CN112915104A - Application of copper sulfide, copper sulfide nanocomposite and preparation method and application thereof - Google Patents
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- A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
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- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
- A61K47/6867—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
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- 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
Abstract
The application discloses application of copper sulfide, a copper sulfide nanocomposite and a preparation method and application thereof. The copper sulfide nano-drug can inhibit growth and relapse of leukemia of mice by inhibiting a tyrosine kinase signal pathway and activating an iron death pathway.
Description
Technical Field
The application relates to application of copper sulfide, a copper sulfide nano composite material, a preparation method and application thereof, and belongs to the technical field of leukemia drugs.
Background
At present, most of the research on the application of copper sulfide nanoparticles is carried out around solid tumors, because the copper sulfide nanoparticles have high permeability and retention effects on the solid tumors, such as photodynamic, photothermal and photoacoustic imaging, and the like, but the application of the copper sulfide nanoparticles to the solid tumors has not been reported for systemic blood diseases such as leukemia. Leukemia is a heterogeneous disease that remains difficult to treat due to factors such as patient and intrinsic biology, and 30% of adults diagnosed with leukemia have mutations in the related tyrosine kinase 3(FLT 3). Midostaurin is a multi-target kinase inhibitor and has remarkable curative effect on leukemia patients. However, the effect of conventional leukemia chemotherapy is far from satisfactory because the cured patient will relapse, and although relapsed leukemia can be treated by hematopoietic stem cell transplantation, there is also a high mortality rate of infection due to incomplete clearance of leukemia cells.
Hematopoietic stem cell development abnormalities are the source of the initiation of leukemia and its drug resistance, and most treatment regimens do not kill leukemia stem cells effectively because they are resting in the hypoxic microenvironment of bone marrow (the bone marrow niche). How to utilize the advantages of the inorganic nano material to radically eliminate leukemia cells and overcome potential drug resistance hidden dangers of leukemia is the primary key scientific problem to be solved by the technology of the application.
Disclosure of Invention
According to one aspect of the present application, there is provided the use of copper sulphide as a medicament for leukemia. The inventor of the application creatively discovers that the copper sulfide nano-drug can inhibit the growth and relapse of leukemia of mice by inhibiting a tyrosine kinase signal pathway and activating an iron death pathway.
An application of copper sulfide as leukemia medicine.
Optionally, the copper sulfide is a hollow copper sulfide.
Optionally, the particle size of the copper sulfide is 180-240 nm.
Optionally, the upper limit of the particle size of the copper sulfide is selected from 200, 210, 220, 230 or 240nm, and the lower limit is selected from 180, 200, 210, 220 or 230 nm.
The technical scheme of the application aims to provide a medicine for treating leukemia, which is a malignant blood system disease, and takes antigen-antibody specificity as a target, copper sulfide which has a unique appearance and is easily taken up by leukemia cells without tissue structures is selected for design and optimization, three mechanism ways are provided for synergistic treatment, and a unique solution is provided for the advanced scientific problem of leukemia treatment. The technology of the application is hopeful to open a new nano-drug development mode and provide a new strategy for cancer treatment. The research suggests that the copper sulfide nano-drug can inhibit growth and relapse of leukemia of mice by inhibiting a tyrosine kinase signal pathway and activating an iron death pathway. This is a feature and innovation of the technology of the present application.
According to another aspect of the present application, there is provided a copper sulfide nanocomposite material, which can precisely target hematopoietic stem cells by modifying hematopoietic stem cell surface-specific antibodies on the surface of copper sulfide. The research on the precise treatment of the systemic blood system diseases such as leukemia is extremely challenging and is one of the characteristics and innovations of the technology.
A copper sulfide nanocomposite, comprising:
copper sulfide nanoparticles;
a first modifier modified on the surface of the copper sulfide nanoparticle;
a second modifier linked to the first modifier through an amide bond;
the first modifier is derived from compound I;
the compound I is at least one selected from a mercapto-containing compound and an amino and/or carboxyl-containing compound;
the second modification is derived from at least one of hematopoietic stem cell surface-specific antibodies.
Alternatively, the compound I comprises SH-PEG-NH2And/or SH-PEG-COOH;
the hematopoietic stem cell surface-specific antibodies include CD34 antibody and/or CD33 antibody.
Optionally, the molar ratio of the copper sulfide nanoparticles to the first modifier to the second modifier is 1: 10-50: 10 to 50.
Optionally, the particle size of the copper sulfide nano-particles is 180-240 nm.
Optionally, the copper sulfide nanoparticles have an upper particle size limit selected from 200, 210, 220, 230, or 240nm and a lower particle size limit selected from 180, 200, 210, 220, or 230 nm.
Optionally, the particle size of the copper sulfide nanocomposite is 200-250 nm.
Optionally, the copper sulfide nanocomposite has a particle size with an upper limit selected from 210, 220, 230, 240, or 250nm and a lower limit selected from 200, 210, 220, 230, or 240 nm.
Optionally, the copper sulfide nanoparticle has a hollow structure, and the kinase inhibitor is loaded in the hollow structure.
The copper sulfide nano material with the hollow structure has the functions of strong plasma resonance effect, low toxicity, biocompatibility, drug loading and the like, is modified with a CD34 antibody on the surface, is internally loaded with a kinase inhibitor, and is expected to radically treat leukemia.
The copper sulfide nano material loaded with a kinase inhibitor (such as PKC412) can obviously reduce FLT3 down, and effectively kill leukemia cells.
Optionally, the kinase inhibitor comprises at least one of midostaurin, imatinib, dasatinib, nilotinib, sorafenib.
Optionally, the mass ratio of the copper sulfide nanoparticles to the kinase inhibitor is 1: 0.5-1.5.
Unlike the interactions between tissues or cells in solid tumors (e.g., angiogenesis, tumor microenvironment, immune cells, inflammatory factors, etc.), the nature of an abnormal evolution of leukemia is driven by an abnormal regulation of epigenetic transcription in the stem cell environment. The deep elucidation of the action of the process and the dynamic rule of the important molecular events in cancer regulation by nano-drugs with clinical prospect has great significance for leukemia treatment, which is an important scientific problem to be solved by the technology of the application.
The nano-drug-based new treatment strategy which is simple and convenient to synthesize, clear in structure, good in stability, small in toxic and side effects, good in curative effect and clear in mechanism is established, the defects of traditional small-molecule chemotherapy drugs, molecular targeted drugs, apparent drugs, immunotherapy drugs and the like are hopefully overcome, a new combined medication scheme is discovered and expanded, and an important clinical transformation basis is provided for curing leukemia and even cancers. This is the key scientific problem that the technology of this application will be solved finally.
The technical scheme has the greatest characteristic that the advantages of interdisciplinary researches of multiple subjects such as materials science, medicine, biology and the like are fully utilized, the accurate treatment of human major diseases is specially researched, and the method has obvious scientific research value.
According to another aspect of the present application, there is provided a method of preparing a copper sulphide nanocomposite material as defined in any one of the preceding claims, comprising the steps of:
(S1) reacting I a solution I containing copper sulfide nanoparticles with a compound I to obtain copper sulfide nanoparticles I having amino groups and/or carboxyl groups on the surface;
(S3) reacting II a mixture II containing the copper sulfide nanoparticles I having amino groups and/or carboxyl groups on the surface with hematopoietic stem cell surface-specific antibodies, and linking the copper sulfide nanoparticles I having amino groups and/or carboxyl groups on the surface with the hematopoietic stem cell surface-specific antibodies through amide bonds to obtain the copper sulfide nanocomposite;
wherein, the compound I is at least one of compounds containing sulfydryl and amino and/or carboxyl.
Optionally, the ratio of the copper sulfide nanoparticles to the compound I is 100: 0.5-50 mg/mmol.
Optionally, the conditions of reaction I are: the time I is 5-30 hours, and the temperature I is 10-35 ℃.
Optionally, the conditions of reaction I are: the time I is 15-25 hours, and the temperature I is 20-30 ℃.
Optionally, the ratio of the copper sulfide nanoparticles I with amino and/or carboxyl on the surface to the hematopoietic stem cell surface specific antibody is 100: 0.5-1.5 mg/mmol.
Optionally, the mixture II also contains EDC and NHS,
the molar ratio of the EDC, the NHS and the hematopoietic stem cell surface-specific antibody is 0.5-1.5: 0.5-1.5: 0.5 to 1.5.
Alternatively, the conditions of reaction II are: the temperature II is 4-15, and the time II is 10-25 hours.
Optionally, the copper sulfide nanoparticles are obtained by the following treatment: loading a kinase inhibitor into the hollow structure of the copper sulfide nanoparticle.
Optionally, the load is specifically: and (3) oscillating the solution III containing the kinase inhibitor and the copper sulfide nano-particles for 16-30 hours at 400-1000 rpm under the conditions of light protection and 16-30 ℃.
Optionally, in the solution III, the mass ratio of the kinase inhibitor to the copper sulfide nanoparticles is 1: 0.5-1.5.
According to another aspect of the present application, there is provided a use of the copper sulfide nanocomposite according to any one of the above or the copper sulfide nanocomposite prepared by the preparation method according to any one of the above as a leukemia drug.
Abbreviations or english in this application correspond to chinese:
CD34-PEG-CUS @ PKC: copper sulphide nanocomposites loaded with midostaurin;
PKC and PKC412 have the same meaning, and are: midostaurin;
P-FLT-3: FMS-like tyrosine kinase 3 phosphorylation
FLT-3: FMS-like tyrosine kinase 3
P-AKT: protein kinase B phosphorylation
AKT: protein kinase B
GPX-4: glutathione peroxidase
CD34 +: bone marrow CD34 positive cell
CD 34-: bone marrow CD34 negative cells
Kasumi-1: human acute myeloblastic leukemia cells
NB 4: acute promyelocytic leukemia cells
Thp 1: acute monocytic leukemia cells
U937: histiocytic lymphoma cells
MOLM 13: human acute myelogenous leukemia cells
MOLM 14: human acute myeloid leukemia cell
MV 4-11: human myelomonocytic leukemia cells
C1498: murine leukemia cells
FLT3 +: FLT3 mutant acute myeloid leukemia
FBS: fetal bovine serum
RPMI 1640: cell culture medium
ICD34-PEG-CUS @ PKC-MS: inductively coupled plasma mass spectrometry
CCK-8: cell Counting Kit-8 Cell Counting reagent
RPMI: the abbreviation of Roswell Park Memorial Institute refers to Rosevine. Park commemorative research institute
PBS: phosphate buffered saline solution
PVP-K30: (polyvinylpyrrolidone-K30);
SH-PEG-NH2: (mercapto-polyethylene glycol-amino;
EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride)
NHS (N-hydroxysuccinimide);
DAPI (4', 6-diamidino-2-phenylindole);
GSH (reduced glutathione);
GSSG (oxidized glutathione)
In the present application, the room temperature is 25 ℃.
The beneficial effects that this application can produce include:
1) the application of the copper sulfide provided by the application as a leukemia drug. The inventor of the application creatively discovers that the copper sulfide nano-drug can inhibit the growth and relapse of leukemia of mice by inhibiting a tyrosine kinase signal pathway and activating an iron death pathway.
2) The copper sulfide nano composite material provided by the application modifies the hematopoietic stem cell surface specific antibody on the surface of copper sulfide, and can accurately target hematopoietic stem cells.
3) The copper sulfide nano composite material provided by the application can utilize the synergistic interaction effect of the medicine and the copper sulfide by utilizing the hollow structure loaded medicine of the copper sulfide, and can effectively kill leukemia cells.
Drawings
FIG. 1 is a TEM image of the copper sulfide nanocomposite CD34-PEG-CUS @ PKC (A) and hollow CuS (B) loaded with midostaurin prepared in example 1 of the present application;
FIG. 2 is an SEM image of CD34-PEG-CUS @ PKC;
FIG. 3 is an infrared spectrum of the copper sulfide nanocomposite CD34-PEG-CUS @ PKC loaded with midostaurin prepared in example 1 of the present application and hollow CuS;
FIG. 4 is a DLS plot of the copper sulfide nanocomposite CD34-PEG-CUS @ PKC and hollow CuS loaded with midostaurin prepared in example 1 of the present application;
FIG. 5 is a Zeta potential plot of the copper sulfide nanocomposite CD34-PEG-CUS @ PKC and hollow CuS loaded with midostaurin prepared in example 1 of the present application;
FIG. 6 is a graph showing the results of toxicity measurements of copper sulfide on normal cells, as determined in example 2; wherein the abscissa is the concentration of copper sulfide and the ordinate is the survival rate of the cells;
FIG. 7 is a graph of the results of the Midostaurin loaded copper sulfide nanocomposite CD34-PEG-CUS @ PKC cell uptake assay determined in example 3, wherein A is the DAPI signal (blue fluorescence) detected in CD34+ cells, B is the Nile Red signal (red fluorescence) detected in CD34+ cells, and C is the combined A and B; d is the DAPI signal detected in CD 34-cells (blue fluorescence), E is the Nile red signal detected in CD 34-cells (red fluorescence), and F is the combined picture of D and E;
FIG. 8 is the cumulative amount of the copper sulfide nanocomposite CD34-PEG-CUS @ PKC loaded with midostaurin measured in example 3 in CD34+, CD 34-cells;
FIG. 9 is a graph showing the cytotoxicity of copper sulfide on leukemia cells at various concentrations as measured by the CCK-8 method in example 4;
FIG. 10 is a graph showing the effect of copper sulfide on the number of clonal leukemia cells, as determined in example 4, wherein A is an image of two clonal colonies of cells. B is data differential analysis of MV4-11 cells, and C is data differential analysis of MOLM13 cells;
FIG. 11 shows the body weight changes of different model mice in different administration groups within 45 days, wherein A is the body weight change of different administration groups of the C1498 model mouse and B is the body weight change of different administration groups of the FLT3+ model mouse;
FIG. 12 is a Kaplan-Meier method for calculating the survival curve of leukemia mice; wherein A is the survival curve of the C1498 model mouse, and B is the survival curve of the FLT3+ model mouse;
FIG. 13 is a Western blot experiment after treatment of leukemia cells with copper sulfide;
FIG. 14 is an infrared spectrum of a sample after copper sulfide and GSH and a commercial GSSG;
FIG. 15 is a nuclear magnetic resonance spectrum of a sample after copper sulfide and GSH and commercial GSSG;
FIG. 16 is a graph of drug loading rate and encapsulation efficiency of CD34-PEG-CUS @ PKC; wherein A is the drug loading rate of CD34-PEG-CUS @ PKC, and B is the encapsulation efficiency of CD34-PEG-CUS @ PKC;
FIG. 17 is a graph of cytotoxicity of copper sulfide and CD34-PEG-CUS @ PKC on leukemia cells at various concentrations;
FIG. 18 is a graph of the effect of PPS, copper sulfide, midostaurin, CD34-PEG-CUS @ PKC on colony clonal number of leukemia cells, wherein the upper part of the graph is an image of two clonal cell populations; b is data differential analysis of MV4-11 cells, and C is data differential analysis of MOLM13 cells;
FIG. 19 is a survival curve of leukemia mice calculated by the Kaplan-Meier method in example 7; wherein A is the survival curve of the C1498 model mouse, and B is the survival curve of the FLT3+ model mouse;
FIG. 20 is the body weight changes of different model mice in different administration groups within 45 days in example 7, wherein A is the body weight change of different administration groups of the C1498 model mouse and B is the body weight change of different administration groups of the FLT3+ model mouse;
FIG. 21 is a Western blot of CD34-PEG-CUS @ PKC treated leukemia cells.
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples.
Unless otherwise specified, the raw materials in the examples of the present application were all purchased commercially; wherein:
cell line: leukemia mouse primary CD34+ bone marrow cells and normal mouse primary CD 34-bone marrow cells are extracted from mouse bone marrow to obtain CD34+ and CD 34-cells respectively; kasumi-1, NB4, Thp1, U937, MOLM13, MOLM14, MV4-11, C1498 were all derived from American type culture collection (atcc).
Antibody: GPX-4 (Ebos Abcam, # ab125066), FLT-3 (Cell Signaling Technology #3462S, Sagnan), p-FLT-3 (Cell Signaling Technology #3464S, Sagnan), p-Akt (Cell Signaling Technology #4060S, Sagnan), Akt (Cell Signaling Technology #4691S, Sagnan) and β -actin (Santa Cruz, # F1819).
Commercial GSSG was purchased from Bogolon Diamond, model A100524-0001.
Example 1 preparation and characterization of copper sulfide nanocomposite (CD34-PEG-CUS @ PKC)
1. Preparation of copper sulfide nanocomposite (CD34-PEG-CUS @ PKC)
(S1) 100. mu.L of CuCl2The aqueous solution (0.5M) was dissolved in 25mL of water containing 0.25g of PVP-K30 (polyvinylpyrrolidone-K30) with stirring at room temperature using a magneton. 25mL of NaOH aqueous solution (pH 9.0) was added thereto, followed by stirring, and 4. mu.L of hydrazine hydrate aqueous solution (hydrazine hydrate content 85 wt.%) was added dropwise thereto, followed by reaction for 10 minutes to form Cu2Bright yellow solution of O spheres. Mixing 200 μ L of Na2Aqueous S solution (0.32g mL)-1) Add to the bright yellow solution. The incubator at 60 ℃ was shaken for 2 hours at a rotation speed of 200rpm under a shaker. The resulting hollow copper sulfide nanomaterial (CuS) was centrifuged at 11000rpm for 15 minutes at room temperature and washed three times with distilled water.
(S2) a DMSO solution of midostaurin PKC412 (0.2ml, 10mg/ml) was mixed with a hollow copper sulfide nanomaterial aqueous solution (20ml, 100 μ g/ml), shaken in the dark at 24 ℃ (1000 rpm) for 24 hours, washed with 10ml PBS (pH 7.4), and centrifuged (10000rpm, 10min) three times.
(S3) NH2-PEG-SH (MW 2000) was added to 20ml of a solution of 100 μ g/ml CuS @ PKC to give a concentration of NH2-PEG-SH of 1mM and incubated at room temperature for 24 hours. Excess NH2-PEG-SH was removed by centrifugation at 6000rpm for 10 minutes to give NH2-PEG-CuS @ PKC. Then NH is added2-PEG-CuS @ PKC was resuspended in deionized water. To prepare CD34-PEG-CuS @ PKC, an aqueous solution containing 1mM EDC and 1mM NHS was incubated for 30 minutes, then CD34 was added to bring the concentration of CD34 to 1mM, and the mixture was shaken for an additional 30 minutes. Addition of NH2-PEG-CuS @ PKC, NH2-PEG-The concentration of CuS @ PKC was 100. mu.g/ml, and after 12 hours of incubation at 4 ℃ two times of centrifugation (5000rpm, 15 min/time) were performed to obtain purified CD34-PEG-CuS @ PKC.
Prepared according to the above method, but omitting step (S2), copper sulfide nanocomposite (CD34-PEG-CuS) not loaded with midostaurin can be obtained.
2. Characterization of hollow copper sulfide nanomaterial, CD34-PEG-CUS @ PKC composite
CD34-PEG-CuS @ PKC was characterized using Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), hydrated particle size (DLS), Zeta Potential (Zeta Potential), and infrared spectroscopy. The hollow copper sulfide nano material or CD34-PEG-CuS @ PKC is subjected to ultrasonic treatment for 0.5 hour, a copper net is dropped into the hollow copper sulfide nano material or CD34-PEG-CuS @ PKC, and then the hollow copper sulfide nano material or CD34-PEG-CuS @ PKC is subjected to air drying and TEM scanning, as shown in figure 1, wherein A is a TEM image of CD34-PEG-CuS @ PKC, and it can be seen that CD34-PEG-CuS @ PKC is a hollow structure (namely, copper sulfide in CD34-PEG-CuS @ PKC is a hollow structure, and the midostaurin loaded in the CD34-PEG-CuS @ PKC is invisible in a transmission microscope), the particle size is about 200nm, and B is. FIG. 2 is an SEM image of CD34-PEG-CuS @ PKC. The composition of CD34-PEG-CUS @ PKC was analyzed by IR spectroscopy, and the result is shown in FIG. 3, 1187cm-1The enhancement peak indicates that copper sulfide is linked to SH-PEG-NH2The mercapto group in the compound is connected to form a C-S bond, indicating NH2The electrostatic connection between-SH in the-PEG-SH and copper sulfide proves that CuS-PEG-NH2And (4) synthesizing. As shown in FIG. 4, DLS showed that the particle size of CD34-PEG-CuS @ PKC (average particle size 230.5. + -. 18.5nm) was larger than that of CuS (average particle size 211.9. + -. 22.3 nm). Zeta potential diagram As shown in FIG. 5, the Zeta potential decreased from-9.95 mV to-13.1 mV after encapsulation of PEG by CD34-PEG-CuS @ PKC. These confirmed the synthesis of CD34-PEG-CuS @ PKC.
Example 2 toxicity assay of copper sulfide on Normal cells
1. The detection method comprises the following steps: in vitro cytotoxicity assay the toxicity of copper sulphide on normal cells of LO2 (human normal liver cells) was determined by the CCK-8 method. Taking LO2 normal cells of logarithmic growth cycle, cell count is adjusted to 5 × 10 cell density4Cells were seeded at 100 ul/ml in 96-well cell culture platesSuspension, 100% humidity, then different concentrations of copper sulfide (62.5, 125, 250, 500, 1000ug/ml, DMEM as solvent) were added to each well. Cells were then incubated at 37 ℃ and 5% CO2Under the conditions of (1), culturing for 24 hours. Then CCK-8 fluid (10uL) was added to each well, followed by 5% CO at 37 deg.C2The cells were incubated for thirty minutes. The absorbance of each well at 450nm was measured using a microplate reader, and the cell growth curve was plotted with the concentration as the abscissa.
2. And (3) detection results: the toxicity of copper sulfide on normal cells was evaluated by in vitro cell level assay, and the results are shown in fig. 6, wherein the abscissa is the concentration of copper sulfide and the ordinate is the survival rate of cells, indicating that the concentration of copper sulfide is within 1000 μ g/ml, the survival rate of normal cells is above 90%, indicating that copper sulfide has almost no effect on normal cells.
Example 3 Targeted assay of CD34-PEG-CUS @ PKC
1. The cells and the culture medium used.
Cell line: leukemia mouse primary CD34+ bone marrow cells, normal mouse primary CD 34-bone marrow cells.
Culture medium: 10% serum total bone serum (FBS) (Life Technology) in RPMI 1640.
2. Cell uptake assay. First, CD34+ and CD 34-cells were incubated at 37 ℃ in 5% CO2Culturing in the incubator for 24h to obtain better cell state, and incubating with Nile red-stained CD34-PEG-CUS @ PKC (final copper sulfide mass concentration in cell sap is 0.2ug/m L) at 37 deg.C and 5% CO20.5h later, the cells were removed, the nuclei were stained by DAPI (4', 6-diamidino-2-phenylindole) staining, and the uptake of the composite material into the cells was observed under an electron fluorescence microscope. The results are shown in FIG. 7, where A is the DAPI signal (blue fluorescence) detected in CD34+ cells, B is the Nile red signal (red fluorescence) detected in CD34+ cells, and C is the combined image of A and B; d is the DAPI signal (blue fluorescence) detected in CD 34-cells, E is the Nile Red signal (red fluorescence) detected in CD 34-cells, F is the combined image of D and E, and the CD34+ is observed in FIG. 7The red fluorescence in the cells is stronger, and the targeting of CD34-PEG-CUS @ PKC to CD34+ cells is proved.
CD34-PEG-CUS @ PKC-MS inductively coupled plasma mass spectrometry test.
First, CD34+ and CD 34-cells were incubated at 37 ℃ in 5% CO2Culturing in the culture box for 24h to obtain better cell state, adding CD34-PEG-CUS @ PKC (copper sulfide mass concentration in final cell liquid is 0, 20, 40, 60, 80 or 100ug/ml), incubating at 37 deg.C and 5% CO2The cells were taken out after 0.5 hour in the incubator of (1), digested with 5ml of a 5% nitric acid-hydrochloric acid (aqua regia) mixed acid, filtered with a filter screen, and then detected at 5 ml. FIG. 8 shows that CD34-PEG-CUS @ PKC accumulates in CD34+ cells in a greater amount of CD34-PEG-CUS @ PKC-MS, again demonstrating targeting of CD34-PEG-CUS @ PKC.
Example 4 toxicity of copper sulfide to leukemia cells
CCK-8 method test. In vitro cytotoxicity assays the toxicity of copper sulfide on leukemia cells (U937 cells, THP1 cells, NB4 cells, MOML13 cells, MOML14 cells, MV4-11 cells, KASUMI-1 cells) was determined by using Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies). Taking leukemia cells with logarithmic growth cycle, adjusting cell density to 5 × 10 by cell count4Each well of a 96-well cell culture plate was seeded with 100ul of cell suspension at 100% humidity, and then copper sulfide (0, 3.125, 6.25, 12.5, 25, 50, 100ug/ml, RPMI1640 as solvent) at various concentrations was added to each well. Cells were then incubated at 37 ℃ and 5% CO2Under the conditions of (1), culturing for 24 hours. Then CCK-8 fluid (10uL) was added to each well, followed by 5% CO at 37 deg.C2Culturing for 30 min. The absorbance of each well at 450nm was measured using a microplate reader, and the cell growth curve was plotted with the concentration as the abscissa. As shown in fig. 9, copper sulfide has the ability to kill leukemia cells.
2. Plate clone formation experiments. MOLM13 cells and MV4-11 cells were seeded at 200/well in 6-well plates in special media containing different concentrations of copper sulfide (0, 10, 50, 100ug/ml)
Culturing for 10 days in the mixture, removing culture solution after colony formation, washing for 2 times by PBS, fixing for 15min by paraformaldehyde, dyeing for 15min by crystal violet, and washing redundant crystal violet dye by PBS. As shown in FIG. 10, A is MV4-11, MOLM13 cell clonal population image is photographed, B is MV4-11 cell clonal population number data statistics, and C is MOLM13 cell clonal population number data statistics, it can be seen from FIG. 10 that copper sulfide can significantly inhibit the number of cell clonal populations, and the number of clonal populations becomes smaller as the concentration of copper sulfide increases. I.e., 10 demonstrates that copper sulfide inhibits the growth of leukemia cells.
3. And (5) animal experiments. All animal experiments were approved by the university of Jilin usage Committee animal Care center. 10 days after C1498 and FLT3+ leukemia model implantation into C57BL/6 mice (male, 4-6 weeks), different doses of copper sulfide were administered on days 10, 12, 14, 18 and 22, respectively (C1498 model: control group, n-6 mice; 5mg kg)-1Copper sulfide, n ═ 6 mice; 10mg kg-1Copper sulfide, n ═ 6 mice; FLT3+ model: control, n ═ 6 mice; 5mg kg-1Copper sulfide, n ═ 6 mice; 10mg kg-1Copper sulfide, n ═ 6 mice). The results are shown in FIGS. 11-12, in which FIG. 11 shows the weight change of different model mice in different administration groups within 45 days, in which A is the weight change of different administration groups of the C1498 model mice, and B is the weight change of different administration groups of the FLT3+ model mice; FIG. 12 is a Kaplan-Meier method for calculating the survival curve of leukemia mice; wherein A is the survival curve of the C1498 model mouse, and B is the survival curve of the FLT3+ model mouse. Animal experiments prove that the copper sulfide has an effect of treating in vivo, and the life cycle of the mouse is prolonged.
Example 5 investigation of the mechanism of action of copper sulphide
1. Polyacrylamide gel electrophoresis Western blotting experiment (Western blotting).
MV4-11, U937 and MOLM13 cells were seeded into 6-well plates, respectively, and CuS was added to the final concentration of 10. mu.g/ml, 50. mu.g/ml, 100. mu.g/ml for co-culture for 24 h. And after 24 hours, centrifuging to collect cells, cleaning twice, adding 1ml of cell lysate into a 1ml centrifuge tube to obtain a sample, taking out the sample, unfreezing the sample at 4 ℃ for one hour, centrifuging (12000rpm, 10 minutes and 4 ℃) to take out supernatant, measuring the protein concentration PLB for quantification, and obtaining the specific protein immunoblot through the processes of 100 ℃ in a high-temperature metal bath for 10 minutes, 80V first in gel electrophoresis, 120V after 30 minutes, one hour, 0.05A overnight in a transmembrane, antibody adsorption (GPX-4, beta actin), substrate color development and the like. As shown in FIG. 13, the GPX-4 gene was down-regulated with increasing concentration of copper sulfide, demonstrating that the copper sulfide cell killing mechanism is due to down-regulation of the GPX-4 gene.
2. Reaction experiment with glutathione
Copper sulfide reacts with GSH (reduced glutathione): at room temperature, 50mM of 10mL GSH solution and 10mg/mL of 10mL CuS solution were mixed by stirring 1000rpm for 4h, filtered and lyophilized.
As shown in fig. 14 to 15, copper sulfide reacts with GSH, and compared with commercial GSSG (oxidized glutathione), both infrared spectroscopy (fig. 14) and nuclear magnetic resonance spectroscopy (fig. 15) demonstrate that copper sulfide can oxidize GSH into GSSG, and has the ability to consume glutathione, indicating that copper sulfide can also kill leukemia cells by consuming glutathione.
Example 6 determination of the drug Loading Capacity of copper sulfide
The hollow porous structure of the copper sulfide can carry medicine. Midostaurin PKC412 was dissolved in DMSO to make solutions of different concentrations (1, 5, 10, 15 or 20mg/ml), 0.2ml of each of which was mixed with hollow copper sulfide nanomaterial (20ml, 100 μ g/ml), shaken in the dark at 24 ℃ for 24 hours, washed with 10ml of pbs (pH 7.4 containing 1% DMSO) and centrifuged (10000rpm, 10min) three times. The amount contained therein was calculated from the standard curve and the load amount was further calculated, as shown in fig. 16, a is a load rate of 31.1 ± 1.4% and B is a packing efficiency of 82.9 ± 2.7%, indicating that copper sulfide has good drug-loading capacity and therapeutic effect.
Example 7 copper sulphide nanocomposite loaded with midostaurin (CD34-PEG-CUS @ PKC) co-therapy trial
CCK-8 test.
The experimental method comprises the following steps: taking leukemic cells of logarithmic growth cycleCell (MV4-11 cell), cell count adjusted cell density to 5X 104Each well of a 96-well cell culture plate was seeded with 100ul of cell suspension at 100% humidity, and then copper sulfide contents of various concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100ug/ml, DMEM as solvent) were added to each well. Cells were then incubated at 37 ℃ and 5% CO2Under the conditions of (1), culturing for 24 hours. Then CCK-8 fluid (10uL) was added to each well, followed by 5% CO at 37 deg.C2The cells were incubated for thirty minutes. The absorbance of each well at 450nm was measured using a microplate reader, and the cell growth curve was plotted with the concentration as the abscissa.
The results are shown in fig. 17, and it can be seen from fig. 17 that copper sulphide nanocomposite loaded with midostaurin (CD34-PEG-CUS @ PKC) has a better killing effect on leukemia cells than copper sulphide alone, indicating a synergistic effect between midostaurin and copper sulphide.
2. Plate clone formation experiments.
MOLM13 cells, MV4-11 cells were seeded at 200/well in 6-well plates in special media containing PBS, 50mg/ml CuS, 10mM PKC or 50mg/ml CD34-PEG-CUS @ PKC, respectively
Culturing for 10 days in the mixture, removing culture solution after colony formation, washing for 2 times by PBS, fixing for 15min by paraformaldehyde, dyeing for 15min by crystal violet, and washing redundant crystal violet dye by PBS.
The results are shown in FIG. 18, where the upper part of the graph is a two-cell colony image. Data differential analysis for MV4-11 cells and MOLM-13 cells in C from FIG. 18, it can be seen that CD34-PEG-CuS @ PKC inhibits cell growth and effectively treats cancer cell spreading.
3. And (5) animal experiments. All animal experiments were approved by the university of Jilin usage Committee animal Care center. PBS, copper sulphide, PKC, CD34-PEG-CUS @ PKC were administered 10 days after implantation of C1498 and FLT3+ leukemia model into C57BL/6 mice (male, 4-6 weeks), respectively on days 10, 12, 14, 18 and 22 and leukocyte counts were measured on day 25 (C1498 model: control group, n ═ 6 mice; 5mg kg-1 copper sulphide, n ═ 6 mice; 0.5mg kg-1PKC, n ═ 6 mice; 5mg kg-1CD34-PEG-CUS @ PKC, n ═ 6 mice; FLT3+ model: control group, n ═ 6 mice; 5mg kg-1 copper sulphide, n ═ 6 mice; 0.5mg kg-1PKC, n ═ 6 mice; 5 mg-1 CD 34-1-s @ 6 mice; 5 mg-1-PEG ═ 6 mice).
The results are shown in FIGS. 19-20, in which FIG. 20 shows the body weight changes of different model mice in different administration groups within 45 days, wherein A is the body weight change of different administration groups of the C1498 model mice, and B is the body weight change of different administration groups of the FLT3+ model mice; FIG. 19 is a Kaplan-Meier method for calculating the survival curves of leukemic mice; wherein A is the survival curve of the C1498 model mouse, and B is the survival curve of the FLT3+ model mouse. Animal experiments also prove that the midostaurin and the copper sulfide have synergistic effect.
Effect of CD34-PEG-CUS @ PKC on P-FLT-3, P-AKT, GPX-4 expression.
MV4-11 was plated in 6-well plates and co-cultured for 24h with the addition of 10. mu.g/ml, 20. mu.g/ml, 50. mu.g/ml CD34-PEG-CUS @ PKC. And after 24 hours, centrifuging to collect cells, washing twice, adding 1ml of cell lysate into a 1ml centrifuge tube to obtain a sample, taking out the sample, unfreezing the sample at 4 ℃ for one hour, centrifuging (12000rpm, 10 minutes and 4 ℃) to take out supernatant, measuring the protein concentration PLB for quantification, performing high-temperature metal bath at 100 ℃, 10 minutes, performing gel electrophoresis at 80V first, after 30 minutes at 120V, one hour, transferring membrane at 0.05A overnight, adsorbing antibodies (P-FLT-3, P-AKT, GPX-4 and beta-actin) and developing a substrate to obtain a specific protein immunoblot, wherein the specific protein immunoblot is shown in figure 21.
In the process of generating and developing leukemia, a p-FlT3/AKT molecular signal pathway is activated and then highly expressed, and an iron death inhibition gene GPX4 is obviously up-regulated, and figure 21 shows that CD34-PEG-CUS @ PKC can inhibit the expression of p-FlT3/AKT and GPX4 so as to inhibit the growth, proliferation and activate iron death of leukemia.
Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
Claims (10)
1. An application of copper sulfide as leukemia medicine.
2. Use according to claim 1, wherein the copper sulphide is a hollow copper sulphide;
preferably, the particle size of the copper sulfide is 180-240 nm.
3. A copper sulfide nanocomposite, comprising:
copper sulfide nanoparticles;
a first modifier modified on the surface of the copper sulfide nanoparticle;
a second modifier linked to the first modifier through an amide bond;
the first modifier is derived from compound I;
the compound I is at least one selected from a mercapto-containing compound and an amino and/or carboxyl-containing compound;
the second modification is derived from at least one of hematopoietic stem cell surface-specific antibodies.
4. The copper sulfide nanocomposite of claim 3, wherein compound I comprises SH-PEG-NH2And/or SH-PEG-COOH;
the hematopoietic stem cell surface-specific antibodies include CD34 antibody and/or CD33 antibody.
5. The copper sulfide nanocomposite according to claim 3, wherein the molar ratio of the copper sulfide nanoparticles to the first and second modifiers is 1: 10-50: 10 to 50.
6. The copper sulfide nanocomposite according to claim 3, wherein the copper sulfide nanoparticles have a particle size of 180 to 240 nm;
preferably, the particle size of the copper sulfide nano composite material is 200-250 nm.
7. The copper sulfide nanocomposite according to claim 3, wherein the copper sulfide nanoparticles have a hollow structure, the hollow structure being loaded with a kinase inhibitor therein;
preferably, the kinase inhibitor comprises at least one of midostaurin, imatinib, dasatinib, nilotinib, sorafenib;
preferably, the mass ratio of the copper sulfide nanoparticles to the kinase inhibitor is 1: 0.5-1.5.
8. A method for preparing the copper sulfide nanocomposite material according to any one of claims 3 to 7, comprising the steps of:
(S1) reacting I a solution I containing copper sulfide nanoparticles with a compound I to obtain copper sulfide nanoparticles I having amino groups and/or carboxyl groups on the surface;
(S3) reacting II a mixture II containing the copper sulfide nanoparticles I having amino groups and/or carboxyl groups on the surface with hematopoietic stem cell surface-specific antibodies, and linking the copper sulfide nanoparticles I having amino groups and/or carboxyl groups on the surface with the hematopoietic stem cell surface-specific antibodies through amide bonds to obtain the copper sulfide nanocomposite;
wherein, the compound I is at least one of compounds containing sulfydryl and amino and/or carboxyl.
9. The preparation method according to claim 8, wherein the ratio of the copper sulfide nanoparticles to the compound I is 100:0.5 to 50 mg/mmol;
preferably, the conditions of the reaction I are: the time I is 5-30 hours, and the temperature I is 10-35 ℃;
preferably, the ratio of the copper sulfide nanoparticles I with amino and/or carboxyl on the surface to the hematopoietic stem cell surface specific antibody is 100: 0.5-1.5 mg/mmol;
preferably, the mixture II also contains EDC and NHS,
the molar ratio of the EDC, the NHS and the hematopoietic stem cell surface-specific antibody is 0.5-1.5: 0.5-1.5: 0.5 to 1.5;
preferably, the conditions of the reaction II are: the temperature II is 4-15 ℃, and the time II is 10-25 hours;
preferably, the copper sulfide nanoparticles are obtained by the following treatment: loading a kinase inhibitor into the hollow structure of the copper sulfide nanoparticle;
preferably, the load is specifically: oscillating the solution III containing the kinase inhibitor and the copper sulfide nano-particles for 16-30 hours at 400-1000 rpm under the conditions of light shielding and 16-30 ℃;
preferably, in the solution III, the mass ratio of the kinase inhibitor to the copper sulfide nanoparticles is 1: 0.5-1.5.
10. Use of the copper sulfide nanocomposite according to any one of claims 3 to 7 or the copper sulfide nanocomposite prepared by the preparation method according to claims 8 to 9 as a medicament for leukemia.
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