Starch hydrogel microsphere and preparation method therefor

Abstract

Provided in the present application are a starch hydrogel microsphere and a preparation method therefor. The preparation method comprises the following steps: the preparation of a water phase, involving: mixing amylopectin, a cross-linking agent and water, so as to obtain the water phase; the preparation of an oil phase, involving: mixing a dispersing agent and an organic solvent, so as to obtain the oil phase; and reversed-phase polymerization, involving: mixing the water phase and the oil phase to form a reversed-phase polymerization system, and subjecting same to polymerization, so as to obtain a starch hydrogel microsphere. In the present application, a starch hydrogel microsphere having good performance is prepared by means of designing the preparation method for a starch hydrogel microsphere.

Description

A kind of starch hydrogel microsphere and preparation method thereof Technical Field

[0001] The present application belongs to the technical field of embolic microspheres, and specifically relates to starch hydrogel microspheres and a preparation method thereof. Background Art

[0002] Embolization, originally developed by Sadek Hilal in 1968, is a minimally invasive surgical technique that aims to block blood flow to a certain area of ​​the body, thereby effectively shrinking tumors or blocking aneurysms. It is currently used in the treatment of various malignant and benign tumors.

[0003] Transcatheter arterial embolization (TAE) has become widely accepted due to its effectiveness in treating a variety of conditions, including tumors, vascular lesions, and bleeding. For safe and effective treatment, it is important to select the appropriate embolic material. Uterine fibroids are benign muscle tumors that grow on the wall of the uterus. They can grow into a single tumor or multiple tumors and can vary in size. Uterine fibroids can grow into a single tumor or multiple tumors. Uterine fibroids can be as small as an apple seed or as large as a grapefruit. In abnormal cases, uterine fibroids can become very large. An increasingly accepted and currently generally accepted treatment for uterine fibroids is uterine fibroid embolization (UFE). The main purpose of UFE is to reduce the size of uterine fibroids and treat heavy uterine bleeding. Essentially, UFE involves the placement of a catheter into the uterine artery and the injection of embolic microspheres into the uterine artery to achieve fibroid devascularization and gradual shrinkage. Different indications have different requirements for the size and material of the embolic agent, so the choice of embolic agent plays a crucial role in interventional embolization treatment.

[0004] Starch, a widely used excipient in pharmaceutical production, is abundantly available, inexpensive, non-toxic, and possesses excellent biocompatibility and biodegradability, meeting all requirements of drug delivery systems. As a novel biodegradable embolic microsphere product, starch microspheres possess numerous properties not found in other embolic microspheres. Their superiority is increasingly evident in research applications as embolic microspheres, making them a hot topic of research both domestically and internationally.

[0005] The starch embolic microspheres currently on the market are Germany's EmboCept (PharmaCept, Berlin, Germany), which have an in vitro degradation half-life of 35 minutes and are only available in one specification of 50μm. They are only suitable for transcatheter arterial chemoembolizatio (TACE) of the liver and lungs, and their product specifications and application areas are very limited. Some other starch microspheres generally have irregular spherical shapes, and their particle size is difficult to accurately control. At the same time, in terms of the performance characterization of starch microspheres, there is a lack of evaluation indicators such as elasticity and catheter permeability. In addition, the strength, elasticity and catheter permeability of the microspheres currently prepared from starch are not good, which affects the stability of the microspheres during use.

[0006] Therefore, how to provide a starch microsphere with excellent comprehensive performance has become a technical problem that needs to be solved urgently.

[0007] Summary of the Invention

[0008] The present application provides starch hydrogel microspheres and a preparation method thereof.

[0009] In a first aspect, the present application provides a method for preparing starch hydrogel microspheres, the preparation method comprising the following steps:

[0010] Preparation of aqueous phase: mixing pullulan, a cross-linking agent and water to obtain the aqueous phase;

[0011] Oil phase preparation: mixing a dispersant and an organic solvent to obtain the oil phase;

[0012] Reverse phase polymerization: mixing the aqueous phase and the oil phase to form a reverse phase polymerization system, and performing polymerization to obtain the starch hydrogel microspheres.

[0013] In a second aspect, the present application provides starch hydrogel microspheres, which are prepared according to the preparation method described in the first aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0015] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0016] FIG1 is a flow chart of the preparation method provided in an embodiment of the present application;

[0017] Figures 2 to 12 are respectively the morphologies of the microspheres provided in Examples 1 to 11 of the present application after replacement;

[0018] Figures 13-23 are respectively the morphologies of the microspheres provided in Examples 1-11 of the present application after sterilization;

[0019] Figures 24-28 are respectively the morphology images of the microspheres provided in Examples 7-11 of the present application after the catheter passability test;

[0020] Figures 29-38 are images of the starch hydrogel embolic microspheres of Comparative Examples 1-2 and Examples 12-19 under an optical microscope;

[0021] FIG39 is an optical microscope image of the prepared starch hydrogel embolic microspheres before elasticity experiment;

[0022] FIG40 is an optical microscope image of the prepared starch hydrogel embolic microspheres in the elasticity experiment. DETAILED DESCRIPTION

[0023] To make the objectives, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Generally, the components of the embodiments of this application described and shown in the drawings herein can be arranged and designed in various different configurations.

[0024] Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments in the present application without creative work are within the scope of protection of the present application.

[0025] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0026] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be construed as a hard limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numbers within the range. For example, the description of a range from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0027] In this application, unless otherwise specified, the directional words used, such as "upper" and "lower", refer specifically to the directions of the drawings in the accompanying drawings. In addition, in the description of the present application specification, the terms "including", "comprising", etc. mean "including but not limited to". In this article, relational terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. In this article, "and / or" describes the association relationship of associated objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. Wherein A and B can be singular or plural. In this article, "at least one" refers to one or more, and "plurality" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e. a and b), ac, bc, or abc, where a, b and c can be single or multiple.

[0028] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0029] In the description of the embodiments of the present application, it should be understood that the indicated orientation or position relationship is based on the orientation or position relationship shown in the accompanying drawings, or the orientation or position relationship in which the product of the application is conventionally placed when in use, or the orientation or position relationship conventionally understood by those skilled in the art. It is only for the convenience of describing the present application and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present application.

[0030] In the description of the embodiments of the present application, embolic microspheres, hydrogel microspheres and microspheres have the same meaning and can all be understood as hydrogel microspheres.

[0031] An embodiment of the present application provides a method for preparing starch hydrogel microspheres, the preparation method comprising the following steps:

[0032] Preparation of aqueous phase: mixing pullulan, a cross-linking agent and water to obtain the aqueous phase;

[0033] Oil phase preparation: mixing a dispersant and an organic solvent to obtain the oil phase;

[0034] Reverse phase polymerization: mixing the aqueous phase and the oil phase to form a reverse phase polymerization system, and performing polymerization to obtain the starch hydrogel microspheres.

[0035] In this application, starch hydrogel microspheres are prepared using amylopectin, which facilitates the production of better-formed microspheres and improves yield. The microspheres prepared through the above steps exhibit excellent performance indicators, such as improved suspension, catheter permeability, in vitro enzymatic hydrolysis time, room temperature stability, and stability at 4°C, resulting in higher overall performance. The microspheres prepared in this application exhibit ultra-compliant properties, excellent catheter permeability, and a longer degradation time, which are beneficial for the application of UFE.

[0036] Wherein, the aqueous phase is prepared by mixing amylopectin, a cross-linking agent and water to obtain the aqueous phase.

[0037] In some embodiments, the amylopectin comprises corn amylopectin.

[0038] The corn amylopectin can be obtained by the following method:

[0039] Accurately weigh 100 g of purchased corn starch, add 3000 mL of dimethyl sulfoxide (DMSO) and stir at 200 rpm at room temperature for 24 hours. After stirring, centrifuge at 2000 rpm, take the supernatant and add it to 2 times the amount of n-butanol, and let it stand for 24 hours; then centrifuge at 4000 rpm, take the precipitate, wash it three times with n-butanol, remove DMSO and filter it, boil and dissolve the filter cake with sufficient distilled water, let it stand and cool to 60°C, add 10 g of thymol and stir it thoroughly, let it stand at room temperature for 3 days, and filter to obtain a filtrate; precool the filtrate in a 4°C refrigerator, then add an appropriate amount of ether, extract it three times to remove thymol, take the aqueous phase and add a large amount of ethanol until the flocculent precipitate no longer increases, let it stand at room temperature for 3 days, filter to obtain a filter cake, and dry it to obtain a white powder, which is the extracted corn amylopectin.

[0040] In other optional embodiments of the present application, the amylopectin may be selected from other amylopectins similar to corn amylopectin, such as cassava amylopectin.

[0041] Furthermore, in some optional embodiments of the present application, the method for preparing the amylopectin comprises:

[0042] The starch is mixed with a solvent, stirred, and centrifuged. The supernatant is collected and added to alcohol. The mixture is allowed to stand and then centrifuged. The precipitate is washed with alcohol, the solvent removed, and then filtered. The filter cake is dissolved, allowed to stand, thymol is added, and stirred evenly. The filtrate is allowed to stand and then collected. The filtrate is extracted with ether to remove the thymol. The aqueous phase is then added with alcohol until no more flocculent precipitates. The mixture is allowed to stand and filtered to obtain a filter cake. After drying, a white powder, i.e., amylopectin, is obtained.

[0043] In other optional embodiments of the present application, the corn amylopectin can also be purchased commercially.

[0044] In some embodiments, the cross-linking agent is selected from at least one of sodium trimetaphosphate and sodium tripolyphosphate.

[0045] In some embodiments, the cross-linking agent is sodium trimetaphosphate and sodium tripolyphosphate.

[0046] In some embodiments, the cross-linking agent is sodium trimetaphosphate and sodium tripolyphosphate, wherein the mass ratio of sodium trimetaphosphate to sodium tripolyphosphate is (10-100):1, for example, it can be 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1, etc.

[0047] In some embodiments, the cross-linking agent is sodium trimetaphosphate and sodium tripolyphosphate, wherein the mass ratio of sodium trimetaphosphate to sodium tripolyphosphate is (10-90):1.

[0048] In some embodiments, the crosslinking agent is sodium trimetaphosphate and sodium tripolyphosphate, wherein the mass ratio of sodium trimetaphosphate to sodium tripolyphosphate is 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or a range between any two of the above ratios.

[0049] In some embodiments, the aqueous phase includes pullulan, sodium trimetaphosphate, and sodium tripolyphosphate.

[0050] In some embodiments, the aqueous phase further comprises an alkaline solution.

[0051] In some embodiments, the alkaline solution comprises a sodium hydroxide solution.

[0052] In some embodiments, the mass ratio of the amylopectin to the alkaline solution is (8-10):1, for example, 8:1, 8.5:1, 9:1, 9.5:1 or 10:1.

[0053] Illustratively, the aqueous phase is prepared by mixing pullulan and an alkaline solution to obtain a pullulan solution, and then mixing the pullulan solution with a cross-linking agent and water to obtain the aqueous phase.

[0054] In some embodiments, the method for preparing the pullulan solution comprises the following steps: mixing the pullulan and sodium hydroxide, stirring and heating until gelatinized and transparent, and standing to cool for later use.

[0055] For example, 10 g of corn amylopectin and 1 g of NaOH were added to 100 mL of distilled water, stirred and heated until gelatinization and transparency, and then allowed to stand and cool for use.

[0056] In other optional embodiments of the present application, the alkaline solution may also be other applicable alkaline solutions in the art.

[0057] For example, the aqueous phase is prepared by adding 12.5 mL of an aqueous solution containing 0.3 g of sodium trimetaphosphate grade III trisodium (STMP) and 0.03 g of sodium tripolyphosphate (STPP) to 12.5 mL of corn starch solution under stirring, and stirring for 5 minutes to mix thoroughly.

[0058] Oil phase preparation: mixing a dispersant and an organic solvent to obtain the oil phase.

[0059] In some embodiments of the present application, the oil phase preparation step includes: mixing a dispersant and an organic solvent to form a continuous oil phase.

[0060] In some embodiments, the dispersant is selected from a combination of sorbitan monostearate (Sorbitan Monostearate, Span-60, Span 60 or Span 60) and polyoxyethylene sorbitan monostearate (Polysorbate 60, Teween-60, Tween 60).

[0061] In some embodiments, the dispersant is selected from a combination of sorbitan monostearate and polyoxyethylene sorbitan monostearate, and the mass ratio of sorbitan monostearate to polyoxyethylene sorbitan monostearate is (2-10):1, for example, it can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1, etc.

[0062] In some embodiments, the dispersant is selected from a combination of sorbitan monostearate and polyoxyethylene sorbitan monostearate, and the mass ratio of sorbitan monostearate to polyoxyethylene sorbitan monostearate is (2-9.9):1.

[0063] Illustratively, in some embodiments, the dispersant is selected from a combination of sorbitan monostearate and polyoxyethylene sorbitan monostearate, and the mass ratio of sorbitan monostearate to polyoxyethylene sorbitan monostearate is 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1.

[0064] In some embodiments, the organic solvent includes at least one of C4-C15 alkanes (e.g., C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, or C15).

[0065] Illustratively, in some embodiments, the organic solvent is selected from at least one of cyclohexane, n-nonane, n-decane, n-undecane, n-dodecane, or n-tridecane.

[0066] In some embodiments, the organic solvent is selected from cyclohexane.

[0067] Illustratively, in some embodiments, the prepared oil phase is prepared according to the following steps: 1 g of sorbitan monostearate (Span-60) and 0.5 g of polyoxyethylene sorbitan monostearate (Tween-60) are added to 100 mL of cyclohexane under stirring to form a continuous oil phase.

[0068] Alternatively, in some embodiments, the organic solvent is selected from a combination of a first organic solvent and a second organic solvent; wherein the first organic solvent is selected from cyclohexane; and the second organic solvent is selected from at least one of n-nonane, n-decane, n-undecane, n-dodecane, and n-tridecane.

[0069] Further, in some embodiments, the organic solvent is selected from a combination of a first organic solvent and a second organic solvent, and the volume ratio of the first organic solvent to the second organic solvent is (0.5-1.5):0.5-1.5, for example, it can be 0.5:0.5, 0.5:0.8, 0.5:1, 0.5:1.2, 0.5:1.5, 1:0.5, 1:0.8, 1:1.2, 1:1.5, 1.5:0.5, 1.5:0.8, 1.5:1, 1.5:1.2, etc.

[0070] In the present application, by using at least two organic solvents together as the solvent of the oil phase, the strength, elasticity, and catheter passability of the prepared microspheres can be effectively improved, thereby facilitating the improvement of the stability of the microspheres during use.

[0071] Reverse phase polymerization: mixing the aqueous phase and the oil phase to form a reverse phase polymerization system, and performing polymerization to obtain the starch hydrogel microspheres.

[0072] In some embodiments, the volume ratio of the oil phase to the water phase is (3-5):1, for example, it can be 3:1, 3.3:1, 3.6:1, 4:1, 4.2:1, 4.4:1, 4.6:1, 4.8:1, 4.9:1 or 5:1, etc.

[0073] Furthermore, the volume ratio of the oil phase to the water phase is (3-4.9):1.

[0074] Illustratively, the volume ratio of the oil phase to the water phase is 3.5:1, 4:1, 4.5:1 or 4.8:1.

[0075] In some embodiments, the volume ratio of the oil phase to the water phase is 100:32.5.

[0076] In some embodiments, the polymerization temperature is 25-70°C, for example, 25°C, 26°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 69°C or 70°C.

[0077] Furthermore, in some embodiments, the polymerization temperature is 26-69°C, for example, 26°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C or 69°C.

[0078] Illustratively, in some embodiments of the present application, the polymerization temperature is 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C or 60°C.

[0079] Furthermore, in some embodiments, the polymerization method specifically comprises the following steps:

[0080] The aqueous phase and the oil phase are mixed to form a reverse phase polymerization system, and the reaction is stirred at 25-70° C. for at least 3 hours (for example, 3 hours, 4 hours, 5 hours, 6 hours, etc.), and then the temperature is naturally lowered while stirring.

[0081] For example, in some embodiments, the aqueous phase and the oil phase are mixed to form a reverse phase polymerization system, which is stirred at 25-70° C. for 3 hours, 4 hours, or 5 hours, and then naturally cooled and stirred.

[0082] Furthermore, in some embodiments, the natural cooling and stirring includes: continuous stirring for at least 12 hours (for example, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 28 hours or 30 hours, etc.).

[0083] Further optionally, in some embodiments, the natural cooling and stirring may last for 12 hours, 15 hours, 18 hours, 20 hours or 24 hours.

[0084] Illustratively, in some embodiments, after the oil phase is stirred for a period of time, the water phase is added to the oil phase to fully mix the oil phase and the water phase to form a reverse phase polymerization system, which is stirred and reacted, and then stirred at room temperature overnight for more than 12 hours (greater than or equal to 12 hours) and allowed to stand.

[0085] In some embodiments, the polymerization further includes a post-processing step, and the post-processing method includes:

[0086] Add water or ethanol to the polymerization reaction system to terminate the reaction, filter, wash and sieve.

[0087] Further optionally, in some embodiments, a certain amount of ethanol is added to the reactant, filtered, washed with ethanol and then dried.

[0088] In other optional embodiments, the ethanol may also be other alcohols or alcohol solutions.

[0089] Further optionally, in some embodiments, the reaction product washed with ethanol and then dried is redissolved and sieved.

[0090] Further optionally, the step of redissolving and sieving the reaction product comprises: adding water to the reaction product, then sieving, and taking the sieve residue.

[0091] Further optionally, in some embodiments, sieves with different pore sizes can be selected for sieving to obtain corn amylopectin hydrogel microspheres with different diameters and particle sizes.

[0092] In a second aspect, the present application provides starch hydrogel microspheres, which are prepared according to the above-mentioned preparation method.

[0093] In a specific embodiment, the present application provides a method for preparing high-strength starch hydrogel microspheres, the flow chart of which is shown in FIG1 . The method for preparing high-strength starch hydrogel microspheres comprises:

[0094] S1. Preparation of oil phase: The oil phase includes a dispersant and an organic solvent;

[0095] S2. aqueous phase preparation: adding a crosslinking agent to the pullulan solution to obtain an aqueous phase; and

[0096] S3. Reverse phase polymerization: the aqueous phase and the oil phase are mixed to form a reverse phase suspension polymerization system, and then a polymerization reaction is carried out to obtain microspheres.

[0097] It should be noted that, in the above-mentioned method S1 for preparing high-strength starch hydrogel microspheres, the organic solvent includes at least one of C4-C15 alkanes, and preferably includes at least two of C4-C15 alkanes.

[0098] At the same time, the present application also provides a high-strength starch hydrogel microsphere, which is prepared by the preparation method provided above.

[0099] The high-strength starch hydrogel microspheres are realized based on the above-mentioned preparation method. The specific steps of the preparation method can refer to the above-mentioned embodiment. Since the high-strength starch hydrogel microspheres adopt part or all of the technical solutions of the above-mentioned embodiments, they at least have all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be repeated here.

[0100] The present application will be further described below in conjunction with specific Examples 1-11. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods in the following examples where specific conditions are not specified are generally determined in accordance with national standards. If there are no corresponding national standards, then the methods are carried out in accordance with general international standards, conventional conditions, or conditions recommended by the manufacturer.

[0101] Preparation of corn amylopectin solution: Add 10g amylopectin and 1g NaOH to every 100mL distilled water, stir and heat until gelatinized and transparent, let it stand and cool for later use.

[0102] Example 1:

[0103] Preparation of aqueous phase: Under stirring conditions, add 7.5 mL of an aqueous solution containing 1.5 g of STMP (sodium trimetaphosphate) and 0.15 g of STPP (sodium tripolyphosphate) to 25 mL of the pullulan solution and stir for 5 minutes to mix thoroughly.

[0104] Oil phase preparation: Add 1 g Span-60 and 0.5 g Tween-60 to 100 mL cyclohexane under stirring to form the continuous oil phase;

[0105] Reverse phase polymerization: After stirring the continuous oil phase for a period of time, add the water phase to the oil phase, mix thoroughly, and stir. The reaction temperature is 50°C and the reaction is carried out for 3 hours. Then, stir at room temperature overnight and let it stand. Add a certain amount of ethanol, filter, wash with ethanol 3 times, and then dry. The resulting powder is starch microspheres. After redissolving in water and sieving, starch hydrogel microspheres of different particle sizes can be obtained.

[0106] Example 2:

[0107] The cyclohexane in Example 1 was replaced by n-nonane.

[0108] Example 3:

[0109] The cyclohexane in Example 1 was replaced by n-decane.

[0110] Example 4:

[0111] The cyclohexane in Example 1 was replaced by n-undecane.

[0112] Example 5:

[0113] The cyclohexane in Example 1 was replaced by n-dodecane.

[0114] Example 6:

[0115] The cyclohexane in Example 1 was replaced by n-tridecane.

[0116] Example 7:

[0117] Preparation of aqueous phase: Under stirring conditions, add 7.5 mL of an aqueous solution containing 1.5 g of STMP (sodium trimetaphosphate) and 0.15 g of STPP (sodium tripolyphosphate) to 25 mL of the pullulan solution, and stir for 5 minutes to mix thoroughly.

[0118] Oil phase preparation: Under stirring conditions, add 1g Span-60 and 0.5g Tween-60 to 100mL of mixed solvent (50mL of cyclohexane and 50mL of n-nonane) to form the continuous oil phase;

[0119] Reverse phase polymerization: After stirring the continuous oil phase for a period of time, add the water phase to the oil phase, mix thoroughly, and stir. The reaction temperature is 50°C and the reaction is carried out for 3 hours. Then, stir at room temperature overnight and let it stand. Add a certain amount of ethanol, filter, wash with ethanol 3 times, and then dry. The resulting powder is starch microspheres. After redissolving in water and sieving, starch hydrogel microspheres of different particle sizes can be obtained.

[0120] Example 8:

[0121] The n-nonane in Example 7 was replaced by n-decane.

[0122] Example 9:

[0123] The n-nonane in Example 7 was replaced by n-undecane.

[0124] Example 10:

[0125] The n-nonane in Example 7 was replaced by n-dodecane.

[0126] Example 11:

[0127] The n-nonane in Example 7 was replaced by n-tridecane.

[0128] The microspheres provided in Examples 1 to 11 were replaced by taking 100 mL of the 300-600 micron microspheres from each group after screening and adding 100 mL of normal saline. After the microspheres were allowed to settle and stratify, the supernatant was removed and 100 mL of normal saline was poured in again. The operation was repeated three or more times to obtain replaced microspheres with normal saline as the storage medium. The morphology of the microspheres was observed by taking photos under an optical microscope. The results are shown in Figures 2 to 12. As can be seen from Figures 2 to 12, each group of microspheres formed a good spherical shape with a good spherical appearance, wherein a small amount of fragments appeared in Examples 4 and 5.

[0129] The replaced microspheres provided in Examples 1 to 11 were subjected to mechanical property testing: the replaced microspheres of 300-600 μm were subjected to stress testing using a texture analyzer to obtain the corresponding strength and elasticity. The results are shown in Table 1 below.

[0130] Table 1

[0131] As can be seen from the above table, the microspheres prepared by the method provided in the examples of the present application have higher strength, and according to the comparison between Examples 1-6 and Examples 7-11, it can be seen that the strength of the microspheres is increased from 0.602g to 14.301g by the specific selection of the organic solvent in the present application.

[0132] The replaced microspheres provided in Examples 1 to 11 were sterilized: the replaced 300-600 μm microspheres were sterilized by high-pressure moist heat sterilization (120°C, 30 minutes), and the sterilized microspheres were removed and imaged using an optical microscope to evaluate the stability of the replaced microspheres after sterilization. The results are shown in Figures 13 to 23. As can be seen from the figures, the microspheres in the groups of Examples 1-6 produced obvious fragments after high-pressure moist heat sterilization. In mechanical testing, it was also found that the microspheres of Examples 1-6 had lower strength. This shows that the lower strength makes these microspheres less stable under moist heat sterilization conditions, which is not conducive to the application of embolization technology. In contrast, Examples 7-11 have higher strength and can maintain a good morphology after moist heat sterilization.

[0133] The microspheres provided by the groups without debris after sterilization (Examples 7 to 11) were subjected to a microsphere suspension test: 2 mL of replaced 300-600 μm microspheres (only for the group without debris after sterilization) were taken, 2 mL of contrast agent (iohexol 300) was added, and after mixing evenly to a stable state, a 20 mL syringe was used for vertical tube timing to observe the suspension state of the microspheres.

[0134] Next, a catheter passability test was conducted: the syringe containing the contrast agent-microsphere suspension was connected to a 2.7F microcatheter. The end of the microcatheter was placed in a centrifuge tube, and then a pulsed injection was performed at a rate of 1 mL / 1 min. The microspheres that passed through the catheter were collected and imaged using an optical microscope to evaluate the stability of the displaced microspheres after catheter injection. The results are shown in Figures 24 to 28. As can be seen from the figures, in the Example 7-11 group, Examples 10-11 produced obvious fragments after suspension and catheter passage, while Example 7-9 had a better morphology and was more suitable for embolization applications.

[0135] In summary, the morphological results of the microspheres provided by Examples 1 to 11 at various stages are shown in Table 2 below.

[0136] Table 2

[0137] From the above content, it can be seen that the present application has prepared starch hydrogel microspheres with excellent performance and high strength by designing the organic solvent.

[0138] In another specific embodiment, the present application provides a method for preparing starch hydrogel embolic microspheres, the preparation method comprising:

[0139] S1: preparing the aqueous phase: mixing pullulan, a cross-linking agent and water to obtain the aqueous phase;

[0140] S2: oil phase preparation: mixing the dispersant and the organic solvent to obtain the oily phase;

[0141] S3: reverse phase polymerization: mixing the aqueous phase and the oil phase to form a reverse phase polymerization system, and performing polymerization to obtain the starch hydrogel microspheres;

[0142] Wherein, the volume ratio of the oil phase to the water phase is (3-5):1;

[0143] S4. Terminate the reverse polymerization reaction, filter, wash and sieve to obtain starch hydrogel embolic microspheres.

[0144] At the same time, the present application also provides a starch hydrogel embolic microsphere, which is prepared by the preparation method provided above.

[0145] Some specific embodiments are listed below to better illustrate the present application.

[0146] Extraction of corn amylopectin:

[0147] Accurately weigh 100g of corn starch, add 3000mL of DMSO, and stir at 200rpm at room temperature for 24 hours. After stirring, centrifuge at 2000rpm, take the supernatant and add it to twice the amount of n-butanol. After standing for 24 hours, centrifuge at 4000rpm. Wash the precipitate three times with n-butanol to remove the DMSO and filter. Boil and dissolve the filter cake with sufficient distilled water, cool it to 60°C, add 10g of thymol and stir thoroughly, let it stand at room temperature for 3 days, and filter to obtain the filtrate. Precool the filtrate in a 4°C refrigerator, then add an appropriate amount of ether to extract it three times to remove the thymol. Take the aqueous phase and add a large amount of ethanol until the flocculent precipitate no longer increases. Let it stand at room temperature for 3 days, filter the filter cake, and dry it to obtain a white powder, which is the extracted corn amylopectin.

[0148] Preparation of corn amylopectin solution:

[0149] Add 10 g corn amylopectin and 1 g NaOH to 100 mL of distilled water, stir and heat until gelatinized and transparent, then let it stand and cool for later use.

[0150] Preparation of corn starch solution:

[0151] Add 10 g corn starch and 1 g NaOH to 100 mL distilled water, stir and heat until gelatinized and transparent, then let it stand and cool for later use.

[0152] The following examples use the corn amylopectin prepared above to prepare starch hydrogel embolic microspheres.

[0153] Example 12

[0154] Under stirring, 1g Span-60 and 0.5g Tween 60 were added to 100mL of cyclohexane to form the continuous oil phase. After stirring for a period of time, 25mL of the previously prepared corn amylopectin solution, 0.3g STMP, and 0.03g STPP were added to the oil phase in sequence. After thorough mixing, the mixture was stirred and reacted at 50°C for 3 hours, then stirred at room temperature overnight and allowed to stand. A certain amount of ethanol was added, filtered, washed three times with ethanol, and dried. The resulting powder was the corn amylopectin microspheres. After re-dissolving in water and sieving, corn amylopectin hydrogel microspheres of different particle sizes were obtained.

[0155] Example 13

[0156] Preparation of aqueous phase: Add 12.5 mL of an aqueous solution containing 0.3 g of STMP to 12.5 mL of corn amylopectin solution under stirring, and stir for 5 minutes to mix thoroughly.

[0157] Oil phase preparation: Add 1 g Span-60 and 0.5 g Tween 60 to 100 mL cyclohexane under stirring to form the continuous oil phase;

[0158] Reverse phase polymerization: After stirring the continuous oil phase for a period of time, add 25 mL of water phase to the oil phase, mix thoroughly, stir, and react at 50°C for 3 hours, then stir at room temperature overnight and let it stand; add a certain amount of ethanol, filter, wash with ethanol 3 times and then dry. The resulting powder is corn amylopectin microspheres. After re-dissolving in water and sieving, corn amylopectin hydrogel microspheres of different particle sizes can be obtained.

[0159] Example 14

[0160] Preparation of aqueous phase: Add 12.5 mL of an aqueous solution containing 0.3 g of STMP and 0.003 g of STPP to 12.5 mL of corn amylopectin solution under stirring, and stir for 5 minutes to mix thoroughly.

[0161] Oil phase preparation: Add 1 g Span-60 and 0.5 g Tween 60 to 100 mL cyclohexane under stirring to form the continuous oil phase;

[0162] Reverse phase polymerization: After stirring the continuous oil phase for a period of time, add 25 mL of water phase to the oil phase, mix thoroughly, stir, and react at 50°C for 3 hours, then stir at room temperature overnight without letting it stand; add a certain amount of ethanol, filter, wash with ethanol three times, and then dry. The resulting powder is corn amylopectin microspheres. After redissolving in water and sieving, corn amylopectin hydrogel microspheres of different particle sizes can be obtained.

[0163] Example 15

[0164] Preparation of aqueous phase: Add 12.5 mL of an aqueous solution containing 0.3 g of STMP and 0.015 g of STPP to 12.5 mL of corn amylopectin solution under stirring, and stir for 5 minutes to mix thoroughly.

[0165] Oil phase preparation: Add 5 g of Span-60 and 0.5 g of Tween 60 to 100 mL of cyclohexane under stirring to form the continuous oil phase;

[0166] Reverse phase polymerization: After stirring the continuous oil phase for a period of time, add 25 mL of water phase to the oil phase, mix thoroughly, stir, and react at 50°C for 24 hours, then stir at room temperature overnight without letting it stand; add a certain amount of ethanol, filter, wash with ethanol three times, and then dry. The resulting powder is corn amylopectin microspheres. After redissolving in water and sieving, corn amylopectin hydrogel microspheres of different particle sizes can be obtained.

[0167] Example 16

[0168] Preparation of aqueous phase: Add 12.5 mL of an aqueous solution containing 0.3 g of STMP and 0.03 g of STPP to 12.5 mL of corn amylopectin solution under stirring, and stir for 5 minutes to mix thoroughly.

[0169] Oil phase preparation: Add 5 g of Span-60 and 1 g of Tween 60 to 100 mL of cyclohexane under stirring to form the continuous oil phase;

[0170] Reverse phase polymerization: After stirring the continuous oil phase for a period of time, 25 mL of water phase was added to the oil phase, mixed thoroughly, stirred, and reacted at 50°C for 24 hours, and then stirred at room temperature overnight; stirring was performed throughout the process without standing, a certain amount of ethanol was added, filtered, washed with ethanol three times, and then dried. The resulting powder was corn amylopectin microspheres, which were re-dissolved in water and sieved to obtain corn amylopectin hydrogel microspheres of different particle sizes.

[0171] Example 17

[0172] Preparation of aqueous phase: Add 12.5 mL of an aqueous solution containing 0.6 g of STMP and 0.06 g of STPP to 12.5 mL of corn amylopectin solution under stirring, and stir for 5 minutes to mix thoroughly.

[0173] Oil phase preparation: Add 1 g Span-60 and 0.5 g Tween 60 to 100 mL cyclohexane under stirring to form the continuous oil phase;

[0174] Reverse phase polymerization: After stirring the continuous oil phase for a period of time, add 20 mL of water phase to the oil phase, mix thoroughly, stir, and react at 50°C for 24 hours, then stir at room temperature overnight without letting it stand; add a certain amount of ethanol, filter, wash with ethanol three times, and then dry. The resulting powder is corn amylopectin microspheres. After redissolving in water and sieving, corn amylopectin hydrogel microspheres of different particle sizes can be obtained.

[0175] Example 18

[0176] Preparation of aqueous phase: Add 12.5 mL of an aqueous solution containing 0.6 g of STMP and 0.06 g of STPP to 12.5 mL of corn amylopectin solution under stirring, and stir for 5 minutes to mix thoroughly.

[0177] Oil phase preparation: Add 2 g Span-60 and 1 g Tween 60 to 75 mL cyclohexane under stirring to form the continuous oil phase;

[0178] Reverse phase polymerization: After stirring the continuous oil phase for a period of time, add 25 mL of water phase to the oil phase, mix thoroughly, stir, and react at 50°C for 24 hours, then stir at room temperature overnight without letting it stand; add a certain amount of ethanol, filter, wash with ethanol three times, and then dry. The resulting powder is corn amylopectin microspheres. After redissolving in water and sieving, corn amylopectin hydrogel microspheres of different particle sizes can be obtained.

[0179] Example 19

[0180] Preparation of aqueous phase: Add 12.5 mL of an aqueous solution containing 0.6 g of STMP and 0.06 g of STPP to 12.5 mL of corn amylopectin solution under stirring, and stir for 5 minutes to mix thoroughly.

[0181] Oil phase preparation: Add 1 g Span-60 and 0.5 g Tween 60 to 100 mL cyclohexane under stirring to form the continuous oil phase;

[0182] Reverse phase polymerization: After stirring the continuous oil phase for a period of time, 25 mL of water phase was added to the oil phase, mixed thoroughly, stirred, and reacted at 70°C for 24 hours, then stirred at room temperature overnight without standing; a certain amount of ethanol was added, filtered, and washed with ethanol three times and then dried. The resulting powder was corn amylopectin microspheres. After redissolving in water and sieving, corn amylopectin hydrogel microspheres of different particle sizes could be obtained.

[0183] Comparative Example 1

[0184] This comparative example provides a method for preparing starch hydrogel embolic microspheres, comprising the following steps:

[0185] Under stirring conditions, 1g Span-60 and 0.5g Tween 60 were added to 100mL cyclohexane to form the continuous oil phase; after stirring for a period of time, 25mL corn starch solution, 0.3g STMP and 0.03g STPP were added to the oil phase in sequence, and after thorough mixing, stirred, reacted at 50°C for 3 hours, then stirred at room temperature overnight and allowed to stand; a certain amount of ethanol was added, filtered, washed with ethanol three times and then dried. The resulting powder was corn starch microspheres, which were re-dissolved in water and sieved to obtain corn starch hydrogel microspheres of different particle sizes.

[0186] Comparative Example 2

[0187] Preparation of aqueous phase: Add 12.5 mL of an aqueous solution containing 0.3 g of STMP and 0.03 g of STPP to 12.5 mL of corn starch solution under stirring, and stir for 5 minutes to mix thoroughly.

[0188] Oil phase preparation: Add 1 g Span-60 and 0.5 g Tween 60 to 100 mL cyclohexane under stirring to form the continuous oil phase;

[0189] Reverse phase polymerization: After stirring the continuous oil phase for a period of time, add 25 mL of water phase to the oil phase, mix thoroughly, stir, react at 50°C for 3 hours, then stir at room temperature overnight and let stand; add a certain amount of ethanol, filter, wash with ethanol 3 times and then dry. The resulting powder is corn starch microspheres. After redissolving in water and sieving, corn starch hydrogel microspheres of different particle sizes can be obtained.

[0190] The properties of the corn starch hydrogel microspheres prepared in Examples 12-19 and Comparative Examples 1-2 were tested.

[0191] Experimental Example 1 Microsphere Suspension Test

[0192] 2 mL of 300-600 μm hydrogel starch microspheres were added to 2 mL of contrast agent (iohexol 300). After mixing until a stable state was achieved, a 20 mL syringe was used for vertical tube timing to observe the suspension state of the microspheres. The results are shown in Table 3.

[0193] Table 3

[0194] Experimental Example 2 Catheter passability test

[0195] The syringe containing the contrast agent-microsphere suspension was connected to a 2.7F microcatheter. The distal end of the microcatheter was placed in a centrifuge tube. A pulsed injection was then performed at a rate of 1 mL / min. During the injection, the microspheres were observed for dispersion, adhesion, clumping, wall adhesion, and tube clogging as they entered the microcatheter and exited the microcatheter into the centrifuge tube. The results are shown in Table 4.

[0196] Table 4

[0197] Experimental Example 3: Microsphere Degradability Test:

[0198] 1 mL of 300-600 μm microspheres was added to 9 mL of artificial plasma and incubated on a shaker at 37°C. α-amylase was then added to a concentration of 100 U / L. Every 24 hours, 0.5 mL of the mixture was diluted to 1 mL. Particle size analysis was performed using dynamic light scattering (DLS). The time it took for the average particle size to drop to half of its initial value was used as an indicator of microsphere degradation. The test results are shown in Table 5.

[0199] Table 5

[0200] Experimental Example 4 Microsphere Stability Test

[0201] Take 1 mL of 300-600 μm microspheres and 9 mL of normal saline, add them to a vial, seal it, and store it in a refrigerator at room temperature or 4°C, respectively. Take samples every 5 days to observe the morphology and particle size.

[0202] The results of the room temperature stability test are shown in Table 6, and the results of the 4°C refrigerator storage stability test are shown in Table 7.

[0203] Table 6

[0204] Table 7

[0205] The summary results of the morphology, agglomeration, yield, suspension, catheter passability, in vitro enzymatic hydrolysis time, room temperature stability and 4°C stability of each example are shown in Table 8.

[0206] In Table 8, (1) the morphology and agglomeration of the starch microspheres prepared in Examples 12 to 19 were detected by optical microscopy, as shown in Figures 31 to 38 of the specification; (2) the yield was the yield of the starch microspheres prepared in each example; (3) the suspension was based on the suspension test results of the microspheres in Experimental Example 1; (4) the duct passability was based on the duct passability test results of Experimental Example 2; (5) the in vitro enzymatic hydrolysis time was based on the degradability test results of the microspheres in Experimental Example 3; and (6) the stability at room temperature and at 4°C was based on the stability test results of the microspheres in Experimental Example 4.

[0207] Table 8

[0208] It can be seen from the results in Table 8 above that the yield of the starch hydrogel embolic microspheres prepared in the examples of the present application is improved, and the suspension, catheter permeability, in vitro enzymatic hydrolysis time, room temperature stability and 4°C stability of the starch hydrogel embolic microspheres are all improved. Therefore, it can be concluded that the comprehensive performance of the starch hydrogel embolic microspheres provided in the present application is improved.

[0209] Furthermore, according to Figures 29 and 30 of the specification, it can be seen that Comparative Examples 1 and 2 did not exhibit a rounded morphology, retained the morphological characteristics of the original starch particles, and failed to produce deep crosslinking. Example 13 obtained relatively round microspheres by premixing the corn amylopectin solution with a crosslinking agent, but problems such as fragmentation and adhesion occurred. Examples 14-19 further improved the experimental parameters, increased the reaction heating time, and eliminated the reaction standing process, achieving the preparation of round, smooth, highly absorbent, and highly translucent corn amylopectin hydrogel microspheres.

[0210] Furthermore, as shown in Figures 31-38, the starch hydrogel microspheres prepared in Examples 12-19 have a particle size distribution covering the range of 20-2000 microns, and have good elasticity and can be compressed by more than 50% under external force without deformation (as shown in Figures 39-40).

[0211] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

Claims

1. A method for preparing starch hydrogel microspheres, comprising the following steps: Preparation of aqueous phase: mixing pullulan, a cross-linking agent and water to obtain the aqueous phase; Preparation of oil phase: mixing a dispersant and an organic solvent to obtain the oil phase; Reverse phase polymerization: the water phase and the oil phase are mixed to form a reverse phase polymerization system, and polymerization is performed to obtain the starch hydrogel microspheres.

2. The method for preparing starch hydrogel microspheres according to claim 1, wherein The cross-linking agent is selected from at least one of sodium trimetaphosphate and sodium tripolyphosphate.

3. The method for preparing starch hydrogel microspheres according to claim 1, wherein The cross-linking agent is sodium trimetaphosphate and sodium tripolyphosphate; Optionally, the cross-linking agent is sodium trimetaphosphate and sodium tripolyphosphate, wherein the mass ratio of the sodium trimetaphosphate to sodium tripolyphosphate is (10-100):

1.

4. The method for preparing starch hydrogel microspheres according to any one of claims 1 to 3, wherein: The aqueous phase also includes an alkaline solution; Optionally, the alkaline solution comprises a sodium hydroxide solution; Optionally, the mass ratio of the amylopectin to the alkaline solution is (8-10):

1.

5. The method for preparing starch hydrogel microspheres according to claim 1, wherein The dispersant is selected from a combination of sorbitan monostearate and polyoxyethylene sorbitan monostearate; Optionally, the dispersant is selected from a combination of sorbitan monostearate and polyoxyethylene sorbitan monostearate, and the mass ratio of the dispersant selected from sorbitan monostearate and polyoxyethylene sorbitan monostearate is (2-10):

1.

6. The method for preparing starch hydrogel microspheres according to claim 1, wherein The organic solvent includes at least one of C4-C15 alkanes; Optionally, the organic solvent is selected from at least one of cyclohexane, n-nonane, n-decane, n-undecane, n-dodecane or n-tridecane.

7. The method for preparing starch hydrogel microspheres according to claim 6, wherein: The organic solvent comprises at least two of C5-C11 alkanes, and the organic solvent is selected from a combination of a first organic solvent and a second organic solvent; The first organic solvent is selected from cyclohexane; The second organic solvent is selected from at least one of n-nonane, n-decane, n-undecane, n-dodecane, and n-tridecane; Optionally, the organic solvent is selected from a combination of a first organic solvent and a second organic solvent, and a volume ratio of the first organic solvent to the second organic solvent is (0.5-1.5): (0.5-1.5).

8. The method for preparing starch hydrogel microspheres according to claim 1, wherein The volume ratio of the oil phase to the water phase is (3-5):1; Optionally, the volume ratio of the oil phase to the water phase is 100:32.

5.

9. The method for preparing starch hydrogel microspheres according to claim 1, wherein The polymerization temperature is 25-70°C; Optionally, the polymerization method specifically comprises the following steps: At 25-70°C, add the water phase to the oil phase, stir and react for at least 3 hours, then cool naturally and stir; Optionally, the natural cooling and stirring comprises: Stir continuously for at least 12 hours.

10. The method for preparing starch hydrogel microspheres according to claim 1, wherein: The polymerization further includes a post-treatment step, wherein the post-treatment method includes: Add water or ethanol to the polymerization reaction system to terminate the reaction, filter, wash and sieve.

11. A starch hydrogel microsphere prepared according to the preparation method according to any one of claims 1 to 10.

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