2018年诺贝尔生理学或医学奖归属于CTLA-4和PD-1免疫检查点的发现者James P.Allison和Tasuku Honjo。
唾液酸结合性免疫球蛋白样凝集素-15(sialic acid-binding immunoglobulin-like lectin-15，Siglec-15)是在CTLA-4和PD-1之后于2019年新发现的，又一备受关注的免疫检查点。Siglec-15最初被鉴定为Siglec基因家族成员之一，具有特征性的唾液酸结合免疫球蛋白型凝集素结构。Siglec-15关于破骨细胞分化和骨重建的作用已有报道，但对其免疫功能的研究较少。陈列平教授构建了高通量体外功能筛选系统—基因组级T细胞活性阵列（TCAA)，发现Siglec-15可以持续抑制T细胞活性，随后进行了一系列实验来验证Siglec-15在肿瘤免疫中的作用，确定Siglec-15为新的免疫检查点。研究表明，Siglec-15 的mRNA在大多数正常人体组织和各种免疫细胞亚群中几乎不表达，骨髓来源的树突细胞（BMDCs）中也未检测到Siglec-15的mRNA，但骨髓衍生的巨噬细胞（BMDMs）中可检测到低水平的Siglec-15 mRNA。巨噬细胞集落刺激因子（M-CSF）可诱导巨噬细胞中Siglec-15 mRNA表达增加。Siglec-15单抗可抑制巨噬细胞中Siglec-15的表达进而起到抑制肿瘤生长的作用。敲除小鼠的Siglec-15基因可显著抑制肿瘤生长，与野生型小鼠巨噬细胞相比，来自Siglec-15基因敲除小鼠的巨噬细胞可更高水平的诱导T细胞的增殖。研究发现，Siglec-15广泛在多种肿瘤细胞中表达，将B16小鼠黑色素肿瘤细胞皮下注射到野生型小鼠和Siglec-15基因敲除小鼠，定期测量肿瘤，肿瘤体积无显著差异，B16-GMCSF（粒细胞-巨噬细胞集落刺激因子GM-CSF基因过表达的B16小鼠黑色素瘤细胞）肿瘤细胞皮下注射到野生型小鼠和Siglec-15基因敲除小鼠，定期测量肿瘤，野生型小鼠的肿瘤体积远高于基因敲除小鼠。由此可知，Siglec-15是巨噬细胞相关的免疫检查点。因此，可通过抑制巨噬细胞免疫检查点Siglec-15的表达实现肿瘤免疫治疗。
聚多巴胺是一种高分子化合物，是多巴胺的自聚体。黑色素的主要成分是聚多巴胺，广泛分布在人体的毛发、皮肤、肝脏、脾脏、脑等器官中，具有抗炎抗氧化的作用。在大鼠脑缺血性中风模型中，黑色素/聚多巴胺可以保护缺血性大脑免受ROS和RNS（活性氧和活性氮）诱导的损害，LPS刺激巨噬细胞，诱导巨噬细胞肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）表达增加，黑色素/聚多巴胺可以抑制巨噬细胞TNF-α和IL-1β的表达。聚多巴胺/聚多巴胺能有效地包覆在几乎任何材料的表面，且具有金属离子螯合性、光热转换性、高分散稳定性以及良好的生物相容性等特点。
目前，黑色素/聚多巴胺纳米颗粒已见报道，其通常是作为药物制剂的载体，通过负载和运输其中包载的活性成分而实现药物载体的用途，同时还可以与作为药物载体的聚多巴胺进行修饰，实现长循环或者靶向的目的。此外，由于黑色素/聚多巴胺具有光热作用，也报道过配合红外光使用被用于光热药物。由于黑色素/聚多巴胺纳米颗粒具有细胞毒性低等特点，更多的应用还有待发现。
 技术问题
鉴于上述问题，本发明的目的在于提供一种黑色素纳米颗粒或聚多巴胺纳米颗粒作为免疫检查点抑制药物的应用，可以通过作用于巨噬细胞杀伤肿瘤。
 技术解决方案
本发明一个方面提供了一种黑色素纳米颗粒或聚多巴胺纳米颗粒在制备治疗肿瘤的免疫检查点抑制药物中的用途。
本发明另一个方面提供了一种黑色素纳米颗粒或聚多巴胺纳米颗粒在制备Siglec-15抑制剂中的用途。
本发明再一个方面提供了一种抗肿瘤的免疫检查点抑制药物，以黑色素纳米颗粒或聚多巴胺纳米颗粒作为唯一活性成分。
本发明再一个方面提供了一种Siglec-15的抑制剂，包括黑色素纳米颗粒或聚多巴胺纳米颗粒中的至少一种，优选地，黑色素纳米颗粒或聚多巴胺纳米颗粒作为唯一活性成分。
本发明再一个方面提供了一种抗肿瘤的免疫治疗药物，包括黑色素纳米颗粒或聚多巴胺纳米颗粒。
本发明再一个方面提供了一种抗肿瘤的免疫方法，包括将黑色素纳米颗粒或聚多巴胺纳米颗粒给予受试者的步骤。
在本发明中，Siglec-15指唾液酸结合免疫球蛋白型凝集素15。
在本发明的技术方案中，所述的肿瘤选自黑色素瘤、宫颈癌、胰腺癌、结肠癌、胃癌、肺癌、肾细胞癌、肝癌、卵巢癌、食管腺癌、胆管上皮癌、前列腺癌、多发性骨肉瘤、肠癌、乳腺癌、食管癌、头颈癌、皮肤癌、肾癌、白血病、结肠癌、卵巢浆液性囊腺癌、子宫内膜癌、甲状腺癌、头颈鳞状细胞癌、多形成性胶质细胞瘤、前列腺癌、胸腺癌、脑低级别胶质瘤、直肠腺癌、嗜铬细胞瘤和副神经节瘤、肾透明细胞癌、腺癌、膀胱尿路上皮癌、肾乳头状细胞癌、胰腺癌、肾嫌色细胞癌、乳腺浸润癌、肺鳞癌、肉瘤、急性髓细胞样白血病。
在本发明的技术方案中，黑色素纳米颗粒或聚多巴胺纳米颗粒在施用过程中不包含以光照射的步骤。
在本发明的技术方案中，黑色素纳米颗粒或聚多巴胺纳米颗粒不作为光疗剂。
在本发明的技术方案中，黑色素纳米颗粒或聚多巴胺纳米颗粒中不包含其他活性物质。
在本发明的技术方案中，黑色素纳米颗粒或聚多巴胺纳米颗粒作为唯一活性成分。
在本发明的技术方案中，黑色素纳米颗粒为以黑色素形成的纳米颗粒，所述的黑色素纳米颗粒中不负载抗肿瘤活性物质。优选地，所述黑色素纳米颗粒为多巴胺与碱性氨基酸形成的聚合物自组装形成的纳米颗粒。更优选地，所述的碱性氨基酸为组氨酸、精氨酸或赖氨酸中的一种或多种。
在本发明的技术方案中，聚多巴胺纳米颗粒指聚多巴胺形成的纳米颗粒，所述的聚多巴胺纳米颗粒中不负载抗肿瘤活性物质。
在本发明的技术方案中，黑色素纳米颗粒或聚多巴胺纳米颗粒的粒径为1nm至1000nm，优选为10-500nm，30-300nm或者30-80nm范围内。
在本发明的技术方案中，活性成分指单独作用于肿瘤，并具有抗肿瘤效果的成分。所述活性成分可以为小分子化合物，蛋白，核苷酸等。
 附图说明
图1 是实施例1制备得到的黑色素纳米颗粒的透射电镜（TEM）图片。
图2 是本发明实施例2针对黑色素纳米颗粒的不同剂量下的相对细胞活动，即细胞毒性检测结果。
图3A是本发明实施例3针对黑色素纳米颗粒对巨噬细胞Siglec-15 mRNA转录水平的抑制作用结果。
图3B是本发明实施例3针对黑色素纳米颗粒对巨噬细胞Siglec-15蛋白水平的这些纳米颗粒
图3C是使用qPCR检测M-CSF和黑色素纳米颗粒对巨噬细胞BMDM Siglec-15 mRNA表达的影响。
图4A为B16-GM-CSF实验组和对照组小鼠体重变化曲线，如图所示，黑色素纳米颗粒对小鼠体重无影响，毒性较小；
图4B为B16-GM-CSF实验组和对照组小鼠肿瘤体积变化曲线，如图所示，黑色素纳米颗粒组小鼠肿瘤体积显著小于对照组；
图4C为B16F10实验组和对照组小鼠肿瘤体积变化曲线，如图所示，黑色素纳米颗粒组小鼠肿瘤体积显著小于对照组；
图4D是B16-GM-CSF实验组和对照组C57BL6小鼠肿瘤组织中Siglec-15转录水平的影响的结果图；
图4E为B16-GM-CSF实验组和对照组Melanin对C57BL6小鼠肿瘤组织中Siglec-15蛋白水平的影响的结果图；
图4F为B16-GM-CSF实验组和对照组Melanin对C57BL6小鼠肿瘤组织中T细胞种群变化情况图；
图4G为B16-GM-CSF实验组和对照组Melanin对C57BL6小鼠肿瘤组织中CD3+T细胞和MDSC种群变化统计图。
 本发明的实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
术语“纳米颗粒”是指粒径在1nm至1000nm范围内的颗粒，其中粒径是指具有与颗粒相同体积的理想球体的直径。在具体实施例中可以为10-500nm，30-300nm或者50-100nm范围内。
术语“聚多巴胺（Polydopamine, PDA）”是指由多巴胺自聚形成的一种高分子化合物或包含该高分子化合物的组合物。具体地，在本发明中聚多巴胺为多巴胺自聚形成的高分子化合物。
术语“黑色素”是指多巴胺与碱性氨基酸形成的聚合物，所述的碱性氨基酸为组氨酸、精氨酸或赖氨酸中的一种或多种。黑色素具有自组装特性能够自组装形成纳米颗粒。黑色素分子中具有聚多巴胺，因此具有与聚多胺类似的性质。
在本发明中，聚多巴胺纳米颗粒是指聚多巴胺为纳米颗粒成分的一种纳米颗粒，聚多巴胺纳米颗粒中不包含其他具有抗肿瘤效果的活性成分。
在本发明中，黑色素纳米颗粒是指以黑色素为纳米颗粒成分的一种纳米颗粒，黑色素纳米颗粒中不包含其他具有抗肿瘤效果的活性成分。
术语“Siglec-15”指唾液酸结合性免疫球蛋白样凝集素家族（Siglec family）基因中的一个，其编码一个短的胞外结构域（ECD）。因此，其作为肿瘤治疗的靶点，通过抑制Siglec-15的转录或翻译，可以实现肿瘤抑制效果。
术语“治疗有效量”是有效治疗、缓解、改善、减轻癌症的一种或多种症状或特征、延迟其发作、抑制其进展、降低其严重程度和/或减少其发生率的量。
术语“免疫检查点抑制药物”是作用于免疫系统，并通过免疫系统的起效，其能够对免疫检查点进行抑制，进而对肿瘤进行抑制的一类药物。其并不直接通过化学或者物理的方法杀死肿瘤细胞，或不直接引起肿瘤细胞的凋亡。免疫检查点抑制药物不同于化疗药物、放疗药物和光热治疗。其中，化疗药物的作用方法为通过将相化疗应药物作用于肿瘤细胞，进而引起肿瘤细胞的死亡或者凋亡。而放疗药物通过外界施加物理射线，引发肿瘤细胞凋亡，有时同时需要施加放疗增敏药物。光热治疗是由近红外光介导的物理治疗，可以产生局部高温杀死肿瘤细胞，并且不引起正常组织的损失。免疫检查点抑制药物需要作用于免疫系统，引发免疫反应，例如免疫系统的激活实现抗肿瘤效果。
本发明的一个实施例提供了一种黑色素纳米颗粒或聚多巴胺纳米颗粒在制备治疗肿瘤的免疫检查点抑制药物中的用途。
本发明另一个实施例提供了一种黑色素纳米颗粒或聚多巴胺纳米颗粒在制备Siglec-15的抑制剂中的用途。
优选地，黑色素纳米颗粒或聚多巴胺纳米颗粒在制备Siglec-15翻译、转录或表达的抑制剂中的用途。
本发明再一个实施例提供了一种抗肿瘤的免疫检查点抑制药物，其包含黑色素纳米颗粒或聚多巴胺纳米颗粒，优选地，以黑色素纳米颗粒或聚多巴胺纳米颗粒作为唯一活性成分。
本发明再一个实施例提供了一种Siglec-15的抑制剂，其包括黑色素纳米颗粒或聚多巴胺纳米颗粒中的至少一种，优选地，黑色素纳米颗粒或聚多巴胺纳米颗粒作为唯一活性成分。
本发明再一个实施例提供了一种治疗肿瘤或延缓肿瘤进程的方法，其包括将黑色素纳米颗粒或聚多巴胺纳米颗粒给予受试者的步骤。更具体的，需要向受试者给与治疗有效量的黑色素纳米颗粒或聚多巴胺纳米颗粒。“治疗有效量”是对癌症或肿瘤的一种或多种症状或特征有效治疗、缓解、改善、减轻、延迟其发作、抑制其进展、降低其严重性和/或降低其发生率的量。
本发明再一个实施例提供了黑色素纳米颗粒或聚多巴胺纳米颗粒作为Siglec-15的抑制剂的用途。
在一些具体的实施例中，黑色素纳米颗粒或聚多巴胺纳米颗粒作为Siglec-15的翻译、转录或表达的抑制剂的用途。
在本发明中，Siglec-15指唾液酸结合免疫球蛋白型凝集素15。
在一些具体的实施例中，所述的肿瘤选自有黑色素瘤、宫颈癌、胰腺癌、结肠癌、胃癌、肺癌、肾细胞癌、肝癌、卵巢癌、食管腺癌、胆管上皮癌、前列腺癌、多发性骨肉瘤、肠癌、乳腺癌、食管癌、头颈癌、皮肤癌、肾癌、白血病、结肠癌、卵巢浆液性囊腺癌、子宫内膜癌、甲状腺癌、头颈鳞状细胞癌、多形成性胶质细胞瘤、前列腺癌、胸腺癌、脑低级别胶质瘤、直肠腺癌、嗜铬细胞瘤和副神经节瘤、肾透明细胞癌、腺癌、膀胱尿路上皮癌、肾乳头状细胞癌、胰腺癌、肾嫌色细胞癌、乳腺浸润癌、肺鳞癌、肉瘤、急性髓细胞样白血病。
在一些具体的实施例中，黑色素纳米颗粒或聚多巴胺纳米颗粒在施用过程中不包含以光照射的步骤。
在一些具体的实施例中，黑色素纳米颗粒或聚多巴胺纳米颗粒不作为光疗剂。
在一些具体的实施例中，黑色素纳米颗粒或聚多巴胺纳米颗粒中不包含其他活性物质。
在一些具体的实施例中，黑色素纳米颗粒或聚多巴胺纳米颗粒作为唯一活性成分。
在一些具体的实施例中，聚多巴胺纳米颗粒指聚多巴胺形成的纳米颗粒所述的聚多巴胺纳米颗粒中不负载其他抗肿瘤活性物质。优选地，所述聚多巴胺纳米颗粒为多巴胺聚合成的聚合物自组装形成的纳米颗粒。
在一些具体的实施例中，黑色素纳米颗粒为以黑色素形成的纳米颗粒，所述的黑色素纳米颗粒中不负载抗肿瘤活性物质。优选地，所述黑色素纳米颗粒为多巴胺与碱性氨基酸形成的聚合物自组装形成的纳米颗粒。更优选地，所述的碱性氨基酸为组氨酸、精氨酸或赖氨酸中的一种或多种。
在一些具体的实施例中，黑色素纳米颗粒或聚多巴胺纳米颗粒的粒径为1nm至1000nm，优选为10-500nm，30-300nm或者30-80nm范围内。
在一些具体的实施例中，聚多巴胺纳米颗粒上还可以包含长循环修饰和或主动靶向修饰，其中长循环的修饰PEG修饰，主动靶向修饰包括但不限于介导类(叶酸,黄素单核苷酸,转铁蛋白等)、多肽类(RGD肽,K237肽等)、糖类(肝磷脂、透明质酸)以及抗体类(单链抗体片段,单克隆抗体AMG 655)。
在一些具体的实施例中，Siglec-15的抑制剂或抗肿瘤的免疫检查点抑制药物为注射剂、冻干粉针剂。
在一些具体的实施例中，黑色素纳米颗粒或聚多巴胺纳米颗粒可以通过制备获得，也可以通过市售产品购买获得。黑色素纳米颗粒或聚多巴胺纳米颗粒的制备方法包括但不限于现有技术中任意公开的制备方法获得。
在一些具体的实施例中，黑色素纳米颗粒可以通过现有技术中任意公开的制备方法获得，例如，将黑色素加入氢氧化钠溶液中，超声振荡至混匀完全溶解；缓慢加入HCl溶液中和，并将细胞粉碎，离心，水洗和冷冻干燥，得到黑色素纳米颗粒。
或者在一些具体的实施例中，黑色素纳米颗粒通过将碱性氨基酸与多巴胺在水溶液中反应聚合，然后通过离心去除沉淀，获得上清液后获得。
在一些具体的实施例中，黑色素纳米颗粒制备过程中，离心方法为以500g以下速度离心去除沉淀，再以800g以上速度离心获得上清液。
在一些具体的实施例中，黑色素纳米颗粒制备过程中，还包括将获得的上清液以醇溶液和水洗涤的步骤。
在一些具体的实施例中，黑色素纳米颗粒制备过程中，碱性氨基酸选自精氨酸、组氨酸或者赖氨酸。
在一些具体的实施例中，黑色素纳米颗粒制备过程中，碱性氨基酸与多巴胺的质量比为5：1-3，优选为5：2。
实施例1黑色素纳米颗粒制备及透射电镜观察
在25°C条件下，将8 mg L-赖氨酸完全溶解在95 mL水溶液中，在搅拌下将5mL 多巴胺（4mg mL -1）缓慢注入上述溶液中。3小时后，通过低速离心（400g）除去所有大的沉淀物，然后通过高速离心（1000g）分离上清液，并用去离子水洗涤3次。经乙醇和去离子水洗涤几次后，获得黑色素纳米颗粒。
拍摄黑色素纳米颗粒的透射电子显微镜（TEM）图片，如图1所示。
实施例2黑色素纳米颗粒毒性研究
本实验采用CCK8法检测黑色素纳米颗粒对小鼠巨噬细胞RAW264.7细胞存活率的影响。将RAW264.7细胞以1×10 4个/孔的密度接种在96孔板中，24 h后，给与黑色素纳米颗粒浓度梯度刺激，浓度梯度设计为5、10、50、100、500 μg/mL，24 h后加入CCK8，37 ℃，5% CO 2培养箱孵育30 min后酶标仪检测450 nm处OD值（OD 450），用如下公式计算细胞存活率。细胞存活率（%）=（实验孔-空白孔）/（对照孔-空白孔）×100%
如图2所示，黑色素纳米颗粒对小鼠巨噬细胞RAW264.7的细胞活力无显著影响，表明黑色素纳米颗粒无明显细胞毒性。
实施例3 评价巨噬细胞集落刺激因子（M-CSF）和黑色素纳米颗粒对巨噬细胞免疫检查点Siglec-15 表达的影响
使用qPCR检测M-CSF和黑色素纳米颗粒对巨噬细胞RAW264.7 中Siglec-15 mRNA表达的影响。将RAW264.7细胞以2×10 5个/孔的密度接种在12孔板中，24 h后，给予黑色素纳米颗粒刺激，浓度分别为0 μg/mL、10μg/mL和100 μg/mL。2 h后在上述孔中分别加入空白对照试剂PBS和刺激试剂M-CSF（50 ng/mL）。
24 h后弃培养基，用PBS清洗两次，加入Trizol （每孔0.5 mL）裂解细胞，待细胞裂解完全后收集到1.5 mL 离心管中，在室温放置5 min，使核酸蛋白复合物完全分离。加入0.2 mL氯仿，反复颠倒15 s，冰上放置3 min，4℃、12000 g离心15 min。吸出上层液体0.3 mL转移到新的1.5 mL离心管，加入0.3 ml异丙醇，反复颠倒15 s，-20℃放置10 min。4℃、12000 g离心15 min，弃上清。加1 mL 75%乙醇。4℃、7500 g离心5 min，弃上清，室温放置干燥，大约需5-10 min。加入30 μLDEPC水，放置10 min待mRNA完全溶解，用NANO drop测mRNA浓度，并记录260/280以判断所提取mRNA质量。使用逆转录试剂盒将RNA逆转录成cDNA，并进行qPCR反应，比较Siglec-15 mRNA的表达量。实验结果如图3A。
使用Western-blot检测M-CSF和黑色素纳米颗粒对巨噬细胞RAW264.7 中Siglec-15 蛋白表达的影响。RAW264.7接种至6孔板中，密度为1×10 6 cells/wall培养过夜，给予黑色素纳米颗粒刺激，浓度分别为0 μg/mL、10μg/mL，37℃培养2 h后在上述孔中分别加入空白对照试剂PBS和刺激试剂M-CSF（50 ng/mL）。24 h后弃培养基，用PBS清洗两次，加入适量RIPA溶液（加入1× PMSF），冰上裂解30 min后，4℃，12000 rpm离心5 min，取上清至新的EP管中。利用BCA试剂盒检测蛋白浓度后，将蛋白利用聚丙烯酰氨凝胶（SDS-PAGE）电泳分离。Bio-Rad电转仪将蛋白转移至PVDF膜上，条件为200 mA，2 h。5%脱脂奶粉封闭1h, GAPDH和Siglec-15一抗孵育过夜。回收一抗，TBST洗3次，每次10min。室温孵育二抗1h，回收二抗，用TBST洗4次，每次10min。利用ECL化学发光超敏显色试剂盒，检测GAPDH和Siglec-15蛋白表达。实验结果如图3B。
使用qPCR检测M-CSF和黑色素纳米颗粒对巨噬细胞BMDM Siglec-15 mRNA表达的影响。实验结果如图3C。
如图3A所示，通过对比未加入M-CSF进行刺激（Unstimulated组）且黑色素纳米颗粒0 μg/mL的数据，加入M-CSF进行刺激（M-CSF组）且黑色素纳米颗粒0 μg/mL的数据，可知M-CSF可以显著增加巨噬细胞中Siglec-15 mRNA的转录表达水平，黑色素颗粒可以显著降低巨噬细胞中Siglec-15 mRNA的转录水平。且降低的幅度与剂量相关，高剂量组100μg/mL优于低剂量组10μg/mL。从未刺激组数据中可知，增加足够浓度的黑色素纳米颗粒（100 μg/mL）也能够降低巨噬细胞中Siglec-15 mRNA的转录水平。以上结果证实了黑色素纳米颗粒能够降低巨噬细胞中Siglec-15 mRNA的转录水平。如图3B所示，利用Western-blot技术检测Siglec-15蛋白表达情况，结果证实了黑色素纳米颗粒能够降低巨噬细胞中Siglec-15蛋白的表达水平。如图3B所示，利用qPCR技术检测Siglec-15 mRNA表达情况，结果证实了黑色素纳米颗粒能够降低原代巨噬细胞中Siglec-15转录水平。
实施例4 评价黑色素纳米颗粒对小鼠肿瘤的抑制作用
构建小鼠的黑色素瘤模型：购买雌性C57BL/6小鼠(五周)，将小鼠随机分成四组，（1）B16-GM-CSF对照组；（2）B16-GM-CSF给药组（黑色素纳米颗粒）；（3）B16F10对照组；（4）B16F10给药组（黑色素纳米颗粒）
实验方法为：（1）和（2）组小鼠背部右侧植入GM-CSF基因过表达的B16F10细胞（1.5×10 5个），（3）和（4）组小鼠背部右侧植入B16F10细胞（1.5×10 5个）。待肿瘤体积达到20 mm 3后每隔一天进行腹腔注射，对于（1）组和（3）组，每天注射生理盐水100 μL；给药组（黑色素纳米颗粒），每天注射100 μL 3 mg/mL的黑色素纳米颗粒，每隔一天用游标卡尺测量肿瘤体积，并按照公式V =AB 2/2 计算肿瘤体积，其中A是肿瘤的长径，B是肿瘤的短径（mm）。每次测量结果绘制成肿瘤体积变化曲线，并且观察每组小鼠的体重变化并绘制体重变化曲线。在对照组肿瘤达到2000 mm 3时结束实验，取肿瘤组织备用，并用流式细胞仪检测小鼠肿瘤CD3+T细胞组分以及肿瘤MDSC组分的变化，对所取B16-GM-CSF肿瘤组织进行Siglec-15的转录水平和蛋白水平的检测（GAPDH为管家基因）实验结果如图4。
图4A为B16-GM-CSF组和给药组小鼠体重变化曲线，如图所示，黑色素纳米颗粒对小鼠体重无影响，可知黑色素纳米颗粒毒性较小；图4B为B16-GM-CSF给药组和B16-GM-CSF对照组小鼠肿瘤体积变化曲线，如图所示，黑色素纳米颗粒组小鼠肿瘤体积显著小于对照组。图4C为B16F10给药组和B16F10对照组小鼠肿瘤体积变化曲线，如图所示，黑色素纳米颗粒组小鼠肿瘤体积显著小于对照组。图4D为给药组和对照组黑色素纳米颗粒（Melanin）对C57BL6小鼠肿瘤组织中Siglec-15转录水平的影响的结果图，如图所示，黑色素纳米颗粒能够降低小鼠肿瘤组织中Siglec-15mRNA的转录水平；图4E为实验组和对照组黑色素纳米颗粒对C57BL6小鼠肿瘤组织中Siglec-15蛋白水平影响的结果图，如图所示，黑色素纳米颗粒能够降低小鼠肿瘤组织中Siglec-15蛋白水平；图4F为实验组和对照组黑色素纳米颗粒对C57BL6小鼠肿瘤组织中的CD3+ T细胞种群组成比例明显升高，MDSC组成比例明显降低，证明黑色素纳米颗粒处理后小鼠的肿瘤免疫杀伤显著增强；图4G为图4F的统计图。
综上所述，本发明所述的黑色素纳米颗粒或聚多巴胺纳米颗粒可以抑制肿瘤生长。本发明所述的黑色素纳米颗粒或聚多巴胺纳米颗粒不影响细胞活力，可以通过抑制巨噬细胞中免疫检查点Siglec-15的表达抑制小鼠肿瘤的生长。同时黑色素纳米颗粒或聚多巴胺作为人体生物色素具有极好的生物相容性，在肿瘤的治疗领域具有良好的应用前景。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，对本领域普通技术人员来说，凡在本发明的精神和原则之内做任何修改，等同替换和改进，均应包含在本发明的保护范围内。

Claims (10)

  1. 一种黑色素纳米颗粒或聚多巴胺纳米颗粒在制备Siglec-15的抑制剂中的用途。
  2. 一种Siglec-15的抑制剂，其包括黑色素纳米颗粒或聚多巴胺纳米颗粒中的至少一种。
  3. 根据权利要求2所述的抑制剂，其特征在于，黑色素纳米颗粒或聚多巴胺纳米颗粒作为唯一活性成分。
  4. 一种黑色素纳米颗粒或聚多巴胺纳米颗粒在制备治疗肿瘤的药物，优选在制备治疗肿瘤的免疫检查点抑制药物中的用途。
  5. 权利要求4所述的用途，所述的肿瘤选自黑色素瘤、宫颈癌、胰腺癌、结肠癌、胃癌、肺癌、肾细胞癌、肝癌、卵巢癌、食管腺癌、胆管上皮癌、前列腺癌、多发性骨肉瘤、肠癌、乳腺癌、食管癌、头颈癌、皮肤癌、肾癌、白血病、结肠癌、卵巢浆液性囊腺癌、子宫内膜癌、甲状腺癌、头颈鳞状细胞癌、多形成性胶质细胞瘤、前列腺癌、胸腺癌、脑低级别胶质瘤、直肠腺癌、嗜铬细胞瘤和副神经节瘤、肾透明细胞癌、腺癌、膀胱尿路上皮癌、肾乳头状细胞癌、胰腺癌、肾嫌色细胞癌、乳腺浸润癌、肺鳞癌、肉瘤、急性髓细胞样白血病。
  6. 一种抗肿瘤的免疫检查点抑制药物，其以黑色素纳米颗粒或聚多巴胺纳米颗粒作为唯一活性成分。
  7. 根据权利要求1，4-5任一项所述的用途，或者权利要求2或3所述的抑制剂，或者权利要求6所述的免疫检查点抑制药物，其特征在于，黑色素纳米颗粒为黑色素形成的纳米颗粒，所述的黑色素纳米颗粒中不负载抗肿瘤活性物质；
    聚多巴胺纳米颗粒指聚多巴胺形成的纳米颗粒，所述的聚多巴胺纳米颗粒中不负载抗肿瘤活性物质。
  8. 根据权利要求1，4-5任一项所述的用途，或者权利要求2或3所述的抑制剂，或者权利要求6所述的免疫检查点抑制药物，其中，黑色素纳米颗粒或聚多巴胺纳米颗粒的粒径为1nm至1000nm。
  9. 一种抗肿瘤的免疫方法，其包括将黑色素纳米颗粒或聚多巴胺纳米颗粒给予受试者的步骤。
  10. 根据权利要求9所述的免疫方法，所述的肿瘤选自黑色素瘤、宫颈癌、胰腺癌、结肠癌、胃癌、肺癌、肾细胞癌、肝癌、卵巢癌、食管腺癌、胆管上皮癌、前列腺癌、多发性骨肉瘤、肠癌、乳腺癌、食管癌、头颈癌、皮肤癌、肾癌、白血病、结肠癌、卵巢浆液性囊腺癌、子宫内膜癌、甲状腺癌、头颈鳞状细胞癌、多形成性胶质细胞瘤、前列腺癌、胸腺癌、脑低级别胶质瘤、直肠腺癌、嗜铬细胞瘤和副神经节瘤、肾透明细胞癌、腺癌、膀胱尿路上皮癌、肾乳头状细胞癌、胰腺癌、肾嫌色细胞癌、乳腺浸润癌、肺鳞癌、肉瘤、急性髓细胞样白血病。
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PERRING JAMES; CRAWSHAY-WILLIAMS FELICITY; HUANG CINDY; TOWNLEY HELEN E: "Bio-inspired melanin nanoparticles induce cancer cell death by iron adsorption", JOURNAL OF MATERIALS SCIENCE: MATERIALS IN MEDICINE, SPRINGER US, NEW YORK, vol. 29, no. 12, 30 November 2018 (2018-11-30), New York, pages 1 - 9, XP036659632, ISSN: 0957-4530, DOI: 10.1007/s10856-018-6190-x *

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