Modified carrageenan / gelatin double network structure composite microspheres capable of loading hydrophobic drugs, and preparation method and application thereof

Abstract

The application discloses modified carrageenan / gelatin double-network structure composite microspheres capable of loading hydrophobic drugs and a preparation method and application thereof. The composite microspheres are formed by crosslinking of acid anhydride esterified carrageenan and gelatin. The acid anhydride esterified carrageenan is formed by esterification reaction of high-concentration carrageenan and acid anhydride, and the substitution degree of the acid anhydride esterified carrageenan is controlled to be 10% to 20%. The substitution degree of the esterification reaction is controlled by controlling process parameters, the impurity content is reduced, the ability of the microspheres to load hydrophobic drugs is endowed, the double-network structure microspheres are prepared by a double-crosslinking technology, drug leakage can be reduced, drug release can be delayed, and the double-network structure microspheres can be applied to the fields of interventional embolization, medical beauty filling, tissue defect repair and drug carriers.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical polymer materials, and particularly relates to modified carrageenan / gelatin double-network structure composite microspheres capable of loading hydrophobic drugs and a preparation method and application thereof. BACKGROUND

[0002] Hydrogel microspheres are a kind of microspheres composed of three-dimensional network structure gels which are extremely hydrophilic, can rapidly swell in water and can keep a large volume of water without dissolving in the swollen state. Any hydrophilic drug can be dissolved and then enter the interior of the microspheres through swelling or capillary phenomenon to realize the loading of water-soluble drugs, such as N-acryloyl-aminoacetaldehyde-dimethyl acetal modified polyvinyl alcohol microspheres, polyacrylic acid sodium vinyl alcohol copolymer microspheres generated by polymerization saponification reaction of vinyl acetate and methyl acrylate, and polyvinyl alcohol microspheres modified with sulfonic acid groups, etc., which can all load hydrophilic bleomycin drugs.

[0003] Hydrophobic drugs are a kind of drugs which are insoluble or slightly soluble in water. Most of the existing compounds are poorly soluble drugs, which need to be first loaded into water-based micro-nano carriers such as microemulsions, liposomes, micelles, etc., and then the micro-nano carriers containing hydrophobic drugs enter the interior of the microspheres through swelling or capillary phenomenon, so as to realize the drug loading of hydrogel microspheres. In this process, there are many problems in the preparation, quality control and drug loading of micro-nano carriers in terms of industrial production implementation. Therefore, how to simply, quickly and efficiently realize the direct loading of hydrophobic drugs by hydrogel microspheres is a difficult problem that researchers have not solved.

[0004] Carrageenan is a hydrophilic colloid composed of galactose and dehydrated galactose sulfate calcium, potassium, sodium and ammonium salt, which has good gel-forming property and mechanical property. Gelatin is a product obtained by incomplete acid hydrolysis, alkaline hydrolysis or enzymatic degradation and purification of collagen in animal skin, bone, tendon and ligament, or a mixture of different gelatin products, which has good biocompatibility and hydrophilicity. Both materials are commonly used materials for preparing hydrogel microspheres loaded with hydrophilic drugs.

[0005] CN 103990185B discloses a carrageenan and gelatin microsphere embolic agent and its preparation method, which mixes carrageenan and gelatin to form an aqueous phase, and then emulsifies and crosslinks the gelatin in an oil phase to obtain microspheres. The microspheres prepared by the method are only chemically crosslinked with a crosslinking agent in the oil phase, and the whole microspheres are still two hydrophilic polymer materials, carrageenan and gelatin, so they can only load hydrophilic drugs in the form of aqueous solution. CN113350295B discloses a preparation method of a carrier micro-particle loaded with a bioactive drug and its application. The method first prepares a methyl methacrylate anhydride modified carrageenan, and then forms a ball in a micro-fluidic electrospray device and performs photo-crosslinking to obtain a modified carrageenan microsphere of a single material. The method uses a low concentration of 1% carrageenan solution for modification, and the modification efficiency is low. The whole microsphere is dominated by hydrophilic properties, so it can only load water-soluble bioactive drugs in the form of aqueous solution, and no control measures for controlling drug leakage are involved. The above microspheres containing carrageenan do not involve the function of loading hydrophobic drugs. SUMMARY

[0006] The purpose of the application is to solve the technical problems of existing hydrogel microspheres that cannot directly load hydrophobic drugs and have drug leakage. The application discloses a modified carrageenan / gelatin double network structure composite microsphere that can load hydrophobic drugs. The double network structure microsphere of the application can reduce drug leakage and delay drug release, and can be used in the fields of interventional embolization, medical beauty filling, tissue defect repair and drug carriers.

[0007] The application also provides a preparation method and application of the modified carrageenan / gelatin double network structure composite microsphere that can load hydrophobic drugs.

[0008] Technical scheme: In order to achieve the above-mentioned purpose, the application discloses a modified carrageenan / gelatin double network structure composite microsphere that can load hydrophobic drugs. The composite microsphere is mainly formed by crosslinking acid anhydride esterified carrageenan and gelatin. The acid anhydride esterified carrageenan is mainly formed by esterification reaction of high-concentration carrageenan and acid anhydride, and the substitution degree of the acid anhydride esterified carrageenan is controlled to be 10%-20%.

[0009] The concentration of the high-concentration carrageenan is 2.0wt%-5.0wt%, and the mass ratio of acid anhydride to kappa-carrageenan is 1:2-2:1.

[0010] The acid anhydride is one or more of 2-methacrylic anhydride, acrylic anhydride and 2-methylene succinic anhydride (itaconic anhydride).

[0011] The mass ratio of the acid anhydride esterified carrageenan to gelatin is 2:5-3:2.

[0012] The modified carrageenan / gelatin double-network structure composite microspheres capable of loading hydrophobic drugs are prepared by the following steps:

[0013] (1) adjusting the high-concentration κ-carrageenan aqueous solution to pH 8.0-8.6 with a base, slowly adding an acid anhydride, in the process, adjusting and maintaining the pH at 6.0-8.6 with the base, after the addition of the acid anhydride, continuing to adjust and maintain the pH at 8.0-8.6 with the base for reaction, after the reaction, purifying, washing, drying, and obtaining the acid anhydride esterified carrageenan;

[0014] (2) mixing the obtained acid anhydride esterified carrageenan with gelatin, performing primary crosslinking in an emulsification system to obtain primary crosslinking microspheres of acid anhydride esterified carrageenan / gelatin single-network structure composite microspheres, and performing secondary crosslinking in an organic solution system to obtain secondary crosslinking microspheres of acid anhydride esterified carrageenan / gelatin double-network structure composite microspheres.

[0015] In step (1), the high-concentration κ-carrageenan aqueous solution is adjusted to pH 8.0-8.6 with a base under stirring at 45-70 ℃, a certain amount of acid anhydride is slowly added, in the process, the pH is adjusted and maintained at 6.0-8.6 with the base, then the pH is continuously adjusted and maintained at 8.0-8.6 with the base, and after the reaction at 45-70 ℃ for 4-12 hours, the product is purified, washed, dried, and the acid anhydride esterified carrageenan is obtained.

[0016] As a preferred embodiment, the base is one or more of sodium hydroxide, potassium hydroxide, and sodium carbonate, and the concentration of the base used for adjusting the pH is 1.0 M-10.0 M; and the pH value is maintained at 6.0-7.0 after the first adjustment after the addition of the acid anhydride.

[0017] As a preferred embodiment, the purification step comprises precipitating or dialyzing the reaction solution with an organic solvent after the pH is stabilized, filtering after standing, and repeatedly washing the precipitate with an organic solvent or an organic solution mixed with water, such as 95% ethanol, anhydrous ethanol, isopropanol, etc. The post-treatment, washing, and drying can be performed in some ways reported in the prior art, such as vacuum drying, freeze drying, pressure filtration drying, or heating drying, and crushing, etc.

[0018] The acid anhydride esterified carrageenan prepared in the above steps can control the degree of substitution of the modified carrageenan within a certain range, provide suitable crosslinking sites for the subsequent primary crosslinking to form single-network microspheres, and maintain the hydrophilic and hydrophobic properties of the whole microspheres within a certain range, so that the microspheres can retain certain hydrophilicity and have good ball-forming properties in the preparation process, and can also be endowed with certain hydrophobicity to successfully load hydrophobic drugs.

[0019] The step (2) is the first crosslinking, which is mixing and dispersing anhydride esterified carrageenan, gelatin, initiator and water to obtain an aqueous phase; adding the aqueous phase into an oil phase containing an emulsifier, sufficiently emulsifying, then adding a catalyst, and performing the first crosslinking at 50-80 DEG C for 1-6 h, then cooling and solidifying after crosslinking, removing the oil phase, washing and drying to obtain the first crosslinking microspheres; wherein the mass ratio of the anhydride esterified carrageenan to the gelatin is 2:5-3:2; the mixed gelatin obtained by mixing the anhydride esterified carrageenan and the gelatin has a concentration of 8.0wt%-14.0wt% in the aqueous phase.

[0020] The initiator is one or more of persulfate, azobisdimethylamid hydrochloride, and the concentration of the initiator in the aqueous phase is 0.02wt%-0.2wt%; the catalyst is one or more of sodium bisulfite and tetramethyl ethylenediamine, and the concentration of the catalyst in the emulsification system is 0.003wt%-0.03wt%.

[0021] Preferably, the initiator is potassium persulfate, and the catalyst is sodium bisulfite.

[0022] Further, the emulsifier is one or more of a Span or a mixture of Span and Tween, and preferably is Span 80; the oil phase is one or more of liquid paraffin, white vaseline, cetyl alcohol and glycerol monostearate; the volume ratio of the oil phase to the emulsifier is 400:1-200:1; and the volume ratio of the aqueous phase to the oil phase is 1:6-1:3.

[0023] The step (2) is the second crosslinking, which is performing the second crosslinking of the first crosslinking microspheres in an organic solution containing a crosslinking agent at 20-50 DEG C, removing the crosslinking agent, washing and drying to obtain the second crosslinking microspheres; wherein the proportion of the first crosslinking microspheres in the organic solution is 5.0wt%-15.0wt%; the crosslinking agent is one or more of glutaraldehyde, genipin and a carbodiimide crosslinking agent; the organic solution is a uniform system mixed by one or more of methanol, ethanol, isopropanol and water; and the proportion of the crosslinking agent in the organic solution is 5.0wt%-12.0wt%.

[0024] Preferably, the crosslinking agent is glutaraldehyde.

[0025] The modified carrageenan / gelatin double network structure composite microspheres of the application can be used for loading hydrophobic drugs and can be applied to interventional embolization, medical beauty filling, tissue defect repair and drug carriers.

[0026] The present application adopts the way of primary cross-linking microspheres in emulsion system, by controlling the process parameters such as the ratio of initiator, catalyst and anhydride esterified carrageenan, gelatin, reaction temperature and time, etc., the anhydride esterified carrageenan in the microspheres is primary cross-linked, a single network structure is formed in the microspheres, and the leakage of hydrophobic drugs is preliminarily controlled, and the release is prolonged. In the above primary cross-linking step, the type, ratio, shear emulsification rate of the emulsifier reported in the prior art can be selected according to the particle size requirement of the microspheres. The oil phase can be removed by some ways reported in the prior art, such as centrifugation, washing, drying, etc.

[0027] The present application adopts the way of secondary cross-linking in organic solution, the type of cross-linking agent, the concentration, temperature and time of reaction, etc., on the basis of the single network structure formed by primary cross-linking, the secondary cross-linking is carried out, and the leakage of hydrophobic drugs is controlled by double network structure, and the release is prolonged. In the prescription and process of secondary cross-linking, the cross-linking agent can be removed by some ways reported in the prior art, such as one or more of amino acids, (hydro) sulfite, etc., removing formaldehyde, washing; the washing can be carried out by some ways reported in the prior art, such as repeatedly washing with organic solvents, water or a mixture of the two in a certain proportion; the drying can be carried out by some ways reported in the prior art, such as vacuum drying, freeze drying or heating drying, etc.

[0028] The present application adopts the way of esterification reaction of high concentration carrageenan aqueous solution in gradient change pH environment, the carrageenan aqueous solution with a concentration of 1.0wt% is used for reaction in the prior art, because the carrageenan forms a certain viscosity hydrogel in water, it is generally considered that the high concentration viscous solution is not conducive to chemical reaction and operation difficulty, etc. However, the present application researches and finds that the high concentration carrageenan with a concentration of 2.0wt%-5.0wt% not only does not appear the operation difficulty under the specific preparation conditions, but also can improve the collision probability between reactants, greatly improve the esterification efficiency. However, when the concentration is greater than 5.0wt%, the reaction solution is too viscous, the molecular movement is limited, and the reaction efficiency is greatly reduced. And the polymerization of anhydride monomer generally adds a polymerization inhibitor to prevent the self-polymerization of monomer, which is different from the prior art which always maintains pH8.0, the present application controls the gradient change of pH value, which can not only ensure the degradation of carrageenan, but also reduce the salt impurities generated by phenol methoxy phenol in alkali, greatly improve the purity of modified carrageenan, and reduce the content of by-products of modified carrageenan in subsequent cross-linking reaction.

[0029] The preparation method of the present application controls the degree of substitution of carrageenan modification within a certain range (10%-20%) by controlling process parameters, which not only greatly reduces the impurity content, but also endows the microspheres with the ability to load hydrophobic drugs. In addition, the double-network structure microspheres prepared by the double-crosslinking technology can reduce drug leakage and delay drug release, and can be used in the fields of interventional embolization, medical aesthetic filling, tissue defect repair and drug carriers. Since the chemical structure, physical solubility and drug release behavior of hydrophobic drugs are completely different from those of hydrophilic drugs, the microsphere preparation method of the present application cannot be used as a reference for the process of loading water-soluble drug microspheres.

[0030] Advantages: Compared with the prior art, the present application has the following advantages:

[0031] 1. The present application uses the esterification reaction of carrageenan aqueous solution at high concentration, and the high concentration of carrageenan aqueous solution within a certain range can greatly improve the esterification efficiency and provide sufficient loading sites for subsequent loading of hydrophobic drugs.

[0032] 2. The present application uses the esterification reaction of carrageenan aqueous solution in a gradient change pH environment, which can not only ensure that the carrageenan is not degraded, but also reduce the generation of salt impurities of the polymerization inhibitor when it meets alkali, greatly improve the purity of the modified carrageenan, and reduce the content of by-products of the modified carrageenan in subsequent crosslinking reaction.

[0033] 3. The present application controls the degree of substitution of modified carrageenan within a certain range, which provides suitable crosslinking sites for the formation of single-network microspheres in subsequent primary crosslinking, and maintains the hydrophilic and hydrophobic properties of the overall microspheres within a certain range, which can not only retain a certain hydrophilicity of the microspheres to improve the ball forming property during preparation, but also endow the microspheres with a certain hydrophobicity to successfully load hydrophobic drugs.

[0034] 4. The present application uses double-crosslinked microspheres, which first perform primary crosslinking on the anhydride esterification carrageenan in the microspheres to form a single-network structure, and then perform secondary crosslinking on the gelatin to form a double-network structure, which controls the leakage and prolongs the release of hydrophobic drugs through the double-network structure.

[0035] 5. The preparation method of the present application is simple, the raw materials are easy to obtain, and the double-network structure microspheres are prepared, which has high drug loading rate, can reduce drug leakage and delay drug release, and can be used in the fields of interventional embolization, medical aesthetic filling, tissue defect repair and drug carriers. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 The microscope photos of the microspheres prepared in Example 5 and Comparative Example 4 are shown, with the left photo being Example 5 and the right photo being Comparative Example 4. DETAILED DESCRIPTION

[0037] The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially. The experimental methods in the examples, unless otherwise specified, are generally carried out according to conventional conditions, or according to the conditions recommended by the manufacturer.

[0038] Example 1

[0039] (1) 200 mL of a 2.0 wt% aqueous solution of K-carrageenan was adjusted to pH 8.0 at 45°C with stirring using 1.0 M sodium hydroxide, 2.0 g of 2-methyl acrylic anhydride was slowly added, the mixture was stirred until uniform, 1.0 M sodium hydroxide was added to adjust the pH to 6.0 while adding the anhydride, then the pH was continuously adjusted to 8.0 using the base, and the reaction was continued at 45°C for 4 hours. After the reaction, 5 times the volume of absolute ethanol was added to precipitate and remove impurities, after standing, the mixture was filtered, the solid portion was repeatedly washed with 95% ethanol, and then absolute ethanol was used to remove excess water, the residue was dried at 50°C for 24 hours to remove residual ethanol and water, and the anhydride-esterified carrageenan was obtained.

[0040] (2) 2.0 g of the anhydride-esterified carrageenan and 5.0 g of gelatin were weighed out, mixed with 90 mL of water, and heated and stirred at 50°C. After 2 mL of an aqueous solution containing 0.02 g of the initiator potassium persulfate was added, an aqueous phase was prepared. 540 mL of liquid paraffin was mixed with 1.4 mL of Span 80, heated and stirred at 50°C until uniform, and an oil phase was obtained. At 50°C, the aqueous phase was slowly added to the oil phase, emulsified at a mechanical stirring rate of 200 rpm for 30 min, 1 mL of a 16% sodium bisulfite solution was added to catalyze, and the reaction was carried out at room temperature for 1 hour. After cooling and solidifying in an ice bath for 1 hour with stirring, the surface liquid paraffin of the microspheres was repeatedly washed and filtered using an appropriate amount of petroleum ether, and then an appropriate amount of absolute ethanol was used to remove residual organic solvents and water from the surface of the microspheres. After drying, the first crosslinked microspheres were obtained.

[0041] (3) 2.5 g of the first crosslinked microspheres were weighed out, 50 mL of a 80% methanol solution and 2.5 g of glutaraldehyde were added, and the mixture was mixed and subjected to secondary crosslinking at 25°C for 6 h. After the microspheres were washed and filtered using an appropriate amount of 80% methanol, 50 mL of a 80% methanol solution and 0.8 g of glycine were added to remove aldehyde for 16 h. After the microspheres were washed and filtered using an appropriate amount of 95% methanol to remove water, the modified carrageenan / gelatin double network structure composite microspheres were obtained by vacuum drying for 12 h.

[0042] Example 2

[0043] Example 2 was the same as the method of Example 1, except that 200 mL of a 5.0 wt% aqueous solution of K-carrageenan was used, and 5.0 g of 2-methyl acrylic anhydride was slowly added.

[0044] Example 3

[0045] Example 3 was the same as Example 1 except that 200 mL of a 3.0 wt% aqueous kappa-carrageenan solution was used and 3.0 g of 2-methacrylic anhydride was added slowly.

[0046] Example 4

[0047] (1) 200 ml of a 5.0 wt% aqueous kappa-carrageenan solution was adjusted to pH 8.6 with 6.0 M sodium carbonate at 70°C with stirring, 20.0 g of acrylic anhydride was added slowly, the mixture was stirred until uniform, 6.0 M sodium carbonate was added slowly to adjust the pH to 7.0 while adding the anhydride, then the pH was adjusted and maintained at 8.6 with the base, the reaction was continued at 70°C for 12 hours, 3 volumes of absolute ethanol was added to the entire system to precipitate and remove impurities, after standing, the solid was filtered, the solid was repeatedly washed with 95% ethanol, then absolute ethanol was used to remove excess water, the solid was dried at 70°C for 6 hours to remove residual ethanol and water, and the anhydride-esterified carrageenan was obtained.

[0048] (2) 3.0 g of the anhydride-esterified carrageenan and 2.0 g of gelatin were weighed out and mixed with 35 ml of water, heated and stirred at 70°C, 1 mL of a 0.07 g solution containing an initiator, azobisdimethylaminoformamide hydrochloride, was added to prepare the aqueous phase; 100 ml of white vaseline was mixed with a 5:1 mixture of 0.5 ml of Span 80 and Tween 80 solution, heated and stirred uniformly at 70°C to obtain the oil phase. The aqueous phase was slowly added to the oil phase at 70°C, emulsified at a mechanical stirring rate of 500 rpm for 2 hours, 1 ml of a 16% tetramethylethylenediamine solution was added for catalysis, the reaction was continued for 6 hours, the mixture was cooled and solidified in an ice bath for 3 hours with stirring, then an appropriate amount of isopropyl alcohol was added to repeatedly wash the surface of the microspheres, centrifuged to remove the white vaseline on the surface of the microspheres, then an appropriate amount of absolute ethanol was used to remove residual organic solvents and water on the surface of the microspheres, centrifuged and dried to obtain the primary crosslinked microspheres.

[0049] (3) 2.5 g of the primary crosslinked microspheres were weighed out, 17 ml of a 90% isopropyl alcohol solution and 2.0 g of a carbodiimide crosslinking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, were added, mixed, and subjected to secondary crosslinking at 40°C for 72 hours. The microspheres were washed with an appropriate amount of 90% isopropyl alcohol, 15 ml of a 90% isopropyl alcohol solution and 1.0 g of glycine were added to remove aldehyde for 24 hours, the microspheres were washed with an appropriate amount of 90% isopropyl alcohol to remove water, and then vacuum dried for 72 hours to obtain the modified carrageenan / gelatin double network structure composite microspheres.

[0050] Example 5

[0051] (1) 200 ml of 3.0 wt% aqueous K-carrageenan solution was adjusted to pH 8.0 with 3.0 M potassium hydroxide at 55 °C with stirring, 6.0 g of 2-methylene succinic anhydride was added slowly, and the mixture was stirred until homogeneous. The pH was adjusted to 8.0 with 3.0 M potassium hydroxide while the anhydride was added, and the pH was then maintained at 8.0 with base. The reaction was continued at 55 °C for 8 h, after which the solution was dialyzed to remove impurities. The solid was isolated by filtration, washed repeatedly with 95% ethanol, and dried at 60 °C for 12 h to remove residual ethanol and water. The anhydride-esterified carrageenan was obtained.

[0052] (2) 4.0 g of the anhydride-esterified carrageenan and 4.0 g of gelatin were mixed with 80 ml of water, and the mixture was heated and stirred at 80 °C. After 2 mL of a solution containing 0.08 g of the initiator sodium persulfate was added, the aqueous phase was prepared. 400 ml of cetyl alcohol was mixed with 1.3 ml of Span 60, and the mixture was heated and stirred at 80 °C until homogeneous. The aqueous phase was slowly added to the oil phase, and emulsification was carried out at 80 °C for 120 min at a mechanical stirring rate of 300 rpm. After 1 ml of a 4.8% sodium bisulfite solution was added as a catalyst, the reaction was continued for 2 h. The mixture was cooled in an ice bath for 4 h, and then an appropriate amount of isopropanol was added to wash the surface of the microspheres. The residual organic solvent and water on the surface of the microspheres were removed with an appropriate amount of anhydrous ethanol. After filtration and drying, the first-stage crosslinked microspheres were obtained.

[0053] (3) 5.0 g of the first-stage crosslinked microspheres were mixed with 50 ml of anhydrous ethanol and 4.0 g of genipin, and the mixture was stirred at 50 °C for 24 h to perform secondary crosslinking. The microspheres were washed with an appropriate amount of anhydrous ethanol, and 50 ml of a 80% ethanol solution and 0.5 g of sodium bisulfite were added to remove aldehydes for 10 h. The microspheres were washed with an appropriate amount of anhydrous ethanol to remove water, and then vacuum dried for 18 h. The modified carrageenan / gelatin double-network structure composite microspheres were obtained.

[0054] Comparative Example 1

[0055] Preparation of low-concentration modified carrageenan / gelatin composite microspheres

[0056] Prepared according to the method of Example 1, except that the concentration of the aqueous K-carrageenan solution in step (1) was 1.0%.

[0057] Comparative Example 2

[0058] Preparation of ultra-high-concentration modified carrageenan / gelatin composite microspheres

[0059] Prepared according to the method of Example 1, except that the concentration of the aqueous K-carrageenan solution in step (1) was 6.0%.

[0060] Comparative Example 3

[0061] Preparation of modified carrageenan under non-pH gradient conditions

[0062] Prepared according to step (1) of Example 1, except that the pH value of the κ-carrageenan solution is always maintained at pH 8.0.

[0063] Comparative Example 4

[0064] Preparation of highly substituted carrageenan / gelatin composite microspheres

[0065] Prepared according to the method of Example 5, except that the amount of acrylic anhydride used in step (1) is 25.0g.

[0066] Comparative Example 5

[0067] Preparation of carrageenan / gelatin single-network composite microspheres

[0068] (1) Weigh 2.0g carrageenan and 5.0g gelatin, mix with 90ml water, heat and stir at 50℃ to prepare the aqueous phase; mix 540ml liquid paraffin with 1.4ml Span 80, heat and stir evenly at 50℃ to obtain the oil phase. Slowly add the above aqueous phase to the oil phase, emulsify at a mechanical stirring rate of 200rpm for 30min, cool and solidify in an ice bath for 1 hour, then add an appropriate amount of petroleum ether to repeatedly wash the liquid paraffin on the surface of the microspheres, filter, and then use an appropriate amount of anhydrous ethanol to remove residual organic solvent and moisture on the surface of the microspheres to obtain uncrosslinked carrageenan / gelatin composite microspheres.

[0069] (2) Weigh 2.5g of uncrosslinked carrageenan / gelatin composite microspheres, add 50ml of 80% methanol solution and 2.5g of glutaraldehyde, mix and perform secondary crosslinking at 25℃ for 6h. Wash the microspheres with an appropriate amount of 80% methanol, filter and add 50ml of 80% methanol solution and 0.8g of glycine to remove aldehydes for 16h. Wash the microspheres with an appropriate amount of 95% methanol, filter and remove water, and vacuum dry for 12h to obtain carrageenan / gelatin single-network structure composite microspheres.

[0070] Example 6

[0071] Determination of the degree of substitution in the modification reaction of carrageenan solutions of different concentrations

[0072] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 The degree of substitution in the modification reaction was determined by ¹H NMR. Appropriate amounts of different modified carrageenan samples were dissolved in D₂O, and the chemical shifts of the samples were detected using a 600MHz NMR spectrometer. The degree of substitution was defined as the ratio of the average intensity integral of the methyl proton peak of the ester group to the proton peak of the methylene group present in carrageenan β-D-galactose. The results are shown in Table 1.

[0073] Table 1. Results of the degree of substitution determination in the modification reaction of carrageenan solutions of different concentrations.

[0074] Modified carrageenan samples Carrageenan concentration (%) Degree of substitution (%) Example 1 Step (1) 2.0 10.2 Example 2 Step (1) 5.0 15.3 Example 3 Step (1) 3.0 13.7 Example 4 Step (1) 3.0 13.6 Example 5 Step (1) 5.0 19.8 Comparative Example 1 Step (1) 1.0 6.1 Comparative Example 2 Step (1) 6.0 5.3

[0075] The results show that the high concentration carrageenan solution of 2.0wt%-5.0wt% can increase the collision probability between reactants, greatly improving the esterification efficiency. The low concentration carrageenan solution of 1.0wt% has low collision probability of reactants and low esterification efficiency, but the carrageenan aqueous solution with a concentration greater than 5% will cause the reaction liquid to be too viscous, the molecular movement is limited, and the reaction efficiency is greatly reduced.

[0076] In summary, it is shown that the modified carrageenan prepared by the method has a relatively optimal degree of substitution, which lays a foundation for the formation of the subsequent microsphere network structure.

[0077] Example 7

[0078] Determination of impurity content in modified carrageenan prepared under different pH conditions

[0079] An appropriate amount of modified carrageenan sample was taken in a 50ml volumetric flask containing 20ml ice acetic acid, 1ml 2% sodium nitrite solution was added to the sample, diluted to the mark with ice acetic acid, and mixed thoroughly. Place for 10min. With ice acetic acid as blank control, the absorbance of the solution was measured at wavelength 420nm. The impurity content corresponding to the obtained absorbance was found from the working curve. The concentration of impurities was expressed by mass fraction (10 -6 ), and the impurity content (10 -6 ) = impurity mass (μg) / sample mass (g).

[0080] The results are shown in Table 2.

[0081] Table 2 Determination results of impurity content in modified carrageenan prepared under different pH conditions

[0082] Modified carrageenan samples pH gradient change Impurity content (10 -6 )] Example 1 Step (1) 8.0 / 6.0 / 8.0 0.08 Example 4 Step (1) 8.3 / 6.5 / 8.3 0.07 Comparative Example 3 Step (1) 8.0 / 8.0 / 8.0 1.31

[0083] Example 8

[0084] Comparison of the ball forming property of modified carrageenan / gelatin composite microspheres with different degrees of substitution

[0085] The degree of substitution of the modified carrageenan in Example 5 and Comparative Example 4 was determined by the method in Example 6, which was 19.8% and 24.3% respectively, and the micrographs of the final microspheres prepared by the two methods are shown in Figure 1 The results show that the degree of substitution exceeding 20% is not good for the formation of microspheres, which is not conducive to the preparation of high-quality microspheres and the loading of drugs in the later stage.

[0086] Example 9

[0087] Determination of drug loading rate and leakage rate of microspheres with different network structures and degrees of substitution loaded with hydrophobic drugs

[0088] There are many commonly used hydrophobic drugs in clinic, such as lenvatinib, sorafenib, regorafenib, paclitaxel, etc. The present application takes regorafenib as an example to illustrate the ability of the microspheres of the present application to load hydrophobic drugs.

[0089] Preparation of drug-loaded microspheres: 200 mg of double-network structure composite microspheres were placed in 1 ml of 50 mg / ml regorafenib DMSO solution, shaken, and allowed to stand for 15 minutes. Then 10 ml of water for injection was added for washing, and the operation was repeated 4 times to obtain drug-loaded microspheres.

[0090] Chromatographic conditions: chromatographic column Gemini C18 chromatographic column (250 mm x 4.6 mm, 5 μm); mobile phase: 0.5% potassium dihydrogen phosphate solution-acetonitrile (30:70); detection wavelength: 260 nm; volume flow rate: 0.5 mL / min; column temperature: 30°C; sample size: 5 μL.

[0091] Drug loading rate determination: the above washing water was combined and diluted to 50 ml, shaken, and an appropriate amount was diluted with the mobile phase, and the drug loading rate was determined by high performance liquid chromatography under the above chromatographic conditions. Drug loading rate = (dose - drug amount in washing solution) / dose x 100%.

[0092] Leakage rate determination: 1 ml of drug-loaded microspheres was taken and placed in 2 ml of normal saline at room temperature for 7 days. The drug amount leaked in the normal saline was determined by diluting a certain proportion with the mobile phase under the above chromatographic conditions. Leakage rate = (total drug amount of microspheres - drug amount in normal saline) / total drug amount of microspheres x 100%.

[0093] The results are shown in Table 3.

[0094] Table 3 Drug loading rate and leakage rate determination results of microspheres with different network structures and degrees of substitution

[0095]

[0096] The results show that the microspheres prepared in Examples 1-5 and Example 1 Step (1) (2) have a large drug loading capacity for hydrophobic drugs, with a maximum of 99.5%, while the unmodified carrageenan / gelatin microspheres have a drug loading capacity of only 12.4%. This shows that the modified carrageenan can efficiently load hydrophobic drugs due to its hydrophobicity. The double network structure formed by the crosslinking of modified carrageenan and gelatin in the microspheres prepared in Examples 1-5 achieves double blocking of the loaded drugs, resulting in a very low drug leakage rate of not more than 2.0%. However, the microspheres prepared in Example 1 Step (1) (2) are only crosslinked with modified carrageenan, which can only form a single network structure, resulting in a weak ability to control drug leakage and a high drug leakage rate of 21.7%.

[0097] The results of Table 3 also show that the modified carrageenan substitution degree is less than 10%, the overall microspheres have limited hydrophobic properties, the drug loading capacity is extremely low, and the drug loading capacity is not significantly improved compared with the unmodified carrageenan / gelatin composite microspheres, indicating that the modified carrageenan substitution degree in the range of 10%-20% loaded with hydrophobic drugs has very good effect.

[0098] In summary, it is shown that the microspheres prepared by the method of the present application have excellent properties of loading hydrophobic drugs and controlling leakage.

Claims

1. A modified carrageenan / gelatin dual-network composite microsphere capable of carrying hydrophobic drugs, characterized in that, The composite microspheres are mainly formed by cross-linking of anhydride-esterified carrageenan and gelatin. The anhydride-esterified carrageenan is mainly formed by the esterification reaction of high-concentration carrageenan and anhydride. The degree of substitution of the anhydride-esterified carrageenan is controlled at 10%-20%. The high-concentration carrageenan has a concentration of 2.0wt%-5.0wt%, and the mass ratio of acid anhydride to κ-carrageenan is 1:2-2:

1. The mass ratio of acid anhydride esterified carrageenan to gelatin is 2:5-3:

2. The high-concentration carrageenan aqueous solution undergoes esterification under a gradient pH environment. The acid anhydride mentioned is one or more of 2-methacrylic anhydride, acrylic anhydride, and 2-methylenesuccinic anhydride; The preparation method of the modified carrageenan / gelatin dual-network structure composite microspheres capable of carrying hydrophobic drugs includes the following steps: (1) Adjust the pH of the high concentration κ-carrageenan aqueous solution to 8.0-8.6 with alkali, slowly add acid anhydride, and adjust and maintain the pH at 6.0-8.6 with alkali during this process. After the acid anhydride is added, continue to adjust and maintain the pH at 8.0-8.6 with alkali for the reaction. After the reaction, purify, wash and dry to obtain acid anhydride esterified carrageenan. (2) The obtained anhydride esterified carrageenan is mixed with gelatin and cross-linked in an emulsion system to obtain primary cross-linked microspheres of anhydride esterified carrageenan / gelatin single network structure composite microspheres. The primary cross-linked microspheres are then cross-linked in an organic solution system to obtain secondary cross-linked microspheres of anhydride esterified carrageenan / gelatin double network structure composite microspheres.

2. A method for preparing the modified carrageenan / gelatin dual-network structure composite microspheres capable of carrying hydrophobic drugs as described in claim 1, characterized in that, Includes the following steps: (1) Adjust the pH of the high concentration κ-carrageenan aqueous solution to 8.0-8.6 with alkali, slowly add acid anhydride, and adjust and maintain the pH at 6.0-8.6 with alkali during this process. After the acid anhydride is added, continue to adjust and maintain the pH at 8.0-8.6 with alkali for the reaction. After the reaction, purify, wash and dry to obtain acid anhydride esterified carrageenan. (2) The obtained anhydride esterified carrageenan is mixed with gelatin and cross-linked in an emulsion system to obtain primary cross-linked microspheres of anhydride esterified carrageenan / gelatin single network structure composite microspheres. The primary cross-linked microspheres are then cross-linked in an organic solution system to obtain secondary cross-linked microspheres of anhydride esterified carrageenan / gelatin double network structure composite microspheres.

3. The preparation method according to claim 2, characterized in that, In step (1), the high concentration κ-carrageenan aqueous solution was adjusted to pH 8.0-8.6 with alkali under stirring at 45-70℃. A certain amount of acid anhydride was slowly added. During this process, the pH was adjusted and maintained at 6.0-8.6 with alkali. Then, the pH was adjusted and maintained at 8.0-8.6 with alkali. After reacting at 45-70℃ for 4-12 hours, the solution was purified, washed, and dried to obtain acid anhydride esterified carrageenan.

4. The preparation method according to claim 2, characterized in that, Step (2) involves mixing and dispersing anhydride-esterified carrageenan, gelatin, initiator, and water to obtain an aqueous phase; adding the aqueous phase to an oil phase containing an emulsifier, fully emulsifying, adding a catalyst, and reacting at 50-80℃ for 1-6 hours to carry out primary crosslinking; cooling and curing after crosslinking and removing the oil phase; washing and drying to obtain primary crosslinked microspheres; wherein the mass ratio of anhydride-esterified carrageenan to gelatin is 2:5-3:2; and the concentration of the mixed adhesive obtained by mixing anhydride-esterified carrageenan and gelatin in the aqueous phase is 8.0wt%-14.0wt%.

5. The preparation method according to claim 4, characterized in that, The initiator is one or more of persulfate and azobisisobutyramidine hydrochloride, and the concentration of the initiator in the aqueous phase is 0.02 wt%-0.2 wt%. The catalyst is one or more of sodium bisulfite and tetramethylethylenediamine, and the concentration of the catalyst in the emulsion system is 0.003 wt%-0.03 wt%.

6. The preparation method according to claim 2, characterized in that, Step (2) involves performing secondary crosslinking on the primary crosslinked microspheres at 25-50°C in an organic solution containing a crosslinking agent, removing the crosslinking agent, washing, and drying to obtain the secondary crosslinked microspheres. In the secondary crosslinking process, the proportion of the primary crosslinked microspheres in the organic solution is 5.0 wt%-15.0 wt%. The crosslinking agent is one or more of glutaraldehyde, genipin, and carbodiimide crosslinking agents. The organic solution is a homogeneous system of one or more of methanol, ethanol, isopropanol, and water. The proportion of the crosslinking agent in the organic solution is 5.0 wt%-12.0 wt%.

7. The application of the modified carrageenan / gelatin dual-network composite microspheres capable of carrying hydrophobic drugs as described in claim 1 in the preparation of interventional embolization, medical aesthetic filling materials, tissue defect repair materials, and drug carriers.

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