A radioactive embolization microsphere loaded with radioactive iodine and a negatively charged drug, its preparation method and application

Abstract

This invention discloses a radioactive embolization microsphere capable of loading radioactive iodine and negatively charged drugs, its preparation method, and its application. The microsphere is a quaternary ammonium salt type containing a catechol structure and iodine, which can load radioactive iodine and negatively charged drugs. This invention prepares quaternary ammonium salt type microspheres containing a catechol structure using polyamino polymers or their hydrochlorides, catechol monomers, and iodine-containing monomers as main raw materials. These microspheres can be loaded with radioactive iodine and negatively charged drugs, enabling combined radiotherapy, chemotherapy, and embolization treatment of tumors, as well as radiographic diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and in particular relates to a radioactive embolization microsphere capable of loading radioactive iodine and negatively charged drugs, its preparation method and application. Background Technology

[0002] Transcatheter arterial chemoembolization (TACE) is a commonly used interventional procedure for treating primary liver cancer. It involves placing a catheter into the hepatic artery in the liver via a minimally invasive approach to deliver chemotherapy and embolize the lesions. Drug-eluting beads (DEBs) can combine embolization and chemotherapy. Currently, the main types on the market include DC beads, Hepasphere, and Callisphere. These microspheres primarily utilize the negative charge of carboxylic acid or sulfonic acid groups to adsorb positively charged chemotherapeutic drugs, enhancing the embolization effect. However, they cannot adsorb negatively charged drugs.

[0003] During chemotherapy, tumor cells often develop drug resistance, reducing the effectiveness of treatment. Radiotherapy, in addition to killing tumor cells, can also reduce their drug resistance. Therefore, combining chemotherapy and radiotherapy can significantly improve the therapeutic effect of embolization. Developing drug-eluting microspheres that can be loaded with radioactive elements is therefore of great significance for reducing drug resistance in embolization therapy and improving its efficacy. Furthermore, if the microspheres can be visualized during or after surgery, doctors can more precisely target the treatment area, detect and determine the endpoint, identify areas of insufficient treatment during surgery, improve patient safety during the procedure, and provide a basis for postoperative diagnosis. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, the present invention provides a contrast-enhancing embolization microsphere and a contrast-enhancing embolization microsphere loaded with radioactive iodine and a negatively charged drug, providing a new reagent or drug for clinical use in the embolization, radiotherapy and chemotherapy synergistic treatment of tumors, significantly improving the therapeutic effect and reducing chemotherapy resistance.

[0005] The present invention also provides a method for preparing and applying the radioactive iodine and negatively charged drug-loadable imaging embolism microspheres.

[0006] Technical solution: In order to achieve the above objectives, the present invention provides a radioactive embolism microsphere, wherein the microsphere is a quaternary ammonium salt type microsphere containing a catechol structure and iodine element.

[0007] The method for preparing the radiopaque embolic microspheres of the present invention includes the following steps:

[0008] (1) Aqueous phase preparation: Dissolve the polyamino polymer or its hydrochloride, catechol monomer, and iodine-containing monomer in water;

[0009] (2) Oil phase preparation: Add emulsifier to low polarity solvent and stir until dissolved;

[0010] (3) Microsphere preparation: The aqueous phase was added to the oil phase under stirring, 3-halo-1,2-epoxypropane was added, the reaction was heated, and then washed and filtered.

[0011] Wherein, the polyamino polymer in step (1) is one or more of polyacrylamide and polyethyleneimine; the hydrochloride of the polyamino polymer is one or more of polyacrylamide hydrochloride and polyethyleneimine hydrochloride; the catechol monomer is one or more of dopamine and dopamine hydrochloride; the iodine-containing monomer is one or more of 5-amino-2,4,6-triiodophthalic acid, 3-acetamido-5-amino-2,4,6-triiodobenzoic acid, 5-amino-N-(2-hydroxyethyl)-2,4,6-triiodobenzoylbenzoic acid, and 3-amino-2,4,6-triiodobenzoic acid.

[0012] In step (1), 100 parts by weight of the polyamino polymer or its hydrochloride, 1-40 parts by weight of the catechol monomer, and 10-200 parts by weight of the iodine-containing monomer are dissolved in 100-1000 parts of pure water and stirred until dissolved. When the polyamino polymer or its hydrochloride is used, 5-60 parts by weight of an alkaline compound are also required. The alkaline compound is one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, pyridine, triethylamine, sodium acetate, sodium hydroxide, and potassium hydroxide.

[0013] The low-polarity solvent mentioned in step (2) includes one or more of liquid paraffin, diethyl ether, petroleum ether, hexane, cyclohexane, and heptane; the emulsifier is one or more of Span, Tween, lauryl polyoxyethylene (120) ether, polyethylene glycol (200) monolaurate, glycerol monolaurate, N,N-dimethylhexamethylene amide, polyglycerol fatty acid ester, polyethylene glycol (200) dilaurate, diglycerol monolaurate, sorbitol monolaurate, and nonylphenoxy polyethylene glycol; wherein 0.5-5 parts of emulsifier are added to a low-polarity solvent with a volume of 2-10 times that of the aqueous phase, wherein the amount of emulsifier added is based on the weight ratio of each substance in the aqueous phase, and the mixture is stirred until dissolved.

[0014] In step (3), the aqueous phase is added to the oil phase under mechanical stirring at 100-500 rpm, and 10-50 parts of 3-halo-1,2-epoxypropane are added, wherein the amount of 3-halo-1,2-epoxypropane added is based on the weight ratio of each substance in the aqueous phase. The temperature is raised to 30-100℃, and the reaction is carried out for 2-24 hours. After washing and filtration, the product is obtained. The 3-halo-1,2-epoxypropane is one or more of epichlorohydrin and epibromopropane.

[0015] The radioactive embolic microspheres loaded with radioactive iodine and negatively charged drugs of the present invention are quaternary ammonium salt type microspheres containing catechol structure and iodine element and loaded with radioactive iodine and negatively charged drugs.

[0016] The radioactive iodine-loaded and negatively charged drug-loaded contrast-enhancing embolic microspheres of the present invention are prepared by the following steps:

[0017] (1) Loading radioactive iodine: The prepared microspheres were placed in a container containing a catalyst, and then a radioactive iodide solution was added. After centrifugation, the supernatant was discarded to obtain microspheres loaded with radioactive iodine.

[0018] (2) Loaded with negatively charged radiotherapy / chemotherapy drugs: The prepared radioactive iodine-loaded microspheres are placed in a solution containing negatively charged drugs, shaken and stirred, and the supernatant is discarded by centrifugation to obtain microspheres loaded with negatively charged radiotherapy / chemotherapy drugs; microspheres prepared by polyamino polymers need to be acidified before loading with negatively charged radiotherapy / chemotherapy drugs: the microspheres are placed in an acidic compound solution, and the supernatant is discarded by centrifugation.

[0019] The acidic compound is one or more of hydrochloric acid, sulfuric acid, acetic acid, and phosphoric acid.

[0020] The radioactive iodide is one or more of sodium iodide

[125] and sodium iodide

[131] ; the catalyst is one or more of chloramine-T and 1,3,4,6-tetrachloro-3α,6α-diphenylglyurea; and the negatively charged drug is one or more of carboxylates, phosphates, phosphates, nucleotides, deoxynucleotides, cyclic dinucleotides, oncolytic viruses, and Na2HPO4

[32] .

[0021] Preferably, the negatively charged drug is Combretastatin disodium phosphate (CA4P), Combretastatin A1 diphosphate tetrasodium salt (OXi-4503sodium, CA1P), STING agonist ADU-S100, MDK-563729, E7766, MK-1454, MSA-2, TAK-676, SR-717, norcantharidin sodium, mRNA-2416, oncolytic adenovirus,

[32] Na2HPO4,

[125] NaI,

[131] NaI, etc.

[0022] Application of the radioactive iodine-loaded and negatively charged drug-loaded contrast-enhancing embolization microspheres of the present invention in the preparation of combined radiotherapy, chemotherapy, and embolization drugs for tumors and contrast-enhancing diagnostic reagents.

[0023] The microspheres described in this invention can be prepared by adjusting the amount of emulsifier, stirring speed, and oil-water ratio, as reported in existing technologies, to obtain microspheres with different particle size ranges. The washing, filtering, and sieving steps can be performed using methods reported in existing technologies.

[0024] The present invention prepares quaternary ammonium salt microspheres containing catechol structures using polyamino polymers or their hydrochlorides, catechol monomers and iodine-containing monomers as the main raw materials. These microspheres can be loaded with radioactive iodine and negatively charged drugs to achieve combined radiotherapy, chemotherapy and embolization treatment of tumors and imaging diagnosis.

[0025] This invention utilizes polyamino polymers or their hydrochlorides, iodine-containing monomers, and catechol monomers as main raw materials to prepare radioactive iodine-loaded and negatively charged drug-loaded microspheres. The polyamino compounds carry a large amount of positive charge, allowing the microspheres prepared from them to adsorb negatively charged drugs. The catechol monomers react with iodides, and grafting them onto the microspheres allows for the loading of radioactive iodine. The iodine-containing monomers increase the density of the microspheres, enabling them to be visualized under standard X-rays. This invention is the first to copolymerize these with polyamino polymers to prepare radioactive iodine-loaded and negatively charged drug-loaded microspheres.

[0026] This invention is the first to prepare radiopaque microspheres using quaternary ammonium salts containing catechol structures and iodine, which are then further loaded with radioactive iodine / phosphorus and negatively charged drugs. Using specific raw materials and preparation methods, this invention not only produces microspheres with good sphericity and radiopaque function, but also exhibits excellent loading performance for both negatively charged radiotherapy and chemotherapy drugs. Furthermore, experiments have shown that the radiopaque microspheres prepared by this invention, simultaneously loaded with low doses of radioactive iodine / phosphorus and chemotherapy drugs, can significantly improve tumor treatment efficacy, significantly exceeding the therapeutic effects of high-dose single-chemoembolization microspheres, single-radioembolization microspheres, and combined radiotherapy and chemotherapy with free drugs.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0028] 1. This invention uses polyamino polymers or their hydrochlorides, iodine-containing monomers and catechol monomers as starting materials to prepare a radioactive iodine and negatively charged drug-loadable imaging embolism microsphere. The microsphere contains or is acidified to have quaternary ammonium groups, which carry a large number of positive charges and can adsorb negatively charged drugs that cannot be loaded by currently commercially available microspheres.

[0029] 2. The microspheres prepared by this invention can also be loaded with radioactive iodine and negatively charged radiotherapy and chemotherapy drugs. A single microsphere can be loaded with multiple radioactive iodine elements and multiple negatively charged drugs, realizing combined treatment of radiotherapy, chemotherapy and embolization, which can significantly improve the therapeutic effect of embolization.

[0030] 3. The microspheres prepared by this invention can be visualized during or after surgery, allowing doctors to more accurately target the treatment area, detect and determine the endpoint, identify areas of insufficient treatment during surgery, improve patient safety during surgery, and provide a basis for postoperative diagnosis. Attached Figure Description

[0031] Figure 1 The image shows the SEM image of the microspheres prepared in Example 1 of this invention.

[0032] Figure 2 The infrared spectrum of the microspheres prepared in Example 1 of this invention is shown. The spectrum displays a 2000 cm⁻¹ infrared spectrum. -1 and 3000cm -1 The presence of characteristic peaks of quaternary ammonium salts indicates that the microspheres were successfully synthesized.

[0033] Figure 3 The microspheres were developed under DSA, and the AD values ​​were for the microspheres prepared in Examples 8, 7, 6 and 1, respectively.

[0034] Figure 4 For the bar chart of tumor size-to-volume ratio after treatment and the analysis of differences, "*" indicates p≤0.05, "**" indicates p≤0.01, "***" indicates p≤0.001, and "****" indicates p≤0.0001. Detailed Implementation

[0035] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0037] The raw materials used in the embodiments of this invention include polyacrylamide hydrochloride (polyacrylamide hydrochloride, polyallylamine hydrochloride, CAS: 71550-12-4, average Mw = 17500, Merck, catalog number 283215), polyethyleneimine (Aladdin, catalog number E107079, average Mw = 10000), polyacrylamide (Aladdin, catalog number P432586, average Mw = 15000); 5-amino-2,4,6-triiodophthalic acid, dopamine hydrochloride (dopamine hydrochloride), liquid paraffin, Span 80, epichlorohydrin, 5-amino-N-(2-hydroxyethyl)-2,4,6-triiodophthaloylbenzoic acid, 3-amino-2,4,6-triiodophthalic acid, and 5-amino-2,4,6-triiodophthalic acid.

[0038] NaI(Na 131 Solution I (from Atomic High-Tech Co., Ltd.), CA1P, 1,3,4,6-tetrachloro-3α,6α-diphenylglycourea, etc. are all commercially available and can be purchased from reagent companies such as Merck, Aladdin, and McLean, or similar reagents from pharmaceutical companies.

[0039] Example 1

[0040] Preparation of microspheres

[0041] 25 mL of liquid paraffin and 10 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 150 rpm, and the mixture was stirred for 0.5 h to obtain the oil phase. Then, 1600 mg of polyacrylamide hydrochloride, 1600 mg of 5-amino-2,4,6-triiodophthalic acid, 200 mg of dopamine hydrochloride, and 300 mg of NaOH were dissolved in 3 mL of pure water to obtain the aqueous phase. This aqueous phase was added dropwise to the flask containing the oil phase, and the mixture was stirred at 150 rpm for 1 h. Then, 200 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 250 mL of isopropanol and 100 mL of ethanol for 1 h, and then filtered. Finally, the filter residue was washed with hydrochloric acid solution, filtered, and dried. The infrared spectrum of the microspheres prepared in this example is shown below. Figure 2 As shown, the spectrum displays 2000 cm⁻¹ -1 and 3000cm -1 The presence of characteristic peaks for quaternary ammonium salts indicates successful synthesis of the microspheres. SEM images of the microspheres are shown below. Figure 1 As shown, the prepared microspheres have regular morphology and good sphericity. The development effect of the microspheres is as follows. Figure 3 As shown in Figure D, the imaging effect is good. DSA imaging: The microspheres were placed in a centrifuge tube, physiological saline was added to swell them for 1 hour, then centrifuged, and the supernatant was removed. The centrifuge tube containing the microspheres was placed under DSA imaging to observe the imaging of the microspheres. After reducing the amount of iodine-containing monomer added, the imaging effect of the microspheres decreased significantly.

[0042] Example 2

[0043] Microsphere preparation

[0044] 25 mL of liquid paraffin and 10 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 150 rpm, and the mixture was stirred for 0.5 h to obtain the oil phase. Then, 1600 mg of polyacrylamide hydrochloride, 600 mg of dopamine hydrochloride, 1600 mg of 3-acetamido-5-amino-2,4,6-triiodobenzoic acid (CAS: 1713-07-1), and 400 mg of NaOH were dissolved in 3 mL of pure water to obtain the aqueous phase. This aqueous phase was added dropwise to the flask containing the oil phase, and the mixture was stirred at 150 rpm for 1 h. Then, 220 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 250 mL of isopropanol and 100 mL of ethanol for 1 h, and then filtered. Finally, the residue was washed with hydrochloric acid solution, filtered, and dried.

[0045] Example 3

[0046] Microsphere preparation

[0047] 25 mL of liquid paraffin and 10 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 170 rpm, and the mixture was stirred for 0.5 h to obtain the oil phase. Then, 1800 mg of polyacrylamide hydrochloride, 200 mg of dopamine hydrochloride, 1450 mg of 5-amino-N-(2-hydroxyethyl)-2,4,6-triiodom-benzoic acid, and 300 mg of NaOH were dissolved in 3 mL of pure water to obtain the aqueous phase. This aqueous phase was added dropwise to the flask containing the oil phase, and the mixture was stirred at 150 rpm for 1 h. Then, 200 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 250 mL of isopropanol, 100 mL of petroleum ether, and 100 mL of ethanol for 1 h, and then filtered. Finally, the residue was washed with hydrochloric acid solution, filtered, and dried.

[0048] Example 4

[0049] Microsphere preparation

[0050] 25 mL of liquid paraffin and 15 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 120 rpm, and the mixture was stirred for 0.5 h to obtain the oil phase. Then, 1684 mg of polyacrylamide hydrochloride, 300 mg of dopamine, 1300 mg of 3-amino-2,4,6-triiodobenzoic acid, and 200 mg of NaOH were dissolved in 3 mL of pure water to obtain the aqueous phase. This aqueous phase was added dropwise to the flask containing the oil phase, and the mixture was stirred at 120 rpm for 1 h. Then, 200 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 300 mL of isopropanol and 100 mL of ethanol for 1 h, and then filtered. Finally, the residue was washed with hydrochloric acid solution, filtered, and dried.

[0051] Example 5

[0052] Microsphere preparation

[0053] 25 mL of liquid paraffin and 15 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 140 rpm, and the mixture was stirred for 0.5 h to obtain the oil phase. Then, 1800 mg of polyacrylamide hydrochloride, 180 mg of dopamine, 1100 mg of 5-amino-2,4,6-triiodophthalic acid, and 250 mg of NaOH were dissolved in 3 mL of pure water to obtain the aqueous phase. This aqueous phase was added dropwise to the flask containing the oil phase, and the mixture was stirred at 140 rpm for 1 h. Then, 250 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 250 mL of isopropanol and 50 mL of ethanol for 1 h, and then filtered. Finally, the residue was washed with sulfuric acid solution, filtered, and dried.

[0054] Example 6

[0055] Microsphere preparation

[0056] 25 mL of liquid paraffin and 20 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 110 rpm, and the mixture was stirred for 0.5 h to obtain the oil phase. Then, 1600 mg of polyacrylamide hydrochloride, 390 mg of dopamine, 800 mg of 5-amino-2,4,6-triiodophthalic acid, and 400 mg of NaOH were dissolved in 3 mL of pure water to obtain the aqueous phase. This aqueous phase was added dropwise to the flask, and the mixture was stirred at 110 rpm for 1 h. Then, 220 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 220 mL of isopropanol and 150 mL of ethanol for 1 h, and then filtered. Finally, the filter residue was washed with hydrochloric acid solution, filtered, and dried. The prepared microspheres were then developed using DSA. Figure 3 As shown in B.

[0057] Example 7

[0058] Microsphere preparation

[0059] 25 mL of liquid paraffin and 15 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 160 rpm, and the mixture was stirred for 0.5 h to obtain the oil phase. Then, 2500 mg of polyethyleneimine, 300 mg of 5-amino-2,4,6-triiodophthalic acid, 390 mg of dopamine, and 100 mg of NaOH were dissolved in 3 mL of pure water to obtain the aqueous phase. This aqueous phase was added dropwise to the oil phase flask, and the mixture was stirred at 160 rpm for 1 h. Then, 210 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 250 mL of isopropanol and 100 mL of methanol for 1 h, and then filtered. Finally, the filter residue was washed with hydrochloric acid solution, filtered, and dried. The prepared microspheres were then developed using DSA. Figure 3 As shown in C.

[0060] Example 8

[0061] Microsphere preparation

[0062] 25 mL of liquid paraffin and 25 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 150 rpm, and the mixture was stirred for 0.5 h to obtain the oil phase. Then, 1000 mg of polyacrylamide, 200 mg of dopamine, 400 mg of 5-amino-2,4,6-triiodophthalic acid, and 270 mg of NaOH were dissolved in 3 mL of pure water to obtain the aqueous phase. This aqueous phase was added dropwise to the flask containing the oil phase, and the mixture was stirred at 150 rpm for 1 h. Then, 200 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 200 mL of petroleum ether and 100 mL of ethanol for 1 h, and then filtered. Finally, the filter residue was washed with sulfuric acid solution, filtered, and dried. The prepared microspheres were developed using DSA as shown in the figure. Figure 3 As shown in Figure A.

[0063] Comparative Example 1

[0064] 25 mL of liquid paraffin and 10 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 150 rpm, and the mixture was stirred for 0.5 h. Then, 1600 mg of polyacrylamide hydrochloride, 400 mg of dopamine hydrochloride, 1600 mg of 5-amino-2,4,6-triiodophthalic acid, and 800 mg of NaOH were dissolved in 3 mL of pure water and added dropwise to the oil phase flask. The mixture was stirred at 150 rpm for 1 h. Then, 200 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 250 mL of isopropanol and 100 mL of ethanol for 1 h, and then filtered. Finally, the residue was washed with hydrochloric acid solution, filtered, and dried. Due to the increased amount of NaOH added, a large amount of NaCl was generated during the reaction, and the emulsion was broken during the reaction, resulting in a large number of fragments in the microspheres.

[0065] Comparative Example 2

[0066] 25 mL of liquid paraffin and 10 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 150 rpm, and the mixture was stirred for 0.5 h. Then, 1600 mg of polyacrylamide hydrochloride, 1600 mg of 5-amino-2,4,6-triiodophthalic acid, 1000 mg of dopamine hydrochloride, and 300 mg of NaOH were dissolved in 3 mL of pure water and added dropwise to the above oil phase flask. The mixture was stirred at 150 rpm for 1 h. Then, 200 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, a large amount of undissolved dopamine hydrochloride remained in the solution.

[0067] Comparative Example 3

[0068] 25 mL of liquid paraffin and 10 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 150 rpm, and the mixture was stirred for 0.5 h. Then, 1600 mg of polyacrylamide hydrochloride, 90 mg of dopamine hydrochloride, 1600 mg of 5-amino-2,4,6-triiodophthalic acid, and 300 mg of NaOH were dissolved in 3 mL of pure water and added dropwise to the oil phase flask. The mixture was stirred at 150 rpm for 1 h. Then, 200 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 250 mL of isopropanol and 100 mL of ethanol for 1 h, and then filtered. Finally, the mixture was washed with hydrochloric acid solution, filtered, and dried.

[0069] Comparative Example 4

[0070] 25 mL of liquid paraffin and 20 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 170 rpm, and the mixture was stirred for 0.5 h. Then, 1600 mg of gelatin, 200 mg of dopamine hydrochloride, 1600 mg of 5-amino-2,4,6-triiodophthalic acid, and 300 mg of NaOH were dissolved in 3 mL of pure water and added dropwise to the oil phase flask above, stirring for 1 h. Then, 200 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 250 mL of isopropanol and 100 mL of ethanol for 1 h, and then filtered. Finally, the mixture was washed with hydrochloric acid solution, filtered, and dried.

[0071] Comparative Example 5

[0072] 25 mL of liquid paraffin and 10 μL of SPAN 80 were added to a 50 mL two-necked flask equipped with a mechanical stirrer. The stirring speed was adjusted to 150 rpm, and the mixture was stirred for 0.5 h. Then, 1600 mg of polyacrylamide hydrochloride, 2400 mg of 5-amino-2,4,6-triiodophthalic acid, 200 mg of dopamine hydrochloride, and 500 mg of NaOH were dissolved in 3 mL of pure water and added dropwise to the oil phase flask. The mixture was stirred for 1 h. Then, 200 μL of epichlorohydrin was added, and the reaction was carried out at 50 °C for 24 h. After the reaction was completed, the mixture was washed with 250 mL of isopropanol and 100 mL of ethanol for 1 h, and then filtered. Finally, it was washed with hydrochloric acid solution, filtered, and dried. Due to the increased amount of NaOH required to increase the amount of catechol monomer, a large amount of NaCl was generated during the reaction. The emulsion was unstable during the reaction, and all the prepared microspheres broke into fragments.

[0073] Example 9

[0074] Study on drug loading capacity of different microspheres

[0075] The drug-eluting microspheres prepared using different embodiments of the present invention, and Comparative Examples 3 and 4, were loaded with CA1P and radionuclides, with drug loading amounts of 0.2 mg / μL and 2.0 μCi / μL microspheres, respectively. The loading method and the method for determining the drug loading amount are as follows:

[0076] (1) Combritin A1 diphosphate tetrasodium salt (CA1P): Prepare an 80 μg / mL CA1P saline solution, dilute it 2, 4, 8, 16, and 32 times respectively, and measure the absorbance at 295 nm using a UV spectrophotometer. Plot a standard curve with concentration as the ordinate and absorbance as the abscissa. Place microspheres in a CA1P saline solution (2.5 mg / mL), shake for 1 h, collect the supernatant, and calculate the drug loading.

[0077] (2) Radioactive iodine (

[131] NaI): Microspheres were placed in a container containing 100 μL of 1,3,4,6-tetrachloro-3α,6α-diphenylglyurea (1 mg / mL DMSO solution), and then a solution of radioactive iodine (NaI) was added. 131 After shaking for 1 hour, the radioactivity count of the supernatant was measured and the loading was calculated.

[0078] And calculate according to the following formula

[0079] Maximum drug loading (CA1P) = (1-M 上清 / M 总 )×0.2

[0080] Maximum drug loading (

[131] NaI) = (1-M) 上清 / M 总 )×2.0

[0081] In the formula M 上清 M represents the total mass of the drug in the supernatant. 总 M represents the total mass of the drug added; if the drug is a radionuclide, M represents the radioactivity count.

[0082] Table 1 shows the drug loading capacity of CA1P and radionuclides on the drug-eluting microspheres prepared in different embodiments of the present invention. The drug-eluting microspheres prepared in Example 1 exhibited the best performance in loading both negatively charged drugs and radioactive iodine. In Comparative Example 3, due to the relatively small amount of dopamine added during preparation, the loading of radioactive iodine was lower. The microspheres in other embodiments all demonstrated good performance in loading both radioactive iodine and negatively charged drugs. Comparative Example 4, prepared by copolymerizing gelatin and dopamine, resulted in microspheres with lower efficiency in loading both radionuclides and negatively charged drugs.

[0083] Table 1. Loading capacity of microspheres prepared in different embodiments and comparative examples for CA1P and radioactive iodine.

[0084]

[0085] Example 10

[0086] Microsphere loading capacity tests for different types of iodine and drugs

[0087] Different types of drugs were loaded onto the microspheres prepared in Example 1, with drug loading amounts of 0.2 mg / μL and 2.0 μmg / μL microspheres, respectively. The loading and drug loading determination methods are as follows:

[0088] (1) CA1P: according to method (1) of Example 9.

[0089] (2) Sodium cantharidate: Accurately measure 1.0, 2.0, 3.0, 5.0, 7.0, and 10.0 ml of sodium cantharidate aqueous solution (1 mg / mL) into 50 ml volumetric flasks, dilute to the mark with mobile phase, and shake well. Inject 20 μL of each solution into the chromatograph and record the chromatogram. Perform linear regression with concentration (C, μg / mL) as the abscissa and peak area (A) as the ordinate (chromatographic conditions: SHISEIDOTYPE MG ODS column (150 mm 4.6 mm, 5 m), column temperature 30℃. Use 0.1 mol / L dipotassium hydrogen phosphate solution - acetonitrile (80:20) (adjust pH to 2.6 with phosphoric acid) as the mobile phase, and the detection wavelength is 210 nm). Place microspheres in sodium cantharidate aqueous solution (1 mg / mL), shake for 1 h, and use the supernatant for liquid chromatography to test and calculate the drug loading.

[0090] (3) ADU-S100 (Interferon Gene Stimulating Factor (STING)): Prepare ADU-S100 saline solutions of 0.01, 0.03, 0.06, and 0.12 mg / mL, respectively, and inject 20 μL into the chromatograph, recording the chromatograms. Perform linear regression with concentration (C, μg / mL) on the x-axis and peak area (A) on the y-axis (chromatographic conditions: mobile phase iPrOH:hexane = 10:90; column temperature 25℃; flow rate 1.0 mL / min; detection wavelength 210 nm). Place microspheres in ADU-S100 saline solution (0.1 mg / mL), shake for 1 h, and use liquid chromatography to test and calculate the drug loading.

[0091] (4) mRNA-2416: Prepare a 0.1 mg / mL physiological saline solution of mRNA-2416, add microspheres, shake for 1 h, take the supernatant and determine the content of mRNA-2416 in the supernatant according to the method of GB / T 34796-2017, and calculate the loading.

[0092] (5) In

[32] Na2HPO4: Take microspheres and place them in

[32] Na2HPO4 solution. After shaking for 0.5 h, measure the radioactivity count of the supernatant and calculate the load.

[0093] (6) Radioactive iodine: according to method (2) of Example 9.

[0094] The formula for calculating the drug loading capacity of microspheres is:

[0095] Drug loading = (1-M) 上清 / M 总 )×100%

[0096] Where: M 上清 M represents the total mass of the drug in the supernatant. 总 M represents the total mass of the drug added; if the drug is a radionuclide, M represents the radioactivity count.

[0097] The drug loading results of the microspheres prepared in Example 1 of the present invention are shown in Table 2.

[0098] Table 2. Drug loading capacity of microspheres for radiotherapy or chemotherapy

[0099] drug Added amount Drug loading CA1P 0.89 mg / μL microspheres 92.3％ Sodium norcanthate 0.22 mg / μL microspheres 85.6％ ADU-S100 1.78ug / μL microspheres 89.5％ mRNA-2416 2.22ug / μL microspheres 85.4％ <![CDATA[

[32] Na2HPO4]]> 53.3 μCi / μL microspheres 98.6％

[125] NaI 88.9 μCi / μL microspheres 62.3％

[131] NaI 88.9 μCi / μL microspheres 63.1％

[0100] As shown in Table 2, the microspheres prepared in Example 1 have good loading performance for various drugs.

[0101] Example 11

[0102] The therapeutic effects of free drugs and drug-loaded microspheres on tumors.

[0103] The microspheres prepared in Example 1 of this invention were loaded with different drugs, and their therapeutic effects on tumors were examined.

[0104] The method for loading a single drug is as described in Examples 9-10.

[0105] Two drug loading methods were employed: Microspheres were placed in a container containing 100 μL of 1,3,4,6-tetrachloro-3α,6α-diphenylglyurea (1 mg / mL DMSO solution), and then a solution of radioactive iodine (

[131] NaI) (88.9 μCi / μL microspheres) was added. After shaking for 1 h, the supernatant was discarded by centrifugation and dried. Then, CA1P solution (0.89 mg / μL microspheres) was added, shaken for 1 h, centrifuged, the supernatant was discarded, and dried.

[0106] Modeling: A rabbit tumor model was established using VX-II cells in the muscle structure of healthy New Zealand rabbits. After the subcutaneous tumor grew to 2 cm in diameter, it was removed and cut into cubes with sides of 3 mm for later use. Healthy New Zealand rabbits were anesthetized, the abdominal cavity was opened, the left lateral lobe of the liver was harvested and incised. The tumor was embedded in the liver tissue, the wound was sealed, and sutured.

[0107] Dosage: The specific dosage is shown in Table 3. The dosage of CA1P+

[131] NaI embolization microspheres is half that of free CA1P,

[131] NaI injection, and single CA1P-loaded embolization microspheres and single

[131] NaI-loaded embolization microspheres.

[0108] The drug-loaded microspheres were administered as follows: DSA-guided interventional embolization of the VX-2 rabbit model – approximately 10-14 days post-procedure, enhanced CT scans were used to observe the volume of the left hepatic lesion in the rabbit model; interventional treatment began when the lesion size was approximately 1cm × 1cm. After anesthesia, the muscle layer was incised at the femoral artery in the right femur of the rabbit, and the femoral artery was dissected. The distal end was ligated, and a 4F sheath was inserted through vascular puncture, into which a 2.7F microcatheter was inserted into the femoral artery. Under DSA guidance, enhanced angiography with iodoxane was performed, and the microcatheter was positioned to the hepatic artery branch anterior to the tumor lesion. Finally, drug-loaded microspheres prepared in Example 1 were used for embolization. The catheter, guidewire, and sheath were then removed, and the proximal vessel was ligated, completing the procedure. In the free group, drug was administered via catheter perfusion, similar to the microsphere group.

[0109] Tumor volume measurement: On days 0 and 7 of treatment, CT scans were performed to measure the length, width, and height of the tumor. The tumor volume was calculated using the following formula:

[0110]

[0111] Where a, b, and c represent the length, width, and height of the tumor, respectively.

[0112] As shown in Table 3 and Figure 4As shown, the therapeutic effect of interventional administration of low-dose radioactive nuclide and chemotherapeutic drug-loaded contrast-enhanced microspheres is significantly better than that of systemic administration of high-dose single chemotherapeutic microspheres, single radiotherapy embolization microspheres, and free drugs combined with radiotherapy and chemotherapy, and the difference is statistically significant.

[0113] Table 3. Tumor size after treatment of New Zealand white rabbit VXII liver cancer model with different drug loadings.

[0114]

[0115]

Claims

1. A visualizing embolizing microsphere, characterized by, The microspheres are quaternary ammonium salt type microspheres containing a catechol structure and iodine; the catechol monomers in the catechol structure are one or more of dopamine and dopamine hydrochloride; the iodine monomers in the quaternary ammonium salt type microspheres containing iodine are one or more of 5-amino-2,4,6-triiodophthalic acid, 3-acetamido-5-amino-2,4,6-triiodobenzoic acid, 5-amino-N-(2-hydroxyethyl)-2,4,6-triiodobenzoylbenzoic acid, and 3-amino-2,4,6-triiodobenzoic acid. The method for preparing the aforementioned imaging embolism microspheres includes the following steps: (1) Aqueous phase preparation: Dissolve the polyamino polymer or its hydrochloride, catechol monomer, and iodine-containing monomer in water; (2) Oil phase preparation: Add emulsifier to low polarity solvent and stir until dissolved; (3) Microsphere preparation: The aqueous phase was added to the oil phase under stirring, 3-halo-1,2-epoxypropane was added, the reaction was heated, and then washed and filtered. Wherein, the polyamine polymer in step (1) is one or more of polyacrylamide and polyethyleneimine; the hydrochloride salt of the polyamine polymer is one or more of polyacrylamide hydrochloride and polyethyleneimine hydrochloride; In step (1), 100 parts by weight of the polyamino polymer or its hydrochloride, 1-40 parts by weight of the catechol monomer, and 10-200 parts by weight of the iodine-containing monomer are dissolved in 100-1000 parts of pure water and stirred until dissolved. When the polyamino polymer hydrochloride is used, 5-60 parts by weight of an alkaline compound are also required. The alkaline compound is one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, pyridine, triethylamine, sodium acetate, sodium hydroxide, and potassium hydroxide.

2. A method of producing the visualizing embolizing microspheres according to claim 1, characterized by, Includes the following steps: (1) Aqueous phase preparation: Dissolve the polyamino polymer or its hydrochloride, catechol monomer, and iodine-containing monomer in water; (2) Oil phase preparation: Add emulsifier to low polarity solvent and stir until dissolved; (3) Microsphere preparation: The aqueous phase was added to the oil phase under stirring, 3-halo-1,2-epoxypropane was added, the reaction was heated, and then washed and filtered. The polyamine polymer mentioned in step (1) is one or more of polyacrylamide and polyethyleneimine; the hydrochloride salt of the polyamine polymer is one or more of polyacrylamide hydrochloride and polyethyleneimine hydrochloride. In step (1), 100 parts by weight of the polyamino polymer or its hydrochloride, 1-40 parts by weight of the catechol monomer, and 10-200 parts by weight of the iodine-containing monomer are dissolved in 100-1000 parts of pure water and stirred until dissolved. When the polyamino polymer hydrochloride is used, 5-60 parts by weight of an alkaline compound are also required. The alkaline compound is one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, pyridine, triethylamine, sodium acetate, sodium hydroxide, and potassium hydroxide.

3. The method of claim 2, wherein the microspheres are formed by a process comprising: The low-polarity solvent mentioned in step (2) includes one or more of liquid paraffin, diethyl ether, petroleum ether, hexane, cyclohexane, and heptane; the emulsifier is one or more of Span, Tween, lauryl polyoxyethylene (120) ether, polyethylene glycol (200) monolaurate, glycerol monolaurate, N,N-dimethylhexamethylene amide, polyglycerol fatty acid ester, polyethylene glycol (200) dilaurate, diglycerol monolaurate, sorbitol monolaurate, and nonylphenoxy polyethylene glycol; wherein, 0.5-5 parts of emulsifier are added to 2-10 times the volume of the low-polarity solvent in the aqueous phase and stirred until dissolved.

4. The method of claim 2, wherein the microspheres are formed by a process comprising: In step (3), the aqueous phase is added to the oil phase under mechanical stirring at 100-500 rpm, and 10-50 parts of 3-halo-1,2-epoxypropane are added. The temperature is raised to 30-100℃ and the reaction is carried out for 2-24 hours. The mixture is then washed and filtered to obtain the final product. The 3-halo-1,2-epoxypropane is one or more of epichlorohydrin and epibromopropane.

5. A radiopaque iodine and negatively charged drug loaded microsphere embolization microsphere characterized in that, The microspheres are quaternary ammonium salt type microspheres containing a catechol structure and iodine element, and loaded with radioactive iodine and a negatively charged drug; the catechol monomers in the catechol structure are one or more of dopamine and dopamine hydrochloride; the iodine monomers in the quaternary ammonium salt type microspheres containing iodine element are one or more of 5-amino-2,4,6-triiodophthalic acid, 3-acetamido-5-amino-2,4,6-triiodobenzoic acid, 5-amino-N-(2-hydroxyethyl)-2,4,6-triiodobenzoylbenzoic acid, and 3-amino-2,4,6-triiodobenzoic acid. The method for preparing the radioactive iodine-loaded and negatively charged drug-loaded contrast-enhancing embolic microspheres includes the following steps: (1) Loading radioactive iodine: The microspheres prepared according to claim 2 are placed in a container containing a catalyst, and then a radioactive iodide solution is added. After centrifugation, the supernatant is discarded to obtain the microspheres loaded with radioactive iodine. (2) Loading negatively charged drugs: Place the radioactive iodine-loaded microspheres obtained in step (2) into a solution containing negatively charged drugs, shake and stir, centrifuge and discard the supernatant to obtain microspheres loaded with radioactive iodine and negatively charged drugs; microspheres prepared by polyamino polymers need to be acidified before loading negatively charged drugs: place the microspheres in an acidic compound solution, centrifuge and discard the supernatant.

6. A method of producing the radiolabeled and negatively charged drug-loaded visualization embolizing microspheres of claim 5, characterized by Includes the following steps: (1) Loading radioactive iodine: The microspheres prepared according to claim 2 are placed in a container containing a catalyst, and then a radioactive iodide solution is added. After centrifugation, the supernatant is discarded to obtain the microspheres loaded with radioactive iodine. (2) Loading negatively charged drugs: Place the radioactive iodine-loaded microspheres obtained in step (2) into a solution containing negatively charged drugs, shake and stir, centrifuge and discard the supernatant to obtain microspheres loaded with radioactive iodine and negatively charged drugs; microspheres prepared by polyamino polymers need to be acidified before loading negatively charged drugs: place the microspheres in an acidic compound solution, centrifuge and discard the supernatant.

7. The method for preparing radioactive iodine-loaded and negatively charged drug-loaded contrast-enhancing embolic microspheres according to claim 6, characterized in that, The radioactive iodide is one or more of [125] sodium iodide and [131] sodium iodide; the catalyst is one or more of chloramine-T and 1,3,4,6-tetrachloro-3α,6α-diphenylglyurea; the negatively charged drug is one or more of carboxylates, phosphates, phosphates, nucleotides, deoxynucleotides, cyclic dinucleotides, oncolytic viruses, and [32] Na2HPO4.

8. The use of the radioactive iodine-loaded and negatively charged drug-loaded contrast-enhancing embolization microspheres as described in claim 5 in the preparation of combined radiotherapy, chemotherapy and embolization therapy drugs for tumors and contrast-enhancing diagnostic reagents.

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