CN111603574A - Oxygen-carrying microsphere and preparation method and application thereof - Google Patents

Oxygen-carrying microsphere and preparation method and application thereof Download PDF

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CN111603574A
CN111603574A CN202010122023.5A CN202010122023A CN111603574A CN 111603574 A CN111603574 A CN 111603574A CN 202010122023 A CN202010122023 A CN 202010122023A CN 111603574 A CN111603574 A CN 111603574A
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oxygen
microspheres
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彭盛
张福君
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Sun Yat Sen University Cancer Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
    • A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention discloses an oxygen-carrying microsphere, which comprises a carrier matrix, a radionuclide and an oxygen-producing material, wherein the oxygen-producing material is wrapped by the carrier matrix, and the radionuclide is wrapped by the carrier matrix or chelated with the surface of the carrier matrix. The invention also discloses a preparation method of the oxygen-carrying microsphere and application of the oxygen-carrying microsphere in preparing a medicament for treating tumor radio-embolism. The oxygen-carrying microsphere has the capability of supplying oxygen by itself, so that the condition of hypoxia in a tumor region caused by embolism effect in the tumor radioactive embolism treatment process is relieved or eliminated, the treatment effect is finally improved, and adverse reactions caused by hypoxia are reduced.

Description

Oxygen-carrying microsphere and preparation method and application thereof
Technical Field
The invention relates to the technical field of tumor treatment, in particular to an oxygen-carrying microsphere and a preparation method and application thereof.
Background
Blood supply to normal liver is 75% from portal vein and 25% from hepatic artery. Whereas the blood supply for hepatocellular carcinoma (HCC) comes almost entirely from the hepatic artery. By using the unique blood supply mode, the radionuclide-labeled microspheres can selectively stay in hepatic artery supplying blood to liver cancer tissue, and the carried nuclide releases radioactive rays to cause the death of peripheral tumor cells, while the normal hepatic cells far away from the microspheres are hardly damaged.
Two types of radioactive microspheres, each developed by NORDION, Canada, are currently in clinical use
Figure BDA0002394621350000011
Developed by Sirtex Medical in Australia
Figure BDA0002394621350000012
The physical properties and mode of production of the two microspheres are different, but they both use β radiation released by the radionuclide yttrium-90 to exert therapeutic effects.
Figure BDA0002394621350000013
Is a glass microsphere containing non-radioactive yttrium-89, the yttrium-89 in the glass microsphere is activated into radioactive yttrium-90 by neutron activation before use, and the specific production process is disclosed in the patent US 4,789,5011And US 5,011,6772。
Figure BDA0002394621350000014
Activated yttrium-90 ions are adsorbed using ion exchange resin microspheres and immobilized at the adsorption sites in their phosphate form (US 20070253898 a1 and US 20100215571 a 1).
In radiotherapy, the permanent destruction of the DNA strands by radiation requires the presence of oxygen, and the radioactive microspheres need to be controlled to a size between 20-40 μm in diameter to avoid significant ischemic and embolic effects, but at the same time are sufficiently large to not pass through the capillary network into the venous circulation. Although the embolization effect of the radiomicrospheres was not expected to be significant by careful design of the size of the embolization microspheres, several studies have reported that the Vascular Endothelial Growth Factor (VEGF) associated with embolization effects increases rapidly and treatment results are poor after radioimmunoassay of tumors in patients3-4. The embolization effect of the microspheres can cause local hypoxia of liver cancer tissues, thereby increasing the radioresistance of liver cancer cells. To kill tumor cells, the radiation dose has to be increased to increase the amount of microspheres injected. Furthermore, the embolization effect of the radio-embolization therapy induces the body to systemically release angiogenic factors that promote the growth of untreated lesions or extrahepatic (micro) metastases and may affect the survival of the patient.
To inhibit the angiogenic response due to embolic effects and to improve therapeutic efficacy, several studies have attempted to combine Sorafenib (Sorafenib) during the course of yttrium-90 radiotherapeutic embolization4-5. Sorafenib is a multi-kinase inhibitor which targets a plurality of factors involved in angiogenesis and hepatoma cell proliferation, including VEGF receptors, PDGF, RAF-1 and the like, and is used to inhibit an angiogenic response caused by an embolic effect and to improve a therapeutic effect.
Conventional radio-embolic microspheres such as
Figure BDA0002394621350000021
And
Figure BDA0002394621350000022
with only the radionuclide and its corresponding carrier matrix. The microspheres can stay in capillaries of a tumor part due to the designed physical size so as to be focused on the tumor part in a targeted manner, but the embolism effect caused by the microspheres can cause local hypoxia of tumor tissues, so that the radioresistance of tumor cells is increased. To kill the tumor cells, the radiation dose has to be increased. In addition, embolic effects induce the body to systemically release angiogenic factors. This factor promotes the growth of untreated lesions and/or extrahepatic (micro) metastases. The invention mainly solves the problems of poor curative effect and adverse reaction caused by local hypoxia of a tumor region in the traditional radio-embolism treatment process.
Disclosure of Invention
Based on the above problems, the present invention aims to overcome the defects of the prior art and provide an oxygen carrying microsphere for tumor radioimmunoassay, which has the ability of supplying oxygen by itself, so as to alleviate or eliminate the hypoxia condition of the tumor region caused by the embolization effect in the process of tumor radioimmunoassay, and finally improve the treatment effect and reduce the adverse reaction caused by hypoxia.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following aspects:
in a first aspect, the present invention provides an oxygen-carrying microsphere comprising a carrier matrix, a radionuclide, and an oxygen-generating material, the carrier matrix encapsulating the oxygen-generating material, the radionuclide being encapsulated by the carrier matrix or chelated to a surface of the carrier matrix. Preferably, the carrier matrix has a hollow cavity, which is filled with an oxygen generating material or/and a radionuclide. Wherein, the oxygen-producing material is used for slowing down or eliminating the local hypoxia condition of the tumor area caused by the embolism effect in the radioactive embolism treatment process, and improving the sensitivity of tumor cells to rays, thereby improving the radiotherapy effect.
Preferably, the content of the carrier matrix in the oxygen-carrying microspheres is 9-90 wt%, the content of the radionuclide is 5-90 wt%, and the content of the oxygen-generating material is 1-80 wt%. More preferably, the carrier matrix in the oxygen-carrying microsphere is poly (lactide-glycolide) copolymer (PLGA, molecular weight: 20,000), the content is about 20 wt%, and the radionuclide is177Lu、166Ho or90The content of one of Y is 55 wt%, the oxygen generating material is calcium peroxide, and the mass ratio of the oxygen generating material to the microspheres is about 25 wt%. Particularly, when the content of the carrier matrix (PLGA) in the oxygen-carrying microsphere is 20 wt%, the content of the radionuclide is 55 wt%, and the content of the oxygen-generating material (calcium peroxide) is 25 wt%, the therapeutic effect is best when the oxygen-carrying microsphere is used for treating tumor radioactive embolism.
In one embodiment, the oxygen-carrying microspheres have a carrier matrix content of 16 wt%, a radionuclide content of 55 wt%, and a calcium peroxide content of 29 wt%.
In one embodiment, the oxygen-carrying microspheres have a carrier matrix content of 23 wt%, a radionuclide content of 55 wt%, and a calcium peroxide content of 22 wt%.
In one embodiment, the oxygen-carrying microspheres have a carrier matrix content of 25.7 wt%, a radionuclide content of 55 wt%, and a calcium peroxide content of 19.3 wt%.
Preferably, the carrier matrix is a high molecular polymer and/or a protein; more preferably, the carrier matrix is a biodegradable polymer, preferably an aliphatic polylactone, a binary or ternary random or block copolymer between lactones, a binary or ternary random or block copolymer between a lactone and a polyether; the protein is preferably human serum albumin or gelatin.
Preferably, the oxygen generating material is at least one of oxygen, an inorganic peroxide and an organic polymer material containing a peroxide bond. More preferably, the inorganic peroxides include, but are not limited to, Sodium Percarbonate (SPC), calcium peroxide (CaO)2) Magnesium peroxide (MgO)2) And hydrogen peroxide (H)2O2) And the like.
Preferably, the radionuclide is:
1) radioactive element90Y、32P、18F、140La、153Sm、165Dy、166Ho、169Er、169Yb、177Lu、186Re、188Re、103Pd、198Au、192Ir、90Sr、111In and67at least one of Ga;
2) an inorganic salt of the radioactive element;
3) a metal oxide of the radioactive element; or
4) An organic complex of the radioactive element.
Preferably, the oxygen generating material is a mixed gas of oxygen and a fluorine-containing gas, and the fluorine-containing gas accounts for 0.5-50% of the mixed gas by volume. Alternatively, the oxygen generating material is preferably a perfluorocarbon having an oxygen adsorbing ability and a medium boiling point (60 to 160 ℃), such as perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorononane, perfluorooctane, perfluorobromooctane, perfluoro-15-crown-5-ether, or the like.
Preferably, the fluorine-containing gas is at least one of sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane.
In a second aspect, the invention provides a preparation method of the oxygen-carrying microsphere, which adopts an emulsion solvent evaporation method, a chemical crosslinking method or a heating protein denaturation method to prepare the oxygen-carrying microsphere.
In a third aspect, the invention provides the use of said oxygen-carrying microspheres in the preparation of a medicament for the treatment of tumor radioimmunoassay.
In a fourth aspect, the present invention provides a medicament for the treatment of tumor radioembolism, which comprises the oxygen carrying microsphere.
In conclusion, the beneficial effects of the invention are as follows:
the invention provides an oxygen-carrying microsphere for tumor radio-embolization treatment for the first time and a preparation method thereof, compared with the existing radio-embolization microsphere such as
Figure BDA0002394621350000041
And
Figure BDA0002394621350000042
the novel embolism microsphere only has radionuclide and corresponding carrier matrix thereof, has the capability of supplying oxygen by itself, thereby relieving or eliminating the hypoxia condition of a tumor area caused by embolism effect in the tumor radio-embolism treatment process, finally improving the treatment effect and reducing the adverse reaction caused by hypoxia.
Drawings
FIG. 1 is a schematic illustration of the encapsulation of various oxygen generating materials, wherein (A) oxygen is filled in the hollow cavity of the microsphere; (B) the perfluorinated oil is wrapped in the middle of the carrier matrix to form microspheres with a core-shell structure; (C) the peroxide nanoparticles are embedded between carrier matrices;
FIG. 2 is a schematic diagram of the embedding of different nuclides, wherein (A) inorganic salts of the nuclides are embedded in the hollow cavities of the microspheres; (B) nuclide nanoparticles or organic complexes thereof are embedded between carrier matrices; (C) nuclides are chelated on the chelating groups on the surfaces of the microspheres;
FIG. 3(A) is a schematic view of a binding structure of a support matrix and a chelating group; (B) chemical structural formulas of different chelating groups;
FIG. 4 is a synthetic scheme of a carrier matrix PLGA-PEG-DOTA incorporating coordinating groups;
FIG. 5 is a drawing of the polymer PLGA-PEG-DOTA1H NMR spectrum;
FIG. 6 is an optical image of an oxygen-carrying embolic microsphere;
FIG. 7 is the oxygen release curve of oxygen-implanted embolized microspheres in phosphate buffer.
Detailed Description
In some embodiments, the present invention provides an oxygen-carrying microsphere for tumor radioimmunoassay treatment, which comprises three components, namely an oxygen generating material, a radionuclide and a carrier matrix, wherein:
1) oxygen generating material
The oxygen generating material refers to a material which can provide oxygen directly or through some chemical reaction, such as oxygen, inorganic peroxide or organic polymer material containing peroxy bond, and the like, and the content of the oxygen generating material in the oxygen carrying microsphere is 1-80 wt%.
In some cases, the oxygen generating material may be oxygen directly, as shown in FIG. 1-A, stored in the interstices of hollow or porous radioactive microspheres, which then slowly releases its entrained oxygen to the tumor environment by physical diffusion. In order to control the release rate of oxygen, one or more fluorine-containing gases, such as sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane, etc., may be mixed into the oxygen in a certain proportion (0.5% to 50%).
In some cases, the oxygen generating material is a perfluorocarbon having oxygen adsorbing capacity and a medium boiling point (60-160 ℃), such as perfluoropentane, perfluorohexane, perfluoroheptane, perfluorooctane, perfluorononane, perfluorooctane, perfluorobromooctane, perfluoro-15-crown-5-ether, and the like. As shown in FIG. 1-B, perfluorocarbons are wrapped in a carrier matrix to form microspheres with a core-shell structure.
In some cases, the oxygen generating material is an inorganic peroxy compound, including but not limited to Sodium Percarbonate (SPC), calcium peroxide (CaO)2) Magnesium peroxide (MgO)2) And hydrogen peroxide (H)2O2) And the like. Inorganic peroxidation as shown in FIG. 1-CThe substance is embedded between microsphere matrixes as solid nanoparticles (such as calcium peroxide nanoparticles) or filled in the cavities of the hollow microspheres as liquid state (such as hydrogen peroxide).
2) Radionuclides
Radionuclide refers to90Y、32P、18F、140La、153Sm、165Dy、166Ho、169Er、169Yb、177Lu、186Re、188Re、103Pd、198Au、192Ir、90Sr、111In and67ga and the like, and the content of the Ga accounts for 5-90 wt% of the oxygen-carrying microspheres. The radionuclide may be encapsulated in the microsphere in the form of its inorganic salt, metal oxide or organic complex.
In some cases, as shown in FIGS. 2-A and 2-B, the nuclide is first encapsulated between the carrier matrices of the microspheres or in the hollow cavities formed by the carrier matrices in the form of its inorganic salts, metal oxides, or organic complexes. Before use, the nuclide carried in the microsphere is excited to be radioactive by neutron radiation treatment.
In some cases, the radionuclide ions are bound to chelating sites on the surface of the microspheres, such as DOTA, DATP, and the like.
3) Carrier matrix
The carrier matrix is used for wrapping or carrying radionuclide and oxygen-producing material, and is generally high molecular polymer (such as polymethyl methacrylate, polyvinyl alcohol, poly (lactide-glycolide) copolymer) or protein (such as human serum albumin, gelatin, etc.), and the content of the high molecular polymer in the oxygen-carrying microspheres is 10-90 wt%.
In some cases, the carrier matrix is a biodegradable polymer, which is an aliphatic polylactone, a binary or ternary random or block copolymer between lactones and polyethers (molecular weight 5000-500000), such as Polylactide (PLA), Polycaprolactone (PCL), poly (lactide-glycolide) copolymer (PLGA), poly (lactide-caprolactone) copolymer (PLC), and poly (lactide-glycolide-polyglycol ether) copolymer (PLGE), and the like.
In some cases, the polymer may require chemical modification to bind one or more chemical structures for chelating radionuclides, as shown in FIG. 3-A, typically with the addition of a spacer group between the chelating group and the polymer. The spacer group is typically polyethylene glycol (PEG) with a molecular weight of 300-500, and the chelating group is typically DOTA, DTAP or derivatives thereof, and the chemical structure thereof is shown in FIG. 3-B.
4) Preparation of embolizing microspheres
The oxygen carrying microsphere for treating embolism is prepared by emulsion solvent evaporation, chemical crosslinking, heating protein denaturation or other methods according to the chemical and physical characteristics of the material containing the nuclear substance, the oxygen generating material and the carrier matrix.
In some cases, the oxygen-carrying microspheres are prepared by an emulsion solvent evaporation process. Generally, the nuclear-containing material (nuclide organic complex or inorganic nanoparticles), the oxygen-generating material, and the carrier matrix are dissolved or uniformly dispersed in a water-insoluble organic solvent; then preparing the organic solution into microdroplets with the diameter of 10-200 mu m in an aqueous solution containing a surfactant by mechanical stirring, ultrasonic emulsification, membrane emulsification or microfluidization emulsification, and the like, and stirring the emulsion to evaporate the organic solvent to finally obtain the oxygen-carrying microspheres.
In some cases, the oxygen-carrying microspheres are prepared by evaporation of a W/O/W multiple emulsion solvent, the core-containing material (inorganic salt) is dissolved in an aqueous phase, emulsified in an organic solvent in which the carrier matrix or the oxygen-generating material is dissolved, and then the emulsion is emulsified a second time in an aqueous solution containing a surfactant to prepare droplets having a diameter of between 10 and 200 μm, and the emulsion is stirred to evaporate the solvent to obtain the oxygen-carrying microspheres.
In some cases, the oxygen-carrying microspheres are prepared by a chemical crosslinking method in which a core-containing material (nuclide organic complex, or inorganic nanoparticles), an oxygen-generating material, and a carrier matrix are dissolved or uniformly dispersed in a water-insoluble organic solvent, and a crosslinking agent is added to the solvent, and then emulsified into droplets in an aqueous solution containing a surfactant, and the droplets are cured to form the oxygen-carrying microspheres by heating or by adding a catalyst to the aqueous phase to trigger a reaction.
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods in the present invention are all conventional methods.
Example 1 preparation of holmium-containing organic complex oxygen-carrying embolizing microspheres
1g of holmium acetylacetonate (HoAcAc, Sigma-Aldrich, USA), 1g of polylactic acid (PDLLA, molecular weight: -20,000, Sigma-Aldrich, USA) and 5ml of cyclooctane (Sigma-Aldrich, USA) were weighed and dissolved in 30ml of chloroform. The chloroform solution was added to 200ml of a 2% (w/v) aqueous solution of polyvinyl alcohol (PVA, molecular weight: 13,000-23,000, 87-89% hydrolysis), and the mixture was stirred at 1000rpm for 20 hours on a magnetic stirrer to obtain microspheres. The microspheres were collected by centrifugation and then washed three times with double distilled water. The washed microspheres were dispersed in 50ml of cryoprotectant (25mM glycerol, 0.5% Pluronic F-127(w/v), 0.1% (w/v) sucrose, 3.0% (w/v) mannitol and 5% (w/v) aqueous polyethylene glycol 4000), and microspheres of 20-40 μm size were separated by stainless steel sieves and then dispensed into glass vials (5 ml per vial). The glass vials were rapidly cooled in liquid nitrogen and then lyophilized for 48h by a lyophilizer (FreeZone 6, Labconco, USA) to remove water and polymer-encapsulated cyclooctane to give dry hollow microspheres. And sealing the small bottle containing the freeze-dried powder by using a rubber plug, then pumping out air in the bottle, and filling oxygen to obtain the oxygen-carrying microspheres.
Example 2 preparation of holmium-containing inorganic salt oxygen-carrying embolizing microspheres
Weighing 1g of holmium chloride (HoCl)3Sigma-Aldrich, USA) was dissolved in 10ml of 0.5% Pluronic F-68(Thermo Fisher, USA) aqueous solution. Adding HoCl3The aqueous solution was added to 30ml of a chloroform solution in which 1g of PLGA (molecular weight: -20,000, Sigma-Aldrich, USA) was dissolved, and stirred for 5min at 2000rpm on a magnetic stirrer to obtain an emulsion. Then, the above emulsion was slowly added to 200ml of a 2% (w/w) aqueous solution of polyvinyl alcohol (PVA, molecular weight: 13,000-23,000, 87-89% hydrolysis, Sigma-Aldrich, USA) and the above mixture was stirred at 1000rpm for 20 hours to obtain microspheres. The microspheres are collected by centrifugationThen adding double distilled water to clean for three times. The washed microspheres were dispersed in 50ml of cryoprotectant (25mM glycerol, 0.5% (w/v) Pluronic F-127, 0.1% (w/v) sucrose, 3.0% (w/v) mannitol and 5% (w/v) aqueous polyethylene glycol 4000), and microspheres of 20-40 μm size were separated by stainless steel sieves and then dispensed into glass vials (5 ml per vial). The glass vial was rapidly cooled in liquid nitrogen and then lyophilized for 48h by a lyophilizer (FreeZone 6, Labconco, USA) and the hollow microspheres were obtained by drawing off the water. And sealing the small bottle containing the freeze-dried powder by using a rubber plug, then pumping out air in the bottle, and filling oxygen to obtain the oxygen-carrying microspheres.
EXAMPLE 3 preparation of Yttrium-containing oxygen-carrying embolic microspheres
Adding 8.4mmol of yttrium nitrate (Y (NO)3)3Sigma-Aldrich, USA) and phosphoric acid (H)3PO4Sigma-Aldrich, USA) was dissolved in 300mL of double distilled water. 150ml of 56mM NaOH solution was slowly added to the yttrium nitrate-phosphoric acid solution with constant stirring, and stirring was continued for 20 minutes to give an opaque suspension. The suspension was centrifuged at 4000rpm for 5 minutes to collect a white precipitate. The precipitate was washed three times with double distilled water. And drying the yttrium phosphate nano-particles in a vacuum environment. 1g of yttrium phosphate nanoparticles was weighed into 10ml of 0.5% Pluronic F-68(Thermo Fisher, USA) aqueous solution, and the yttrium phosphate nanoparticles were uniformly dispersed in the aqueous solution by sonication for 30min or more, and then larger particles were removed by passing through a 0.45 μm pore size filter. The yttrium phosphate nanoparticle suspension was added to 30ml of a chloroform solution in which 1g of PLGA (molecular weight: -20,000) was dissolved, and stirred on a magnetic stirrer at 2000rpm for 5min to obtain an emulsion. Then, 200ml of a 2% (w/v) aqueous solution of polyvinyl alcohol (PVA, molecular weight: 13,000-23,000, 87-89% hydrolysis) was slowly added to the above emulsion and the above mixture was stirred at 1000rpm for 20 hours to obtain microspheres. The microspheres were collected by centrifugation and then washed three times with double distilled water. The washed microspheres were dispersed in 50ml of cryoprotectant (25mM glycerol, 0.5% Pluronic F-127(w/v), 0.1% (w/v) sucrose, 3.0% (w/v) mannitol and 5% (w/v) aqueous polyethylene glycol 4000), and microspheres of 20-40 μm size were separated by stainless steel sieves and dispensed into sample vials (5 ml per vial). Placing the sample bottle inThe microspheres were rapidly cooled in liquid nitrogen and then lyophilized for 48h by a lyophilizer (FreeZone 6, Labconco, USA) to remove water and obtain hollow microspheres. And sealing the small bottle containing the freeze-dried powder by using a rubber plug, then pumping out air in the bottle, and filling oxygen to obtain the oxygen-carrying microspheres.
Example 4 calcium peroxide nanoparticle preparation
Weighing 10g of calcium chloride (CaCl)2Sigma-Aldrich, USA) was added to 100ml of double distilled water, followed by 50ml of 1M ammonia water (Sigma-Aldrich, USA) and 300ml of polyethylene glycol (molecular weight: 200, Sigma-Aldrich, USA) for 30 minutes. 50ml of 35% hydrogen peroxide solution (Sigma-Aldrich) was added dropwise to the above mixture and stirring was continued for 3h, after which 0.1M sodium hydroxide solution (Sigma-Aldrich) was added to adjust the pH of the mixture to 11.5 and the solution gradually changed from clear to a white emulsion. Stirring was continued for 20min and the suspension was centrifuged at 8000rpm for 5min to collect a white precipitate. The precipitate was washed three times with double distilled water. And drying in a vacuum environment to obtain the calcium peroxide nanoparticles.
Example 5 Synthesis of holmium-containing calcium peroxide embolic microspheres
1g of the calcium peroxide nanoparticles prepared in example 4 was weighed into 20ml of chloroform, and the calcium peroxide nanoparticles were uniformly dispersed in chloroform by sonication for 30 minutes, and then larger particles were removed through a filter having a pore size of 0.45. mu.m. In addition, 1g of holmium acetylacetonate (HoAcAc, Sigma-Aldrich, USA) and 1g of polylactic acid (PDLLA, molecular weight: -20,000, Sigma-Aldrich, USA) were weighed and dissolved by adding 10ml of chloroform. The two solutions were mixed together, sonicated for 5 minutes, then 200ml of a 2% (w/v) aqueous solution of polyvinyl alcohol (PVA, molecular weight: 13,000-23,000, 87-89% hydrolyzed, Sigma-Aldrich, USA) was slowly added and the mixture was stirred at 1000rpm for 20h to obtain microspheres. The microspheres were collected by centrifugation and then washed three times with double distilled water. The washed microspheres were dispersed in 50ml of cryoprotectant (25mM glycerol, 0.5% Pluronic F-127(w/v), 0.1% (w/v) sucrose, 3.0% (w/v) mannitol and 5% (w/v) aqueous polyethylene glycol 4000), and microspheres of 20-40 μm size were separated by stainless steel sieves and dispensed into sample vials (5 ml per vial). The glass vial was rapidly cooled in liquid nitrogen and then lyophilized for 48h by a lyophilizer (FreeZone 6, Labconco, USA) to obtain lyophilized powder of the oxygen-carrying microspheres.
Example 6 Synthesis of Polymer containing chelating sites PLGA-PEG-DOTA
PLGA-PEG-DOTAP was prepared according to the synthetic route shown in FIG. 4. Specifically, first, 2g of PLGA (lactide: glycolide 50:50, Mw. about.10,000, 0.2mmol, Sigma-Aldrich, USA), 46mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC,0.24mmol, Sigma-Aldrich, USA) and 52mg of N-hydroxythiosuccinimide (NHS,0.24mmol, Sigma) were weighed out and dissolved in 10ml of anhydrous dimethyl sulfoxide (DMSO), followed by stirring under a nitrogen atmosphere for 24 hours to activate the carboxylic acid to obtain PLGA-NHS. The above solution was added dropwise to 5ml of an anhydrous DMSO solution in which polyethylene glycol (PEG-bisamine, Mw-1000, 0.4mmol Sigma) was dissolved, and after stirring for further 24 hours, the reaction solution was added dropwise to 100ml of double distilled water stirred at 1000rpm, and a white precipitate was precipitated. The suspension was centrifuged at 4000rpm for 20 minutes to collect a white precipitate. And washing the precipitate for three times by using double distilled water, removing excessive PEG-bisamine and other impurities, and drying in a vacuum environment to obtain PLGA-PEG-amine powder. 91.38mg of DOTA-NHS ester (0.12mmol, Sainta Rexi, China) was weighed and dissolved in 3ml of anhydrous dimethyl sulfoxide (DMSO), 5ml of a solution in which 1.1g of PLGA-PEG-amine (0.1mmol) was dissolved in anhydrous dimethyl sulfoxide was added dropwise to the above solution, and after stirring for 24 hours under a nitrogen atmosphere, the reaction solution was added dropwise to 50ml of double distilled water stirred at 1000rpm, and a white precipitate was precipitated. The suspension was centrifuged at 4000rpm for 20 minutes to collect a white precipitate. The precipitate was washed three times with double distilled water to remove excess DOTA-NHS ester and other impurities. Drying in a vacuum environment to obtain PLGA-PEG-DOTA powder; the chemical structure of the synthesized powder is as follows1H NMR as shown in FIG. 5.
Example 7 Synthesis of oxygen-Carrier PLGA microspheres containing chelating sites DOTA
1g of calcium peroxide nanoparticles (prepared in example 4) was weighed into 20ml of chloroform, sonicated for 30 minutes to uniformly disperse the calcium peroxide nanoparticles in the chloroform, and then larger particles were removed through a 0.45 μm pore size filter. In addition, 1g of PLGA/PLGA-PEG-DOTA (9:1mol/mol) was weighed and dissolved in 10ml of chloroform. The two solutions were mixed together, sonicated for 5 minutes, then 200ml of a 2% (w/v) aqueous solution of polyvinyl alcohol (PVA, molecular weight: 13,000-23,000, 87-89% hydrolyzed, Sigma-Aldrich) was slowly added and the mixture was stirred at 1000rpm for 20h to obtain microspheres. The microspheres were collected by centrifugation and then washed three times with double distilled water. Dispersing the cleaned microspheres into 50ml of cryoprotectant solution [ 25mM glycerol, 0.5% Pluronic F-127(w/v), 0.1% (w/v) sucrose, 3.0% (w/v) mannitol and 5% (w/v) aqueous polyethylene glycol 4000 ], separating the microspheres with the size of 20-40 μm by a stainless steel sieve, and then obtaining freeze-dried powder of the microspheres by freeze-drying.
Example 8 PLGA microsphere Synthesis
1g of PLGA/PLGA-PEG-DOTA (9:1mol/mol) was weighed out and dissolved in 30ml of chloroform, and then 200ml of a 2% (w/v) aqueous solution of polyvinyl alcohol (PVA, molecular weight: 13,000-23,000, 87-89% hydrolysis, Sigma-Aldrich, USA) was slowly added and the mixture was stirred at 1000rpm for 20 hours to obtain microspheres. The microspheres were collected by centrifugation and then washed three times with double distilled water. The washed microspheres were dispersed in 50ml of a cryoprotectant (25mM glycerol, 0.5% Pluronic F-127(w/v), 0.1% (w/v) sucrose, 3.0% (w/v) mannitol and 5% (w/v) aqueous polyethylene glycol 4000), microspheres of 20-40 μm in size were separated by stainless steel sieve, and lyophilized powder of microspheres was obtained by lyophilization.
EXAMPLE 9 radionuclide labeling of embolic microspheres
100mg of microspheres prepared in example 7 were weighed and dispersed in 25ml of 0.5M ammonium acetate buffer (pH 5.0, Sigma-Aldrich, USA).177LuCl3(500. mu.l, 5000MBq) solution was added to the microsphere suspension and the solution was hot-water-bathed at 90 ℃ for 15 minutes, then cooled to room temperature, warmed to 90 ℃ for 15 minutes, then cooled to room temperature, and so on, in triplicate. Microspheres were collected by centrifugation (1000g,5 min) and washed three times with double distilled water to remove free177Lu3+。
EXAMPLE 10 characterization of embolic microspheres
The embolized microspheres prepared in example 8, which were in situ prepared, were diluted to 1mg/mL and dropped onto a glass slide and observed under an inverted optical microscope, the optical picture of which is shown in FIG. 6, and the particle size of which was 26.3 μm as analyzed by Image analysis software Image J.
EXAMPLE 11 in vitro oxygen Release from embolic microspheres
20mg of the oxygen-carrying embolizing microspheres prepared in example 7 was dispersed in 20mL of phosphate buffer solution (PBS, pH 7.4), and the whole was placed in a hypoxic environment with the oxygen concentration controlled at 0.05 mmol/L. The oxygen concentration of the PBS solution was measured by a dissolved oxygen meter (JPB-607A, Shanghai Reye, China) at a specific time point for 15 days. In addition, embolizing microspheres (ordinary microspheres) without calcium peroxide nanoparticles prepared in example 8 were used as a control. The oxygen release curve is shown in fig. 7, which shows that the oxygen-carrying embolism microsphere of the invention can release oxygen continuously within 10 days, and improve the hypoxic environment of the microsphere.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Reference documents:
(1)Day,D.E.；Ehrhardt,G.J.,Glass Microspheres.Google Patents:1988.
(2)Day,D.E.；Ehrhardt,G.J.,Radioactive Glass Microspheres.GooglePatents:1991.
(3)Carpizo,D.R.；Gensure,R.H.；Yu,X.；Gendel,V.M.；Greene,S.J.；Moore,D.F.；Jabbour,S.K.；Nosher,J.L.J.J.o.V.；Radiology,I.Pilot Study of AngiogenicResponse to Yttrium-90 Radioembolization with Resin Microspheres.2014,25(2),297-306.e1.
(4)Rosenbaum,C.；Van Den Hoven,A.；Braat,M.；Koopman,M.；Lam,M.；Zonnenberg,B.；Verkooijen,H.；van den Bosch,M.J.E.r.Yttrium-90Radioembolization for Colorectal Cancer Liver Metastases:A Prospective CohortStudy on Circulating Angiogenic Factors and Treatment Response.2016,6(1),92.
(5)Lencioni,R.；Llovet,J.M.；Han,G.；Tak,W.Y.；Yang,J.；Guglielmi,A.；Paik,S.W.；Reig,M.；Chau,G.-Y.；Luca,A.J.J.o.h.Sorafenib or Placebo Plus Tace withDoxorubicin-Eluting Beads for Intermediate Stage Hcc:The Space Trial.2016,64(5),1090-1098.

Claims (10)

1. an oxygen-carrying microsphere comprising a carrier matrix, a radionuclide and an oxygen generating material, said carrier matrix encapsulating said oxygen generating material, said radionuclide being encapsulated by said carrier matrix or chelated to a surface of said carrier matrix.
2. The oxygen-carrying microsphere of claim 1, wherein the oxygen-carrying microsphere comprises from 9 to 90 wt% of the carrier matrix, from 5 to 90 wt% of the radionuclide, and from 1 to 80 wt% of the oxygen generating material.
3. The oxygen-carrying microsphere of claim 1, wherein the carrier matrix is a high molecular polymer and/or a protein.
4. The oxygen-carrying microsphere of claim 1, wherein the oxygen generating material is at least one of oxygen, an inorganic peroxide and an organic polymeric material containing a peroxide bond.
5. The oxygen-carrying microsphere of claim 1, wherein the radionuclide is:
1) radioactive element90Y、32P、18F、140La、153Sm、165Dy、166Ho、169Er、169Yb、177Lu、186Re、188Re、103Pd、198Au、192Ir、90Sr、111In and67at least one of Ga;
2) an inorganic salt of the radioactive element;
3) a metal oxide of the radioactive element; or
4) An organic complex of the radioactive element.
6. The oxygen-carrying microsphere of claim 1, wherein the oxygen generating material is a mixed gas of oxygen and a fluorine-containing gas, and the fluorine-containing gas accounts for 0.5-50% of the mixed gas by volume.
7. The oxygen-carrying microsphere of claim 6, wherein the fluorine-containing gas is at least one of sulfur hexafluoride, perfluoropropane, perfluorobutane, perfluoropentane, and perfluorohexane.
8. The method for preparing oxygen-carrying microspheres of any one of claims 1 to 7, wherein the oxygen-carrying microspheres are prepared by an emulsion solvent evaporation method, a chemical crosslinking method or a heated protein denaturation method.
9. Use of the oxygen-carrying microspheres of any one of claims 1 to 7 for the preparation of a medicament for the treatment of tumor radioembolism.
10. A medicament for the treatment of tumor radioembolism, which comprises the oxygen-carrying microspheres as claimed in any one of claims 1 to 7.
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