CN115463129A - Application of melanin or polydopamine nanoparticles as immune checkpoint Siglec-15 inhibitor in tumor resistance - Google Patents
Application of melanin or polydopamine nanoparticles as immune checkpoint Siglec-15 inhibitor in tumor resistance Download PDFInfo
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Abstract
The invention relates to an application of melanin or polydopamine nanoparticles as an anti-tumor immune checkpoint Siglec-15 inhibitor, and particularly discloses an application of the melanin or polydopamine nanoparticles in preparation of the immune checkpoint Siglec-15 inhibitor. The invention overcomes the technical prejudice, and finds that the melanin nanoparticles or polydopamine nanoparticles can be used as an inhibitor of Siglec-15 and can reduce the mRNA and protein levels of the melanin nanoparticles or the polydopamine nanoparticles. Since Siglec-15 can act as an immune checkpoint, regulating tumor immune killing, the present invention further finds its use alone as an immune checkpoint inhibitor for anti-tumor therapy. Since these nanoparticles do not act directly on tumors, but activate anti-tumor immune responses by inhibiting immune checkpoints, they are expected to have a broad spectrum of anti-tumor therapeutic effects.
Description
Technical Field
The invention relates to the field of biological science and medical nano materials, in particular to application of melanin nano particles or polydopamine nano particles.
Background
Cancer is one of the leading causes of death in humans today. The current commonly used anti-tumor methods mainly comprise targeted therapy, chemotherapy, radiotherapy, surgery and the like. Novel anti-tumor immunotherapy based on immune checkpoints, which relies on the body's own immune system to kill tumor cells, is receiving wide attention. Sites with inhibitory immunomodulatory effects in the immune response are called immune checkpoints. The immune killing of tumor by activating body through inhibiting immune checkpoint is the immune checkpoint blocking therapy. The first antibody, ipilimumab, targeting the immune checkpoint CTL4 was approved for marketing in 2011, opening a new era in tumor immunotherapy. The nobel physiological or medical reward in 2018 was assigned to James p. Allison and Tasuku Honjo, the discoverers of CTLA-4 and PD-1 immune checkpoints.
Sialic acid-binding immunoglobulin-like lectin-15 (sialic acid-binding immunoglobulin-like lectin-15, siglec-15) was newly discovered in 2019 after CTLA-4 and PD-1, yet another immune checkpoint of interest. Siglec-15 was originally identified as one of the Siglec gene family members, with a characteristic sialic acid binding immunoglobulin-type lectin structure. Siglec-15 has been reported on the role of osteoclast differentiation and bone remodeling, but its immune function has been less studied. Professor shoji constructs a high-throughput in vitro functional screening system, a genome-level T Cell Activity Array (TCAA), finds that Siglec-15 can continuously inhibit T cell activity, and then carries out a series of experiments to verify the role of Siglec-15 in tumor immunity and determine that Siglec-15 is a new immune checkpoint. Studies have shown that Siglec-15mRNA is hardly expressed in most normal human tissues and various immune cell subsets, and Siglec-15mRNA is not detected in bone marrow-derived dendritic cells (BMDCs) either, but low levels of Siglec-15mRNA are detected in bone marrow-derived macrophages (BMDMs). Macrophage colony-stimulating factor (M-CSF) induces increased Siglec-15mRNA expression in macrophages. The Siglec-15 monoclonal antibody can inhibit the expression of Siglec-15 in macrophages so as to play a role in inhibiting tumor growth. The Siglec-15 gene of the knockout mouse can obviously inhibit tumor growth, and macrophages from the Siglec-15 gene knockout mouse can induce the proliferation of T cells at a higher level compared with wild mouse macrophages. The research shows that Siglec-15 is widely expressed in various tumor cells, B16 mouse melanoma cells are subcutaneously injected into a wild-type mouse and a Siglec-15 gene knockout mouse, the tumors are measured periodically, the tumor volume is not obviously different, B16-GMCSF (granulocyte-macrophage colony stimulating factor GM-CSF gene overexpressed B16 mouse melanoma cells) tumor cells are subcutaneously injected into the wild-type mouse and the Siglec-15 gene knockout mouse, the tumors are measured periodically, and the tumor volume of the wild-type mouse is far higher than that of the gene knockout mouse. It follows that Siglec-15 is a macrophage-associated immune checkpoint. Thus, tumor immunotherapy can be achieved by inhibiting the expression of the macrophage immune checkpoint, siglec-15.
Polydopamine is a high molecular compound and is a self-polymer of dopamine. The main component of melanin is polydopamine, which is widely distributed in human hair, skin, liver, spleen, brain, etc., and has antiinflammatory and antioxidant effects. In a rat cerebral ischemic stroke model, melanin/polydopamine can protect ischemic brain from damage induced by ROS and RNS (reactive oxygen species and reactive nitrogen species), LPS stimulates macrophages, macrophage tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) expression increase is induced, and melanin/polydopamine can inhibit macrophage TNF-alpha and IL-1 beta expression. The polydopamine/polydopamine can be effectively coated on the surface of almost any material, and has the characteristics of metal ion chelation, light-heat conversion, high dispersion stability, good biocompatibility and the like.
At present, melanin/polydopamine nanoparticles are reported, and are usually used as carriers of pharmaceutical preparations, the application of the pharmaceutical carriers is realized by loading and transporting active ingredients contained in the nanoparticles, and meanwhile, the nanoparticles can be modified with polydopamine serving as the pharmaceutical carriers to realize the purposes of long circulation or targeting. In addition, since melanin/polydopamine has a photothermal effect, it has also been reported that it is used in combination with infrared light for photothermal drugs. More applications are yet to be found due to the characteristics of low cytotoxicity and the like of the melanin/polydopamine nanoparticles.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a use of melanin nanoparticles or polydopamine nanoparticles as an immune checkpoint inhibitor, which can kill tumors by acting on macrophages.
One aspect of the present invention provides a use of a melanin nanoparticle or a polydopamine nanoparticle for the preparation of an immune checkpoint inhibitory drug for the treatment of a tumor.
Another aspect of the invention provides a use of melanin nanoparticles or polydopamine nanoparticles in the preparation of a Siglec-15 inhibitor.
In still another aspect, the present invention provides an anti-tumor immune checkpoint inhibitor, which comprises melanin nanoparticles or polydopamine nanoparticles as the only active ingredient.
In yet another aspect, 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.
In still another aspect, the present invention provides an anti-tumor immunotherapeutic agent comprising melanin nanoparticles or polydopamine nanoparticles.
In yet another aspect, the present invention provides an anti-tumor immunization method comprising the step of administering melanin nanoparticles or polydopamine nanoparticles to a subject.
In the present invention, siglec-15 refers to sialic acid binding to immunoglobulin-type lectin 15.
In the technical scheme of the invention, 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 sarcoma, intestinal 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, thymus cancer, brain low-grade glioma, rectal adenocarcinoma, pheochromocytoma and paraganglioma, renal clear cell carcinoma, adenocarcinoma, bladder urothelial carcinoma, renal papillary cell carcinoma, pancreatic cancer, renal chromophobe cancer, breast infiltration cancer, lung squamous cell carcinoma, sarcoma and acute myeloid leukemia.
In the technical solution of the present invention, the melanin nanoparticles or polydopamine nanoparticles do not include a step of irradiating with light during the application process.
In the technical scheme of the invention, the melanin nanoparticles or polydopamine nanoparticles are not used as phototherapy agents.
In the technical scheme of the invention, the melanin nanoparticles or the polydopamine nanoparticles do not contain other active substances.
In the technical scheme of the invention, the melanin nanoparticles or polydopamine nanoparticles are used as the only active ingredients.
In the technical scheme of the invention, the melanin nano-particles are nano-particles formed by melanin, and anti-tumor active substances are not loaded in the melanin nano-particles. Preferably, the melanin nanoparticles are nanoparticles formed by self-assembly of a polymer formed by dopamine and a basic amino acid. More preferably, the basic amino acid is one or more of histidine, arginine or lysine.
In the technical scheme of the invention, the polydopamine nanoparticles refer to nanoparticles formed by polydopamine, and the polydopamine nanoparticles are not loaded with anti-tumor active substances.
In the technical scheme of the invention, the particle size of the melanin nano-particles or the polydopamine nano-particles is 1nm to 1000nm, preferably 10-500nm,30-300nm or 30-80 nm.
In the technical scheme of the invention, the active ingredients are ingredients which act on tumors independently and have an anti-tumor effect. The active ingredients can be small molecule compounds, proteins, nucleotides and the like.
Advantageous effects
Melanin nanoparticles or polydopamine nanoparticles are generally considered photothermal active and can be used as photothermal agents, or as carriers, containing other active ingredients, or modifying other targeting ingredients on their surfaces for tumor treatment or detection. The invention overcomes the technical prejudice, and finds that the melanin nanoparticles or polydopamine nanoparticles can be used as an inhibitor of Siglec-15 and can reduce the mRNA and protein levels of the melanin nanoparticles or the polydopamine nanoparticles. Since Siglec-15 is an immune checkpoint, the present invention further finds its use alone as an immune checkpoint inhibitor for anti-tumor therapy. Specifically, the anti-tumor immune activity of the organism can be further improved by inhibiting the expression of Siglec-15, tumor cells can be killed by immune cells, and the effect of treating tumors can be achieved without loading other active ingredients or matching with light therapy. Moreover, since it does not act directly on tumors but acts by activating the immune system, it can be expected to have a broad spectrum of antitumor effects.
Drawings
Fig. 1 is a Transmission Electron Microscope (TEM) picture of the melanin nanoparticles prepared in example 1.
Fig. 2 shows the results of the cytotoxicity test, relative cell activity, at different doses of melanin nanoparticles, in example 2 of the present invention.
FIG. 3A shows the result of the inhibition of macrophage Siglec-15mRNA transcription level by melanin nanoparticles of the present invention in example 3.
FIG. 3B are the nanoparticles of example 3 of the present invention against the level of macrophage Siglec-15 protein by melanin nanoparticles
FIG. 3C is a qPCR assay to examine the effect of M-CSF and melanin nanoparticles on macrophage BMDM Siglec-15mRNA expression.
FIG. 4A is a graph showing the body weight change curves of mice in the B16-GM-CSF test group and the control group, as shown in the figure, the melanin nanoparticles have no influence on the body weight of the mice and have low toxicity;
FIG. 4B is a graph showing the tumor volume change curves of mice in the experimental group B16-GM-CSF and the control group, as shown in the figure, the tumor volume of the mice in the melanin nanoparticle group is significantly smaller than that of the control group;
fig. 4C is a curve showing the change in tumor volume of mice in the B16F10 experimental group and the control group, as shown in the figure, the tumor volume of mice in the melanin nanoparticle group is significantly smaller than that of the control group;
FIG. 4D is a graph showing the results of the effect of Siglec-15 transcription levels in tumor tissues of C57BL6 mice in B16-GM-CSF experimental and control groups;
FIG. 4E is a graph showing the results of the effect of Melanin on Siglec-15 protein levels in tumor tissues of C57BL6 mice in B16-GM-CSF experimental and control groups;
FIG. 4F is a graph of the change in T cell populations in tumor tissue of B16-GM-CSF experimental and control Melanin vs C57BL6 mice;
FIG. 4G is a statistical plot of the change in CD3+ T cell and MDSC populations in tumor tissue of B16-GM-CSF experimental and control Melanin versus C57BL6 mice.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below, but the present invention is not to be construed as being limited to the implementable range thereof.
The term "nanoparticle" refers to a particle having a size in the range of 1nm to 1000nm, where particle size refers to the diameter of an ideal sphere having the same volume as the particle. In particular embodiments, it may be in the range of 10-500nm,30-300nm or 50-100 nm.
The term "Polydopamine (PDA)" refers to a polymer compound formed by the self-polymerization of dopamine or a composition comprising the polymer compound. Specifically, polydopamine in the present invention is a polymeric compound formed by self-polymerization of dopamine.
The term "melanin" refers to a polymer of dopamine with a basic amino acid that is one or more of histidine, arginine, or lysine. The melanin has self-assembly properties and can self-assemble to form nanoparticles. Melanin has polydopamine in its molecule and thus has properties similar to those of polypolyamines.
In the present invention, the polydopamine nanoparticle refers to a nanoparticle in which polydopamine is a nanoparticle component, and the polydopamine nanoparticle does not contain other active ingredients having an anti-tumor effect.
In the present invention, the melanin nanoparticle refers to a nanoparticle containing melanin as a nanoparticle component, and the melanin nanoparticle does not contain other active ingredients having an anti-tumor effect.
The term "Siglec-15" refers to one of the sialic acid binding immunoglobulin-like lectin family (Siglec family) genes, which encodes a short extracellular domain (ECD). Therefore, the compound can be used as a target of tumor treatment, and can realize a tumor inhibition effect by inhibiting the transcription or translation of Siglec-15.
The term "therapeutically effective amount" is an amount effective to treat, ameliorate, reduce one or more symptoms or characteristics of, delay the onset of, inhibit the progression of, reduce the severity of, and/or reduce the incidence of cancer.
The term "immune checkpoint inhibitory drugs" is a class of drugs that act on and through the action of the immune system, and are capable of inhibiting immune checkpoints and thus tumors. It does not directly kill tumor cells by chemical or physical means or directly cause apoptosis of tumor cells. Immune checkpoint inhibitory drugs are different from chemotherapeutic drugs, radiotherapeutic drugs and photothermal therapy. The chemotherapy medicament acts on the tumor cells through the corresponding chemotherapy medicament, and further causes the death or apoptosis of the tumor cells. The radiotherapy medicine induces the apoptosis of tumor cells by applying physical rays from the outside, and sometimes radiotherapy sensitizing medicines are required to be applied at the same time. Photothermal therapy is a physical therapy mediated by near infrared light, can produce local hyperthermia to kill tumor cells, and does not cause loss of normal tissues. Immune checkpoint suppressive drugs need to act on the immune system, triggering an immune response, e.g. activation of the immune system to achieve an anti-tumor effect.
One embodiment of the invention provides a use of a melanin nanoparticle or a polydopamine nanoparticle for the preparation of an immune checkpoint inhibitor medicament for the treatment of a tumor.
Another embodiment of the invention provides a use of melanin nanoparticles or polydopamine nanoparticles in the preparation of an inhibitor of Siglec-15.
Preferably, the use of a melanin nanoparticle or a polydopamine nanoparticle for the preparation of an inhibitor of Siglec-15 translation, transcription or expression.
Yet another embodiment of the present invention provides an anti-tumor immune checkpoint inhibitor comprising melanin nanoparticles or polydopamine nanoparticles, preferably as the only active ingredient.
Yet another embodiment of the 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 sole active ingredient.
Yet another embodiment of the present invention provides a method of treating or delaying progression of a tumor comprising the step of administering to a subject a melanin nanoparticle or a polydopamine nanoparticle. More specifically, it is desirable to administer a therapeutically effective amount of melanin nanoparticles or polydopamine nanoparticles to a subject. A "therapeutically effective amount" is an amount effective to treat, alleviate, ameliorate, reduce, delay the onset of, inhibit the progression of, reduce the severity of, and/or reduce the incidence of one or more symptoms or characteristics of a cancer or tumor.
Yet another embodiment of the invention provides the use of melanin nanoparticles or polydopamine nanoparticles as an inhibitor of Siglec-15.
In some specific embodiments, the use of a melanin nanoparticle or a polydopamine nanoparticle as an inhibitor of translation, transcription, or expression of Siglec-15.
In the present invention, siglec-15 refers to sialic acid binding to immunoglobulin-type lectin 15.
In some specific embodiments, the tumor is selected from the group consisting of melanoma, cervical cancer, pancreatic cancer, colon cancer, gastric cancer, lung cancer, renal cell carcinoma, liver cancer, ovarian cancer, esophageal adenocarcinoma, cholangiocarcinoma, prostate cancer, multiple sarcoma, intestinal 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, thymus cancer, brain low-grade glioma, rectal adenocarcinoma, pheochromocytoma and paraganglioma, renal clear cell carcinoma, adenocarcinoma, urinary bladder urothelial carcinoma, renal papillary cell carcinoma, pancreatic cancer, renal chromophobe cancer, breast infiltrating cancer, lung squamous cell carcinoma, sarcoma, acute myeloid leukemia.
In some specific embodiments, the melanin nanoparticles or polydopamine nanoparticles do not comprise a step of irradiation with light during administration.
In some specific embodiments, the melanin nanoparticles or polydopamine nanoparticles do not act as a phototherapeutic agent.
In some specific embodiments, the melanin nanoparticles or polydopamine nanoparticles do not contain other active substances.
In some specific embodiments, melanin nanoparticles or polydopamine nanoparticles are the only active ingredients.
In some specific embodiments, polydopamine nanoparticles refer to polydopamine-forming nanoparticles wherein no other anti-tumor active is loaded. Preferably, the polydopamine nanoparticle is a nanoparticle formed by self-assembly of a polymer formed by polymerization of dopamine.
In some embodiments, the melanin nanoparticles are nanoparticles formed with melanin, which are not loaded with an anti-tumor active substance. Preferably, the melanin nanoparticles are nanoparticles formed by self-assembly of a polymer formed by dopamine and a basic amino acid. More preferably, the basic amino acid is one or more of histidine, arginine or lysine.
In some specific embodiments, the melanin nanoparticles or polydopamine nanoparticles have a particle size in the range of 1nm to 1000nm, preferably 10-500nm,30-300nm, or 30-80 nm.
In some specific embodiments, the polydopamine nanoparticle may further comprise long-circulating modifications and/or active targeting modifications, wherein the long-circulating modifications are PEG modifications, and the active targeting modifications include, but are not limited to, mediating species (folic acid, flavin mononucleotide, transferrin, etc.), polypeptides (RGD peptide, K237 peptide, etc.), carbohydrates (heparin, hyaluronic acid), and antibodies (single-chain antibody fragment, monoclonal antibody AMG 655).
In some specific embodiments, the inhibitor of Siglec-15 or the anti-tumor immune checkpoint inhibitor is an injection, a lyophilized powder for injection.
In some specific embodiments, the melanin nanoparticles or polydopamine nanoparticles can be obtained by preparation, or can be obtained by purchase from commercially available products. The preparation method of the melanin nanoparticles or the polydopamine nanoparticles comprises but is not limited to the preparation method disclosed in any of the prior art.
In some specific embodiments, the melanin nanoparticles can be obtained by any preparation method disclosed in the prior art, for example, adding melanin into a sodium hydroxide solution, and ultrasonically oscillating until the melanin is uniformly mixed and completely dissolved; and slowly adding HCl solution for neutralization, and crushing, centrifuging, washing and freeze-drying the cells to obtain the melanin nano-particles.
Or in some specific embodiments, the melanin nanoparticles are obtained by polymerizing a basic amino acid with dopamine in an aqueous solution, and then removing the precipitate by centrifugation to obtain a supernatant.
In some specific embodiments, the melanin nanoparticles are prepared by centrifuging at a speed of less than 500g to remove the precipitate, and centrifuging at a speed of more than 800g to obtain the supernatant.
In some specific embodiments, the melanin nanoparticle preparation process further comprises a step of washing the obtained supernatant with an alcohol solution and water.
In some embodiments, the basic amino acid is selected from arginine, histidine, or lysine during the preparation of the melanin nanoparticles.
In some specific embodiments, the mass ratio of the basic amino acid to the dopamine in the melanin nanoparticle preparation process is 5:1-3, preferably 5:2.
example 1 preparation of melanin nanoparticles and Transmission Electron microscopy
Completely dissolve 8mg L-lysine in 95mL of aqueous solution at 25 deg.C, and add 5mL of dopamine (4 mg mL) with stirring -1 ) Slowly injecting into the solution. After 3 hours, all large precipitates were removed by low speed centrifugation (400 g) and the supernatant was separated by high speed centrifugation (1000 g) and washed 3 times with deionized water. After washing several times with ethanol and deionized water, melanin nanoparticles were obtained.
A Transmission Electron Microscope (TEM) picture of the melanin nanoparticles was taken as shown in fig. 1.
Example 2 toxicity study of melanin nanoparticles
The CCK8 method is adopted in the experiment to detect the influence of the melanin nano-particles on the survival rate of mouse macrophage RAW264.7 cells. RAW264.7 cells were plated at 1X 10 4 The density of each well is inoculated in a 96-well plate, after 24h, the melanin nanoparticles are given a concentration gradient stimulation, the concentration gradient is designed to be 5, 10, 50, 100, 500 mug/mL, after 24h CCK8, 37, DEG C5 CO is added 2 Incubating the culture box for 30min, and detecting OD (OD) at 450nm with enzyme-labeling instrument 450 ) The cell viability was calculated by the following formula. Cell viability (%) = (experimental well-blank well)/(control well-blank well) × 100%
As shown in fig. 2, the melanin nanoparticles had no significant effect on the cell viability of mouse macrophage RAW264.7, indicating that the melanin nanoparticles had no significant cytotoxicity.
Example 3 evaluation of the Effect of macrophage colony-stimulating factor (M-CSF) and melanin nanoparticles on the expression of the macrophage immune checkpoint Siglec-15
qPCR was used to examine the effect of M-CSF and melanin nanoparticles on Siglec-15mRNA expression in macrophage RAW 264.7. RAW264.7 cells were plated at 2X 10 5 The density of each well was inoculated in 12-well plates and 24h later, melanin nanoparticle stimulation was given at concentrations of 0. Mu.g/mL, 10. Mu.g/mL and 100. Mu.g/mL, respectively. After 2h, the blank reagent PBS and the stimulating reagent M-CSF (50 ng/mL) were added to the wells, respectively.
After 24h, the culture medium is discarded, the cells are washed twice by PBS, trizol (0.5 mL per well) is added to lyse the cells, the cells are collected into a 1.5mL centrifuge tube after complete lysis, and the centrifuge tube is placed at room temperature for 5min to completely separate the nucleic acid protein complex. 0.2mL of chloroform was added, the mixture was inverted repeatedly for 15 seconds, placed on ice for 3min, and centrifuged at 12000g at 4 ℃ for 15min. Aspirate 0.3mL of supernatant and transfer to a new 1.5mL centrifuge tube, add 0.3mL of isopropanol, reverse repeatedly for 15s, and stand at-20 ℃ for 10min. Centrifuge at 12000g for 15min at 4 ℃ and discard the supernatant. Add 1ml 75% ethanol. Centrifuging at 7500g for 5min at 4 deg.C, discarding supernatant, and drying at room temperature for 5-10min. Adding 30 μ L DEPC water, standing for 10min until mRNA is completely dissolved, measuring mRNA concentration with NANO drop, and recording 260/280 to judge the quality of the extracted mRNA. The RNA was reverse transcribed into cDNA using a reverse transcription kit, and qPCR reaction was performed to compare the expression amount of Siglec-15mRNA. The results of the experiment are shown in FIG. 3A.
Western-blot was used to examine the effect of M-CSF and melanin nanoparticles on Siglec-15 protein expression in macrophage RAW 264.7. RAW264.7 was seeded into 6-well plates at a density of 1X 10 6 cells/wall were incubated overnight at concentrations of 0. Mu.g/mL and 10. Mu.g/mL for melanin nanoparticle stimulation, and blank control reagent PBS and stimulating reagent M-CSF (50 ng/mL) were added to the wells after incubation at 37 ℃ for 2h. Discarding the culture medium after 24h, washing with PBS twice, adding appropriate amount of RIPA solution (adding 1 × PMSF), cracking on ice for 30min, centrifuging at 4 deg.C 12000rpm for 5min, and collecting the supernatantIn the EP tube of (1). After the protein concentration was detected using the BCA kit, the proteins were separated by polyacrylamide gel electrophoresis (SDS-PAGE). The Bio-Rad electrotransfer instrument transfers the proteins to PVDF membrane under the conditions of 200mA,2h.5% skim milk powder blocked for 1h, GAPDH and Siglec-15 primary antibody were incubated overnight. Primary antibody was recovered and washed 3 times with TBST for 10min each. The secondary antibody was incubated at room temperature for 1h, recovered and washed 4 times with TBST for 10min each. And detecting the expression of GAPDH and Siglec-15 proteins by using an ECL chemiluminescence hypersensitive color development kit. The experimental results are shown in fig. 3B.
The effect of M-CSF and melanin nanoparticles on macrophage BMDM Siglec-15mRNA expression was examined using qPCR. The results of the experiment are shown in FIG. 3C.
As shown in FIG. 3A, by comparing the data of 0 μ g/mL melanin nanoparticles without M-CSF (Unstimulated group) and with M-CSF (M-CSF group), the data of 0 μ g/mL melanin nanoparticles with M-CSF (M-CSF group) can significantly increase the transcription expression level of Siglec-15mRNA in macrophages, and the data of 0 μ g/mL melanin nanoparticles with M-CSF can significantly decrease the transcription level of Siglec-15mRNA in macrophages. And the magnitude of the decrease was dose-dependent, with the high dose group at 100 μ g/mL being better than the low dose group at 10 μ g/mL. From the unstimulated data, it was shown that increasing sufficient concentrations of melanin nanoparticles (100 μ g/mL) also reduced the level of Siglec-15mRNA transcription in macrophages. The above results demonstrate that melanin nanoparticles are able to reduce the transcription level of Siglec-15mRNA in macrophages. As shown in FIG. 3B, the expression of Siglec-15 protein is detected by using Western-blot technology, and the result proves that the melanin nanoparticles can reduce the expression level of Siglec-15 protein in macrophages. As shown in FIG. 3B, the expression of Siglec-15mRNA was detected by qPCR, and the result confirmed that melanin nanoparticles can reduce the transcription level of Siglec-15 in primary macrophages.
Example 4 evaluation of inhibitory Effect of melanin nanoparticles on mouse tumors
Constructing a melanoma model of mice: female C57BL/6 mice were purchased (five weeks) and randomly divided into four groups, (1) B16-GM-CSF control group; (2) B16-GM-CSF administration group (melanin nanoparticle); (3) B16F10 control group; (4) B16F10 administration group (melanin nanoparticles)
The experimental method comprises the following steps: (1) And (2) group mice were implanted on the right side of the back with B16F10 cells (1.5X 10) in which GM-CSF gene was overexpressed 5 Mice of groups (3) and (4) were implanted with B16F10 cells (1.5X 10 cells) on the right side of the back 5 One). When the tumor volume reaches 20mm 3 Intraperitoneal injection is carried out every other day, and 100 mu L of normal saline is injected every day for the group (1) and the group (3); administration group (melanin nanoparticles), 100 μ L of 3mg/mL melanin nanoparticles were injected daily, tumor volume was measured every other day with a vernier caliper, and the formula V = AB was followed 2 The tumor volume was calculated where A is the major diameter of the tumor and B is the minor diameter (mm) of the tumor. Each measurement was plotted as a tumor volume change curve, and the change in body weight of each group of mice was observed and a body weight change curve was plotted. The tumor reached 2000mm in the control group 3 After the experiment was completed, tumor tissues were taken for use, and changes in mouse tumor CD3+ T cell fraction and tumor MDSC fraction were detected by flow cytometry, and the transcription level and protein level of Siglec-15 (GAPDH is housekeeping gene) were measured on the taken B16-GM-CSF tumor tissues as shown in FIG. 4.
FIG. 4A is a graph showing the body weight change curves of mice in the group B16-GM-CSF and the group administered with the melanin nanoparticles, as shown in the figure, the melanin nanoparticles have no influence on the body weight of the mice, and it can be seen that the toxicity of the melanin nanoparticles is low; FIG. 4B is a graph showing the change in tumor volume of mice in the B16-GM-CSF group and the B16-GM-CSF control group, as shown in the figure, the tumor volume of mice in the melanin nanoparticle group is significantly smaller than that of the control group. Fig. 4C is a curve showing the change of tumor volume in mice in the B16F10 administration group and B16F10 control group, and as shown in the figure, the tumor volume in mice in the melanin nanoparticle group is significantly smaller than that in the control group. FIG. 4D is a graph of the results of the effect of Melanin nanoparticles (Melanin) on Siglec-15 transcript levels in tumor tissues of C57BL6 mice in the dosing and control groups, as shown, the Melanin nanoparticles were able to reduce the Siglec-15mRNA transcript levels in tumor tissues of mice; FIG. 4E is a graph showing the effect of melanin nanoparticles on Siglec-15 protein levels in tumor tissues of C57BL6 mice in experimental and control groups, as shown in the figure, melanin nanoparticles were able to lower Siglec-15 protein levels in tumor tissues of mice; fig. 4F shows that the ratio of melanin nanoparticles to CD3+ T cell population in tumor tissue of C57BL6 mice in the experimental group and the control group is significantly increased, and the ratio of MDSC composition is significantly decreased, which proves that the tumor immune killing of mice treated by melanin nanoparticles is significantly enhanced; fig. 4G is a statistical chart of fig. 4F.
In conclusion, the melanin nanoparticles or polydopamine nanoparticles of the present invention can inhibit tumor growth. The melanin nanoparticles or polydopamine nanoparticles do not affect cell viability, and can inhibit the growth of mouse tumors by inhibiting the expression of an immune checkpoint Siglec-15 in macrophages. Meanwhile, the melanin nano-particles or polydopamine serving as human body biological pigment has excellent biocompatibility and has good application prospect in the field of tumor treatment.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements that are within the spirit and principles of the present invention will be apparent to those of ordinary skill in the art.
Claims (10)
1. Use of a melanin nanoparticle or a polydopamine nanoparticle for the preparation of an inhibitor of Siglec-15.
2. An inhibitor of Siglec-15, comprising at least one of a melanin nanoparticle or a polydopamine nanoparticle.
3. Inhibitor according to claim 2, characterized in that melanin nanoparticles or polydopamine nanoparticles are the only active ingredient.
4. Use of a melanin nanoparticle or a polydopamine nanoparticle for the preparation of a medicament for the treatment of a tumor, preferably for the preparation of an immune checkpoint inhibitor for the treatment of a tumor.
5. The use of claim 4, wherein the tumor is selected from the group consisting of melanoma, cervical cancer, pancreatic cancer, colon cancer, gastric cancer, lung cancer, renal cell carcinoma, liver cancer, ovarian cancer, esophageal adenocarcinoma, cholangiocarcinoma, prostate cancer, multiple sarcoma, intestinal 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, thymus cancer, brain low-grade glioma, rectal adenocarcinoma, pheochromocytoma and paraganglioma, renal clear cell carcinoma, adenocarcinoma, urinary bladder urothelial carcinoma, renal papillary cell carcinoma, pancreatic cancer, renal chromophobe cancer, breast infiltrating cancer, lung squamous carcinoma, sarcoma, acute myeloid leukemia.
6. An anti-tumor immune checkpoint inhibitory drug, which has melanin nanoparticles or polydopamine nanoparticles as the only active ingredient.
7. The use of any one of claims 1,4-5, or the inhibitor of claim 2 or 3, or the immune checkpoint inhibitor of claim 6, wherein the melanin nanoparticles are melanin-forming nanoparticles that are not loaded with an anti-tumor active;
the polydopamine nanoparticles refer to nanoparticles formed by polydopamine, and the polydopamine nanoparticles are not loaded with anti-tumor active substances.
8. The use of any one of claims 1,4-5, or the inhibitor of claim 2 or 3, or the immune checkpoint inhibitory drug of claim 6, wherein the particle size of the melanin nanoparticles or polydopamine nanoparticles is from 1nm to 1000nm.
9. An anti-tumor immunization method comprising the step of administering a melanin nanoparticle or a polydopamine nanoparticle to a subject.
10. The immunological method of claim 9, wherein the tumor is selected from the group consisting of melanoma, cervical cancer, pancreatic cancer, colon cancer, gastric cancer, lung cancer, renal cell carcinoma, liver cancer, ovarian cancer, esophageal adenocarcinoma, cholangiocarcinoma, prostate cancer, multiple sarcoma, intestinal 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, thymus cancer, brain low-grade glioma, rectal adenocarcinoma, pheochromocytoma and paraganglioma, renal clear cell carcinoma, adenocarcinoma, bladder urothelial cancer, renal papillary cell carcinoma, pancreatic cancer, renal chromophobe cancer, breast infiltrating cancer, lung squamous cell carcinoma, sarcoma, acute myeloid leukemia.
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