Preparation method of camptothecin-loaded chitosan nanocellulose gamma-cyclodextrin metal organic framework porous composite microspheres

Abstract

The invention provides a preparation method of chitosan nanocellulose gamma-cyclodextrin metal organic framework composite microspheres loaded with camptothecin. The gamma-cyclodextrin metal organic framework with uniform particle size is prepared by taking ethanol as a solvent and polyethylene glycol 20000 as a surfactant through a steam diffusion method. Due to the fact that camptothecin is unstable under the alkaline condition, the gamma-cyclodextrin metal organic framework is neutralized to be neutral through acetic acid, the medicine is loaded into a cavity of the gamma-cyclodextrin metal organic framework through an impregnation method, and the solubility, stability and bioavailability of the camptothecin medicine are improved. In order to solve the problem that the water solubility of the gamma-cyclodextrin metal organic framework is too high, Span 85 is adopted for surface hydrophobic modification. And loading a drug-loaded gamma-cyclodextrin metal organic framework modified by Span 85 by taking the chitosan / nanocellulose porous microspheres prepared by an alkaline-urine system as a carrier. The composite material has good biocompatibility, biodegradability and drug slow release performance, the preparation process is simple and environmentally friendly, and the composite material has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of preparation of anti-tumor drug delivery systems, and specifically relates to a method for preparing chitosan nano-cellulose gamma-cyclodextrin metal organic framework porous composite microspheres loaded with camptothecin. Background Art

[0002] Cancer is a major disease that threatens human health. The "Global Cancer Research General Report" released by the World Health Organization's cancer research department in 2020 shows that there are about 19.3 million new cases of various cancers and about 9.96 million deaths each year in the world. Cancer, cardiovascular disease, and accidents are the main causes of human death. Unlike acute infectious diseases and routine malnutrition, cancer will not be reduced due to human technological progress and rapid economic development, so sufficient attention must be paid to the treatment of cancer.

[0003] Camptothecin (CPT) is considered to be one of the main effective anticancer prodrugs in the 21st century, and it has shown good antitumor activity in in vitro activity experiments of tumor cells. Unfortunately, camptothecin (CPT) has some major disadvantages in therapeutic applications, such as poor water solubility and low bioavailability. In addition, at physiological pH, the lactone ring of CPT is easily hydrolyzed to produce inactive carboxylate forms. Therefore, it is very necessary to design new carriers for delivering CPT. The development of nanotechnology in recent years has provided new possible solutions for drug formulation and administration. Various nano-delivery systems can achieve high drug encapsulation rate and high cell uptake efficiency through surface modification and ligand coupling targeted delivery. Therefore, the emergence of drug nano-delivery systems provides new ideas and methods for improving the solubility, permeability and stability of CPT preparations, improving drug efficacy, reducing the incidence of adverse reactions, and reversing drug resistance.

[0004] Cyclodextrin metal organic framework (CD-MOFs) is a new type of metal organic framework material synthesized with biomass material cyclodextrin as organic ligand. It has the characteristics of hydrophilicity on the outside and hydrophobicity on the inside, which enables it to carry other guest molecules. After forming a stable inclusion complex with the guest molecule, it can change the physical and chemical properties of the guest molecule, making it have properties such as controlled release, improved stability, antioxidant and water solubility. Therefore, cyclodextrin is widely used in food, medicine, environment, agriculture and other fields. Among them, α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin are the three most common cyclodextrins, which are composed of 6, 7 and 8 glucose units respectively. α-cyclodextrin has the smallest cavity size and is easy to form stable supramolecules with monocyclic aromatic hydrocarbons, such as azobenzene and its derivatives; β-cyclodextrin has a moderate cavity size and is easy to form inclusion complexes with anthraquinone, cholesterol, ferrocene, adamantane, cyclodiene, azo compounds and their derivatives; γ-cyclodextrin has the largest cavity size and usually forms inclusion complexes with larger guest molecules, such as pyrene, anthracene, phenanthrene, steroids, etc. Due to the better symmetry of γ-cyclodextrin, the cyclodextrin metal organic framework prepared with γ-cyclodextrin as raw material has higher structural stability; on the other hand, γ-cyclodextrin metal organic framework has a higher specific surface area and richer pores than α-cyclodextrin metal organic framework and β-cyclodextrin metal organic framework, and is more suitable for application in the field of drug delivery.

[0005] Due to the powdery crystal structure, high brittleness and instability in water of γ-cyclodextrin metal organic framework, it faces great challenges in processing and molding, which limits its application range. Natural polymers such as cellulose and chitosan have the advantages of being degradable, renewable, flexible and easy to process and mold, and can be used as composite substrate materials for MOFs to improve their processing and molding properties and broaden their application range. As a derivative of chitin, chitosan has attracted widespread attention due to its special properties: (1) chitosan has good biocompatibility and can be biodegraded, and the degradation products are non-toxic; (2) chitosan and its derivatives have many unique properties, such as antibacterial, antimicrobial, chelating of heavy metal ions, antiviral, anticancer, and promoting wound healing. Therefore, they are expected to be used in drug release systems, wound healing materials, sewage treatment, heavy metal recovery, membrane separation, cosmetics, anticoagulant drugs, daily chemicals, etc.

[0006] Cellulose is the most abundant natural polymer compound in the world and the most widely distributed and abundant polysaccharide in nature. Nanocellulose is a nanoscale fiber or crystal extracted from natural cellulose. It has the advantages of being degradable, renewable, non-toxic and high-strength. It can be used to prepare various high-value-added advanced functional materials. According to the preparation method and source, nanocellulose can be divided into three categories: cellulose nanocrystals (CNC), cellulose nanofibers (CNF) and bacterial cellulose (BC). Cellulose nanofibers (CNF) have a small diameter and have multiple advantages such as high activity, large specific surface area, high transparency, renewable and degradable. Nanocellulose can be used to prepare nanocomposites, aerogels, hydrogels, biomedicine and other fields, and has broad application prospects.

[0007] However, most of the processing and molding of natural polymers uses aqueous solution as the medium, and the instability of cyclodextrin metal organic framework in aqueous solution makes it difficult to compound into natural polymers with a complete framework structure. Sorbitan trioleate (Span 85) is a nonionic surfactant composed of a hydrophilic hydroxyl segment and a hydrophobic long alkyl chain. Span 85 can be bound to the outer surface of the γ-cyclodextrin metal organic framework through the physical adsorption of its polar hydroxyl end and the metal center of the γ-cyclodextrin metal organic framework, and the hydrophobic long alkyl chain of Span 85 on the outer surface of the γ-cyclodextrin metal organic framework can effectively prevent water molecules from contacting MOF, thereby enhancing its moisture resistance. Since Span 85 has a long chain structure, it can be prevented from entering the internal pores of the γ-cyclodextrin metal organic framework, resulting in a decrease in the porosity of the material. A layer of Span 85 was deposited on the outer surface of the MOF through a simple solution immersion process to obtain a water-stable γ-cyclodextrin metal organic framework. This method is simple and does not require complicated chemical synthesis. In addition, Span 85 is non-chemically toxic, inexpensive, and can achieve large-scale production of moisture-resistant γ-cyclodextrin metal-organic frameworks.

[0008] In summary, we have successfully prepared a chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere loaded with camptothecin. Its particle size is uniform, with abundant specific surface area and pores, the preparation method is safe and convenient, the preparation process is green and environmentally friendly, it has excellent physicochemical properties and significant acid-induced swelling ability, and can be used as an excellent drug delivery system for camptothecin. Summary of the invention

[0009] In order to solve the problems of the prior art, the purpose of the present invention is to provide a method for preparing chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres loaded with camptothecin. The camptothecin drug delivery system prepared by the present invention has high biocompatibility, good drug loading rate, and excellent physicochemical properties. It can achieve slow release of camptothecin in the tumor microenvironment and reduce the toxicity of camptothecin itself to a certain extent.

[0010] In order to achieve the above objectives, the present invention provides the following technical solutions:

[0011] The present invention provides a method for preparing chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres loaded with camptothecin, comprising the following steps:

[0012] (1) Dissolve γ-cyclodextrin and potassium hydroxide in 50 mL of deionized water, mix them evenly by ultrasonication, filter the solution into a beaker through an aqueous disposable mixed cellulose ester filter, quickly pour in 5 mL of ethanol, place the beaker into a large beaker with ethanol solution at the bottom, seal the top of the large beaker with a sealing film, heat in a water bath for 6 hours, centrifuge to remove micron-sized crystals, quickly add polyethylene glycol 20000 and ethanol to the centrifuge, crystallize at room temperature overnight, and collect the crystals by centrifugation; wash with isopropanol and ethanol three times respectively, vacuum dry for 24 hours, and activate with dichloromethane for three days to obtain an activated basic γ-cyclodextrin metal organic framework.

[0013] (2) The prepared basic γ-cyclodextrin metal-organic framework was added to a mixed solution of acetic acid and ethanol (glacial acetic acid: anhydrous ethanol = 1:9, v / v), and shaken at room temperature for 30 minutes. The precipitate was collected by centrifugation, washed with anhydrous ethanol 5 times, and finally vacuum dried at 60°C for 24 hours to obtain a neutral γ-cyclodextrin metal-organic framework.

[0014] (3) The prepared neutral γ-cyclodextrin metal organic framework and camptothecin were dissolved in methanol solution, and the drug was loaded into the hollow neutral γ-cyclodextrin metal organic framework by a simple soaking process. The precipitate was obtained by centrifugation and vacuum dried for 12 hours. 10 mL of Span 85 was dissolved in 20 mL of dichloromethane, and then 1 g of the camptothecin-loaded γ-cyclodextrin metal organic framework was added to the solution and allowed to stand for 48 hours. After the reaction was completed, the precipitate was obtained by filtration and washed with dichloromethane several times, and finally dried under vacuum at 40°C for 12 hours.

[0015] (4) Chitosan was dissolved by freeze-thawing steps using an alkaline urine system, and 5%wt carboxylated cellulose nanofibers and epichlorohydrin were added thereto to obtain a chitosan / nanocellulose pregel solution. A certain amount of chitosan pregel solution was slowly added to an oil phase containing 200mL isooctane and 2mL Span 85, and emulsified at room temperature at 1500rpm to completely gel the chitosan / nanocellulose pregel droplets. Subsequently, the emulsion was transferred to a 2L ethanol / water (7:3, v / v) mixed solvent for demulsification, and the microspheres were collected by centrifugation. The obtained gel microspheres were repeatedly washed in anhydrous ethanol and deionized water to remove residual emulsifiers and alkaline solvents. Finally, the purified microspheres were refrigerated (4°C).

[0016] (5) The camptothecin-loaded neutral γ-cyclodextrin metal organic framework modified with Span 85 of the above step (3) is mixed with the chitosan / nanocellulose mixed solution of the above step (4) to prepare chitosan / nanocellulose / γ-cyclodextrin metal organic framework composite microspheres loaded with camptothecin by an emulsification method.

[0017] In the above technical solution, the combination of the anti-solvent and the surfactant used in step (1) is ethanol and polyethylene glycol 20000.

[0018] In the above technical solution, the size of the γ-cyclodextrin metal organic framework prepared in step (1) is 200-350nm.

[0019] In the above technical scheme, the solvents for removing excess polyethylene glycol 20000 in step (1) are isopropanol and ethanol (3 times each, 100 mL each time).

[0020] In the above technical solution, the ratio of glacial acetic acid to anhydrous ethanol in step (2) is 1:9.

[0021] In the above technical solution, the drug carried by the neutral γ-cyclodextrin metal organic framework modified by Span 85 in step (3) is camptothecin.

[0022] In the above technical solution, carboxylated cellulose nanofibers are added in step (4).

[0023] In the above technical solution, in step (4), an alkaline urine system is used as a solvent and carboxylated cellulose nanocellulose is used as a reinforcing agent to prepare chitosan / nanocellulose porous microspheres.

[0024] In the above technical solution, in step (5), the camptothecin-loaded γ-cyclodextrin metal organic framework modified with Span 85 is compounded into the chitosan / nanocellulose porous microspheres as a complete framework.

[0025] The present invention relates to a chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere loaded with camptothecin prepared by the above preparation method, wherein the particle size of the γ-cyclodextrin metal organic framework before drug loading is 200-350nm, and the particle size of the γ-cyclodextrin metal organic framework after camptothecin loading is 300-400nm, and the specific surface area of ​​the prepared activated neutral γ-cyclodextrin metal organic framework is 900-1050m 2 / g.

[0026] The chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere loaded with camptothecin prepared by the preparation method has a drug loading rate of 22%-30%.

[0027] A chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere loaded with camptothecin prepared by the above preparation method, the crystal structure and physicochemical properties of the γ-cyclodextrin metal organic framework after modification with Span 85 are not changed, and Span 85 can synergize with the γ-cyclodextrin metal organic framework to a certain extent to improve the solubility of camptothecin.

[0028] The chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres loaded with camptothecin prepared by the above preparation method have excellent mechanical properties and significant acid-induced swelling behavior.

[0029] The present invention provides a method for preparing chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres loaded with camptothecin. An alkaline γ-cyclodextrin metal organic framework with uniform particle size and excellent morphology is prepared by an improved vapor diffusion method, using ethanol as an anti-solvent and polyethylene glycol 20000 as a surfactant. Since camptothecin is unstable under alkaline conditions, acetic acid is used to neutralize the alkaline γ-cyclodextrin metal organic framework prepared above, and camptothecin is loaded in the hydrophobic cavity of the neutral γ-cyclodextrin metal organic framework by an impregnation method, which effectively improves the solubility, stability and bioavailability of the camptothecin drug. In order to solve the problem that the high solubility of γ-cyclodextrin metal organic framework in water makes it impossible to achieve slow release of camptothecin, the surface hydrophobic coating of γ-cyclodextrin metal organic framework was carried out using Span 85, and chitosan / nanocellulose porous microspheres were prepared using alkaline urine system as solvent. The chitosan / nanocellulose porous microspheres were used as matrix to load Span 85-modified neutral γ-cyclodextrin metal organic framework loaded with camptothecin. The composite material has the advantages of good biocompatibility, biodegradability and drug sustained release. The preparation method is simple and the preparation process is green and environmentally friendly, so it has good application prospects.

[0030] Compared with the prior art, the invention has the following beneficial effects: the introduction of a green and safe neutral γ-cyclodextrin metal organic framework with an internal hydrophobic cavity enhances the solubility, stability and bioavailability of camptothecin; the modification of Span 85 improves the stability of the prepared neutral γ-cyclodextrin metal organic framework in aqueous solution, ensuring that the neutral γ-cyclodextrin metal organic framework loaded with camptothecin in the subsequent water environment can be composited into the chitosan / nanocellulose porous microspheres as a complete framework. The prepared chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres loaded with camptothecin have good biocompatibility, a safe and environmentally friendly preparation process, and can achieve the slow release of camptothecin in a tumor environment. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods:

[0032] Example 1: Preparation of a neutral γ-cyclodextrin metal organic framework loaded with camptothecin, comprising the following steps:

[0033] (1) Dissolve γ-cyclodextrin (1.62 g) and potassium hydroxide (0.5611 g) in 50 mL of deionized water, mix them by ultrasonication for 30 min, filter the solution through a 0.45 μm aqueous disposable mixed cellulose ester filter into a beaker, quickly pour in 5 mL of ethanol, place the beaker into a large beaker with ethanol solution at the bottom, seal the top of the large beaker with a sealing film, heat in a 60°C water bath for 6 h, centrifuge at 12000 rpm to remove micron-sized crystals, quickly add polyethylene glycol 20000 (0.44 g) and ethanol (50 mL) to the centrifuge, crystallize overnight at room temperature, collect the crystals by centrifugation (8000 rpm), wash three times with isopropanol and ethanol, 100 mL each time, collect the crystals again, vacuum dry for 24 h, activate with dichloromethane for three days, and collect the precipitate by centrifugation (5000 rpm) to obtain the activated basic γ-cyclodextrin metal-organic framework.

[0034] (2) The basic γ-cyclodextrin metal organic framework prepared in the above (1) was neutralized with acetic acid to prepare a neutral γ-cyclodextrin metal organic framework. Weigh 0.6 g of the basic γ-cyclodextrin metal organic framework, add 20 mL of a mixed solution of acetic acid and ethanol (glacial acetic acid: anhydrous ethanol = 1:9, v / v), and shake at 400 rpm for 40 min at room temperature. Collect the precipitate by centrifugation (5000 rpm), wash it with anhydrous ethanol 5 times, and finally vacuum dry it at 60°C for 24 h to obtain a neutral γ-cyclodextrin metal organic framework. Weigh 0.1 g of the neutral γ-cyclodextrin metal organic framework, add 10 mL of pure water to dissolve it, and the pH value is about 7.0.

[0035] (3) Camptothecin (0.005 g) was dissolved in 50 mL of methanol solution, and 0.03 g of the prepared neutral γ-cyclodextrin metal organic framework was added thereto. The mixture was magnetically stirred at 25°C for 12 h. After the reaction was completed, the precipitate was collected by centrifugation (12000 rpm). The precipitate was washed with methanol 5 times, the supernatant was discarded, and the precipitate was freeze-dried to obtain the neutral γ-cyclodextrin metal organic framework loaded with camptothecin.

[0036] Example 2: A neutral γ-cyclodextrin metal organic framework loaded with camptothecin modified by Span 85, comprising the following steps:

[0037] (1) Dissolve γ-cyclodextrin (1.62 g) and potassium hydroxide (0.5611 g) in 50 mL of deionized water, mix well by ultrasonication for 30 min, filter the solution through a 0.45 μm aqueous disposable mixed cellulose ester filter into a beaker, quickly pour in 5 mL of ethanol, place the beaker into a large beaker with ethanol solution at the bottom, seal the top of the large beaker with a sealing film, heat in a 60°C water bath for 6 h, centrifuge at 12,000 rpm to remove micron-sized crystals, and quickly add polyethylene glycol to the centrifuge. Alcohol 20000 (0.44g) and ethanol (50mL), crystallize overnight at room temperature, collect crystals by centrifugation (8000rpm), wash three times with isopropanol and ethanol, 100mL each time, collect crystals again, vacuum dry for 24h, activate with dichloromethane for three days, collect precipitates by centrifugation (5000rpm) to obtain activated basic γ-cyclodextrin metal organic framework; neutralize the prepared basic γ-cyclodextrin metal organic framework with acetic acid to prepare neutral γ-cyclodextrin metal organic framework. Weigh 0.6g basic γ-cyclodextrin metal organic framework, add 20mL of acetic acid and ethanol mixed solution (glacial acetic acid: anhydrous ethanol = 1:9, v / v), and shake at 400rpm for 40min at room temperature. Collect precipitates by centrifugation (5000rpm), wash 5 times with anhydrous ethanol, and finally vacuum dry at 60℃ for 24h to obtain neutral γ-cyclodextrin metal organic framework. Weigh 0.1g of neutral γ-cyclodextrin metal organic framework, add 10mL of pure water to dissolve it, and the pH value is about 7.0. Dissolve camptothecin (0.005g) in 50mL of methanol solution, add 0.01g of the prepared neutral γ-cyclodextrin metal organic framework, stir magnetically at 25°C for 12h, and after the reaction is completed, collect the precipitate by centrifugation (12000rpm). Wash the precipitate with methanol 5 times, discard the supernatant, and freeze-dry the precipitate to obtain the neutral γ-cyclodextrin metal organic framework loaded with camptothecin.

[0038] (2) 10 mL of Span 85 was dissolved in 20 mL of dichloromethane, and then 1 g of neutral γ-cyclodextrin metal organic framework loaded with camptothecin was added to the solution and allowed to stand for 48 h. After the reaction was completed, the precipitate was filtered and washed with dichloromethane several times, and finally dried under vacuum at 40 ° C for 12 h to obtain Span 85 modified neutral γ-cyclodextrin metal organic framework loaded with camptothecin.

[0039] Example 3: Cytotoxicity test of neutral γ-cyclodextrin metal organic framework, comprising the following steps:

[0040] 100 μL of human breast cancer MDA-MB-231 cells (5x10 4After culturing for 24 hours, add γ-cyclodextrin and γ-cyclodextrin metal organic framework aqueous solution with concentrations of 0.100, 0.200, 0.400, 0.800, and 1.200 mg / mL to each well, and continue incubation for 48 hours. Then add an equal amount of MTT solution (25μL, 0.80 mg / mL) to each well, incubate for 3 hours, centrifuge at 3000 speed for 5 minutes, and remove the supernatant culture medium. Add 150uL of dimethyl sulfoxide to each well, shake for 5 minutes until the deposited purple solid is completely dissolved, and read the absorbance of the solution in each well at 562nm and 620nm using an enzyme reader (recorded as A 566 and A 620 ), the difference of absorbance at two locations is recorded as A = A 566 -A 620 , the cell survival rate was calculated according to the A value, and the calculation formula is as follows:

[0041] Cell survival rate (%) = experimental group A value x 100% / blank group A value

[0042] The results showed that the survival rate of human tumor cells MDA-MB-231 was as low as 80%, so the neutral γ-cyclodextrin metal organic framework is a safe and low-toxic material.

[0043] Example 4: Preparation of chitosan / nanocellulose porous microspheres, comprising the following steps:

[0044] (1) 4 g of chitosan was dispersed in 96 g of an alkali / urea solvent system (4.5% wt lithium hydroxide, 7% wt potassium hydroxide, 8% wt urea, 80.5% wt deionized water) and stirred for 10 min, frozen at -30°C for 6 h, then thawed with stirring at room temperature, and the completely thawed solution was centrifuged and degassed at 8000 rpm and 4°C for 10 min to obtain a clear and transparent 4% wt chitosan alkaline solution.

[0045] (2) To 100 g of the chitosan alkaline solution prepared in step (1), 1% wt of carboxylated cellulose nanofibers and 2 mL of epichlorohydrin were added successively to obtain a chitosan / nanocellulose pregel solution.

[0046] (3) 50 g of chitosan / nanocellulose pregel solution was slowly added to the oil phase containing 500 mL of isooctane and 5 mL of Span 80, and emulsified at room temperature for 3 h at 1500 rpm to completely gel the chitosan / nanocellulose pregel droplets. Subsequently, the emulsion was transferred to 2 L of ethanol / water (7:3, volume ratio) mixed solvent for demulsification, and the microspheres were collected by centrifugation. The obtained microspheres were repeatedly washed in anhydrous ethanol and deionized water to remove residual emulsifiers and alkaline solvents. Finally, the purified microspheres were refrigerated (4°C) for later use.

[0047] Example 5: Preparation of chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres loaded with camptothecin, comprising the following steps:

[0048] (1) 4 g of chitosan was dispersed in 96 g of an alkali / urea solvent system (4.5% wt lithium hydroxide, 7% wt potassium hydroxide, 8% wt urea, 80.5% wt deionized water) and stirred for 10 min, frozen at -30°C for 6 h, then thawed with stirring at room temperature, and the completely thawed solution was centrifuged and degassed at 8000 rpm and 4°C for 10 min to obtain a clear and transparent 4% wt chitosan alkaline solution.

[0049] (2) Dissolve γ-cyclodextrin (1.62 g) and potassium hydroxide (0.5611 g) in 50 mL of deionized water, mix well by ultrasonication for 30 min, filter the solution through a 0.45 μm aqueous disposable mixed cellulose ester filter into a beaker, quickly pour in 5 mL of ethanol, place the beaker into a large beaker with ethanol solution at the bottom, seal the top of the large beaker with a sealing film, heat in a 60°C water bath for 6 h, centrifuge at 12,000 rpm to remove micron-sized crystals, and quickly add polyethylene glycol to the centrifuge. Alcohol 20000 (0.44g) and ethanol (50mL), crystallize overnight at room temperature, collect crystals by centrifugation (8000rpm), wash three times with isopropanol and ethanol, 100mL each time, collect crystals again, vacuum dry for 24h, activate with dichloromethane for three days, collect precipitates by centrifugation (5000rpm) to obtain activated basic γ-cyclodextrin metal organic framework; neutralize the prepared basic γ-cyclodextrin metal organic framework with acetic acid to prepare neutral γ-cyclodextrin metal organic framework. Weigh 0.6g basic γ-cyclodextrin metal organic framework, add 20mL of acetic acid and ethanol mixed solution (glacial acetic acid: anhydrous ethanol = 1:9, v / v), and shake at 400rpm for 40min at room temperature. Collect precipitates by centrifugation (5000rpm), wash 5 times with anhydrous ethanol, and finally vacuum dry at 60℃ for 24h to obtain neutral γ-cyclodextrin metal organic framework. Weigh 0.1g of neutral γ-cyclodextrin metal organic framework, add 10mL of pure water to dissolve it, and the pH value is about 7.0. Camptothecin (0.005g) is dissolved in 50mL of methanol solution, and 0.01g of the prepared neutral γ-cyclodextrin metal organic framework is added thereto. The solution is stirred magnetically at 25°C for 12h. After the reaction is completed, the precipitate is collected by centrifugation (12000rpm). The precipitate is washed with methanol 5 times, the supernatant is discarded, and the precipitate is freeze-dried to obtain a neutral γ-cyclodextrin metal organic framework loaded with camptothecin. 10mL of Span 85 is dissolved in 20mL of dichloromethane, and then 1g of neutral γ-cyclodextrin metal organic framework loaded with camptothecin is added to the solution and allowed to stand for 48h. After the reaction is completed, the precipitate is obtained by filtration and washed several times with dichloromethane, and finally dried under vacuum at 40°C for 12h to obtain a neutral γ-cyclodextrin metal organic framework loaded with camptothecin modified by Span 85.

[0050] (3) To 100 g of the chitosan alkaline solution prepared in step (1), 1% wt of carboxylated cellulose nanofibers, 1 g of the neutral γ-cyclodextrin metal organic framework loaded with camptothecin modified by Span 85 prepared in step (2), and 2 mL of epichlorohydrin were added successively to obtain a chitosan / nanocellulose / γ-cyclodextrin metal organic framework@camptothecin solution.

[0051] (4) 50 g of chitosan / nanocellulose / γ-cyclodextrin metal organic framework @ camptothecin pre-solution was slowly added to the oil phase containing 500 mL of isooctane and 5 mL of Span 80, and emulsified at room temperature for 3 h at 1500 rpm to completely gel the chitosan / nanocellulose / γ-cyclodextrin metal organic framework @ camptothecin microdroplets. Subsequently, the emulsion was transferred to 2 L of ethanol / water (7:3, volume ratio) mixed solvent for demulsification, and the microspheres were collected by centrifugation. The obtained microspheres were repeatedly washed in anhydrous ethanol and deionized water to remove residual emulsifiers and alkaline solvents. Finally, the purified microspheres were refrigerated (4°C) for use.

Claims

1. A method for preparing chitosan nanocellulose γ-cyclodextrin metal organic framework porous composite microspheres loaded with camptothecin, the preparation method comprising the following steps: (1) Dissolve γ-cyclodextrin and potassium hydroxide in 50 ml of deionized water, mix them evenly by ultrasonication, filter the solution into a beaker through an aqueous disposable mixed cellulose ester filter, quickly pour in 5 ml of ethanol, place the beaker into a large beaker with ethanol solution at the bottom, seal the top of the large beaker with a sealing film, heat in a water bath for 6 hours, centrifuge to remove micron-sized crystals, quickly add polyethylene glycol 20000 and ethanol to the centrifuge, crystallize at room temperature overnight, collect the crystals by centrifugation; wash with isopropanol and ethanol three times respectively, vacuum dry for 24 hours, and activate with dichloromethane for three days to obtain an activated basic γ-cyclodextrin metal organic framework. (2) The prepared basic γ-cyclodextrin metal-organic framework was added to a mixed solution of acetic acid and ethanol (glacial acetic acid: anhydrous ethanol = 1:9, v / v), and shaken at room temperature for 40 minutes. The precipitate was collected by centrifugation, washed with anhydrous ethanol 5 times, and finally vacuum dried at 60°C for 24 hours to obtain a neutral γ-cyclodextrin metal-organic framework. (3) The prepared neutral γ-cyclodextrin metal organic framework and camptothecin were dissolved in a methanol solution, and the drug was loaded into the hollow neutral γ-cyclodextrin metal organic framework by a simple soaking process. The precipitate was obtained by centrifugation and freeze-dried to obtain the neutral γ-cyclodextrin metal organic framework loaded with camptothecin. 10 ml of Span 85 was dissolved in 20 ml of dichloromethane, and then the γ-cyclodextrin metal organic framework loaded with camptothecin was added to the solution and allowed to stand for 48 hours. After the reaction was completed, the precipitate was obtained by filtration and washed with dichloromethane several times, and finally dried under vacuum at 40°C for 12 hours. (4) Chitosan was dissolved by freeze-thawing steps using an alkaline urine system, and 5%wt carboxylated cellulose nanofibers and epichlorohydrin were added thereto to obtain a chitosan / nanocellulose pregel solution. A certain amount of chitosan pregel solution was slowly added to an oil phase containing 200ml isooctane and 2ml Span 80, and emulsified at room temperature at 1500rpm / min to completely gel the chitosan / nanocellulose pregel droplets. Subsequently, the emulsion was transferred to a 2L ethanol / water (7:3, v / v) mixed solvent for demulsification, and the microspheres were collected by centrifugation. The obtained gel microspheres were repeatedly washed in anhydrous ethanol and deionized water to remove residual emulsifiers and alkaline solvents. Finally, the purified microspheres were refrigerated (4°C). (5) The camptothecin-loaded neutral γ-cyclodextrin metal organic framework modified with Span 85 of the above step (3) is mixed with the chitosan / nanocellulose mixed solution of the above step (4) to prepare chitosan / nanocellulose / γ-cyclodextrin metal organic framework composite microspheres loaded with camptothecin by an emulsification method.

2. The method for preparing a camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere according to claim 1, characterized in that: The combination of the anti-solvent and the surfactant used in step (1) is ethanol and polyethylene glycol 20000.

3. The method for preparing a camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere according to claim 1, characterized in that: The size of the γ-cyclodextrin metal organic framework prepared in step (1) is 200-350 nm.

4. The method for preparing a camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere according to claim 1, characterized in that: The solvents for removing excess polyethylene glycol 20000 in step (1) are isopropanol and ethanol (3 times each, 100 ml each time).

5. The method for preparing a camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere according to claim 1, characterized in that: In step (2), the ratio of glacial acetic acid to anhydrous ethanol is 1:

9.

6. The method for preparing a camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere according to claim 1, characterized in that: In step (3), the drug carried in the neutral γ-cyclodextrin metal organic framework modified by Span 85 is camptothecin.

7. The method for preparing a camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere according to claim 1, characterized in that: In step (4), carboxylated cellulose nanofibers are added.

8. The method for preparing a camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere according to claim 1, characterized in that: In step (4), an alkaline urine system is used as a solvent and carboxylated cellulose nanocellulose is used as a reinforcing agent to prepare chitosan / nanocellulose porous microspheres.

9. The method for preparing a camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microsphere according to claim 1, characterized in that: Step (5) The camptothecin-loaded γ-cyclodextrin metal organic framework modified by Span 85 is composited into the chitosan / nanocellulose porous microspheres as a complete framework.

10. The camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres prepared by the preparation method according to any one of claims 1 to 9, characterized in that: The particle size of the γ-cyclodextrin metal organic framework before drug loading is 200-350nm, while the particle size of the γ-cyclodextrin metal organic framework after camptothecin loading is 300-400nm. (1) The chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres loaded with camptothecin prepared by the preparation method according to any one of claims 1 to 9, characterized in that: The specific surface area of ​​the activated neutral γ-cyclodextrin metal organic framework prepared is 900-1050m 2 / g. (2) The camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres prepared by the preparation method according to any one of claims 1 to 9, characterized in that the drug loading rate is 22%-30%. (3) The camptothecin-loaded chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres prepared by the preparation method according to any one of claims 1 to 9 are characterized in that the crystal structure and physicochemical properties of the γ-cyclodextrin metal organic framework after modification with Span 85 are not changed, and Span 85 can synergize with the γ-cyclodextrin metal organic framework to a certain extent to improve the solubility of camptothecin. (4) The chitosan / nanocellulose / γ-cyclodextrin metal organic framework porous composite microspheres loaded with camptothecin prepared by the preparation method according to any one of claims 1 to 9, characterized in that the prepared chitosan / nanocellulose porous microspheres have excellent mechanical properties and significant acid-induced swelling behavior.