CN114010783B

CN114010783B - Multifunctional boron-rich nano targeting preparation based on covalent organic framework material, and preparation method and application thereof

Info

Publication number: CN114010783B
Application number: CN202111291736.5A
Authority: CN
Inventors: 邵堃; 李帮健; 李广哲
Original assignee: Dalian University of Technology
Current assignee: Dalian University of Technology
Priority date: 2021-11-03
Filing date: 2021-11-03
Publication date: 2022-10-18
Anticipated expiration: 2041-11-03
Also published as: CN114010783A

Abstract

A multifunctional boron-rich nano targeting preparation based on a covalent organic framework material, a preparation method and application thereof belong to the technical field of medicines. The multifunctional boron-rich nano targeting preparation is used as an excellent boron nano medicament through a boron-rich covalent organic framework material COF, and realizes high-efficiency load on immune adjuvants, photosensitizers, sonosensitizers, chemotherapeutic drugs and the like due to unique high specific surface area, pi-pi stacking and pore channel interaction; DSPE-PEG by surface modification _2k -X molecules cross the barrier, enabling intratumoral implementation ¹⁰ B. Co-delivery of drug molecules and photosensitizers. The multifunctional boron-rich nano targeting preparation observes the distribution condition of the drug in the cells through a fluorescence microscope, and monitors the distribution in the nano sheets in real time so as to formulate the optimal neutron irradiation condition; intratumoral delivery by simultaneous delivery ¹⁰ B and the drug molecules realize the targeted radiation and immune combined treatment of BNCT.

Description

Multifunctional boron-rich nano targeting preparation based on covalent organic framework material, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a multifunctional boron-rich nano targeting preparation based on a covalent organic framework material, and a preparation method and application thereof.

Background

Brain gliomas are the most common primary craniocerebral tumors arising from brain and spinal cord glial cell carcinogenesis. The characteristics of strong transferability, high infiltration, strong drug resistance and the like cause that the recurrence rate and the fatality rate are higher. The annual incidence rate of brain glioma in China is reported to be 5 to 8/10 ten thousand people, and the 5-year fatality rate is ranked 3 rd in the median line of the whole tumor, and is only second to pancreatic cancer and lung cancer. The traditional treatment modalities mainly include surgical resection, radiation therapy and chemotherapy. However, the development process of brain glioma is accompanied by the problems of tumor cell diffusion infiltration and unclear infiltration boundary, and the like, so that the brain glioma is difficult to completely remove by an operation. Radiotherapy is limited by the radiation dose of the brain and is easily tolerated, resulting in poor therapeutic effect. In addition, the blood-brain barrier (BBB) in the brain limits the enrichment of over 95% of small-molecule drugs and biological macromolecules into brain tissue, making drug therapy difficult to achieve. Therefore, the development of a high-efficiency and low-toxicity treatment strategy for the brain glioma is of great significance.

A novel Boron Neutron Capture Therapy (BNCT) which is a noninvasive tumor therapy method is a binary chemoradiotherapy with strong targeting property and high energy transmission linear density in a cell scale, and has the advantages of strong tissue penetration capacity, high controllability, short treatment time and the like. The principle of action is the stable isotope boron 10: ( ¹⁰ B) Upon irradiation with low-energy (0.025 eV) or epithermal neutrons (10,000 eV) a neutron capture-fission reaction occurs, followed by instability ¹¹ B isotope undergoes nuclear fission reaction to produce high-energy and short-range alpha particles ( ⁴ He ²⁺ ) And ⁷ Li ³⁺ the particles further exert a tumor cell killing effect. Alpha particles and ⁷ Li ³⁺ the range of the particles is respectively 10 mu m and 5 mu m, the particles are limited within the target cell scale (20 mu m), the influence on non-target cells is small, and the damage is low. It is worth mentioning that BNCT provides a valuable solution for deep tumor therapy since high energy neutron beams can penetrate thick tissues and bones.

Advantages of BNCT treatment also include:

the generated alpha rays can kill the tumor cells in the mitotic period and the resting period at the same time (the traditional radiotherapy and chemotherapy are mainly used in the prior art)Acting on actively dividing cells, and being insensitive to quiescent tumor cells);

the tumor cells in an anaerobic state can be killed at the same time without additional oxygenation (the traditional radiotherapy has low treatment sensitivity on the anaerobic cells);

the generated lethal injury and potential lethal injury can not be subjected to DNA repair, and the composition has obvious curative effect on refractory tumors capable of repairing DNA injury after chemotherapy and radiotherapy and can effectively inhibit tumor recurrence.

Studies have shown that tumor cells often use multiple mechanisms to escape immune surveillance, and a single therapeutic strategy is often difficult to effectively overcome tumor metastasis. Although physical therapies such as BNCT can trigger immunogenic cell death to a certain extent, the anti-tumor immune response generated by radiotherapy-induced organisms is not enough to effectively prevent tumors from developing metastases to the far end. Therefore, the combined application of radiotherapy and immunotherapy provides a new idea for tumor treatment, namely, the combined application of radiotherapy and immunotherapy activates an organism to generate anti-tumor immune response while killing in-situ solid tumors, blocks tumor immune escape, breaks through immune suppression caused by tumors, exerts the optimal anti-tumor effect and finally prevents tumor recurrence. DMXAA (2, 5-pentoxifylline, vadimezan) is an agonist of the interferon gene stimulating factor (STING) expressed in antigen presenting cells. DMXAA binds to STING proteins on the endoplasmic reticulum of dendritic cells, stimulates dendritic cell maturation and antigen presentation. In the tumor immunotherapy, the compound can be used as an immune agonist to start the anti-tumor immune response of the body.

Currently, treatment of brain gliomas by BNCT is not fully generalized, primarily due to the limited ability of current boron delivery agents to enrich for brain gliomas. To improve the therapeutic effect of BNCT on brain gliomas, solving the problem of intracerebral delivery of boron atoms becomes a crucial issue. Nanocarriers have the advantage of being unique as boron delivery agents. On one hand, the nano-carrier can improve the water solubility of the medicine, realize intravenous injection, enhance the curative effect of the medicine and reduce toxic and side effects; on the other hand, the natural tumor passive targeting characteristic and the surface-modifiable active targeting characteristic of the nano-carrier are utilized to realize crossing the blood brain barrier and specific accumulation in brain glioma cells.

At present, the covalent organic framework structure bonded by boron-oxygen bonds is used as a nano boron-rich drug combined delivery immune agonist and a fluorescent probe to realize the combined treatment of BNCT and immunotherapy, and is not reported at home and abroad.

Disclosure of Invention

The invention aims to provide a tumor-targeted multifunctional boron-rich nano-drug, and the realization ¹⁰ The high-efficiency delivery of the B atom and the activation of anti-tumor immune response are applied to BNCT-immune combination treatment, and the targeting of different types of tumors can be realized by replacing different targeting peptides. The boron-rich covalent organic framework which modifies the targeting molecule and simultaneously loads the immune agonist and the fluorescent probe is self-assembled into the multifunctional boron-rich nano-drug, so that enough boron-rich nano-drug can be efficiently and safely transported ¹⁰ B to tumor cells, and simultaneously starting body specific immune response through the delivered immune adjuvant, and monitoring in tumors through fluorescent tracing and PET imaging ¹⁰ And (4) the content of B. The basic process is as follows: the targeting peptide modified on the surface of the nano preparation can cross blood brain barrier, target tumor sites and then perform the following steps ¹⁰ B content is irradiated by neutron and radiotherapy is carried out. The carrier releases immunologic adjuvant to activate Dendritic Cells (DCs) to mature and promote presentation of tumor-associated antigen, and enhance CD8 ⁺ T cells infiltrate into the tumor area, inhibiting in situ tumor and tumor metastasis and recurrence. Multifunctional boron-rich nano targeting preparation realizes ¹⁰ The dual role of precise localization at the B atom targeting organ and cell level and activation of specific anti-tumor immune responses, thus enabling the innovative tumor treatment strategy of BNCT and immune combination.

The technical scheme adopted by the invention is as follows: the multifunctional boron-rich nano targeting preparation is a nano-drug suspension with the diameter of 80 to 180nm, which is formed by self-assembly of a boron-rich covalent organic framework material COF loaded with a drug and a photosensitizer and a target.

The boron-rich covalent organic framework material COF is synthesized by taking terephthalic acid diboronic acid and (3-aminopropyl) -triethoxysilane as raw materials through a tube-sealing solvothermal method.

The drug is selected from 2, 5-pentoxifylline (DMXAA), doxorubicin (DOX), and Paclitaxel (PTX). DOX is a broad-spectrum antitumor drug, can inhibit the synthesis of RNA and DNA, and has effect on various tumors. Is suitable for malignant lymphoma, breast cancer, bronchogenic carcinoma, neuroblastoma, bladder cancer, thyroid cancer, prostatic cancer, head and neck squamous carcinoma, gastric cancer, and hepatocarcinoma. PTX is a natural anticancer drug that can destabilize the microtubules and tubulin dimers that make up microtubules from dynamic equilibrium, stabilize microtubules and inhibit the mitosis and trigger apoptosis of cancer cells, thereby effectively preventing the proliferation of cancer cells and playing a role in anticancer. Has been widely used for treating breast cancer, ovarian cancer, partial head and neck cancer and lung cancer in clinic.

The photosensitizer is selected from chlorin e6 (Ce 6) and IR780 dye.

The target comprises DSPE-PEG _2k -X and DSPE-PEG _2k ，DSPE-PEG _2k -X has the following structure:

wherein X is selected from the following structures:

Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Try-Cys

、

Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys

、

Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys

、

Arg-Gly-Asp-Cys

、

Cys-Asn-Gly-Arg-Val-Ser-Thr-Asn-Gly-Arg-Cys

；

each milliliter of the suspension contains 3 to 5mg of boron-rich covalent organic framework material COF, and each milliliter of the suspension is loaded with 0.25 to 1mg of photosensitizer, 0.5 to 2mg of medicine and 1.2 to 12mg of DSPE-PEG _2k -X。

A preparation method of a multifunctional boron-rich nano-targeting preparation based on a covalent organic framework material comprises the following steps:

(1) taking terephthalic acid diborate (BDBA) and (3-aminopropyl) -triethoxysilane (APTES) as raw materials, 1,3, 5-trimethylbenzene and 1, 4-dioxane as solvents, synthesizing a boron-rich covalent organic framework material (COF) by a sealed tube solvothermal method, and drying in vacuum to obtain white solid powder.

(2) And mixing the white solid powder obtained in the step with a DMSO solution in which a photosensitizer and a medicament are dissolved, carrying out ultrasonic oscillation, and adsorbing the medicament and the photosensitizer through a pore channel of the boron-rich covalent organic framework material COF and pi-pi action to obtain the bulk boron-rich covalent organic framework material COF loaded with the medicament and the photosensitizer.

(3) To the step

Adding dissolved target (DSPE-PEG) _2k -X and DSPE-PEG _2k ) Mixing the ethanol solution, and ultrasonically oscillating. Stripping the blocky boron-rich covalent organic framework material COF under the action of ultrasonic, and slowly adding deionized water to emulsify the blocky boron-rich covalent organic framework material COF.

(4) Will be described in detail

Dialyzing the obtained drug-loaded nanosheet suspension in a phosphate buffer solution, sampling at intervals, passing through a sephadex chromatographic column, monitoring the position and number of peaks, and completing purification when only a single peak is formed to obtain a suspension of the nano-drug with the diameter of 80 to 180nm, thus obtaining the multifunctional boron-rich nano-targeting preparation.

The definitions of the medicine, the photosensitizer and the target substance are the same as those of the multifunctional boron-rich nano-targeting preparation.

The steps are

The method adopts a three-time freezing-pump-melting circulation method to remove the gas in the mixed solvent of mesitylene and 1, 4-dioxane, and comprises the following specific steps: freezing the solvent by using liquid ammonia, vacuumizing a sealed pipe by using an oil pump, heating in a water bath to melt the solvent, releasing gas in the solvent, freezing the solvent by using liquid nitrogen again, and circulating for three times. The prepared boron-rich covalent organic framework material COF is dried in vacuum to form white solid powder, the molar ratio of terephthalic diboronic acid to (3-aminopropyl) -triethoxysilane is 1 to 3 to 4.5, and the volume ratio of trimethylbenzene to 1, 4-dioxane is 1 to 1.5.

The steps are

The mass ratio of the traditional Chinese medicine to the photosensitizer to the boron-rich covalent organic framework material COF is (1) - (4). Preferably, the mass ratio of the medicine to the photosensitizer to the boron-rich covalent organic framework material COF is 2 to 4:2 to 5:15 to 20, and most preferably, the mass ratio of the medicine to the photosensitizer to the boron-rich covalent organic framework material COF is 2:1:10.

the steps are

The mass ratio of the medium-target substance to the boron-rich covalent organic framework material COF is 3 to 1, DSPE-PEG _2k The mass fraction of X in the target substance is 10 to 90 percent, and the ethanol solution of the target substance is dispersed in the step

In the obtained solution, stripping is carried out under the action of ultrasound, and a boron-rich covalent organic framework material COF loaded with a medicament and a photosensitizer and a target are self-assembled to form a suspension.

The steps are as follows

Ultrasonic emulsification is adopted for 1-3 h.

The steps are

The adding speed of the deionized water in the ultrasonic emulsification process is 20uL/min.

The multifunctional boron-rich nano targeting preparation based on the covalent organic framework material is used for preparing anti-tumor drugs.

The tumor comprises brain glioma, breast cancer, ovarian cancer, liver cancer, kidney cancer, lung cancer, colon cancer, bladder cancer, pancreatic cancer, metrocarcinoma, gastric cancer, and rectal cancer.

The boron-rich covalent organic framework material COF is a planar network structure which is synthesized by taking p-phenylboronic acid (BDBA) as a raw material and taking 3-aminopropyl) -triethoxysilane (APTES) as a stabilizer through a tube-sealing solvothermal method, is connected by rigid building units and is stacked through the action of pi-pi between layers. As a boron nano-drug for BNCT, the boron nano-drug has high boron content and extremely low cytotoxicity to normal cells. The boron-rich covalent organic framework material COF can adsorb various hydrophobic and aromatic molecular drugs (such as photosensitizer, sonosensitizer, immunologic adjuvant and the like) through pi-pi conjugated or porous channels. After the fluorescent probe is stably adsorbed, the in-vivo tracing of the boron nano-drug and the determination of the peak reaching time in the tumor are facilitated. The stable adsorbed molecular medicine such as immunologic adjuvant can induce body specific anti-tumor immune response.

DSPE-PEG _2k Is an amphiphilic polymer composed of phospholipid and polyethylene glycol 2000, has the characteristics of biocompatibility and biosafety, is widely applied to drug delivery, gene transfection and vaccine delivery, can obviously improve the circulation time in vivo and stabilizeAnd (5) packaging the medicament. The end of PEG can be connected with an active group (such as maleimide and-Mal) for coupling antibody and polypeptide to realize targeted drug delivery. Boron-rich covalent organic framework materials COF and DSPE-PEG _2k -X and DSPE-PEG _2k Self-assembly process: amphiphilic block polymer DSPE-PEG with hydrophilic group and hydrophobic group _2k And DSPE-PEG _2k Dissolving X in an organic solvent, by preliminary sonication, DSPE-PEG _2k And DSPE-PEG _2k Wrapping the surface and the pore canal of a boron-rich covalent organic framework by X, and then slowly dripping deionized water, DSPE-PEG under the ultrasonic action _2k And DSPE-PEG _2k The hydrophilic end of the-X is dispersed in the water phase, and the hydrophobic end is tightly attached to the surface of the boron-rich covalent organic framework of the hydrophobic core and a pore channel, so that a nano suspension with good dispersibility is formed.

The DSPE-PEG _2k X is by distearoylphosphatidylethanolamine-maleimide polyethylene glycol 2000 (DSPE-PEG) _2k -Mal) and maleimide (-Mal) at the end of polyethylene glycol and low density lipoprotein receptor-related protein Angiopep-2, tumor cell-penetrating peptide HS-TAT, HS-iRGD, HS-RGD, or thiol (-SH) on tumor neovascular endothelial cell targeting peptide HS-NGR, and the synthesis comprises the following steps: 3 equivalents of DSPE-PEG _2k mixing-Mal and 1 equivalent of Angiopep-2, HS-TAT, HS-iRGD, HS-RGD or HS-NGR, adding PBS, stirring in dark at room temperature for 10 to 12 hours, dialyzing in ice water bath for 3 hours, changing liquid for 1 time per hour, removing PBS, and freeze-drying to obtain solid powder DSPE-PEG _2k -X. The polypeptide can be obtained by Hangzhou Zhongji peptide biochemistry Co Ltd, and has the following structure:

HS-TAT：Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-SH；

HS-iRGD：Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys-SH;

HS-RGD：Arg-Gly-Asp-Cys-SH;

HS-NRG：Cys-Asn-Gly-Arg-Val-Ser-Thr-Asn-Gly-Arg-Cys-SH；

angiopep-2 (24 KDa) is a novel polypeptide consisting of 19 amino acid molecules that can be endocytosed into brain tumors mediated by a low density lipoprotein receptor-related protein (LRP) expressed on the BBB. Research shows that human glioma cells and mouse glioma cells both express LRP receptor and have application prospect of clinical transformation.

DSPE-PEG modified with targeting molecule Angiopep-2 _2k The specific preparation method of-X is as follows: 3mg of distearoylphosphatidylethanolamine-maleimide polyethylene glycol 2000 (DSPE-PEG) _2k -Mal) and 1mg of Angiopep-2, 5mL of phosphate buffer (pH 7.4) was added, and the mixture was stirred at room temperature for 12 hours in the dark to allow coupling reaction between thiol (-SH) of Angiopep-2 and maleimide (Mal-) at the end of polyethylene glycol. 2,4,6,8, 10 and 12 h of reaction solution and Ellman's reagent analysis solution are respectively taken to react, and OD value at 412 nm is measured to monitor the reaction. Experiments prove that after 12 hours of reaction, the reaction is basically complete, ice-water bath dialysis is carried out for 3 hours, liquid is changed for 1 time per hour, and white solid powder DSPE-PEG is obtained by freeze drying _2k -Ang, and stored at-20 ℃ until use.

The multifunctional boron-rich nano targeting preparation comprises a carrier module, a medicine carrying module and a targeting module. By DSPE-PEG in the target _2k -X enables targeting to specific tumor cells or lesions, simultaneously enabling co-delivery of drug molecules and photosensitizers, visualization tracking by photosensitizers, and innovative tumor treatment strategies with combination of chemotherapy and radiotherapy by drug molecules. For example: boron-rich covalent organic framework material COF loaded with immune adjuvant DMXAA and photosensitizer Ce6 and targeting agent (DSPE-PEG) _2k -Ang and DSPE-PEG _2k ) The self-assembled multifunctional boron-rich nano targeting preparation realizes the effect of the brain glioma internal section by the Blood Brain Barrier (BBB) crossing of the Angiopep-2 molecule modified by the nano preparation ¹⁰ B. Co-delivery of the immunoadjuvant DMXAA and the imaging molecule Ce 6. The fluorescence imaging capability of the Ce6 molecule can realize in-vivo tracing of the nanosheets, and meanwhile, the nitrogen heterocyclic structure of the Ce6 molecule can be effectively combined ⁶⁴ Cu ²⁺ Chelation, realizing in vivo Positron Emission Computed Tomography (PET) imaging, and monitoring in vivo distribution of the nanosheets in real time to formulate optimal neutron irradiation conditions. Co-delivered immunoadjuvantsThe DMXAA can be combined with STING protein on endoplasmic reticulum of DCs to release Interferon-beta (Interferon-beta, INF-beta), promote DCs cell maturation, promote presentation of tumor-associated antigen, and enhance infiltration of Cytotoxic T Lymphocyte (CTL) to tumor region, thereby killing tumor cells.

The invention has the beneficial effects that: the multifunctional boron-rich nano targeting preparation based on the covalent organic framework material is prepared by self-assembling a boron-rich covalent organic framework material COF loaded with a medicament and a photosensitizer and a target, and the boron-rich covalent organic framework material COF is formed by connecting organic construction elements through dynamic covalent bonds and has a periodic two-dimensional (2D) or three-dimensional (3D) network structure, so that the multifunctional boron-rich nano targeting preparation has the characteristics of large specific surface area, small density, high porosity, good stability, uniform pore diameter, adjustable structure, diversified functions and the like. The covalent organic framework bonded by the boron-oxygen bond after being modified by adding the stabilizer contains abundant boron atoms, and can be used as an excellent boron nano-drug. In addition, due to the unique high specific surface area, pi-pi stacking and pore channel interaction, the covalent organic framework has very high loading and encapsulating capacity on hydrophobic drugs and aromatic drugs, and has unique advantages and potential application values in the aspect of efficient loading and delivery of immune adjuvants, photosensitizers, sonosensitizers, chemotherapeutic drugs and the like. The multifunctional boron-rich nano targeting preparation is prepared by surface modification of DSPE-PEG _2k -X molecules cross the barrier, enabling intratumoral implementation ¹⁰ B. Co-delivery of drug molecules and photosensitizers. Observing the distribution condition of the drug in the cell through a fluorescence microscope, and monitoring the distribution in the nanosheet body in real time so as to establish the optimal neutron irradiation condition; intratumoral delivery by simultaneous delivery ¹⁰ B and the drug molecules realize the targeted radiation and immune combined treatment of BNCT.

In conclusion, the multifunctional boron-rich nano targeting preparation has clear advantages and wide application prospect, realizes BNCT and immune combined treatment, activates specific anti-tumor immune response while killing tumor cells, plays a synergistic anti-cancer role, has good targeting property and small toxic and side effects, and lays a foundation for thoroughly treating tumors.

Drawings

FIG. 1 is a representation diagram of multifunctional boron-rich nano-targeting agent DM & Ce6@ COF-Ang 1.

Wherein (a) is DSPE-PEG _2k -graph of reaction rates for Mal and Angiopep-2;

(b) Is a powder X-ray diffraction (PXRD) characterization chart of APTES-COF-1;

(c) Is a purification analysis chart of DM & Ce6@ COF-Ang 1;

(d) Thermogravimetric analysis diagram for DM & Ce6@ COF-Ang 1;

(e) Is an ultraviolet visible absorption spectrogram of DM & Ce6@ COF-Ang 1;

(f) Fluorescence spectrum for DM & Ce6@ COF-Ang 1.

Fig. 2 is an ultraviolet lamp and infrared spectrum of the multifunctional boron-rich nano-targeting agent.

Wherein (a) is an ultraviolet lamp diagram of the boron-rich nano preparation;

(b) Is an infrared spectrum of DM & Ce6@ COF-Ang 1.

Fig. 3 is an XPS energy level diagram and elemental distribution diagram of a multifunctional boron-rich nano-targeting formulation.

Wherein (a) is an XPS energy level diagram of DM & Ce6@ COF-Ang 1;

(b) The distribution diagram of the elements B, C, N and O of DM & Ce6@ COF-Ang 1.

Fig. 4 is a graph for investigating the appearance and stability of the multifunctional boron-rich nano preparation.

(a) TEM image for DM & Ce6@ COF-Ang 1;

(b) AFM images for DM & Ce6@ COF-Ang 1;

(c) Is a particle size change diagram of the nano preparation in water for 48 hours;

(d) A 7-day stability investigation graph of DM & Ce6@ COF-Ang1 in water;

(e) A 7-day stability profile for DM & Ce6@ COF-Ang1 in different concentrations of Fetal Bovine Serum (FBS);

(f) Is zeta potential diagram of nano preparation in water.

Fig. 5 is a graph of the results of in vitro cell uptake experiments for multifunctional boron-rich nanoformulations.

Wherein (a) is an uptake graph of the boron-rich nano preparation by a fluorescence microscope monitoring cell;

(b) Is a quantitative analysis chart of the cell uptake of the boron-rich nano preparation;

(c) Evaluating the toxicity graph of the boron-rich nano preparation on the bEND.3 cells and the GL261 cells by an MTT method;

(d) The uptake of the nanoplatelets by the cells was examined for ICP-OES.

Fig. 6 is a distribution plot of boron-rich nano-formulations in cells.

Wherein, (a) is the distribution map of the boron-rich nano preparation in the organelle;

(b) The intracellular distribution profile of DM & Ce6@ COF-Ang1 was monitored for fluorescence microscopy.

FIG. 7 is a diagram of the construction and evaluation of an in vitro blood brain barrier model.

(a) Is an in vitro blood brain barrier model leakage test experimental graph;

(b) Is an in vitro blood brain barrier model fluorescein sodium penetration experimental diagram;

(c) An in vitro blood brain barrier crossing force diagram was evaluated for boron-rich nanoformulations.

Figure 8 is a graph of the effect of boron-rich nanoformulations on immune cells in vitro.

(a) Activation map of dendritic cells (BMDCs) derived from bone marrow in vitro;

(b) Activation and repolarization patterns of macrophages (M.phi.) of bone marrow origin in vitro.

Figure 9 is a graph of the effect of boron-rich nanoformulations on microglia.

(a) An identification map of primary cultured Microglia cells (Microglia) in vitro;

(b) Activation and repolarization profiles for primary cultured Microglia cells (Microglia) in vitro.

FIG. 10 is a graph of in situ model evaluation of mouse brain glioma using boron-rich nano-formulations.

(a) Analyzing maps for in vivo imaging

(b) Is a boron content tissue distribution map;

(c) DMXAA content profile in brain gliomas was determined for HPLC.

The invention is described in detail below with reference to specific embodiments. The scope of the invention is not to be limited by the specific embodiments but by the scope of the appended claims.

Detailed Description

Example 1

Preparation of amino functionalized covalent organic framework material-1 (APTES-COF-1):

the APTES-COF-1 is prepared by the following steps which are shown as the formula (I):

(I)

1) 0.165 g of 1, 4-terephthaloylic acid (BDBA) and 0.05 g of (3-aminopropyl) -triethoxysilane (APTES) are mixed in 5mL of a mixed solution of mesitylene and 1, 4-dioxane in a volume ratio of 1.

2) The reactants are treated by ultrasonic (40 KHz, 100W) for 60 min, then vacuumized, heated to 75 ℃ and reacted for 20 h.

3) After the reaction, the reaction mixture was repeatedly washed with acetone and filtered to obtain a white powder, which was dried under vacuum at 120 ℃ and stored at room temperature for further use.

Example 2

Preparation of amino functionalized covalent organic framework material-2 (APTES-COF-2):

1) 0.165 g of 1, 4-terephthaloylic acid (BDBA) and 0.08 g of (3-aminopropyl) -triethoxysilane (APTES) are mixed in 5mL of a mixed solution of mesitylene and 1, 4-dioxane in a volume ratio of 1.25.

Example 3

Preparation of amino functionalized covalent organic framework material-3 (APTES-COF-3):

1) 0.165 g of 1, 4-terephthaloyldiboronic acid (BDBA) and 0.10 g of (3-aminopropyl) -triethoxysilane (APTES) are mixed in 5ml of a mixed solution of mesitylene and 1, 4-dioxane in a volume ratio of 1.5.

Example 4

DSPE-PEG _2K Preparation of Ang:

DSPE-PEG for modifying targeting molecules _2K -Ang is by DSPE-PEG _2K The maleimide at the Mal terminal and the sulfhydryl (-SH) at the Angiopep-2 terminal are subjected to coupling reaction in a buffer solution. The method comprises the following specific steps:

1) 3mg of distearoylphosphatidylethanolamine-maleimide polyethylene glycol 2000 (DSPE-PEG) _2k Mal) and 1mg of Angiopep-2, 5mL of Phosphate Buffered Saline (PBS) was added, and stirred at room temperature for 12 hours in the dark.

2) Taking reaction liquid of 2,4,6,8, 10 and 12 hours to react with Ellman's reagent analysis liquid respectively, and measuring the OD value at 412 nm to monitor the reaction process.

3) Preparing Angiopep-2 solution (0.2 mg/mL) with the same concentration, measuring the ultraviolet absorption of the two at 412 nm under an ultraviolet spectrophotometer after reacting with Ellman's reagent, and calculating the yield according to the following formula:

yield% = (OD) _Ang-SH -OD _Ang-PEG-DSPE )/(OD _Ang-SH -OD _Blank )×100%

Wherein OD _Ang-SH 、OD _Ang-PEG-DSPE 、OD _Blank Respectively, the ultraviolet absorption at 412 nm after the reaction of Ang-SH, ang-PEG-DSPE and a blank solvent with Ellman's reagent.

4) As shown in FIG. 1 (a), the reaction rate reached 92.5% in 12 hours, the solution was dialyzed for 3 hours in an ice-water bath, and the solution was changed 1 time per hour, and freeze-dried to obtain a white solid powder, which was stored at-20 ℃ for further use.

Example 5

The Angiopep-2 was replaced with HS-TAT, HS-iRGD, HS-RGD or HS-NGR, and DSPE-PEG was prepared respectively by the method of reference example 4 _2K -TAT、DSPE-PEG _2K -iRGD、DSPE-PEG _2K -RGD、DSPE-PEG _2K The raw materials HS-TAT, HS-iRGD, HS-RGD and HS-NGR are purchased from Hangzhou Zhongji peptide Biochemical Co.

Example 6

Preparation of multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-Ang 1:

the multifunctional boron-rich nano targeting preparation is prepared according to the following steps, wherein the adopted blocky COF-1 is prepared in the first embodiment. The preparation method of the specific multifunctional boron-rich nano targeting preparation comprises the following steps:

1) mu.L of DMXAA (15 mg/mL) and 20. Mu.L of Ce6 (20 mg/mL) in DMSO were added to a centrifuge tube containing 5mg of bulk COF-1 and sonicated for 15min.

2) Adding a mixture of DSPE-MPEG containing 13 mg _2k And 2mg DSPE-PEG _2k 200 μ L of ethanol from Ang, shaking for 30min.

3) 700 μ L of deionized water (20 uL/min) was slowly added under sonication (40 KHz, 200W), and the emulsion was ultrasonically stripped.

4) Purifying by dialysis to obtain a suspension, DM & Ce6@ COF-Ang1

In addition, the same method as above is adopted to remove DMXAA, ce6, DSPE-PEG in the prescription _2k The Ang component is prepared into a targeting group DM @ COF-Ang (Ce 6 is removed) and a non-targeting group DM respectively&Ce6@ COF (removal of target), DM @ COF (removal of target and Ce 6) and COF (removal of DMXAA, target and Ce 6) several nano-formulations.

Example 7

Preparation of multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-Ang 2:

the multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-Ang2 is prepared by adopting the bulk COF-1 in the first embodiment, and the preparation steps of the multifunctional boron-rich nano-targeting preparation are as follows:

1) mu.L of DMXAA (15 mg/mL) and 20. Mu.L of Ce6 (20 mg/mL) in DMSO were added to a centrifuge tube containing 3mg of bulk COF-1 and sonicated for 15min.

2) Adding DSPE-MPEG solution containing 12mg _2k And 3mg DSPE-PEG _2k 200 μ L of ethanol from Ang, shaking for 30min.

4) Purification by dialysis gave a suspension, DM & Ce6@ COF-Ang2.

Example 8

Preparation of multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-Ang 3:

the multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-Ang3 is prepared by adopting the bulk COF-1 prepared in the first embodiment. The preparation method of the specific multifunctional boron-rich nano targeting preparation comprises the following steps:

1) 50 μ L of DMXAA (15 mg/mL) and 30 μ L of Ce6 (20 mg/mL) in DMSO was added to a centrifuge tube containing 4 mg of bulk COF-1 and sonicated for 15min.

2) Adding DSPE-MPEG solution containing 14 mg _2k And 1mg DSPE-PEG _2k 200 μ L of ethanol from Ang, shaking for 30min.

3) 700 μ L of deionized water (20 μ L/min) was slowly added under sonication (40 KHz, 200W), and the emulsion was ultrasonically stripped.

4) Purification by dialysis gave a suspension, DM & Ce6@ COF-Ang3 being obtained.

Example 9

Preparation of multifunctional boron-rich nano-targeting preparation DOX & Ce6@ COF-Ang:

the multifunctional boron-rich nano-targeting preparation DOX & Ce6@ COF-Ang is prepared by adopting the bulk COF-2 in the second embodiment. The preparation method of the specific multifunctional boron-rich nano targeting preparation comprises the following steps:

1) mu.L DOX (20 mg/mL) and 40. Mu.L Ce6 (20 mg/mL) in DMSO was added to a centrifuge tube containing 5mg of bulk COF-2 and sonicated for 20min.

4) Purification by dialysis gave a suspension, DOX & Ce6@ COF-Ang was obtained.

Example 10

Preparation of multifunctional boron-rich nano-targeting preparation DM & IR780@ COF-Ang:

the multifunctional boron-rich nano-targeting preparation DM & IR780@ COF-Ang is prepared by adopting the bulk COF-3 of the third embodiment. The preparation method of the specific multifunctional boron-rich nano targeting preparation comprises the following steps:

1) mu.L of DMXAA (15 mg/mL) and 25mL of IR780 (20 mg/mL) in DMSO was added to a centrifuge tube containing 5mg of bulk COF-3 and sonicated for 25min.

4) Purification by dialysis gave a suspension, DM & IR780@ COF-Ang was obtained.

Example 11

Preparation of multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-TAT:

the multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-TAT is prepared by adopting the bulk COF-1 in the first embodiment. The preparation method of the specific multifunctional boron-rich nano targeting preparation comprises the following steps:

1) 50 μ L of DMXAA (15 mg/mL) and 35 μ L of Ce6 (20 mg/mL) in DMSO was added to a centrifuge tube containing 5mg of bulk COF-1 and sonicated for 15min.

2) Adding a mixture of DSPE-MPEG containing 13 mg _2k And 2mg DSPE-PEG _2k 200 μ L ethanol of TAT, shaking for 30min.

4) Purification by dialysis gave a suspension, DM & Ce6@ COF-TAT being obtained.

Example 12

Preparing a multifunctional boron-rich nano targeting preparation DM & Ce6@ COF-iRGD:

the multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-iRGD is prepared by adopting the bulk COF-1 of the first embodiment. The preparation method of the specific multifunctional boron-rich nano targeting preparation comprises the following steps:

1) 50 μ L of DMXAA (15 mg/mL) and 40 μ L of Ce6 (20 mg/mL) in DMSO was added to a centrifuge tube containing 5mg of bulk COF-1 and sonicated for 15min.

2) Adding DSPE-MPEG solution containing 13 mg _2k And 2mg DSPE-PEG _2k 200 μ L ethanol of-iRGD, shaking for 30min.

4) The suspension obtained is purified by dialysis to obtain DM & Ce6@ COF-iRGD.

Example 13

Preparing a multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-RGD:

the multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-RGD is prepared by adopting the bulk COF-1 of the first embodiment. The preparation method of the specific multifunctional boron-rich nano targeting preparation comprises the following steps:

1) 50 μ L of DMXAA (15 mg/mL) and 45 μ L of Ce6 (20 mg/mL) in DMSO was added to a centrifuge tube containing 5mg of bulk COF-1 and sonicated for 15min.

2) Adding a mixture of DSPE-MPEG containing 13 mg _2k And 2mg DSPE-PEG _2k 200 μ L of ethanol with RGD, shaking for 30min.

4) The suspension was purified by dialysis to obtain DM & Ce6@ COF-RGD.

Example 14

Preparation of multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-NGR:

the multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-NGR is prepared by adopting the bulk COF-1 of the first embodiment. The preparation method of the specific multifunctional boron-rich nano targeting preparation comprises the following steps:

1) 50 μ L of DMXAA (15 mg/mL) and 30 μ L of Ce6 (20 mg/mL) in DMSO was added to a centrifuge tube containing 5mg of bulk COF-1 and sonicated for 15min.

2) Adding a mixture of DSPE-MPEG containing 13 mg _2k And 3mg DSPE-PEG _2k 200. Mu.L of ethanol from NGR, shaking for 30min.

4) The suspension was purified by dialysis to obtain DM & Ce6@ COF-NGR.

Example 15

The amino functionalized covalent organic framework material-1 (APTES-COF-1) and the multifunctional boron-rich nano targeting preparation prepared in the

embodiments

1 and 6 are characterized:

(1) Performance test experiment 1: the characterization of APTES-COF-1 by X-ray diffractometer (Japan science, smartLab 9 kw) is carried out by X-ray diffractometer (PXRD), as shown in figure 1 (b), the result shows that the characteristic peak is consistent with the report in the literature, and APTES-COF-1 is successfully synthesized.

(2) Performance test experiment 2: purification and thermogravimetric analysis of DM & Ce6@ COF-Ang1

A sephadex chromatographic column method is adopted to verify whether the free medicine can be completely removed by dialysis. As shown in fig. 1 (c), the results indicate that purified boron-rich nanoformulations can be obtained after dialysis. The dialyzed and purified nano preparation is subjected to freeze drying and thermogravimetric analysis (SDTA 851, mettler Switzerland), the temperature rise range is 50-500 ℃, the temperature rise speed is 10 ℃/min, and as shown in figure 1 (d), the result shows that the mass fraction of the covalent organic framework accounts for 25% of the whole nano material.

(3) Performance test experiment 3: ultraviolet and fluorescence spectrometry of DM & Ce6@ COF-Ang1

A3 ml quartz cuvette was charged with a solution of DM & Ce6@ COF-Ang1 sample dispersed in water, and the absorption curve of the sample was measured with a UV-visible spectrophotometer (Perkinelmer, lambda 750S). Fig. 1 (e) illustrates that DMXAA and Ce6 are successfully loaded simultaneously. The 1 mLDM &Ce6@ COF-Ang1 sample solution was added to the quartz cuvette, and the emission curve (Ex: 345 nm) of the sample was tested by a fluorescence spectrometer (Hitachi, F7000), as shown in FIG. 1 (F), and DMXAA and Ce6 were successfully loaded at the same time. FIG. 2 (a) shows a fluorescent photograph under UV irradiation (365 nm).

(4) Performance test experiment 4: infrared spectrum of multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-Ang1

Functional groups of the multifunctional boron-rich nano-targeting agent DM & Ce6@ COF-Ang1 were analyzed by Fourier transform infrared spectroscopy (FTIR, thermo Fisher 6700), as shown in FIG. 2 (b). FTIR spectra show that DMXAA and Ce6 are simultaneously supported on a boron-rich covalent organic framework material COF-1.

(5) Performance test experiment 5: elemental analysis of DM & Ce6@ COF-Ang1

The elements of the multifunctional boron-rich nano-targeting agent DM & Ce6@ COF-Ang1 were analyzed by X-ray photoelectron spectroscopy (XPS, thermo Fisher), as shown in FIG. 3 (a). Mapping analysis was performed using a high-resolution transmission electron microscope for DM & Ce6@ COF-Ang1 elemental composition, as shown in FIG. 3 (b). Elemental analysis showed that DM & Ce6@ COF-Ang1 is rich in a sufficient number of boron atoms.

Example 16

The multifunctional boron-rich nano-targeting preparation prepared in example 6 and the DM & ce6@ COF, DM @ COF and COF nano-formulations of the non-targeting group were subjected to morphology and stability investigation:

TEM images of the functional boron-rich nano-targeted agent DM & Ce6@ COF-Ang1 were obtained by transmission electron microscopy (TEM, HT7700 EXALENS), as shown in FIG. 4 (a), with a diameter of about 161 nm. DM & Ce6@ COF-Ang1 was diluted 20-fold with deionized water, dispersed on a clean silicon wafer, dried and measured for thickness by atomic force microscope (Bruker, dimension Icon), as shown in FIG. 4 (b), which was about 5 nm thick. A dynamic light scattering particle size plot was obtained from 1mL of the DM & Ce6@ COF-Ang1 solution, as measured by a Zeta potential and particle size analyzer (Malvern Zetasizer, nanozs 90), after DM & Ce6@ COF-Ang1 was dispersed in the liquid for 48 hours, and the particle size in water was about 171.4 nm, in PBS about 162.5 nm, and in 1640 complete medium was about 160.4 nm, as shown in FIG. 4 (c). 48 DLS test after h shows that COF, DM @ COF, DM & Ce6@ COF-Ang1 have good stability. The particle size changes of the nanosheets COF, DM @ COF, DM & Ce6@ -COF and DM & Ce6@ -COF-Ang 1 in water within 7 days are measured by the same method, and as shown in FIG. 4 (d), the boron-rich nano preparation is relatively stable within 7 days. The particle size changes of the nanosheets COF, DM @ COF, DM & ce6@ COF-Ang1 in different concentrations of FBS within 7 days were measured by the same method, as shown in fig. 4 (e), demonstrating that the boron-rich nanoformulation is stable in different environments within 7 days. The results of the potential test are shown in FIG. 4 (f), and the potential of DM & Ce6@ COF-Ang1 was about-15.3 mV.

Example 17

In vitro cell uptake experiments were performed on the multifunctional boron-rich nano-targeting formulation prepared in example 6 and the DM & ce6@ COF, DM @ COF and COF nano-formulations of the non-targeting group

(1) Monitoring uptake of boron-rich nano-preparation by cells by fluorescence microscope

Selecting GL261 cells and bEND.3 cells with good growth state in logarithmic growth phase, digesting and collecting, adjusting cell density, and mixing 1mL cell suspension (3 × 10) ⁵ cell/mL) were evenly and slowly seeded in 35 mm petri dishes and cultured routinely for 12 h until the cells were adherent and fully spread. Taking out the dish, removing supernatant, washing with PBS, and adding DM&Ce6@COF-Ang1、DM&Ce6@ COF, DM @ COF and COF nanoformulations were incubated with light for various periods of time to give a final Ce6 concentration of 5. Mu.g/mL. At 0.5 h,1 h, 2h after addition of the nanopreparative, cells were taken out and the nuclei were labeled with DAPI. The fluorescence microscope was used to observe the enrichment of Ce6 with red fluorescence in each cell group, and images were collected as shown in fig. 5 (a). Quantitative analysis of fluorescence intensity As shown in FIG. 5 (b), the results of fluorescence imaging and analysis showed that GL261 cells and bEND.3 cells were directed to DM in the target group&The uptake of Ce6@ COF-Ang1 was significantly higher than that of the non-targeted group.

(2) MTT method for evaluating in-vitro cytotoxicity of boron-rich nano preparation

Determination of DM by UV-Vis spectrophotometry&Ce6@COF-Ang1、DM&Ce6@COF. DMXAA content in DM @ COF. Mouse brain microvascular endothelial cells (bEND.3) were plated in 96-well plates at 4X 10 per well ³ The density of individual cells was cultured at 37 ℃ for 24 hours. Thereafter, the medium was replaced with complete medium containing different concentrations of the nanoformulation of DMXAA (0. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 75. Mu.g/mL, 100. Mu.g/mL). After 4 hours of incubation drug was withdrawn, fresh medium was added and incubated for 20 hours, after which the medium was discarded and 100 μ L of MTT was added to each well and further incubated for 4 hours. Subsequently, MTT (0.5 mg/mL) was replaced with 150. Mu.L of dimethyl sulfoxide (DMSO), and the absorbance (OD) of each well was measured at 570 nm using a microplate reader, and an average of 6 duplicate wells was taken for each sample. The relative survival rate of the cells was calculated according to the following formula: (Experimental group OD) ₅₇₀ Control group OD ₅₇₀ ) X 100%, experiment was repeated 3 times. Toxicity of boron-rich nanopreparation on mouse glioma cells (GL 261) was determined in the same manner. As shown in fig. 5 (c), it can be seen that the drug is not significantly toxic to the xend.3 cells within a concentration range of 75 μ g/mL without neutron irradiation. The drug was not significantly toxic to GL261 cells at a concentration range of 100 μ g/mL in the absence of neutron irradiation.

(3) ICP-OES detection of uptake of nanosheet by cells

Collecting GL261 cells with good growth state in logarithmic growth phase by digestion, adjusting cell density, and mixing 1mL cell suspension (1 × 10) ⁶ cell/mL) were seeded evenly and slowly in six-well plates and cultured routinely for 12 h until the cells were adherent and fully expanded. The dish was removed and the supernatant discarded, washed with PBS and then 1mL of cell culture medium containing COF, DM @, DMXAA @ COF-Ang1 nanosheets was added. After adding the nano-sheets for 0.5 h,1 h, 2h and 4 h, removing supernatant, washing with PBS for three times, and collecting cells. 1mL of a solution containing 68% HNO ₃ And 30% H ₂ O ₂ （V _{68% HNO3} : V _{30% H2O2} Digestion of mixed solution of = 3). As shown in FIG. 5 (d), the GL261 cells added with DM @ COF-Ang1 group reached a maximum concentration of 74.47 + -3.16 ppm at 2h, which is higher than the intracellular concentration required by BNCT ¹⁰ B content20 ppm。

(4) Co-localization analysis of boron-rich nano preparation by monitoring cells with fluorescence microscope

Using free Ce6, DM&Ce6@ COF and DM&The subcellular localization in GL261 cells was explored by incubating Ce6@ COF-Ang1 with GL261 cells for 2 hours, respectively. The distribution of several substances in GL261 cells was collected by confocal laser microscopy, as shown in FIG. 6 (a), DM&Ce6@ COF-Ang1 enters cells mainly through a lysosome pathway, and has high green fluorescence coincidence degree with a lysosome probe LTG, and the Pearson coefficient is 0.918. Free Ce6 and DM&The co-localization coefficients of ce6@ cof with lysosomes were 0.758 and 0.625, respectively. And counting DM by using a fluorescence microscope&Intracellular distribution of Ce6@ COF-Ang1, shown in FIG. 6 (b), 39.8% DM&Ce6@ COF-Ang1 is distributed within 5 μm distance from nucleus, and is more favorable for alpha particle and ⁷ Li ³⁺ killing of the nucleus by the particle.

Example 18

Construction and identification of in-vitro blood brain barrier model and evaluation of blood brain barrier crossing capacity of multifunctional boron-rich nano targeting preparation

(1) Construction and leakage test of in vitro blood brain barrier model

The Transwell chamber was removed, DMEM medium was added and placed in a 24-well plate, and left to stand in a cell incubator for 20min for activation. bEND.3 cells in logarithmic growth phase at 1X 10 ⁴ cell/well density was seeded in upper Transwell chamber and 0.6 mL of medium was added to the 24-well plate in the lower Transwell chamber and placed in an incubator for 5 days until the cells were confluent (during which time the upper Transwell chamber cell medium was periodically changed to allow tight adherent growth of bEND.3 cells). mu.L and 200. Mu.L of the culture medium were added to the upper and lower chambers of bEND.3, respectively, at a liquid level difference of 1 cm in the Transwell chamber, and the medium was placed in an incubator to continue the culture, and the liquid level heights of 1,4, and 9 hours were recorded to observe whether or not the liquid level difference between the two chambers remained at the original level. As shown in fig. 7 (a), the liquid level difference did not change significantly on day 5, i.e. the BBB in vitro modeling was initially judged to be successful.

(2) In vitro blood brain barrier model sodium fluorescein penetration experiment

Detecting BBB model in vitro culture by using fluorescein sodium (FLU), selecting bEND.3 independent culture model group and blank group, each group having 3 models, replacing whole solution with serum-free DMEM culture solution before measurement, adding 50 μ g/mL fluorescein sodium into the supply tank, placing at 37 deg.C and 0.5% CO ₂ After culturing in an incubator for 5min, 10 min, 20min, 40 min and 80 min, 50 μ L of culture solution is taken from the receiving pool and the FLU amount passing through different models is measured by a fluorescence microplate reader. As shown in FIG. 7 (b), the bEND.3 culture model group alone blocked the permeation of sodium fluorescein well.

(3) Evaluation of boron-rich Nanoparticulate Capacity crossing blood brain Barrier in vitro

And selecting a blood brain barrier model group qualified in a leakage test and a fluorescein sodium permeation test to evaluate the capability of free Ce6, DM & Ce6@ COF and DM & Ce6@ COF-Ang1 in crossing the blood brain barrier. Adding solution 300 μ L of free Ce6, DM & Ce6@ COF and DM & Ce6@ COF-Ang1 (containing 10 μ g/mL Ce 6) into the donor pool, incubating for 3 h, observing the uptake by fluorescence microscope, as shown in FIG. 7 (c), the targeting group DM & Ce6@ COF-Ang1 has obvious ability of crossing blood brain barrier.

Example 19

The effect of the multifunctional boron-rich nano-targeting preparation prepared in example 6 on dendritic cells, macrophages and microglia induced and cultured in vitro was examined.

(1) Activation and maturation of bone marrow-derived dendritic cells (BMDCs)

Extraction and culture of BMDCs: after the mice are killed at the dislocation of cervical vertebrae, the mice are soaked in ethanol for disinfection, and thighbones and shinbones of the mice are dissected out and muscles attached to the thighbones and the shinbones are stripped. The femoral and tibial ends are carefully cut with scissors to expose the medullary cavity. The bone marrow contents were blown down with a 10 mL sterile syringe needle containing the culture medium. 1800 Centrifuging at rpm for 5min, and collecting cells. The isolated cells were cultured on sterile plates using LCM medium containing 20 ng/mL GM-CSF and 20 ng/mL IL-4. Fluid changes were made on

days

3 and 6, and used for subsequent experiments on day 7.

Biomarker analysis of BMDCs: cells were physically mixed with free DMXAA, COF, dm @ COF-Ang, DMXAA + COF (each group containing 25 μ g/mL DMXAA), incubated at 37 ℃ for 4 hours, then stained with PE-Cy7-anti-mouse CD11c, PE-anti-mouse CD80, APC-anti-mouse CD86 at room temperature for 60 minutes, centrifuged and washed, then fixed with 1% PFA cell fixative at 4 ℃ for 90 minutes, and surface molecule expression was detected by flow cytometry, 20,000 cells were collected each time, and data analysis was performed using FlowJo 9.0 software.

As shown in FIG. 8 (a), DM @ COF-Ang has a significant activation effect on dendritic cells in vitro when the content of DMXAA is 25. Mu.g/mL.

(2) Activation and repolarization of in vitro bone marrow derived macrophages (M phi)

Bone marrow-derived M phi cell induction: mouse bone marrow cells were collected using the procedure for extraction of BMDCs. 37. Culture was performed in 24-well plates at 20 ng/mL M-CSF in LCM medium at 6 days, and media was supplemented on day 3. Stimulating and culturing with IFN-gamma (100 ng/ml) and LPS (25 ng/ml) for 24 h at 7 days to induce M1 type macrophage; m2-type macrophages were induced by stimulation of culture with IL-4 (10 ng/ml) for 24 h.

Biomarker analysis of bone marrow-derived M phi cells: cells were incubated with free DMXAA, COF, dm @ COF-Ang, DMXAA + COF physically mixed groups (each group containing 25 μ g/mL DMXAA) at 37 ℃ for 48 hours, then stained with PE-anti-mouse F4/80, PE-Cy7-anti-mouse CD206, APC-anti-mouse CD80 at room temperature for 60 minutes, centrifuged and washed, then fixed with 1% PFA cell fixative at 4 ℃ for 90 minutes, and expression of surface molecules was detected by flow cytometry, 20,000 cells were collected at a time, and data analysis was performed using FlowJo 9.0 software.

As shown in FIG. 8 (b), DM @ COF-Ang has significant activation and repolarization effects on macrophages in vitro at DMXAA levels of 25. Mu.g/mL.

(3) In vitro mouse primary Microglia (Microglia) culture and activation

1) Sterilizing clean mice in 24 h with 75% alcohol, cutting head under aseptic condition, cutting scalp and skull, taking out brain tissue, and placing in cold D-Hank's solution with pH of 7.2 and no calcium or magnesium.

2) Aseptically stripping brain tissue, removing cerebellum, separating cerebral cortex, and carefully stripping meninges and blood vessels.

3) Cutting the tissue into 1mm by iris scissors ³ The left and right tissue blocks are digested by 0.125 percent trypsin, and are acted for 20min at 37 ℃, and are shaken for 2 to 3 times.

4) The supernatant was discarded and the complete inoculum was added to stop digestion and rinsed twice. The pipette is gently blown and beaten until no obvious brain tissue block appears by visual observation, and is kept stand for 2 min.

5) The suspension was collected in a new centrifuge tube, centrifuged (1000 r/min,10 min,4 ℃) and the supernatant discarded. Adding complete culture solution for re-suspension, and filtering with a 200-mesh filter screen. 3 mice/dish were inoculated in 10 cm dishes.

6) Placing at 37 ℃ and 5% CO ₂ The culture medium was changed once after 24 h in the incubator, and the solution was changed every 3d later, and the growth and survival of the cells were observed under a mirror.

7) The cell culture is about 14 to 16 days, the cell stratification phenomenon can be observed in the mixed glial cell culture under an inverted phase contrast microscope, namely the bottom layer is flat and is provided with a plurality of protruded astrocytes, and the microglia are obviously smaller and are in a similar circle shape, have strong refractivity and are attached to the surfaces of the astrocytes to grow.

8) Pouring out the culture solution, digesting with 2 to 3 mL of 0.05 percent pancreatin, gently shaking the culture bottle while observing, transferring the digestion solution containing the floating microglia into a 10 mL centrifuge tube when the microglia attached to the astrocytes are separated, and immediately stopping digestion by using the complete culture medium.

Microglia were seeded on slides in 24-well plates, stained by Hoechst

Cells with nuclei marked essentially all marked with the minigel marker CD11b, CD11b positive rate (CD 11 b) ⁺ Hoechst ⁺ /Hoechst ⁺ ) > 90%, as shown in FIG. 9 (a).

Surface marker molecular analysis of Microglia: cells were incubated with free DMXAA, COF, dm @ COF-Ang, DMXAA + COF, respectively, physically mixed groups (each group was pulled up with 25 μ g/mL DMXAA) at 37 ℃ for 48 hours, then stained with PE-anti-mouse F4/80, PE-Cy7-anti-mouse CD206, APC-anti-mouse CD80 at room temperature for 60 minutes, centrifuged and washed, then fixed with 1% PFA cell fixative at 4 ℃ for 90 minutes, surface molecule expression was detected by flow cytometry, 20,000 cells were collected each time, and data analysis was performed using FlowJo 9.0 software. As shown in FIG. 9 (b), when the amount of DMXAA is 25. Mu.g/mL, DMXAA @ COF-Ang has significant activation and repolarization effects on Microglia in vitro.

Example 20

In-situ model evaluation of mouse glioma in situ model of multifunctional boron-rich nano-targeting preparation DM & Ce6@ COF-Ang1 prepared in example 6 and DM & Ce6@ COF, DM @ COF and COF nano-preparations of non-target group

(1) Establishing glioma in-situ model

1) Cell preparation: selecting GL261 cell in logarithmic growth phase and good growth state for trypsinization, obtaining single cell suspension according to passage step, regulating cell density to 4 × 10 ⁷ cell/mL, mixing the cell suspension evenly and placing the cell suspension in an incubator at 37 ℃ for later use;

2) Anesthetizing the mice: 5 percent chloral hydrate is used for carrying out intraperitoneal injection anesthesia according to the weight (300 mg/kg) of the mouse, and the prone position of the mouse is fixed on a mouse brain stereotaxic apparatus after the anesthesia is successful;

3) Mice were treated: the head of the mouse is unhaired and disinfected by iodophor, and the connection line of the inner canthus intersects with the sagittal midline of the head;

4) A longitudinal incision is 1 cm at the junction, a hydrogen peroxide cotton swab is used for wiping the surface of the skull, a bregma point is fully exposed, and a small hole with the diameter of 1mm is drilled at a striatum part (0.6 mm in front of the bregma point and 1.8 mm on the right);

5) Inoculation: sucking 5 μ L of glioma cell suspension with a micro-injector, vertically and slowly inserting needle 4.0 mm, then withdrawing needle 1.0 mm, staying for 5min, slowly injecting the cell suspension into mouse brain at uniform speed, standing for 5min, and slowly pulling out;

6) And (3) sewing: bone holes were closed with sterile bone wax, the mouse scalp was sutured with sterile thread, the mice were housed under normal conditions after iodine-containing sterilization, and 200 μ L ampicillin was injected subcutaneously for 3 consecutive days.

(2) In vivo imaging analysis

In order to further confirm the targeting effect and the BBB crossing capability of DM & Ce6@ COF-Ang1, an In Vivo Imaging System (IVIS) is utilized to carry out near infrared fluorescence imaging on free Ce6, a non-targeting group DM & Ce6@ COF and a targeting group DM & Ce6@ COF-Ang1 nano preparation. As shown in fig. 10 (a), ce6 fluorescence of the tumor area DM & ce6@ cof-Ang1 nano-formulation reached a maximum at 2 hours (circled in the figure), and in vitro imaging of tumor and other normal organs collected at 2 hours after injection showed that the targeting group DM & ce6@ cof-Ang1 nano-formulation had significant targeting effect and ability to cross BBB.

(3) Structural distribution of boron content

When the tumor is 2 weeks, the tail vein of the tumor-bearing mice is injected with the targeting group DM&Ce6@ COF-Ang1 nano-formulation (50 mg/kg), mice were sacrificed 2h after injection. Rapidly taking heart, liver, spleen, lung, kidney, brain and tumor tissues, and adding 5mL of the solution containing 68% HNO ₃ And 30% H ₂ O ₂ （V _{68% HNO3} : V _{30% H2O2} Digestion of a mixed solution of = 3). As shown in FIG. 10 (b), DM was injected&In tumors of Ce6@ COF-Ang1 nano-preparation group ¹⁰ The content of B is 100.44 mu g/g, which is much higher than the treatment requirement of BNCT ¹⁰ B concentration (> 20. Mu.g/g), and ¹⁰ the concentration ratio of B at the tumor site and the normal site (T: N = 6.9) is much larger than the required 3.

(4) HPLC determination of DMXAA content in brain glioma

When the tumor is 2 weeks, the tumor-bearing mice are injected with DM intravenously&Ce6@ COF-Ang1 nano preparation (50 mg/kg), mice were sacrificed 2h after injection, tumor tissues were taken, and 1mL of deionized water was added for homogenization. 100 μ L of the tissue homogenate was added to a tube containing 1mL of chromatographic grade methanol, sonicated for 1 h (40 KHz, 100W), and then centrifuged at 10000 rpm for 5min using a centrifuge. Taking supernatant obtained by centrifugation, and filtering with a 0.22 μm filter membrane; standard curves were prepared with 0.25, 0.5, 1, 2, 2.5. Mu.g/mL standard solutions in chromatographic grade methanol. The mobile phase adopts 40 percent CH ₃ OH+60% H ₂ O, flow rate 2 mL/min, UV absorbance at 345 nm, DMXAA @ COF group and PBS group were treated the same. As shown in fig. 10 (c), multifunctional boron-rich nano targeting agent DM was injected&The content of DMXAA in the brain glioma of Ce6@ COF-Ang1 reaches 148.79 ppm.

The results indicate that the targeted group boron-rich nano-formulation is able to cross the cell membrane more rapidly and accumulate inside the cell via the lysosomal pathway than the non-targeted group nano-formulation. In a mouse brain glioma in-situ model, a living body imaging system is used for observing that the fluorescence intensity of a mouse brain tumor part of a targeting boron-rich nano preparation group is obviously higher than that of a non-targeting boron-rich nano preparation group and a free fluorescence probe group after intravenous injection of a boron-rich nano preparation for 2 hours. Grinding and analyzing normal brain tissue and glioma tissue, and collecting glioma tissue ¹⁰ The content of B atoms and DMXAA is far higher than the distribution in normal brain tissue, and the treatment requirements of BNCT are met ( ¹⁰ B is more than 20 ppm), the requirements of targeted radiation and immune combined treatment of BNCT are met.

Claims

1. The multifunctional boron-rich nano targeting preparation based on the covalent organic framework material is characterized in that the multifunctional boron-rich nano targeting preparation is a nano-drug suspension with the diameter of 80 to 180nm, which is self-assembled by a boron-rich covalent organic framework material COF loaded with a drug and a photosensitizer and a target;

the boron-rich covalent organic framework material COF is synthesized by taking terephthalic acid diboronic acid and (3-aminopropyl) -triethoxysilane as raw materials through a tube-sealing solvothermal method;

the drug is selected from 2, 5-pentoxifylline and adriamycin;

the photosensitizer is selected from chlorin e6 (Ce 6) and IR780 dye;

wherein X is selected from the following structures:

Thr-Phe-Phe-Tyr-Gly-Gly-Ser-Arg-Gly-Lys-Arg-Asn-Asn-Phe-Lys-Thr-Glu-Glu-Try-Cys

、

Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys

、

Cys-Arg-Gly-Asp-Lys-Gly-Pro-Asp-Cys

、

Arg-Gly-Asp-Cys

、

Cys-Asn-Gly-Arg-Val-Ser-Thr-Asn-Gly-Arg-Cys

；

each milliliter of suspension contains 3 to 5mg of boron-rich covalent organic framework material COF, and each milliliter of suspension is loaded with 0.25 to 1mg of photosensitizer, 0.5 to 2mg of medicine and 1.2 to 12mg of DSPE-PEG _2k -X。

2. The preparation method of the multifunctional boron-rich nano-targeting agent based on the covalent organic framework material as claimed in claim 1, characterized by comprising the following steps:

(1) taking terephthalic acid diborate, (3-aminopropyl) -triethoxysilane as raw materials, mesitylene and 1, 4-dioxane as solvents, and synthesizing a boron-rich covalent organic framework material COF by a tube-sealed solvothermal method;

(2) will be described in detail

Mixing the prepared boron-rich covalent organic framework material COF with a DMSO solution in which a medicament and a photosensitizer are dissolved, and performing ultrasonic oscillation to obtain the boron-rich covalent organic framework material COF loaded with the medicament and the photosensitizer;

(3) to the step

The reaction solution is added with DSPE-PEG dissolved with a target substance _2k -X and DSPE-PEG _2k The ethanol solution is used for stripping the block-shaped boron-rich covalent organic framework material COF under the ultrasonic action, and deionized water is slowly added to emulsify the block-shaped boron-rich covalent organic framework material COF;

(4) will be described in detail

Dialyzing the obtained nanosheet suspension in a phosphate buffer solution to obtain a suspension of the nano-drug with the diameter of 80 to 180nm;

the definition of the medicine, the photosensitizer and the target substance is the same as the claim 1.

3. The method for preparing the multifunctional boron-rich nano-targeting preparation based on the covalent organic framework material as claimed in claim 2, wherein the steps are as follows

The method comprises the following steps of treating a mixed solvent of mesitylene and 1, 4-dioxane by a three-time freeze-pump-melt circulation method, drying the prepared boron-rich covalent organic framework material COF in vacuum to obtain white solid powder, wherein the molar ratio of terephthalic acid diboronic acid to (3-aminopropyl) -triethoxysilane is 1 to 4.5, and the volume ratio of the trimethylbenzene to the 1, 4-dioxane is 1 to 1.5.

4. The method for preparing the multifunctional boron-rich nano-targeting preparation based on the covalent organic framework material as claimed in claim 2, wherein the steps are as follows

Chinese medicinal composition and photosensitizerAnd the mass ratio of the boron-rich covalent organic framework material COF is 1 to 4 to 2 to 8.

5. The method for preparing the multifunctional boron-rich nano-targeting formulation based on covalent organic framework material according to claim 2, wherein the step of

The mass ratio of the medium-target substance to the boron-rich covalent organic framework material COF is 3 to 1, DSPE-PEG _2k The mass fraction of the-X in the target is 10 to 90 percent.

6. The method for preparing the multifunctional boron-rich nano-targeting preparation based on covalent organic framework material according to claim 5, characterized by comprising the following steps

Ultrasonic emulsification is adopted for 1-3 h.

7. The method for preparing the multifunctional boron-rich nano-targeting preparation based on covalent organic framework material according to claim 6, characterized by comprising the following steps

The speed of adding deionized water in the ultrasonic emulsification process of (2) is 20uL/min.

8. The use of the multifunctional boron-rich nano-targeting agent based on covalent organic framework materials according to claim 1, wherein the multifunctional boron-rich nano-targeting agent is used for the preparation of anti-tumor drugs.

9. The use of claim 8, wherein the tumor comprises brain glioma, breast cancer, ovarian cancer, liver cancer, kidney cancer, lung cancer, colon cancer, bladder cancer, pancreatic cancer, uterine cancer, stomach cancer, rectal cancer.