Cyclodextrin hyperbranched microspheres and preparation method thereof

Abstract

The invention relates to a cyclodextrin hyperbranched microsphere and a preparation method thereof, and belongs to the technical field of embolism microspheres. The preparation method of the cyclodextrin hyperbranched microspheres comprises the following steps: grafting glycidol on cyclodextrin in a hyperbranched manner to obtain a hyperbranched polymer; dissolving a hyperbranched polymer and a cross-linking agent in an alkaline solution to obtain a water phase solution; and adding the water-phase solution into the oil-phase solution to obtain a water-in-oil reversed-phase suspension polymerization system, and reacting to obtain the cyclodextrin hyperbranched microspheres. Cyclodextrin is grafted with glycidol to modify hydrophilic hydroxyl, and hydrophobic drug molecules are captured in combination with hydrophobic cavities of cyclodextrin molecules, so that the microspheres can effectively load drugs; moreover, the dense pore structure of the hyperbranched microsphere is combined with the embedding effect of the hydrophobic cavity, so that the microsphere has a good drug sustained-release effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of embolization microspheres, and particularly relates to a cyclodextrin hyperbranched microsphere and a preparation method thereof. BACKGROUND

[0002] In transcatheter arterial chemoembolization (TACE), drug-eluting beads (DEB) can adsorb antitumor drugs (such as doxorubicin, irinotecan, topotecan and sorafenib) through ion exchange, so as to release the chemotherapy drugs smoothly, so that the tumor maintains a high blood drug concentration and the whole body has a low blood drug concentration, thereby reducing side effects.

[0003] However, the in vitro test of the existing drug-eluting microspheres shows that these microspheres release all or most of the loaded drugs in a single burst release, that is, the drug sustained release cannot be effectively achieved, resulting in a to-be-improved treatment effect. Therefore, achieving better drug sustained release is an important problem to be solved for the current drug-eluting microspheres. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a cyclodextrin hyperbranched microsphere and a preparation method thereof, so as to slow down the release of drugs and improve the drug sustained release effect.

[0005] In a first aspect, the present application provides a preparation method of a cyclodextrin hyperbranched microsphere, comprising the following steps: S1: hyperbranched grafting of epoxy propanol on cyclodextrin to obtain a hyperbranched polymer; S2: dissolving the hyperbranched polymer and a crosslinking agent in an alkaline solution to obtain an aqueous phase solution; S3: adding the aqueous phase solution into an oil phase solution to obtain a water-in-oil reverse suspension polymerization system, and obtaining the cyclodextrin hyperbranched microsphere after reaction.

[0006] In the above technical solution, the cyclodextrin hyperbranched microsphere is obtained by grafting epoxy propanol on cyclodextrin to form a hyperbranched polymer, and then adding a crosslinking agent to crosslink through reverse suspension polymerization. The hydrophobic cavity of the cyclodextrin molecule can capture hydrophobic drug molecules and embed the drug molecules through non-covalent bond force. The cyclodextrin surface is modified with rich terminal hydroxyl groups by grafting epoxy propanol, forming a hyperbranched hydrophilic structure, which can efficiently load hydrophobic drugs with positive ions. Moreover, the hyperbranched microsphere formed by crosslinking has a dense pore structure, which, in combination with the embedding effect of the cyclodextrin hydrophobic cavity on the drug, can effectively delay the release of the drug and achieve better drug treatment effect.

[0007] In a second aspect, the embodiments of the present application provide a cyclodextrin hyperbranched microsphere, which comprises a three-dimensional network structure obtained by crosslinking a hyperbranched polymer and a crosslinking agent, and the hyperbranched polymer is a hyperbranched polyglycidyl ether formed by grafting epoxypropanol on cyclodextrin.

[0008] In the above technical solution, the cyclodextrin hyperbranched microsphere in the present application is obtained by crosslinking a hyperbranched polyglycidyl ether formed by grafting epoxypropanol on cyclodextrin, wherein the cyclodextrin molecule has a hydrophobic cavity, can capture hydrophobic drug molecules, and embed the drug molecules through non-covalent bond force, and the cyclodextrin surface is modified with abundant terminal hydroxyl groups by grafting epoxypropanol, forming a hyperbranched hydrophilic structure, which can effectively load hydrophobic drugs with cations. Moreover, the three-dimensional network structure formed by crosslinking has a dense pore structure, which, in combination with the embedding effect of the cyclodextrin molecule cavity on the drug, can effectively delay the release of the drug and achieve better drug treatment effect. When the cyclodextrin hyperbranched microsphere is applied as a drug-eluting microsphere in TACE, it can make the blood drug concentration stable, avoid or reduce the peak-valley phenomenon, reduce the systemic toxic side effects and achieve better killing effect on tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0009] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0010] Figure 1 A process flow chart of a preparation method of a cyclodextrin hyperbranched microsphere provided by the embodiments of the present application. DETAILED DESCRIPTION

[0011] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0012] It should be noted that in the present application, "and / or", such as "feature 1 and / or feature 2", means "feature 1" alone, "feature 2" alone, or "feature 1" plus "feature 2".

[0013] In addition, in the description of the present application, unless otherwise specified, the meaning of "multiple" in "one or more" refers to two and more than two; the range of "value a ~ value b" includes both end values "a" and "b", and "unit of measurement" in "value a ~ value b + unit of measurement" represents the "unit of measurement" of both "value a" and "value b".

[0014] Figure 1 A process flow chart of a preparation method of a cyclodextrin hyperbranched microsphere provided by an embodiment of the present application is provided. Please see Figure 1 The preparation method of the cyclodextrin hyperbranched microsphere provided by the first aspect of the present application comprises the following steps: S1: hyperbranched grafting of glycidol on cyclodextrin to obtain a hyperbranched polymer.

[0015] In the present application, cyclodextrin (CD) is a cyclic oligosaccharide formed by 6-8 glucose units connected by α-1,4 glycosidic bonds (divided into α-CD, β-CD, γ-CD), the outer surface of the molecule is rich in multiple hydrophilic hydroxyl groups (each glucose unit contains 3 hydroxyl groups), and the internal cavity is composed of C-H bonds and oxygen atoms of glycosidic bonds, which is hydrophobic, and can form a stable "inclusion compound" with hydrophobic drugs through non-covalent bonding. Glycidol (also known as glycidol), which contains both epoxy groups (three-membered epoxy ring) and hydroxyl groups (-OH) in its molecular structure, is a "bifunctional monomer". By using the hydroxyl groups on the surface of cyclodextrin as "initiation sites" and forming hyperbranched segments through ring-opening polymerization of glycidol, a hyperbranched structure of "cyclodextrin core-hyperbranched arm" can be obtained.

[0016] Preferably, the cyclodextrin in the present application is β-cyclodextrin (β-CD), which has an internal cavity size that can adapt to most hydrophobic drugs, which is beneficial to expand the application range.

[0017] In some embodiments, the mass percentage of cyclodextrin in glycidol is 20 wt% to 85 wt%. As an example, the mass percentage of cyclodextrin in glycidol is 20 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 85 wt% or any range between any two of the above values.

[0018] By controlling the amount of cyclodextrin added, the degree of branching of the hyperbranched polymer can be adjusted accordingly, so as to adjust the content of hyperbranched terminal hydroxyl groups in the final synthesized microspheres. Controlling the amount of cyclodextrin added within a suitable range is beneficial to increasing the degree of branching and the number of hyperbranched terminal hydroxyl groups of the microspheres, thereby improving the drug loading capacity and the drug release effect.

[0019] In some embodiments, step S1 comprises: S11: stirring and dissolving the cyclodextrin in a methanol-potassium methoxide mixed solution under 60-90℃ and anaerobic conditions to obtain a cyclodextrin solution; S12: adding propylene glycol to the cyclodextrin solution and reacting at 80-120℃ for 8-20h, and then purifying and drying to obtain the hyperbranched polymer.

[0020] In the present application, a methanol-potassium methoxide mixed solution is used as the solvent, wherein the methanol is beneficial to dissolving the cyclodextrin, the potassium methoxide can provide an alkaline environment to activate the hydroxyl groups on the surface of the cyclodextrin, and the anaerobic environment is beneficial to avoiding the oxidation of active groups, so that the cyclodextrin solution can react with propylene glycol to obtain the hyperbranched polymer.

[0021] For example, the temperature in step S11 can be 60℃, 70℃, 80℃, 90℃ or any range between any two of the above values. This is beneficial to improving the solubility of the cyclodextrin and promoting the activation of the hydroxyl groups.

[0022] For example, the temperature in step S12 can be 80℃, 90℃, 100℃, 110℃, 120℃ or any range between any two of the above values; and the reaction time can be 8h, 10h, 12h, 15h, 20h or any range between any two of the above values. Controlling the reaction temperature and time in the appropriate range is beneficial to accelerating the reaction rate, promoting the activation of the newly generated propylene glycol segment hydroxyl groups to form more branched sites, and facilitating the grafting reaction of the cyclodextrin and propylene glycol.

[0023] Further, in the methanol-potassium methoxide mixed solution, the mass percentage of potassium methoxide is 5-20 wt%. By controlling the amount of potassium methoxide added, it is beneficial to maintain the matching with the amount of cyclodextrin added, and to promote the activation of the hydroxyl groups while dissolving.

[0024] Further, after the reaction in step S12 is completed, methanol can be added to stir and dissolve the residual substances, and then mixed with the acidified resin to remove impurities, followed by dialysis purification and freeze-drying.

[0025] After obtaining the hyperbranched polymer of cyclodextrin by grafting propylene glycol on cyclodextrin, the present application further comprises the following steps: S2: dissolving the hyperbranched polymer and the crosslinking agent in an alkaline solution to obtain an aqueous solution; S3: adding the aqueous solution to an oil phase solution to obtain a water-in-oil inverse suspension polymerization system, and after reaction, obtaining cyclodextrin hyperbranched microspheres.

[0026] In some embodiments, the mass ratio of the hyperbranched polymer and the crosslinking agent is 1: (1~6). For example, the mass ratio of the hyperbranched polymer and the crosslinking agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, or any range between any two of the above values. Controlling the amount of the crosslinking agent added in a suitable range is conducive to improving the crosslinking density. The mechanical strength of the microspheres is maintained, while the drug loading capacity and the sustained release effect are improved.

[0027] In some embodiments, the crosslinking agent includes at least one of ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether (Mn=200), polyethylene glycol diglycidyl ether (Mn=1000), polyethylene glycol diglycidyl ether (Mn=2000), or polyethylene glycol diglycidyl ether (Mn=3000). Here, Mn represents the number average molecular weight, which further corresponds to the chain length of the crosslinking agent. The shorter the chain length of the crosslinking agent, the higher the crosslinking density of the hyperbranched structure, and the drug loading capacity of the microspheres is reduced; while the chain length of the crosslinking agent is too long, which will block the cavity of the cyclodextrin, and also reduce the drug loading utilization rate of the cavity in the microspheres. By controlling the type and molecular weight of the crosslinking agent, matching the crosslinking agent with a suitable chain length is conducive to controlling the crosslinking density to a suitable degree, while improving the drug loading capacity of the microspheres.

[0028] In some embodiments, the step S2 includes: stirring and dissolving the hyperbranched polymer in the alkaline solution to obtain a mixed solution; adding the crosslinking agent to the mixed solution and stirring to obtain an aqueous phase solution.

[0029] Further, the alkaline solution includes a sodium hydroxide solution with a mass percentage of 10 wt%~30 wt%.

[0030] In some embodiments, the step S3 includes: under stirring, adding the aqueous phase solution to the oil phase solution, and performing emulsion reaction at a first temperature for 1 h~3 h; then increasing the temperature to a second temperature and reacting for 6 h~12 h, wherein the first temperature is lower than the second temperature; and after the reaction is completed, performing filtration and washing to obtain the cyclodextrin hyperbranched microspheres. Here, performing emulsion at a lower first temperature can prevent explosive polymerization.

[0031] Further, the first temperature is 2 ℃~10 ℃, and the second temperature is 40 ℃~80 ℃.

[0032] For example, the temperature of the emulsion reaction is 2 ℃, 4 ℃, 5 ℃, 8 ℃, 10 ℃, or any range between any two of the above values; and the time is 1 h, 1.5 h, 2 h, 2.5 h, 3 h, or any range between any two of the above values.

[0033] As an example, the temperature of the inverse suspension polymerization reaction is 40℃, 50℃, 60℃, 70℃, 80℃, or any range between any two of the above values; the time is 6h, 8h, 10h, 12h, or any range between any two of the above values.

[0034] In some embodiments, the oil phase solution comprises an oily dispersant and an oily solvent. The oily dispersant comprises at least one of Span 20, Span 40, Span 60, Span 80, Span 20, Tween 20, Tween 40, Tween 60, or Tween 80; the oil phase solvent comprises at least one of liquid paraffin, n-hexane, n-decane, or cyclohexane.

[0035] It can be understood that after the reaction is completed, the product solution is further filtered, washed, and sieved.

[0036] In the embodiments of the present application, the hyperbranched polymer is further crosslinked by a crosslinking agent, and the cyclodextrin hyperbranched microspheres are prepared by inverse suspension polymerization, so that the microspheres have a relatively dense crosslinked network structure, which endows the microspheres with excellent mechanical properties and greatly improves the sustained release effect of the drug-loaded microspheres.

[0037] In order to further improve the drug loading capacity of the above-mentioned microspheres, the present application modifies and protects the primary hydroxyl groups on the cyclodextrin before preparing the hyperbranched polymer.

[0038] In some embodiments, before step S1, there is further a step S0 of modifying the primary hydroxyl groups on the cyclodextrin with a protective agent.

[0039] In the present application, the hydrophobic inner cavity of the cyclodextrin can load drugs through the channel on the narrow side, and the primary hydroxyl groups at the channel on the narrow side have high reactivity. By modifying the primary hydroxyl groups with a protective agent, the hyperbranched and crosslinking agents are prevented from blocking the drug loading channel in the cavity, which is conducive to keeping the drug channel on one side of the cavity relatively unobstructed and avoiding the influence of hyperbranched crosslinking on the drug loading capacity of the microspheres. In the present application, the drug release of the microspheres is delayed, and the drug loading capacity of the microspheres is taken into account, so that the microspheres have high drug loading capacity and excellent drug sustained release effect in the application scenario of post-drug loading, greatly improving the practical application value of the microspheres.

[0040] Further, the molar ratio of the cyclodextrin to the protective agent is 1:(1.2-1.5). As an example, the molar ratio of the cyclodextrin to the protective agent is 1:1.2, 1:1.3, 1:1.4, 1:1.5, or any range between any two of the above values. By controlling the amount of the protective agent added, the primary hydroxyl groups are efficiently and directionally protected and modified, and the side reactions are reduced.

[0041] Further, the protecting agent comprises at least one of tert-butyldimethylsilyl chloride, triphenylmethyl chloride, tert-butyldiphenylsilyl chloride or 1-(tert-butoxy carbonyl) imidazole. These protecting agents have high selectivity and can selectively react with the primary hydroxyl group, thereby efficiently achieving protection modification and facilitating reduction of side reactions.

[0042] Further, the step S0 comprises: dissolving the cyclodextrin and the protecting agent in an organic solvent, adding a basic auxiliary agent, and reacting at 20-50°C for 48-120h to modify and protect the primary hydroxyl group of the cyclodextrin. The organic solvent facilitates complete dissolution of the cyclodextrin and the protecting agent, the basic auxiliary agent facilitates activation of the primary hydroxyl group, and further controlling the reaction temperature and time in a suitable range facilitates efficient and selective modification of the primary hydroxyl group.

[0043] For example, the reaction temperature in the step S0 is 20°C, 30°C, 40°C, 50°C or any range between any two of the above values; and the reaction time is 48h, 60h, 80h, 100h, 120h or any range between any two of the above values.

[0044] Further, the organic solvent comprises N,N-dimethylformamide; and the basic auxiliary agent comprises triethylamine.

[0045] The embodiments of the present application can effectively avoid side reactions of the primary hydroxyl group of the cyclodextrin in the hyperbranched reaction in the step S1 and the crosslinking agent reaction in the step S2 by modifying and protecting the primary hydroxyl group of the cyclodextrin with the protecting agent, and then sequentially performing the steps S1, S2 and S3 of the present application, thereby keeping the drug loading channel of the cyclodextrin unblocked and improving the drug loading capacity of the microspheres.

[0046] In some embodiments, after modifying and protecting the primary hydroxyl group of the cyclodextrin with the protecting agent and then sequentially performing the steps S1, S2 and S3 of the present application, the method further comprises a step S4, wherein, The step S4 comprises: removing the protecting agent on the microspheres by a removing agent. Specifically, the microspheres are dissolved in a removing agent solution, hydrolyzed by stirring and then filtered and dried, so as to remove the protecting agent on the microspheres, thereby exposing the drug loading channel of the microspheres and facilitating loading of specific drugs by the microspheres.

[0047] Optionally, the drug loaded by the microspheres comprises doxorubicin.

[0048] Further, different removal agents can be selected according to different protective agents. For example, when the protective agent is tert-butyl dimethyl chlorosilane or tert-butyl diphenyl chlorosilane, the removal agent is tetrabutylammonium fluoride (TBAF); when the protective agent is triphenyl chloromethane, the removal agent is p-toluenesulfonic acid or acetic acid; when the protective agent is 1-(tert-butoxy carbonyl) imidazole, the removal agent is trifluoroacetic acid (TFA).

[0049] In the present application, the cyclodextrin is grafted with propylene oxide to form a hyperbranched polymer, and the molecular chain of the hyperbranched polymer contains a large number of hydrophilic hydroxyl groups. Then, a crosslinking agent is added to perform inverse suspension polymerization crosslinking to form cyclodextrin hyperbranched microspheres with a three-dimensional network structure. The hydrophobic cavity of the cyclodextrin molecule can capture hydrophobic drug molecules and embed the drug molecules through non-covalent bond force. The surface of the cyclodextrin is modified with a large number of terminal hydroxyl groups by grafting propylene oxide, forming a hyperbranched hydrophilic structure, which can effectively load hydrophobic drugs with cations. The hyperbranched microspheres formed by crosslinking have a dense pore structure, and in combination with the embedding effect of the cyclodextrin molecular cavity on the drug, the release of the drug can be effectively delayed, achieving a better drug treatment effect.

[0050] The second aspect of the present application also provides a cyclodextrin hyperbranched microsphere, which includes a three-dimensional network structure obtained by crosslinking a hyperbranched polymer with a crosslinking agent. The hyperbranched polymer is a hyperbranched polyglycidyl ether formed by grafting propylene oxide onto cyclodextrin.

[0051] The cyclodextrin hyperbranched microsphere in the present application is obtained by crosslinking a hyperbranched polyglycidyl ether formed by grafting propylene oxide onto cyclodextrin. The cyclodextrin molecule has a hydrophobic cavity, which can capture hydrophobic drug molecules and embed the drug molecules through non-covalent bond force. The surface of the cyclodextrin is modified with a large number of terminal hydroxyl groups by grafting propylene oxide, forming a hyperbranched hydrophilic structure, which can effectively load cationic drugs. The three-dimensional network structure formed by crosslinking has a dense pore structure, and in combination with the embedding effect of the cyclodextrin molecular cavity on the drug, the release of the drug can be effectively delayed, achieving a better drug treatment effect. The cyclodextrin hyperbranched microsphere can be used as a drug-eluting microsphere in TACE, which can stabilize the blood drug concentration, avoid or reduce the peak-trough phenomenon, reduce the systemic toxic side effects, and achieve a better killing effect on tumor cells.

[0052] The features and performance of the technical solutions of the present application are further described in detail below in combination with examples.

[0053] Example 1 The present example provides a cyclodextrin hyperbranched microsphere, and a preparation method thereof includes the following steps: (1) Preparation of hyperbranched polymer In a double-tube reactor, 3 g of β-CD was weighed into a 100 mL round-bottom flask, 5 mL of 10% methanol-methanolic potassium solution was added under stirring at 80°C, and after the β-CD was completely dissolved, 10 mL of propylene oxide was slowly added dropwise into the flask, and the reaction was carried out at 100°C for 20 h. After the reaction was completed, heating was stopped, 15 mL of methanol solution was added for further dissolution, and after the dissolution was completed, the obtained product was mixed with acidified resin and stirred for 48 h, and then dialyzed with a 3000 Da dialysis bag for 3 days, and freeze-dried to obtain a hyperbranched polyglycidyl ether (denoted as β-CD-HPG).

[0054] (2) Preparation of an aqueous phase solution The β-CD-HPG was dissolved in pure water to prepare a 0.4 g / mL solution; 0.6 g of NaOH was weighed into a reaction flask, and then 5 mL of the β-CD-HPG solution was added and stirred until the NaOH was completely dissolved. 6 g (0.012 mol) of polyethylene glycol diglycidyl ether (Mn=500) was slowly added into the above mixture, and stirring was continued at room temperature to obtain an aqueous phase solution.

[0055] (3) Inverse suspension polymerization 50 mL of light liquid paraffin, 50 mL of cyclohexane, 1 g of Span 60, and 0.1 g of Tween 60 were added into a three-necked flask, and stirring was carried out at a speed of 200 rpm to obtain an oil phase solution.

[0056] The oil phase solution was placed in a 5°C low-temperature reaction tank, and then the aqueous phase solution was slowly added dropwise into the oil phase solution, and after the addition was completed, stirring was continued for 1 h to uniformly disperse and emulsify, and then the temperature was increased to 50°C for overnight reaction. After the reaction was completed, the solution was poured into a beaker and placed in a fume hood for standing, and after the solution was layered, the upper oil phase was discarded, and after filtration, washing, and screening, a cyclodextrin hyperbranched microsphere was obtained.

[0057] Example 2 The cyclodextrin hyperbranched microsphere provided in this example differs from that of Example 1 in that in step (1), the mass of β-CD is 6 g.

[0058] Example 3 The cyclodextrin hyperbranched microsphere provided in this example differs from that of Example 1 in that in step (1), the mass of β-CD is 9 g.

[0059] Example 4 The cyclodextrin hyperbranched microsphere provided in this example differs from that of Example 2 in that in step (2), the crosslinking agent used is 2.4 g (0.012 mol) of polyethylene glycol diglycidyl ether (Mn=200). Example 5 This example provides a cyclodextrin hyperbranched microspheres, the preparation method of which is different from that of Example 2 in that in step (2), 12 g (0.012 mol) of polyethylene glycol diglycidyl ether (Mn=1000) is used as the crosslinking agent. Example 6 This example provides a cyclodextrin hyperbranched microspheres, the preparation method of which is different from that of Example 2 in that: Before step (1), β-CD and tert-butyldimethylsilyl chloride are weighed and dissolved in DMF, followed by the addition of triethylamine. The reaction is carried out at 30 °C for 96 h to protect the primary alcohol at position 6 of the cyclodextrin. Subsequently, the solvent is removed by vacuum distillation, and the product is purified by silica gel column chromatography using gradient elution with methanol and dichloromethane, and vacuum dried to obtain modified cyclodextrin with tert-butyldimethylsilyloxy groups modified on the primary hydroxyl groups. In step (1), 6 g of the above-mentioned modified cyclodextrin is weighed instead of 3 g of β-CD.

[0060] After step (3), step (4) is further included: the cyclodextrin hyperbranched microspheres are placed in an excess of aqueous tetrabutylammonium fluoride solution at room temperature and stirred to hydrolyze and remove the tert-butyldimethylsilyloxy groups.

[0061] Example 7 This example provides a cyclodextrin hyperbranched microspheres, the preparation method of which is different from that of Example 3 in that: Before step (1), β-CD and tert-butyldimethylsilyl chloride are weighed and dissolved in DMF, followed by the addition of triethylamine. The reaction is carried out at 30 °C for 96 h to protect the primary alcohol at position 6 of the cyclodextrin. Subsequently, the solvent is removed by vacuum distillation, and the product is purified by silica gel column chromatography using gradient elution with methanol and dichloromethane, and vacuum dried to obtain modified cyclodextrin with tert-butyldimethylsilyloxy groups modified on the primary hydroxyl groups. In step (1), 6 g of the above-mentioned modified cyclodextrin is weighed instead of 3 g of β-CD.

[0062] In step (2), 9 g (0.018 mol) of polyethylene glycol diglycidyl ether (Mn=500) is used.

[0063] After step (3), step (4) is further included: the cyclodextrin hyperbranched microspheres are placed in an excess of aqueous tetrabutylammonium fluoride solution at room temperature and stirred to hydrolyze and remove the tert-butyldimethylsilyloxy groups.

[0064] Comparative Example 1 The preparation method of this comparative example is different from that of Example 2 in that: In step (1), the mass of β-CD is 0.5 g. Comparative Example 2 The preparation method of the present comparative example is different from that of Example 2 in that: In step (1), the mass of β-CD was 15 g. Comparative Example 3 The preparation method of the present comparative example is different from that of Example 2 in that: In step (2), 24 g (0.0048 mol) of polyethylene glycol diglycidyl ether (Mn = 5000) was used as the crosslinking agent.

[0065] Performance test and result analysis 1. Mechanical properties In Examples 1-7 and Comparative Example 2, cyclodextrin hyperbranched microspheres can be prepared, while in Comparative Example 1, the amount of β-CD used is too small relative to the amount of epoxy propanol, which causes the epoxy propanol to self-aggregate and fail to crosslink into a ball; in Comparative Example 3, the chain length of the crosslinking agent is too long, resulting in a too loose crosslinking structure, and thus a complete spherical structure cannot be obtained.

[0066] Displacement of the cyclodextrin hyperbranched microspheres prepared in Examples 1-7 and Comparative Example 2 was performed: 100 mL of each group of microspheres with a size of 100 μm-300 μm was taken and added to 100 mL of normal saline, and after the microspheres were allowed to stand and separate, the supernatant was discarded; 100 mL of normal saline was added again, and the above operation was repeated until the volume of the microspheres no longer changed, obtaining the displacement microspheres with normal saline as the storage medium. The displaced 100 μm-300 μm microspheres were subjected to stress testing by a texture analyzer to obtain the corresponding strength and elasticity, and the results are shown in Table 1.

[0067] Table 1: Mechanical property test results of the microspheres in Examples 1-7 and Comparative Example 2

[0068] As can be seen from Table 1, compared with Comparative Example 2, the microspheres prepared in Examples 1-7 of the present application have good compression elasticity (higher than 40%), which is beneficial to improve the catheter passing performance of the microspheres. As can be seen from Table 1, by adjusting the mass of cyclodextrin, the type and mass of the crosslinking agent, the strength and elasticity of the microspheres can be synergistically controlled, and microspheres with high strength and elasticity can be obtained, which is beneficial to the smooth passing of the microspheres through the catheter, can reduce the damage to the tissue wall, and will not rupture prematurely.

[0069] 2. Drug loading performance 1 mL of 100 μm-300 μm microspheres provided by Examples 1-7 was mixed with 4 mL of 20 mg / mL doxorubicin, and samples were taken at regular time intervals, and the residual amount of doxorubicin in the supernatant was tested by high performance liquid chromatography, and then the drug loading amount of the microspheres was calculated. The drug loading amount test results are shown in Table 2.

[0070] Table 2: Drug loading test results of microspheres in Examples 1-7

[0071] As can be seen from Table 2, the cyclodextrin hyperbranched microspheres prepared in Examples 1-7 all have good drug loading performance, and the drug loading capacity for doxorubicin can be as high as 36.69 mg / mL.

[0072] By comparing the drug loading data of Examples 1-3, it can be seen that the feeding ratio of β-CD-HPG can significantly affect the drug loading capacity of the microspheres. It is speculated that the following reasons lead to this: by controlling the feeding ratio, the branching degree and molecular weight of β-CD-HPG will be different. In Example 1, the feeding ratio of branched core β-CD is small, and the hyperbranched polymer obtained has a higher branching degree and more hydroxyl groups, so it has the highest drug loading capacity. In Example 3, the feeding ratio of β-CD is too high, and the polymer multi-core branches. In the same reaction time, the molecular weight of the polymer is smaller, the branching degree is lower, and the number of hydroxyl groups is fewer, so the drug loading capacity is reduced.

[0073] By comparing Examples 2, 4, and 5, it can be seen that in Example 4, the crosslinking agent has a shorter chain length (polyethylene glycol diglycidyl ether Mn=200), and the drug loading capacity of the microspheres is greatly reduced. This may be because the crosslinking network formed by the shorter crosslinking agent is more dense, which to some extent affects the entry of drug molecules into the network structure. There is no obvious difference between Examples 2 and 5, but a longer crosslinking agent is used in Example 5. This may be because the free movement of the chain segment of the crosslinking agent that is too long occupies part of the cavity position, thereby affecting the entry of the drug into the cavity.

[0074] By comparing Examples 2 and 6, the protection of the primary hydroxyl group on the cyclodextrin significantly improves the overall drug loading capacity of the microspheres. In addition, by comparing Examples 3 and 7, the protection of the primary hydroxyl group on the cyclodextrin also effectively improves the overall drug loading capacity of the microspheres. This may be because in the cyclodextrin without protection of the primary hydroxyl group, the cavity is blocked by the dense crosslinking network, which blocks the entry of the hydrophobic drug into the cavity.

[0075] 3. Drug release performance Take 2 mL of the above drug-loaded microspheres, and use normal saline to perform drug elution experiment on the microspheres: load the drug-loaded microspheres into a dialysis bag, and place the dialysis bag in a sufficient volume of normal saline and shake. At different time points, sample and test the residual amount of doxorubicin in the eluent by high performance liquid chromatography. The drug release data is shown in Table 3.

[0076] Table 3: Drug release performance test results of microspheres in Examples 1-7

[0077] As can be seen from Table 3, the cyclodextrin hyperbranched microspheres prepared in Examples 1-7 of the present application all have good drug release performance, with a release rate of less than 15% in 3 h, a release rate of less than 80% in 48 h, and a complete release time of more than 135 h.

[0078] As can be seen from Comparative Examples 1-3, the branching degree of the β-CD-HPG can greatly affect the drug release rate, and Examples 1 and 2 both have good drug release effect, while the branching degree of the microspheres in Example 3 is lower, and the drug release effect is slightly reduced. By controlling the branching degree of the hyperbranched polymer, the drug release performance can be further improved.

[0079] As can be seen from Comparative Examples 2, 4 and 5, the length of the chain of the crosslinking agent also affects the drug release effect. By adjusting the length of the chain of the crosslinking agent, the drug release effect can be further improved.

[0080] As can be seen from the comparison between Comparative Example 2 and Example 6, and the comparison between Comparative Example 3 and Example 7, the modification of the primary hydroxyl group affects the branching degree of the hyperbranched polymer to some extent. Therefore, the drug release effect of Example 2 is relatively better than that of Example 6, but the difference is not large, and Example 6 has a higher drug loading capacity than Example 2.

[0081] The above-described examples are part of the examples of the present application, rather than all the examples. The detailed description of the examples of the present application is not intended to limit the scope of the claimed application, but only represents selected examples of the present application. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application.

Claims

1. A method for preparing cyclodextrin hyperbranched microspheres, characterized in that, Includes the following steps: S1: Hyperbranching of glycidol onto cyclodextrin yields a hyperbranched polymer; S2: Dissolve the hyperbranched polymer and crosslinking agent in an alkaline solution to obtain an aqueous solution; S3: The aqueous solution is added to the oil solution to obtain a water-in-oil reverse suspension polymerization system. After the reaction, cyclodextrin hyperbranched microspheres are obtained.

2. The preparation method according to claim 1, characterized in that, Step S1 includes: S11: The cyclodextrin is dissolved in a methanol-potassium methoxide mixed solution under anaerobic conditions at 60 ℃~90 ℃ to obtain a cyclodextrin solution. S12: Add the glycidol dropwise to the cyclodextrin solution and react at 80 ℃~120 ℃ for 8 h~20 h. After the reaction, purify and dry to obtain the hyperbranched polymer. Optionally, in the methanol-potassium methoxide mixed solution, the mass percentage of potassium methoxide is 5 wt% to 20 wt%.

3. The preparation method according to claim 1, characterized in that, The cyclodextrin accounts for 20 wt% to 85 wt% of the glycidol by mass. And / or, the mass ratio of the hyperbranched polymer to the crosslinking agent is 1:(1~6).

4. The preparation method according to claim 1, characterized in that, Step S2 includes: stirring and dissolving the hyperbranched polymer in the alkaline solution to obtain a mixture; The crosslinking agent is added to the mixture, and the mixture is stirred to obtain the aqueous solution. Optionally, the alkaline solution comprises a sodium hydroxide solution with a mass percentage of 10 wt% to 30 wt%.

5. The preparation method according to claim 1, characterized in that, The crosslinking agent includes at least one of ethylene glycol diglycidyl ether, polyethylene glycol diglycidyl ether (Mn=200), polyethylene glycol diglycidyl ether (Mn=1000), polyethylene glycol diglycidyl ether (Mn=2000), or polyethylene glycol diglycidyl ether (Mn=3000).

6. The preparation method according to claim 1, characterized in that, Step S3 includes: Under stirring conditions, the aqueous phase solution is added dropwise to the oil phase solution, and the emulsification reaction is carried out at a first temperature for 1 h to 3 h; then the temperature is raised to a second temperature for 6 h to 12 h, wherein the first temperature is lower than the second temperature; after the reaction is completed, the mixture is filtered and washed to obtain the cyclodextrin hyperbranched microspheres. Optionally, the first temperature is 2 ℃ to 10 ℃, and the second temperature is 40 ℃ to 80 ℃.

7. The preparation method according to claim 1, characterized in that, The process includes step S0 before step S1: modifying the primary hydroxyl groups on the cyclodextrin with a protective agent.

8. The preparation method according to claim 7, characterized in that, The molar ratio of the cyclodextrin to the protective agent is 1:(1.2~1.5); And / or, the protective agent includes at least one of tert-butyldimethylchlorosilane, triphenylchloromethane, tert-butyldiphenylchlorosilane, or 1-(tert-butoxycarbonyl)imidazole.

9. The preparation method according to claim 7, characterized in that, Step S0 includes: The cyclodextrin and the protecting agent are dissolved in an organic solvent, and an alkaline auxiliary is added. The reaction is carried out at 20 °C to 50 °C for 48 h to 120 h, thereby modifying the primary hydroxyl group of the cyclodextrin with a protecting group. Optionally, the organic solvent includes N,N-dimethylformamide; optionally, the alkaline auxiliary includes triethylamine; and / or... Step S3 is followed by step S4: removing the protective agent from the cyclodextrin hyperbranched microspheres by a removing agent, optionally including at least one of tetrabutylammonium fluoride, p-toluenesulfonic acid, or acetic acid and trifluoroacetic acid.

10. A cyclodextrin hyperbranched microsphere, prepared according to any one of claims 1 to 9, characterized in that, The cyclodextrin hyperbranched microspheres comprise a three-dimensional network structure obtained by crosslinking a hyperbranched polymer with a crosslinking agent. The hyperbranched polymer is a hyperbranched polyglycidyl ether formed by grafting glycidol onto a cyclodextrin core.

Images