CN111603575A - A core-shell structure radioembolization microsphere and its preparation method and application - Google Patents

Abstract

The invention provides a radioembolization microsphere with a core-shell structure, which uses a biodegradable polymer material as a shell to wrap a radionuclide as an inner core to form a core-shell structure microsphere. In the biodegradable core-shell structure radioembolization microsphere provided by the present invention, the radionuclide is wrapped inside the microsphere by a 1-30 μm thick biodegradable polymer shell. Due to the thick polymer shell and all the radionuclides located in the center of the microspheres, the destruction of the polymer shell surface will not lead to the leakage of radionuclides. After the treatment, the radionuclide loses its radioactivity, and the biodegradable property of the shell gradually dissolves the embolic microspheres, thereby re-opening the blood vessels in the treatment area. In addition, since the core-shell microspheres in the present invention have a certain proportion of cavities (5%-80% v/v), their overall density is closer to the density of water and thus has better dispersibility.

Description

A core-shell structure radioembolization microsphere and its preparation method and application

technical field

The invention belongs to the field of medicine and medicine, and particularly relates to a radioembolization microsphere with a core-shell structure and a preparation method and application thereof.

Background technique

Due to the unique blood supply of hepatocellular carcinoma (HCC), which is almost entirely derived from the hepatic artery, whereas the blood supply of the normal liver is composed of ~75% from the portal vein and ~25% from the hepatic artery, radionuclide-labeled microspheres can pass through The hepatic artery supplying blood to the liver cancer tissue is selectively retained in the hepatocellular carcinoma area. The radionuclide release rays carried by the microspheres are generally beta rays, and their action distance is generally several millimeters to more than ten millimeters, which can cause the death of surrounding tumor cells, but hardly damage the normal liver cells far away from the microspheres. .

和澳大利亚的Sirtex Medical开发的

这两种微球都是利用钇-90释放的β射线发挥治疗作用，但其物理性质和生产方式不同。

是一种含有非放射性钇-89的玻璃微球，使用前通过中子活化使玻璃微球中的钇-89活化为放射性的钇-90(US 4,789,501和US 5,011,677)。

使用离子交换树脂微球吸附活化的钇-90离子，并以其磷酸盐形式固化在吸附位点(US 20070253898 A1)。玻璃微球携带的反射性核素较吸附微球多，但是其密度远高于水，在肝脏血管中的沉积效果不好。而密度接近水的树脂微球有较好效果但其核素携带量不高。除了放射性核素⁹⁰Y，专利CN1080266A公布了一种使用³²P的放射玻璃微球。专利WO 2018,028644A1公布了一种同时使用³²P和⁹⁰Y的磷酸钇碳微球。上述微球都是使用不可生物降解材料来制作栓塞微球，会造成即使接受治疗的病变消失后，相关的区域的毛细血管网永久堵塞。There are currently two types of radiation microspheres in clinical use, which are developed by NORDION in Canada.

Developed with Australia's Sirtex Medical

Both types of microspheres use beta rays released by yttrium-90 to exert therapeutic effects, but their physical properties and production methods are different.

It is a glass microsphere containing non-radioactive yttrium-89. The yttrium-89 in the glass microsphere is activated to radioactive yttrium-90 by neutron activation before use (US 4,789,501 and US 5,011,677).

Activated yttrium-90 ions are adsorbed using ion exchange resin microspheres and immobilized at the adsorption sites in their phosphate form (US 20070253898 A1). Glass microspheres carry more reflective nuclides than adsorbed microspheres, but their density is much higher than that of water, and the deposition effect in liver blood vessels is not good. The resin microspheres with a density close to water have better effect but their nuclide carrying capacity is not high. In addition to the radionuclide ⁹⁰ Y, patent CN1080266A discloses a radioactive glass microsphere using ³² P. Patent WO 2018,028644A1 discloses a yttrium phosphate carbon microsphere using both ³² P and ⁹⁰ Y. The above-mentioned microspheres are all made of non-biodegradable materials to make embolic microspheres, which will cause permanent blockage of the capillary network in the relevant area even after the treated lesions disappear.

In order to allow the capillary network to reopen after treatment to restore blood flow to normal tissues, some radioactive microspheres use biodegradable materials such as poly(lactide-co-glycolide) (PLGA), polylactide (PLLA) and human serum albumin (HSA). For example, patent US 8,691,280 B2 discloses a PLA microsphere containing ¹⁶⁶ Ho and other documents disclose a ¹⁸⁸ Re-labeled PLGA microsphere. Biodegradable radioactive microspheres can be generally divided into two categories according to the way of nuclide labeling. A kind of organic ligands that chelate radionuclides on the surface of microspheres or encapsulate the nuclides, and then activate the encapsulated nuclides to have radioactivity through neutron activation. Another type of radionuclide dose carried by surface chelated nuclide microspheres is limited by surface chelation sites, and its nuclide carrying is generally small, and it is necessary to increase the injection volume of microspheres to meet the therapeutic dose requirements. By encapsulating microspheres such as organic complexes of nuclides, a large carrying amount of nuclides can be achieved, and the carrying rate of nuclides is between 10-30wt%, but such microspheres, in the neutron activation step, The surface structure and surface polymer materials of the polymer microspheres are easily damaged by the neutron beam, resulting in the scission of the polymer chains, resulting in the radionuclide wrapped between the polymers close to the surface of the microspheres losing the polymer coating and leaking after injection. Damage to normal organs that are sensitive to radiation, such as the kidneys.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide a biodegradable core-shell structure radioembolization microsphere, the structure of which is shown in schematic diagram 1 . Radionuclides in the form of inorganic salts, oxides or organic complexes are surrounded by a biodegradable polymer shell with a thickness of 1-30 μm. Due to the limitation of the biodegradable polymeric shell, the radionuclide in the middle of the microspheres will not leak until it loses radioactivity, and the shell will gradually degrade by its biodegradable properties. After the treatment, the blood vessels can be re-dredged.

To achieve the above object, the technical scheme adopted in the present invention is:

The invention discloses a radioembolization microsphere with a core-shell structure, which is formed by using a biodegradable polymer material as a shell to wrap a radionuclide as an inner core to form a core-shell structure microsphere. The radioembolization microspheres in the present invention encapsulate the radionuclide in a biodegradable polymer material to ensure that the microspheres have a high nuclide carrying rate and the radionuclide will not leak.

Preferably, the radionuclide is selected from ⁹⁰ Y, ³² P, ¹⁸ F, ¹⁴⁰ La, ¹⁵³ Sm, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁶⁹ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁰³ Pd, ¹⁹⁸ A combination of one or more nuclides of Au, ¹⁹² Ir, ⁹⁰ Sr, ¹¹¹ In and ⁶⁷ Ga.

Preferably, the carrying rate of the radionuclide is 10-30 wt%.

Preferably, the biodegradable polymer material is polylactide (PLA), polycaprolactone (PCL), poly(lactide-glycolide) copolymer (PLGA), poly(lactide- either caprolactone) copolymer (PLC) or poly(lactide-glycolide-polyglycol ether) copolymer (PLGE). The degradation life cycle of the polymer material must be at least five times the half-life of the radionuclide to ensure the integrity of the physical form of the microspheres to prevent the leakage of the radionuclide.

Preferably, the thickness of the shell composed of the polymer material in the microsphere is 1-30 μm.

The invention also provides the application of the radioembolization microspheres prepared by the invention in the treatment of tumors.

The present invention also provides a preparation method of the above-mentioned core-shell structure radioembolization microspheres, comprising preparing the core-shell structure microspheres with the radionuclide-containing complex and biodegradable polymer material by an emulsion evaporation method. The spheres and the steps of obtaining radioembolized microspheres by making the nuclides encapsulated in the microspheres radioactive by neutron excitation.

Preferably, the preparation method further comprises preparing the obtained embolic microsphere solution into embolic microsphere dry powder.

Preferably, in the preparation method, the radionuclide is selected from ⁹⁰ Y, ³² P, ¹⁸ F, ¹⁴⁰ La, ¹⁵³ Sm, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁶⁹ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ A combination of one or more nuclides among Re, ¹⁰³ Pd, ¹⁹⁸ Au, ¹⁹² Ir, ⁹⁰ Sr, ¹¹¹ In and ⁶⁷ Ga. The carrying rate of the radionuclide is 10-30 wt%.

Preferably, in the preparation method, the radionuclides are water-soluble inorganic salts of radionuclides, water-insoluble inorganic salts of radionuclides, ion chelates of radionuclides, oxides of radionuclides or organic compounds thereof. Coordinate.

Preferably, when the radionuclide is selected from its water-insoluble inorganic salts, oxides and organic complexes, it is first prepared into micro/nano particles with a particle size of 10 nm-20 μm.

Preferably, the water-soluble inorganic salt of the radionuclide is its chloride or nitrate.

Preferably, the water-insoluble inorganic salt of the radionuclide is its phosphate, carbonate or sulfate.

F68,

F108或

188等)、棕榈酸、硬脂酸聚烃氧(40)酯(PEG-40-Stearate)、聚乙烯醇(PVA)、二棕榈酰磷脂酰胆碱(DPPC)、二棕榈酰磷脂酰甘油(DPPG)或二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-mPEG2000)中的任意一种或者多种。Preferably, in the preparation method, in order to help dissolve or disperse radionuclides, a certain amount of surfactant is added to the solution, and the active surfactant is selected from polyoxyethylene polyoxypropylene ether block copolymers (such as

F68,

F108 or

188, etc.), palmitic acid, polyoxy (40) stearate (PEG-40-Stearate), polyvinyl alcohol (PVA), dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl glycerol ( DPPG) or any one or more of distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-mPEG2000).

Preferably, the preparation method further comprises freeze-drying the embolized microspheres, and the freeze-drying protective agent includes the following components: glucose, galactose, fructose, sucrose, trehalose, dextran, soluble starch, polyethylene Any one or more of glycol or mannitol.

Preferably, the lyoprotectant is a mixture of polyethylene glycol, mannitol and sucrose.

F-127、0.1％w/v蔗糖、3％w/v甘露醇和5％w/v聚乙二醇4000。Preferably, the lyoprotectant comprises the following components: 25 mM glycerol, 0.5% w/v

F-127, 0.1% w/v sucrose, 3% w/v mannitol and 5% w/v polyethylene glycol 4000.

Beneficial effects of the present invention: In the biodegradable core-shell structure radioembolization microspheres provided by the present invention, the radionuclide is wrapped inside the microspheres by a biodegradable polymer shell with a thickness of 1um-30um. Due to the thicker polymer shell and all the radionuclides located in the center of the microspheres, the destruction of the surface layer of the polymer shell by neutron activation will not lead to the leakage of radionuclides. After the treatment, the radionuclide loses its radioactivity, and the biodegradable property of the shell gradually dissolves the embolic microspheres, thereby re-opening the blood vessels in the treatment area. In addition, since the core-shell microspheres of the present invention have a certain proportion of cavities (porosity is 5%-80%), their overall density is closer to that of water and thus has better dispersibility.

Description of drawings

FIG. 1 is a schematic diagram of the core-shell structure of the radioembolized microspheres of the present invention.

FIG. 2 is a scanning electron microscope image of the radioembolized microspheres prepared in Example 2. FIG.

3 is a graph showing the release curve of ¹⁷⁷ Lu in human serum from the embolized microspheres prepared in Example 1 and Comparative Example.

Detailed ways

In order to show the technical solutions, objects and advantages of the present invention more concisely and clearly, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

The present invention mainly includes a combination of two aspects: biodegradable polymer materials and radionuclides.

Biodegradable polymer materials include: polylactide (PLA), polycaprolactone (PCL), poly(lactide-co-glycolide) copolymer (PLGA), poly(lactide-caprolactone) Either copolymer (PLC) or poly(lactide-glycolide-polyglycol ether) copolymer (PLGE). The colostrum is prepared by mixing it with radionuclides in the form of an organic solution. The colostrum is then emulsified to obtain a water/oil/water re-emulsion. After the organic solvent is volatilized, the macromolecular polymer is precipitated to form a shell, and impurities are removed to obtain an embolization microsphere solution. , the embolization microsphere solution can be lyophilized to obtain the embolization microsphere dry powder, the average thickness of which is 1 μm-30 μm.

The radionuclide is selected from ⁹⁰ Y, ³² P, ¹⁸ F, ¹⁴⁰ La, ¹⁵³ Sm, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹ Er, ¹⁶⁹ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁰³ Pd, ¹⁹⁸ Au, ¹⁹² A combination of one or more nuclides of Ir, ⁹⁰ Sr, ¹¹¹ In and ⁶⁷ Ga. Among them, radionuclides are encapsulated inside the microspheres in the form of water-soluble inorganic salts, organic complexes, ion chelates, oxides, etc. 10 to 30%. The embolic microspheres can directly encapsulate the radionuclide complex during the preparation process to have radioactivity. Alternatively, the embolizing microspheres first encapsulate the non-radioactive nuclide complex, and before use, the embolizing microsphere dry powder excites the encapsulated nuclide through neutrons. Among them, neutron excitation is preferred.

F68、

F108和

188等)、棕榈酸、硬脂酸聚烃氧(40)酯(PEG-40-Stearate)、聚乙烯醇(PVA)、二棕榈酰磷脂酰胆碱(DPPC)、二棕榈酰磷脂酰甘油(DPPG)或二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-mPEG2000)中的任意一种。其中以

F68为优选。In order to help improve the solubility or dispersibility of radionuclides in solution, a certain amount of surfactant is added to the solution, and the active surface area is selected from polyoxyethylene polyoxypropylene ether block copolymers (such as

F68,

F108 and

188, etc.), palmitic acid, polyoxy (40) stearate (PEG-40-Stearate), polyvinyl alcohol (PVA), dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl glycerol ( DPPG) or distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-mPEG2000). of which

F68 is preferred.

In order to freeze-dry the obtained microsphere solution during the preparation of microspheres, the freeze-drying protective agent mainly uses one or more of the following carbohydrates, such as glucose, galactose, fructose, sucrose, trehalose, dextran, soluble Starch, polyethylene glycol and mannitol, etc. Among them, the mixture of polyethylene glycol, mannitol and sucrose is the preferred lyoprotectant.

Several embodiments are listed below to illustrate the invention in more detail.

Example 1 Preparation of lutetium chloride-containing embolization microspheres

2 g of lutetium chloride (LuCl ₃ ) was weighed and dissolved in 1 ml of 0.5% w/v Pluronic F-68 aqueous solution. The LuCl ₃ aqueous solution was added to 30 ml of chloroform solution in which 2 g of poly(lactide-glycolide) copolymer (PLGA) (molecular weight: ~20,000, Sigma, USA) was dissolved, and placed on a magnetic stirrer and stirred at 2000 rpm for 5 min An emulsion is obtained. Then, the above emulsion was slowly added to 200 ml of 2% (w/w) polyvinyl alcohol (PVA, molecular weight: 13,000-23,000, 87-89% hydrolyzed) aqueous solution and stirred at 1000 rpm for 20 h to obtain microscopic ball. The microspheres were collected by centrifugation and then washed three times with double distilled water to remove excess PVA. Disperse the washed microspheres into 50 ml of cryoprotectant (25 mM glycerol, 0.5% w/v Pluronic F-127, 0.1% w/v sucrose, 3% w/v mannitol and 5% w/v polyethylene glycol). 4000 aqueous solution), the microspheres with a size of 20-40 μm were separated through a stainless steel sieve, and then divided into sample bottles, 5 ml per bottle. The sample vial was rapidly cooled in liquid nitrogen, and then lyophilized by a lyophilizer to obtain a dry powder of microspheres. Embolized microspheres for stability testing, the embolized microspheres were prepared using ¹⁷⁷ LuCl ₃ .

This example only exemplifies the radioactive plug microspheres prepared in the form of water-soluble inorganic salts of the radionuclide lutetium, but any one of the nuclides exemplified above can be used to achieve the same purpose, and microspheres with the same structure can be obtained .

Similarly, according to the needs of the actual situation, the combination of different nuclides is completely operable, which can achieve the purpose of synergy and obtain synergistic technical effects.

Example 2 Preparation of embolic microspheres containing yttrium phosphate nanoparticles

F-68(ThermoFisher,USA)水溶液中，超声30min以上使磷酸钇纳米颗粒均匀分散在水溶液里，然后通过过滤器(孔径0.45μm)除去较大颗粒。将磷酸钇纳米颗粒悬浮液加入30ml溶解有1g的PLGA(分子量：～30,000,Sigma-Aldrich,USA)的氯仿溶液中，放置磁力搅拌器上以2000rpm搅拌5分钟min获得乳浊液。然后，将上述乳浊液缓慢加入200ml的2％w/v的聚乙烯醇(PVA,分子量：13,000-23,000,87-89％水解,Sigma-Aldrich,USA)水溶液并以1000rpm搅拌上述混合液20h获得微球。微球通过离心进行收集，然后加入双蒸水清洗三次。将清洗好的微球分散到50ml的冷冻保护液(25mM甘油、0.5％w/v Pluronic F-127、0.1％w/v蔗糖、3％w/v甘露醇和5％w/v聚乙二醇4000水溶液)中，通过不锈钢筛分离尺寸在20-40μm的微球，然后分装到样品瓶中(每瓶5ml)。将样品瓶置于液氮中迅速冷却，然后通过冻干机进行冻干48h获得栓塞微球的冻干粉。8.4 mmol of yttrium chloride (YCl ₃ , Sigma-Aldrich, USA) and phosphoric acid (H ₃ PO ₄ , Sigma-Aldrich, USA) were dissolved in 300 mL of double distilled water. Under constant stirring, 150 ml of 56 mM NaOH solution was slowly added to the above yttrium nitrate-phosphoric acid solution, and continued stirring for 20 min to obtain an opaque suspension. The suspension was centrifuged at 4000 rpm for 5 min to collect the white precipitate. The precipitate was washed three times with double distilled water. In a vacuum environment, the above-mentioned yttrium phosphate nanoparticles are dried. Weigh 2.0g of yttrium phosphate nanoparticles and add 10ml of 0.2% w/v

In F-68 (ThermoFisher, USA) aqueous solution, ultrasonication for more than 30min made the yttrium phosphate nanoparticles uniformly dispersed in the aqueous solution, and then the larger particles were removed through a filter (pore size 0.45 μm). The yttrium phosphate nanoparticle suspension was added to 30 ml of a chloroform solution in which 1 g of PLGA (molecular weight: ~30,000, Sigma-Aldrich, USA) was dissolved, and an emulsion was obtained by stirring at 2000 rpm for 5 min on a magnetic stirrer. Then, the above emulsion was slowly added to 200 ml of 2% w/v polyvinyl alcohol (PVA, molecular weight: 13,000-23,000, 87-89% hydrolyzed, Sigma-Aldrich, USA) aqueous solution and the above mixture was stirred at 1000 rpm for 20 h Obtain microspheres. The microspheres were collected by centrifugation and then washed three times with the addition of double distilled water. Disperse the washed microspheres into 50 ml of cryoprotectant (25 mM glycerol, 0.5% w/v Pluronic F-127, 0.1% w/v sucrose, 3% w/v mannitol and 5% w/v polyethylene glycol). 4000 aqueous solution), the microspheres with a size of 20-40 μm were separated through a stainless steel sieve, and then distributed into sample vials (5 ml per vial). The sample vial was placed in liquid nitrogen to rapidly cool, and then lyophilized by a freeze dryer for 48 hours to obtain a freeze-dried powder of embolized microspheres.

This example only exemplifies the radioembolization microspheres prepared in the form of insoluble inorganic salts of the radionuclide yttrium, but any one of the nuclides exemplified above can be used to achieve the same purpose, and microbeads with the same structure can be obtained. ball.

Similarly, according to the needs of the actual situation, the combination of different nuclides is completely operable, which can achieve the purpose of synergy and obtain synergistic technical effects.

Example 3 Preparation of phospholipid-coated holmium acetylacetonate embolization microspheres

200 mg of dipalmitoyl phosphatidyl choline (DPPC, Avanti, USA), 150 mg of dipalmitoyl phosphatidyl glycerol (DPPG, Avanti, USA) and 150 mg of distearoyl phosphatidyl ethanolamine-polyethyl acetate were measured with an analytical balance. Diol (DSPE-mPEG2000, Avanti, USA) was placed in a flask, 25 ml of propylene glycol (Sigma-Aldrich, USA) and 25 ml of glycerol (Sigma-Aldrich, USA) were added, and heated in a water bath at 65 °C for 20 min to dissolve the phospholipids , then add 150ml of distilled water (65°C) and continue heating in a water bath for 20min at 65°C to obtain a uniform phospholipid solution, and then cool to room temperature to obtain a phospholipid solution (2.5mg/ml).

Weigh 2 g of holmium acetylacetonate (HoAcAc, Sigma-Aldrich, USA) into 3 ml of chloroform to dissolve. The chloroform solution was added to 15ml of the phospholipid solution (2.5mg/ml) prepared above, placed on a magnetic stirrer and stirred at 2500rpm for 5 minutes to obtain an emulsion, and 45ml of phospholipid dilution (0.5mg/ml) was slowly added to the obtained emulsion. ml) and placed on a magnetic stirrer to stir for 2h to evaporate the dichloromethane in the emulsion and solidify the holmium acetylacetonate into microspheres. The HoAcAc microsphere solution was collected by centrifugation (3000 x g, 10 min) and resuspended to wash away excess phospholipids and other small molecules. The above-mentioned microspheres were re-dispersed into 10 ml of phospholipid solution (0.2 mg/ml) and ultrasonicated for 5 min, so that the above-mentioned microspheres were uniformly dispersed in the phospholipid solution to obtain holmium acetylacetonate microspheres.

The prepared HoAcAc microsphere suspension was added to 30 ml of chloroform solution in which 2 g of PLGA (molecular weight: ~30,000) was dissolved, and placed on a magnetic stirrer and stirred at 2000 rpm for 5 min to obtain an emulsion. Then, the above emulsion was slowly added to 200 ml of 2% w/v polyvinyl alcohol (PVA, molecular weight: 13,000-23,000, 87-89% hydrolyzed, Sigma-Aldrich, USA) aqueous solution and the above mixture was stirred at 1000 rpm for 5 h Obtain microspheres. The microspheres were collected by centrifugation and then washed three times with the addition of double distilled water. Disperse the washed microspheres into 50 ml of cryoprotective solution (25 mM glycerol, 0.5% w/v Pluronic F-127, 0.1% w/v sucrose, 3.0% w/v mannitol and 5% w/v polyethylene glycol 4000). In aqueous solution), the microspheres with a size of 20-40μm were separated by stainless steel sieves, and then divided into sample bottles (5ml per bottle), the sample bottles were placed in liquid nitrogen to cool rapidly, and then lyophilized by a lyophilizer for 48h A lyophilized powder of the embolic microspheres was obtained.

This example only exemplifies the radioembolization microspheres prepared in the form of organic complexes of the radionuclide holmium, but using any of the nuclides exemplified above can achieve the same purpose and obtain microspheres with the same structure.

Similarly, according to the needs of the actual situation, the combination of different nuclides is completely operable, which can achieve the purpose of synergy and obtain synergistic technical effects.

Example 4 Preparation of embolic microspheres labeled by chelation (comparative example)

2 g of PLGA (molecular weight: ~20,000, Sigma, USA) was weighed and dissolved in 10 ml of chloroform. , and then slowly add 200 ml of 2% w/v polyvinyl alcohol (PVA, molecular weight: 13,000-23,000, 87-89% hydrolyzed, Sigma-Aldrich) aqueous solution and stir the above mixture at 2000 rpm for 20h to obtain microspheres. The microspheres were collected by centrifugation and then washed three times with the addition of double distilled water. Microspheres with a size of 20-40 μm were separated through a stainless steel sieve, dispersed in 60 ml of 0.1 M morpholine ethanesulfonic acid buffer (MES, pH 6.5) and soaked for 10 minutes, and 600 mg of 1-ethyl-(3-dimethicone) were weighed. Methylaminopropyl)carbodiimide hydrochloride (EDC, Sigma-Aldrich, USA) and 360 mg of N-hydroxythiosuccinimide (NHS, Sigma-Aldrich, USA) were added to the above mixture to activate Carboxyl groups on the surface of the microspheres. Five minutes later, 10 ml of MES buffer in which 5 g of bisamino-modified polyethylene glycol (PEG-bis amine, Mw-1000, 5 mmol, Sigma-Aldrich, USA) was dissolved was added and stirred overnight. The microspheres were collected by centrifugation and then washed three times with double distilled water to remove excess PEG-bisamine and other impurities. The washed microspheres were dispersed in 60ml of 0.1M MES buffer and soaked for ten minutes. Weigh 150 mg of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (DOTA -NHS ester, 0.2 mmol, Xi'an Ruixi, China) was dissolved in 3 ml of anhydrous dimethyl sulfoxide (DMSO), then slowly added dropwise to the microsphere suspension and stirred overnight. After the reaction, the microspheres were collected by centrifugation, and then washed three times by adding double distilled water. Disperse the washed microspheres into 50 ml of cryoprotective solution (25 mM glycerol, 0.5% w/v Pluronic F-127, 0.1% w/v sucrose, 3.0% w/v mannitol and 5% w/v polyethylene glycol). 4000 aqueous solution), the microspheres with a size of 20-40 μm were separated through a stainless steel sieve, and then divided into sample vials (5ml per vial), the sample vials were placed in liquid nitrogen to rapidly cool, and then lyophilized by a lyophilizer 48h to obtain the lyophilized powder of the embolized microspheres. Weigh 100 mg of embolized microspheres and disperse them into 20 ml of 0.05% w/v Pluronic F-68 ammonium acetate buffer (0.5 M, pH 5.0), and then add 2 ml of LuCl ₃ aqueous solution (0.1 mml , pH 5, adjusted with dilute hydrochloric acid), and the mixture was incubated in a water bath at 60 °C for 15 min to label the surface of the microspheres with radionuclides. After labeling, the microspheres were collected by centrifugation, and then washed three times by adding a 0.05% w/v aqueous solution of Pluronic F-68 to remove free Lu ³⁺ . Embolized microspheres for stabilization testing, labeled with radioactive ¹⁷⁷ LuCl ₃ solution.

Example 5 Measurement of particle size of nanoparticles and embolized microspheres, characterization of surface morphology, and measurement of nuclide carrying rate of embolized microspheres

1. Measure the particle size of micro/nano particles and embolic microspheres

Take 100ul of the nanoparticles prepared in Example 2 and Example 3 and add them to 2ml of phosphate buffered saline (PBS) dilution respectively, and measure the size of micro/nanoparticles by particle size measuring instrument Mastersize 2000 (Malvern, UK) using direct light scattering method. path. The results are shown in Table 1:

Table 1: Particle size of nuclide micro/nanoparticles for encapsulation prepared by different examples

Take 100ul of the embolization microspheres of Example 1, Example 2, Example 3, and Example 4 (comparative example) and add them to the 2ml PBS solution dilution respectively, then drop them into a hemocytometer, take the optical photos of the microspheres through a microscope, and pass The image processing software Image J analyzes and obtains the particle size measurement of the microspheres, as shown in Table 2:

Table 2: Particle size of embolic microspheres prepared in different examples

It can be seen from Table 2 that the particle diameters of the embolic microspheres prepared in Examples 1-4 are all between 20-35 μm.

2. Surface morphology characterization of embolized microspheres

The embolic microspheres prepared in Example 2 were diluted with double distilled water, then collected by centrifugation, re-dispersed in double distilled water, washed away the lyoprotectant, and then dropped on a silicon wafer. After the sample was completely dried, it was put into a scanning electron microscope (SEM, JSM-7200F, JEOL, Japan) to obtain SEM pictures, as shown in Figure 2. The surface of the embolized microspheres was smooth and morphologically intact.

3. Measure the radionuclide carrying rate of embolic microspheres

The nuclide carrying rate of the embolized microspheres was measured by inductively coupled plasma mass spectrometry. In order to exclude the interference of lyoprotectant on the measurement of carryover rate, the embolized microspheres were prepared without adding lyoprotectant during the lyophilization process. Weigh 5 mg of the embolized microspheres prepared in Examples 1 to 4, and use inductively coupled plasma mass spectrometry (ICP-quadrupole-MS, Varian 810-MS, USA) to measure the signal intensity of the corresponding nuclide and compare it with the standard curve of the corresponding nuclide to determine the final nuclide content. The nuclide carrying rate %=the mass of the nuclide/the total mass of the embolized microspheres×100%. The results are shown in Table 3, indicating that the embolization microspheres of the core-shell structure of the present invention have a higher nuclide carrying rate, which (Examples 1-3) are obtained by chelating the combined microspheres (Example 4). 7 times or more.

Table 3: Nuclide Carrying Rate of Embolized Microspheres Prepared in Different Examples

4. Measuring the porosity and calculating the density of the embolized microspheres

The porosity of the plugged microspheres was determined by mercury porosimetry. The porosity was measured by weighing 30 mg of the embolized microspheres prepared in Examples 1-3 by a porosimeter (PASCAL 140-400, POROTEC, Germany). The density ρ _particle of the microspheres is calculated by the following formula:

where ρ _air : air density (0.0013 g/cm ³ ); p: porosity of microspheres; a: ratio of nuclide to its related complexes; ρ _complex : density of nuclide complexes; ρ _{pol ymer :} shell Density of polymer (PLGA: 1.3 g/cm ³ ); e: nuclide carrying rate.

The porosity and density of the microspheres prepared in Examples 1-3 are shown in Table 4, and their density is close to the density of water (0.996 g/cm ³ ), thus having better dispersibility in aqueous solution.

Table 4: Porosity and calculated density of embolized microspheres prepared in different examples

Example 6 In vitro stability test of embolized microspheres

10 mg of ¹⁷⁷ Lu embolization microspheres prepared in Example 1 and Example 4 (comparative example) were weighed and dispersed into 20 ml of human serum solution, and the microsphere suspension was placed in a shaking incubator set at 37°C for continuous shaking. Take 10ml of the solution, centrifuge (2000×g, 5min) and take 8ml of the supernatant, measure the radiation intensity by a scintillation counter (Beckman LS6500, USA), continue to measure for 15 days, and put the supernatant back into the original solution after each measurement.

The amount of ¹⁷⁷ Lu released from the microspheres was expressed as a percentage of the radiation intensity entering the solution and the radiation intensity of the microspheres. Its release curve is shown in Figure 3. During the measurement period, the ¹⁷⁷ Lu in the microspheres prepared in Example 1 hardly leaked in human serum, and compared with the microspheres in Example 4, the core-shell structure prepared in Example 1 had no leakage. The microspheres showed better stability. Since the radionuclide of the microspheres of Example 4 is labeled on the surface of the microspheres by chelation, the slight degradation of the polymer on the surface of the microspheres will directly cause the release of radionuclides, while the radionuclides of the microspheres prepared in Example 1 are polymerized The outer shell of the material is wrapped in the core, and the pre-degradation of the polymer on the surface of the microsphere does not lead to the release of the radionuclide wrapped in the core.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (10)

1. A radioembolization microsphere of a core-shell structure is characterized in that, a core-shell structure microsphere is formed by encapsulating a radionuclide as an outer shell of a biodegradable macromolecular material. 2. The radioembolization microsphere of core-shell structure as claimed in claim 1, wherein the radionuclide is selected from the group consisting of ⁹⁰ Y, ³² P, ¹⁸ F, ¹⁴⁰ La, ¹⁵³ Sm, ¹⁶⁵ Dy, ¹⁶⁶ Ho, A combination of one or more nuclides among ¹⁶⁹ Er, ¹⁶⁹ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁰³ Pd, ¹⁹⁸ Au, ¹⁹² Ir, ⁹⁰ Sr, ¹¹¹ In and ⁶⁷ Ga. 3 . The core-shell structure radioembolization microsphere according to claim 1 or 2 , wherein the carrying rate of the radionuclide is 10-30 wt %. 4 . 4. The radioembolization microsphere of core-shell structure as claimed in claim 1, wherein the biodegradable macromolecular material is polylactide, polycaprolactone, poly(lactide-glycolide) ) copolymer, poly(lactide-caprolactone) copolymer or one or more of poly(lactide-glycolide-polyethylene glycol ether) copolymer. 5 . The radioembolization microsphere with a core-shell structure according to claim 1 or 4 , wherein the thickness of the shell composed of the polymer material in the microsphere is 1-30 μm. 6 . 6 . The application of the radioembolization microspheres according to any one of claims 1 to 5 in the treatment of tumors. 7. A preparation method of the radioembolization microspheres of the core-shell structure as described above, characterized in that, comprising forming the microspheres of the core-shell structure with the radionuclide and biodegradable macromolecular materials by an emulsion evaporation method and The step of obtaining radioembolized microspheres by making the nuclide encapsulated in the microspheres radioactive by neutron excitation. 8 . The preparation method according to claim 7 , further comprising preparing the obtained embolic microsphere solution into a dry powder of embolic microspheres. 9 . 9. The preparation method of claim 7, wherein the radionuclide in the preparation method is selected from the group consisting of ⁹⁰ Y, ³² P, ¹⁸ F, ¹⁴⁰ La, ¹⁵³ Sm, ¹⁶⁵ Dy, ¹⁶⁶ Ho, A combination of one or more nuclides in ¹⁶⁹ Er, ¹⁶⁹ Yb, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁰³ Pd, ¹⁹⁸ Au, ¹⁹² Ir, ⁹⁰ Sr, ¹¹¹ In and ⁶⁷ Ga; the radionuclide The carryover rate is 10-30 wt%. 10. The preparation method of claim 7, wherein the radionuclide in the preparation method is a water-soluble inorganic salt of a radionuclide, a water-insoluble inorganic salt of a radionuclide, or an ion of a radionuclide Chelates, oxides of radionuclides or their organic complexes; when said radionuclides are selected from their water-insoluble inorganic salts, oxides and organic complexes, they are first prepared into a particle size of 10nm- 20 μm nano/micro particles.

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