CT-traced tumor radiotherapy embolism microspheres and preparation method and application thereof

Abstract

The invention relates to preparation and application of a tumor radiotherapy embolism microsphere preparation capable of achieving CT imaging tracing. The microsphere is prepared by taking a polyvinyl alcohol derivative as a framework material, polymerizing, cross-linking and curing the polyvinyl alcohol derivative and an N-(aminoalkyl) methacrylamide hydrochloride monomer, and further covalently connecting a metal chelating agent and a CT imaging agent 2, 3, 5-triiodobenzaldehyde. The microsphere can mark radionuclides 111ln, 68Ga, Al18F, 64Cu, 177Lu and 90Y, embolism treatment and radionuclide internal irradiation treatment are combined, meanwhile, the microsphere CT tracing function is beneficial to monitoring the distribution condition of the microsphere in real time, and ectopic embolism is reduced.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to a CT-traced tumor radiotherapy embolization microsphere and a preparation method and application thereof. Background Art

[0002] Since most malignant tumors have abundant blood vessels, these blood vessels play an important role in the supply of nutrients and oxygen to the tumor, as well as the recurrence and metastasis of advanced tumors. Tumor embolization therapy is a minimally invasive local treatment method, which generally uses a superselective catheter to deliver embolic agents specifically to the tumor blood vessels through the femoral artery, and eventually completely blocks the blood vessels, thereby blocking the tumor's nutrient and oxygen supply to achieve the purpose of inhibiting tumor growth. In order to further enhance the effect of embolization therapy, chemotherapy drugs or radioactive therapeutic nuclides are usually combined with embolization therapy in clinical practice to increase the synergistic treatment effect.

[0003] In order to confirm the injection endpoint of the embolic agent, clinicians usually mix the CT contrast agent and the embolic agent and inject them simultaneously. However, since the contrast agent and the embolic agent are simply physically mixed, it is difficult for the contrast agent to accurately indicate the actual position of the embolic agent and the embolic effect under the flushing of blood in the body. In view of such clinical problems, covalently linking the iodine-containing contrast agent to the embolic agent will solve the above clinical dilemma.

[0004] Currently, the radioembolic agents available for clinical use are mainly 90 Y-labeled resin microspheres and glass microspheres are of a single variety and 90 As a pure β-nuclides, Y has limited imaging capability. Therefore, there is an urgent need in the art for a tumor radiotherapy embolization microsphere capable of CT imaging and tracing to improve the imaging capability. Summary of the invention

[0005] The present invention relates to a CT-traced tumor radiotherapy embolization microsphere and its preparation method and application. The microsphere is made of polyvinyl alcohol derivatives as the skeleton material, and is polymerized, cross-linked and solidified with N-(aminoalkyl) methacrylamide hydrochloride monomers, and is further covalently linked to a metal chelator and a CT imaging agent 2,3,5-triiodobenzaldehyde. The microsphere can be labeled with radionuclides. 111 ln, 68 Ga, Al 18 F, 64 Cu, 177 Lu and 90 Y. The multifunctional microsphere preparation can realize embolization therapy combined with radionuclide internal irradiation therapy for tumors, and can also perform CT tracing on the microspheres. Specifically, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a CT-traced tumor radiotherapy embolization microsphere, which is formed by polymerizing and cross-linking N-(aminoalkyl)methacrylamide hydrochloride monomers and polyvinyl alcohol derivative macromonomers in proportion, and the molar ratio of N-(aminoalkyl)methacrylamide hydrochloride monomers to polyvinyl alcohol derivative macromonomers is 50:1 to 1000:1;

[0007] Furthermore, the CT-traced tumor radiotherapy embolization microspheres are also covalently linked to metal chelators and CT imaging agents;

[0008] Furthermore, the structure of the N-(aminoalkyl)methacrylamide hydrochloride monomer is shown in Formula I:

[0009]

[0010] Wherein, R is selected from any one or more of -NH2, -COOH, -NCO, -N3, -DBCO;

[0011] Furthermore, the polyvinyl alcohol derivative macromonomer is formed by acetalization of N-(2,2-dimethoxyethyl)-2-acrylamide and the hydroxyl group of polyvinyl alcohol, wherein the degree of substitution of N-(2,2-dimethoxyethyl)-2-acrylamide is in the range of 0.1% to 30%;

[0012] Furthermore, the polyvinyl alcohol molecule in the polyvinyl alcohol derivative macromonomer is 50 to 100 kD;

[0013] Further, the covalently linked metal chelator is selected from any one or more of DOTA-Tri(BOC)-COOH, NOTA-di(BOC)-COOH, DOTAGA-Tetra(BOC)-COOH, and NOTAGA-Tri(BOC)-COOH; further, the mass ratio of the metal chelator to the microsphere is 0.05:1 to 2:1....

[0014] Further, the covalently linked CT imaging agent is 2,3,5-triiodobenzaldehyde;

[0015] Furthermore, the mass ratio of the CT imaging agent to the microspheres is 0.1:1 to 10:1;

[0016] The second aspect of the present invention provides a method for preparing any of the above-mentioned CT-traced tumor radiotherapy embolization microspheres, comprising the following steps:

[0017] S1: preparing an aqueous phase solution, preparing an aqueous solution of N-(aminoalkyl)methacrylamide hydrochloride monomer, a polyvinyl alcohol derivative macromonomer, and an aqueous solution of an initiator potassium persulfate;

[0018] S2: an oil phase solution containing an emulsifier, wherein the oil phase is liquid paraffin, butyl acetate, soybean oil or silicone oil, preferably liquid paraffin; the emulsifier is Span 80, Span 60 or Hypermer 2296, preferably Span 80;

[0019] S3: adding the aqueous phase solution prepared in S1 dropwise to the oil phase prepared in S2 to form an emulsion;

[0020] S4: adding an aqueous solution containing a catalyst N,N,N',N'-tetramethylethylenediamine to the above emulsion to catalyze a polymerization cross-linking reaction, solidify to form microspheres, and then wash with ethyl acetate, anhydrous ethanol, ultrapure water, and acetone and dry;

[0021] S5: dispersing the dried microspheres in anhydrous dimethyl sulfoxide (DMSO), adding a metal chelating agent, a condensing agent and triethylamine according to a ratio, and stirring the reaction at room temperature for 12 to 16 hours to form chelated microspheres, wherein the molar ratio of the metal chelating agent, the condensing agent and the triethylamine is 1:1 to 2:1 to 3; preferably, the molar ratio of the metal chelating agent, the condensing agent and the triethylamine is 1:1.5:2;

[0022] S6: washing the chelated microspheres obtained in S5 with DMSO, mixing with a DMSO solution of 2,3,5-triiodobenzaldehyde, dispersing the mixture in a DMSO solution of oxalic acid, stirring the mixture in a metal bath at 90-120° C. for 12-16 hours, and centrifuging the reaction solution for 3-10 minutes after the reaction is completed, wherein the mass ratio of the chelated microspheres to 2,3,5-triiodobenzaldehyde is 1:0.1-1:10, and the concentration of oxalic acid is 1.5M-3M;

[0023] S7: washing the microspheres obtained in S6 with DMSO, dispersing them in trifluoroacetic acid and stirring them at room temperature for 1 to 4 hours to obtain microspheres after reaction;

[0024] S8: washing the microspheres obtained in S7 with DMSO, ethanol and acetone, and then sieving to collect microspheres with different particle size ranges;

[0025] Further, in S1, the concentration of the aqueous solution of N-(aminoalkyl)methacrylamide hydrochloride monomer is 100 mg / mL to 1 g / mL;

[0026] Furthermore, in S1, the concentration of the aqueous solution of the polyvinyl alcohol derivative macromolecular monomer is 7.5wt% to 20wt%;

[0027] Further, in S1, the concentration of the potassium persulfate aqueous solution is 100 mg / mL to 200 mg / mL;

[0028] Further, in S2, the mass volume of the emulsifier: oil phase is 1g: 0.01-0.02mL;

[0029] Further, in S3, the mass ratio of the aqueous phase solution to the oil phase is 330-350:0.7-0.1;

[0030] Further, in S4, the volume ratio of the catalyst N,N,N',N'-tetramethylethylenediamine water: oil phase is 1:30-50; further, the concentration of the catalyst N,N,N',N'-tetramethylethylenediamine aqueous solution is 8-12 v / v%;

[0031] Further, in S5, the condensing agent is selected from any one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid (TBTU), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC); preferably 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU);

[0032] Further, the particle size range of the CT-traced tumor radiotherapy embolization microspheres is 20 to 1300 μm; further, the particle size range of the CT-traced tumor radiotherapy embolization microspheres is preferably 20 to 120 μm, 120 to 200 μm, 200 to 300 μm, 300 to 500 μm, 500 to 700 μm, 700 to 900 μm, 900 to 1100 μm or 1100 to 1300 μm; further, the particle size range of the CT-traced tumor radiotherapy embolization microspheres is preferably 20 to 120 μm;

[0033] The third aspect of the present invention provides a method for radionuclide labeling of any of the above-mentioned microspheres, comprising the following steps:

[0034] (2) Weigh 1 mg to 500 mg of the above microsphere powder and disperse it in 0.4 mL to 4 mL of 1 M sodium acetate buffer to form a microsphere suspension;

[0035] (2) adding a radionuclide solution having a radionuclide dose of 0.1 mCi to 1000 mCi to the microsphere suspension prepared in step (1), oscillating and mixing to obtain a mixed solution; preferably, the radionuclide dose is 1 mCi to 100 mCi;

[0036] (3) adjusting the pH of the mixed solution to a range of 3 to 7, and then heating the mixed solution in a metal bath at a temperature of 80° C. to 130° C. for a heating time of 10 to 60 min. After the heating is completed, washing the heated microspheres with PBS until no radioactivity is detected in the supernatant;

[0037] The fourth aspect of the present invention provides the use of any of the above-mentioned microspheres in the preparation of a radiotherapy drug for treating CT-traced tumors;

[0038] Furthermore, the CT tracing of the tumor radiotherapy embolization microspheres is SPECT / CT imaging or PET / CT imaging;

[0039] Furthermore, the CT-traced tumor radiotherapy embolization microspheres are used to label radioisotopes. 111 In, 68 Ga、Al 18 F. 64 Cu, 177 Lu, 90 Any one or more of Y; further, the labeling rate of the labeling is 90% to 100%;

[0040] Furthermore, when the labeled nuclide is 111 In or 177 Lu, SPECT / CT imaging was performed;

[0041] Or further, when the labeled nuclide is 68 Ga、Al 18 F or 64 Cu, PET / CT imaging was performed;

[0042] Or further, when the labeled nuclide is 177 Lu or 90 Y, intratumoral irradiation therapy;

[0043] Beneficial Effects

[0044] The present invention provides a multifunctional microsphere that can be used for tumor radiotherapy, chemotherapy, embolization and imaging, and a preparation method and application thereof. The microsphere uses a polyvinyl alcohol derivative as a skeleton material, and is polymerized, cross-linked and solidified with an N-acryloylamino acid monomer. The microsphere can be labeled with radioactive nuclide iodine and can adsorb chemotherapy drugs at the same time. The multifunctional microsphere preparation can achieve embolization and combined radiotherapy and chemotherapy of tumors, and simultaneously perform emission computed tomography or single photon emission computed tomography imaging. Compared with the prior art, more types of nuclides can be labeled, real-time imaging can be achieved, and the imaging effect is better. In addition, its combined radiotherapy and chemotherapy tumor treatment effect is better, and it has a wide range of clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 . nat Field emission scanning electron microscopy image of Lu-DPT microspheres;

[0046] Figure 2 . nat Particle size distribution of Lu-DPT microspheres;

[0047] Figure 3 . 177 Lu-DPT microsphere labeling rate diagram;

[0048] Figure 4 . 177 The labeling stability of Lu-DPT microspheres in phosphate buffered saline (PBS) and fetal bovine serum (FBS);

[0049] Figure 5 After 72 hours of incubation, 177 IC of Lu-DPT microspheres in N1S1 cells and HepG2 cells 50 value;

[0050] Figure 6 A CT imaging of DPT microspheres with gradient concentrations; Figure 6 B Linear regression line of DPT microsphere HU value and concentration; Figure 6 Distribution diagram of CDPT microspheres in liver cancer model rats;

[0051] Figure 7 .SPECT / CT images of model rats at predetermined time points after hepatic artery embolization;

[0052] Figure 8 . Figure 8 A indicates that rats with orthotopic liver cancer in each treatment group were injected with caudal vein before (0 days) and 3 and 10 days after hepatic artery embolization. 18 F-FDG, small animal PET / CT imaging to monitor tumor location 18 Results of changes in F-FDG uptake; Figure 8 B indicates the tumor location in each treatment group 18 The curve of the maximum F-FDG uptake value (SUVmax) changing with time (mean±SD, n=3, *p<0.05, **p<0.01, ***p<0.001); Figure 8 C represents the changes in body weight of animals in each treatment group; Figure 8 D shows HE staining, Ki67 and Tunel staining of each treatment group. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with embodiments and drawings.

[0054] Example 1

[0055] Weigh 300 mg of N-(aminopropyl)methacrylamide hydrochloride, dissolve in 200 μL ultrapure water, and adjust the pH to neutral with 100 μL 10M sodium hydroxide solution. Weigh 40 mg of N,N'-methylenebisacrylamide and ultrasonically disperse it in 150 μL ultrapure water, then add 1.334 mL of 10% polyvinyl alcohol derivative (polyvinyl alcohol molecular weight: 75 kDa, N-(2,2-dimethoxyethyl)-2-acrylamide substitution degree: 12% to 3%), and continue to ultrasonically dissolve. Add N-(aminopropyl)methacrylamide solution to polyvinyl alcohol derivative solution, mix well, add 87μL 150mg / mL potassium persulfate solution, mix well, add to 8mL liquid paraffin containing 0.12g Span 80, stir and emulsify at 600rpm for 10min in a 37℃ water bath under nitrogen, then add 200μL 10% (v / v) N,N,N',N'-tetramethylethylenediamine aqueous solution dropwise. The reaction solution is placed under nitrogen and stirred for 12h. After the reaction is completed, the reaction solution is centrifuged at 5000rpm for 5min to remove the liquid paraffin, and the lower precipitate is washed twice with ethyl acetate, anhydrous ethanol, and ultrapure water. After dehydration and centrifugation with acetone for 2 times, dry overnight in a vacuum drying oven at room temperature to obtain dry microsphere powder.

[0056] Weigh 100 mg of microsphere powder and suspend it in 1 mL of anhydrous DMSO. Weigh 50 mg of DOTA-Tri(BOC)-COOH (87.3 μmol), 49.8 mg of HATU (131 μmol) and 174.6 μmol of triethylamine, dissolve them in 0.5 mL of anhydrous DMSO, and add them to the microsphere suspension one by one. Continue stirring and reacting at room temperature overnight. After the reaction is completed, the reaction solution is centrifuged at 5000 rpm for 5 minutes, washed three times with DMSO, and set aside.

[0057] The washed microspheres were suspended in 1 mL of anhydrous DMSO, 200 mg of 2,3,5-triiodobenzaldehyde was weighed and dispersed in 1 mL of anhydrous DMSO, 630 mg of oxalic acid was weighed and dissolved in 2 mL of anhydrous DMSO, and the above three were mixed and placed in a 100°C metal bath for stirring and reaction overnight. After the reaction was completed, the reaction solution was centrifuged at 5000 rpm for 5 min, washed three times with DMSO, and then washed twice with anhydrous ethanol and acetone in sequence, and dried in a vacuum drying oven at room temperature overnight to obtain dry microsphere powder.

[0058] Take 2μL 1mCi / μL 177LuCl3 solution was added to 200 μL 1M sodium acetate solution, 1 mg microspheres were weighed and added to the mixed solution, mixed evenly, placed in a 100°C metal bath, and labeled for 10 min. After labeling, 5 μL of microsphere solution was taken for radioactive paper chromatography to determine the labeling rate of the microspheres. The remaining labeled microspheres were centrifuged at 5000 rpm for 5 min, the supernatant was removed, and the lower precipitate was continued to be washed with ultrapure water until no radioactivity was detected in the supernatant, and 5 μL of microsphere solution was taken for radioactive paper chromatography to determine the radiochemical purity of the washed microspheres.

[0059] The microspheres are uniform in size and have a rounded surface under field emission scanning electron microscopy ( Figure 1 The average particle size of the microspheres is about 45 μm ( Figure 2 ). The results of the radioactive paper chromatography experiment showed that the microsphere labeling rate was above 90% ( Figure 3 In the in vitro labeling stability study, the radiochemical purity was maintained at more than 95% after 28 days ( Figure 4 ). This indicates that the microsphere preparation of the present invention has remarkable stability.

[0060] 177 Lu-DPT MPs were co-incubated with rat hepatoma cell N1S1 and human hepatoma cell HepG2 for 72 h. 177 Lu's IC 50 15.78±9.91μCi / mL and 9.42±3.29μCi / mL respectively (see Figure 5 ). The results show that: 177 Lu-DPT MPs have a good killing effect on two types of liver cancer cells.

[0061] Different concentrations of DPT MPs agarose mimetics were prepared in vitro and imaged under small animal CT, showing that the HU value of the microspheres was linearly correlated with the concentration. The results of in vivo animal experiments showed that the distribution of microspheres could be clearly located under small animal CT images when DPTMPs were injected into the hepatic artery of rats bearing N1S1 liver cancer in situ (see Figure 6 ). The results showed that DPT MPs have CT tracing ability.

[0062] Similarly, rats bearing orthotopic N1S1 hepatocellular carcinoma were injected via the hepatic artery 177 Lu-DPT MPs can accurately monitor the distribution of radioactive microspheres in model rats under small animal SPECT / CT imaging and provide accurate liver tumor images up to 30 days after injection ( Figure 7 ), arrows indicate the positions of microspheres.

[0063] Intravenous injection 18F-FDG, using small animal PET / CT imaging to monitor the growth of N1S1 liver cancer rats in situ after different treatments. The results showed that compared with the saline group and the embolization group, the radioembolization group could significantly reduce the tumor uptake value. The tissue sections of each group also showed that the radioembolization group could significantly reduce the expression of ki67 in the tumor and significantly increase the expression of Tunel ( Figure 8 ); arrows indicate the location of the tumor.

[0064] Figure 8 A: rats with orthotopic liver cancer in each treatment group were injected with caudal vein before (0 days) and 3 and 10 days after hepatic artery embolization. 18 F-FDG, small animal PET / CT imaging to monitor tumor location 18 The results of F-FDG uptake changes (arrows indicate the location of the tumor, and the dosages of each treatment group are as follows: the Control group was injected with 0.2 mL of normal saline; the TAE group was injected with 0.2 mL of DPT microsphere suspension containing 10 mg of microspheres; the TARE group was injected with 177 Lu-DPT microsphere suspension 0.2mL, containing 10mg microspheres, 177 Lu 1000μCi; Figure 8 B is the tumor location in each treatment group 18 The curve of the maximum F-FDG uptake value (SUVmax) changing with time (mean±SD, n=3, *p<0.05, **p<0.01, ***p<0.001); Figure 8 C is the weight change of animals in each treatment group; Figure 8 D is HE staining, Ki67 and Tunel staining of each treatment group. The experimental results show that 177 Lu-DPTMPs radioembolization can significantly improve the therapeutic effect of in situ liver cancer compared with the DPTMPs embolization group and the normal saline control group.

[0065] Example 2

[0066] Weigh 189.5 mg of N-(aminopropyl)methacrylamide hydrochloride, dissolve in 200 μL ultrapure water, and adjust the pH to neutral with 124 μL 10M sodium hydroxide solution. Weigh 40 mg of N,N'-methylenebisacrylamide and ultrasonically disperse it in 150 μL ultrapure water, then add 1.334 mL of 7.5% polyvinyl alcohol derivative (polyvinyl alcohol molecular weight: 60 kDa, N-(2,2-dimethoxyethyl)-2-acrylamide substitution degree: 12% to 13%), and continue ultrasonic dissolution. Add N-(aminopropyl)methacrylamide solution to polyvinyl alcohol derivative solution, mix well, add 87μL 100mg / mL potassium persulfate solution, mix well, add to 8mL butyl acetate containing 0.12g Span 60, stir and emulsify at 600rpm for 10min in a 37℃ water bath under nitrogen, then add 200μL 8% (v / v) N,N,N',N'-tetramethylethylenediamine aqueous solution dropwise. The reaction solution is placed under nitrogen and stirred for 14h. After the reaction is completed, the reaction solution is centrifuged at 5000rpm for 5min to remove liquid paraffin, and the lower precipitate is washed twice with ethyl acetate, anhydrous ethanol, and ultrapure water. After dehydration and centrifugation with acetone for 2 times, dry overnight in a vacuum drying oven at room temperature to obtain dry microsphere powder.

[0067] Weigh 100 mg of microsphere powder and suspend it in 1 mL of anhydrous DMSO. Weigh 5 mg DOTAGA-Tetra(BOC)-COOH (7.13 μmol), 4.064 mg HBTU (10.69 μmol) and 14.26 μmol triethylamine, dissolve them in 0.5 mL of anhydrous DMSO, and add them to the microsphere suspension one by one. Continue stirring and reacting at room temperature overnight. After the reaction is completed, the reaction solution is centrifuged at 5000 rpm for 5 minutes, washed three times with DMSO, and set aside.

[0068] The washed microspheres were suspended in 1 mL of anhydrous DMSO, 10 mg of 2,3,5-triiodobenzaldehyde was weighed and dispersed in 1 mL of anhydrous DMSO, 540 mg of oxalic acid was weighed and dissolved in 2 mL of anhydrous DMSO, the above three were mixed, and placed in a 90°C metal bath for stirring and reaction overnight. After the reaction was completed, the reaction solution was centrifuged at 5000 rpm for 5 minutes, washed three times with DMSO, and then washed twice with anhydrous ethanol and acetone in sequence, and dried in a vacuum drying oven at room temperature overnight to obtain dry microsphere powder.

[0069] Take 2μL 1mCi / μL 90YCl3 solution was added to 200 μL 1M sodium acetate solution, 1 mg microspheres were weighed and added to the mixed solution, mixed evenly, placed in a metal bath at 80°C, and labeled for 10 min. After labeling, 5 μL of microsphere solution was taken for radioactive paper chromatography to determine the labeling rate of the microspheres. The remaining labeled microspheres were centrifuged at 5000 rpm for 5 min, the supernatant was removed, and the lower precipitate was continued to be washed with ultrapure water until no radioactivity was detected in the supernatant, and 5 μL of microsphere solution was taken for radioactive paper chromatography to determine the radiochemical purity of the washed microspheres.

[0070] The microspheres were uniform in size and rounded in surface under field emission scanning electron microscope. The average particle size of the microspheres was about 50 μm. The results of radioactive paper chromatography experiments showed that the microsphere labeling rate was above 95%. In the in vitro labeling stability study, the radiochemical purity was still above 98% at 28 days. This shows that the microsphere preparation of the present invention has significant stability.

[0071] 90 Y-DPT MPs were co-incubated with rat hepatoma cell N1S1 and human hepatoma cell HepG2 for 72 h. 90 IC of Y 50 The results showed that: 90 Y-DPT MPs have a good killing effect on two types of liver cancer cells.

[0072] Different concentrations of DPT MPs agarose mimetics were prepared in vitro and imaged under small animal CT, showing that the HU value of the microspheres was linearly correlated with the concentration. The results of in vivo animal experiments showed that the distribution of microspheres could be clearly located under small animal CT images when DPTMPs were injected into rats bearing N1S1 liver cancer in situ through the hepatic artery. The results showed that DPT MPs have CT tracing capabilities.

[0073] Similarly, rats bearing orthotopic N1S1 hepatocellular carcinoma were injected via the hepatic artery 90 Y-DPT MPs can accurately monitor the distribution of radioactive microspheres in model rats under small animal SPECT / CT imaging and can provide accurate liver tumor images up to 10 days after injection.

[0074] Intravenous injection 18 F-FDG, using small animal PET / CT imaging to monitor the growth of in situ N1S1 liver cancer rats after different treatments. The results showed that compared with the saline group and the embolization group, the radioembolization group could significantly reduce the tumor uptake value. The tissue sections of each group also showed that the radioembolization group could significantly reduce the expression of ki67 in the tumor and significantly increase the expression of Tunel. The experimental results show that 177Lu-DPTMPs radioembolization can significantly improve the therapeutic effect of in situ liver cancer compared with the DPTMPs embolization group and the normal saline control group.

[0075] Example 3

[0076] Weigh 2.2g of N-(aminopropyl)methacrylamide hydrochloride, dissolve in 500μL ultrapure water, and adjust the pH to neutral with 200μL 10M sodium hydroxide solution. Weigh 40mg of N,N'-methylenebisacrylamide and ultrasonically disperse it in 150μL ultrapure water, then add 1.334mL of 20% polyvinyl alcohol derivative (polyvinyl alcohol molecular weight: 75kDa, N-(2,2-dimethoxyethyl)-2-acrylamide substitution degree: 12% to 13%), and continue ultrasonic dissolution. Add N-(aminopropyl)methacrylamide solution to polyvinyl alcohol derivative solution, mix well, add 100μL 200mg / mL potassium persulfate solution, mix well, add to 8mL silicone oil containing 0.12g Hypermer2296, stir and emulsify at 600rpm for 10min in a 37℃ water bath under nitrogen, then add 200μL 12% (v / v) N,N,N',N'-tetramethylethylenediamine aqueous solution dropwise. The reaction solution was placed under nitrogen and continued to stir for 16h. After the reaction was completed, the reaction solution was centrifuged at 5000rpm for 5min to remove liquid paraffin, and the lower precipitate was washed twice with ethyl acetate, anhydrous ethanol, and ultrapure water. After dehydration and centrifugation with acetone for 2 times, it was dried overnight at room temperature in a vacuum drying oven to obtain dry microsphere powder.

[0077] Weigh 100 mg of microsphere powder and suspend it in 1 mL of anhydrous DMSO. Weigh 200 mg NOTAGA-Tri(BOC)-COOH (367.85 μmol), 209.8 mg HATU (551.78 μmol) and 735.7 μmol triethylamine, dissolve them in 0.5 mL of anhydrous DMSO, and add them to the microsphere suspension one by one. Continue stirring and reacting at room temperature overnight. After the reaction is completed, the reaction solution is centrifuged at 5000 rpm for 5 minutes, washed three times with DMSO, and set aside.

[0078] The washed microspheres were suspended in 1 mL of anhydrous DMSO, 10 mg of 2,3,5-triiodobenzaldehyde was weighed and dispersed in 1 mL of anhydrous DMSO, 540 mg of oxalic acid was weighed and dissolved in 2 mL of anhydrous DMSO, the above three were mixed, and placed in a 90°C metal bath for stirring and reaction overnight. After the reaction was completed, the reaction solution was centrifuged at 5000 rpm for 5 minutes, washed three times with DMSO, and then washed twice with anhydrous ethanol and acetone in sequence, and dried in a vacuum drying oven at room temperature overnight to obtain dry microsphere powder.

[0079] Take 2μL 1mCi / μL 64 CuCl2 solution was added to 200 μL 1M sodium acetate solution, 1 mg microspheres were weighed and added to the mixed solution, mixed evenly, placed in a 130°C metal bath, and labeled for 10 min. After labeling, 5 μL of microsphere solution was taken for radioactive paper chromatography to determine the labeling rate of the microspheres. The remaining labeled microspheres were centrifuged at 5000 rpm for 5 min, the supernatant was removed, and the lower precipitate was continued to be washed with ultrapure water until no radioactivity was detected in the supernatant, and 5 μL of microsphere solution was taken for radioactive paper chromatography to determine the radiochemical purity of the washed microspheres.

[0080] The microspheres were uniform in size and rounded in surface under field emission scanning electron microscope. The average particle size of the microspheres was about 55 μm. The results of radioactive paper chromatography experiments showed that the microsphere labeling rate was above 98%. In the in vitro labeling stability study, the radiochemical purity was still above 99% at 20 days. This shows that the microsphere preparation of the present invention has significant stability.

[0081] Different concentrations of DPT MPs agarose mimetics were prepared in vitro and imaged under small animal CT, showing that the HU value of the microspheres was linearly correlated with the concentration. The results of in vivo animal experiments showed that the distribution of microspheres could be clearly located under small animal CT images when DPTMPs were injected into rats bearing N1S1 liver cancer in situ through the hepatic artery. The results showed that DPT MPs have CT tracing capabilities.

[0082] Similarly, rats bearing orthotopic N1S1 hepatocellular carcinoma were injected via the hepatic artery 64 Cu-DPT MPs can accurately monitor the distribution of radioactive microspheres in model rats under small animal PET / CT imaging and can provide accurate liver tumor images up to 20 days after injection.

[0083] Intravenous injection 18 F-FDG, using small animal PET / CT imaging to monitor the growth of in situ N1S1 liver cancer rats after different treatments. The results showed that the embolization group could significantly reduce the tumor uptake value compared with the saline group. The tissue sections of each group also showed that the embolization group could significantly reduce the expression of ki67 in the tumor and significantly increase the expression of Tunel. The experimental results show that 64 Cu-DPT MPs radioembolization can significantly improve the therapeutic effect of in situ liver cancer compared with the normal saline control group.

[0084] Comparative Example

[0085] Weigh 120 mg of N-acryloyltyrosine (NAT) and dissolve it in 120 μL of 400 mg / mL sodium hydroxide solution, and adjust the pH to neutral with concentrated hydrochloric acid. Weigh 40 mg of N,N'-methylenebisacrylamide and ultrasonically disperse it in 150 μL of ultrapure water, then add 1.334 mL of 7.5% polyvinyl alcohol derivative (polyvinyl alcohol molecular weight: 75 kDa, N-(2,2-dimethoxyethyl)-2-acrylamide substitution degree: 12% to 13%), and continue to ultrasonically dissolve. Add N-acryloyltyrosine solution to polyvinyl alcohol derivative solution, mix well, add 87μL 150mg / mL potassium persulfate solution, mix well, add to 8mL liquid paraffin containing 0.12g Span 80, stir and emulsify at 600rpm for 10min in a 37℃ water bath under nitrogen, then add 200μL 10% (v / v) N,N,N',N'-tetramethylethylenediamine aqueous solution dropwise. The reaction solution is placed under nitrogen and continues to stir for 12h. After the reaction is completed, the reaction solution is centrifuged at 5000rpm for 5min to remove the liquid paraffin, and the lower precipitate is washed twice with ethyl acetate, anhydrous ethanol, and ultrapure water in sequence. Subsequently, the precipitate is dispersed with a small amount of ultrapure water and sieved through 70μm and 40μm filter screens in sequence, and the 70μm filter retention part and the 40μm sieve filter part are filtered out. Collect the precipitate between the 40-70 μm sieves, continue to dehydrate with acetone and centrifuge twice, and then dry in a vacuum drying oven at room temperature overnight to obtain dry NAT-PVA microsphere powder. Weigh 5 mg of microspheres and suspend them in 100 μL 0.01M pH 7.4 PBS. Take 10 μL 10mCi / mL Na131I solution and add it to the microsphere suspension. After mixing evenly, add 10 μL 10mg / mL chloramine-T PBS solution, shake gently to mix, and place the microsphere suspension in a 37°C water bath for 30 minutes. After labeling, take 5 μL of the microsphere solution for radioactive paper chromatography to determine the labeling rate of the microspheres. The remaining labeled microspheres are centrifuged at 5000 rpm for 5 minutes, the supernatant is removed, and the lower precipitate is continued to be washed with ultrapure water until no radioactivity is detected in the supernatant. Take another 5 μL of the microsphere solution for radioactive paper chromatography to determine the radiochemical purity of the washed microspheres.

[0086] The comparison between Example 1 of this patent and the comparative example is as follows:

[0087]

[0088]

[0089] It can be seen that, compared with the prior art, the microspheres prepared by the present invention can label more types of radionuclides, have better imaging effects, can perform real-time imaging, and have better tumor treatment effects.

Claims

1. A CT-traced tumor radiotherapy embolization microsphere, characterized in that: The invention is prepared by polymerizing and cross-linking N-(aminoalkyl)methacrylamide hydrochloride monomer and polyvinyl alcohol derivative macromonomer in proportion, and the molar ratio of the N-(aminoalkyl)methacrylamide hydrochloride monomer and the polyvinyl alcohol derivative macromonomer is 50:1-1000:

1.

2. The CT-traced tumor radiotherapy embolization microspheres according to claim 1, characterized in that: The CT-traced tumor radiotherapy embolization microspheres are also covalently linked to metal chelators and CT imaging agents.

3. The CT-traced tumor radiotherapy embolization microspheres according to claim 1 or 2, characterized in that: The structure of the N-(aminoalkyl)methacrylamide hydrochloride monomer is shown in Formula I: Wherein, R is selected from any one or more of -NH2, -COOH, -NCO, -N3, and -DBCO.

4. The CT-traced tumor radiotherapy embolization microspheres according to claim 1 or 2, characterized in that: The polyvinyl alcohol derivative macromonomer is formed by acetalization of N-(2,2-dimethoxyethyl)-2-acrylamide and the hydroxyl group of polyvinyl alcohol, wherein the degree of substitution of N-(2,2-dimethoxyethyl)-2-acrylamide ranges from 0.1% to 30%; further, the polyvinyl alcohol molecule in the polyvinyl alcohol derivative macromonomer is 50 to 100 kD.

5. The CT-traced tumor radiotherapy embolization microspheres according to claim 2, characterized in that: The covalently linked metal chelator is selected from any one or more of DOTA-Tri(BOC)-COOH, NOTA-di(BOC)-COOH, DOTAGA-Tetra(BOC)-COOH, and NOTAGA-Tri(BOC)-COOH; further, the mass ratio of the metal chelator to the microsphere is 0.05:1 to 2:

1.

6. The CT-traced tumor radiotherapy embolization microspheres according to claim 2, characterized in that: The covalently linked CT imaging agent is 2,3,5-triiodobenzaldehyde; further, the mass ratio of the CT imaging agent to the microspheres is 0.1:1 to 10:

1.

7. A method for preparing the CT-traced tumor radiotherapy embolization microspheres according to any one of claims 2 to 6, comprising the following steps: S1: preparing an aqueous phase solution, preparing an aqueous solution of N-(aminoalkyl)methacrylamide hydrochloride monomer, a polyvinyl alcohol derivative macromonomer, and an aqueous solution of an initiator potassium persulfate; S2: an oil phase solution containing an emulsifier, wherein the oil phase is liquid paraffin, butyl acetate, soybean oil or silicone oil, preferably liquid paraffin; the emulsifier is Span 80, Span 60 or Hypermer 2296, preferably Span 80; S3: adding the aqueous phase solution prepared in S1 dropwise to the oil phase prepared in S2 to form an emulsion; S4: adding an aqueous solution containing a catalyst N,N,N',N'-tetramethylethylenediamine to the above emulsion to catalyze a polymerization cross-linking reaction, solidify to form microspheres, and then wash with ethyl acetate, anhydrous ethanol, ultrapure water, and acetone and dry; S5: dispersing the dried microspheres in anhydrous dimethyl sulfoxide (DMSO), adding a metal chelating agent, a condensing agent and triethylamine according to a ratio, and stirring the reaction at room temperature for 12 to 16 hours to form chelated microspheres, wherein the molar ratio of the metal chelating agent, the condensing agent and the triethylamine is 1:1 to 2:1 to 3; preferably, the molar ratio of the metal chelating agent, the condensing agent and the triethylamine is 1:1.5:2; S6: washing the chelated microspheres obtained in S5 with DMSO, mixing with a DMSO solution of 2,3,5-triiodobenzaldehyde, dispersing the mixture in a DMSO solution of oxalic acid, stirring the mixture in a metal bath at 90-120° C. for 12-16 hours, and centrifuging the reaction solution for 3-10 minutes after the reaction is completed, wherein the mass ratio of the chelated microspheres to 2,3,5-triiodobenzaldehyde is 1:0.1-1:10, and the concentration of oxalic acid is 1.5M-3M; S7: washing the microspheres obtained in S6 with DMSO, dispersing them in trifluoroacetic acid and stirring them at room temperature for 1 to 4 hours to obtain microspheres after reaction; S8: washing the microspheres obtained in S7 with DMSO, ethanol and acetone, and then sieving to collect microspheres with different particle size ranges; Further, in S1, the concentration of the aqueous solution of N-(aminoalkyl)methacrylamide hydrochloride monomer is 100 mg / mL to 1 g / mL; Furthermore, in S1, the concentration of the aqueous solution of the polyvinyl alcohol derivative macromolecular monomer is 7.5wt% to 20wt%; Further, in S1, the concentration of the potassium persulfate aqueous solution is 100 mg / mL to 200 mg / mL; Further, in S2, the mass volume of the emulsifier: oil phase is 1g: 0.01-0.02mL; Further, in S3, the mass ratio of the aqueous phase solution to the oil phase is 330-350:0.7-0.1; Further, in S4, the volume ratio of the catalyst N,N,N',N'-tetramethylethylenediamine water: oil phase is 1:30-50; further, the concentration of the catalyst N,N,N',N'-tetramethylethylenediamine aqueous solution is 8-12 v / v%; Further, in S5, the condensing agent is selected from any one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroboric acid (TBTU), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC); preferably 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); Furthermore, the particle size range of the CT-traced tumor radiotherapy embolization microspheres is 20 to 1300 μm; further, the particle size range of the CT-traced tumor radiotherapy embolization microspheres is preferably 20 to 120 μm, 120 to 200 μm, 200 to 300 μm, 300 to 500 μm, 500 to 700 μm, 700 to 900 μm, 900 to 1100 μm or 1100 to 1300 μm; further, the particle size range of the CT-traced tumor radiotherapy embolization microspheres is preferably 20 to 120 μm.

8. A method for radionuclide labeling of the CT tracer tumor radiotherapy embolization microspheres according to any one of claims 1 to 6, comprising the following steps: (1) Weigh 1 mg to 500 mg of the above microsphere powder and disperse it in 0.4 mL to 4 mL of 1 M sodium acetate buffer to form a microsphere suspension; (2) adding a radionuclide solution having a radionuclide dose of 0.1 mCi to 1000 mCi to the microsphere suspension prepared in step (1), oscillating and mixing to obtain a mixed solution; preferably, the radionuclide dose is 1 mCi to 100 mCi; (3) The pH of the mixed solution is adjusted to 3-7, and then heated in a metal bath at 80°C-130°C for 10-60 min. After heating, the heated microspheres are washed with PBS until no radioactivity is detected in the supernatant.

9. Use of the CT-traced tumor radiotherapy embolization microspheres according to any one of claims 1 to 6 in the preparation of radiotherapy drugs for treating CT-traced tumors; Furthermore, the CT tracing of the tumor radiotherapy embolization microspheres is SPECT / CT imaging or PET / CT imaging; Furthermore, the CT-traced tumor radiotherapy embolization microspheres are used to label radioisotopes. 111 In, 68 Ga、Al 18 F. 64 Cu, 177 Lu, 90 Any one or more of Y; further, the labeling rate of the labeling is 90% to 100%; Furthermore, when the labeled nuclide is 111 In or 177 Lu, SPECT / CT imaging was performed; Or further, when the labeled nuclide is 68 Ga、Al 18 F or 64 Cu, PET / CT imaging was performed; Or further, when the labeled nuclide is 177 Lu or 90 Y, undergo intratumoral irradiation therapy.

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