Porous organic cage nano-drug delivery system as well as preparation method and medical application thereof

Abstract

The invention discloses a porous organic cage nano-drug delivery system as well as a preparation method and medical application thereof. The porous organic cage is formed by taking porous organic cage molecules loaded with drug molecules as a main body, and the porous organic cage nano-drug delivery system is simple in preparation method, has the characteristics of high biological safety, high drug loading capacity, pH responsiveness and slow release effect, and is particularly suitable for precise treatment of tumors.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the construction of responsive nano drug carriers using porous organic cages (POCs) materials and their application in biomedicine. Background Art

[0002] Cancer is one of the main causes of death in humans, and its incidence rate has continued to rise in recent years. However, there is still no efficient treatment method that can completely kill tumor cells without damaging normal cells or organs. During tumor treatment, certain systemic toxic side effects often occur, affecting the survival rate of cancer patients. Therefore, it is necessary to develop tumor in situ responsive treatment. An important feature of tumors is the enhanced permeability and retention (EPR) effect. The cell junctions of blood vessels in normal tissues are less than 6nm, but due to the EPR effect, the cell junctions of solid tumor blood vessels can range from 200 to 600nm. Therefore, nanoparticles smaller than 200nm can passively accumulate at the tumor site during blood circulation, thereby targeting drugs to cancer cells. In addition, at the tumor site, lactic acid or other acids produced by hypoxia can cause its pH to be lower than that of normal tissues. The extracellular pH of tumors is about 6.8, while the intracellular pH can be between 4.5 and 6.5. Therefore, designing a pH-responsive smart nanodrug carrier through the pH difference between tumors and normal tissues has become a hot research direction. To date, porous nanomaterials, including metal organic framework materials, covalent organic framework materials, porous organic polymers, porous carbon, porous silica and other materials have been developed and used for targeted drug delivery. However, how to design and prepare responsive nano drug carriers to improve anti-tumor efficiency is still full of challenges.

[0003] Porous organic cages (POCs) are a new class of porous nanomaterials. They are self-assembled into organic macromolecules with specific cavity sizes by covalently bonded organic synthons. These discrete macromolecules are stacked into an ordered structure through weak interactions, and their pores are composed of intra-cage cavities and stacked through-holes. Porous organic cages have the advantages of low density, high specific surface area, high porosity, good biosafety, and good chemical and thermal stability. Porous organic cages are different from metal organic frameworks (MOFs) and covalent organic frameworks (COFs). Porous organic cages (POCs) are three-dimensional cage structures (with specific) internal cavities formed by self-assembly of light elements such as C, H, N, O, and B through the formation of covalent bonds. They are stable and can exist for a long time. At present, porous organic cages have potential application prospects in the fields of selective guest inclusion, gas adsorption storage and separation, selective separation of organic small molecules, and nanocavity catalysis. So far, the application of such porous materials as nano-drug carriers in biomedicine is still blank. This special nanocage material can encapsulate drugs into cavities or pores, carry the drugs in the body to participate in circulation, and then open the cage under specific conditions to release the drugs. Due to its unique characteristics, porous organic cage nanodrug carriers are expected to overcome the limitations of anti-cancer chemotherapy or high toxicity caused by the nanocarriers themselves, while also being expected to achieve high drug loading and responsive drug release, thereby achieving precise treatment of tumors and effectively reducing the toxic side effects of drugs. Summary of the invention

[0004] The present invention provides a method of using porous organic cages as nano drug carriers to prepare drug nanocapsules with high biosafety, high drug loading, pH responsiveness and sustained release effect through a simple method, and apply the drug nanocapsules to the precise treatment of tumors.

[0005] In order to achieve the above-mentioned object of the invention, the specific technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides the use of porous organic cages as nano drug carriers for loading and delivering drug molecules.

[0007] According to an embodiment of the present invention, the porous organic cage and loaded drugs are prepared into nanocapsules for drug delivery.

[0008] According to an embodiment of the present invention, the porous organic cage is a calixarene-based porous organic cage, and further, is selected from a calix[4]arene-based porous organic cage, for example, more specifically, selected from a resorcinol calix[4]arene-based porous organic cage, such as C-methylresorcinol calix[4]arene; C-ethylresorcinol calix[4]arene; C-propylresorcinol calix[4]arene; C-butylresorcinol calix[4]arene; C-isobutylresorcinol calix[4]arene, etc.;

[0009] According to an embodiment of the present invention, the porous organic cage is selected from one, two or more of resorcinol calix[4]arene organic cages: CPOC-101, CPOC-201, CPOC-301, HPOC-101, HPOC-201, HPOC-301, HPOC-401.

[0010] In a second aspect, the present invention provides the use of porous organic cages as nano drug carriers for preparing anti-tumor drugs.

[0011] According to an embodiment of the present invention, the porous organic cage and the loaded anticancer drug are prepared into nanocapsules for drug delivery.

[0012] According to an embodiment of the present invention, the porous organic cage is a calix[4]arene-based porous organic cage, and further, is selected from calix[4]arene-based porous organic cages, for example, more specifically, selected from resorcinol calix[4]arene-based porous organic cages, such as C-methylresorcinol calix[4]arene; C-ethylresorcinol calix[4]arene; C-propylresorcinol calix[4]arene; C-butylresorcinol calix[4]arene; C-isobutylresorcinol calix[4]arene and the like.

[0013] The porous organic cage is selected from one, two or more of the resorcinol calix[4]arene organic cages: CPOC-101, CPOC-201, CPOC-301, HPOC-101, HPOC-201, HPOC-301, and HPOC-401.

[0014] According to an embodiment of the present invention, the anticancer drug is selected from: one, two or more of doxorubicin (DOX), camptothecin (CPT), 5-fluorouracil (5-FU), and paclitaxel (PTX).

[0015] According to an embodiment of the present invention, the cancer can be adrenocortical carcinoma (ACC), colon cancer (COAD), lung adenocarcinoma (LUAD), liver cancer cells (HpeG2), melanoma cells (B16-F10), etc. As an example, liver cancer cells (HpeG2) and melanoma cells (B16-F10) are selected as models.

[0016] In a third aspect, the present invention provides a method for preparing a drug delivery system, the method comprising the following steps:

[0017] (1) mixing the porous organic cage and the drug molecules in a solution so that the drug molecules enter the porous organic cage;

[0018] (2) dissolving the porous organic cage loaded with drug molecules obtained in step (1) in the oil phase of the surfactant, adding water, and allowing the oil phase to diffuse into the water phase to form nanocapsules.

[0019] According to an embodiment of the present invention, the porous organic cage material is a calix[4]arene-based porous organic cage, and further, is selected from calix[4]arene-based porous organic cages, for example, more specifically, selected from resorcinol calix[4]arene-based porous organic cages, such as C-methylresorcinol calix[4]arene; C-ethylresorcinol calix[4]arene; C-propylresorcinol calix[4]arene; C-butylresorcinol calix[4]arene; C-isobutylresorcinol calix[4]arene and the like.

[0020] The porous organic cage is selected from one, two or more of the resorcinol calix[4]arene organic cages: CPOC-101, CPOC-201, CPOC-301, HPOC-101, HPOC-201, HPOC-301, and HPOC-401.

[0021] As an example, the porous organic cage (POC) is selected from calixarene-based porous organic cage CPOC-301, whose molecular formula is C 360 H 384 N 24 O 48 , having a molecular structure as shown in Formula I.

[0022]

[0023] According to an embodiment of the present invention, the drug molecule is selected from one, two or more of doxorubicin (DOX), camptothecin (CPT), 5-fluorouracil (5-FU) and paclitaxel (PTX). As an example, the drug molecule selected is paclitaxel (PTX).

[0024] According to an embodiment of the present invention, the surfactant is selected from one, two or more of Tween 20, Tween 80, sodium linear alkylbenzene sulfonate (LAS), cocamidopropyl betaine (CAB-35) and polyoxyethylene hydrogenated castor oil (RH40). As an example, polyoxyethylene hydrogenated castor oil (RH40) is selected.

[0025] According to an embodiment of the present invention, in step (1), the porous organic cage is immersed in the drug molecule solution to achieve mixing. Further, the drug-loaded porous organic cage is separated from the solution by vacuum filtration.

[0026] Preferably, the mass ratio of the porous organic cage to the drug molecule is 5:1 to 1:1, for example, 3:1, 2:1, 1.5:1, 1.4:1, 1.35:1, 1.3:1.

[0027] Preferably, the drug molecule solution is a solution of drug molecules dissolved in an organic solvent. The solvent may be a C1-C6 alcohol, such as methanol, ethanol, propanol, etc.

[0028] Preferably, the concentration of the drug molecule solution is 10-30 mg / mL, further, it can be said to be 10-20 mg / mL.

[0029] Preferably, after the porous organic cage is immersed in the drug molecule solution, it is stirred for 10-30 hours, for example, 15 hours.

[0030] Preferably, the step (1) further comprises purifying the sample obtained after filtration, for example, washing and drying.

[0031] For example, the drying method may be vacuum drying. Preferably, the drying temperature may be 60-150° C., exemplarily 100° C.; the drying time may be 6-20 hours, for example 12 hours.

[0032] Preferably, the step (1) further comprises pre-treating the porous organic cage by vacuum drying.

[0033] According to an embodiment of the present invention, in the step (2), the porous organic cage loaded with drug molecules obtained in the step (1) is mixed with a surfactant, dissolved in a constant temperature water bath, an aqueous phase is added, and the temperature is continued to be constant to form drug-loaded nanocapsules.

[0034] Preferably, the mass ratio of the porous organic cage loaded with drug molecules to the surfactant is 1:100 to 1:400, such as 1:150 to 1:300, 1:170 to 1:250, and exemplarily 1:200.

[0035] Preferably, the temperature of the constant temperature water bath is 30-100°C, for example, 50°C.

[0036] Preferably, stirring is performed during dissolution in a constant temperature water bath, and the stirring time is 10-30 hours, for example, 15 hours.

[0037] Preferably, the aqueous phase is added by dropwise addition of ultrapure water, and the ratio (ml / mg) of the volume of ultrapure water to the mass of the porous organic cage loaded with drug molecules obtained in step (1) is 1:2 to 1:10, for example 1:3 to 1:8, 1:4 to 1:6, 1:5.

[0038] Preferably, stirring is continued at a constant temperature for 1-3 hours, for example, 2 hours.

[0039] The preparation method according to the present invention further comprises a step of preparing a porous organic cage before step (1).

[0040] Furthermore, the above-mentioned step of preparing a porous organic cage is to prepare a calixarene-based porous organic cage CPOC-301 (whose molecular formula is C 360 H 384 N 24 O 48 ) step, which is obtained by reacting tetraaldehyde resorcinol calix[4]arene (C4RACHO) and p-phenylenediamine (PDA).

[0041] Preferably, the molar ratio of tetraaldehyde resorcinol calix[4]arene (C4RACHO) to p-phenylenediamine (PDA) is 1:(1-3), exemplified by 1:1, 1:2, 1:3.

[0042] Preferably, the reaction temperature is 40-100° C., for example, 65° C.; the reaction time is 1-3 days, for example, 2 days.

[0043] Preferably, the reaction is carried out in an organic solvent. For example, the solvent may be chloroform.

[0044] Preferably, the preparation method further comprises: after the reaction is completed, the step of collecting the product porous organic cage from the reaction solution.

[0045] Preferably, the collecting comprises: evaporating the reaction solution obtained after the reaction is completed at a constant temperature to obtain porous organic cage crystals. For example, the evaporation temperature is room temperature; for another example, the evaporation time is 6-20 hours, for example, 12 hours.

[0046] Preferably, the preparation method further comprises purifying the crystal product, for example, by filtering, washing with methanol, exchanging methanol several times, and drying to obtain a porous organic cage from which the guest solvent molecules are removed.

[0047] Preferably, the drying method may be vacuum drying. The drying temperature may be 60-130° C., such as 100° C., and the drying time may be 6-20 hours, such as 12 hours.

[0048] In a fourth aspect, the present invention provides a drug delivery system prepared by the above preparation method.

[0049] According to an embodiment of the present invention, the drug delivery system is formed mainly by porous organic cage molecules loaded with drug molecules. According to an embodiment of the present invention, the drug delivery system is a nanocapsule.

[0050] According to an embodiment of the present invention, the particle size of the nanocapsule is less than 25 nm, further, less than 23 nm, 22 nm, and within the range of 20±2 nm, 20±1 nm.

[0051] The present invention proposes for the first time a solution for preparing drug nanocapsules using porous organic cages as drug delivery carriers, and conducts in-depth research on the application of the porous organic cage drug delivery system in anti-tumor aspects.

[0052] First, according to the implementation scheme of the present invention, in terms of in vitro anti-tumor evaluation, liver cancer cells (HpeG2) and melanoma cells (B16-F10) were used as models, the MTT method was used for detection, and paclitaxel (PTX) raw material was used as the control group to determine the proliferation inhibitory effect of porous organic cage-paclitaxel nanocapsules at different concentrations and times on the two types of cells, and the half inhibitory concentration (IC50) was used as an indicator to evaluate its in vitro cellular anti-tumor activity.

[0053] Secondly, in terms of in vivo antitumor pharmacodynamics, a tumor-bearing mouse model was established: 4T1 cells with good growth in the logarithmic phase were digested with trypsin, counted, centrifuged at 1000 rpm, and redispersed with 1640 culture medium. The cells were counted, centrifuged at 1000 rpm, and redispersed with PBS and stored in an ice box for later use. The cell number was 1×10 7 Cell / mL depilated the backs of healthy mice, and 100 μL was injected on the right back of the mice to establish a tumor-bearing mouse model. After inoculation, when the tumor volume grew to 100 mm3, 24 mice were randomly divided into 4 groups (6 mice in each group): normal saline group, porous organic cage group, porous organic cage-paclitaxel nanocapsule group and free paclitaxel group (the dosage was 10 mg / kg, calculated as paclitaxel). The drugs were injected through the tail vein every other day for a period of 14 days. From the start of drug administration, the weight and survival of the mice were observed every other day, the long and short diameters of the tumors were measured, and the average tumor volume and tumor burden of each group of mice were calculated.

[0054] Beneficial effects of the present invention:

[0055] The present invention provides a method of using porous organic cages as nano drug carriers to prepare nano drug capsules with high biosafety, high drug loading, pH responsiveness, and sustained release through a simple method, and apply the nano drug capsules to the precise treatment of tumors. At present, the application of porous organic cages as nano drug carriers in tumor treatment is still blank. In addition, the use of porous organic cages as nano drug carriers has the following advantages in anti-tumor applications: good biosafety, high drug loading, pH responsiveness, and sustained release. Loading drugs on porous organic cages can also help achieve long drug circulation and improve targeting to the micro-acid environment of tumors, thereby improving the anti-tumor efficacy of drugs and reducing the toxic side effects of normal tissues and organs. In summary, the preparation process of the present invention is simple, reproducible, and has good application transformation prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1Cell viability of MCF-7 (A) and MCF-10A cells after incubation with POC for 24 h (B).

[0057] Figure 2 It is the hemolysis rate after incubation of blank POC nanocapsule solution with different concentrations and 50% whole blood at 37℃ for 1 hour.

[0058] Figure 3 The figure shows the body weight changes of mice 6 days after administration.

[0059] Figure 4 PTX standard curve (A), PTX fitting curve (B) and PTX concentration in supernatant before and after reaction (C).

[0060] Figure 5 For infrared spectrum.

[0061] Figure 6 In vitro release of POC-PTX.

[0062] Figure 7 Particle size, PDI changes (a) and particle size distribution (b) of POC-PTX nanocapsules stored at 2-8°C, electron micrographs of blank POC nanocapsules (C) and POC-PTX nanocapsules (D).

[0063] Figure 8 POC solid powder (A); RH 40 aqueous solution (B); appearance of blank POC nanocapsule solution (C) and POC-PTX nanocapsule solution (D).

[0064] Fig. 9 Tyndall effect diagram of blank POC nanocapsule solution (A); POC nanocapsule solution loaded with PTX (B).

[0065] Fig.10 UV spectrum.

[0066] Fig.11 is the XRD pattern.

[0067] Fig.12 In vitro release of POC-PTX nanocapsules at pH 7.4, 6.5, 5.5 and 1.2.

[0068] Fig.13 The changes in particle size and PDI of POC-PTX nanocapsule solution at different times.

[0069] Fig.14 The changes in encapsulation efficiency and drug loading rate of POC-PTX nanocapsule solution at 2-8°C for 72 hours.

[0070] Fig.15To compare the pharmacokinetic concentration-time curves of PTX and POC-PTX after intravenous injection.

[0071] Fig.16 This is a comparison of the in vitro MTT antitumor effects of POC-PTX and free PTX.

[0072] Fig.17 Comparison of cellular uptake of POC-C6, Free C6 and MCF-7 cells after incubation for 0.5h (A) and 2h (B), as well as Image J semi-quantitative results (C).

[0073] Fig.18 In vivo fluorescence imaging of Free DIR and POC-DIR NCs at 2, 4, 8, and 24 hours.

[0074] Fig.19 Design for experiments.

[0075] Fig. 20 The weight changes of mice.

[0076] Fig.21 Changes in tumor volume.

[0077] Fig. 22 Photo of the tumor.

[0078] Fig.23 Tumor weight and tumor inhibition rate.

[0079] Fig.24 HE staining of tissues, organs and tumor sections in each group after treatment.

[0080] Fig.25 Comparison of serum indicators in each group after treatment.

[0081] Fig.26 Schematic diagram of the construction and application of porous organic cage nanodrug delivery system. DETAILED DESCRIPTION

[0082] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0083] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0084] Example 1: Preparation and biological performance characterization of POC

[0085] Synthesis of porous organic cage material (POC): tetraaldehyde resorcinol calix [4] arene (C4RACHO, 82 mg), p-phenylenediamine (PDA, 21.5 mg), chloroform (15 mL) and nitrobenzene (5 mL) were placed in a 50 mL pressure bottle, ultrasonicated for 10 minutes, sealed, and heated at 65 ° C for 2 days. After cooling to room temperature, it was divided into 20 mL glass bottles and placed in a 500 mL wide-mouth bottle. The crystals were collected by methanol diffusion, washed with a large amount of methanol in turn, and vacuum dried at 100 ° C for 10 hours to obtain a red-black microcrystalline powder for use. The synthesized POC was subjected to cell safety experiments, hemolysis tests, and animal acute toxicity tests, see attached. Figure 1-3 .

[0086] Figure 1 It shows that the MTT method was used to evaluate the safety effects of POC on MCF-10A (normal breast cells) and MCF-7 (breast cancer cells). The two cells were cultured in DMEM medium (10% fetal bovine serum) and 10A special complete medium, respectively, at 37°C, 5% CO2 incubator. Cells in the logarithmic growth phase were inoculated in a 96-well culture plate and incubated for 24 hours, with a cell number of 3000 / well. After adding the drug, they were incubated for 24 hours, and MTT 20μL (5mg / mL) was added to each well. The cells were cultured in the dark for 4 hours, and the culture was terminated and the culture medium in the well was discarded. 150μL of dimethyl sulfoxide was added to each well, and the cells were shaken at a low speed on a shaker for 10 minutes to fully dissolve the crystals. The absorbance of each well was measured at 570nm on an enzyme-linked immunosorbent assay. The experiment was divided into three groups: a blank group (culture medium containing cells and RH40), a drug-added group, and a background group (culture medium only). Calculate the cell survival rate and draw a curve with concentration as the horizontal axis and cell survival rate as the vertical axis ( Figure 1 ). When the POC was administered at a concentration of 200 μg / mL, the survival rates of both cell lines were high, and it was basically non-toxic to the cells and had a high safety.

[0087] Figure 2 It shows that the mother liquor is diluted with PBS in a gradient manner and mixed with 50% whole blood in a test tube and then placed in a 37°C constant temperature water bath. After 1 hour, transfer to a centrifuge tube and centrifuge at 3000rpm for 15 minutes. Take 150μL of the supernatant in a 96-well plate, set up 3 replicate wells, and put it into an enzyme-linked immunosorbent assay instrument to measure the OD value of each group of samples at a wavelength of 570nm. Ultrapure water was used as the positive control group and PBS as the negative control group. The hemolysis rate was calculated according to the following formula: Hemolysis rate (%) = (D 样 -D 阴 ) / (D 阴 -D 阳 )×100%, where: D 样 is the absorbance value of the POC sample group; D 阴 is the absorbance value of the negative control group; D 阳is the absorbance value of the positive control group. As shown in the figure, when the POC dosage concentration is 200μg / mL, the survival rate of the two cells is high, it is basically non-toxic to the cells and has high safety; when the concentration is 400μg / mL, the hemolysis rate is less than 5%, and there is no hemolysis, indicating that POC has good biocompatibility at the indicated dosage concentration.

[0088] Figure 3 It shows that, using ICR mice as the research object, a POC solution (suspension) with a concentration of 10mg / mL was prepared, and multiple intraperitoneal administration was performed within 48h (the dosing interval was 2h, and the dosing volume was 0.1mL / 10g), and the survival rate and health status of the mice were observed. The experiment was divided into two groups, namely the drug administration group (POC) and the control group (same proportion RH40), with 6 mice in each group, half male and half female. Results: The cumulative dose within 48h was 2000mg / kg, and there was no death in the mice, the survival rate was 100%, and there was no significant change in the weight of the mice within 6 days ( Figure 3 ).

[0089] Example 2: Preparation and physical properties of POC-PTX nanocapsules

[0090] 116 mg of POC was immersed in 5 mL of paclitaxel (PTX) methanol solution and stirred for 15 h. The drug-loaded sample was separated from the solution by vacuum filtration, washed with methanol, and dried in a vacuum drying oven. The drug loading was calculated by absorbing the supernatant of the drug solution before and after the reaction. The synthesized POC-PTX was characterized by UV-Vis spectrophotometry, infrared spectroscopy, and in vitro release. See Appendix Figure 4-6 .

[0091] Figure 4 The results show that the drug loading of POC was 24.63% as measured by UV-Vis spectrophotometer. The drug loading of POC was 24.63% as measured by the formula: drug loading (%) = (drug content in supernatant before reaction - drug content in supernatant after reaction) / [(drug content in supernatant before reaction - drug content in supernatant after reaction) + organic cage feed amount] × 100%.

[0092] Figure 5 It is shown that 1-2 mg of PTX, POC and POC-PTX solids were weighed, ground into powder clockwise with KBr (100-200 mg) in a clean mortar, and pressed into thin sheets with good light transmittance. After deducting the potassium bromide background, the scanning analysis was performed in the range of 400-4000 cm-1. When POC was loaded with PTX, its -OH 3365 cm -1 The stretching vibration peak of 815cm -1 The peak at 1118 cm -1The appearance of the absorption peak may be due to the formation of non-covalent bonds such as molecular hydrogen bonds and π-π conjugation between PTX and POC.

[0093] Figure 6 It is shown that the in vitro release behavior of paclitaxel from drug-loaded POC-PTX solid in 5% SDS aqueous solution at different pH (5.5, 7.4) was studied by dynamic dialysis. 1 mL of POC-PTX solid solution was put into a dialysis bag (with a cutoff relative molecular mass of 3500D), and then the dialysis bag was placed in a dialysis tube filled with 50 mL of 5% SDS PBS. The oscillator speed was set to 200 r / min and the temperature was set to 37°C. The in vitro release behavior of drug-loaded POC was investigated. 1 mL of sample was taken at the specified time points (0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 96, 120 h), and 1 mL of fresh release medium was immediately added. The release was quantified by UV detection and the release-time curve was drawn. POC-PTX is released faster in an acidic environment than in a neutral environment and has a sustained-release effect. The release amount reaches 82.57% within 48 hours, indicating that its drug-loaded POC solid has an acid-responsive and sustained-release mechanism.

[0094] Example 3: Preparation and physical properties of POC-PTX nanocapsules

[0095] Weigh the prepared POC-PTX solid into a vial, add an appropriate amount of surfactant (such as polyoxyethylene hydrogenated castor oil, RH 40, RH40), where POC: surfactant (1:150~1:200, w / w). Place it in a constant temperature water bath, stir at 50℃ for 2h until completely dissolved, slowly add ultrapure water under stirring, the oil phase solvent diffuses into the water phase, and stir at constant temperature for 1h to form drug-loaded POC nanocapsules. At the same time, dilute it 10 times with ultrapure water, measure its particle size, PDI, and Zeta potential at different times with a laser particle size potential analyzer, and investigate its physical and chemical properties and stability. Without adding paclitaxel, the rest of the operations are the same as above to prepare a blank POC nanocapsule solution without drugs.

[0096] 1mL of POC-PTX nanocapsule solution was placed in an ultrafiltration tube and centrifuged at low speed (5000rpm, 4℃) for 20min. The free paclitaxel was centrifuged in the supernatant of the ultrafiltration tube. 200μL of the free solution was diluted with 10 times methanol (2mL) and subjected to high-performance liquid phase determination (i.e., free drug content = unencapsulated drug content). The mother solution was diluted to a certain multiple and then quantitatively determined by liquid phase in the same way. The drug loading and encapsulation efficiency were calculated. The encapsulation efficiency (EE) and drug loading (DL) at different times were determined by the same method to investigate the stability.

[0097] The encapsulation efficiency was calculated using the following formula (3):

[0098] Encapsulation efficiency (%) = (total amount of drug - free drug content) / total amount of drug × 100% Formula (3)

[0099] The drug loading was calculated using the following formula (4):

[0100] Drug loading (%) = (total drug content - free drug content) / (total POC content + total drug content) × 100% Formula (4)

[0101] Figure 7 The results showed that the particle size of POC-PTX nanocapsules was about 20nm, with an obvious normal distribution and a uniform spherical appearance. The particle size increased slightly after drug loading, which may be the result of the interaction between PTX and POC. It has good stability when stored at 2-8℃ for 120h.

[0102] Figure 8 The results showed that the POC solid powder was brown, and the RH 40 aqueous solution was clear and transparent; the blank POC nanocapsule solution was translucent reddish brown; and the appearance of the POC nanocapsule solution loaded with paclitaxel did not change significantly.

[0103] Fig. 9 It shows that both the blank POC nanocapsule solution and the POC nanocapsule loaded with paclitaxel (PTX) have the Tyndall effect, indicating that both are colloids with particle diameters between 1-100 nm.

[0104] Fig.10 It shows that by weighing POC-PTX nanocapsules, POC nanocapsules and paclitaxel (PTX) solution, ultrapure water solvent was used as blank, and UV scanning was performed. The absorption peaks of POC-PTX nanocapsules after drug loading were red-shifted, indicating that the two may have non-covalent binding such as π-π conjugation, hydrogen bonding, hydrophobic interaction, etc.

[0105] Fig.11 It is shown that X-ray diffraction (XRD) was performed on PTX, POC solid, PTX+POC physical mixture, POC nanocapsule freeze-dried, and POC-PTX nanocapsule freeze-dried samples. The scanning speed was 5 (°)·min-1, and the scanning angle range was 3° to 50°. The test voltage and current were 20kV and 20mA, respectively, and the emission source was Cu-Kα. POC-PTX nanocapsule retained most of the characteristic peaks of POC, while several strong characteristic peaks of PTX almost disappeared. It is speculated that the two may be combined by non-covalent bonding, and the spectrum of the POC+PTX physical mixture shows a simple superposition of the absorption peaks of the two.

[0106] Fig.12 It was shown that the in vitro paclitaxel release behavior of the nanocapsules at different pH values ​​(1.2, 5.5, 6.5 and 7.4) was studied by dynamic dialysis. 1 mL of the POC-PTX nanocapsule solution was put into a dialysis bag (with a relative molecular mass cutoff of 3500D), and then the dialysis bag was placed in a dialysis tube containing 50 mL of a 5% sodium dodecyl sulfate (SDS) aqueous solution. The oscillator speed was set to 200 r / min and the temperature was set to 37°C. The in vitro release behavior of the nanocapsules was investigated. 1 mL of samples were taken at specified time points (0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 h), and 1 mL of fresh release medium was immediately added. The sample was diluted with 5 times methanol, and the quantification was performed by high performance liquid chromatography to draw the release curve. Fig.12 It can be seen that the POC nanocapsule releases PTX faster in an acidic environment than in a neutral environment. In addition, the PTX release amount is higher in an acidic environment, and both have the characteristics of sustained release, reaching 82.41% after 48 hours. This shows that the prepared POC-PTX nanocapsule has pH responsiveness, which may be because the imine bond in POC is unstable in an acidic environment, thereby destroying its structure and releasing the drug. Therefore, the pH responsiveness of the POC carrier structure enables it to have the effect of targeting the slightly acidic environment of the tumor, thereby improving the efficacy of the drug and reducing the toxic side effects of normal tissues and organs.

[0107] Fig.13 The results show that the particle size and PDI change tests show that the particle size of the POC-PTX nanocapsule solution fluctuates around 20 nm within 120 hours, and the PDI has basically not changed, indicating that it has strong stability.

[0108] Fig.14 The results show that the encapsulation rate and drug loading rate of POC-PTX nanocapsule solution are 83.57% and 36.72% respectively. The encapsulation rate and drug loading rate of POC-PTX nanocapsule solution are not changed much when stored at 2-8℃ for 72h, and RE is 6.7% and 11.5%, respectively, indicating that POC-PTX nanocapsule is relatively stable. In summary, POC-PTX nanocapsule solution has the characteristics of high drug loading and good stability.

[0109] Fig.15The results show that the content of paclitaxel in rat plasma changes with time after PTX (Taxol) and POC-PTX nanocapsules (POC-PTX NC) were injected into the rat tail vein. SD rats weighing 180-220g were randomly divided into free PTX group and POC-PTX group, with 6 rats in each group. They were fasted before the experiment and had free access to water. PTX solution control group and POC-PTX nanocapsule solution group were injected into the tail vein, respectively, with a dose of 15mg / kg. 0.5mL of blood was collected from the eye sockets at 0, 0.083, 0.25, 0.75, 1, 2, 3, 6, 8, 10, and 12, transferred to heparinized EP tubes, centrifuged (5000rpm, 4℃, 5min), and the upper plasma was frozen and stored in a -4℃ refrigerator. For subsequent sample analysis. DAS was used to fit the non-compartmental model. 100 μL of rat plasma was taken into a 1.5 mL ep tube, and 500 μL of ethyl acetate was added to extract the plasma sample. After vortexing for 5 minutes, the sample was centrifuged (10000 rpm, 10 minutes, 4°C). The supernatant was dried with a nitrogen gas meter, and 100 μL of mobile phase was reconstituted. The sample was vortexed for 1 minute, centrifuged (12000 rpm, 10 minutes, 4°C), and 80 μL was taken into the inner tube for injection analysis. HPLC was used for determination. In addition, Table 1 shows that the POC-PTX group t 1 / 2 It was 1.67 times that of the free PTX group, and AUC 0-t is 3.51 times, MRT 0-t The drug-loaded POC nanocapsules can prolong the circulation time of paclitaxel in the blood and improve its bioavailability.

[0110] Table 1 Pharmacokinetic parameters

[0111]

[0112] *P<0.05VS Free PTX

[0113] Example 4: In vitro anti-tumor evaluation of POC-PTX nanocapsules

[0114] In this study, liver cancer cells (HpeG2) and melanoma cells (B16-F10) were used as models, and the MTT method was used to detect the proliferation inhibition of the two cells. Paclitaxel raw materials were used as the control group to determine the inhibitory effect of POC-PTX nanocapsules at different concentrations and different times on the proliferation of the two cells. The half inhibitory concentration (IC 50 ) were used as indicators to evaluate its in vitro antitumor activity.

[0115] Fig.16 The results showed that POC-PTX exhibited better tumor inhibitory effects in both cell lines than free PTX, and its effect of inhibiting tumor cell growth became more obvious as the incubation time with cells increased.

[0116] Fig.17 It shows that in the cell uptake experiment, 1.8 mL of MCF-7Cell in the logarithmic phase was cultured in a 6-well plate, with 10,000 cells per well. After 24 hours of culture, 200 μL of coumarin-6 API and POC-C6 nanocapsules (in terms of C6) were added, and the culture was continued for 30 minutes and 2 hours. 20 μL of Hoechst 33258 (346 / 460 nm) was added to stain the cell nucleus for 15 minutes, the culture medium was removed, and the cells were washed three times with PBS. Finally, they were fixed with 4% (w / w) paraformaldehyde aqueous solution for 10 minutes, and then washed three times with PBS. The fluorescence characteristics of coumarin 6 were used to observe the cell uptake under a fluorescent inverted microscope. POC-PTX NC can enhance the cell's ability to uptake PTX, and the uptake capacity is higher as the incubation time with cells increases.

[0117] Fig.18 It was shown that in the characterization of tissue distribution, the in vivo distribution of the fluorescent probe group (DIR) and the fluorescently labeled DIR-POC-PTX nanocapsule group was evaluated by using the ICR mouse breast cancer model (4T1): the 4T1 cells with good logarithmic growth were digested with trypsin, counted, and collected by centrifugation. The cells were re-dispersed with PBS and stored in an ice box for later use. The cell number was 1×107Cell / mL. 100μL was injected into the right posterior back of the mouse to establish a tumor-bearing mouse model. After inoculation, when the tumor volume grew to 100mm3, 1 0 tumor-bearing mice were randomly divided into two groups (n=5): DIR-labeled POC-PTX nanocapsule group and free PTX-DIR group. The mice were placed in a small animal in vivo imaging instrument for fluorescence tissue distribution analysis (excitation wavelength 740nm, emission wavelength 790nm) 4h after administration (with isoflurane anesthesia during the period) by tail vein injection, and in vivo imaging pictures were obtained. The mice were killed by carbon dioxide gas anesthesia for 24h, and their hearts, livers, spleens, lungs, kidneys and tumors were removed for small animal imaging to observe the fluorescence intensity of DIR in ex vivo tissues. The POC nanocapsule group showed higher fluorescence intensity, longer systemic circulation time and more accumulation in the tumor site. This should be the result of the combined effects of passive targeted drug delivery of nanoformulations based on the EPR effect and the specific drug release of the carrier in the micro-acidic environment of the tumor.

[0118] Example 5: In vivo antitumor pharmacodynamics of POC-PTX nanocapsules

[0119] Establishment of tumor-bearing mouse model: 4T1 cells with good growth in logarithmic phase were digested with trypsin, counted, centrifuged at 1000 rpm, re-dispersed with 1640 culture medium, counted, centrifuged at 1000 rpm, and re-dispersed with PBS and stored in an ice box for later use. The number of cells was 1×10 7Cell / mL: Depilate the back of healthy mice, take 100 μL and inject it on the right back of the mice to establish a tumor-bearing mouse model. After inoculation, wait until the tumor grows to 100 mm 3 24 mice were randomly divided into 4 groups (6 mice in each group): saline group, POC group, POC-PTX nanocapsule group and free PTX group (dosage was 10 mg / kg, calculated as PTX). The mice were injected into the tail vein every other day for 14 days. Fig.19 ), and the body weight and survival of mice were observed every other day from the start of administration ( Fig. 20 ), measure the long diameter and short diameter of the tumor, and calculate the average volume and tumor burden of each group of mice. (Use a vernier caliper to measure the long diameter (L) and short diameter (W) of the tumor, and calculate the formula V = 1 / 2 (L×W 2 ) The tumor volume of mice was calculated. After the experiment, the mice were killed by cervical dislocation, and the normal tissues and organs as well as the tumor were dissected, immersed in formalin, embedded in paraffin, sectioned, stained with hematoxylin-eosin (HE), and observed under a fluorescence microscope.

[0120] Figure 21-23 It shows that the tumor volume change curve shows that the tumor volume of mice in the saline and POC groups continues to increase over time, indicating that POC does not have a pharmacological effect, but only acts as a carrier. The Free PTX and POC-PTX nanocapsule preparation groups showed tumor inhibition effects. Compared with Free PTX, the POC-PTX nanocapsule preparation group showed a stronger anti-tumor effect, indicating that the nanocapsule preparation with POC as a carrier can better enhance the efficacy of drugs. The reason is that the nanocapsule is small in size and can enhance the EPR effect, making it easier for drugs to enter tumor cells. At the same time, the POC carrier releases drugs in response to the weakly acidic microenvironment of the tumor, thereby enhancing drug targeting. Combined with the comparison of the tumor weight and tumor inhibition rate of each group of mice after the treatment, it can be inferred that paclitaxel loaded in POC nanocapsules can improve its anti-tumor effect. In addition, the weight of mice in each group did not change significantly during the treatment, indicating that POC and its nanoformulations have good biosafety.

[0121] Fig.24 and 25The results of HE staining showed that the organ tissue morphology of all groups was similar, no obvious glomerular damage was observed in the kidney sections, and no hepatic cord rupture was observed in the liver tissue. It was proved that POC had no tissue organ damage at the corresponding dosage and had good safety. In addition, compared with the saline group and the POC blank carrier group, the PTX and POC-PTX nanocapsule groups significantly inhibited the growth of tumor cells, while the POC-PTX group had a wider range of tumor necrosis, and the number of cell nuclei atrophy and fragmentation was greater, showing a stronger tumor killing effect than Free PTX.

Claims

1. Use of porous organic cages as nano drug carriers for loading and delivering drug molecules; Optionally, the porous organic cage and loaded drug are prepared into nanocapsules for drug delivery; Optionally, the porous organic cage is a calixarene-based porous organic cage, and further, is selected from calix[4]arene-based porous organic cages, for example, more specifically, selected from resorcinol calix[4]arene-based porous organic cages, such as C-methylresorcinol calix[4]arene; C-ethylresorcinol calix[4]arene; C-propylresorcinol calix[4]arene; C-butylresorcinol calix[4]arene; C-isobutylresorcinol calix[4]arene, etc. Optionally, the porous organic cage is selected from one, two or more of resorcinol calix[4]arene organic cages: CPOC-101, CPOC-201, CPOC-301, HPOC-101, HPOC-201, HPOC-301, HPOC-401.

2. Use of porous organic cages as nano drug carriers for the preparation of anti-tumor drugs; Optionally, the porous organic cage and the loaded anticancer drug are prepared into nanocapsules for drug delivery; Optionally, the porous organic cage is a calixarene-based porous organic cage, and further, is selected from a calix[4]arene-based porous organic cage, for example, more specifically, selected from a resorcinol calix[4]arene-based porous organic cage, such as C-methylresorcinol calix[4]arene; C-ethylresorcinol calix[4]arene; C-propylresorcinol calix[4]arene; C-butylresorcinol calix[4]arene; C-isobutylresorcinol calix[4]arene, etc. The porous organic cage is selected from one, two or more of the resorcinol calix[4]arene organic cages: CPOC-101, CPOC-201, CPOC-301, HPOC-101, HPOC-201, HPOC-301, and HPOC-401.

3. The use according to claim 2, wherein the anticancer drug is selected from: one, two or more of doxorubicin (DOX), camptothecin (CPT), 5-fluorouracil (5-FU), and paclitaxel (PTX); Optionally, the cancer can be adrenocortical carcinoma (ACC), colon cancer (COAD), lung adenocarcinoma (LUAD), hepatoma cells (HpeG2), melanoma cells (B16-F10), etc. As an example, hepatoma cells (HpeG2) and melanoma cells (B16-F10) are selected as models.

4. A method for preparing a drug delivery system, the method comprising the following steps: (1) mixing the porous organic cage and the drug molecules in a solution so that the drug molecules enter the porous organic cage; (2) dissolving the porous organic cage loaded with drug molecules obtained in step (1) in the oil phase of the surfactant, adding water to diffuse the oil phase into the water phase, and forming nanocapsules; Optionally, the porous organic cage is a calixarene-based porous organic cage, and further, is selected from a calix[4]arene-based porous organic cage, for example, more specifically, selected from a resorcinol calix[4]arene-based porous organic cage, such as C-methylresorcinol calix[4]arene; C-ethylresorcinol calix[4]arene; C-propylresorcinol calix[4]arene; C-butylresorcinol calix[4]arene; C-isobutylresorcinol calix[4]arene, etc. Optionally, the porous organic cage is selected from one, two or more of resorcinol calix[4]arene organic cages: CPOC-101, CPOC-201, CPOC-301, HPOC-101, HPOC-201, HPOC-301, HPOC-401; As an example, the porous organic cage is selected from the calixarene-based porous organic cage CPOC-301, whose molecular formula is C 360 H 384 N 24 O 48 , having a molecular structure as shown in Formula I: Optionally, the drug molecule is selected from one, two or more of doxorubicin (DOX), camptothecin (CPT), 5-fluorouracil (5-FU) and paclitaxel (PTX). As an example, the drug molecule is paclitaxel (PTX); Optionally, the surfactant is selected from one, two or more of Tween 20, Tween 80, sodium linear alkylbenzene sulfonate (LAS), cocamidopropyl betaine (CAB-35) and polyoxyethylene hydrogenated castor oil (RH40). As an example, polyoxyethylene hydrogenated castor oil (RH40) is selected.

5. The preparation method according to claim 4, wherein in step (1), the porous organic cage is immersed in a drug molecule solution to achieve mixing; further, the drug-loaded porous organic cage is separated from the solution by vacuum filtration; Optionally, the mass ratio of the porous organic cage to the drug molecule is 5:1 to 1:1, for example, 3:1, 2:1, 1.5:1, 1.4:1, 1.35:1, 1.3:1; Optionally, the drug molecule solution is a solution of drug molecules dissolved in an organic solvent; The solvent may be a C1-C6 alcohol, for example, methanol, ethanol, propanol, etc.; Optionally, the concentration of the drug molecule solution is 10-30 mg / mL, further, it can be said that it is 10-20 mg / mL; Optionally, after immersing the porous organic cage in the drug molecule solution, stirring for 10-30 hours, for example, 15 hours; Optionally, the step (1) further comprises purifying the sample obtained after filtration, for example, washing and drying; For example, the drying method may be vacuum drying. Preferably, the drying temperature may be 60-150° C., exemplarily 100° C.; the drying time may be 6-20 hours, for example 12 hours; Optionally, the step (1) further comprises pre-treating the porous organic cage by vacuum drying.

6. The preparation method according to claim 4, wherein in step (2), the porous organic cage loaded with drug molecules obtained in step (1) is mixed with a surfactant, dissolved in a constant temperature water bath, and an aqueous phase is added, and the temperature is continued to be constant to form drug-loaded nanocapsules; Optionally, the mass ratio of the porous organic cage loaded with drug molecules to the surfactant is 1:100 to 1:400, such as 1:150 to 1:300, 1:170 to 1:250, and exemplarily 1:200; Optionally, the temperature of the thermostatic water bath is 30-100° C., for example, 50° C.; Optionally, stirring is performed during dissolution in a constant temperature water bath, and the stirring time is 10-30 hours, for example, 15 hours; Optionally, the aqueous phase is added by dropwise addition of ultrapure water, and the ratio (ml / mg) of the volume of ultrapure water to the mass of the porous organic cage loaded with drug molecules obtained in step (1) is 1:2 to 1:10, for example, 1:3 to 1:8, 1:4 to 1:6, 1:5; Optionally, stirring is continued at constant temperature for 1-3 hours, for example, 2 hours.

7. The preparation method according to claim 4, further comprising the step of preparing a porous organic cage before step (1); Furthermore, the above-mentioned step of preparing a porous organic cage is to prepare a calixarene-based porous organic cage CPOC-301 (whose molecular formula is C 360 H 384 N 24 O 48 ), which is obtained by reacting tetraaldehyde resorcinol calix[4]arene (C4RACHO) and p-phenylenediamine (PDA); Optionally, the molar ratio of tetraaldehyde resorcinol calix[4]arene (C4RACHO) to p-phenylenediamine (PDA) is 1:(1-3), exemplified by 1:1, 1:2, 1:3; Optionally, the reaction temperature is 40-100° C., for example, 65° C.; the reaction time is 1-3 days, for example, 2 days; Optionally, the reaction is carried out in an organic solvent; for example, the solvent may be chloroform; Optionally, the preparation method further comprises: after the reaction is completed, collecting the product porous organic cage from the reaction solution; Optionally, the collecting comprises: evaporating the reaction solution obtained after the reaction is completed at a constant temperature to obtain porous organic cage crystals; for example, the evaporation temperature is room temperature; for another example, the evaporation time is 6-20 hours, for example, 12 hours; Optionally, the preparation method further comprises purifying the crystallized product; for example, by filtering, washing with methanol, exchanging methanol several times, and drying, to obtain a porous organic cage from which the guest solvent molecules are removed; Optionally, the drying method may be vacuum drying; the drying temperature may be 60-130° C., such as 100° C., and the drying time may be 6-20 h, such as 12 h.

8. A drug delivery system prepared by the preparation method according to any one of claims 4 to 7. 9 . The drug delivery system according to claim 8 , which is mainly formed by porous organic cage molecules loaded with drug molecules.

10. The drug delivery system according to any one of claims 8 to 9, which is a nanocapsule; Optionally, the particle size of the nanocapsule is less than 25 nm, further, less than 23 nm, 22 nm, and within the range of 20±2 nm, 20±1 nm.

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