Preparation and application of active oxygen response type cyclodextrin covalent skeleton

Abstract

The invention provides preparation and application of a cyclodextrin covalent skeleton with active oxygen sensitivity. Specifically, the invention designs a cyclodextrin covalent skeleton bonded by thioketal, and aims to construct an intelligent drug delivery system with active oxygen response and removal. The cyclodextrin covalent skeleton disclosed by the invention can realize active oxygen responsive disintegration in an active oxygen environment, can effectively remove the active oxygen level, has good biological safety and inflammation targeting property, and can enrich anti-inflammatory drugs at colon focus parts to relieve the development and deterioration of colitis diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical biomaterials, and more particularly to the preparation and application of a reactive oxygen species-responsive cyclodextrin covalent framework. Background Art

[0002] Excessive and uncontrolled inflammatory responses can cause various oxidative damages to cells, tissues, and organs, and then cause systemic harm to the body. In particular, inflammatory bowel disease is an inflammatory disease with a complex pathogenesis and recurrence, and has now become a major global health problem. A large amount of reactive oxygen species (ROS) is generated at the inflammatory site of ulcerative colitis, and its concentration is as high as 10-100 times that of normal tissues. Excessive ROS will accelerate cell apoptosis and speed up the occurrence and development of inflammation. Therefore, reducing the ROS level to relieve oxidative stress is an important strategy for treating inflammatory diseases. Limited by the characteristics of low transport efficiency and non-specific targeting of traditional drug systems, stimulus-responsive drug carriers have been widely used for precise drug delivery to reduce systemic drug exposure, improve the safety and effectiveness of drugs, and have important clinical significance for the precise treatment of inflammatory diseases. In recent years, ROS-stimulated responsive drug delivery has been used to respond to the physiological oxidative microenvironment, not only limitedly relieving the oxidative stress level, but also improving the enrichment and retention of drugs at the inflammatory site.

[0003] Metal-organic frameworks (MOFs) are highly competitive in drug loading in porous materials. However, tens of thousands of MOFs are prepared from toxic metal ions and chemical materials whose safety remains to be verified, and their "drugability" is poor. Cyclodextrin (CD)-metal-organic framework (CD-MOF) assembled by coordination of CD with potassium ions has the characteristics of good biocompatibility, high porosity, and amphiphilic nano-spaces on the basis of retaining the inherent advantages of MOF. Its drug delivery characteristics are novel and efficient, and it can be developed into a new type of pharmaceutical excipient and drug carrier. However, the coordination bond formed between the organic ligand CD and potassium ions is unstable in water, resulting in the rapid disintegration of CD-MOF in a humid environment, which greatly limits the application of CD-MOF in the controlled release delivery of drugs in a physiological environment. It is worth noting that the hydroxyl groups on CD in CD-MOF can provide modification and functionalization sites, and preparing crosslinked CD-MOF is one of the effective strategies to improve the water stability of CD-MOF.

[0004] However, there is no porous MOF-based material in the art that is responsive to reactive oxygen species, has excellent biocompatibility, and can perform controlled release delivery of drugs. Summary of the Invention

[0005] The object of the present invention is to provide a ROS-responsive covalent cyclodextrin framework material TOF for drug delivery in inflammatory diseases.

[0006] In a first aspect of the present invention, there is provided a cyclodextrin covalent framework prepared by crosslinking using a thiosulfonate crosslinking agent with cyclodextrin or cyclodextrin metal-organic framework as a basic structural unit.

[0007] In another preferred embodiment, the thiosulfonate crosslinking agent is a compound of formula I as follows:

[0008]

[0009] Wherein, n1 and n2 are each independently 1, 2, 3 or 4.

[0010] In another preferred embodiment, n1 is equal to or not equal to n2.

[0011] In another preferred embodiment, the compound of formula I is a symmetric compound.

[0012] In another preferred embodiment, the crosslinking agent is selected from the group consisting of:

[0013]

[0014] In another preferred embodiment, the crosslinking is prepared by the method described in the third aspect of the present invention.

[0015] In another preferred embodiment, the thiosulfonate crosslinking agent crosslinks the hydroxyl groups in cyclodextrin or cyclodextrin metal-organic framework to obtain a cyclodextrin covalent framework.

[0016] In another preferred embodiment, the cyclodextrin covalent framework is in an amorphous state.

[0017] In another preferred embodiment, the cyclodextrin covalent framework remains stable within 150 - 220 °C (preferably 200 °C).

[0018] In another preferred embodiment, the cyclodextrin covalent framework removes the coordination ions in the basic structural unit of the cyclodextrin metal-organic framework, preferably K + .

[0019] In another preferred embodiment, the cyclodextrin covalent framework is prepared by the method described in the second aspect of the present invention.

[0020] In another preferred embodiment, the cyclodextrin is selected from the group consisting of: γ-CD, β-CD, α-CD, or a combination thereof.

[0021] In another preferred embodiment, the cyclodextrin metal-organic framework is a cyclodextrin metal-organic framework formed by cyclodextrin and alkali metal ions.

[0022] In another preferred example, the alkali metal ion is selected from the group consisting of: K + , Li + , Na + , Mg 2+ , Ca 2+ , or a combination thereof.

[0023] In another preferred example, the cyclodextrin metal-organic framework is a micron-scale cyclodextrin metal-organic framework or a nano-scale cyclodextrin metal-organic framework.

[0024] In another preferred example, the cyclodextrin metal-organic framework is the cyclodextrin metal-organic framework prepared by the method according to Patent CN201610125456.X.

[0025] In another preferred example, the size of the micron-scale cyclodextrin metal-organic framework is 1 - 10 μm.

[0026] In another preferred example, the size of the nano-scale cyclodextrin metal-organic framework is 100 - 1000 nm.

[0027] In another preferred example, the cyclodextrin metal-organic framework is a metal-organic framework formed by the coordination of KOH and γ-CD.

[0028] In another preferred example, the cyclodextrin covalent framework is a cyclodextrin covalent framework responsive to reactive oxygen species.

[0029] In another preferred example, the cyclodextrin covalent framework rapidly degrades in an environment with H2O2.

[0030] In another preferred example, the cyclodextrin covalent framework rapidly degrades in an environment with H2O2 in a H2O2 concentration-dependent manner, that is, the higher the H2O2 concentration, the higher the degradation rate.

[0031] In another preferred example, in an environment with H2O2, the degradation rate of the cyclodextrin covalent framework exceeds 75% within 12 h, preferably exceeds 80%, and more preferably exceeds 90%.

[0032] In another preferred example, a large number of sulfhydryl groups are exposed after the degradation of the cyclodextrin covalent framework.

[0033] In another preferred example, the sulfhydryl group content after the degradation of the cyclodextrin covalent framework is 50 - 500 μmol / g, preferably 70 - 200 μmol / g.

[0034] In another preferred example, substantial dissociation occurs after the degradation of the cyclodextrin covalent framework. Preferably, it changes from the previous regular cubic structure to an irregular morphology.

[0035] In another preferred embodiment, the cyclodextrin covalent framework has no obvious cytotoxicity, and preferably has a cell survival rate higher than 80% at 1 - 500 μg / mL (preferably 2 - 400 μg / mL).

[0036] In another preferred embodiment, since the cyclodextrin covalent framework has reactive oxygen species-stimulating responsiveness, it has targeting ability to inflammatory sites, especially targeting ability to colon inflammation.

[0037] In another preferred embodiment, the cyclodextrin covalent framework has good biocompatibility. Preferably, after continuous administration 5 times, no pathological changes are observed in each organ and / or the blood cell-related counts do not change.

[0038] In a second aspect of the present invention, there is provided a method for preparing the cyclodextrin covalent framework as described in the first aspect of the present invention, comprising the following steps:

[0039] In a solvent, an activated crosslinking agent is mixed with cyclodextrin or a cyclodextrin metal-organic framework, and the reaction gives the cyclodextrin covalent framework.

[0040] In another preferred embodiment, the solvent, the first solvent, and the second solvent are each independently selected from the group consisting of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane, chloroform, toluene, or a combination thereof.

[0041] In another preferred embodiment, the first solvent and the second solvent are the same or different.

[0042] In another preferred embodiment, the cyclodextrin and the cyclodextrin metal-organic framework are each independently as described in the first aspect of the present invention.

[0043] In another preferred embodiment, the method comprises the following steps:

[0044] (s1) In a second solvent, in the presence of a first catalyst, an activated crosslinking agent is mixed with cyclodextrin or a cyclodextrin metal-organic framework, and the reaction gives the cyclodextrin covalent framework.

[0045] In another preferred embodiment, the method two optionally further comprises the step of preparing an activated crosslinking agent.

[0046] In another preferred embodiment, the step of preparing the activated crosslinking agent comprises the following steps:

[0047] (s0) In a first solvent, a crosslinking agent is mixed with an activator, and the reaction gives an activated crosslinking agent.

[0048] In another preferred embodiment, in step (s0), the activator is selected from the group consisting of acyl chlorinating reagents, N,N-carbonyldiimidazole, diphenyl carbonate, triphosgene, or a combination thereof.

[0049] In another preferred example, the acyl chloride reagent is selected from the group consisting of: oxalyl chloride, phosphorus trichloride, phosphorus pentachloride, thionyl chloride, or a combination thereof.

[0050] In another preferred example, in step (s0), the activator is oxalyl chloride or N,N-carbonyldiimidazole.

[0051] In another preferred example, in step (s0), when the activator is an acyl chloride reagent, the molar ratio of the crosslinking agent to the activator in the feed is 1:2 to 1:15, such as 1:5, 1:8, 1:10, 1:12.

[0052] In another preferred example, in step (s0), when the activator is selected from the group consisting of: N,N-carbonyldiimidazole, diphenyl carbonate, triphosgene, or a combination thereof, the molar ratio of the crosslinking agent to the activator in the feed is 1:1 to 1:5, such as 1:2, 1:3, 1:4.

[0053] In another preferred example, in step (s0), the reaction is carried out at 10 - 50 °C, preferably 20 - 45 °C.

[0054] In another preferred example, in step (s0), the reaction time of the reaction is 2 - 24 h, preferably 2 - 12 h, such as 2 - 4 h.

[0055] In another preferred example, step (s0) includes the following steps: dissolving the crosslinking agent and the activator in a first solvent, reacting at a certain temperature for a certain time to obtain an activated crosslinking agent, and optionally performing a simple post-treatment before proceeding to the next step of the reaction.

[0056] In another preferred example, the simple post-treatment includes rotary evaporation to remove the solvent and excess reactants.

[0057] In another preferred example, when the activator is selected from the group consisting of: N,N-carbonyldiimidazole, diphenyl carbonate, triphosgene, or a combination thereof, the reaction mixture containing the activated crosslinking agent obtained in step (s0) directly proceeds to the next step of the reaction.

[0058] In another preferred example, in step (s1), the cyclodextrin is γ-CD, β-CD, α-CD, or a combination thereof, preferably γ-CD.

[0059] In another preferred example, in step (s1), the cyclodextrin metal-organic framework is a micron-scale cyclodextrin metal-organic framework or a nano-scale cyclodextrin metal-organic framework.

[0060] In another preferred example, in step (s1), when the activator is an acyl chloride reagent, the first catalyst is N,N-dimethylformamide.

[0061] In another preferred example, in step (s1), when the activator is selected from the group consisting of N,N-carbonyldiimidazole, diphenyl carbonate, triphosgene, or a combination thereof, the first catalyst is selected from the group consisting of pyridine, triethylamine, or a combination thereof.

[0062] In another preferred example, in step (s1), the molar ratio of the cyclodextrin or cyclodextrin metal-organic framework to the activated crosslinking agent is 1:2 to 1:20, preferably 1:4 to 1:18, such as 1:6, 1:8, 1:10, 1:12, 1:14.

[0063] In another preferred example, in step (s1), the reaction is carried out at -5 to 100 °C, preferably at 0 to 35 °C or 50 to 80 °C.

[0064] In another preferred example, in step (s1), when the activator is an acyl chloride reagent, the reaction is carried out at 0 to 35 °C.

[0065] In another preferred example, in step (s1), when the activator is selected from the group consisting of N,N-carbonyldiimidazole, diphenyl carbonate, triphosgene, or a combination thereof, the reaction is carried out at 45 to 80 °C.

[0066] In another preferred example, in step (s1), the reaction time of the reaction is 10 to 48 h, preferably 12 to 32 h, such as 12 to 24 h.

[0067] In another preferred example, step (s1) includes the steps of: adding cyclodextrin or cyclodextrin metal-organic framework to an activated crosslinking agent solution, adding a first catalyst, reacting at a certain temperature for a certain time, and post-treating to obtain the cyclodextrin covalent framework.

[0068] In another preferred example, the post-treatment includes one or more of the following steps: cooling, quenching the reaction with ethanol, precipitation by centrifugation, washing, and drying.

[0069] In another preferred example, the washing detergent includes one or more of dichloromethane, ethanol, 50% ethanol, and water.

[0070] In the third aspect of the present invention, a preparation method of a thioacetal-based crosslinking agent is provided, including the following steps:

[0071] In the presence of a second catalyst, mixing a compound of formula A, a compound of formula B with acetone, and reacting to obtain a compound of formula I;

[0072]

[0073] In another preferred example, the compound of formula A and the compound of formula B are the same or different.

[0074] In another preferred embodiment, the molar ratio of the compound of formula A, the compound of formula B, and acetone is (0.5 - 1):(0.5 - 1):1, preferably 1:1:2.

[0075] In another preferred embodiment, the second catalyst is selected from the group consisting of benzenesulfonic acid, p-toluenesulfonic acid, sulfuric acid, phosphoric acid, hydrochloric acid, or a combination thereof.

[0076] In another preferred embodiment, the reaction is carried out at 5 - 45 °C, preferably 10 - 35 °C.

[0077] In another preferred embodiment, the reaction time of the reaction is 2 - 10 h, preferably 3 - 8 h, for example 6 h.

[0078] In another preferred embodiment, the method further comprises a post-treatment step: quenching the reaction, crystallization, filtration, washing, and drying.

[0079] In a fourth aspect of the present invention, a drug delivery system is provided, which comprises:

[0080] (a) The cyclodextrin covalent backbone described in the first aspect of the present invention;

[0081] (b) A drug loaded in the cyclodextrin covalent backbone.

[0082] In another preferred embodiment, the drug is a drug for preventing and / or treating inflammation, such as dexamethasone and mometasone furoate.

[0083] In another preferred embodiment, the loading amount of the drug delivery system is 1% - 30%, preferably 3% - 15%, for example 5%, 6%, 7%, 8%, 9%, 10%.

[0084] In another preferred embodiment, the drug in the drug delivery system has the property of active oxygen-responsive release.

[0085] In another preferred embodiment, the drug in the drug delivery system is released slowly in the gastrointestinal environment.

[0086] In another preferred embodiment, the release of the drug in the drug delivery system in an environment with H2O2 is increased by at least 10%, preferably 20%, preferably in the presence or absence of H2O2 in the colon.

[0087] In another preferred embodiment, the drug delivery system has no obvious cytotoxicity. Preferably, the cell survival rate is higher than 80% at 1 - 500 μg / mL (preferably 2 - 400 μg / mL).

[0088] In another preferred embodiment, the mucosal adhesiveness of the drug delivery system under the stimulation of reactive oxygen species is increased by at least 1.3 times, preferably at least 1.6 times, compared with that without the stimulation of reactive oxygen species.

[0089] In another preferred embodiment, due to the reactive oxygen species-stimulus responsiveness, the drug delivery system has targeting property to the inflammatory site, especially targeting property to the colon inflammatory site. After 24 hours of administration, it still resides at the inflammatory site.

[0090] In another preferred embodiment, the drug delivery system has good biocompatibility. Preferably, after continuous administration 5 times, no pathological changes are observed in each organ and / or the blood cell-related counts do not change.

[0091] In another preferred embodiment, the drug delivery system has targeting property to the inflammatory site, preferably targeting property to the colon inflammatory site.

[0092] In another preferred embodiment, the drug delivery system does not accumulate in other organs, such as major organs including the heart, liver, spleen, lung, and kidney.

[0093] In another preferred embodiment, the content of the drug delivery system at the colon inflammatory site of patients with colitis is 1.5 times higher, preferably 2 times higher, than the content in the colon of normal subjects.

[0094] In another preferred embodiment, after 24 hours of administration, the drug delivery system still remains at the colon inflammatory site.

[0095] In the fifth aspect of the present invention, there is provided a method for preparing the drug delivery system as described in the fourth aspect of the present invention, comprising the steps of:

[0096] Mixing the cyclodextrin covalent framework as described in the first aspect of the present invention with a drug to obtain the drug delivery system.

[0097] In another preferred embodiment, the drug is as described in the fourth aspect of the present invention.

[0098] In another preferred embodiment, the method is carried out in a solvent selected from the group consisting of methanol, ethanol, tetrahydrofuran, chloroform, dichloromethane, or a combination thereof.

[0099] In another preferred embodiment, the mass ratio of the cyclodextrin covalent framework to the drug is (0.5 - 1.5):(0.5 - 1.5), preferably (0.8 - 1.2):(0.8 - 1.2), such as 1:1.

[0100] In the sixth aspect of the present invention, there is provided a pharmaceutical composition comprising: the drug delivery system as described in the fourth aspect of the present invention; and a pharmaceutically acceptable carrier.

[0101] In the seventh aspect of the present invention, there is provided the use of the cyclodextrin covalent framework as described in the first aspect of the present invention, the drug delivery system as described in the fourth aspect of the present invention, or the pharmaceutical composition as described in the sixth aspect of the present invention in the preparation of a drug for (a) scavenging reactive oxygen species; (b) anti-inflammation; and / or (c) preventing and / or treating colitis.

[0102] In another preferred example, the colitis is ulcerative colitis, more preferably acute ulcerative colitis.

[0103] In another preferred example, the reactive oxygen species include one or more of superoxide anion, DPPH·, and OH·.

[0104] In another preferred example, the scavenging of reactive oxygen species is dose-dependent scavenging of reactive oxygen species.

[0105] In another preferred example, the anti-inflammation has one or more characteristics selected from the following group:

[0106] (a) reducing the content of IL-1β in cells;

[0107] (b) reducing the secretion of NO by cells.

[0108] In another preferred example, the prevention and / or treatment of colitis has one or more characteristics selected from the following group:

[0109] (a) increasing the colon length;

[0110] (b) reducing the decrease in body weight;

[0111] (c) reducing the disease index.

[0112] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 It is the mass spectrum of TK7 in Example 1.

[0114] Figure 2 It is the 1H NMR spectrum of TK7 in Example 1.

[0115] Figure 3 It is the infrared spectrum of TK7 in Example 1.

[0116] Figure 4 It is the 1H NMR spectrum of TK9 in Example 1.

[0117] Figure 5 It is the infrared spectrum of oxalyl chloride-activated TOF in Example 1.

[0118] Figure 6 Powder crystal diffraction pattern of TOF activated by oxalyl chloride in Example 1.

[0119] Figure 7 Thermogravimetric diagram of TOF activated by oxalyl chloride in Example 1.

[0120] Figure 8 X-ray photoelectron spectroscopy of TOF activated by oxalyl chloride in Example 1.

[0121] Figure 9 Infrared spectrum of TOF activated by CDI in Example 1.

[0122] Figure 10 X-ray photoelectron spectroscopy of TOF activated by CDI in Example 1.

[0123] Figure 11 Infrared spectrum of COF in Example 2.

[0124] Figure 12 H2O2-responsive degradation curve of TOF activated by oxalyl chloride in Example 3.

[0125] Figure 13 H2O2-responsive degradation curve of TOF activated by CDI in Example 3.

[0126] Figure 14 In vitro cumulative release curve of dexamethasone in Example 4.

[0127] Figure 15 Superoxide anion scavenging curves of TOF activated by oxalyl chloride and DEX@TOF in Example 4.

[0128] Figure 16 DPPH· scavenging curves of TOF activated by oxalyl chloride and DEX@TOF in Example 4.

[0129] Figure 17 OH· scavenging curves of TOF activated by oxalyl chloride and DEX@TOF in Example 4.

[0130] Figure 18 DPPH· scavenging curve of TOF activated by CDI in Example 4.

[0131] Figure 19 OH· scavenging curve of TOF activated by CDI in Example 4.

[0132] Figure 20 Cytotoxicity evaluation of TOF, COF, DEX@COF or DEX@TOF against RAW 264.7, Caco-2, HT-29 cells in Example 4.

[0133] Figure 21 To investigate the effects of TOF, COF, DEX@COF, or DEX@TOF in Example 4 on the release of inflammatory factors from lipopolysaccharide-stimulated RAW 264.7 cells.

[0134] Figure 22 To show the fluorescence images of rhodamine B-labeled DEX@COF or DEX@TOF adhering to the intestinal mucosa of ex vivo mice in Example 5.

[0135] Figure 23 To present the representative in vivo imaging pictures of the heart, liver, spleen, lung, kidney, stomach, small intestine, and colon tissues of colitis mice at different time points after intragastric administration of Cy5-labeled DEX@TOF in Example 6.

[0136] Figure 24 To display the representative in vivo imaging pictures of Cy5-labeled DEX@TOF and DEX@COF in the colon of colitis mice at 6 h and 12 h after intragastric administration in Example 6.

[0137] Figure 25 To illustrate the schematic diagram of the dosing regimen for efficacy evaluation (A), pictures of the colon of mice in each group (B), quantitative analysis of colon length (C), changes in the percentage of mouse body weight (D), and changes in the disease index (E) in Example 7.

[0138] Figure 26 To show the changes in body weight of mice in each group for safety evaluation in Example 8.

[0139] Figure 27 To present the blood indices of mice in each group for safety evaluation in Example 8.

[0140] Figure 28 To show the HE pictures of tissues of mice in each group for safety evaluation in Example 8. Detailed implementation mode

[0141] After extensive and in-depth research, the present inventors have first discovered a cyclodextrin covalent framework with excellent reactive oxygen species (ROS) stimulus responsiveness, which is obtained by crosslinking using a thioacetal crosslinker with cyclodextrin or cyclodextrin metal-organic framework as the basic structural unit.

[0142] This cyclodextrin covalent framework has excellent ROS stimulus responsiveness and ROS scavenging ability. It can not only respond to degrade under ROS stimulation and thus release drugs in a responsive manner after loading drugs, but also has a strong ability to scavenge ROS even without loading drugs, showing excellent anti-inflammatory effects.

[0143] The cyclodextrin covalent framework has excellent targeting to colon inflammation, and due to the formation of sulfhydryl groups after degradation, it forms disulfide bonds with proteins, thus having good mucosal adhesiveness and further increasing the residence time at the colon inflammation site. Therefore, it becomes an efficient treatment plan for colon inflammation and has broad application prospects. On this basis, the inventor completed the present invention.

[0144] Term

[0145] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0146] As used herein, the terms "comprising", "including", "containing" can be used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the said terms include "consisting of" and "consisting essentially of".

[0147] The cyclodextrin covalent framework of the present invention

[0148] In the present invention, a thioacetal molecule responsive to reactive oxygen species (ROS) is used as a cross-linking agent to covalently cross-link the active hydroxyl groups on CD-MOF, and a thioacetal-embedded cyclodextrin covalent organic framework (TOF) is prepared. TOF retains the supramolecular loading capacity in CD-MOF and can achieve the delivery of various drugs. By regulating different synthesis strategies, thioacetal groups with ROS responsiveness can be successfully introduced into CD-MOF, which greatly improves the water stability of CD-MOF and also endows it with ROS responsiveness and scavenging ability to meet the required drug delivery. More importantly, ROS at the inflammation site oxidizes and cleaves the thioacetal groups in TOF, exposing sulfhydryl groups with mucosal adhesion ability, which can prolong the residence time of nanoparticles at the inflammation site. On the other hand, compared with cross-linked CD-MOF without ROS sensitivity, TOF has good retention ability at the inflammation lesion and excellent anti-inflammatory and antioxidant abilities, effectively alleviating the development process of inflammatory diseases.

[0149] The TOF carrier described in the present invention is different from the reported functionalized materials of cross-linked CD-MOF. For example, Furukawa et al. (Angew Chem Int Edit, 2012, 51(42): 10566-10569) introduced the cross-linking agent ethylene glycol diglycidyl ether, and Singh et al. (Rsc Adv, 2017, 7(34): 20789-20794) introduced the cross-linking agent diphenyl carbonate to prepare cubic cross-linked CD-MOF, which only improved the water stability of CD-MOF but did not have ROS responsiveness; Huang et al. (Carbohyd Polym, 2022, 285: 119252) prepared cross-linked CD-MOF using borate ester bond as the cross-linking agent, which had ROS responsiveness, but the product morphology was damaged, being spherical particles of about 100 nm, and the dosage was large; He et al. (Acs Appl Mater Inter, 2022, 14(34): 38421-38435) Although the ROS-responsive cross-linked CD-MOF prepared with oxalyl chloride as the cross-linking agent had controllable morphology, its stability in the physiological environment was weak and it was difficult to achieve inflammation retention. In current research, no ROS-responsive cross-linked CD-MOF constructed with thioacetal as the cross-linking agent and CD-MOF as the backbone has been found.

[0150] The present invention provides a cyclodextrin covalent framework prepared with a thioacetal cross-linking agent, and the preparation method includes the following steps:

[0151] (1) Prepare CD-MOF with different particle sizes according to Patent CN201610125456.X;

[0152] (2) Preparation of thioacetal cross-linking agent: 2-mercaptoacetic acid or 3-mercaptopropionic acid, anhydrous acetone and a catalytic amount of p-toluenesulfonic acid are respectively added to a round-bottom flask, reacted at room temperature, and after the reaction is completed, the reaction solution is quenched in an ice bath until crystals completely precipitate, filtered by suction, washed three times with ice hexane and cold water, and then freeze-dried to obtain a white powder thioacetal cross-linking agent.

[0153] (3) Activation of thioacetal cross-linking agent: The thioacetal cross-linking agent and oxalyl chloride are combined and dissolved in a certain amount in Solvent 1, and a catalytic amount of N,N-dimethylformamide is added dropwise, and reacted at a certain temperature 1 for a certain time 1, and the residual solvent and excessive reactants are removed to obtain thioacetal activated cross-linking agent 1. Alternatively, the thioacetal cross-linking agent and the activator are combined and added to Solvent 2, and reacted at a certain temperature 2 for a certain time 2 to obtain thioacetal activated cross-linking agent 2;

[0154] (4) Preparation of cyclodextrin covalent framework: Dissolve the thioacetal bond crosslinking agent 1 and CD-MOF in a certain amount in solvent 3 and react at a certain temperature 3 for a certain time 3 to obtain the oxalyl chloride-activated cyclodextrin covalent framework TOF. Alternatively, in solvent 4, add cyclodextrin or cyclodextrin metal-organic framework, thioacetal-activated crosslinking agent 2, and add a certain amount of catalyst. By controlling the reaction temperature 4 and reaction time 4, obtain the cyclodextrin covalent framework TOF.

[0155] The cyclodextrin described above includes one or more of γ-CD, β-CD, and α-CD.

[0156] The CD-MOF described above includes the CD-MOF formed by CD and alkali metal ions, preferably the CD-MOF composed of γ-CD and potassium ions.

[0157] The particle size of the nanoscale CD-MOF is 100 - 1000 nm.

[0158] The particle size of the micron-scale CD-MOF is 1 - 10 μm.

[0159] In step (3), the molar ratio of the thioacetal bond crosslinking agent to oxalyl chloride is 1:2 - 1:10, preferably 1:10.

[0160] In step (3), the solvent 1 is selected from the following group: dichloromethane, tetrahydrofuran, or a combination thereof.

[0161] In step (3), the temperature 1 is 0 - 25 °C, preferably 25 °C.

[0162] In step (3), the time 1 is 2 - 12 h, preferably 4 h.

[0163] In step (3), the linker or activator includes but is not limited to N,N-carbonyldiimidazole, diphenyl carbonate, triphosgene.

[0164] In step (3), the ratio range of the thioacetal crosslinking agent to the linker or activator used is 1:1 - 1:6.

[0165] In step (3), the solvent 2 includes but is not limited to tetrahydrofuran, dichloromethane, N,N-dimethylformamide.

[0166] In step (3), the reaction temperature 2 is 25 - 40 °C, and the reaction time 2 is 2 - 24 h.

[0167] In step (4), the molar ratio of the CD-MOF to the thioacetal bond crosslinking agent 1 is 1:6 - 1:18, preferably 1:12.

[0168] In step (4), the solvent 3 is selected from the following group: dichloromethane, tetrahydrofuran, or a combination thereof.

[0169] In step (4), the temperature 3 is 0 - 30 °C, preferably 25 °C.

[0170] In step (4), the time 3 is 12 - 48 h, preferably 24 h.

[0171] In step (4), the solvent 4 includes but is not limited to N,N - dimethylformamide and dimethyl sulfoxide.

[0172] In step (4), the molar ratio range of the CD - MOF or CD to the activated thioacetal cross - linker 2 is 1:2 - 1:12.

[0173] In step (4), the catalyst includes but is not limited to pyridine and triethylamine.

[0174] In step (4), the reaction temperature 4 ranges from 25 - 100 °C, and the reaction time 4 is 10 - 48 h.

[0175] The drug - loading system of the present invention

[0176] The cyclodextrin covalent framework of the present invention is used for drug delivery.

[0177] The present invention provides a drug - loading system, comprising:

[0178] (1) The cyclodextrin covalent framework of the present invention as a drug carrier;

[0179] (2) A drug for treating inflammatory diseases, wherein the anti - inflammatory drug is loaded on the cyclodextrin covalent framework.

[0180] Preferably, the anti - inflammatory drugs loaded on the cyclodextrin covalent framework include but are not limited to dexamethasone and mometasone furoate.

[0181] The present invention has the following main advantages:

[0182] (1) The cyclodextrin covalent framework prepared by the present invention has a simple preparation process and good biosafety.

[0183] (2) The cyclodextrin covalent framework prepared by the present invention has the ability to scavenge reactive oxygen species, synergistically exerts a therapeutic effect with drug molecules, and can controllably release drug molecules in the inflammatory microenvironment, thereby reducing the reactive oxygen species level at the inflammatory site and alleviating the process of inflammatory diseases.

[0184] (3) The cyclodextrin covalent framework prepared by the present invention degrades to expose sulfhydryl groups in the inflammatory environment, has excellent mucoadhesive ability, and enhances retention at the inflammatory site.

[0185] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0186] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the method of the present invention. The preferred methods and materials described herein are for illustrative purposes only.

[0187] Example 1: Construction and Characterization of Cyclodextrin Covalent Organic Frameworks

[0188] (1) Synthesis and Characterization of Thioacetal Crosslinkers

[0189] Synthesis of thioacetal crosslinker (TK7): 2-Mercaptoacetic acid (5.52 g, 60 mmol), anhydrous acetone (6.96 g, 120 mmol) and a catalytic amount of p-toluenesulfonic acid (10 mg) were added to a round-bottom flask and reacted at room temperature for 6 h. After the reaction was completed, the reaction solution was quenched in an ice bath until crystals completely precipitated, filtered by suction, washed three times with ice hexane and cold water, and then freeze-dried to obtain white powder TK.

[0190] Synthesis of thioacetal crosslinker (TK9): 3-Mercaptopropionic acid (6.36 g, 60 mmol), anhydrous acetone (6.96 g, 120 mmol) and a catalytic amount of p-toluenesulfonic acid (100 mg) were added to a round-bottom flask and reacted at room temperature for 6 h. After the reaction was completed, the reaction solution was quenched in an ice-salt bath until crystals completely precipitated, filtered by suction, washed three times with ice hexane and cold water, and then freeze-dried to obtain white powder TK9.

[0191] Characterization of thioacetal crosslinker: The relative molecular mass of TK7 was determined by a high-resolution mass spectrometer. The mass spectrum showed that the ion peak (m / z) of TK7 was 223.3 [M-H] - , which was consistent with the theoretical relative molecular mass of 224.3 ( Figure 1 ). The chemical structure of TK7 was characterized by 1H NMR. Its chemical shift was 1 1H NMR (600 MHz, DMSO-d6, δ): 3.38 (s, 4H), 1.55 (s, 6H), 12.62 (s, 2H), corresponding to the characteristic peaks of TK7 ( Figure 2 ). The structural formula of TK7 was characterized by infrared spectroscopy, Figure 3 showing that 2944 cm -1 was the O-H stretching vibration in the carboxyl group, and 2905 cm -1are the stretching vibrations of -CH3 and -CH2. In addition, the stretching vibration of the carbonyl group (C=O), the single-bond vibration of -S-CH2-, and the stretching vibration of C-O are clearly visible at 1710 cm -1 , 1208 cm -1 , 1179 cm -1 respectively. The chemical structure of TK9 was characterized by 1H NMR, and its chemical shift was 1 1H NMR (600 MHz, D2O, δ): 2.82 (t, 4H), 2.63 (s, 4H), 1.55 (s, 6H), corresponding to the characteristic peaks of TK9 ( Figure 4 ).

[0192] (2) Preparation and Characterization of Thioacetal Activated Crosslinkers

[0193] Synthesis of Oxalyl Chloride Activated Thioacetal Crosslinker: The crosslinker TK7 or TK9 and the activator oxalyl chloride were added to a round-bottom flask containing 75 mL of dichloromethane according to a certain molar concentration feed ratio (1:2 - 1:10), and 15 μL of the catalyst N,N-dimethylformamide was added dropwise. After the solution became clear, the reaction was continued at a certain temperature for a certain time. After the reaction was completed, the residual solvent and the excess reactants were removed by a rotary evaporator to obtain the oxalyl chloride activated thioacetal crosslinker 1 liquid.

[0194] Synthesis of N,N-Carbonyldiimidazole (CDI) Activated Thioacetal Crosslinker: The crosslinker TK7 or TK9 and the activator N,N-carbonyldiimidazole (CDI) were dissolved in 4 mL of N,N-dimethylformamide according to a certain molar feed ratio (1:2.5 - 1:4), and activated at a certain temperature (25 - 40 °C) for a certain time (2 - 4 h) to obtain the CDI activated thioacetal crosslinker 2 liquid.

[0195] (3) Synthesis and Characterization of Thioacetal-Embedded Cyclodextrin Covalent Frameworks

[0196] Synthesis of Oxalyl Chloride Activated Cyclodextrin Covalent Framework (TOF): As shown in Table 1, 1 g of CD-MOF powder was weighed into a round-bottom flask, 15 mL of dichloromethane was added, and it was ultrasonically dispersed. After stirring in an ice-water bath for 15 min, the thioacetal activated crosslinker 1 with a certain molar feed ratio to CD-MOF was slowly added, and the reaction was carried out at a certain temperature for a certain time. After the reaction was completed, the obtained precipitate was centrifuged and washed twice with dichloromethane, ethanol, 50% ethanol, and pure water respectively, and then freeze-dried to obtain the oxalyl chloride activated cyclodextrin covalent framework TOF.

[0197] Synthesis of CDI-activated cyclodextrin covalent framework (TOF): In the solution of activated thioacetal-activated crosslinker 2, micron-sized CD-MOF or γ-CD was added according to a certain molar ratio of feed materials (1:6 - 1:8), and 150 μL of triethylamine as a catalyst was added. At a certain temperature (50 - 70 °C), magnetic stirring was carried out at 350 rpm for a certain time (12 - 24 h). After the reaction ended, when the system cooled to room temperature, 15 mL of 95% ethanol was added to quench the reaction, and then centrifuged. The lower layer precipitate was washed twice with 15 mL of anhydrous ethanol, 50% ethanol, and pure water respectively. After freeze-drying the product, CDI-activated cyclodextrin covalent framework TOF could be obtained.

[0198] Table 1: Synthesis of thioacetal-embedded cyclodextrin covalent framework

[0199]

[0200]

[0201]

[0202] Characterization of TOF: Taking No. 9 in Table 1 as an example, the particle size of micron-sized oxalyl chloride-activated TOF is about 2 - 3 μm, and the particle size of nano-sized oxalyl chloride-activated TOF is about 250 - 500 nm. Fourier transform infrared spectroscopy was used to analyze the functional group composition of TOF obtained by oxalyl chloride activation. The spectrum showed a characteristic peak of carbonyl (C=O) at 1754 cm -1 . At this position, there is no carbonyl characteristic peak in CD-MOF, which proves that the TK7 crosslinker has successfully crosslinked the hydroxyl groups between γ-CDs in CD-MOF ( Figure 5 ). Powder X-ray diffraction was used to analyze the crystal properties of the sample. The results showed that TOF lost the crystallinity of the original CD-MOF and was in an amorphous state ( Figure 6 ). Thermogravimetry was used to analyze the thermal stability of TOF obtained by oxalyl chloride activation. Compared with CD-MOF, within 200 °C, the weight loss was more (about 20%), mainly free water molecules, while TOF had less weight loss within 200 °C and had good stability ( Figure 7 ). X-ray photoelectron spectroscopy was used to determine the elemental composition of nano-TOF obtained by oxalyl chloride activation. The spectrum showed that sulfur element in the TK crosslinker was successfully introduced into TOF ( Figure 8 ), with a proportion of 0.78%. It should be noted that the percentage content of potassium element in TOF decreased significantly, indicating that coordinated potassium ions were removed during the crosslinking process. In addition, the content of chlorine element in TOF was almost zero, indicating that excess crosslinker was removed during the washing process. Taking No. 49 in Table 1 as an example, the particle size of CDI-activated TOF was about 1.5 μm. Fourier transform infrared spectroscopy was used to analyze the functional group composition of CDI-activated TOF, Figure 9showed a characteristic peak of the carbonyl group at 1737 cm -1 which proved that the TK9 crosslinker had successfully crosslinked the hydroxyl groups between γ-CDs in CD-MOF. The elemental composition of CDI-activated TOF was determined by X-ray photoelectron spectroscopy, Figure 10 showing that sulfur element in the TK9 crosslinker was successfully introduced into TOF, and the proportion of sulfur element was 5.2%.

[0203] According to Table 1, the preparation of TOF was carried out according to Example 1. Similar to Example 1, all the prepared TOFs showed that the thioacetal groups were embedded in them, and the formation of ester bonds in the crosslinked products was found.

[0204] Example 2: Synthesis and Characterization of Non-ROS Responsive Cyclodextrin Covalent Framework

[0205] 1,7-Heptanedicarbonyl chloride was selected as the carbon chain linker to synthesize non-ROS responsive COF as a control carrier. The specific operation was as follows: Weigh 1 g of nano-CD-MOF powder into a round-bottom flask, add 15 mL of dichloromethane, disperse it by ultrasonic wave, stir it in an ice-water bath for 15 min, then slowly add 7.5 mmol of 1,7-heptanedicarbonyl chloride liquid, and react at room temperature for 24 h. After the reaction was completed, the reactant was centrifuged (4000 rpm, 5 min), and the lower layer precipitate was washed twice with dichloromethane, ethanol, 50% ethanol, and pure water respectively, and then freeze-dried to obtain white non-ROS responsive cyclodextrin covalent framework powder (COF).

[0206] Fourier transform infrared spectroscopy was used to analyze the functional group composition of COF. As Figure 11 shown, a characteristic peak of the ester bond appeared at 1750 cm -1 which confirmed the successful preparation of COF. The particle size of COF was about 250 - 500 nm.

[0207] Example 3: ROS Degradation Evaluation of Cyclodextrin Covalent Organic Framework

[0208] H2O2-responsive degradation of oxalyl chloride-activated TOF: About 1 mg of nano oxalyl chloride-activated TOF powder was ultrasonically dispersed in 4 mL of 0, 1, 10, 100 mM H2O2 solutions, incubated in a shaker at 100 rpm and 37 °C, and 3 mL of liquid was taken out at specific time points (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h), and the transmittance of each sample at 500 nm was immediately measured by an ultraviolet-visible spectrophotometer to plot its degradation curve. The H2O2-responsive degradation curve of TOF was as Figure 12It was shown that, compared with the environment without H2O2, the degradation of TOF was obvious in the H2O2 medium. With the increase of the H2O2 concentration, the dissociation degree of TOF became larger. The degradation rates of TOF incubated in the environments of 1, 10, and 100 mM H2O2 for 12 h were 80%, 92%, and 102% respectively.

[0209] H2O2-responsive degradation of CDI-activated TOF: About 1 mg of nano-TOF powder was ultrasonically dispersed in 4 mL of 0, 1, 10 mM H2O2 solutions, incubated in a shaker at 100 rpm and 37 °C. At specific time points (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h), 3 mL of the liquid was taken out and immediately the transmittance of each sample at 500 nm was measured with a UV-visible spectrophotometer to plot its degradation curve. Figure 13 It was shown that with the increase of the H2O2 concentration, the dissociation degree of the thioacetal bond became larger. The degradation rates of TOF incubated in the environments of 0, 1, 10 mM H2O2 for 12 h were 37%, 62%, and 85% respectively.

[0210] Example 4: In vitro evaluation of dexamethasone-loaded cyclodextrin covalent organic framework

[0211] (1) Loading of dexamethasone

[0212] Activation of TOF with oxalyl chloride for loading dexamethasone: 300 mg of dexamethasone (DEX) was dissolved in 10 mL of anhydrous methanol, 300 mg of TOF or COF was added, and after ultrasonic dispersion, it was placed in a 40 °C water bath and magnetically stirred at 300 rpm for 2 h. After the drug loading was completed, the suspension was filtered by a Buchner funnel, and the filter cake was washed twice with anhydrous methanol and twice with pure water, and then freeze-dried to obtain the dexamethasone-loaded nanoparticles (the oxalyl chloride-activated TOF nanoparticles loaded with dexamethasone are abbreviated as DEX@TOF, and the COF nanoparticles loaded with dexamethasone are abbreviated as DEX@COF). The drug loading amounts of DEX@TOF and DEX@COF were measured by high performance liquid chromatography to be 8.26% and 10.37% respectively.

[0213] Activation of CDI-activated TOF for loading dexamethasone: Dexamethasone (300 mg) was dissolved in 10 mL of anhydrous methanol, 300 mg of CDI-activated TOF was added, and after ultrasonic dispersion, it was placed in a 40 °C water bath and magnetically stirred at 300 rpm for 2 h. After the drug loading was completed, the suspension was filtered by a Buchner funnel, and the filter cake was washed twice with anhydrous methanol and twice with pure water, and then freeze-dried to obtain the dexamethasone-loaded cyclodextrin covalent organic framework. The drug loading amount was measured by high performance liquid chromatography to be 7.56%.

[0214] (2) In vitro release of dexamethasone-loaded nanoparticles

[0215] To investigate the in vitro release of DEX@TOF and DEX@COF prepared in Example 4 in the gastrointestinal tract, the drug release contents of DEX@TOF and DEX@COF were measured at different stages in phosphate buffer solutions with pH 1.2 (artificial gastric juice), pH 6.8 (artificial intestinal juice), and pH 7.4 (artificial colonic juice, with or without 100 mM H2O2). 5 mg / mL suspensions of DEX@TOF and DEX@COF were sealed in dialysis bags (MWCO = 3500 Da) and sequentially placed in 15 mL of artificial gastric juice (0 - 2 h), artificial intestinal juice (2 - 6 h), and artificial colonic juice (6 - 24 h). They were shaken in a constant temperature water bath shaker at 37°C at a constant speed of 100 rpm. At different time points (0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 24 h), 1 mL of the release sample was taken and an equal volume of isothermal release medium was replenished. The taken samples were centrifuged (12000 rpm, 5 min) and then analyzed by high performance liquid chromatography, and the cumulative release percentage of dexamethasone was calculated. The in vitro cumulative release curve of dexamethasone ( Figure 14 ) showed that the release of DEX@TOF and DEX@COF was slow in various simulated gastric and intestinal juices, and the trends of the two were quite similar, indicating that they had good stability in the gastrointestinal environment. When the artificial colonic juice medium was replaced with a solution containing 1 mM H2O2 and incubated for 24 h, the drug release of DEX@TOF increased by 20%, while there was no significant difference in the drug release of the DEX@COF group, indicating that DEX@TOF had good ROS-responsive drug release behavior.

[0216] (3) Evaluation of the in vitro ROS scavenging ability of TOF nanoparticles loaded with dexamethasone

[0217] Superoxide anion (O2 - ) scavenging of oxalyl chloride-activated TOF and DEX@TOF: Dispersions of TOF and DEX@TOF with a series of concentrations of 5, 2, 1, 0.5, and 0.25 mg / mL were mixed with the reagents prepared in advance according to the instructions of the superoxide anion kit. They were incubated in a water bath at 37°C for 40 min. After adding the color reagent, the liquid was thoroughly mixed using a vortex mixer. The absorbance values of each liquid at 550 nm were measured using a microplate reader, and the superoxide anion scavenging ability of each sample was calculated according to the instructions of the kit. As Figure 15 can be seen, the superoxide anion scavenging abilities of TOF and DEX@TOF were quite similar and showed dose-dependent scavenging.

[0218] DPPH· scavenging by oxalyl chloride-activated TOF and DEX@TOF: 500 μL of TOF and DEX@TOF aqueous dispersions with concentrations of 0.2, 0.5, 1, 1.5, and 2 mg / mL were mixed with 500 μL of 0.2 mM DPPH· ethanol solution. At the same time, the DPPH· ethanol solution and pure water were mixed in equal proportions as the control group. The mixtures were incubated in a shaker at 37 °C and 100 rpm for 2 h, centrifuged (12,000 rpm, 5 min), 200 μL of the supernatant was taken and placed in a 96-well plate, and the absorbance value at 517 nm was measured using a microplate reader, and the DPPH· scavenging rate of each sample was calculated. Through Figure 16 It can be seen that TOF and DEX@TOF have excellent DPPH· scavenging ability. When the concentration is 1 mg / mL, the DPPH· scavenging rates of both are about 88%. In addition, the DPPH· scavenging abilities of TOF and DEX@TOF are comparable, showing dose-dependent scavenging.

[0219] OH· scavenging by oxalyl chloride-activated TOF and DEX@TOF: In this experiment, the phenanthroline method was used to investigate the OH· scavenging ability of each sample. 250 μL of 1.5 mM phenanthroline solution was fully mixed with 250 μL of TOF and DEX@TOF aqueous dispersions with concentrations of 0.4, 1, 2, 3, and 4 mg / mL. Then, 250 μL of FeSO4·7H2O solution and 250 μL of 0.03% H2O2 solution were added respectively. At the same time, the mixture without the sample group was used as the control group, and the mixture without H2O2 was used as the blank group. Each group of samples was incubated in a shaker at 37 °C and 100 rpm for 1 h, centrifuged (12,000 rpm, 5 min), 200 μL of the supernatant was taken and placed in a 96-well plate, and the absorbance value at 536 nm was measured using a microplate reader, and the OH· scavenging rate of each sample was calculated. Through Figure 17 It can be seen that the OH· scavenging abilities of TOF and DEX@TOF are comparable, both showing dose-dependent scavenging, indicating that the ability of the samples to scavenge OH· is mainly attributed to the thioxanthone group in TOF.

[0220] DPPH· scavenging by CDI-activated TOF: 500 μL of CDI-activated TOF aqueous dispersions with concentrations of 0.2, 0.5, 1, 1.5, and 2 mg / mL were mixed with 500 μL of 0.2 mM DPPH· ethanol solution. At the same time, the DPPH· ethanol solution and pure water were mixed in equal proportions as the control group. The mixtures were incubated in a shaker at 37 °C and 100 rpm for 2 h, centrifuged (12,000 rpm, 5 min), 200 μL of the supernatant was taken and placed in a 96-well plate, and the absorbance value at 517 nm was measured using a microplate reader, and the DPPH· scavenging rate of each sample was calculated. Through Figure 18It can be seen that the TOF activated by CDI has excellent scavenging ability. When the concentration is 1 mg / mL, the DPPH· scavenging rate is about 80%, showing dose-dependent scavenging.

[0221] OH· scavenging of CDI-activated TOF: Mix 250 μL of 1.5 mM o-phenanthroline solution with 250 μL of reticular TOF aqueous dispersions with concentrations of 0.4, 1, 2, 3, and 4 mg / mL respectively. Then add 250 μL of FeSO4·7H2O solution and 250 μL of 0.03% H2O2 solution respectively. At the same time, use the mixture without the sample group as the control group and the mixture without H2O2 as the blank group. Incubate each group of samples in a shaker at 37 °C and 100 rpm for 1 h, centrifuge (12,000 rpm, 5 min), take 200 μL of the supernatant and place it in a 96-well plate, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value at 536 nm, and calculate the OH· scavenging rate of each sample. The results show ( Figure 19 ) that the CDI-activated TOF shows dose-dependent scavenging of OH·. When the concentration is 1 mg / mL, the scavenging rate of OH· is as high as 50%.

[0222] (4) Cytotoxicity evaluation of dexamethasone-loaded nanoparticles

[0223] Select RAW 264.7, Caco-2, and HT-29 cells in good growth condition and logarithmic growth phase and inoculate them into 96-well plates at a density of 1×10 4 cells / well. The final volume in each well is 200 μL. Add sterile PBS to the edge wells, and place them in a cell culture incubator for 24 h to allow them to adhere and grow. The next day, aspirate the original culture medium, and add different concentrations of oxalyl chloride-activated TOF, COF, DEX@COF, or DEX@TOF samples in equal volumes, which are 2, 5, 10, 20, 50, 100, 200, and 400 μg / mL respectively. At the same time, set up a blank group containing only the culture medium and a control group containing cells and the culture medium. After continuing to incubate in the incubator for 24 h, add 15 μL of CCK-8 solution to each well, continue to incubate for 1.5 h, use an ELISA reader to measure the absorbance at 450 nm wavelength of each well, and calculate the cell survival rate. The results show ( Figure 20 ) that within the range of 2 - 400 μg / mL, the cell survival rate of each nanoparticle is above 80%, without obvious cytotoxicity, and has good cell safety.

[0224] (5) Evaluation of the in vitro anti-inflammatory ability of dexamethasone-loaded nanoparticles

[0225] Select RAW 264.7 cells in logarithmic growth phase at 1.5×10 5Cells were inoculated at a density of [number] per well in a 24-well plate. After overnight culture, the culture medium was discarded, and a lipopolysaccharide solution at a concentration of 1 μg / mL, free DEX, oxalyl chloride-activated TOF, or DEX@COF or DEX@TOF solution (the concentration of DEX was 8 μg / mL) was added. At the same time, the group with lipopolysaccharide and culture medium was used as the positive control group, and the group with only culture medium was used as the negative control group. After continued culture in an incubator for 12 h, the cell supernatant was aspirated, centrifuged (1500 rpm, 10 min), the supernatant suspension was removed, and the content of IL-1β in the supernatant was measured using an ELISA kit, and the content of NO in the supernatant was measured using a nitric oxide detection kit. The results showed that ( Figure 21 ), compared with the blank control, after stimulation with lipopolysaccharide, the amounts of IL-1β and NO secreted by the cells could be significantly increased. Both the blank oxalyl chloride-activated TOF and the group containing dexamethasone preparation could effectively reduce the secretion of IL-1β and NO by the cells, and the DEX@TOF group had the strongest anti-inflammatory ability, mainly due to the synergistic effect of dexamethasone and blank TOF.

[0226] Example 5: In vitro mucoadhesion evaluation of dexamethasone-loaded cyclodextrin covalent organic framework

[0227] (1) Determination of the thiol content in the ROS degradation product of TOF

[0228] The Ellman's method was used to determine the free thiol content of the ROS degradation product of oxalyl chloride-activated TOF. Weighed 25 mg of TOF and dispersed it in 5 mL of an aqueous solution of 100 mM H2O2. After incubation in a shaker at 37 °C for 12 h, the clarified crude reaction product was dialyzed against pure water for 24 h (the molecular weight cut-off of the dialysis bag was 1500 Da), and pure water was changed several times during the period to maximize the removal of small molecule impurities. After freeze-drying the dialyzed liquid, the ROS degradation product of TOF could be obtained. Dissolved 3 mg of the ROS degradation product of TOF in 0.5 mL of pure water, added an equal volume of 0.75 mM DTNB solution freshly prepared with 0.05 M PBS (pH = 8), vortexed and mixed well, and left to stand in the dark at room temperature for 2 h. The absorbance of each sample solution at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and at the same time, the solution tube without the sample to be measured was used as the blank control, in triplicate. Among them, the standard curve of free thiol was established based on the thiol content in cysteine at different concentrations, and the content unit of free thiol was μmol / g. The thiol content in the ROS degradation product of TOF was determined to be 104 μmol / g by an ELISA reader.

[0229] (2) In vitro mucoadhesion evaluation of TOF

[0230] The fresh colon of a mouse was cleaned and cut into mucosal segments approximately 3×1 cm at 5 cm from the anus. 50 μL of 0.5 mg / mL rhodamine B-labeled DEX@TOF or DEX@COF solution (with or without H2O2 degradation) was incubated with the mouse mucosa at 37 °C for 30 min, then the mucosa was washed with physiological saline for 15 min, and imaging was performed using a small animal in vivo fluorescence imager to calculate its fluorescence intensity. The quantitative results are as Figure 22 shown. The fluorescence intensities of the DEX@COF groups with or without H2O2 degradation were comparable, indicating that DEX@COF does not have mucosal adhesion ability. The fluorescence intensity of the DEX@TOF group treated with H2O2 degradation was the highest, and its fluorescence intensity was 1.6 times that of the DEX@TOF group without H2O2 degradation, indicating that DEX@TOF has good mucosal adhesiveness in an inflammatory environment. This experimental phenomenon is attributed to the disruption of the thioxanthone bond in DEX@TOF in an inflammatory environment, further exposing the thiol groups, which can form disulfide bonds with mucin in intestinal mucus.

[0231] Example 6: Evaluation of the colitis targeting of dexamethasone-loaded cyclodextrin covalent organic framework

[0232] Healthy male C57BL / 6 mice were intragastrically administered a suspension of Cy5-labeled DEX@TOF. At 4 h, 6 h, 8 h, 12 h, and 24 h after administration, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, stomach, small intestine, and colon tissues were removed, and imaging analysis was performed using a small animal in vivo fluorescence imager. In addition, after the successful establishment of an acute ulcerative colitis C57BL / 6 mouse model, a suspension of Cy5-labeled DEX@TOF or Cy5-labeled DEX@COF with the same dose as the healthy group was intragastrically administered. At 4 h, 6 h, 8 h, 12 h, and 24 h after administration, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, stomach, small intestine, and colon tissues were removed, and imaging analysis was performed using a small animal in vivo fluorescence imager. The distribution of the microparticles in each tissue is as Figure 23 shown. At 6 h after intragastric administration, the distribution of DEX@TOF in the mouse colon tissue was the highest, and its content in the colon of colitis mice was approximately 2.6 times that of the colon tissue of healthy group mice, indicating that DEX@TOF has a certain targeting ability to the colon inflammatory site. After 24 h of administration, there was still a signal in the colon of the DEX@TOF group, indicating that the nanoparticles had a strong retention ability at the inflammatory site, and there was no obvious signal in the main organs of the heart, liver, spleen, lung, and kidney, indicating that the DEX@TOF nanoparticles did not accumulate in other organs. In particular, at 6 h after administration, the fluorescence intensity of the DEX@TOF nanoparticles in the colon of acute ulcerative colitis mice was significantly higher than that of DEX@COF ( Figure 24 ). Even at 24 h, the fluorescence intensity of the DEX@TOF group was higher than that of DEX@COF, indicating that compared with the non-ROS-responsive DEX@COF nanoparticles, the ROS-responsive DEX@TOF nanoparticles had stronger targeting ability to the inflammatory site.

[0233] Example 7: Pharmacodynamic Evaluation of Dexamethasone-Loaded Cyclodextrin Covalent Organic Framework in the Treatment of Acute Ulcerative Colitis

[0234] C57BL / 6 mice with acute ulcerative colitis were allowed to freely drink a 2.5% DSS solution aqueous solution within 7 days, and healthy mice were allowed to freely drink distilled water. All mice were administered drugs according to the experimental groups from the 3rd day to the 7th day, once a day. Mice in the healthy group were gavaged with normal saline. Mice with acute ulcerative colitis (UC) were randomly divided into 5 groups, with 6 mice in each group, and were respectively gavaged with normal saline (NS), free DEX (dose 2 mg / kg), TOF, DEX@TOF (DEX dose 2 mg / kg), and DEX@COF (DEX dose 2 mg / kg). The body weights of the mice were recorded daily during the experiment, and the fecal morphology and blood in the stool were observed, and their disease index DAI was scored. On the 8th day, the mice were euthanized and the entire colon was collected, measured for colon length and gently washed with normal saline, and the colon length was measured with a ruler and recorded. The distal part of the colon tissue of 1 cm was used for HE staining. The remaining part was prepared into a colon homogenate for the determination of various tissue factors.

[0235] The pharmacodynamic results showed ( Figure 25 ) that within 7 days, the body weights of the mice in the blank control group did not decrease, while the body weights of the mice in the acute ulcerative colitis group decreased significantly. After the intervention of each group of preparations, the decrease in the body weights of the mice was improved. Compared with the normal group, the colon of the mice in the model group was significantly shortened, with local congestion and bloody stools visible in the intestinal lumen. It is worth noting that all of DEX, TOF, and DEX@COF could improve the condition of the mice in the acute ulcerative colitis group, and their effects were comparable. After the intervention of DEX@TOF, the remission degree of the development course of acute colitis was the largest, the feces were mostly yellow semi-thin stools, the condition of bloody stools was inhibited, and the disease index decreased significantly, mainly due to the synergistic effect of DEX and TOF. By measuring various inflammatory factors in the colon tissue of the mice, DEX, TOF, DEX@COF, and DEX@TOF could all reduce the oxidative stress level of each tissue, among which DEX@TOF had the strongest effect, further confirming the ROS scavenging ability of the TOF material.

[0236] Example 8: Preliminary Safety Evaluation of Dexamethasone-Loaded Cyclodextrin Covalent Organic Framework

[0237] Healthy male C57BL / 6 mice were intragastrically administered with normal saline, free DEX (dose: 2 mg / kg), oxalyl chloride-activated TOF, DEX@TOF (DEX dose: 2 mg / kg), and DEX@COF (DEX dose: 2 mg / kg) once a day for 5 consecutive days. Twenty-four hours after the last administration, whole blood was collected using EDTAK2 as an anticoagulant for routine blood tests. Meanwhile, the main organs of the heart, liver, spleen, lungs, and kidneys were subjected to HE staining.

[0238] During the intragastric administration of DEX@TOF, no animal deaths or abnormal behaviors were observed, and there were no significant differences in the body weight changes of the mice compared to the control group ( Figure 26 ). After multiple administrations, there were no significant changes in the white blood cell, red blood cell, and platelet counts of the mice ( Figure 27 ), indicating that each delivery system had good biosafety. In addition, HE sections of the various organs of the mice showed no observed relevant pathological changes in the heart, liver, spleen, lungs, and kidneys ( Figure 28 ). The above preclinical safety study results indicate that the DEX@TOF drug delivery system has good biocompatibility and is expected to achieve clinical translation.

[0239] All documents mentioned in this invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A cyclodextrin covalent framework is prepared by using a cyclodextrin or a cyclodextrin metal-organic framework as a basic structural unit and crosslinking with a thiosulfonate crosslinking agent.

2. The cyclodextrin covalent framework according to claim 1, wherein The thiosulfonate crosslinking agent is a compound of the following formula I: wherein n1 and n2 are each independently 1, 2, 3 or 4.

3. The cyclodextrin covalent framework according to claim 1, wherein The cyclodextrin is selected from the group consisting of: γ-CD, β-CD, α-CD, or a combination thereof; the cyclodextrin metal-organic framework is a cyclodextrin metal-organic framework formed by cyclodextrin and an alkali metal ion.

4. The cyclodextrin covalent framework according to claim 1, wherein, The cyclodextrin covalent framework is prepared by the following method, including the following steps: In a solvent, an activated crosslinking agent is mixed with a cyclodextrin or a cyclodextrin metal-organic framework, and reacted to obtain the cyclodextrin covalent framework.

5. A method for preparing the cyclodextrin covalent framework according to claim 1, characterized in that, Including the following steps: In a solvent, an activated crosslinking agent is mixed with a cyclodextrin or a cyclodextrin metal-organic framework, and reacted to obtain the cyclodextrin covalent framework.

6. The preparation method according to claim 5, characterized in that, The method includes the following steps: (s1) In a second solvent, in the presence of a first catalyst, an activated crosslinking agent is mixed with a cyclodextrin or a cyclodextrin metal-organic framework, and reacted to obtain the cyclodextrin covalent framework.

7. A drug delivery system, characterized in that, The drug delivery system includes: (a) The cyclodextrin covalent framework according to claim 1; (b) A drug loaded in the cyclodextrin covalent framework.

8. The drug delivery system according to claim 7, characterized in that, The drug is a drug for preventing and / or treating inflammation, such as dexamethasone and mometasone furoate.

9. A pharmaceutical composition, characterized in that, Including: The drug delivery system according to claim 7; And a pharmaceutically acceptable carrier.

10. Use of the cyclodextrin covalent framework according to claim 1, the drug delivery system according to claim 7, or the pharmaceutical composition according to claim 9 in the preparation of a drug for (a) scavenging reactive oxygen species; (b) anti-inflammatory; and / or (c) preventing and / or treating colitis.

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