Radionuclide nano-carrier as well as preparation method and application thereof

Abstract

The invention relates to the technical field of tumor diagnosis and treatment, and discloses a radionuclide nano-carrier as well as a preparation method and application thereof. According to the preparation method, a mode of thermally decomposing a single Cd (S2CNBut2) precursor is adopted to obtain a hydrophobic non-spherical multi-foot CdS nano material with a natural bionic structure, and then the hydrophobic non-spherical multi-foot M2-xS nano material is obtained through cation exchange. Experimental evidences show that the non-spherical multi-legged M < 2-x > S nano material can achieve the marking stability of 90% or above, and the long-time stability characteristic is achieved. Meanwhile, after marking, excellent radioactive killing toxicity is shown, the cell survival rate is only 20%, long-acting stability as long as 96 hours is achieved, compared with a spherical nano-carrier and a rod-shaped nano-carrier, the tumor residence time is remarkably prolonged, and good tumor diagnosis and treatment application prospects are shown.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor diagnosis and treatment, and in particular to a radionuclide nanocarrier and a preparation method and application thereof. Background Art

[0002] Radioactive nuclides cause direct DNA ionizing radiation damage through their own decay of α, β, and γ particles, as well as indirect water decomposition free radical (·OH) damage, and show excellent application prospects in micrometastatic tumors, prostate cancer, and blood diseases. However, a key bottleneck in the development of radioactive drugs is that radioactive nuclides are not sufficiently retained in target lesions, often causing indifferent damage to normal tissues and blood, and have certain radiotoxicity. Therefore, the rational design of radionuclide delivery carriers and the reduction of side effects such as the nuclide's own blood toxicity have become important issues in the current design and research of radioactive drugs.

[0003] The basic principles of designing radiopharmaceuticals mainly include three important components: specific tumor targeting groups, linkers, and chelating groups for radionuclide labeling. Current radioactive delivery carrier platforms mainly include antibodies, peptides, small molecules, and nanoparticle carriers. Considering the decay characteristics of radionuclides themselves, such as the decay process of radioactive α and β particles or Auger electrons produced during delivery, the design of radionuclide delivery systems needs to balance factors such as the radioactive half-life of the nuclide itself, the blood circulation half-life, and the retention time in the target lesion. However, due to the limitation of the retention time of these traditional carrier delivery platforms in the tumor site, these radioactive delivery carriers cannot achieve effective treatment in the tumor, and often need to increase the radiation dose to increase the therapeutic efficacy, which has increased toxic side effects. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a radionuclide nanocarrier and a preparation method thereof, so that it can prolong the retention time of radionuclide in tumors, reduce the self-toxicity of radionuclide and the damage effect on normal tissues, and improve the tumor treatment effect;

[0005] Another object of the present invention is to provide a series of applications of the above-mentioned radionuclide nanocarrier in the preparation of radionuclide medicine and radionuclide combined carrier.

[0006] In order to solve the above technical problems or at least partially solve the above technical problems, the present invention provides a method for solving the above technical problems or at least partially solving the above technical problems. As a first aspect of the present invention, a radionuclide nanocarrier is provided, which is a non-spherical multi-pod structure M 2-xS is a nanomaterial; 0≤x≤1, and M is selected from one or more of Cu, Zn, Fe, Ag and Mn.

[0007] Optionally, in the M 2-x The non-spherical multi-pod structure of the S nanomaterial is coupled to one or more of Au, Pd, Pt, Ag and Fe2O3 on its surface to form a heterojunction nanocarrier. Further optionally, metal atoms are coupled to the surface of the heterojunction nanocarrier to form a ternary nanocarrier.

[0008] As a second aspect of the present invention, a method for preparing the radionuclide nanocarrier is provided, comprising:

[0009] Using oleic acid and octadecene as morphology modifiers, CdS non-spherical multi-pod nanomaterials were obtained by thermal decomposition of a single precursor Cd(S2CNBut2);

[0010] [MeCN]4MPF6 is added to the CdS non-spherical multi-pod nanomaterial to obtain a non-spherical multi-pod nanomaterial by cation exchange. 2-x S is a nanomaterial, 0≤x≤1, and M is selected from one or more of Cu, Zn, Fe, Ag and Mn.

[0011] Optionally, the preparation method further comprises: the non-spherical multi-pod M 2-x The S nanomaterial reacts with a precursor containing one or more than two selected from Au, Pd, Pt, Ag and Fe2O3 to obtain a heterojunction nanocarrier.

[0012] As a third aspect of the present invention, provided is the use of the radionuclide nanocarrier in the preparation of radionuclide medicine or radionuclide combination carrier.

[0013] As a fourth aspect of the present invention, a radioactive nuclide medicine is provided, comprising the radioactive nuclide nanocarrier, a linker, a chelating ligand, a tumor targeting group and a nuclide described in the present invention; the linker is selected from a reactive phospholipid PEG reagent, which is coupled to the surface of the radioactive nuclide nanocarrier; the chelating ligand and the tumor targeting group are coupled to the linker respectively; and the tumor targeting group is chelated and fixed to the chelate.

[0014] Optionally, the reactive phospholipid PEG reagent includes one or more of DSPE-PEG-MAL, DSPE-PEG-SH, DSPE-PEG-NH2, DSPE-PEG-COOH, DSPE-PEG-OH, and DSPE-PEG-NHS; the chelating ligand includes one or more of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triacetyltriamine (NOTA), 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP), and N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED); the tumor targeting group includes one or more of HA, PSMA, CEA, AFP, HCG, CA153, CA199, CA125, EBV, and VCA; the nuclide includes therapeutic α-nuclide, diagnostic β-nuclide, + -Nuclide, therapeutic beta - -nuclide, diagnostic gamma-nuclide and therapeutic Auger electron type nuclide, or two or more thereof.

[0015] As a fifth aspect of the present invention, a method for preparing the radioactive nuclide is provided, comprising:

[0016] The radionuclide nanocarrier of the present invention is incubated with a linker to obtain a carrier-linker product; the tumor targeting group and the chelating ligand-PEG-NH2 are coupled under the action of a crosslinker to obtain a tumor targeting group-chelating ligand product; the carrier-linker product and the tumor targeting group-chelating ligand product are coupled under the action of a crosslinker to obtain a carrier-linker-tumor targeting group-chelating ligand product;

[0017] The carrier-connector-tumor targeting group-chelating ligand product is added with a nuclide for incubation and labeling to obtain the radioactive nuclear medicine.

[0018] As the sixth aspect of the present invention, a radionuclide combined carrier is provided, comprising the radionuclide nanocarrier described in the present invention and a conventional radionuclide carrier; the conventional radionuclide carrier comprises one or more of small molecules, polypeptides, antibodies, and spherical nanoparticles.

[0019] The present invention adopts a method of thermally decomposing a single Cd (S2CNBut2) precursor to obtain a hydrophobic natural bionic structure non-spherical multi-pod CdS nanomaterial, and then obtains a hydrophobic non-spherical multi-pod M by cation exchange. 2-x S nanomaterials. Experimental evidence of the present invention shows that non-spherical multi-pod-shaped M 2-xS nanomaterials can achieve more than 90% labeling stability and long-term stability. At the same time, after labeling, they show excellent radioactive killing toxicity, with a cell survival rate of only 20%, and achieve long-term stability of up to 96 hours. Compared with spherical nanocarriers and rod-shaped nanocarriers, the tumor retention time is significantly increased, showing good application prospects for tumor diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0021] Figure 1 The non-spherical multi-pod Cu 2-x Preparation process of S nanocarrier and non-spherical multi-pod CdS material and non-spherical multi-pod Cu 2-x Transmission electron microscopy characterization image of S nanocarrier;

[0022] Figure 2 The non-spherical multi-pod Cu 2-x Preparation process of S@Au nanocarrier and non-spherical multi-pod Cu 2-x S nanocarrier and Cu 2-x Transmission electron microscopy characterization image of S@Au nanocarrier;

[0023] Figure 3 The non-spherical multi-pod Cu 2-x Preparation process of radioactive nucleopharmaceuticals prepared with S@Au nanomaterials as carriers; the red line in the figure represents DSPE-PEG-HA-DOTA (DSPE-HA-DOTA can also be used in this application);

[0024] Figure 4 The carrier-linker-tumor targeting group-chelating ligand product Cu prepared in Example 1 is shown. 2-x S-DSPE-HA-DOTA and carrier-linker-tumor targeting group-chelating ligand product Cu 2-x S@Au-DSPE-HA-DOTA hydrated particle size results (A); carrier-linker product Cu 2-x S@Au-DSPE and carrier-linker-tumor targeting group-chelating ligand product Cu 2-x Zeta potential results of S@Au-DSPE-HA-DOTA (B); CdS, Cu 2-x S and Cu 2-x UV-Vis-NIR absorption curve (C) and powder diffraction pattern (D) of S@Au;

[0025] Figure 5 Shown are the results of radiolabeling stability (A), radioactive cytotoxicity (B) and blood half-life (C, D) of the radioactive nucleopharmaceutical prepared in Example 1;

[0026] Figure 6 The figure shows the verification results of the long tumor retention time after radionuclide labeling of the quasi-spherical nanocarriers, rod-shaped nanocarriers and non-spherical multi-pod heterojunction nanocarriers prepared in Comparative Example 1 and Example 1 (SPECT / CT imaging comparison diagram); Quasi-sphere represents quasi-spherical nanocarriers, Nano rods represents rod-shaped nanocarriers, and Tetrapod-branched represents non-spherical multi-pod heterojunction nanocarriers;

[0027] Figure 7 The figure shows the in vitro ROS verification experiment of the tumor radiotherapy-chemodynamics combined technology of the nanocarrier of the present invention; A, the curve diagram of the degradation of MB by Fenton-like reaction mediated by Cu2S / Cu2S@Au-DSPE-PEG-MAL nanocomposite; B, the curve diagram of the degradation of DPBF by Fenton-like reaction mediated by Cu2S / Cu2S@Au-DSPE-PEG-MAL nanocomposite; C, the chemical kinetic cell ROS staining results of Cu2S / Cu2S@Au under the same dose of X-ray irradiation, the horizontal axis is bright field, ROS fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH2-DA) and the composite of the two, and the vertical axis is PBS, Cu2S and Cu2S@Au incubated with cells; D, Cu under different doses of X-ray irradiation 2-x Chemical dynamics of S@Au carriers and cellular ROS staining results;

[0028] Figure 8 Shown is the small animal level verification experiment of the tumor radiotherapy-chemodynamics combined technology; mouse body weight (A), survival curve (B), tumor anatomical diagram (C), tumor volume on the 14th day (D) and the biological tissue distribution curve of the radionuclide (E). DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0030] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0031] The existing radioactive carrier platforms are mainly antibodies, peptides, small molecules and spherical nanoparticle carrier platforms, while the research on non-spherical multipod-shaped radioactive nanocarriers and their tumor radiotherapy-chemodynamics combined technology is still in the early stage, and its application value and system research need to be further deepened. Most of the existing multipod-shaped nanomaterials are CdS material systems with potential toxicity. 2-x The research on the precise synthesis and preparation methods of S's nanomaterial carrier platform is still immature.

[0032] In a first aspect of the present invention, a radionuclide nanocarrier is provided, which is a non-spherical multi-pod structure. 2-x S nanomaterials, also known as pine-leaf structures, have multi-legged branching structures and abundant surface edge sites; wherein 0≤x≤1, M is selected from one or more of Cu, Zn, Fe, Ag and Mn. In certain embodiments of the present invention, a non-spherical multi-legged Cu 2-x S nanomaterials, the structural diagram and transmission electron microscope characterization pictures are shown in Figure 1 , non-spherical multi-pod-like structure of Cu 2-x The S nanomaterial carrier exhibits remarkable stability during circulation and is degradable in the tumor microenvironment, which is beneficial to the release of copper ions, thereby significantly enhancing the chemodynamic therapy (CDT) effect.

[0033] In certain embodiments of the present invention, in order to increase the functionality of the radionuclide nanocarrier of the present invention, for example, adding Au to increase radiosensitization, adding Pd to regulate the charge electron nucleophilicity of the carrier, etc., in the M 2-x The non-spherical multi-pod structure of S nanomaterials is coupled with one or more of Au, Pd, Pt, Ag and Fe2O3 on the surface to form a heterojunction nanocarrier, such as Cu 2-x S@Au、Cu 2-x S@Ag、Cu 2-x S@Pd、Cu 2-x S@Pt、Cu 2-x S@Au / Ag、Cu 2-x S@Fe2O3, etc. In some other embodiments of the present invention, a Cu 2-x S@Au heterojunction nanocarrier, structural schematic diagram and transmission electron microscopy characterization pictures are shown in Figure 2In some other embodiments of the present invention, in order to further functionalize the nanocarrier provided by the present invention, metal atoms may be coupled to the surface of the heterojunction nanocarrier. The metal atoms may be selected according to actual needs to form a ternary nanocarrier, such as Cu 2-x S@Fe2O3@Au、Cu 2-x S@Fe2O3@Au, etc.

[0034] In a second aspect of the present invention, a method for preparing the radionuclide nanocarrier is provided, comprising:

[0035] Using oleic acid and octadecene as morphology modifiers, CdS non-spherical multi-pod nanomaterials were obtained by thermal decomposition of a single precursor Cd(S2CNBut2);

[0036] [MeCN]4MPF6 is added to the CdS non-spherical multi-pod nanomaterial to obtain a non-spherical multi-pod nanomaterial by cation exchange. 2-x S is a nanomaterial, 0≤x≤1, and M is selected from one or more of Cu, Zn, Fe, Ag and Mn.

[0037] In certain embodiments of the present invention, the volume mass ratio of oleic acid, octadecene and precursor Cd(S2CNBut2) is 1-4mL:6-9mL:50-100mg; the mass volume ratio of CdS non-spherical multipodal nanomaterial to [MeCN]4MCuPF6 is 1-10mg:12-120mg.

[0038] In an embodiment of the present invention, the reaction temperature during the thermal decomposition is controlled at 145-210°C. Exceeding this temperature range is not conducive to obtaining a monodisperse material with a foot-like morphology. It is preferably carried out by injecting the precursor Cd(S2CNBut2) into oleic acid and octadecene.

[0039] In certain embodiments of the present invention, during the cation exchange, the reaction temperature is controlled at 20-50°C, and it is preferably carried out by injecting [MeCN]4MPF6 into the CdS non-spherical multipodal nanomaterial, and the injection speed can be selected to be 1-10 mL / h.

[0040] In certain embodiments of the present invention, the obtained CdS non-spherical multi-pod nanomaterials can be dissolved and collected by using chloroform, and the related reactions can be carried out by adding toluene solvent.

[0041] In some other embodiments of the present invention, Cu 2-x S is used as an example to specifically provide the following preparation method:

[0042] a. Mix 1-4 mL of oleylamine (OAm) and 6-9 mL of octadecene (ODE) and stir slowly, inject 50-100 mg of Cd(S2CNBut2) precursor at 150-210°C, and keep at 145-200°C for 10-60 min.

[0043] b. After the reaction is cooled, the reaction solution is taken out and centrifuged, and the precipitate is retained and dissolved and collected with chloroform to obtain CdS non-spherical multi-pod nanomaterials.

[0044] c. Take 1-10 mg of CdS non-spherical multipod nanomaterials, add toluene, mix and slowly stir, and inject [MeCN]4CuPF6 at 1-10 mL / h (12 mg / mL) at 20-50°C.

[0045] d. After the reaction is completed, the reaction solution is taken out and centrifuged, and the precipitate is retained and dissolved in chloroform to collect the product to obtain non-spherical multi-pod Cu 2-x S nano material, that is, the radionuclide nano carrier of the present invention.

[0046] In certain embodiments of the present invention, the value of x is related to the amount of the [MeCN]4CuPF6 precursor, and the value of x can be controlled by the amount of the precursor. For example, when the amount of [MeCN]4MCuPF6 is 10 mL (12 mg / mL), x=0, and the obtained carrier is Cu2S;

[0047] In certain embodiments of the present invention, the preparation method further comprises: the non-spherical multi-pod M 2-x The S nanomaterial reacts with a precursor containing one or more of Au, Pd, Pt, Ag and Fe2O3 to obtain a heterojunction nanocarrier. 2-x The precursor used for S@Au can be HAuCl4 to prepare Cu 2-x The precursor used by S@Ag can be AgNO3 to prepare Cu 2-x The precursor used by S@Pd can be K2PdCl4 to prepare Cu 2-x The precursors used in S@Pt can be K2PtCl4, Cu 2-x The precursors used in S@Au / Ag can be HAuCl4 and AgNO3 to prepare Cu 2-x The precursor of S@Fe2O3 can be Fe(C5H7O2)2. In some other embodiments of the present invention, the non-spherical multi-pod M 2-x The mass ratio of S nanomaterial to precursor can be 1-10 mg:5-15 mg, and the reaction can be carried out by adding toluene solvent. 2-x This is done by injecting precursors into S nanomaterials.

[0048] In the third aspect of the present invention, experimental verification shows that the non-spherical multi-pod nanocarrier of the present invention can achieve a labeling stability of more than 90% and a long-term stability of 6 days. At the same time, after labeling, it exhibits excellent radioactive killing toxicity, and the cell survival rate is only 20%, showing a good tumor killing application prospect, and the relevant carrier blood circulation time is 4h. In addition, long-term stability of up to 96 hours can be achieved, and compared with spherical carriers and rod-shaped carriers, the tumor retention time is significantly increased. Based on this, the present invention provides the use of the radionuclide nanocarrier in the preparation of radionuclide medicines or radionuclide combination carriers.

[0049] In the fourth aspect of the present invention, a radioactive nuclide medicine is provided, comprising the radioactive nuclide nanocarrier, a linker, a chelating ligand, a tumor targeting group and a nuclide described in the present invention; the linker is selected from a reactive phospholipid PEG reagent, which is coupled to the surface of the radioactive nuclide nanocarrier; the chelating ligand and the tumor targeting group are coupled to the linker respectively; and the tumor targeting group is chelated and fixed to the chelate.

[0050] In certain embodiments of the present invention, the reactive phospholipid PEG reagent includes one or more of DSPE-PEG-MAL, DSPE-PEG-SH, DSPE-PEG-NH2, DSPE-PEG-COOH, DSPE-PEG-OH, and DSPE-PEG-NHS; the chelating ligand includes one or more of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triacetyltriamine (NOTA), 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid) (NOTP), and N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED); the tumor targeting group includes one or more of HA, PSMA, CEA, AFP, HCG, CA153, CA199, CA125, EBV, and VCA; the nuclide includes therapeutic α-nuclide, diagnostic β-nuclide, + -Nuclide, therapeutic beta - -nuclide, diagnostic gamma-nuclide and therapeutic Auger electron type nuclide, or two or more thereof.

[0051] In some other embodiments of the present invention, the therapeutic α-nuclides include but are not limited to 212 Bi, 211 At 230 U. 225 Ac, 223 Ra, etc.; diagnostic type β + - Nuclides include but are not limited to: 18 F. 64 Cu,68 Ga, 82 Rb, 83 Sr. 86 Y. 89 Zr, etc.; therapeutic β - - Nuclides include but are not limited to: 32 P. 66 Cu, 90 Y. 131 I. 166 Ho, 177 Lu et al.; Diagnostic gamma-nuclides include but are not limited to: 99 mTc, etc. Therapeutic Auger electron nuclides include but are not limited to: 111 In, 123 I. 125 I etc.

[0052] In other embodiments of the present invention, the molecular weight of PEG in the reactive phospholipid PEG reagent is 500-10000, and optimal anti-tissue protein adsorption can be achieved within this molecular weight range.

[0053] In a fifth aspect of the present invention, a method for preparing the radioactive nuclide is provided, comprising:

[0054] The radionuclide nanocarrier of the present invention is incubated with the linker to react,

[0055] Obtaining a carrier-linker product; coupling the tumor targeting group and the chelating ligand-PEG-NH2 under the action of a crosslinking agent to obtain a tumor targeting group-chelating ligand product; coupling the carrier-linker product with the tumor targeting group-chelating ligand product under the action of a crosslinking agent to obtain a carrier-linker-tumor targeting group-chelating ligand product;

[0056] The carrier-connector-tumor targeting group-chelating ligand product is added with a nuclide for incubation and labeling to obtain the radioactive nuclear medicine.

[0057] In certain embodiments of the present invention, the cross-linking agent is selected from NHS and EDCI.

[0058] In certain embodiments of the present invention, in order to improve the water solubility of the radionuclide nanocarrier, sodium hydroxide can also be added to the incubation reaction of the radionuclide nanocarrier and the linker; in certain embodiments of the present invention, for every 1-10 mg of the radionuclide nanocarrier, 10 mL of 0.01-10 mM NaOH aqueous solution and 5-100 mg of the linker can be added, and the incubation reaction can be assisted by ultrasound.

[0059] In certain embodiments of the present invention, in order to improve the labeling efficiency of radionuclides, the nuclides may be acidified in advance, for example, by mixing the nuclides with an acidic buffer solution, wherein the acidic buffer solution may be a NaAc-HAc buffer solution.

[0060] In certain embodiments of the present invention, each 1-10 μg of the non-spherical multipodal nanocarrier coupled with a linker, a tumor targeting group and a chelating ligand can be labeled with 50-200 μCi of radionuclide.

[0061] In some other embodiments of the present invention, Cu 2-x S or Cu 2-x Taking S-Au non-spherical multipod nanocarrier as an example, the following radioactive nuclear medicine preparation method is specifically provided, and its preparation schematic diagram is shown in Figure 3 :

[0062] a. Take 1-10 mg of the non-spherical multi-pod nanocarrier material Cu of the present invention. 2-x S or Cu 2-x S@Au, add 10 mL of 0.01-10 mM NaOH aqueous solution, then add 5-100 mg of DSPE-PEG-MAL solution, and sonicate.

[0063] b. Stir at 30-60℃ for 2h to evaporate the solvent and centrifuge to obtain hydrophilic nanocrystalline Cu 2-x S / Cu 2-x S@Au-DSPE-PEG-MAL and redispersed it in a certain amount of deionized water.

[0064] c. Under room temperature, 10-50 mg HA (MW 3000) was dissolved in water, activated by NHS and EDCI for 0.5-2 h, and then 20-100 mg DOTA-PEG-NH2 was added thereto for reaction for 1-10 h to obtain HA-DOTA; after the reaction, centrifuged (5000-15000 rpm) for 5-20 min and redispersed in a certain amount of deionized water.

[0065] d. The above HA-DOTA solution was activated with NHS and EDCI for 0.5-2h, and then the Cu prepared in step b was 2-x S / Cu 2-x S@Au-DSPE-PEG-MAL was added and reacted for 1-10h to obtain Cu 2-x S / Cu 2-x S@Au-DSPE-PEG-HA-DOTA hydrophilic nanocrystals; after the reaction is completed, centrifuge (5000-15000 rpm) for 5-20 min and redisperse it in a certain amount of deionized water.

[0066] e. 50-200μCi 177 LuCl3 was acidified by adding 50-200 μL of NaAc-HAc buffer solution at pH 4-5.5.

[0067] f. Add 1-10 μg of the hydrophilic nanocrystals prepared in step d to the above buffer solution.

[0068] g.React at 37-100℃ for 30-60min.

[0069] h. After the reaction is completed, the reaction solution is taken out and ultrafiltered and centrifuged, and the supernatant is collected to obtain the radioactive nuclide labeled with the nuclide.

[0070] In the sixth aspect of the present invention, a radionuclide combination carrier is also provided, comprising the radionuclide nanocarrier described in the present invention and a conventional radionuclide carrier; the conventional radionuclide carrier comprises one or more of small molecules, polypeptides, antibodies, and spherical nanoparticles.

[0071] In addition, the radionuclide nanocarriers and radionuclides provided in this application can also be used in combination with chemokinetics. In vitro experiments show that the nanocarriers of the present invention have excellent tumor microenvironment response characteristics, can achieve effective release of Cu ions in a weak acid environment in the tumor, enhance the chemokinetic treatment effect, and show excellent chemokinetic properties in the tumor microenvironment. The ROS probe test shows that the ROS type of the probe is OH. At the same time, studies have shown that under the synergistic mechanism of chemokinetics and radionuclides internal irradiation, tumor growth is effectively inhibited, the weight of mice is stable, and the tumor volume is significantly reduced.

[0072] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified. In the specific embodiments of the present invention, unless otherwise specified, the experimental environment and parameter conditions of each group in the specific embodiment test are consistent with each other except for the differences clearly stated.

[0073] The following is a further description of a radionuclide nanocarrier provided by the present invention and its preparation method and application.

[0074] Embodiment 1:

[0075] (1) Preparation of non-spherical multipod Cu2S support

[0076] a. Mix 1 mL of oleylamine (OAm) and 9 mL of octadecene (ODE) and stir slowly, inject 90 mg of Cd (S2CNBut2) precursor at 150-210°C, and keep at 145-200°C for 10-60 min.

[0077] b. After the reaction is cooled, the reaction solution is removed and centrifuged (5000-15000 rpm) for 5-20 min, and the precipitate is dissolved and collected with chloroform to obtain a non-spherical multi-pod CdS nanomaterial;

[0078] c. Take 10 mg of CdS non-spherical multipod nanomaterials, add 30 mL of toluene, mix and stir slowly, and inject 10 mL of [MeCN]4CuPF6 at 3 mL / h at 20-50°C to a concentration of 12 mg / mL.

[0079] d. After the reaction is completed, the reaction solution is centrifuged (5000-15000 rpm) for 5-20 min, and the precipitate is retained and dissolved in chloroform to collect the product to obtain a non-spherical multi-pod Cu2S nanocarrier, that is, the radionuclide nanocarrier of the present invention, and the schematic diagram of its preparation process is shown in Figure 1 . Figure 1 The electron microscopy results in the figure show that the shape and structure of the "multi-pod-like nanomaterial" remain unchanged before and after ion exchange.

[0080] e. Take 1-10 mg of the above non-spherical multi-pod Cu2S nanocarrier, add 5-30 mL of toluene, mix and slowly stir, inject 5-15 mg of HAuCl4 precursor at 20-50 ° C, keep at 20-60 ° C for 10-60 min. Then disperse it into the solution by centrifugation to obtain non-spherical multi-pod Cu2S@Au heterojunction nanocarrier. The schematic diagram of its preparation process is shown in Figure 2 , Figure 2 The TEM and STEM results in the images showed that the gold nanoparticles were successfully attached to the surface of the non-spherical multipodal Cu2S nanocarriers.

[0081] (2) Preparation of radioactive nuclides:

[0082] a. Take 1-10 mg of the above non-spherical multipod Cu2S nanocarrier or non-spherical multipod Cu2S@Au heterojunction nanocarrier, add 10 mL of 0.01-10 mM NaOH aqueous solution, then add 5-100 mg of DSPE-PEG-MAL solution, and ultrasonicate (100-500 W, 2-10 minutes).

[0083] b. Stir at 30-60°C for 2 hours to evaporate the chloroform. Centrifuge (5000-15000 rpm) for 5-20 minutes to obtain hydrophilic nanocrystalline Cu2S / Cu2S@Au-DSPE-PEG-MAL, and redisperse it in a certain amount of deionized water.

[0084] c. Under room temperature, 10-50 mg HA (MW 3000) was dissolved in water, activated by NHS and EDCI for 0.5-2 h, and then 20-100 mg DOTA-PEG-NH2 was added thereto for reaction for 1-10 h to obtain HA-DOTA; after the reaction, centrifuged (5000-15000 rpm) for 5-20 min and redispersed in a certain amount of deionized water.

[0085] d. The above HA-DOTA solution was activated with NHS and EDCI for 0.5-2h, and then the Cu prepared in step b was 2-x S / Cu 2-x S@Au-DSPE-PEG-MAL was added and reacted for 1-10h to obtain Cu 2-x S / Cu 2-x S@Au-DSPE-PEG-HA-DOTA hydrophilic nanocrystals; after the reaction is completed, centrifuge (5000-15000 rpm) for 5-20 min and redisperse it in a certain amount of deionized water.

[0086] e. Add 50-200 μCi of 177LuCl3 to 50-200 μL of NaAc-Hac buffer solution at pH 4-5.5 to acidify.

[0087] f. Add 1-10 μg of the hydrophilic nanocrystals prepared in step d to the above buffer solution.

[0088] g. Use a constant temperature oscillator (37-100°C, 800-1000 rpm) to react for 30-60 min.

[0089] h. After the reaction is completed, the reaction solution is taken out and ultrafiltered and centrifuged (3000-5000 rpm) for 5-20 min, and the supernatant is collected 2-3 times to obtain the radioactive nuclide labeled radionuclide. The preparation process diagram is shown in Figure 3 .

[0090] Comparative Example 1:

[0091] (1) Preparation of quasi-spherical nanocarriers

[0092] 4 ml of oleylamine (OAM), 6 ml of octadecene (ODE), 100 mg of thioacetamide (TA) and 0.4 ml of dodecanethiol (DDT) were mixed and stirred slowly, and the Cu(S2CNBut2) precursor was injected at 150-210°C, and kept at 145-190°C for 10-60 min. After the reaction was cooled naturally, the reaction solution was taken out and centrifuged (5000-15000 rpm) for 5-20 min, and the precipitate was retained and dissolved and collected with chloroform to obtain quasi-spherical Cu2S nanocarriers.

[0093] The preparation methods of other heterojunction carriers and radionuclide labels refer to Example 1;

[0094] (2) Preparation of rod-shaped nanocarriers

[0095] 4 ml of oleylamine (OAM), 6 ml of octadecene (ODE), 10 mg of thioacetamide (TA) and 0.4 ml of dodecanethiol (DDT) were mixed and stirred slowly, and the Cu(S2CNBut2) precursor was injected at 150-210°C, and kept at 145-190°C for 10-30 min. After the reaction was cooled naturally, the reaction solution was taken out and centrifuged (5000-15000 rpm) for 5-20 min, and the precipitate was retained and dissolved and collected with chloroform to obtain a rod-shaped Cu2S nanocarrier.

[0096] The preparation methods of other heterojunction carriers and radionuclide labels refer to Example 1;

[0097] Experimental example:

[0098] (1) The non-spherical multi-pod CdS nanomaterials and non-spherical multi-pod Cu prepared in Example 1 2-x S nanocarrier, non-spherical multipod Cu 2-x The S-Au heterojunction nanocarriers were tested for Zeta potential, hydrated particle size, UV-visible-near infrared absorption, and powder diffraction. The results are shown in Figure 4 ;

[0099] Figure 4 It shows that relative to the support-linker product Cu 2-x S-Au-DSPE, carrier-linker-tumor targeting group-chelating ligand product Cu 2-x The Zeta potential of S-Au-DSPE-HA-DOTA decreased. Since DOTA contains carboxyl groups, the potential decreased after successful coupling, indicating that the product was successfully coupled. The hydrated particle size test showed that Cu 2-x S-DSPE-HA-DOTA and Cu 2-x The size of S-Au-DSPE-HA-DOTA in water phase is about 190nm; compared with the standard card, Cu 2-x S and Cu 2-x The S-Au contrast is consistent, and the UV absorption of both is consistent with Cu 2-x S absorption, and secondly, the xrd comparison with the standard card is also correct, which indicates that Cu 2-x S and Cu 2-x S-Au was successfully synthesized.

[0100] (2) Testing the radiolabeling stability, radioactive cytotoxicity and blood half-life of the radioactive nuclide prepared in Example 1;

[0101] Figure 5 The results showed that the non-spherical multi-pod Cu2S nanocarrier and the non-spherical multi-pod Cu2S@Au heterojunction nanocarrier were used to 177 After Lu labeling and ultrafiltration, the non-spherical multipod nuclide nanocarriers of the present invention were stored in physiological saline and 10% FBS, and then ultrafiltered at different times, and measured and calculated using an activity meter, which verified that the non-spherical multipod nuclide nanocarriers of the present invention had good stability in both physiological saline and 10% FBS, and could achieve a labeling stability of more than 90%, and a long-term stability of 6 days;

[0102] Radioactive cytotoxicity The same number of HepG2 cells were incubated in a 96-well plate and then incubated with different doses of radionuclides. The CCK-8 experiment verified that the radionuclides of the present invention had good tumor killing ability.

[0103] Blood half-life: The same dose of radioactive nucleoside was injected into nude mice through the tail vein, blood was collected from the eyeballs at different times, and the radioactivity count was measured by a γ radioimmunoassay counter. The results showed that the radioactive nucleoside of the present invention had a good circulation time in the body.

[0104] The results of radioactive cytotoxicity and blood half-life can both indicate that the radioactive nanocarrier of the present invention has better tumor killing ability and better circulation characteristics in the body.

[0105] (3) verification of long tumor retention time;

[0106] Figure 6 The SPECT / CT imaging comparison chart shows that as time goes by, the signal intensity of the quasi-spherical and rod-shaped nanocarriers at 48 h is lower than that of the non-spherical multipod heterojunction nanocarriers of the present invention. Therefore, it can be seen that the nanocarriers of the present invention have a longer retention ability in the tumor than conventional quasi-spherical and rod-shaped nanocarriers.

[0107] (4) In vitro ROS verification experiment using tumor radiotherapy-chemodynamics combined technology:

[0108] In vitro chemical kinetics of ROS validation. Figure 7 A and B are curves of the degradation of MB and DPBF by Fenton-like reaction mediated by the encapsulated Cu2S / Cu2S@Au-DSPE-PEG-MAL nanocomposite when the concentration of methylene blue (MB) is 10 μg / mL and the concentration of the Cu2S / Cu2S@Au carrier is 4 μg / mL; the Cu2S / Cu2S@Au carrier is fixed by the Fenton reagent and the methylene blue solution. +The absorbance of methylene blue decreases significantly with the concentration of methylene blue and the dose of x-ray, which can quantitatively detect the concentration of hydroxyl radicals produced to a certain extent. DPBF is a singlet oxygen-indicating fluorescent probe with high specificity for singlet oxygen (1O2), which can form endoperoxides and decompose into 1,2-dibenzoylbenzene. 1,3-Diphenylisobenzofuran can be used to detect the generation of reactive oxygen species (ROS). Once combined with singlet oxygen (1O2), DPBF is irreversibly oxidized, and the absorption intensity at UV-visible light (410nm) decreases rapidly. Results A and B show that under different doses of X-ray, the heterojunction "multipod nanomaterial" can produce hydroxyl radicals and singlet oxygen through UV-visible-near infrared absorption.

[0109] In vitro chemokinetic cell ROS staining verification. The same number of HepG2 cells were incubated in a confocal dish, incubated with the same concentration of Cu2S / Cu2S@Au for the same time, and then incubated with the ROS fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH2-DA) after X-ray irradiation. The confocal microscopy results verified that the tumor radiotherapy-chemokinetic combination of the nanocarrier of the present invention has a high killing ability. Among them, it can be seen from Figure D that with the increase of irradiation dose, the green fluorescence increases, which fully demonstrates that the tumor radiotherapy-chemokinetic combination of the Cu2S@Au nanocarrier of the present invention has a high killing ability;

[0110] (5) Small animal verification experiment of tumor radiotherapy-chemodynamics combined technology

[0111] The non-spherical multipod-shaped Cu2S nanocarriers, non-spherical multipod-shaped Cu2S@Au heterojunction nanocarriers and radionuclides labeled with them prepared in Example 1 were subjected to a small animal level verification experiment of the tumor radiotherapy-chemokinetic coupling technology. After establishing a subcutaneous liver cancer model, nude mice were randomly divided into 6 groups, namely PBS, Cu2S, Cu2S@Au, 177 Lu, Cu2S- 177 Lu, Cu2S@Au- 177 Lu (40μg, 200μCi). The body weight of mice treated with the combined technique was observed in detail ( Figure 8 -A), survival curve ( Figure 8 -B) Schematic diagram of tumor anatomy ( Figure 8 -C), tumor volume on day 14 ( Figure 8 -D) and the biological tissue distribution curve of radioactive nuclides ( Figure 8 -E).

[0112] The weight of the mice was detected on the 1st, 3rd, 5th, 7th, 9th, 11th and 13th days, and the size of the tumor was measured with a vernier caliper. The weight results showed that there was no significant change in the weight of the mice in each group, indicating that the radionuclide nanocarrier and the nuclear medicine labeled with the radionuclide of the present invention are highly safe and can reduce the autotoxicity of the radionuclide and the damaging effect on normal tissues.

[0113] After 14 days, the mice were euthanized and their major organ tissues, including heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, muscle, bone, brain and tumor, were removed by dissection. The mass was weighed and the radioactivity count was determined using a γ radioimmunoassay counter to evaluate the in vivo biodistribution of the material. Figure 8 The results show that the non-spherical multipod-shaped nanocarrier of the present invention has excellent therapeutic effect and can effectively shrink liver cancer tumors.

[0114] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0115] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A radionuclide nanocarrier, characterized in that: It is a non-spherical multi-pod structure. 2-x S is a nanomaterial; 0≤x≤1, and M is selected from one or more of Cu, Zn, Fe, Ag and Mn.

2. The radionuclide nanocarrier according to claim 1, characterized in that: In the M 2-x The non-spherical multi-pod structure surface of the S nanomaterial is coupled with one or more of Au, Pd, Pt, Ag and Fe2O3 to form a heterojunction nanocarrier.

3. The radionuclide nanocarrier according to claim 2, characterized in that: Metal atoms are coupled on the surface of the heterojunction nanocarrier to form a ternary nanocarrier.

4. The method for preparing the radionuclide nanocarrier according to claim 1, characterized in that: include: Using oleic acid and octadecene as morphology modifiers, CdS non-spherical multi-pod nanomaterials were obtained by thermal decomposition of a single precursor Cd(S2CNBut2); [MeCN]4MPF6 is added to the CdS non-spherical multi-pod nanomaterial to obtain a non-spherical multi-pod nanomaterial by cation exchange. 2-x S is a nanomaterial, 0≤x≤1, and M is selected from one or more of Cu, Zn, Fe, Ag and Mn.

5. The preparation method according to claim 4, characterized in that: Also includes: The non-spherical multi-pod M 2-x The S nanomaterial reacts with a precursor containing one or more than two selected from Au, Pd, Pt, Ag and Fe2O3 to obtain a heterojunction nanocarrier.

6. Use of the radionuclide nanocarrier according to any one of claims 1 to 3 in the preparation of radionuclide medicine or radionuclide combined carrier.

7. A radioactive nuclear medicine, characterized in that: It comprises the radionuclide nanocarrier, linker, chelating ligand, tumor targeting group and nuclide as described in any one of claims 1-3; the linker is selected from reactive phospholipid PEG reagent, which is coupled to the surface of the radionuclide nanocarrier; the tumor targeting group is coupled to the chelating ligand and the linker respectively; the chelating ligand chelates and fixes the nuclide.

8. The radioactive nuclide according to claim 7, characterized in that: The reactive phospholipid PEG reagent includes one or more of DSPE-PEG-MAL, DSPE-PEG-SH, DSPE-PEG-NH2, DSPE-PEG-COOH, DSPE-PEG-OH, and DSPE-PEG-NHS; the chelating ligand includes one or more of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, 1,4,7-triacetyltriamine, 1,4,7-triazacyclononane-1,4,7-tri(methylenephosphonic acid), and N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid; the tumor targeting group includes one or more of HA, PSMA, CEA, AFP, HCG, CA153, CA199, CA125, EBV, and VCA; the nuclide includes therapeutic α-nuclide, diagnostic β-nuclide + -Nuclide, therapeutic beta - -nuclide, diagnostic γ-nuclide and therapeutic Auger electron type nuclide, or two or more thereof.

9. The method for preparing the radioactive nucleopharmaceutical according to claim 7, characterized in that: include: The radionuclide nanocarrier according to any one of claims 1 to 3 is incubated with a linker to obtain a carrier-linker product; the tumor targeting group and the chelating ligand-PEG-NH2 are coupled under the action of a cross-linking agent to obtain a tumor targeting group-chelating ligand product; the carrier-linker product and the tumor targeting group-chelating ligand product are coupled under the action of a cross-linking agent to obtain a carrier-linker-tumor targeting group-chelating ligand product; The carrier-connector-tumor targeting group-chelating ligand product is added with a nuclide for incubation and labeling to obtain the radioactive nuclear medicine.

10. A radionuclide combined carrier, characterized in that: It comprises the radionuclide nanocarrier described in any one of claims 1 to 3 and a conventional radionuclide carrier; the conventional radionuclide carrier comprises one or more of small molecules, polypeptides, antibodies, and spherical nanoparticles.

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