Drug-loading biodegradable embolism microsphere as well as preparation method and application thereof

Abstract

The invention provides a drug-loading biodegradable embolism microsphere as well as a preparation method and application thereof, and belongs to the field of biomedical materials. According to the drug-loading biodegradable embolism microsphere prepared by the invention, gelatin molecules with good biocompatibility and low immunogenicity are used as a skeleton, a carboxylated aldosaccharide derivative is used as a cross-linking agent, a large amount of carboxylic acid groups are introduced in the chemical cross-linking process, the microsphere is formed by cross-linking and curing, and the microsphere can be biodegraded and absorbed, so that the drug-loading biodegradable embolism microsphere has the advantages of good biocompatibility, good biocompatibility and good immunogenicity. And the microspheres are endowed with electronegativity through dissociation of carboxylic acid groups, so that carrying of chemotherapeutic drugs with positive charges is realized. By introducing the carboxyl-rich cross-linking reagent, in the process of forming the microspheres through chemical cross-linking, the content of carboxyl in the microspheres is greatly increased, the loading efficiency of the microspheres on antitumor drugs can be effectively improved, and the microspheres are endowed with the capability of realizing drug sustained release at local lesions. The embolism microsphere prepared by the invention has biological absorbability and high-efficiency drug loading property at the same time.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical materials, in particular to a drug-loaded biodegradable embolization microsphere and a preparation method and application thereof. BACKGROUND

[0002] Transcatheter arterial chemoembolization (TACE) is a milestone interventional technique for the treatment of advanced hepatocellular carcinoma, and its core principle is based on the pathological feature that more than 90% of the blood supply of hepatocellular carcinoma is derived from the hepatic artery. By precisely delivering embolic agents and chemotherapeutic drugs to the tumor-feeding artery through a catheter, TACE can achieve the dual goals of treatment: on the one hand, it blocks the blood supply of the tumor by embolic agents, causing ischemic necrosis; on the other hand, it uses the chemotherapeutic drugs (such as doxorubicin and irinotecan) loaded by the embolic agents for sustained release, maintaining a high local drug concentration and reducing systemic toxicity.

[0003] As the core carrier of TACE efficacy, vascular embolic agents need to meet the three requirements of mechanical embolization strength, drug controlled release ability and biological safety. According to the material properties, embolic agents can be divided into two categories: non-degradable and biodegradable. Non-degradable materials are represented by DCBead® and Hepasphere®. Their technical solutions are based on polyvinyl alcohol (PVA) or polyacrylate matrix, and the drug loading is achieved through the charge interaction of ion exchange groups such as sulfonic acid and carboxylic acid with chemotherapeutic drugs (such as doxorubicin), with high drug loading rate, but long-term retention in the body can easily cause chronic inflammation of blood vessels, foreign body granuloma formation, and even require secondary surgical intervention. Although biodegradable materials (such as PLA, PLGA, and gelatin) can avoid foreign body residue by gradually degrading, the existing technology has bottlenecks such as low drug loading efficiency and mismatch between degradation rate and drug release curve.

[0004] Therefore, the development of an embolic microsphere with both drug loading performance and biodegradable characteristics can effectively fill the gap in the clinical need for such vascular embolic agents, effectively improve the efficacy of vascular embolization of the lesion site, and at the same time reduce the immunological risks brought by long-term foreign body retention.

[0005] Chinese patent CN109316626A proposes a preparation method of drug-loaded gelatin embolic microspheres, which can synthesize gelatin microspheres with uniform particle size and narrow particle size range in a single batch. The reaction process is controllable, and different specifications of gelatin microspheres can be synthesized by controlling the reaction conditions, with a particle size range of 50-2500 μm. Chinese patent CN102585258B provides a gelatin embolic microsphere and a preparation method thereof, and in the examples, the preparation of doxorubicin-loaded microspheres and drug loading tests are carried out, and the results show that the microspheres have certain drug loading capacity.

[0006] However, the above prepared gelatin embolization microspheres have biodegradable properties, but the drug loading mechanism mainly depends on the physical adsorption of the porous structure of the microspheres and the ion exchange between the small amount of carboxyl groups in the gelatin molecules and the drug molecules. Therefore, the above method leads to generally low drug loading efficiency, and the drug release process is difficult to control, which cannot meet the actual needs of clinical high drug loading rate and controllable release performance of embolization microspheres. SUMMARY

[0007] Therefore, the purpose of the present application is to provide a preparation method of drug-loaded biodegradable embolization microspheres with high drug loading efficiency and excellent biodegradable properties. The present application uses carboxylated polyaldehyde sugar derivatives as crosslinking agents and introduces them into the crosslinking process of gelatin sponge to prepare drug-loaded biodegradable embolization microspheres based on ion exchange mechanism. The microspheres can significantly improve the drug loading efficiency and accurately control the drug release rate, providing a new solution for clinical interventional vascular embolization treatment.

[0008] In order to achieve the above purpose, the present application provides the following technical solutions:

[0009] A preparation method of drug-loaded biodegradable embolization microspheres, comprising the following steps:

[0010] (1) Synthesis of carboxylated sugar derivatives

[0011] The sugar is dissolved in an acetate buffer, a catalyst and a laccase are added, and an enzymatic reaction is carried out in an oxygen atmosphere. After the reaction is completed, an ethanol aqueous solution is added to terminate the reaction, the pH is adjusted to 7.0, the catalyst and the laccase are removed by a membrane separation system, and the filtrate is freeze-dried to obtain a carboxylated sugar derivative powder;

[0012] (2) Synthesis of carboxylated polyaldehyde sugar derivatives

[0013] The carboxylated sugar derivative powder obtained in step (1) is dissolved in deionized water, and sodium periodate is added under a nitrogen atmosphere to carry out an oxidation reaction. After the reaction is completed, barium chloride is added to precipitate the reaction, and after filtration and freeze-drying, a carboxylated polyaldehyde sugar derivative powder is obtained;

[0014] (3) Preparation of gelatin solution

[0015] Gelatin is added to deionized water, and stirring is carried out in a water bath until the gelatin is completely dissolved to obtain a gelatin solution;

[0016] (4) Preparation of gelatin water-in-oil emulsion

[0017] The emulsifier is dispersed in the oil phase, and the gelatin solution is slowly added to the oil phase under water bath heating conditions. Stirring reaction forms a stable gelatin water-in-oil emulsion, and temperature reduction stirring obtains an emulsion system containing microsphere precursors;

[0018] (5) Cross-linking reaction of carboxylated polyalditols derivatives and gelatin

[0019] The carboxylated polyalditols derivatives powder prepared in step (2) is dissolved in a phosphate buffer solution, and then added into the emulsion system of step (4), and the cross-linking reaction is carried out under stirring in a low temperature environment to form microspheres with three-dimensional cross-linked network;

[0020] (6) Washing and drying of microspheres

[0021] The upper oil phase is removed by decantation, the microspheres are washed with a washing solution to completely remove residual oil, and then the microspheres are washed with distilled water until the pH is neutral, and then filtered and freeze-dried to obtain drug-loadable biodegradable embolization microspheres.

[0022] Preferably, the saccharide in step (1) is any one of monosaccharide, disaccharide and polysaccharide; the catalyst is 2,2,6,6-tetramethylpiperidine oxide (TEMPO); the concentration of ethanol in the aqueous ethanol solution is 95%; and the pH value of the acetate buffer solution is 5.0.

[0023] The concentration of saccharide in the enzymatic reaction system is 1-100 g / L, the concentration of catalyst is 5-1000 mg / L, and the concentration of laccase is 0.1-10 mg / L; and the volume / mass ratio of the aqueous ethanol solution to saccharide is 50-250 mL:1-10 g.

[0024] The temperature of the enzymatic reaction in step (1) is 25-40℃, and the reaction time is 45-120 min.

[0025] Preferably, the concentration of carboxylated saccharide derivatives in step (2) is 20-100 g / L, the concentration of sodium periodate is 40-200 g / L, and the concentration of barium chloride is 50-250 g / L; the reaction temperature of the oxidation reaction is 5-15℃, and the reaction time is 15-30 h; and the time of the precipitation reaction is 30 min.

[0026] The number of carboxyl groups in the carboxylated polyalditols functional groups in step (2) is 1-40, and the number of aldehyde groups is 2-40.

[0027] Preferably, the gelatin in step (3) is at least one of acid gelatin, alkali gelatin, enzyme gelatin and modified gelatin; and the concentration of the gelatin solution is 20-40% (w / v).

[0028] Preferably, the oil phase in step (4) is at least one of mineral oil, vegetable oil, alkane, silicone oil; the emulsifier is at least one of sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyglycerol fatty acid ester, lecithin, gum arabic; the volume ratio of the oil phase to the gelatin solution is 5:1-10:1; the volume ratio of the emulsifier to the oil phase is 1:10-1:100.

[0029] The temperature of the water bath heating in step (4) is 50-60℃, the stirring speed is 300-500 rpm, and the reaction time is 10-15 min.

[0030] Preferably, the ratio of the carboxylated polyaldehyde sugar derivative powder, the phosphate buffer solution and the emulsion system in step (5) is 1.5-2.5 g:30-50 mL:100-300 mL; the temperature of the cross-linking reaction is 2-10℃, and the time is 12-48 h.

[0031] The pH of the phosphate buffer solution is 8.7.

[0032] Preferably, the cleaning liquid in step (6) is at least one of acetone, ethanol, propylene glycol, isopropyl alcohol and ethyl acetate.

[0033] Another object of the present application is to provide a drug-loadable biodegradable embolization microsphere prepared by the above method, which uses gelatin molecules with good biocompatibility and low immunogenicity as a skeleton, adopts carboxylated polyaldehyde sugar derivatives as a cross-linking agent, introduces a large number of carboxylic acid groups in the process of chemical cross-linking, and forms microspheres through cross-linking and solidification, so that the microspheres are biodegradable and absorbable, and the microspheres are endowed with negative electric properties through the dissociation of carboxylic acid groups, so as to realize the loading of positively charged chemotherapeutic drugs.

[0034] Preferably, the porosity of the microspheres is 50%-95%, the swelling rate is 100%-500%, the elastic modulus is 0.1-5.0 MPa, and the compression deformation recovery rate is ≥80%; the particle size of the microspheres ranges from 50 to 2000 μm; and the biodegradation time of the drug-loadable biodegradable embolization microspheres is 7-180 days. The gradient control can be realized by adjusting the amount of cross-linking agent and the cross-linking time.

[0035] The present application also provides an application of the drug-loadable biodegradable embolization microspheres, and the drug loading type is at least one of doxorubicin, epirubicin, oxaliplatin, irinotecan and mitoxantrone.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] The prepared drug-loadable biodegradable embolization microspheres can effectively improve the drug loading efficiency of the microspheres on the anti-tumor drugs by introducing a carboxyl-rich cross-linking reagent in the process of chemical cross-linking to form the microspheres, and can also endow the microspheres with the ability of realizing drug sustained release at a local lesion. DETAILED DESCRIPTION

[0038] The application provides a preparation method of drug-loadable biodegradable embolization microspheres, and the steps are as follows:

[0039] (1) Synthesis of carboxylated saccharide derivative

[0040] 1-100 g / L saccharide is dissolved in an acetate buffer solution with a pH value of 5.0, 5-1000 mg / L catalyst TEMPO and 0.1-10 mg / L laccase are added, and an enzymatic reaction is carried out in an oxygen atmosphere at 25-40 °C for 45-120 min; then 95% ethanol aqueous solution is added in a volume-mass ratio of 50-250 mL:1-10 g to terminate the reaction, 0.5 mol / L HCl is used to adjust the pH value to 7.0, the catalyst and the laccase are removed through a membrane separation system, and the filtrate is freeze-dried to obtain a carboxylated saccharide derivative powder;

[0041] (2) Synthesis of carboxylated polyaldehyde saccharide derivative

[0042] The carboxylated saccharide derivative powder obtained in step (1) is dissolved in deionized water to make the concentration of the carboxylated saccharide derivative powder 20-100 g / L, and 40-200 g / L sodium periodate is added under a nitrogen atmosphere at 5-15 °C to react for 15-30 h for oxidation; after the reaction is completed, 50-250 g / L barium chloride is added to precipitate for 30 min, and then filtration and freeze-drying are carried out to obtain a carboxylated polyaldehyde saccharide derivative powder; wherein the number of carboxyl groups in the carboxylated polyaldehyde saccharide functional group is 1-40, and the number of aldehyde groups is 2-40.

[0043] (3) Preparation of gelatin solution

[0044] Gelatin is added to deionized water, and stirring is carried out in a water bath until the gelatin is completely dissolved to obtain a gelatin solution with a concentration of 20-40 w / v%;

[0045] (4) Preparation of gelatin water-in-oil emulsion

[0046] According to the volume ratio of the oil phase to the gelatin solution is 5:1~10:1; the volume ratio of the emulsifier to the oil phase is 1:10~1:100, the emulsifier is dispersed in the oil phase, the gelatin solution is slowly added into the oil phase under the condition of heating in a water bath at 50~60℃, stirring at a speed of 300~500 rpm, and the gelatin oil-in-water stable emulsion is formed after reaction for 10~15 min, and the emulsion system containing the microsphere precursor is obtained by cooling and stirring; the system is in the emulsion state during the continuous stirring in the cooling process, but the cooling is beneficial to the formation of the microsphere precursor (part of the components is solidified into solid);

[0047] (5) Cross-linking reaction of the carboxylated polyaldose derivative and the gelatin

[0048] The carboxylated polyaldose derivative powder prepared in step (2) is dissolved in a phosphate buffer (pH is 8.7), and then added into the emulsion system of step (4), stirring at a temperature of 2~10℃, and the cross-linking reaction is carried out for 12~48 h to form the microspheres with three-dimensional cross-linking network; wherein the ratio of the carboxylated polyaldose derivative powder, the phosphate buffer and the emulsion system is 1.5~2.5 g: 30~50 mL: 100~300 mL;

[0049] (6) Washing and drying of the microspheres

[0050] The upper oil phase is removed by decantation, the microspheres are washed with the washing liquid to completely remove the residual oil, and then the microspheres are washed with distilled water until the pH is neutral, and the drug-loaded biodegradable embolization microspheres are obtained by filtration and freeze-drying.

[0051] The saccharide in step (1) is any one of monosaccharide, disaccharide and polysaccharide; in specific embodiments of the present application, the saccharide is any one of glucose, sucrose and chitosan.

[0052] In specific embodiments of the present application, the gelatin in step (3) is at least one of acid gelatin (manufacturer: Sigma-Aldrich, model: G1890), alkali gelatin (manufacturer: Rose Bengal Gelatin Co., Ltd., model: 180LB8), enzyme gelatin (manufacturer: Sigma-Aldrich, model: G7765) and modified gelatin (manufacturer: Advanced BioMatrix, model: PhotoHA™-Stiff).

[0053] The oil phase in step (4) is at least one of mineral oil, vegetable oil, alkane and silicone oil; in specific embodiments of the present application, the oil phase is any one of liquid paraffin, paraffin oil, olive oil, soybean oil and corn oil.

[0054] The emulsifier in step (4) is at least one of sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyglycerol fatty acid ester, lecithin, gum arabic; in specific embodiments of the application, the emulsifier is any one of Span 60, Span 80, Tween 60, Tween 80.

[0055] In specific embodiments of the application, the cleaning liquid in step (6) is at least one of acetone, ethanol, propylene glycol, isopropyl alcohol, ethyl acetate.

[0056] Another object of the application is to provide a drug-loadable biodegradable embolization microsphere prepared by the above method, the porosity of the microsphere being 50% to 95%, the swelling rate being 100% to 500%, the elastic modulus being 0.1 to 5.0 MPa, and the compression deformation recovery rate being ≥80%; the particle size of the microsphere being 50 to 2000 μm; and the biodegradation time of the drug-loadable biodegradable embolization microsphere being 7 to 180 days. Gradient control can be achieved by adjusting the amount of crosslinking agent and the crosslinking time.

[0057] The application further provides a drug-loadable biodegradable embolization microsphere, the drug load type being at least one of doxorubicin, epirubicin, oxaliplatin, irinotecan, and mitoxantrone.

[0058] The application will be further described below with reference to examples.

[0059] Example 1

[0060] (1) Synthesis of carboxylated glucose

[0061] Glucose (10.0 g) was dissolved in an acetate buffer solution (2.0 L) with pH 5.0, TEMPO (80 mg) and laccase (6 mg) were added, and the mixture was stirred magnetically under an oxygen atmosphere at 38℃ for 60 minutes. The reaction was terminated by adding 95% ethanol (250 mL), the pH was adjusted to 7.0 with 0.5 mol / L HCl, and the catalyst was removed through a 10 kDa ultrafiltration membrane. The 6-carboxylated glucose powder was obtained by freeze-drying.

[0062] (2) Synthesis of carboxylated polyaldehyde glucose

[0063] The 6-carboxylated glucose (34.2 g) was mixed with deionized water (1.0 L), and oxygen was removed by nitrogen bubbling for 30 minutes. The temperature was lowered to 10℃, and sodium periodate (12.8 g) was added. The mixture was stirred for 24 hours, and after the reaction was completed, barium chloride (73.35 g) was added and stirred for 30 minutes to precipitate the by-product. After filtration, the carboxylated polyaldehyde glucose powder was obtained by freeze-drying. The structural formula of the carboxylated polyaldehyde glucose is shown as formula (1):

[0064] (1)

[0065] (3) Preparation of gelatin solution

[0066] Modified gelatin (3 g) was added into deionized water (10 mL) and dissolved in a 55 °C water bath with stirring at 350 rpm to prepare a 30% w / v gelatin solution.

[0067] (4) Preparation of gelatin W / O emulsion

[0068] Tween 60 (3 mL) was dispersed in olive oil (150 mL) and heated in a water bath to 55 °C; the gelatin solution was slowly added to the oil phase and stirred at 400 rpm for 10 min to form a stable emulsion, which was then cooled to 5 °C and stirred for another 25 min to obtain an emulsion system containing microsphere precursors.

[0069] (5) Cross-linking reaction of carboxylated polyoxymethylene glucose

[0070] Carboxylated polyoxymethylene glucose (1.8 g) was dissolved in a pH 8.7 phosphate buffer (50 mL) and added to the emulsion system prepared in step (4); the cross-linking reaction process is shown in formula (2):

[0071] (2)

[0072] (6) Washing and drying of microspheres

[0073] The oil phase was removed by decantation, and the microspheres were washed with ethanol to completely remove residual oil; then the microspheres were washed with distilled water until the pH was neutral, and then filtered; after pre-freezing for 2 hours (-80 °C), the microspheres were vacuum dried for 24 hours to obtain drug-loaded biodegradable embolization microspheres.

[0074] Example 2

[0075] (1) Synthesis of carboxylated sucrose

[0076] Sucrose (1.2 g) was dissolved in a pH 5.0 acetate buffer (200 mL), and TEMPO (8 mg) and laccase (0.12 mg) were added; the mixture was stirred magnetically under an oxygen atmosphere at 35 °C for 120 min; then 95% ethanol (50 mL) was added to terminate the reaction, the pH was adjusted to 7.0 with 0.5 mol / L HCl, the catalyst was removed by a 10 kDa ultrafiltration membrane, the filtrate was collected, and the freeze-dried powder was obtained as 6,6'-carboxylated sucrose.

[0077] (2) Synthesis of carboxylated polyoxymethylene sucrose

[0078] Carboxylated polyaldehyde sucrose powder was obtained by mixing 6,6'-carboxyl sucrose (6.84 g) with deionized water (200 mL), bubbling nitrogen for 30 minutes to remove oxygen, cooling to 10 °C, adding sodium periodate (8.2 g), stirring for 22 hours; after the reaction was completed, barium chloride (14.67 g) was added and stirred for 30 minutes, the precipitate was removed by filtration, and freeze-dried to obtain carboxylated polyaldehyde sucrose powder. The structural formula of carboxylated polyaldehyde sucrose is shown in formula (3):

[0079] (3)

[0080] (3) Preparation of gelatin solution

[0081] Acid gelatin (4 g) was added to deionized water (16 mL) and dissolved in a water bath at 55 °C with stirring at 350 rpm to prepare a 25% (w / v) gelatin solution.

[0082] (4) Preparation of gelatin water-in-oil emulsion

[0083] Span 80 (1.5 ml) was dispersed in liquid paraffin (120 ml) and heated in a water bath to 55 °C; the gelatin solution was slowly added to the oil phase and stirred at 450 rpm for 10 minutes to form a stable emulsion. The temperature was lowered to 3 °C and stirring was continued for 30 minutes to obtain an emulsion system containing microsphere precursors.

[0084] (5) Crosslinking reaction of carboxylated polyaldehyde sucrose and gelatin

[0085] Carboxylated polyaldehyde sucrose powder (2.2 g) was dissolved in phosphate buffer with a pH of 8.7 (30 mL) and added to the emulsion system prepared in step (4); the reaction was stirred at 200 rpm for 18 hours at 4 °C. The crosslinking reaction process is shown in formula (4):

[0086] (4)

[0087] (6) Washing and drying of microspheres

[0088] The oil phase was removed by decanting, and the microspheres were washed with acetone to completely remove residual oil; then the microspheres were washed with distilled water until the pH was neutral, and filtered; after pre-freezing for 2.5 hours (-80 °C), the microspheres were vacuum dried for 36 hours to obtain drug-loaded biodegradable embolization microspheres.

[0089] Example 3

[0090] (1) Synthesis of carboxylated chitosan

[0091] Chitosan (10 g) was dissolved in acetate buffer (2.0 L, pH 5.0), TEMPO (100 mg) and laccase (8 mg) were added, and the mixture was stirred at 40 °C for 45 min under oxygen atmosphere. Then the reaction was terminated by adding 95% ethanol (250 mL), and the pH was adjusted to 7.0 by 0.5 mol / L HC1. The catalyst was removed by 10 kDa ultrafiltration membrane, and the filtrate was collected and freeze-dried to obtain 6-carboxyl chitosan powder.

[0092] (2) Synthesis of carboxylated polyaldehyde chitosan

[0093] Chitosan (10 g) was dissolved in acetate buffer (2.0 L, pH 5.0), TEMPO (100 mg) and laccase (8 mg) were added, and the mixture was stirred at 40 °C for 45 min under oxygen atmosphere. Then the reaction was terminated by adding 95% ethanol (250 mL), and the pH was adjusted to 7.0 by 0.5 mol / L HC1. The catalyst was removed by 10 kDa ultrafiltration membrane, and the filtrate was collected and freeze-dried to obtain 6-carboxyl chitosan powder.

[0094] (5)

[0095] (3) Preparation of gelatin solution

[0096] Gelatin (10 g) was added to deionized water (about 20 mL) and dissolved by stirring at 350 rpm in a 55 °C water bath. The solution was transferred to a 25 mL volumetric flask and diluted to the calibration line with water to prepare a 40% w / v gelatin solution.

[0097] (4) Preparation of gelatin water-in-oil emulsion

[0098] Span 60 (2 mL) was dispersed in silicone oil (180 mL) and heated to 55 °C in a water bath. The gelatin solution was slowly added to the oil phase, and the mixture was stirred at 350 rpm for 12 min to form a stable emulsion. The emulsion was cooled to 4 °C and stirred for another 20 min to obtain an emulsion system containing microsphere precursors.

[0099] (5) Crosslinking reaction of carboxylated polyaldehyde chitosan and gelatin

[0100] Carboxylated polyaldehyde chitosan powder (2.1 g) was dissolved in phosphate buffer (50 mL, pH 8.7) and added to the emulsion system prepared in step (4). The mixture was stirred at 200 rpm at 4 °C for 18 h. The crosslinking reaction process is shown in formula (6).

[0101] (6)

[0102] (6) Washing and drying of microspheres

[0103] The oil phase was removed by decantation, and the microspheres were washed with propylene glycol to completely remove residual oil, and then washed with distilled water until neutral, and filtered. After pre-freezing for 3 hours (-80°C), the microspheres were vacuum dried for 48 hours to obtain drug-loadable biodegradable embolization microspheres.

[0104] Comparative Example 1

[0105] A commercially available cross-linked embolization microsphere product, Biodex (size: 100-300 μm), was used as Comparative Example 1.

[0106] Experiment 1: Drug release performance test

[0107] Drug loading experiments were performed on the drug-loadable biodegradable embolization microspheres prepared in Examples 1-3 and Comparative Example 1, according to the following method:

[0108] 25 mg (particle size 100-300 μm, lyophilized state) of the carboxylated polyaldehyde embolization microspheres prepared in Examples 1, 2, and 3, and Comparative Example 1 were added to 16 mL of an adriamycin solution with a concentration of 2.5 mg / mL. Every 5 min, 15 min, 30 min, and 60 min, 100 μL of the sample was taken, and the drug concentration was detected at a wavelength of 483 nm using a UV spectrophotometer. The drug loading rate was calculated according to Formula (7) to reflect the absorption and loading of the microspheres to the drug.

[0109] (7)

[0110] The results are shown below

[0111] Table

[0112] Test sample Drug loading 5 min Drug loading 15 min Drug loading 30 min Drug loading 60 min Example 1 70.47% 81.52% 89.81% 95.08% Example 2 79.27% 86.14% 92.21% 97.31% Example 3 88.34% 95.16% 98.02% 98.74% Comparative Example 1 86.01% 94.22% 96.51% 98.23%

[0113] As shown in Table 1, the overall drug loading rate and drug loading rate of Examples 1 to 3 gradually increased, which may be related to the number of carboxyl groups and activity of the cross-linking agent used in different examples. The overall drug loading rate and drug loading rate of Examples 1 to 3 were comparable to those of Comparative Example 1.

[0114] Experiment 2: Drug release performance test

[0115] 0.5 g of the microspheres of Example 1, Example 2, Example 3 (particle size 100-300 μm, lyophilized state), and commercially available Biodex microspheres were weighed and placed in a 1.0 mL centrifuge tube. 1 mL of an adriamycin solution with a concentration of 2.5 mg / mL was added, and the microspheres were uniformly dispersed by ultrasonic dispersion for 3 min.

[0116] ​Take 0.5 mL of the above dispersed microsphere suspension, add 1 mL of adriamycin solution with a concentration of 2.5 mg / mL, vortex for 1 min to make the microspheres fully adsorb the drug. Then centrifuge for 5 min, take the supernatant to measure the absorbance at 483 nm wavelength, and calculate the initial drug loading.

[0117] Transfer the remaining microsphere suspension to a dialysis bag, seal it and immerse it in 50 mL of adriamycin solution with a concentration of 2.5 mg / mL, and place it in a 37°C constant temperature shaking table to start the drug release experiment. The sampling time points are 1, 2, 5, 10, 15, 20, 30 days, 5 mL of supernatant is taken from the release medium (while supplementing with fresh release medium to maintain constant volume) to remove particulate impurities after centrifugation for 5 min, and the OD value of the supernatant is measured, and the cumulative drug release rate is calculated according to formula (8).

[0118] (8)

[0119] The results are as follows

[0120] Table

[0121] Time (d) Cumulative drug release rate (%) of Example 1 Cumulative drug release rate (%) of Example 2 Cumulative drug release rate (%) of Example 3 Cumulative drug release rate (%) of Comparative Example 1 1 10.2±0.5 11.5±0.6 13.5±1.2 12.8±0.7 2 15.8±0.7 16.1±0.8 18.0±1.0 17.5±0.9 5 25.4±1.2 23.0±1.3 25.0±1.8 24.8±1.4 10 43.9±2.9 41.3±3.8 48.7±3.9 32.2±3.4 15 65.7±6.5 67.5±5.3 69.0±5.0 38.5±2.7 20 80.2±5.7 79.4±4.9 82.1±2.2 38.8±3.2 30 88.3±6.2 89.7±5.0 93.6±8.3 37.9±4.7

[0122] As can be seen from Table 2, the embolization microspheres of Examples 1-3 exhibit significant sustained release characteristics within a 30-day drug release period: the cumulative drug release rate is 10.2%-13.5% at 1 day, 79.4%-82.1% at 20 days, and the total drug release amount is further increased to 88.3%-93.6% at 30 days, showing a sustained and stable drug release capacity. Compared with the non-degradable microspheres of Comparative Example 1, the total drug release amount of the degradable microsphere system is higher, and this difference is attributed to the material degradation characteristics of the degradable microspheres, which can achieve sustained release of the drug during the gradual disintegration process in the body.

[0123] Experiment 3: In vitro degradation performance test

[0124] Take 50 mg (particle size 100-300 μm) of microspheres of Example 1, Example 2, Example 3 and Comparative Example 1 respectively, and add 250 ml of PBS buffer with pH 7.2. Constant temperature oscillation treatment at 37±1°C for 24 h.

[0125] Then take out the microspheres at 0, 7, 14, 21, 28, 35, 42 days respectively, wash them with PBS for 3 times and freeze-dry them, and calculate the mass loss rate according to formula (9).

[0126] (9)

[0127] In formula (9), W0 is the initial mass, Wt is the mass at time t, and t is the time. t ​To degrade the quality after.

[0128] The results are shown in Table 3 below.

[0129] Table

[0130] Time Example 1 (%) Example 2 (%) Example 3 (%) 0 0.0 0.0 0.0 7 10.2±0.5 12.7±0.6 11.5±0.6 14 35.1±1.8 30.2±1.5 27.1±1.8 21 51.6±2.1 55.3±1.9 48.2±2.1 28 90.6±5.9 94.3±4.3 91.2±6.1 35 92.3±5.2 93.8±7.8 94.4±6.0 42 94.8±9.0 95.5±7.4 95.7±8.9

[0131] As shown in Table 3, the microspheres of Examples 1-3 exhibit significant degradation characteristics in vitro in PBS buffer (pH 7.2, 37℃). The initial mass loss rate is 0 at 0 day, reaches 10.2%-12.7% after 7 days, rapidly rises to 27.1%-35.1% at 14 days, has been degraded by more than 90% at 28 days, and the mass loss rate is stable at 94.8%-95.7% at 42 days, indicating that the degradable microspheres can gradually disintegrate in the simulated physiological environment, and ultimately achieve material degradation.

[0132] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing drug-loadable biodegradable embolic microspheres, characterized by, The method comprises the following steps: (1) synthesis of carboxylated saccharide derivative The saccharide is dissolved in acetate buffer, a catalyst and laccase are added, and the enzymatic reaction is carried out in an oxygen atmosphere. After the reaction is completed, an ethanol aqueous solution is added to terminate the reaction, the pH is adjusted to 7.0, the catalyst and laccase are removed by a membrane separation system, and the filtrate is freeze-dried to obtain carboxylated saccharide derivative powder; (2) synthesis of carboxylated polyaldehyde saccharide derivative The carboxylated saccharide derivative powder obtained in step (1) is dissolved in deionized water, and sodium periodate is added for oxidation reaction under a nitrogen atmosphere. After the reaction is completed, barium chloride is added to precipitate the reaction, and the precipitate is filtered and freeze-dried to obtain carboxylated polyaldehyde saccharide derivative powder; (3) preparation of gelatin solution Gelatin is added to deionized water, and stirred in a water bath until completely dissolved to obtain a gelatin solution; (4) preparation of gelatin water-in-oil emulsion The emulsifier is dispersed in the oil phase, and the gelatin solution is slowly added to the oil phase under water bath heating to form a stable gelatin water-in-oil emulsion. The emulsion system containing microsphere precursors is obtained by cooling and stirring. (5) crosslinking reaction of carboxylated polyaldehyde saccharide derivative and gelatin The carboxylated polyaldehyde saccharide derivative powder prepared in step (2) is dissolved in phosphate buffer, and then added to the emulsion system of step (4). The crosslinking reaction is carried out under low temperature stirring to form three-dimensional crosslinked network microspheres. (6) microsphere cleaning and drying The upper oil phase is removed by decantation, the microspheres are washed with a cleaning solution to completely remove residual oil, and then washed with distilled water until the pH is neutral. The drug-loaded biodegradable embolic microspheres are obtained by filtration and freeze-drying.

2. The method of claim 1, wherein the drug-loaded biodegradable embolic microspheres are prepared by the steps of: The saccharide in step (1) is any one of monosaccharide, disaccharide or polysaccharide; the catalyst is 2,2,6,6-tetramethylpiperidine oxide; the concentration of ethanol in the ethanol aqueous solution is 95%; and the pH value of the acetate buffer is 5.

0. In the enzymatic reaction system, the concentration of saccharide is 1-100 g / L, the concentration of catalyst is 5-1000 mg / L, and the concentration of laccase is 0.1-10 mg / L. The volume-to-mass ratio of the ethanol aqueous solution to saccharide is 50-250 mL:1-10 g. The temperature of the enzymatic reaction in step (1) is 25-40℃, and the reaction time is 45-120 min.

3. The method for preparing drug-loadable biodegradable embolic microspheres according to claim 1, characterized in that, In step (2), the concentration of carboxylated saccharide derivative is 20-100 g / L, the concentration of sodium periodate is 40-200 g / L, and the concentration of barium chloride is 50-250 g / L. The reaction temperature of the oxidation reaction is 5-15℃, and the reaction time is 15-30 h. The precipitation reaction time is 30 min. In step (2), the number of carboxyl groups in the carboxylated polyaldehyde saccharide functional group is 1-40, and the number of aldehyde groups is 2-40.

4. The method for preparing drug-loadable biodegradable embolic microspheres according to claim 1, characterized in that, In step (3), the gelatin is at least one of acid gelatin, alkali gelatin, enzyme gelatin, and modified gelatin. The concentration of the gelatin solution is 20-40%(w / v).

5. The method for preparing drug-loadable biodegradable embolic microspheres according to claim 1, characterized in that, The oil phase in step (4) is at least one of mineral oil, vegetable oil, alkane, and silicone oil; the emulsifier is at least one of sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyglycerol fatty acid ester, lecithin, and gum arabic; the volume ratio of the oil phase to the gelatin solution is 5:1-10:1; and the volume ratio of the emulsifier to the oil phase is 1:10-1:

100. The temperature of the water bath heating in step (4) is 50-60℃, the stirring speed is 300-500 rpm, and the reaction time is 10-15 min.

6. The method for preparing drug-loadable biodegradable embolic microspheres according to claim 1, characterized in that, The ratio of the carboxylated polyalditol derivative powder, the phosphate buffer, and the emulsion system in step (5) is 1.5-2.5 g:30-50 mL:100-300 mL; the cross-linking reaction temperature is 2-10℃, and the time is 12-48 h. The pH of the phosphate buffer is 8.

7.

7. The method for preparing drug-loadable biodegradable embolic microspheres according to claim 1, characterized in that, The cleaning liquid in step (6) is at least one of acetone, ethanol, propylene glycol, isopropyl alcohol, and ethyl acetate.

8. A drug-loadable biodegradable embolizing microsphere, characterized by, Prepared according to the method in any one of claims 1-7.

9. The drug-loadable biodegradable embolizing microspheres according to claim 8, wherein, The porosity of the microspheres is 50%-95%, the swelling rate is 100%-500%, the elastic modulus is 0.1-5.0 MPa, and the compression deformation recovery rate is ≥80%; the particle size of the microspheres is 50-2000 μm; and the biodegradation time of the drug-loaded biodegradable embolization microspheres is 7-180 days.

10. The use of the drug-loadable biodegradable embolization microspheres according to claim 8, characterized in that, The drug-loaded type is at least one of doxorubicin, epirubicin, oxaliplatin, irinotecan, and mitoxantrone.