A high-permeability polymer nanocapsule, a preparation method and application thereof

Abstract

The application discloses a high-permeability polymer nanocapsule and a preparation method and application thereof, and the polymer nanocapsule has a core-shell structure, wherein the inner core of the nanocapsule comprises a functional molecular structure with antioxidant activity, and the polymer shell is a polytertiary amine oxide with phenylboronic acid modification. The application is prepared by in-situ polymerization of a polytertiary amine oxide shell and an active oxygen-unstable crosslinking agent on a macromolecular surface, wherein cationic monomers are combined with the macromolecule through electrostatic interaction and boronic acid nitrogen coordination, and interface polymerization on the molecular surface is realized; due to the active oxygen instability of the phenylboronic acid, the release of the inner core molecules at the damage site can be promoted. The nanocapsule has oral stability and mucus permeability in vitro and in vivo, can be rapidly internalized by cells, can alleviate radioactive damage by releasing antioxidants in the intestinal tract, and can significantly reduce the oxidative stress level of intestinal tissue after radiotherapy and reduce the degree of intestinal inflammation and ulcer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemical biology medicine, and particularly relates to a high-permeability polymer nanocapsule and a preparation method and application thereof. BACKGROUND

[0002] In the current medical field, the treatment of diseases such as inflammation and tumor is still a challenging task. Traditional treatment methods have a series of limitations. For example, traditional drugs have certain toxicity to normal tissues, which can easily cause serious side effects such as nausea, vomiting, hair loss, etc., affecting the quality of life of patients; tumor cells and inflammatory tissues are prone to multi-drug resistance, which greatly reduces the treatment effect of traditional drugs. The uneven distribution of drugs in the body makes it difficult to achieve sufficient concentration in the inflammatory and tumor tissues, resulting in poor treatment effect. The characteristics of the tumor microenvironment such as acidity, hypoxia, and high pressure can affect the penetration and effect of drugs, reducing the treatment effect. The prognosis is not ideal, and the recurrence of inflammation and tumor is easy, and the treatment effect is not lasting.

[0003] In recent years, the mode of drug delivery using delivery systems has attracted widespread attention due to its unique advantages. Nanodrugs can achieve targeted delivery of drugs by adjusting particle size, surface properties and functional molecules, reducing damage to normal tissues, improving drug solubility and stability, and local concentration at the lesion site (Ji G. Application of nanotechnology in drug delivery [M]. Chemical Industry Press, 2015.). Nanoparticles can overcome complex biological barriers, improve the penetration ability of drugs in dense tissues, and enhance the treatment effect.

[0004] ROS is a common active oxygen in the body, and its level in the lesion tissue is high. Nanodrugs release drugs by reacting with ROS to achieve targeted treatment, thereby improving the treatment effect (Guo W, Han M, Guo, et al. Research progress of ROS-responsive nanodrug delivery system [J]. China Modern Applied Pharmacy, 2017, 34(5): 5. DOI: 10.13748 / j.cnki.issn1007-7693.2017.05.032.).

[0005] Although nanocapsules have great potential in theory, they still face many challenges in clinical application. First, how to effectively design a suitable delivery platform to achieve efficient release of biological macromolecules such as proteins and nucleic acids is still a problem to be solved. Second, how to ensure the biological safety, permeability and stability of the nanodrug delivery system to avoid adverse reactions and side effects is also a direction that needs in-depth research. At present, it is urgent to design a ROS-responsive nanocarrier that can achieve intelligent release of drugs, adjust the drug release rate according to the ROS level of the intestinal tissue, and improve the targeting and efficiency of treatment. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a permeable polymer nanocapsule, a preparation method and application thereof, which is safe and effective, can effectively protect the functional molecular structure of the core with antioxidant activity, improve the stability of the molecule, has excellent internalization efficiency on cells, and can be efficiently released in the ROS environment, and has a wide application prospect in the field of drug delivery.

[0007] To solve the above technical problems, the present application proposes a permeable polymer nanocapsule for delivery by initiating interfacial polymerization on the surface of biomolecular drugs, which can provide good stability for multi-mode delivery of biomolecules, and has excellent cross-biological barrier and cell internalization promotion efficiency.

[0008] Inside the nanocapsule, drugs such as antioxidants are carried, and the surface is modified with ROS-sensitive groups, so that it can sense the presence of ROS in the intestinal tract and achieve intelligent release. Through this design, we can realize the directional release of drugs in the intestinal tissue after radiotherapy, reduce oxidative damage and inflammatory response, and improve the treatment effect of radiation enteritis.

[0009] The specific technical solutions of the present application are as follows:

[0010] The first object of the present application is to provide a high permeable polymer nanocapsule, which is a core-shell structure, wherein the core of the core-shell structure is a bioactive molecule, and the bioactive molecule includes at least one of a protein, a nucleic acid, and a CRISPR with antioxidant effect,

[0011] The shell of the core-shell structure includes a polymer polymerized from a monomer material, and the monomer material includes a cationic monomer, a tertiary amine oxide monomer, and a crosslinking agent;

[0012] The cationic monomer is any one of the compounds shown in the following structural formula (1a)-(1d), and the specific structure is as follows:

[0013]

[0014] (1a){[4-(Dihydroxyboronyl)phenyl]methyl}diethyl{2-[(1-oxo-prop-2-enyl)oxy]ethyl}ammonium, (1b){[4-(Dihydroxyboronyl)phenyl]methyl}diethyl{2-[(2-methyl-1-oxo-prop-2-enyl)oxy]ethyl}ammonium,

[0015]

[0016] (1c) benzyl diethyl {2-[(1-oxoprop-2-enyl)oxy]ethyl} ammonium, (1d) benzyl diethyl {2-[(2-methyl-1-oxoprop-2-enyl)oxy]ethyl} ammonium;

[0017] The crosslinking agent is any one of the compounds represented by the following structural formulae (2a)-(2h), and the specific structures are as follows:

[0018]

[0019] (2a) ({3-[(diethyl {2-[(1-oxoprop-2-enyl)oxy]ethyl} azoniidinyl radical) methyl]-5- methyl-2-({[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl] methyl} oxy) phenyl} methyl) diethyl {2-[(1-oxoprop-2-enyl)oxy]ethyl} ammonium, (2b) ({3-[(diethyl {2-[(2-methyl-1-oxoprop-2-enyl)oxy]ethyl} azoniidinyl radical) methyl]-5- methyl-2-({[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl] methyl} oxy) phenyl} methyl) diethyl {2-[(2-methyl-1-oxoprop-2-enyl)oxy] ethyl} ammonium,

[0020] ethyl} ammonium,

[0021]

[0022] (2c) prop-2-enoic acid-(5-methyl-3-{[(1-oxoprop-2-enyl)oxy]methyl}-2-({[4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl] methyl} oxy) phenyl) methyl ester, (2d) 2-methylprop-2-enoic acid-(5-methyl-3-{[(2-methyl-1-oxoprop-2-enyl)oxy]methyl}-2-({[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl] methyl} oxy) phenyl) methyl ester,

[0023]

[0024] (2e) ({3-[(diethyl {2-[(1-oxoprop-2-enyl)oxy]ethyl}azaniumylradical)methyl]-2- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl}methyl)diethyl {2-[(1- oxoprop-2-enyl)oxy]ethyl}azaniumyl;

[0025] {2-[(2-methyl-1-oxoprop-2-enyl)oxy]ethyl}azaniumyl,

[0026]

[0027] (2g) ({3-[(diethyl {2-[(1-oxoprop-2-enyl)oxy]ethyl}azaniumylradical)methyl]phenyl} methyl)diethyl {2-[(1-oxoprop-2-enyl)oxy]ethyl}azaniumyl; and (2h) ({3-[(diethyl {2-[(2-methyl-1-oxoprop-2-enyl)oxy]ethyl}azaniumylradical)methyl] phenyl}methyl)diethyl {2-[(2-methyl-1-oxoprop-2-enyl)oxy]ethyl}azaniumyl.

[0028] The structure of the tertiary amine oxide monomer is shown in formula (3):

[0029]

[0030] wherein R is a hydrogen atom, an alkyl group, and X is a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group or an isobutyl group;

[0031] The molar ratio of the bioactive molecule, the cationic monomer, the tertiary amine oxide monomer and the crosslinking agent is 1:10-3000:10-3000:1-1000.

[0032] The present application is prepared by in-situ polymerization of a polytertiary amine oxide shell and an active oxygen-labile crosslinking agent on the surface of a macromolecule, wherein the cationic monomer is combined with the macromolecule through electrostatic interaction and boronic acid nitrogen (N-B) coordination to realize interface polymerization on the surface of the molecule; in addition, the active oxygen lability of phenylboronic acid can promote the release of the core molecules at the damage site. The nanocapsule has oral stability and mucus permeability in vivo and in vitro, and can be rapidly internalized by cells, thereby realizing the prevention and treatment of diseases such as digestive tract inflammation, tumors, arthritis, neurogenic inflammation, immunity, etc.

[0033] In particular, the nanocapsules can alleviate radiation damage by releasing antioxidants in the intestinal tract, significantly reduce the level of oxidative stress of intestinal tissue after radiation therapy, and reduce the degree of intestinal inflammation and ulceration.

[0034] Preferably, R is a hydrogen atom, and X is an ethyl group.

[0035] The molar ratio of the bioactive molecule, the cationic monomer, the tertiary amine oxide monomer, and the crosslinking agent is 0.6:60:150:10.

[0036] In some embodiments of the present application, the protein is at least one of serum albumin, uric acid oxidase, catalase, glucose oxidase, horseradish peroxidase, superoxide dismutase, lactate dehydrogenase, acetaldehyde dehydrogenase, alcohol oxidase, acetaldehyde oxidase, antibodies, insulin, and cytokines.

[0037] The nucleic acid is at least one of siRNA, mRNA, shRNA, microRNA, ASO, and DNA.

[0038] In an embodiment of the present application, the biomolecule is selected as a model protein bovine serum albumin BSA as an example, but is not limited thereto.

[0039] A second object of the present application is to provide a preparation method of the high-permeability polymer nanocapsule, comprising the following steps:

[0040] (1) mixing the bioactive molecule, the cationic monomer, the tertiary amine oxide monomer, and the crosslinking agent to produce aggregation of the reaction monomers and the molecules;

[0041] (2) adding ammonium persulfate and N,N,N',N'-tetramethyl ethylenediamine to step (1) to initiate interfacial polymerization to obtain the high-permeability polymer nanocapsule.

[0042] In some embodiments of the present application, in step (2), the concentration of the ammonium persulfate is 0.1-10 mg / mL; and the concentration of the N,N,N',N'-tetramethyl ethylenediamine is 0.1-10 mg / mL.

[0043] In some embodiments of the present application, in step (2), the interfacial polymerization is performed under the following conditions: reaction time is 0.1-24 hours, and reaction temperature is 4-37°C.

[0044] In some embodiments of the present application, in step (2), the pH of the interfacial polymerization is 8-10.

[0045] In some embodiments of the present application, the prepared polymer nanocapsule has a particle size of 5-200 nm.

[0046] The application also provides application of the high-permeability polymer nanocapsule in preparation of a macromolecular drug delivery carrier.

[0047] The dosage form of the polymer nanocapsule is selected from one or more of the following: a gastrointestinal administration dosage form, an injection administration dosage form, a respiratory tract administration dosage form, a skin administration dosage form, a mucosa administration dosage form, and a cavity administration dosage form.

[0048] The application also provides application of the high-permeability polymer nanocapsule in preparation of a drug for preventing and / or treating a related disease, such as digestive tract inflammation, pathogen infection, tumor, tumor complication, precancerous syndrome, arthritis, neurogenic inflammation, immune response, allergy or autoimmune disease.

[0049] Preferably, the related disease is induced radiation disease.

[0050] Preferably, the digestive tract inflammation is induced radiation colitis or ulcerative colitis.

[0051] The application can reduce radiation damage by releasing antioxidants in the intestinal tract, can significantly reduce the oxidative stress level of intestinal tissue after radiation therapy, and can reduce the degree of intestinal inflammation and ulceration.

[0052] The above technical solutions of the application have the following advantages compared with the prior art:

[0053] 1. The nanocapsule prepared by the method of interfacial polymerization after the molecular surface aggregation of monomers has a shell structure on the surface, which can keep the drug highly stable in the body.

[0054] 2. The nanocapsule has a smaller particle size than a conventional gel system prepared by polymerization, is more easily passed through a biological barrier, reduces non-specific adsorption between the carrier and mucus, and greatly improves the permeability of the capsule.

[0055] 3. The nanocapsule has good endocytosis ability, effectively improves the transport effect of the drug between tissues, and has good biological responsiveness of the cross-linked structure, which can make the drug be released smoothly in the cell and successfully act on the target cell.

[0056] 4. The overall preparation process is simple and efficient, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 Figure 8 is a proportion screening result diagram of Example 8 of the application; wherein a is a screening formula; b is the particle size of each formula determined by dynamic light scattering (DLS); and c is the cell uptake rate of each nanocapsule.

[0058] Figure 2Particle size and potential graph of the nanocapsules in Example 9 and Comparative Example 1 of the present application.

[0059] Figure 3 TEM graph of the nanocapsules in Example 9 and Comparative Example 1 of the present application.

[0060] Figure 4 Degradation rate of Test Example 1 of the present application under H2O2; wherein a is HPLC time course analysis of ROS-responsive degradation of B-DEAEA, BDD, PBD and PBA; b is ROS-responsive degradation rate.

[0061] Figure 5 Particle tracking experiment graph of the nanocapsules in Test Example 2 of the present application in mucus.

[0062] Figure 6 Result graph of the nanocapsules in Test Example 2 of the present application in mucus layer transport experiment.

[0063] Figure 7 Result graph of the nanocapsules in Test Example 2 of the present application in Caco-2 cell monolayer transport experiment.

[0064] Figure 8 Protein activity experiment result graph of the nanocapsules in Test Example 3 of the present application after release.

[0065] Figure 9 Result graph of the nanocapsules in Test Example 3 of the present application in resisting gastrointestinal juice digestion experiment.

[0066] Figure 10 Protein activity experiment result graph of the nanocapsules in Test Example 4 of the present application after release.

[0067] Figure 11 Result graph of the nanocapsules in Test Example 4 of the present application in resisting gastrointestinal juice digestion experiment.

[0068] Figure 12 Result graph of the body weight change and DAI score in Test Example 5 of the present application in the treatment process of the mouse ulcerative colitis model.

[0069] Figure 13 Result graph of the colon length in Test Example 5 of the present application after the treatment of the mouse ulcerative colitis model.

[0070] Figure 14 Result graph of the colon ROS concentration in Test Example 5 of the present application after the treatment of the mouse ulcerative colitis model.

[0071] Figure 15 Result graph of the body weight change in Test Example 6 of the present application in the treatment process of the radiation enteritis model.

[0072] Figure 16 Figure 6-2 is a graph showing the results of H&E staining of the colon after treatment with the nanocapsule of Example 6 of the present application in a model of radiation enteritis.

[0073] Figure 17 Figure 6-3 is a graph showing the results of ROS concentration in the colon after treatment with the nanocapsule of Example 6 of the present application in a model of radiation enteritis. DETAILED DESCRIPTION

[0074] The experimental methods in the following examples of the present application, unless otherwise specified, are generally carried out according to the conventional conditions, or according to the conditions suggested by the manufacturers. The various common chemical reagents and biological reagents used in the examples are commercially available products,

[0075] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0076] The terms "comprising" and "having" and any variations thereof in the present application are intended to cover the non-exclusive inclusion such that a process, method, article, or apparatus that comprises a list of steps or modules not only comprises those steps or modules but can also include other steps or modules not expressly listed or inherent to such process, method, article, or apparatus.

[0077] The "multiple" mentioned in the present application refers to two or more. The "and / or" describes the association between the associated objects, which means that there can be three relationships, for example, A and / or B can represent the following three cases: A exists alone, A and B exist together, and B exists alone. The character " / " generally represents that the associated objects before and after it are in an "or" relationship.

[0078] Example 1

[0079] 2-(Diethylamine)ethyl methacrylate (3.7 g, 20 mmol) was dissolved in 80 mL of anhydrous CH2Cl2 in an ice bath. mCPBA (5.2 g, 30 mmol) was added over 30 minutes and stirred vigorously. The solution was allowed to warm to room temperature and stirred for another 2 hours. After that, the solvent was removed by evaporation under vacuum and the residue was purified by a column of neutral alumina to obtain ODEA (3.4 g, yield: 84.6%) as a gray-white waxy solid. 1H-NMR (D20, 600 MHz) δ 7.79 (d, J = 7.6 Hz, 2H), 7.50 (d, J = 7.7 Hz, 2H), 6.43 (d, J = 17.3 Hz, IH), 6.20 (dd, J = 17.3, 10.5 Hz, IH), 6.00 (d, J = 10.5 Hz, IH), 4.64 (t, J = 4.9 Hz, 2H), 4.53 (s, 2H), 3.64-3.51 (m, 2H), 3.35 (q, J = 7.2 Hz, 4H), 1.42 (t, J = 7.1 Hz, 6H). 13 C-NMR (D20, 151 MHz) δ 167.27, 134.03, 133.30, 131.90, 128.28, 126.80, 61.46, 57.86, 54.99, 53.87, 7.30.

[0080] Example 2

[0081] 4-(Bromethyl)phenylboronic acid (1.0 g, 4.6 mmol) and 2-(diethylamino)ethyl acrylate (1.0 mL, 5.4 mmol) were mixed in 12 mL of N,N-dimethylformamide (DMF). The reaction was stirred at room temperature under N2protection for 24 hours, then the mixture was poured into ether to precipitate the product, which was washed twice with ether. After drying under reduced pressure, the resulting B-DEAEA was a white solid (1.3 g, yield 87.9%). 1 H-NMR (D20, 600 MHz) δ 7.79 (d, J = 7.6 Hz, 2H), 7.50 (d, J = 7.7 Hz, 2H), 6.43 (d, J = 17.3 Hz, IH), 6.20 (dd, J = 17.3, 10.5 Hz, IH), 6.00 (d, J = 10.5 Hz, IH), 4.64 (t, J = 4.9 Hz, 2H), 4.53 (s, 2H), 3.64-3.51 (m, 2H), 3.35 (q, J = 7.2 Hz, 4H), 1.42 (t, J = 7.1 Hz, 6H). 13 C-NMR (D20, 151 MHz) δ 167.27, 134.03, 133.30, 131.90, 128.28, 126.80, 61.46, 57.86, 54.99, 53.87, 7.30.

[0082] Example 3

[0083] 2,6-bis-(hydroxymethyl)-p-cresol (10.0 g, 58.3 mmol) was dissolved in anhydrous DMF (50 mL), followed by the addition of imidazole (8.9 g, 129.4 mmol). The mixture was transferred to an ice bath, and TBDMSCl (tert-butyldimethylsilyl chloride) (19.9 g, 128.3 mmol) was added dropwise to anhydrous DMF (30 mL). After stirring at room temperature for 3 h, the mixture was diluted with ether (100 mL) and washed three times with water. The organic layers were combined, dried over anhydrous sodium sulfate (Na2SO4), filtered, and concentrated by rotary evaporation. The crude product was purified on a silica gel column using n-hexane / ethyl acetate (95 / 5, v / v) as the eluent to obtain compound 1 as a colorless oil (19.9 g, 86.2% yield). 1 H-NMR (CDCl3, 600MHz) δ (s, 2H), 4.85 (s, 4H), 2.29 (s, 3H), 0.97 (s, 18H), 0.15 (s, 12H).

[0084] Compound 1 (4.5 g, 11.4 mmol) and K2Co3 (2.0 g, 14.4 mmol) were mixed in anhydrous DMF (30 mL) and stirred in an ice bath for 10 min. 4-Bromomethylphenylboronic acid pinyl ester (3.6 g, 11.4 mmol) was then added dropwise to DMF (30 mL) and stirred at room temperature overnight. The mixture was then diluted with ether (100 mL) and washed three times with saturated ammonium chloride (NH4Cl) and sodium chloride (NaCl) solutions. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude product was purified on a silica gel column using n-hexane / ethyl acetate (95 / 5, v / v) as the eluent to obtain compound 2 as a white solid (5.0 g, yield 71.7%). 1 H-NMR (CDCl3, 600MHz) δ7.86-7.79 (m, 2H), 7.45-7.39 (m, 2H), 7.20-7.12 (m, 2H), 4.88(s, 2H), 4.69(s, 4H), 2.34(s, 3H), 1.36(s, 12H), 0.91(s, 18H), 0.06(s, 12H).

[0085] Compound 2 (4.0 g, 6.6 mmol) was dissolved in 40 mL of methanol and TsOH (252 mg, 1.2 mmol) was added. The mixture was stirred at room temperature for 3 h and then concentrated by rotary evaporation. The crude product was purified by silica gel column (hexane / ethyl acetate = 50 / 50, v / v) to afford compound 3 as a white solid (2.1 g, 82.1% yield). 1H-NMR (CDC13, 600 MHz) δ 7.84 (d, J = 7.5 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.16 (s, 2H), 4.97 (s, 2H), 4.66 (s, 4H), 2.33 (s, 3H), 1.35 (s, 12H).

[0086] Compound 3 (1.0 g, 2.6 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran (THF) and cooled in an ice bath. Then PBr3 (1.2 mL, 13.0 mmol) was added dropwise in 28 mL of dry THF and stirred at 0 °C for 20 h under N2 protection. The reaction solution was then concentrated and purified by silica gel column (hexane / ethyl acetate = 85 / 15, v / v) to obtain compound 4 as a yellow solid (1.0 g, yield 78.7%). 1 H-NMR (CDC13, 600 MHz) δ 7.84 (d, J = 7.5 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.16 (s, 2H), 4.97 (s, 2H), 4.66 (s, 4H), 2.33 (s, 3H), 1.35 (s, 12H).

[0087] Compound 4 (1.0 g, 2.0 mmol) and 2-(diethylamine)ethyl acrylate (500 μL, 2.7 mmol) were dissolved in 6 mL of DMF. The reaction was stirred at room temperature for 24 h under N2 protection. The mixture was then poured into ether to precipitate the sediment, which was washed with ether twice after centrifugation. After drying under reduced pressure, BDD was obtained as a white solid (1.2 g, yield 86.4%). 1 H-NMR (CDC13, 600 MHz) δ 7.84 (d, J = 7.5 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.16 (s, 2H), 4.97 (s, 2H), 4.66 (s, 4H), 2.33 (s, 3H), 1.35 (s, 12H). 13 C-NMR (CDC13, 151 MHz) δ 165.10, 162.54, 138.60, 137.57, 135.64, 132.81, 128.44, 127.09, 121.79, 84.11, 80.04, 57.81, 56.58, 54.53, 36.50, 31.42, 24.88, 20.96, 8.60.

[0088] Example 4

[0089] The compound 3 prepared in Example 3 (500 mg, 1.3 mmol) and triethylamine (542 μL, 3.9 mmol) were dissolved in anhydrous THF (50 mL) and cooled to 0 °C. Acryloyl chloride (315 μL, 3.9 mmol) was added dropwise in dry THF (15 mL) and the reaction was stirred at 25 °C for 20 h. The precipitate was filtered and purified by silica gel column (hexane / ethyl acetate = 50 / 50, v / v) and concentrated to give white crystals of PBA (296 mg, yield 46.3%). 1 H-NMR (CDC13, 600 MHz) δ 7.83 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.22 (s, 2H), 6.42 (dd, J = 17.2, 1.4 Hz, 2H), 6.18-6.08 (m, 2H), 5.83 (dd, J = 10.5, 1.4 Hz, 2H), 5.24 (s, 4H), 4.97 (s, 2H), 2.33 (s, 3H), 1.35 (s, 12H). 13 C-NMR (CDC13, 151 MHz) δ 165.97, 154.06, 139.88, 135.11, 134.32, 131.79, 131.20, 129.37, 128.29, 126.96, 83.85, 77.32, 61.62, 24.89, 20.85.

[0090] Example 5

[0091] Pinacol 2,6-dimethylphenylboronate (purchased from Acros, CAS No.: 138500-85-3) (3.5 g, 15.1 mmol), N-bromosuccinimide (NBS) (5.9 g, 33.3 mmol) and azobisisobutyronitrile (AIBN) (0.5 g, 3.0 mmol) were dissolved in 25 mL of tetrachloromethane (CCl4) and sealed in a Schlenk tube. After thoroughly deoxygenated, it was heated at 90 °C for 4 h and cooled to room temperature and filtered. The filtrate was purified by silica gel column (hexane / ethyl acetate = 20 / 1, v / v) to give compound 5 as colorless crystals (5.3 g, yield 90.6%). 1 H-NMR (CDC13, 600 MHz) δ 7.29 (s, 3H), 4.82 (s, 4H), 1.47 (s, 12H).

[0092] Compound 5 (0.8 g, 2.0 mmol) was dissolved in 6 mL DMF. 2-(Diethylamine)ethyl acrylate (0.5 mL, 2.7 mmol) was added and the reaction was stirred at room temperature under N2protection for 24 h. The reaction mixture was poured into ether and the product was precipitated, washed with ether twice. After drying under reduced pressure, PBD was obtained as a white solid (986 mg, yield 86.2%). 1 H-NMR (CDC13, 600 MHz) δ 7.81 (d, J = 7.8 Hz, 2H), 7.59 (t, J = 7.9 Hz, 1H), 6.46 (d, J = 17.3 Hz, 2H), 6.12 (dd, J = 11.2, 6.2 Hz, 2H), 5.94 (d, J = 10.4 Hz, 2H), 5.42 (s, 4H), 4.64 (t, J = 5.3 Hz, 4H), 3.96 (t, J = 5.5 Hz, 4H), 3.74 (q, J = 7.1 Hz, 8H), 1.52 (t, J = 7.1 Hz, 12H), 1.41 (s, 12H). 13 C-NMR (CDC13, 151 MHz) δ 165.35, 162.54, 137.08, 135.90, 132.99, 132.48, 127.12, 85.62, 62.04, 57.93, 56.02, 54.82, 25.03, 9.01.

[0093] Example 6

[0094] ABDA was synthesized according to a similar procedure of B-DEAEA in Example 2. The difference was that benzyl bromide and 2-(diethylamine)ethyl acrylate were mixed in N,N-dimethylformamide (DMF) and stirred at room temperature under N2protection for 24 h.

[0095] ABDA: 1 H-NMR (CDC13, 600 MHz) δ 7.64-7.46 (m, 5H), 6.49 (dd, J = 17.3, 1.2 Hz,

[0096] 1H), 6.12 (dd, J = 17.3, 10.5 Hz, 1H), 5.96 (dd, J = 10.6, 1.2 Hz, 1H), 5.03 (s, 2H), 4.67 (t, J = 5.4 Hz, 2H), 3.92 (t, J = 5.4 Hz, 2H), 3.60 (q, J = 7.2 Hz, 4H), 1.53 (t, J = 7.2 Hz, 6H). 13 C-NMR (CDC13, 151 MHz) δ 165.22, 132.83, 130.94, 63.02, 56.28, 54.50, 8.90.

[0097] Example 7

[0098] ADB was synthesized according to a similar procedure to B-DEAEA in Example 2, except that 1,3-bis(bromomethyl)benzene and ethyl 2-(diethylamino)acrylate were mixed in N,N-dimethylformamide (DMF) and stirred at room temperature for 24 hours under N2 protection.

[0099] ADB: 1 H-NMR (CDCl3, 600MHz) 7.70-7.46 (m, 4H), 6.48 (ddd, J=17.3, 7.2, 1.2Hz,

[0100] 2H), 6.13 (dd, J=17.3, 10.5Hz, 2H), 5.96 (ddd, J=10.4, 3.1, 1.1Hz, 2H), 5.09 (d, J=4.3Hz, 4H), 4.71 (dt, J=1 4.4, 5.3Hz, 4H), 3.88 (dt, J=11.4, 5.4Hz, 4H), 3.62 (dq, J=31.5, 7.2Hz, 8H), 1.54 (dt, J=17.1, 7.1Hz, 12H). 13 C-NMR (CDCl3, 151MHz) 6165.27, 162.59, 139.57, 135.19, 133.35, 133.10, 132.76, 131.54 , 130.05, 128.45, 127.64, 127.06, 62.64, 62.05, 58.33, 57.59, 56.37, 55.01, 54.64, 8.91.

[0101] Example 8

[0102] Protein BSA was dissolved in ultrapure water (800 μL, 0.5 mg / mL). Figure 1 As shown, the newly prepared cationic monomer B-DEAEA, tertiary ammonia oxide monomer ODEA, and cross-linker BDD were added to the protein solution in sequence. After each addition, the solution was mixed evenly for 1 minute by repeated pipetting, and then freshly prepared APS (12.5 μL, 2%) and TEMED (12.5 μL, 5%) solutions were added in sequence to initiate polymerization. After stirring at 4°C for 10 minutes, the reaction was dialyzed on PBS (MWCO 10 kDa) to remove unreacted monomers, cross-linkers, and initiators to obtain nanocapsule solutions with different ratios. The formulation screening results for nanocapsule optimization are shown in Figure 2. Figure 1 The molar ratio of protein / B-DEAEA / ODEA / BDD in the optimal formula is 0.6 / 60 / 150 / 10.

[0103] Example 9

[0104] Protein BSA was dissolved in ultrapure water (800 μL, 0.5 mg / mL). Freshly prepared cationic monomer B-DEAEA (50 μL), tertiary aminoxide monomer ODEA (30 μL), crosslinker BDD (15 μL) were added sequentially to the protein solution. After each addition, the solution was mixed well by repeated pipetting for 1 min, then freshly prepared APS (12.5 μL, 2%) and TEMED (12.5 μL, 5%) solutions were added sequentially to initiate polymerization. After stirring at 4 °C for 10 min, the reaction was dialyzed against PBS (MWCO 10 kDa) to remove unreacted monomers, crosslinker and initiator, resulting in a nanocapsule poBSA solution. The particle size and potential of the resulting example nanocapsule poBSA were as shown in Table 1, and the electron micrograph is shown in FIG. 1. Figure 2 Figure 3

[0105] Comparative Example 1

[0106] Protein BSA was dissolved in ultrapure water (800 μL, 0.5 mg / mL). Freshly prepared cationic monomer B-DEAEA (50 μL), crosslinker BDD (15 μL) were added sequentially to the protein solution. After each addition, the solution was mixed well by repeated pipetting for 1 min, then freshly prepared APS (12.5 μL, 2%) and TEMED (12.5 μL, 5%) solutions were added sequentially to initiate polymerization. After stirring at 4 °C for 10 min, the reaction was dialyzed against PBS (MWCO 10 kDa) to remove unreacted monomers, crosslinker and initiator, resulting in a nanocapsule pBSA solution. The molar ratio of protein / B-DEAEA / BDD in the optimal formulation was 0.6 / 60 / 10. The particle size and potential of the resulting comparative example nanocapsule pBSA were as shown in Table 1, and the electron micrograph is shown in FIG. 1. Figure 2 Figure 3

[0107] Comparative Example 2

[0108] Protein BSA was dissolved in ultrapure water (800 μL, 0.5 mg / mL). Freshly prepared cationic monomer ABDA (50 μL), tertiary aminoxide monomer ODEA (30 μL), crosslinker ADB (15 μL) were added sequentially to the protein solution. After each addition, the solution was mixed well by repeated pipetting for 1 min, then freshly prepared APS (12.5 μL, 2%) and TEMED (12.5 μL, 5%) solutions were added sequentially to initiate polymerization. After stirring at 4 °C for 10 min, the reaction was dialyzed against PBS (MWCO 10 kDa) to remove unreacted monomers, crosslinker and initiator, resulting in a nanocapsuleupoBSA solution.

[0109] Example 10 ​​​​

[0110] Catalase CAT was dissolved in ultrapure water (800 μL, 0.5 mg / mL). Freshly prepared cationic monomer B-DEAEA (50 μL), tertiary amine oxide monomer ODEA (30 μL), crosslinker BDD (15 μL) were added sequentially to the protein solution. After each addition, the solution was mixed homogeneously by repeated pipetting for 1 min, then freshly prepared APS (12.5 μL, 2%) and TEMED (12.5 μL, 5%) solutions were added sequentially to initiate polymerization. After stirring for 10 min at 4 °C, the reaction was dialyzed against PBS (MWCO 10 kDa) to remove unreacted monomers, crosslinker and initiator, resulting in a nanocapsule poCAT solution. The molar ratio of protein / B-DEAEA / ODEA / BDD in the optimal formulation was 0.6 / 60 / 150 / 10.

[0111] Comparative Example 3

[0112] Catalase CAT was dissolved in ultrapure water (800 μL, 0.5 mg / mL). Freshly prepared cationic monomer B-DEAEA (50 μL), crosslinker BDD (15 μL) were added sequentially to the protein solution. After each addition, the solution was mixed homogeneously by repeated pipetting for 1 min, then freshly prepared APS (12.5 μL, 2%) and TEMED (12.5 μL, 5%) solutions were added sequentially to initiate polymerization. After stirring for 10 min at 4 °C, the reaction was dialyzed against PBS (MWCO 10 kDa) to remove unreacted monomers, crosslinker and initiator, resulting in a nanocapsule pCAT solution. The molar ratio of protein / B-DEAEA / BDD in the optimal formulation was 0.6 / 60 / 10.

[0113] Comparative Example 4

[0114] Catalase CAT was dissolved in ultrapure water (800 μL, 0.5 mg / mL). Freshly prepared cationic monomer ABDA (50 μL), tertiary amine oxide monomer ODEA (30 μL), crosslinker ADB (15 μL) were added sequentially to the protein solution. After each addition, the solution was mixed homogeneously by repeated pipetting for 1 min, then freshly prepared APS (12.5 μL, 2%) and TEMED (12.5 μL, 5%) solutions were added sequentially to initiate polymerization. After stirring for 10 min at 4 °C, the reaction was dialyzed against PBS (MWCO 10 kDa) to remove unreacted monomers, crosslinker and initiator, resulting in a nanocapsule upoCAT solution.

[0115] Example 11

[0116] Superoxide dismutase SOD was dissolved in ultrapure water (800 μL, 0.5 mg / mL). Freshly prepared cationic monomer B-DEAEA (50 μL), tertiary amine oxide monomer ODEA (30 μL), crosslinker BDD (15 μL) were added sequentially to the protein solution. After each addition, the solution was mixed well by repeated pipetting for 1 min, then freshly prepared APS (12.5 μL, 2%) and TEMED (12.5 μL, 5%) solutions were added sequentially to initiate polymerization. After stirring at 4°C for 10 min, the reaction was dialyzed against PBS (MWCO 10 kDa) to remove unreacted monomers, crosslinker and initiator, to obtain a nano-capsule poSOD solution. The molar ratio of protein / B-DEAEA / ODEA / BDD in the optimal formulation was 0.6 / 60 / 150 / 10.

[0117] Comparative Example 5

[0118] Superoxide dismutase SOD was dissolved in ultrapure water (800 μL, 0.5 mg / mL). Freshly prepared cationic monomer B-DEAEA (50 μL), crosslinker BDD (15 μL) were added sequentially to the protein solution. After each addition, the solution was mixed well by repeated pipetting for 1 min, then freshly prepared APS (12.5 μL, 2%) and TEMED (12.5 μL, 5%) solutions were added sequentially to initiate polymerization. After stirring at 4°C for 10 min, the reaction was dialyzed against PBS (MWCO 10 kDa) to remove unreacted monomers, crosslinker and initiator, to obtain a nano-capsule pSOD solution. The molar ratio of protein / B-DEAEA / BDD in the optimal formulation was 0.6 / 60 / 10.

[0119] Comparative Example 6

[0120] Superoxide dismutase SOD was dissolved in ultrapure water (800 μL, 0.5 mg / mL). Freshly prepared cationic monomer ABDA (50 μL), tertiary amine oxide monomer ODEA (30 μL), crosslinker ADB (15 μL) were added sequentially to the protein solution. After each addition, the solution was mixed well by repeated pipetting for 1 min, then freshly prepared APS (12.5 μL, 2%) and TEMED (12.5 μL, 5%) solutions were added sequentially to initiate polymerization. After stirring at 4°C for 10 min, the reaction was dialyzed against PBS (MWCO 10 kDa) to remove unreacted monomers, crosslinker and initiator, to obtain a nano-capsule uSOD solution.

[0121] Test Example 1

[0122] The ROS response rates of several organic matters were explored by H2O2 degradation. B-DEAEA, BDD, PBD and PBA of Example 2, Example 3, Example 4 and Example 5 were dissolved in ultrapure water (0.5 mg / mL), and then incubated with H2O2 (10 mM) at room temperature, respectively. Every certain time, 20 μL solution was drawn for HPLC analysis. 4-hydroxybenzyl alcohol and / or 2,6-bis(hydroxymethyl)-p-cresol or 1,3-benzenedimethanol were used as standard controls. The results of degradation rates are shown in Table 1. Figure 4 Since the monomer B-DEAEA and crosslinking agents (BDD, PBA and PBD) are unstable in the presence of ROS due to the presence of phenylboronic acid, we analyzed the degradation rate of crosslinking agents in response to reactive oxygen and determined the most sensitive crosslinking agent BDD.

[0123] Test Example 2

[0124] The Brownian motion of particles in mucus was studied by the method of multiple particle tracking. The wider the range of motion trajectory, the smaller the resistance of the particle in the motion of the mucus, and the easier it is to penetrate the mucus layer. FITC-labeled nanocapsules of Example 1, Comparative Example 1 and Comparative Example 2 were added to the mucus, then transferred to a small petri dish and equilibrated at 37°C for 30 minutes. The motion trajectory of the nanocapsules in the mucus within 10 seconds was obtained using a confocal fluorescence microscope. Then imageJ software was used for analysis, and the length and displacement of each trajectory were calculated. The results are shown in Table 2. Figure 5 The length and displacement of the motion trajectory of Example in the mucus within the same time are greater than those of the comparative examples, indicating that the addition of tertiary aminoxyl ODEA increases the migration rate of nanocapsules in the mucus.

[0125] An in vitro simulation of the mucus layer biological barrier was first scraped fresh rabbit intestinal mucus in the top side of Transwell, about 500 μm thick, FITC-labeled nanocapsules of Example 1, Comparative Example 1 and Comparative Example 2 were added and covered the mucus layer, and 600 μL PBS was added in the basal outside chamber. The device was placed in a constant temperature oven at 37°C. At certain time intervals, 50 μL solution was taken from the top and bottom chambers, and the fluorescence intensity was measured at 488 nm excitation and 525 nm emission. The results are shown in Table 3. Figure 6

[0126] An in vitro model simulating cell transport was first established by seeding Caco-2 cells at a density of 80000 cells / well in the top side of Transwell, and then cultured for 15-21 days to form a single layer of cells. The medium on the top and outside was changed every day, and when the trans-epithelial electrical resistance (TEER) value of the single layer was greater than 5000 Ω / / cm 2 ​The time indicates the model establishment is completed. Before the formal experiment, the medium on the top and bottom of the Transwell was replaced with PBS buffer. After the system was balanced for 20 min, the FITC-labeled nanocapsules of Example 1, Comparative Example 1 and Comparative Example 2 were added to the top chamber, and the device was placed in a thermostat at 37°C. At certain time intervals, 50 μL of solution was sampled from the top and bottom chambers, and the fluorescence intensity was measured at 488 nm excitation and 525 nm emission. The results are shown in Figure 7 . The results show that poBSA FITC and upoBSA FITC do exhibit significantly enhanced permeability across the mucus barrier of the intestine or the epithelial cell barrier compared to pBSA FITC and BSA FITC .

[0127] Test Example 3

[0128] Hydrogen peroxide release and consumption assay of nanocapsules. The nanocapsules of Example 2, Comparative Example 3 and Comparative Example 4 were incubated with H2O2 (1 mM) at 37°C for 1 hour. B-DEAEA and BDD were also incubated under the same conditions. The unspent H2O2 content was determined using a hydrogen peroxide assay kit, and the results are shown in Figure 8 . The results show that free CAT, poCAT and pCAT eliminated about 76.2%, 79.8% and 76.3% of the substrate, respectively, while upoCAT containing non-degradable crosslinker only eliminated 35.2%.

[0129] To evaluate the protection of nanocapsules on proteins in the gastrointestinal tract, the nanocapsules of Example 2, Comparative Example 3 and Comparative Example 4 were incubated in 1 mL simulated gastric fluid (SGF) containing 3.2% pepsin (pH 1.2) or simulated intestinal fluid (SIF) containing 10% trypsin (pH 6.8) for 1 hour. Subsequently, the solution was neutralized to pH 7, and 1 mM protease inhibitor was added to inhibit proteolytic activity. Finally, CAT activity was detected using a kit, and the results are shown in Figure 9 . As shown in the figure, free CAT, pCAT and upoCAT essentially lost or failed to initiate catalytic activity, but poCAT retained about 85% and 70% of the original activity after SGF or SIF incubation, respectively.

[0130] Test Example 4

[0131] Hydrogen peroxide release and protein activity assay of nanocapsules. The nanocapsules of Example 3, Comparative Example 5 and Comparative Example 6 were incubated with H2O2 (1 mM) at 37°C for 1 hour, and the activity of released SOD was determined according to the instructions using an SOD kit, and the results are shown in Figure 10 . Compared with Figure 7Results were similar, free SOD, poSOD and pSOD had strong activity on the substrate, while the non-degradable upoSOD had weak effect.

[0132] To evaluate the protection of nanocapsules on proteins in the gastrointestinal tract, the nanocapsules of Example 3, Comparative Example 5 and Comparative Example 6 were incubated in 1 mL of SGF containing 3.2% pepsin (pH 1.2) or SIF containing 10% trypsin (pH 6.8) for 1 hour. Subsequently, the solution was neutralized to pH 7 and 1 mM protease inhibitors were added to inhibit proteolytic activity. Finally, the SOD activity was detected using a kit and the results are shown in Figure 11 . Similar to the results shown in Figure 2, free SOD, poSOD and pSOD had strong activity on the substrate after incubation in SGF or SIF, while the non-degradable upoSOD had weak effect. Figure 8 Results were similar, after incubation in SGF or SIF, free SOD, pSOD and upoSOD basically lost or failed to induce catalytic activity, only poSOD retained a high level of activity.

[0133] Test Example 5

[0134] The mouse model of ulcerative colitis (UC) was established by free drinking 3% dextran sodium sulfate (DSS). On the third day of modeling, the nanocapsules described in Example 3 and Example 4 were mixed at the same proportion, the nanocapsules described in Comparative Example 3 and Comparative Example 5 were mixed at the same proportion, and the nanocapsules poCAT and poSOD described in Comparative Example 4 and Comparative Example 6 were mixed at the same proportion (poSC). Male C57BL / 6 mice were orally gavaged at a dose of 4 mg / kg equivalent CAT and SOD (6 mice per group), and the commercially available therapeutic drug mesalazine (5-aminosalicylic acid, 5-Aminosalicylic Acid, abbreviated as ASA) enteric-coated tablets were used as a positive drug control group, and mice without drug administration were used as a healthy control group. Body weight, stool consistency and fecal bleeding were monitored daily to determine the disease activity index (DAI). DAI = weight loss score + diarrhea score + rectal bleeding score. On the 8th day, the mice were euthanized and the tissues were collected for further analysis. The results of body weight loss and DAI score are shown in Figure 12 , the results of colon length are shown in Figure 13 , and the results of colon ROS concentration are shown in Figure 14 The disease activity index (DAI) is a comprehensive score reflecting the severity of colitis, which showed a roughly linear increase in UC mice treated with physiological saline, pSC, upoSC or poSC, but this trend basically stopped after poSC treatment. At the same time, the colon of UC mice treated with physiological saline, pSC, upoSC or poSC was significantly shorter than that of normal mice. DSS-treated mice showed an increase in ROS levels, which returned to the basal level after poSC treatment, indicating that poSC can effectively alleviate oxidative stress in vivo.

[0135] Test Example 6

[0136] Oral treatment was performed for induced radiation colitis. C57BL / 6 mice were anesthetized and irradiated with 6-Gy x-rays on the abdomen. Normal saline or poSC (2 mg / kg for both SOD and CAT) was administered orally before and 1 hour after irradiation, and then the same dose was administered orally on day 2 and day 3, once a day. The mice were euthanized on day 4 and the colon was collected for further analysis. The results of body weight loss are shown in Figure 15 , the results of small intestine H&E staining are shown in Figure 16 , and the results of colon ROS concentration are shown in Figure 17 After treatment with poSC, the body weight change of the mice was significantly controlled, and the induced radiation colitis was also significantly improved, tending to be normal mice. The 6-Gy x-ray irradiated mice showed an increase in ROS levels, which returned to the basal level after poSC treatment, indicating that poSC can effectively alleviate oxidative stress caused by induced radiation in vivo.

Claims

1. A high permeable polymeric nanocapsule characterized in that, The high-permeability polymer nanocapsule is a core-shell structure, wherein the inner core of the core-shell structure is a bioactive molecule, and the bioactive molecule comprises at least one of a protein and a nucleic acid, The shell of the core-shell structure comprises a polymer polymerized from monomer materials, and the monomer materials comprise a cationic monomer, a tertiary amine oxide monomer and a crosslinking agent; The cationic monomer is any one of the compounds shown in the following structural formula: The crosslinking agent is any one of the compounds shown in the following structural formula: The tertiary amine oxide monomer has a structure shown in formula (3): The molar ratio of the bioactive molecule, the cationic monomer, the tertiary amine oxide monomer and the crosslinking agent is 1:10-3000:10-3000:1-1000.

2. The high permeable polymeric nanocapsule according to claim 1, characterized in that, The molar ratio of the bioactive molecule, the cationic monomer, the tertiary amine oxide monomer and the crosslinking agent is 0.6:60:150:

10.

3. The high permeable polymeric nanocapsule according to claim 1, characterized in that, The protein is at least one of serum albumin, uric acid oxidase, catalase, glucose oxidase, horseradish peroxidase, superoxide dismutase, lactate dehydrogenase, acetaldehyde dehydrogenase, alcohol oxidase, acetaldehyde oxidase, antibody, insulin, and cytokine, The nucleic acid is at least one of siRNA, mRNA, shRNA, microRNA, ASO and DNA.

4. The method for preparing the highly permeable polymer nanocapsules according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: (1) mixing the bioactive molecule, the cationic monomer, the tertiary amine oxide monomer and the crosslinking agent to make the reaction monomers and the molecules aggregate; (2) adding ammonium persulfate and N,N,N',N'-tetramethyl ethylenediamine to initiate interfacial polymerization in step (1) to obtain the high-permeability polymer nanocapsule.

5. The method for preparing highly permeable polymer nanocapsules according to claim 4, characterized in that: In step (2), the concentration of the ammonium persulfate is 0.1-10 mg / mL, and the concentration of the N,N,N',N'-tetramethyl ethylenediamine is 0.1-10 mg / mL.

6. The method for preparing highly permeable polymer nanocapsules according to claim 4, characterized in that: In step (2), the interfacial polymerization is performed under the following conditions: the reaction time is 0.1-24 hours, the reaction temperature is 4-37°C, and the pH of the interfacial polymerization is 8-10.

7. The method for preparing highly permeable polymer nanocapsules according to claim 4, characterized in that: The prepared polymer nanocapsule has a particle size of 5-200 nm.

8. Use of the high-permeability polymer nanocapsule of any one of claims 1-3 in the preparation of a macromolecular drug delivery carrier.

9. Use of the high-permeability polymer nanocapsule of any one of claims 1-3 in the preparation of a drug for preventing and / or treating a related disease, wherein the related disease is induced radiation colitis or ulcerative colitis.

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