Degradable drug-loaded microspheres and preparation method thereof

Abstract

The invention relates to the technical field of interventional medicine, in particular to degradable drug-loaded microspheres and a preparation method thereof. The preparation method comprises the following steps: adding gelatin and a biological material into a buffer solution, heating, stirring and dissolving to obtain a water phase, adding a dispersing agent into an oil phase matrix solution, stirring to obtain an oil phase, transferring the water phase into the oil phase, stirring, and cooling to obtain gel microspheres; the preparation method comprises the following steps: primarily cleaning gel microspheres, freeze-drying, adding the gel microspheres into normal saline, adding a sulfonation reagent, a catalyst and an initiator for sulfonation reaction, after the reaction is finished, adding a cross-linking agent for cross-linking reaction, and finally carrying out secondary cleaning on the microspheres to obtain the degradable drug-loaded microspheres. The biological material is selected from one or more of sodium alginate, chitosan or carboxymethyl chitosan. The gelatin and the biological material containing the anion group are mixed to prepare the degradable drug-loaded microspheres, so that the mechanical strength of the microspheres can be improved, the degradation time of the microspheres can be prolonged, and meanwhile, drugs can be loaded.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of interventional medical technology, in particular to a degradable drug-loaded microsphere and a preparation method thereof. BACKGROUND

[0002] Primary liver cancer is one of the most common and most malignant tumors in the world. In 2022, 316,500 people died of primary liver cancer, and the number of deaths and mortality rate ranked second. The best treatment for liver cancer patients is to remove or transplant the liver cancer in the early stage of liver cancer. However, most liver cancer patients are found in the late stage. In addition to ablation, chemotherapy, and radiotherapy, transcatheter arterial chemoembolization (TACE) treatment can also be selected. Drug-loaded embolization microspheres are three types of interventional medical devices used to block the blood flow to the main blood vessels of tumor cells during TACE surgery, thereby blocking the blood and oxygen supply to tumor cells, making the tumor smaller or necrotic. By loading chemotherapeutic drugs in embolization microspheres, the chemotherapeutic drugs can be released at the same time as the blood and oxygen supply is blocked, accelerating tumor cell death.

[0003] Currently marketed drug-loaded embolization microspheres, such as TANDEM, DC Beads, CalliSpheres, etc., all use negatively charged groups to graft modify non-degradable high molecular weight materials. The electrostatic interaction between the negatively charged groups and the positively charged drugs is used to achieve drug loading. However, non-degradable embolization materials have certain safety risks, such as irreversible permanent damage to normal organs in the event of ectopic embolization; for tumor tissue, new blood vessel branches are easily produced after permanent embolization, damaging the original blood vessels and making it impossible to perform secondary embolization. However, degradable materials can gradually degrade into harmless small molecular substances in the body and be absorbed or metabolized and excreted from the body, thereby avoiding the risks associated with long-term foreign body retention.

[0004] However, the drug-loaded embolization microspheres in the prior art have poor mechanical properties and weak degradation performance. By adding a biological material containing anionic groups to gelatin and further sulfonating modification, the drug loading performance of the microspheres is further improved, and the degradation performance of the microspheres is also improved. SUMMARY

[0005] To solve the above technical problems, the present application provides a degradable drug-loaded microsphere and a preparation method thereof. The present application prepares a degradable drug-loaded microsphere by mixing gelatin and a biological material containing anionic groups. The combination of the two biological materials can improve the mechanical strength of the microspheres, prolong the degradation time of the microspheres, and also load drugs. The preparation process is simple and easy to industrialize.

[0006] Therefore, the present application provides the following technical solutions,

[0007] In a first aspect, the present invention provides, in optional embodiments, a method for preparing biodegradable drug-loaded microspheres, comprising the following steps:

[0008] S1: Add gelatin and biomaterials to a buffer solution, heat and stir to dissolve, and obtain an aqueous phase. Add a dispersant to an oil phase matrix solution and stir to obtain an oil phase. Transfer the aqueous phase to the oil phase, stir, and cool to obtain gel microspheres.

[0009] S2: After initial cleaning of the gel microspheres, freeze-dry them in a freezing medium. Then, add the freeze-dried gel microspheres to physiological saline and add sulfonating reagent, catalyst and initiator to carry out sulfonation reaction. After the sulfonation reaction is completed, add crosslinking agent to carry out crosslinking reaction. Finally, wash the microspheres a second time to obtain degradable drug-loaded microspheres.

[0010] The biomaterial is selected from one or more of sodium alginate, chitosan, or carboxymethyl chitosan.

[0011] Preferably, in step S1, the mass of the biomaterial is 0.1-0.5 times the mass of the gelatin; and / or, the buffer solution is selected from physiological saline or phosphate buffer solution or a mixture thereof, and the pH value of the phosphate buffer solution is 6-7; and / or the gelatin is selected from one or more of pig skin gelatin, pig bone gelatin or bovine bone gelatin; and / or, the mass ratio of the gelatin to the buffer solution is 1:2-19; ​​and / or, the heating and stirring dissolution temperature is 50-70℃. The oil phase matrix solution is selected from one or more of light liquid paraffin, silicone oil or butyl acetate; and / or, the dispersant is selected from one or more of sodium carboxymethyl cellulose, cellulose acetate butyrate, cellulose acetate, Span 80 or Span 85; and / or, the volume-to-mass ratio of the oil phase matrix solution to the dispersant is 1000mL:11-55g; and / or, the volume-to-mass ratio of the oil phase matrix solution to the gelatin is 1000mL:20-50g. The temperature during stirring of the dispersant added to the oil phase matrix solution is 30-60℃; and / or, the stirring speed for transferring the aqueous phase to the oil phase is 200-2000 r / min, and the time is 15-150 min; and / or, the cooling temperature is 0-10℃.

[0012] Preferably, in step S2, the reagent used for the preliminary cleaning is physiological saline; and / or, the freezing medium is a 5% mannitol solution; and / or, the freeze-drying procedure is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 4 h; 0℃, 25 Pa, 4 h; 10℃, 25 Pa, 2 h; 15℃, 25 Pa, 2 h; 25℃, 25 Pa, 4 h, for a time of 46-50 h. The sulfonating agent is selected from one or more of potassium 3-sulfopropylacrylate, sodium allyl sulfonate, or sodium 2-acrylamido-2-methyl-1-propanesulfonate; and / or, the mass of the sulfonating agent is 0.5-5 times the mass of the gelatin; and / or, the catalyst is selected from one or more of tetramethylethylenediamine, ethylenediamine, or ammonia; and / or, the mass of the catalyst is 0.1-1 times the mass of the sulfonating agent. The initiator is selected from one or more of ammonium persulfate, potassium persulfate, or hydrogen peroxide; and / or, the mass of the initiator is 0.01-0.1 times the mass of the sulfonating agent; and / or, the sulfonation reaction is carried out for 5-7 hours at a temperature of 4-10°C. The crosslinking agent is selected from one of formaldehyde or glutaraldehyde; and / or, the mass of the crosslinking agent is 0.0125-0.125 times the mass of the gelatin; and / or, the crosslinking reaction is carried out for 1.5-2.5 hours at a temperature of 25-30°C. The reagents used for the secondary cleaning are an organic solvent and water for injection; the organic solvent is selected from one or two of acetone, isopropanol, or ethanol.

[0013] Secondly, in an optional embodiment, the present invention provides a biodegradable drug-loaded microsphere prepared using the above-described preparation method.

[0014] Compared with the prior art, the present invention has one of the following beneficial effects:

[0015] 1. This invention prepares biodegradable drug-loaded microspheres by mixing gelatin with a biomaterial containing anionic groups. The combination of the two biomaterials can improve the mechanical strength of the microspheres, prolong the degradation time of the microspheres, and can also load drugs. The preparation process is simple and easy to carry out industrial production.

[0016] 2. Compared with gelatin microspheres that have only undergone sulfonation modification, the biodegradable drug-loaded microspheres prepared in this invention introduce -SO3H groups, which increases the hydrophilicity of the microspheres and thus leads to an increase in the swelling rate of the microspheres. This causes the microspheres to shrink in volume when they are transferred from the low-osmotic-pressure preservation solution to the high-osmotic-pressure drug solution, resulting in an increase in the amount of microspheres required for embolization surgery. In contrast, the gelatin microspheres prepared in this invention have added sodium alginate, whose rigid chain structure enhances the microspheres' resistance to compression. The microspheres do not swell in either the preservation solution or the drug solution.

[0017] 3. Compared with gelatin microspheres that have only undergone sulfonation modification, the biodegradable drug-loaded microspheres prepared in this invention have a simpler way of binding with drugs (mainly relying on electrostatic adsorption). In this invention, the carboxyl groups (-COOH) in the biomaterial can form hydrogen bonds or ionic bonds with the drug, which improves the stability of drug loading and extends the drug release time. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 These are images of the biodegradable drug-loaded microspheres prepared in Example 1 of this invention under a microscope;

[0020] Figure 2 These are images of the biodegradable drug-loaded microspheres prepared in Example 2 of this invention under a microscope;

[0021] Figure 3 These are images of the biodegradable drug-loaded microspheres prepared in Example 3 of this invention under a microscope;

[0022] Figure 4 These are images of the biodegradable drug-loaded microspheres prepared in Example 4 of this invention under a microscope;

[0023] Figure 5 These are images of the biodegradable drug-loaded microspheres prepared in Example 5 of this invention under a microscope;

[0024] Figure 6 These are images of the biodegradable drug-loaded microspheres prepared in Example 6 of this invention under a microscope;

[0025] Figure 7 These are images of the biodegradable drug-loaded microspheres prepared in Example 7 of this invention under a microscope;

[0026] Figure 8 These are images of the biodegradable drug-loaded microspheres prepared in Example 8 of this invention under a microscope;

[0027] Figure 9 These are images of the biodegradable drug-loaded microspheres prepared in Example 9 of this invention under a microscope;

[0028] Figure 10 These are images of the biodegradable drug-loaded microspheres prepared in Comparative Example 1 of this invention under a microscope;

[0029] Figure 11These are images of the biodegradable drug-loaded microspheres prepared in Comparative Example 2 of this invention under a microscope;

[0030] Figure 12 These are images of the biodegradable drug-loaded microspheres prepared in Comparative Example 3 of this invention under a microscope;

[0031] Figure 13 These are images of the biodegradable drug-loaded microspheres prepared in Comparative Example 4 of this invention under a microscope;

[0032] Figure 14 These are images of the biodegradable drug-loaded microspheres prepared in Comparative Example 5 of this invention under a microscope;

[0033] Figure 15 These are images of the biodegradable drug-loaded microspheres prepared in Comparative Example 6 of this invention under a microscope;

[0034] Figure 16 These are images of the biodegradable drug-loaded microspheres prepared in Comparative Example 7 of this invention under a microscope;

[0035] Figure 17 These are images of the biodegradable drug-loaded microspheres prepared in Comparative Example 8 of this invention under a microscope;

[0036] Figure 18 These are images of the biodegradable drug-loaded microspheres prepared in Comparative Example 9 of this invention under a microscope;

[0037] Figure 19 These are images under a microscope of the biodegradable drug-loaded microspheres prepared in Example 1 of this invention after drug loading;

[0038] Figure 20 This is a microscopic image of the biodegradable drug-loaded microspheres prepared in Comparative Example 1 of this invention after drug loading. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0040] The technical solution of the present invention will be described below with reference to embodiments.

[0041] Example 1

[0042] This embodiment provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0043] (1) Weigh 20g of pigskin gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0044] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 1 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃, and add 30 g of potassium 3-sulfopropylacrylate solution, 3 g of ethylenediamine, and 1 g of ammonium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0045] Example 2

[0046] This embodiment provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0047] (1) Weigh 30g of pigskin gelatin and 6g of carboxymethyl chitosan, add them to 164g of phosphate buffer solution (pH 7), heat to 60℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 15g of Span85 and 1.5g of cellulose acetate into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0048] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze-drying for 36 h (the freeze-drying procedure is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); the freeze-dried gelatin microspheres The microspheres were added to physiological saline, and 1.5 g of glutaraldehyde solution was added to the above reaction system for a cross-linking reaction for 2 h (the temperature of the cross-linking reaction was 25℃). After the cross-linking reaction was completed, the temperature of the reaction system was adjusted to 10℃, and 45 g of potassium 3-sulfopropylacrylate solution, 4.5 g of ethylenediamine, and 1.5 g of ammonium persulfate were added for a sulfonation reaction for 6 h. After the sulfonation reaction was completed, the liquid was collected and washed with ethanol, isopropanol, and purified water to obtain degradable drug-loaded microspheres.

[0049] Example 3

[0050] This embodiment provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0051] (1) Weigh 20g of bovine bone gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 65℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0052] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 1 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃, and add 30 g of sodium allyl sulfonate solution, 3 g of tetramethylethylenediamine, and 1 g of potassium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with ethanol and purified water to obtain degradable drug-loaded microspheres.

[0053] Example 4

[0054] This embodiment provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0055] (1) Weigh 20g of pork bone gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain the aqueous phase; take 1000mL of butyl acetate into the reactor, take 10g of Span80 and 1g of sodium carboxymethyl cellulose into the above reactor (the reactor temperature is controlled at 50℃), and stir until completely dissolved to obtain the oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reactor temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0056] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 1 g of formaldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 30℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃, and add 30 g of potassium 3-sulfopropylacrylate solution, 3 g of ethylenediamine, and 1 g of ammonium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with acetone and purified water to obtain degradable drug-loaded microspheres.

[0057] Example 5

[0058] This embodiment provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0059] (1) Weigh 50g of pork bone gelatin and 5g of sodium alginate, add them to 145g of phosphate buffer solution (pH 7), heat to 60℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of butyl acetate into a reaction vessel, take 50g of Span85 and 5g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0060] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze-drying for 36 h (the freeze-drying procedure is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); the freeze-dried gelatin microspheres The microspheres were added to physiological saline, and 2.5 g of glutaraldehyde solution was added to the above reaction system for a cross-linking reaction for 2 h (the temperature of the cross-linking reaction was 25℃). After the cross-linking reaction was completed, the temperature of the reaction system was adjusted to 10℃, and 75 g of potassium 3-sulfopropylacrylate solution, 7.5 g of ethylenediamine, and 2.5 g of ammonium persulfate were added for a sulfonation reaction for 6 h. After the sulfonation reaction was completed, the liquid was collected and washed with acetone, isopropanol, and purified water to obtain biodegradable drug-loaded microspheres.

[0061] Example 6

[0062] This embodiment provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0063] (1) Weigh 10g of pigskin gelatin and 1g of sodium alginate, add them to 189g of phosphate buffer solution (pH 7), heat to 55℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into the reaction vessel, take 10g of Span85 and 1g of cellulose acetate butyrate into the above reaction vessel (the freeze-drying procedure is: -50℃, 0pa, 4h; -35℃, 0pa, 4h; -10℃, 0pa, 4h; 0℃, 25pa) The aqueous phase was stirred until completely dissolved to obtain the oil phase. The aqueous phase solution was added dropwise to the oil phase using a pipette while the stirring speed was slowly increased to 300 r / min. After the aqueous phase was completely added to the oil phase, the stirring speed was maintained at 300 r / min for 20 min. After stirring for 20 min, the temperature of the reactor was lowered to 10℃ and the stirring was continued for 30 min to obtain gel microspheres.

[0064] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze-drying for 36 h (the freeze-drying procedure is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); the freeze-dried gelatin microspheres The microspheres were added to physiological saline, and 0.5 g of glutaraldehyde solution was added to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction was 25℃). After the cross-linking reaction was completed, the temperature of the reaction system was adjusted to 10℃, and 15 g of potassium 3-sulfopropylacrylate solution, 1.5 g of ethylenediamine, and 0.5 g of ammonium persulfate were added for sulfonation reaction for 6 h. After the sulfonation reaction was completed, the liquid was collected and washed with acetone, ethanol and purified water to obtain degradable drug-loaded microspheres.

[0065] Example 7

[0066] This embodiment provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0067] (1) Weigh 20g of pigskin gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0068] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 2.5 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃, and add 30 g of potassium 3-sulfopropylacrylate solution, 3 g of ethylenediamine, and 1 g of ammonium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0069] Example 8

[0070] This embodiment provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0071] (1) Weigh 20g of pigskin gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0072] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 1 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃, and add 60 g of potassium 3-sulfopropylacrylate solution, 6 g of ethylenediamine, and 2 g of ammonium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0073] Example 9

[0074] This embodiment provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0075] (1) Weigh 20g of pigskin gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0076] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 1 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 4℃, and add 30 g of potassium 3-sulfopropylacrylate solution, 3 g of ethylenediamine, and 1 g of ammonium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0077] Comparative Example 1

[0078] This comparative example provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0079] (1) Weigh 20g of pigskin gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0080] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, adjust the reaction system temperature to 25℃, add 1 g of glutaraldehyde solution for cross-linking reaction for 2 h. After the cross-linking reaction is completed, collect the liquid and wash with acetone, isopropanol and purified water to obtain gelatin microspheres.

[0081] Comparative Example 2

[0082] This comparative example provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0083] (1) Weigh 20g of pigskin gelatin and add it to 175g of phosphate buffer solution (pH 7). Heat to 50℃ and stir until completely dissolved to obtain an aqueous phase. Take 1000mL of light liquid paraffin into a reaction vessel. Take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃). Stir until completely dissolved to obtain an oil phase. Pipette the aqueous phase solution into the oil phase while slowly increasing the rotation speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min. After stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres.

[0084] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 1 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃, and add 30 g of potassium 3-sulfopropylacrylate solution, 3 g of ethylenediamine, and 1 g of ammonium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0085] Comparative Example 3

[0086] This comparative example provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0087] (1) Weigh 20g of pigskin gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0088] (2) After washing the gel microspheres with physiological saline, add them to physiological saline and add 1g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2h (the temperature of the cross-linking reaction is 50℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃ and add 30g of potassium 3-sulfopropylacrylate solution, 3g of ethylenediamine and 1g of ammonium persulfate for sulfonation reaction for 6h. After the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0089] Comparative Example 4

[0090] This comparative example provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0091] (1) Weigh 5g of pigskin gelatin and 5g of sodium alginate, add them to 190g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at a speed of 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0092] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 48 h. The freeze drying procedure is as follows: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 4 h; 0℃, 25 Pa, 4 h; 10℃, 25 Pa, 2 h; 15℃, 25 Pa, 2 h; 25℃, 25 Pa, 4 h. Add the freeze-dried gelatin microspheres to physiological saline and add 1 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃ and add 30 g of potassium 3-sulfopropylacrylate solution, 3 g of ethylenediamine, and 1 g of ammonium persulfate for sulfonation reaction for 6 h. After the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0093] Comparative Example 5

[0094] This comparative example provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0095] (1) Weigh 20g of pigskin gelatin and 15g of sodium alginate, add them to 165g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into the reactor, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reactor (the reactor temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reactor temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0096] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 1 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃, and add 30 g of potassium 3-sulfopropylacrylate solution, 3 g of ethylenediamine, and 1 g of ammonium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0097] Comparative Example 6

[0098] This comparative example provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0099] (1) Weigh 20g of pigskin gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 60℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0100] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 0.2 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃, and add 30 g of potassium 3-sulfopropylacrylate solution, 3 g of ethylenediamine, and 1 g of ammonium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0101] Comparative Example 7

[0102] This comparative example provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0103] (1) Weigh 20g of pigskin gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0104] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 1 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃, and add 5 g of potassium 3-sulfopropylacrylate solution, 3 g of ethylenediamine, and 1 g of ammonium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0105] Comparative Example 8

[0106] This comparative example provides a method for preparing biodegradable drug-loaded microspheres, including the following steps:

[0107] (1) Weigh 20g of pigskin gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain an aqueous phase; take 1000mL of light liquid paraffin into a reaction vessel, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reaction vessel (the reaction vessel temperature is controlled at 50℃), and stir until completely dissolved to obtain an oil phase; add the aqueous phase solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min. After the aqueous phase is completely added into the oil phase, continue stirring at 300r / min for 20min; after stirring for 20min, lower the reaction vessel temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0108] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 1 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 50℃, and add 30 g of potassium 3-sulfopropylacrylate solution, 3 g of ethylenediamine, and 1 g of ammonium persulfate for sulfonation reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain degradable drug-loaded microspheres.

[0109] Comparative Example 9

[0110] This comparative example provides a method for preparing sulfonated gelatin microspheres, including the following steps:

[0111] (1) Weigh 30g of potassium 3-sulfopropylacrylate solution, 20g of pigskin gelatin and 5g of sodium alginate, add them to 175g of phosphate buffer solution (pH 7), heat to 50℃, and stir until completely dissolved to obtain sulfonated gelatin solution; take 1000mL of light liquid paraffin into the reactor, take 10g of Span85 and 1g of sodium carboxymethyl cellulose into the above reactor (the reactor temperature is controlled at 50℃), stir until completely dissolved to obtain oil phase; add the sulfonated gelatin solution dropwise into the oil phase through a pipette, while slowly increasing the speed to 300r / min, and after the aqueous phase is completely added into the oil phase, continue to maintain the speed of 300r / min for 20min; after stirring for 20min, lower the reactor temperature to 10℃ and continue stirring for 30min to obtain gel microspheres;

[0112] (2) After washing the gel microspheres with physiological saline, add 200 mL of 5% mannitol solution and transfer to a freeze dryer for freeze drying for 36 h (the freeze drying program is: -50℃, 0 Pa, 4 h; -35℃, 0 Pa, 4 h; -10℃, 0 Pa, 8 h; 0℃, 25 Pa, 8 h; 10℃, 25 Pa, 4 h; 15℃, 25 Pa, 4 h; 25℃, 25 Pa, 4 h); add the freeze-dried gelatin microspheres to physiological saline, add 1 g of glutaraldehyde solution to the above reaction system for cross-linking reaction for 2 h (the temperature of the cross-linking reaction is 25℃). After the cross-linking reaction is completed, adjust the temperature of the reaction system to 10℃, add 3 g of ethylenediamine and 1 g of ammonium persulfate, and continue the reaction for 6 h; after the sulfonation reaction is completed, collect the liquid and wash it with acetone, isopropanol and purified water to obtain sulfonated gelatin microspheres.

[0113] Experimental Example 1

[0114] Microspheres from each example and comparative example were sieved to obtain microspheres with a particle size of 100–300 μm, and examined under natural light and a microscope (see [reference]). Figures 1-18 The appearance of the microspheres was observed, and the results are shown in Table 1.

[0115] Table 1. Appearance of the microspheres

[0116]

[0117] Conclusion: The gelatin microspheres prepared in the embodiments of the present invention are light yellow to dark yellow, relatively round, well dispersible, without agglomeration, and have smooth surfaces. The microspheres prepared in comparative examples 8-9 are too dark in color, the microspheres prepared in comparative examples 4 and 6 are too light in color, the microspheres prepared in comparative examples 1, 7 and 9 show agglomeration, and the microspheres prepared in comparative example 9 have rough surfaces.

[0118] Experiment Example 2

[0119] Microspheres from each example and comparative example were taken, and microspheres with a particle size of 100-300 μm were sieved out. The deformation resistance of the microspheres was tested using a texture analyzer. The microspheres were compressed to a certain ratio and held for 10 seconds. The microspheres were observed to see if they broke. The results are shown in Table 2.

[0120] Table 2 Deformation resistance of microspheres

[0121]

[0122]

[0123] Conclusion: As shown in Table 2, the gelatin microspheres prepared in the embodiments of the present invention did not rupture (a rupture rate of <5% is considered as no rupture) and did not deform after being compressed by 50% and 80% respectively (the deformation rate is the ratio of the average particle size after compression to the average particle size before compression; when the deformation rate is 90-110%, the microspheres are considered as not deforming). The microspheres prepared in Comparative Examples 2, 3, and 8 did not rupture or deform after being compressed by 50% and 80% respectively, while the remaining comparative examples all showed partial rupture and deformation.

[0124] Experimental Example 3

[0125] Gelatin microspheres from each embodiment were taken, and microspheres with a particle size of 100-300 μm were sieved out. 1 mL of microspheres and 9 mL of physiological saline were added to a 20 mL syringe, and 9 mL of contrast agent was added. After being evenly mixed, the mixture was slowly injected into a 1.7 Fr catheter through a three-way valve and a 1 mL syringe. The smooth passage of the microspheres through the catheter was observed. The same method was used to inject commercially available gelatin sponge particles into another custom-made catheter. The smoothness of the passage of the microspheres through the catheter was observed, and the morphology of the microspheres after passing through the catheter was observed. The results are shown in Table 3.

[0126] Table 3. Catheter permeability of microspheres

[0127]

[0128]

[0129] Conclusion: As shown in Table 3, the gelatin microspheres prepared in the embodiments of the present invention can all pass through the conduit smoothly, and the microspheres do not rupture after passing through the conduit and can recover their original shape; although the microspheres prepared in Comparative Examples 1-2, 4-7, and 9 can pass through the conduit smoothly, the microspheres all rupture and deform.

[0130] Experiment Example 4

[0131] Microspheres from each example and comparative example were taken, and microspheres with a particle size of 100-300 μm were sieved. 100 mg of each microsphere was placed in 10 mL of 1% pepsin (activity of approximately 3000 U / mg) hydrochloric acid solution (0.1 M) preheated to 37 °C. The solution was kept at 37 °C in a constant temperature shaker and shaken slowly. The time for complete degradation of the microspheres was recorded. The results are shown in Table 4.

[0132] Table 4 Degradation time of microspheres

[0133] Sample Degradation time (h) Sample Degradation time (h) Example 1 8.0h Comparative Example 1 4.7h Example 2 9.0h Comparative Example 2 4.9h Example 3 8.1h Comparative Example 3 5.0h Example 4 7.8h Comparative Example 4 2.5h Example 5 10.5h Comparative Example 5 1.8h Example 6 6.5h Comparative Example 6 2.6h Example 7 11.0h Comparative Example 7 4.5h Example 8 8.5h Comparative Example 8 15h Example 9 7.0h Comparative Example 9 4.1h

[0134] Conclusion: As shown in Table 4, the gelatin microspheres prepared in the embodiments of the present invention have a relatively long degradation time by pepsin, ranging from 6.5 to 11 hours; while the microspheres prepared in comparative examples 4-6 have a shorter degradation time, and the microspheres prepared in comparative example 8 have a much longer degradation time than those in the embodiments. The microspheres prepared in the other comparative examples have a degradation time of 4-5 hours, which is shorter than that in the embodiments.

[0135] Experimental Example 5

[0136] Take doxorubicin hydrochloride for injection, add water for injection, and dilute to 1 mg / mL. Then dilute to prepare standard solutions of 0.2 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL, respectively. Measure the absorbance of the standard solutions at 480 nm using a UV spectrophotometer and plot a standard curve.

[0137] Prepare 0.5g of microsphere samples for each example and comparative example, and prepare 20mg / mL doxorubicin hydrochloride injection solution. Drug loading is carried out at a ratio of 100mg drug per g of microspheres, i.e., 2.5mL of drug solution is added to 0.5g sample. The drug solution and microspheres are suspended every 5min. The supernatant is collected at 5min, 15min and 30min, and the absorbance is measured by ultraviolet spectrophotometer to calculate the drug loading rate of microspheres at different time points.

[0138] Table 5. Drug loading rate of microspheres

[0139] Sample Drug loading 5 min Drug loading 15 min Drug loading 30 min Example 1 97.59％ 98.77％ 99.69％ Example 2 97.87％ 98.67％ 99.65％ Example 3 96.58％ 98.95％ 99.91％ Example 4 96.64％ 98.87％ 99.77％ Example 5 96.88％ 98.43％ 99.90％ Example 6 90.22％ 95.79％ 99.38％ Example 7 96.09％ 97.98％ 99.16％ Example 8 98.35％ 98.98％ 99.67％ Example 9 93.56％ 95.37％ 97.62％ Comparative Example 1 56.87％ 60.69％ 67.58％ Comparative Example 2 74.55％ 78.64％ 83.31％ Comparative Example 3 88.14％ 89.46％ 91.43％ Comparative Example 4 73.99％ 78.73％ 83.59％ Comparative Example 5 87.88％ 89.27％ 90.93％ Comparative Example 6 59.47％ 65.68％ 70.77％ Comparative Example 7 67.65％ 75.48％ 80.33％ Comparative Example 8 96.23％ 97.18％ 99.75％ Comparative Example 9 53.79％ 60.63％ 66.97％

[0140] Note: The formula for calculating drug loading rate is as follows:

[0141] Drug loading efficiency = (Total drug dosage - Total drug content in supernatant) / Total drug dosage × 100%

[0142] Conclusion: As shown in Table 5, the gelatin microspheres prepared in the embodiments of the present invention have fast drug loading and large drug loading capacity, with a drug loading rate of over 90% after 5 minutes, over 95% after 15 minutes, and over 99% after 30 minutes; the microspheres prepared in comparative examples 1-7 and 9 have slow drug loading and low drug loading capacity, with the highest drug loading rate being less than 91%.

[0143] The drug-loaded microspheres from Example 1 and Comparative Example 1 were observed under a microscope, and the results are shown in [reference needed]. Figures 19-20 .

[0144] Experimental Example 6

[0145] After the drug loading experiment was completed, the drug-loaded samples of each example and comparative example were transferred to dialysis bags (with a molecular weight cutoff of 8000-14000 Da) and placed in the dissolution apparatus basket. 500 mL of phosphate buffer solution (pH = 7.4) was added, the temperature was set to 37℃, and the rotation speed was 50 rpm. Samples were taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 180 h, and 240 h to calculate the drug release rate of the microspheres. The results are shown in Tables 6-7.

[0146] Table 6. Drug release rate of microspheres

[0147]

[0148]

[0149] Table 7 Drug release rate of microspheres

[0150]

[0151] Note: The formula for calculating drug release rate is as follows:

[0152] Drug release rate = (concentration 1 × sampling volume 1 + concentration 2 × sampling volume 2 + ... + concentration n × sampling volume n) / total drug load × 100%

[0153] Conclusion: As shown in Tables 6-7, the drug release rate of the gelatin microspheres prepared in the embodiments of the present invention is about 2%-6% within 0.5 hours, less than 40% (i.e., non-burst release), the time for drug release rate to reach 20% is 36-48 hours, ≤48 hours (i.e., drug release of more than 20% can be achieved within 48 hours), and drug release of more than 60% at 240 hours. The total drug release time exceeds 240 hours, which can achieve the effect of sustained release.

[0154] The microspheres prepared in Comparative Examples 1-4 exhibited relatively rapid drug release in the early stages, followed by a gradual slowdown in the later stages. They reached over 60% drug release in approximately 48 hours, plateaued after 96 hours, and almost ceased release after 240 hours. The microspheres prepared in Comparative Example 5 showed slightly faster drug release in the early stages, followed by a gradual slowdown in the later stages. They reached over 60% drug release in approximately 120 hours, plateaued after 120 hours, and almost ceased release altogether. The microspheres prepared in Comparative Examples 6, 7, and 9 exhibited rapid drug release, with a release rate of 27%-33% in 0.5 hours, approaching a burst release phenomenon, and exceeding 90% release within 240 hours. The microspheres prepared in Comparative Example 8 exhibited excessively slow drug release, reaching 20% ​​release in 96-120 hours, exceeding 48 hours, and only reaching slightly over 40% release after 240 hours.

[0155] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions of the present invention without departing from the spirit and scope of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing degradable drug-loaded microspheres, characterized in that, The method comprises the following steps: S1: adding gelatin and biological material in a buffer solution, heating and stirring to dissolve, to obtain an aqueous phase; adding a dispersing agent in an oil phase matrix solution and stirring, to obtain an oil phase; transferring the aqueous phase to the oil phase and stirring and cooling, to obtain gel microspheres; S2: after the gel microspheres are preliminarily cleaned, they are placed in a freezing medium for freeze-drying; then the freeze-dried gel microspheres are added to physiological saline, and a crosslinking agent is added for crosslinking reaction, to obtain crosslinked microspheres; S3: the crosslinked microspheres are cleaned, and then a sulfonating agent, a catalyst and an initiator are added for sulfonating reaction; after the sulfonating reaction is completed, the microspheres are cleaned, to obtain degradable drug-loaded microspheres. The biological material is selected from one or more of sodium alginate, chitosan or carboxymethyl chitosan.

2. The method for preparing biodegradable drug-loaded microspheres according to claim 1, characterized in that, In step S1, the mass of the biological material is 0.1-0.5 times the mass of the gelatin; and / or, The buffer solution is selected from one or a mixture of two of physiological saline or a phosphate buffer solution, and the pH value of the phosphate buffer solution is 6-7; and / or, The gelatin is selected from one or more of pigskin gelatin, pig bone gelatin or cow bone gelatin; and / or, The mass ratio of the gelatin to the buffer solution is 1:2-19; and / or, The temperature for heating and stirring to dissolve is 50-70℃.

3. The method for preparing biodegradable drug-loaded microspheres according to claim 1, characterized in that, In step S1, the oil phase matrix solution is selected from one or more of light liquid paraffin, silicone oil or butyl acetate; and / or, The dispersing agent is selected from one or more of carboxymethyl cellulose sodium, cellulose acetate butyrate, cellulose acetate, Span 80 or Span 85; and / or, The volume-mass ratio of the oil phase matrix solution to the dispersing agent is 1000 mL:11-55 g; and / or, The volume-mass ratio of the oil phase matrix solution to the aqueous phase matrix solution is 5:

1.

4. The method for preparing biodegradable drug-loaded microspheres according to claim 1, characterized in that, In step S1, when the dispersing agent is added in the oil phase matrix solution and stirred, the temperature is 30-60℃; and / or, The stirring speed for transferring the aqueous phase to the oil phase is 200-2000 r / min, and the time is 15-150 min; and / or, The cooling temperature is 0-10℃.

5. The method for preparing biodegradable drug-loaded microspheres according to claim 1, characterized in that, In step S2, the reagent used for the preliminary cleaning is physiological saline; and / or, The freezing medium is a 5% mannitol solution; and / or, The freeze-drying procedure is: -50℃, 0 pa, 4 h; -35℃, 0 pa, 4 h; -10℃, 0 pa, 4 h; 0℃, 25 pa, 4 h; 10℃, 25 pa, 2 h; 15℃, 25 pa, 2 h; 25℃, 25 pa, 4 h.

6. The method for preparing biodegradable drug-loaded microspheres according to claim 1, characterized in that, In step S2, the crosslinking agent is selected from one of formaldehyde or glutaraldehyde; and / or, The mass of the crosslinking agent is 0.0125-0.125 times the mass of the gelatin; and / or, The crosslinking reaction time is 1.5-2.5 h, and the temperature is 25-30℃.

7. The method for preparing biodegradable drug-loaded microspheres according to claim 1, characterized in that, In step S2, the sulfonating agent is selected from one or more of 3-sulfopropyl acrylate potassium, sodium allylsulfonate or 2-acrylamido-2-methyl-1-propane sulfonic acid sodium; and / or, the sulfonating agent is in an amount of 0.5-5 times the amount of gelatin; and / or, the catalyst is selected from one or more of tetramethylethylenediamine, ethylenediamine or ammonia; and / or, the catalyst is in an amount of 0.1-1 times the amount of sulfonating agent.

8. The method for preparing biodegradable drug-loaded microspheres according to claim 1, characterized in that, in step S2, the secondary washing uses an organic solution and water for injection; the organic solution is selected from one or both of acetone, isopropyl alcohol or ethanol.

9. A degradable drug-loaded microsphere, characterized in that, prepared using the method of any one of claims 1-8.

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