CN114306205B

CN114306205B - Heparin-polypeptide dual-grafted cyclodextrin framework composition with lung targeting function, and preparation method and application thereof

Info

Publication number: CN114306205B
Application number: CN202011062733.XA
Authority: CN
Inventors: 张继稳; 何亚平; 熊婷; 伍丽; 何思钰; 周咏; 周盼盼; 陈加才
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2023-10-31
Anticipated expiration: 2040-09-30
Also published as: WO2022068927A1; CN114306205A

Abstract

The invention relates to a heparin-polypeptide dual-grafted cyclodextrin skeleton composition with lung targeting, a preparation method and application thereof. Specifically, the invention discloses an LMWH-polypeptide-COF composition and a drug-loaded LMWH-polypeptide-COF composition, and the composition has excellent safety, high-efficiency targeting and excellent anti-tumor curative effect on tumors (especially lung cancer).

Description

Heparin-polypeptide dual-grafted cyclodextrin framework composition with lung targeting function, and preparation method and application thereof

Technical Field

The invention relates to the field of medical biological materials, in particular to an antitumor heparin-polypeptide dual-grafted cyclodextrin framework composition with a lung targeting effect, and preparation and application thereof.

Background

Currently, lung cancer is the tumor with highest global morbidity and mortality, and targeted treatment of lung cancer is a scientific difficulty to be broken through urgently. Because of the characteristics of complex pathogenesis, difficult early diagnosis, easy metastasis, high malignancy degree, high death rate and the like, chemotherapy is still a common means for tumor treatment, however, most of chemotherapy drugs mainly use cytotoxicity drugs, and the chemotherapy drugs have stronger toxic and side effects on normal tissues and organs while obtaining treatment effects. There is therefore a need to develop low-toxicity low-dose targeted, intelligent drug delivery systems.

Most lung cancer nano targeting nano delivery systems (liposome, polymer nanoparticles, polymer micelle, solid lipid nanoparticles, magnetic nanoparticles, metal nanoparticles and the like) are used for realizing lung targeting by utilizing tumor microenvironment or high permeation long retention effect (EPR effect), and most of lung targeting nano delivery systems are cell surface receptors which are highly expressed at tumor sites, do not have the specificity of lung tissue targeting, often have unsatisfactory lung targeting effect, and also have short plates with high toxicity, difficult degradation and the like.

Therefore, in order to solve the problems, the design of a safe and efficient lung cancer targeting drug delivery system is a scientific problem to be broken through in the urgent need of lung cancer treatment.

Disclosure of Invention

The invention aims to provide an LMWH-polypeptide-COF composition with excellent safety, high-efficiency targeting and excellent anti-tumor curative effect and a drug-loaded LMWH-polypeptide-COF composition.

In a first aspect of the invention there is provided an LMWH-polypeptide-COF composition comprising the following components:

1) Crosslinking cyclodextrin organic frameworks COFs;

2) A polypeptide covalently linked to the cross-linked cyclodextrin organic backbone COF; and

3) A low molecular heparin LMWH covalently linked to the cross-linked cyclodextrin organic backbone COF.

In another preferred embodiment, the crosslinked cyclodextrin organic framework COF has lung targeting.

In another preferred embodiment, the crosslinked cyclodextrin organic framework COF has a cubic morphology.

In another preferred embodiment, the cross-linked cyclodextrin organic backbone COF has a size of 10-1000nm, preferably 50-800nm, more preferably 100-500nm.

In another preferred embodiment, the polypeptide is selected from the group consisting of: integrin binding peptide (RGD), CD13 metallopeptidase binding peptide (NGR), or a combination thereof.

In another preferred embodiment, the RGD is selected from the group consisting of: linear group a species, cyclic c (RGDfK), or a combination thereof.

In another preferred embodiment, the group a substance is selected from the group consisting of: RGD, DRGDS, GRGD, RGDS, GRGDS, GRGDSP, GRADSPK, GRGDSPK, or a combination thereof.

In another preferred embodiment, the NGR is selected from the group consisting of: linear group B species, cyclic group C species, or a combination thereof.

In another preferred embodiment, the group B substance is selected from the group consisting of: TNGRGP, NGRSRF, RSRNGR, NGRNTV, or a combination thereof.

In another preferred embodiment, the group C substance is selected from the group consisting of: GGCNGRC, CNGRC, or a combination thereof.

In another preferred embodiment, the polypeptide is linked to the crosslinked cyclodextrin organic backbone COF as follows: covalent linkage is carried out through carboxyl or amino at one end of the polypeptide and hydroxyl on the surface of the crosslinked cyclodextrin organic framework COF.

In another preferred embodiment, the low molecular heparin LMWH is selected from the group consisting of: low molecular heparin, low molecular heparin sodium, low molecular heparin calcium, or a combination thereof.

In another preferred embodiment, the low molecular heparin LMWH has a molecular weight of 2000-8000Da, preferably 3000-6000Da, more preferably 4000-5000Da.

In another preferred embodiment, the low molecular heparin LMWH is linked to the cross-linked cyclodextrin organic backbone COF as follows:

a1 Using cystamine to modify the low molecular heparin LMWH to obtain cystamine modified low molecular heparin LMWH;

a2 The amino group at one end of the cystamine modified low molecular heparin LMWH obtained in the step a 1) is covalently connected with the hydroxyl group on the surface of the crosslinked cyclodextrin organic framework COF.

In another preferred embodiment, a 2) the amino group is linked to the hydroxyl group by a carbonyl group of an activated linking arm, resulting in an-O- (c=o) -NH-linkage, thereby effecting the linking of the cystamine modified low molecular heparin LMWH to the crosslinked cyclodextrin organic framework COF.

In another preferred embodiment, the size of the composition is from 10 to 1000nm, preferably from 50 to 800nm, more preferably from 100 to 500nm.

In another preferred embodiment, the mass ratio of the crosslinked cyclodextrin organic framework COF to the polypeptide is 1:0.001-0.1 (preferably 1:0.01-0.1, more preferably 1:0.02-0.08); and/or

The mass ratio of the crosslinked cyclodextrin organic framework COF to the low molecular heparin LMWH is 1:0.001-0.05 (preferably 1:0.005-0.02, more preferably 1:0.008-0.01).

In a second aspect of the present invention there is provided a process for the preparation of the composition of the first aspect of the present invention comprising the steps of:

1) Preparing a polypeptide modified cross-linked cyclodextrin organic framework COF comprising the steps of: in the presence of a first solvent and an activating agent A, enabling the polypeptide and the crosslinked cyclodextrin organic framework COF to fully react to obtain the crosslinked cyclodextrin organic framework COF modified by the polypeptide;

2) Preparing cystamine modified low molecular heparin LMWH comprising the steps of: fully reacting low molecular heparin LMWH with cystamine in the presence of a second solvent and an activating agent B to obtain cystamine modified low molecular heparin LMWH;

3) An activated polypeptide-modified crosslinked cyclodextrin organic framework COF comprising the steps of: activating the polypeptide modified crosslinked cyclodextrin organic framework COF at a first temperature for a first time in the presence of a third solvent, an activated connecting arm and an activated catalyst to obtain an activated polypeptide modified crosslinked cyclodextrin organic framework COF;

4) Preparing a composition according to the first aspect of the invention, comprising the steps of: reacting the cystamine modified low molecular heparin LMWH obtained in step 2) and the activated polypeptide modified cross-linked cyclodextrin organic backbone COF obtained in step 3) in a fourth solvent at a second temperature for a second time to obtain the composition of the first aspect of the invention.

In another preferred embodiment, the method has one or more features selected from the group consisting of:

1) The first solvent is selected from the group consisting of: dimethylformamide, acetonitrile, acetone, or a combination thereof;

2) The activator A is selected from the group consisting of: 4-dimethylaminopyridine, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, triethylamine, N '-disuccinimidyl carbonate, N-hydroxysuccinimide, N' -carbonyldiimidazole, or a combination thereof;

3) The second solvent is selected from the group consisting of: phosphate buffer, water, or a combination thereof;

4) The activator B is selected from the group consisting of: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 1-hydroxybenzotriazole, or a combination thereof;

5) In the cystamine modified low molecular heparin LMWH, the feeding mass ratio of cystamine to the low molecular heparin LMWH is 1:10-20 (preferably 1:12-18, more preferably 1:12-16);

6) The third solvent is selected from the group consisting of: acetonitrile, formamide, dimethylformamide, acetone, methanol, or a combination thereof;

7) The activating linker arm is selected from the group consisting of: n, N '-disuccinimidyl carbonate, N' -carbonyldiimidazole, succinyl chloride, isocyanate, or a combination thereof;

8) The activated catalyst is selected from the group consisting of: triethylamine, pyridine, N-hydroxysuccinimide, or a combination thereof;

9) The molar ratio of polypeptide modified cross-linked cyclodextrin organic backbone COF, the activating linker arm and the activating catalyst is 1:1-10:1-7 (preferably 1:1-8:1-5);

10 The first temperature is 10-100 ℃ (preferably 20-80 ℃);

11 The first time is 3-50h (preferably 5-25 h);

12 The fourth solvent is selected from the group consisting of: formamide, dimethylformamide, acetonitrile, acetone, methanol, or a combination thereof;

13 The second temperature is 10-100 ℃ (preferably 20-80 ℃);

14 The second time is 8-60 hours (preferably 10-50 hours).

In a third aspect of the present invention, there is provided a pharmaceutically active ingredient comprising:

(1) LMWH-polypeptide-COF compositions of the first aspect of the invention as pharmaceutical carriers; and

(2) An anti-tumor drug or a lung targeting drug, which is carried in the drug carrier.

In another preferred embodiment, the antitumor drug comprises an antitumor drug for treating lung cancer.

In another preferred embodiment, the pharmaceutically active ingredient has lung targeting.

In another preferred embodiment, the antineoplastic agent is selected from the group consisting of: cytotoxic drugs, molecular targeted drugs, adjuvant drugs, or combinations thereof.

In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: doxorubicin, doxorubicin hydrochloride, epirubicin, carboplatin, oxaliplatin, 5-fluoropyrimidine, capecitabine, gemcitabine, paclitaxel, docetaxel, topotecan, 10-hydroxycamptothecin, irinotecan, or a combination thereof.

In another preferred embodiment, the molecular targeted drug is selected from the group consisting of: gefitinib, imatinib, erlotinib, or a combination thereof.

In another preferred embodiment, the auxiliary drug is selected from the group consisting of: resveratrol, quercetin, baicalein, curcumin, or a combination thereof.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) The pharmaceutically active ingredient of the third aspect of the invention; and

(b) A pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition comprises: injection, lyophilized preparation, oral preparation, liquid dosage form, solid dosage form, or combination thereof.

In a fifth aspect of the present invention, there is provided a method for preparing a pharmaceutically active ingredient according to the third aspect of the present invention, comprising the steps of:

(i) The LMWH-polypeptide-COF composition of the first aspect of the present invention and an antitumor drug are mixed as a drug carrier, and the antitumor drug is carried on the drug carrier, thereby obtaining the pharmaceutically active ingredient of the third aspect.

In another preferred embodiment, in step (i), after mixing the drug carrier with the anti-neoplastic drug, the drug carrier is loaded with the neoplastic drug by incubation at a suitable temperature (e.g. 10-80 ℃, preferably 20-65 ℃).

In another preferred embodiment, in step (i), the incubation time is from 0.5 to 72 hours, preferably from 1 to 36 hours, more preferably from 2 to 24 hours.

In a sixth aspect of the invention there is provided the use of a cross-linked cyclodextrin organic framework COF and/or an LMWH-polypeptide-COF composition of the first aspect of the invention and/or a pharmaceutically active ingredient of the third aspect, for the manufacture of a medicament for the prophylaxis and/or treatment of a tumour or a medicament for targeting the lung.

In another preferred embodiment, the tumor comprises lung cancer.

In another preferred embodiment, the lung-targeting agent is used for treating a disease selected from the group consisting of: pulmonary infection, pulmonary inflammation, pulmonary fibrosis, COPD, or a combination thereof.

In another preferred embodiment, the pulmonary infection comprises: bacterial infection, viral infection (e.g., influenza infection, coronavirus infection, or infection by other pathogens).

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 is a tissue distribution fluorescence plot of cyclodextrin scaffold composition COF of example 1 with high lung targeting.

FIG. 2 is a scheme showing the synthesis of low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin backbone (LMWH-GS 5-COF) in example 2.

FIG. 3 is an electron micrograph of a low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin backbone (LMWH-GS 5-COF) of example 2.

FIG. 4 is an infrared spectrum of a low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin backbone (LMWH-GS 5-COF) of example 2.

FIG. 5 is a nuclear magnetic resonance spectrum of a low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin backbone (LMWH-GS 5-COF) of example 2.

FIG. 6 is an evaluation of blood compatibility of low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin skeleton LMWH-GS5-COF with low molecular heparin LMWH modified cube cyclodextrin skeleton LMWH-COF in example 2. (a) microscopic images of erythrocytes after different sample treatments; (B) a sample plot after centrifugation; (C) ratio of hemolysis (n=3).

FIG. 7 is an evaluation of B16F10 cytotoxicity of low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin backbone LMWH-GS5-COF and low molecular heparin LMWH modified cube cyclodextrin backbone LMWH-COF in example 2.

FIG. 8 is an A549 cytotoxicity evaluation of low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin backbone LMWH-GS5-COF and low molecular heparin LMWH modified cube cyclodextrin backbone LMWH-COF in example 2.

FIG. 9 is an in vitro release evaluation of redox response of doxorubicin-loaded low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin backbone (RCLD) in example 3,

fig. 10 shows cytotoxicity evaluation of doxorubicin-loaded low molecular heparin LMWH modified cube cyclodextrin backbone CLD and doxorubicin-loaded low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin backbone RCLD in example 3. Fig. 11 shows the inhibition effect of doxorubicin-loaded low molecular heparin LMWH modified cubic cyclodextrin backbone CLD and doxorubicin-loaded low molecular heparin LMWH and GS5 peptide double modified cubic cyclodextrin backbone RCLD on tumor cell migration and invasion in example 3. (A) And (C) representative pictures of wound healing experiments and wound healing rate analysis (n=3). (B) And (D) representative pictures and quantitative relative invasiveness of the cell invasion assay (n=3). * P <0.001.

Fig. 12 shows lung cancer targeting and biodistribution of doxorubicin-loaded low molecular heparin LMWH modified cubic cyclodextrin backbone CLD and doxorubicin-loaded low molecular heparin LMWH and GS5 peptide double modified cubic cyclodextrin backbone RCLD of example 3.

Fig. 13 shows the antitumor effect of doxorubicin-loaded low molecular heparin LMWH modified cubic cyclodextrin backbone CLD and doxorubicin-loaded low molecular heparin LMWH and GS5 peptide double modified cubic cyclodextrin backbone RCLD in the a549 lung cancer model in example 3. (a) lung pictures collected from an a549 lung cancer mouse model; (B) a treatment regimen for RCLD; (C) Quantitative analysis of lung a549 metastatic nodule number (n=5); (D) H & E staining analysis of lung tissue. * p <0.05, < p <0.001, ns means no significant difference between the two groups.

FIG. 14 shows the change in body weight of A549-loaded BALB/c mice in example 3.

Fig. 15 shows the therapeutic effect of doxorubicin-loaded low molecular heparin LMWH modified cubic cyclodextrin backbone CLD and doxorubicin-loaded low molecular heparin LMWH and GS5 peptide double modified cubic cyclodextrin backbone RCLD in the B16F10 metastatic lung cancer model of example 3. (A) Lung pictures collected from B16F10 metastatic lung cancer mouse models; (B) a treatment regimen for RCLD; (C) area of B16F10 metastatic lung cancer (n=5). * P <0.01, p <0.001, ns represents no significant difference between the two groups.

FIG. 16 is an H & E staining analysis of the major organs after the end of treatment in the B16F10 metastatic lung cancer model mice of example 3. Black arrows point to atrophic cardiomyocytes.

Fig. 17 shows the blood cell analysis and serum biochemical analysis of mice after five times of intravenous injection of doxorubicin-loaded low molecular heparin LMWH and GS5 peptide double modified cubic cyclodextrin backbone RCLD in example 3 (n=4). * p <0.05, < p <0.01, < p <0.001.

Fig. 18 is an in vitro release evaluation of topotecan (TPT) -loaded low molecular heparin LMWH and GS5 peptide double modified cubic cyclodextrin backbone RCLT with topotecan-loaded cyclodextrin organic backbone composition tpt@cof in example 50.

Fig. 19 is an evaluation of cytotoxicity of low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin backbone RCLT loaded with topotecan and B16F10 of cyclodextrin organic backbone composition tpt@cof loaded with topotecan in example 50.

FIG. 20 shows the therapeutic effect of topotecan-loaded low molecular heparin LMWH and GS5 peptide double modified cube cyclodextrin backbone RCLT and topotecan-loaded cyclodextrin backbone composition TPT@COF in a B16F10 metastatic lung cancer model in example 50. (A) Lung pictures collected in RCLT treated B16F10 metastatic lung cancer mouse model; (B) treatment regimen of tpt@cof and RCLT; (C) area of RCLT treated B16F10 metastatic lung cancer; (D) Lung pictures collected in a tpt@cof treated B16F10 metastatic lung cancer mouse model; (E) Area of B16F10 metastatic lung cancer treated with tpt@cof (n=5). * P <0.01, p <0.001.

Fig. 21 is a change in body weight of C57BL/6 mice treated with topotecan-loaded low molecular heparin LMWH and GS5 peptide double modified cubic cyclodextrin backbone RCLT with topotecan-loaded cyclodextrin backbone composition tpt@cof in example 50, B16F10 loaded. (A) Weight change in RCLT-treated C57BL/6 mice bearing B16F 10; (B) Weight change in the C57BL/6 mice treated with TPT@COF in B16F 10.

Detailed Description

The inventor has studied intensively for a long time, and found that the nano-scale cyclodextrin skeleton composition COF has high lung targeting property, based on the fact that the nano-scale cyclodextrin skeleton composition COF is subjected to double modification by adopting functional polypeptide and low-molecular heparin, and a composition with excellent safety, high-efficiency targeting property and excellent curative effect (such as anti-tumor curative effect) is obtained after further drug loading. On this basis, the inventors completed the present invention.

Polypeptides

RGD polypeptide refers to polypeptide containing arginine-glycine-aspartic acid tripeptide sequence (Arg-Gly-Asp, RGD) and capable of specifically recognizing integrin alpha _v β ₃ A receptor. Integrin alpha _v β ₃ Receptor proteins play an important role in the processes of tumor generation and metastasis, are highly expressed on the surfaces of various tumor cells and on the endothelium of tumor neovasculature, and are underexpressed or not expressed in normal cells, so that vectors containing RGD polypeptides can target tumor sites through ligand-receptor mediated active targeting. Polypeptides comprising an "RGD" sequence are referred to as RGD polypeptides, and the terms "RGD", "DRGDS", "GRGD", "RGDs", "GRGDs" (also known as "GS 5"), "GRGDSP", "GRADSPK", "GRGDSPK" are all within the family of RGD polypeptides of the invention.

The NGR polypeptide refers to polypeptide containing asparagine-glycine-arginine tripeptide sequence (Asn-Gly-Arg, NGR), can be combined with CD13 metallopeptidase in tumor cells and tumor neovascular endothelial cells in a specific manner, not only can actively target tumor sites, but also can inhibit generation of tumor neovascular. Polypeptides comprising "NGR" sequences are referred to as NGR polypeptides, and the terms "TNGRGP", "NGRSRF", "RSRNGR", "NGRNTV", "GGCNGRC", "CNGRC" are within the NRG polypeptide series of the present invention.

Heparin

The low molecular heparin (Low molecular weight heparin, LMWH) is low molecular heparin which is formed by degrading common heparin through physical, chemical, biological and other modes. LMWH not only inhibits the interaction between tumor cells and platelets, but also affects the alignment of the tumor cell actin cytoskeleton, impeding epithelial-mesenchymal transition of tumor cells.

Heparin-polypeptide double-grafted cyclodextrin skeleton composition

As used herein, a heparin-polypeptide dual grafted cyclodextrin backbone composition is a low molecular heparin LMWH covalently linked polypeptide to a cross-linked cyclodextrin organic backbone COF.

Reduced glutathione

Reduced Glutathione (GSH) is an active thiol-containing tripeptide consisting of glutamic acid, cysteine and glycine, and has a higher concentration of GSH at the tumor site than normal tissue (even 100-fold higher), and can specifically cleave disulfide bonds.

Lung cancer is a tumor with highest global morbidity and mortality, lung metastasis is a major challenge in clinical treatment of cancer, the existing treatment means mainly adopt chemotherapy, and the lung cancer has stronger toxic and side effects on normal tissues and organs while obtaining treatment effects. Most lung cancer targeting nano-delivery systems realize lung targeting by utilizing tumor microenvironment or high permeation long retention effect (EPR effect), and most of the lung cancer targeting nano-delivery systems only target cell surface receptors with high expression at tumor sites and do not have the specificity of lung tissue targeting. On the basis that the nano cyclodextrin framework material has high lung tissue targeting, the surface of the nano cyclodextrin framework material is subjected to double grafting of polypeptide and low-molecular heparin, so that the active targeting and synergistic treatment effects of the carrier are provided, the safety is high, the biocompatibility is good, the targeting is high, and the clinical requirements are met.

The cyclodextrin-metal organic framework (CD-MOF) formed by taking the medicinal auxiliary material cyclodextrin as an organic ligand and taking potassium ions as inorganic metal ion centers not only keeps the biological safety of the cyclodextrin, but also has the advantages of good biocompatibility, cubic morphology, surface modification and the like, and can be applied to the field of drug delivery.

LMWH-polypeptide-COF composition and preparation method and application thereof

The applicant of the invention carries out cross-linking on nano-scale CD-MOF in the early stage to obtain a cross-linked cubic Cyclodextrin Organic Framework (COF) which stably exists in a water system, adopts an arginine-glycine-aspartic acid tripeptide sequence (Arg-Gly-Asp, RGD) to carry out surface modification on the COF, and synthesizes the obtained RGD-COF material which can be used as an artificial platelet carrier and has the effect of high-efficiency hemostasis. The present invention also found and confirmed that nanoscale COFs have high lung targeting characteristics, with in vivo lung targeting coefficients of COFs of 31.7 (mice), 10.1 (rats), 129.0 (rabbits), respectively, after intravenous injection, whereas the lung targeting coefficients reported in the literature for other modes of administration are generally lower than 10, as reported in literature (Biomaterials, 2019, 218:119331) for discoid polymer particles with uptake in the lungs 8.7 times that of the liver. The invention constructs a novel and efficient targeting drug delivery system on the basis of RGD-COF aiming at COF with lung tissue targeting property.

The polypeptide mediated ligand-receptor active targeting treatment utilizes the high affinity between the polypeptide ligand and the over-expressed receptor in tumor cells to directionally deliver the medicine into specific tumor tissues, increase the accumulation of the medicine at the tumor sites and reduce the toxic and side effects on normal tissues. Integrin alpha _v β ₃ The receptor protein plays an important role in the processes of tumor generation and metastasis, is highly expressed on the surfaces of various tumor cells and the neovascular endothelium of tumors, is expressed or not expressed in normal cells, and can specifically recognize integrin alpha by RGD-containing vectors _v β ₃ Receptors, whereby vectors containing RGD polypeptides can mediate actively targeted drug delivery systems. The growth and metastasis of tumor cells require the improvement of nutrients by new blood vessels, and CD13 metallopeptidase is an important regulator of new blood vessel generation, and promotes the proliferation and migration of cells and the formation of new blood vessels. The NGR carrier can specifically recognize CD13 metallopeptidase, so that the NGR carrier can improve the concentration of the drug in tumor tissues through targeted mediation function.

The low molecular heparin (LMWH) is heparin with lower molecular weight, which is degraded by common heparin through physical, chemical, biological and other modes, has the traditional anticoagulation function, and simultaneously has the characteristics of inhibiting tumor metastasis, inhibiting tumor cell proliferation, having better biocompatibility and the like. The LMWH skeleton contains a large number of active modifiable free radicals, and can be endowed with new properties through chemical modification. LMWH can inhibit tumor neovascularization by inhibiting its activity through binding to growth factors associated with neovascularization that is overexpressed at the tumor site. The LMWH has affinity with heparinase and P-/L-selectin to inhibit tumor cell escape and metastasis and other key processes, so as to reach the aim of tumor treatment. In addition, the LMWH molecule has repeated carboxyl, hydroxyl, amino and other groups, and the LMWH molecule can be grafted on the carrier through a simple chemical reaction to endow the carrier with biological modification characteristics. Because the concentration of reduced Glutathione (GSH) at the tumor part is 100 times higher than that of normal tissues, disulfide bonds in cystamine can be specifically broken under the stimulation of high-concentration GSH of tumor cells, and LMWH can be specifically released in the tumor tissues, so that the directional treatment effect is achieved.

The invention provides an LMWH-polypeptide-COF composition, such as LMWH-RGD-COF, wherein a COF material with a cubic structure is arranged in the LMWH-polypeptide-COF composition, and the surface of the LMWH-polypeptide-COF composition is subjected to biological modification by adopting LMWH and RGD. COF is used as a basic drug carrier unit, RGD is used as a target head, and can be specifically combined with a plurality of kinds of integrin alpha on the surfaces of tumor cells and on the epidermis of new blood vessels _v β ₃ The receptor, LMWH is used as a coating layer, and the LMWH-coated COF taking cystamine as a connecting arm can specifically drop the shell LMWH under the stimulation of high-concentration GSH in tumor cells to expose the loaded medicine. The nanoscale intravenous injection cube LMWH-RGD-COF composition can evade phagocytosis and clearance of macrophages, reaches the lung through autonomous lung targeting, further can reduce the administration dosage, reduce the toxic and side effects of chemotherapeutic drugs, promote enrichment of the drugs at tumor positions, and improve the anti-tumor curative effect.

The LMWH-RGD-COF composition is different from the existing functional nanoparticle report, and the existing method for simultaneously modifying LMWH and RGD on the surface of gold nanoparticles has high specific apoptosis activity on cancer cells; the existing PLGA nano particles modified by LMWH function find that the preparation can improve the accumulation of the nano particles in tumor in vitro, but the two nano particles do not show lung targeting. The LMWH-RGD-COF composition provided by the invention has good biocompatibility, safety and lung targeting.

At present, a lung targeting drug delivery system (including an anti-tumor drug and/or a drug targeting the lung) is not established based on a cubic cyclodextrin framework material. Based on the earlier stage, the inventor grafts LMWH on RGD-COF by adopting a reduction sensitive chemical bond, realizes targeted drug delivery as a carrier, and carries out anti-lung cancer treatment by means of the synergistic effect of a therapeutic drug LMWH and the drug. Heparin and RGD are simultaneously modified on the surface of the gold nanoparticles, and the gold nanoparticles have high-specificity apoptosis activity on cancer cells selectively over-expressing RGD receptors, but do not have lung targeting. In-vivo anti-melanin lung metastasis is carried out by adopting nanoparticle (LH-DOX) of low molecular heparin loaded with Doxorubicin (DOX), and the result shows that the intravenous injection and the free DOX are treated at the same dosage (2.5 mg/kg), the curative effect is higher than that of free drug DOX and LMWH groups, and compared with a control group, the lung metastasis area of melanoma is reduced by 70.92%. The existing doxorubicin liposome (LMWH-DOX-Lip) wrapped by low molecular weight heparin shows that compared with DOX and LMWH groups, the result of a mouse lung melanoma metastasis model shows that the LMWH-DOX-Lip can effectively inhibit metastasis at the same DOX (5 mg/kg) dose. Compared with the nano preparation, the carrier designed by the invention can remarkably inhibit the growth and metastasis of lung cancer (almost no lung metastasis nodules of A549 cells and 70% reduction of lung metastasis area of melanoma) under the condition of reducing the drug dosage (such as DOX dosage is reduced from 2.5mg/kg to 1 mg/kg) by utilizing the lung tissue targeting property of the carrier.

The invention provides a low molecular heparin LMWH and polypeptide double-grafted cube cyclodextrin framework composition (LMWH-polypeptide-COF) with lung targeting, and a preparation method and application thereof. Specifically, the invention combines the polypeptide, the low molecular heparin with anti-tumor activity and the anti-tumor drug in the cubic cyclodextrin framework COF to form a multi-component composition, and aims to construct an intelligent drug delivery system with functions of long circulation, lung targeting and the like. The LMWH-polypeptide-COF nanoparticle can evade phagocytosis and clearance of macrophages, has good safety and high-efficiency targeting, enriches the drug at a tumor part, and releases the drug through redox responsiveness at the tumor part, thereby greatly reducing toxic and side effects on normal tissues and enhancing the therapeutic effect of the drug.

Taking an anti-tumor drug as an example, the invention provides an anti-tumor LMWH-polypeptideA COF composition is prepared through covalent linking of hydroxy (-OH) in CD-MOF with proper cross-linking agent to form COF material with high stability in water, and linking arm to make carboxyl (-COOH) or amino (-NH) of polypeptide ₂ ) Covalently linked with hydroxyl (-OH) on the outer surface of COF, and further adopts activator to modify amino (-NH) of low molecular heparin LMWH with cystamine ₂ ) Covalent attachment to hydroxyl (-OH) groups activated on the outer surface of the COF, double biological modification of the surface of the COF is achieved. The COF can be used as a high-efficiency load antitumor drug carrier, the polypeptide is used as a target head, and the COF is specifically combined with a receptor highly expressed by tumor cells to actively target the tumor part; LMWH with disulfide-sensitive release is used as a coating layer for synergistic treatment with chemotherapeutic agents.

The invention provides a preparation method for preparing LMWH-polypeptide-COF, which comprises the following steps:

(I) Providing a nanoscale CD-MOF material;

(II) chemically crosslinking the hydroxyl (-OH) groups of the nano-scale CD-MOF by a crosslinking agent to obtain a COF which is well stable in water;

(III) covalently attaching a functional polypeptide to the surface of said COF, resulting in said polypeptide-COF composition;

and (IV) wrapping cystamine modified low molecular heparin LMWH on the surface of the polypeptide-COF composition, thereby obtaining the LMWH-polypeptide-COF.

Synthesis of LMWH-cystamine: after grafting cystamine to LMWH, introducing terminal amino group, which forms amide bond with carbonyl of polypeptide-COF after activation, this method can raise substitution degree of LMWH on polypeptide-COF. The specific method comprises the following steps: dissolving LMWH in phosphate buffer solution (pH=7.4), sequentially adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in a certain proportion, activating at room temperature for 20-30min, adding cystamine for dissolving, dialyzing, and freeze-drying to obtain LMWH-cystamine.

Activation of polypeptide-COF: dispersing the polypeptide-COF in the step (I II) in an organic solvent, adding a certain amount of connecting arms and a catalyst, and reacting for a period of time at a certain temperature to obtain an activated cube polypeptide-COF composition reaction solution;

preparation of LMWH-polypeptide-COF: slowly dripping the reaction liquid in the step (2) into an organic solution of LMWH-cystamine, adding a certain amount of catalyst, reacting for a period of time at a certain temperature, dialyzing the reaction liquid, and freeze-drying to obtain the LMWH and polypeptide double-grafted cube cyclodextrin skeleton composition.

The preparation method of the pharmaceutical composition is summarized as follows: stirring and incubating the antitumor drug solution and the LMWH-polypeptide-COF for a certain time at a certain temperature according to a certain feeding molar ratio, centrifuging, washing and drying to obtain the antitumor drug.

The invention provides a preparation method of a heparin/polypeptide dual-grafted cyclodextrin skeleton composition with lung targeting, and a preparation method and application thereof. Specifically, the LMWH/RGD double-grafted cyclodextrin skeleton composition has a lung targeting function, can obviously reduce the survival rate of B16F10 tumor cells in vitro after being loaded with a cytotoxic drug (such as doxorubicin), and can also obviously inhibit the migration and invasion of the tumor cells. For the mice A549 human lung adenocarcinoma metastasis model and B16F10 melanoma metastatic lung cancer model, the carrier can reduce the dosage of the doxorubicin by 5 times under the same therapeutic effect of the doxorubicin, reduces the cardiotoxicity and side effects of the doxorubicin, and is expected to realize the attenuation and synergy of lung cancer treatment. The nanometer lung tissue delivery system has good biological safety, main organs do not generate acute toxicity or tissue necrosis after intravenous injection, and hematological researches show that the nanometer lung tissue delivery system does not influence the main blood cell level in a mouse body, and has good clinical conversion value.

Compared with the prior art, the invention has the following main advantages:

(1) The nano material (namely the composition) prepared by the invention has the advantages of simple and controllable preparation process, no need of expensive equipment, mass production, high carrier safety, good biocompatibility, no immunogenicity and good lung targeting.

(2) The nano material prepared by the invention has regular cube shape and smaller particle size, breaks through the limit of the spherical shape of the traditional carrier, effectively evades the phagocytosis and clearance of macrophages, and prolongs the internal circulation time.

(3) The nano material prepared by the invention is modified by a polypeptide sequence in the construction process, so that the cube cyclodextrin organic framework composition COF active targeting characteristic is endowed, the tumor drug targeting treatment is realized, and the toxic and side effects on normal organs and tissues are reduced.

(4) The nano material prepared by the invention adopts low molecular heparin LMWH coating in the construction process, and the nano particles are positioned and released to release the medicine through a redox sensitive release mechanism.

(5) The nano preparation prepared by the invention loads the anti-tumor drug and the low molecular heparin simultaneously, realizes synergistic administration and greatly improves the treatment effect.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

Example 1

Preparation of nanoscale CD-MOF: the mixed system of gamma-CD, KOH aqueous solution and methanol solvent is heated by using a solvothermal mode. 163.0mg of gamma-CD and 56.0mg of KOH are weighed according to the mol ratio of 1:8 and dissolved in 5mL of water, and the mixture is filtered by a 0.8 mu m filter membrane to obtain gamma-CD-KOH mother liquor. 3mL of methanol is added into the mother solution, after heating in a water bath at 60 ℃ for 20min, the methanol with the same volume is added, 64mg of PEG 20000 is added, and heating is continued for 20min. After the reaction system is kept stand for 2 hours, the reaction system is centrifuged for 5 minutes at 4000rpm, ethanol (10 mL multiplied by 2 times) and methanol (10 mL multiplied by 2 times) are respectively used for washing, the obtained product is dried in vacuum at 80 ℃ for 2 hours, and the nano-grade CD-MOF crystal is obtained, and the scanning electron microscope and dynamic light scattering result shows that the obtained CD-MOF is in a regular cube shape and the size is 100-500nm.

Preparation of nanoscale COF: 778.3mg of nano-grade CD-MOF powder is weighed into a round bottom flask, fixed on a magnetic stirrer, 10mL of dimethylformamide is added, heating is carried out at 80 ℃, 771mg of diphenyl carbonate serving as a crosslinking agent (the molar ratio of CD-MOF to diphenyl carbonate is 1:6) and 450 mu L of triethylamine serving as a catalyst are added under the stirring condition of 400rpm, after 24 hours of reaction, the mixture is cooled to room temperature, 20mL of 95% ethanol is adopted to terminate the reaction, centrifugation is carried out for 5 minutes at 4000rpm, 50% ethanol (10 mL multiplied by 2 times), pure water (10 mL multiplied by 2 times) and acetone (10 mL multiplied by 2 times) are respectively used for 2 times, the obtained crystal is dried in vacuum at 40 ℃ for 4 hours to obtain stable COF in water, the yield is about 85%, and the results of a scanning electron microscope and dynamic light scattering result show that the obtained COF is in a regular cube form and the size is 100-500nm.

COF has a high degree of lung targeting in mice: cy 5-labeled COF nanoparticle suspensions (40 mg/kg,10 mL/kg) were intravenously injected into the tail of the Kunming mice, 5min before administration, 0min, 5min, 10min, 15min, 1h, 2h, 4h, 8h and 24h after administration, the mice were sacrificed, and the major organs of heart, liver, spleen, lung and kidney were removed, and the fluorescence intensities of the organs at different time points were measured by using a small animal in vivo imager to investigate the lung targeting of the COF nanoparticles in the mice. After mice are injected with fluorescent dye Cy5 and COF nanoparticles marked by the fluorescent dye Cy5 through tail veins for 1h, the fluorescent dye Cy5 is mainly distributed in the kidneys and livers, and the COF nanoparticles are mainly distributed in the lungs; 15min after administration, cof begins to aggregate in the lungs; 2h after administration, the distribution of COF nanoparticles in the lung reaches the highest value, and the fluorescence signal of the mouse lung is 30 times that of the liver part, so that the COF has high lung targeting to the mouse.

COF has a high degree of lung targeting in rats: the rat tail was intravenously injected with Cy 5-labeled COF nanoparticle suspensions (28 mg/kg,10 mL/kg), 5min before administration, 0min, 5min, 10min, 15min, 1h, 2h, 4h, 8h, and 24h after administration, the rat was sacrificed, and the heart, liver, spleen, lung, and kidney major organs were removed, and the fluorescence intensities of the respective organs at different time points were measured using a small animal biopsy imager, and the lung targeting of COF nanoparticles in the rat body was studied. After rats are injected with fluorescent dye Cy5 and Cy5 marked gamma-CD and COF nano particles for 1h through tail vein, the gamma-CD and Cy5 fluorescent dye are mainly distributed in kidney and liver; COF nanoparticles are predominantly distributed in the lungs; 15min after administration, cof begins to aggregate in the lungs; 2h after administration, the distribution of COF nanoparticles in the lung reaches the highest value, and the fluorescence signal of the rat lung is 10 times that of the liver part, which indicates that the COF has high lung targeting to the rat.

COF has a high degree of lung targeting in rabbits: and (3) carrying out intravenous injection on Cy 5-marked COF nanoparticle suspension on the ear margin of a rabbit, respectively 5min before administration, 0min, 5min, 10min, 15min, 1h, 2h, 4h, 8h and 24h after administration, killing the rabbit, taking out heart, liver, spleen, lung and kidney main organs, measuring the fluorescence intensity of each organ at different time points by adopting a small animal living body imager, and researching the lung targeting of the COF nanoparticles in the rabbit. After 15min of the COF nanoparticles are injected into the rabbit by the ear margin vein, the distribution of the COF in the lung reaches the highest value, and the fluorescence signal of the lung is 120 times that of the liver part, so that the COF has obvious lung targeting function in the rabbit.

The specific results are shown in FIG. 1, wherein A is a tissue fluorescence intensity map of COF in mice, rats and rabbits, B is an in vivo tissue distribution map of COF in mice at different time points, and C is a fluorescence intensity map of COF in mice at different times.

As can be seen from fig. 1A: COF can realize high targeting to lung tissues in mice, rats and rabbits, and fluorescence signals of the lung are 30 times, 10 times and 120 times that of liver parts.

As can be seen from fig. 1B: after COF intravenous administration to the tail of the mice, the COF was distributed mainly in the lungs.

As can be seen from fig. 1C: after 2h of COF intravenous injection in the tail of the mice, the distribution of COF in the lungs reached the highest value, but with time, the enrichment of COF in the lungs gradually decreased.

Example 2

(1) Preparation of double modified composition carrier

Preparation of nanoscale GS 5-COF: 230mg of the nanoscale COF prepared in example 1 and 10mg of GRGDS (also called GS 5) pentapeptide (molar ratio of COF to GS 5: 1) were weighed, placed in a round-bottomed flask, 5mL of dimethylformamide was added, and after stirring uniformly, 5mg of 4-Dimethylaminopyridine (DMAP) and 6mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) were added, and placed on a magnetic stirrer and stirred at 37℃for 24 hours at 600rpm, so that the COF and the GS5 polypeptide were sufficiently coupled. After the reaction is finished, centrifuging at 4000rpm for 5min, respectively washing with 10mL of dimethylformamide and 10mL of pure water for 2 times, and freeze-drying at-50 ℃ for 24h to obtain GS5 modified COF (the product is abbreviated as GS 5-COF), wherein the scanning electron microscope and dynamic light scattering result show that the obtained GS5-COF is in a regular cube form, the size is 100-500nm, and the mass percentage of the cyclodextrin framework material to the GS5 polypeptide is 1:0.05 as measured by High Performance Liquid Chromatography (HPLC).

Preparation of low molecular heparin (LMWH) and GS5 peptide double modified cubic cyclodextrin backbone (COF), i.e. LMWH-GS 5-COF:

Synthesis of low molecular heparin-cystamine (LMWH-CYS): 400mg of low molecular heparin LMWH (0.71 mmol) was weighed out and sonicated in 100mL of phosphate buffer (PBS, pH=7.4), then 3.56mmol of 1-ethyl- (1-dimethylaminopropyl) carbodiimide hydrochloride (EDC) 681.60mg and N-hydroxysuccinimide (NHS) 409.20mg, corresponding to 5 times the molar equivalent of carboxyl groups in LMWH, were added and magnetically stirred at 25℃for 15min to fully activate the carboxyl groups on LMWH. Then 5.6g cystamine CYS (24.8 mmol) was added, and after complete dissolution, stirring was continued for 48h at 25℃to give a crude low molecular heparin-cystamine (LMWH-CYS) product. The crude product was dialyzed against 0.1M NaCl solution (5.85 g NaCl dissolved in pure water to a volume of 1000 mL) for 12h (dialysis bag cut-off molecular weight 3500 Da), and then against pure water for 48h. Filtering the liquid obtained after dialysis through a 0.22 mu m filter membrane and freeze-drying to obtain an intermediate product LMWH-CYS.

Activation of GS 5-COF: 34.13mg (0.133 mol) of N, N' -disuccinimidyl carbonate (DSC) was weighed out and sonicated in 3mL of Acetonitrile (ACN), 28. Mu.L of Triethylamine (TEA) was added, 96.33mg (0.067 mol) of GS5-COF powder was added to the above solution in a molar ratio of COF: DSC: TEA=1:2:3, and activated by stirring at 25℃for 12 hours at 400 rpm.

Preparation of LMWH-GS 5-COF: 300mg of LMWH-CYS is weighed, 18mL of formamide is added, and the mixture is dissolved under magnetic stirring to obtain an LMWH-CYS solution. The activated GS5-COF solution was added to the LMWH-CYS solution and reacted at 25℃for 48 hours under magnetic stirring at 400rpm to give a crude LMWH-GS5-COF product. In order to remove unreacted micromolecules and byproducts thereof, the crude product is dialyzed for three days by acetonitrile/water mixed solvent (4:1, v:v), then is dialyzed for two days by pure water, and is centrifuged and freeze-dried at-50 ℃ to obtain the LMWH-GS5-COF.

The preparation process of the specific LMWH-GS5-COF is shown in FIG. 2.

FIG. 3 is an electron micrograph of LMWH-GS5-COF of example 2, as can be seen from FIG. 3: the LMWH-GS5-COF still maintains a regular cubic morphology with a particle size of 100-500nm.

(2) Preparation of RGD-free low molecular heparin-grafted support:

a nanoscale crosslinked cyclodextrin backbone (COF) was prepared as in example 1 and low molecular heparin-cystamine ((LMWH-CYS) was prepared as in example 2.

Activation of COF: DSC 34.13mg (0.133 mol) was weighed out and sonicated in 3mL of ACN, and 28. Mu.L of TEA was added, to the above solution was added 96.33mg (0.067 mol) of COF powder in a molar ratio of COF: DSC: TEA=1:2:3, and activated for 12h with stirring at 25℃at 400 rpm.

Preparation of heparin-crosslinked cyclodextrin backbone composition (LMWH-COF): 300mg of LMWH-CYS is weighed, 18mL of formamide is added, and the mixture is dissolved under magnetic stirring to obtain an LMWH-CYS solution. The activated COF solution is added into LMWH-CYS solution, and the mixture is reacted for 48 hours at 25 ℃ under the magnetic stirring of 400rpm, so as to obtain the crude product of the heparin-crosslinked cyclodextrin framework composition (LMWH-COF). In order to remove unreacted micromolecules and byproducts thereof, the crude product is dialyzed for three days by acetonitrile/water mixed solvent (4:1, v:v), and then is dialyzed for two days by pure water continuously, and is centrifuged and freeze-dried at-50 ℃ to obtain the LMWH-COF.

(3) Characterization of the vector

Fourier transform infrared spectroscopy analysis of the functional groups of the dual modified support composition: measuring sample by using Thermo Nicolet IS infrared spectrometer, mixing sample and potassium bromide according to mass ratio of 1:10, grinding, tabletting, measuring sample at wave number of 4000-400cm ^-1 Infrared absorption spectrum in the range.

FIG. 4 is an infrared spectrum of LMWH-GS5-COF in example 2.

The infrared results of FIG. 4 show that the productsThe LMWH-GS5-COF product contains a COF of 1751cm ^-1 Characteristic peaks at 1540cm for GS5 are also retained ^-1 And 1203cm ^-1 Characteristic peaks at this point, indicating successful modification of GS5 to COF surface. At the same time, the synthesized LMWH-GS5-COF also shows that the LMWH is 1622cm ^-1 The infrared characteristic absorption peak at the site demonstrates that GS5-COF has successfully modified LMWH.

Characteristic peaks of the dual modified vector were analyzed by nuclear magnetic resonance hydrogen spectroscopy: the sample was subjected to nmr hydrogen spectroscopy using a Bruker AVANCE NEO model nmr hydrogen spectrometer. GS5 polypeptide and LMWH were dissolved in 600. Mu.L of D ₂ In O, LMWH-GS5-COF was dispersed in 600. Mu.L of D ₂ In O, 10. Mu.L of NaOD was added to degrade the mixture. Each sample solution was then placed in a capped nuclear magnetic tube and the spectra collected under a magnetic field of 500 MHz.

FIG. 5 is a nuclear magnetic resonance spectrum of LMWH-GS5-COF in example 2.

From FIG. 5, the product LMWH-GS5-COF is known ¹ H-NMR contained both the characteristic peaks of LMWH (δ=2.1 ppm; δ=5.1 to 5.8 ppm) and GS5 (δ=3.2 ppm), and also retained the characteristic peaks of cyclodextrin protons in COF at δ=5.0 ppm, indicating successful synthesis of both supports.

(4) Biological safety evaluation of vectors

Hemolytic toxicity investigation of the vector: fresh C57BL/6 fresh blood was taken and 2mL of the blood was centrifuged at 2500rpm for 10min in a 3.2% sodium citrate-infiltrated tube to pellet red blood cells, and the upper plasma was discarded and the red blood cells were washed 3 times with physiological saline. The erythrocytes were diluted to a 2% erythrocyte suspension with physiological saline. The LMWH-GS5-COF and LMWH-COF prepared in example 2 above were prepared as suspensions of 5, 10, 25, 100, 200, 400. Mu.g/mL with physiological saline, and an equal volume of 2% erythrocyte suspension was added thereto and shaken well, and incubated in a water bath at 37℃for 1 hour. Pure water and physiological saline with the same volume are taken as positive control and negative control respectively. After incubation, the supernatant was centrifuged at 1500rpm for 10min, and the absorbance at 540nm was measured by UV spectrophotometry and run in parallel for 3 times.

FIG. 6 is an evaluation of blood compatibility between LMWH-GS5-COF and LMWH-COF in example 2. (a) microscopic images of erythrocytes after different sample treatments; (B) a sample plot after centrifugation; (C) ratio of hemolysis (n=3).

The results in FIGS. 6A-C show that: when the concentration of LMWH-GS5-COF and LMWH-COF was increased to 600. Mu.g/mL, the hemolysis rate was still less than 1%. A hemolysis ratio of less than 5% is generally considered safe, indicating that LMWH-MOF and LMWH-GS5-COF nanoparticles have good blood compatibility.

B16F10 cytotoxicity evaluation of vector: B16F10 cells were cultured in DMEM medium (containing 10% fetal bovine serum) at 37 ℃ in a 5% CO2 constant temperature and humidity incubator. Selecting cells in logarithmic phase for experiment, the cells in logarithmic phase were grown at 5×10 ³ The density of each/well was inoculated in a 96-well plate and cultured for 12 hours. After removing the upper layer of culture solution, 200. Mu.L of LMWH, LMWH-COF, LMWH-GS5-COF samples of different concentrations were added, respectively, while a blank group (containing only culture solution) and a control group (containing cells and culture solution) were set. The experiment was set up at 8 different concentrations of 0.0067, 0.033, 0.067, 0.33, 0.67, 1.33, 13.3 and 133. Mu.g.mL, respectively ^-1 After a further incubation period of 24 hours in the incubator, 15. Mu.L of CCK-8 solution was added to each well and incubation was continued for 2 hours at 37 ℃. And (3) stopping culturing, detecting an absorbance value at 450nm by adopting an enzyme-labeled instrument, and calculating the cell survival rate according to a formula (1).

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Wherein A is _Sample Absorbance values for the samples; a is that _control Absorbance values for the control group; and A _blank Absorbance values for the blank group.

FIG. 7 is an evaluation of B16F10 cytotoxicity of LMWH-GS5-COF and LMWH-COF in example 2, wherein A is B16F10 cytotoxicity of LMWH and B is B16F10 cytotoxicity of LMWH-COF and LMWH-GS5-COF nanoparticles.

The results in FIG. 7A show that when the LMWH concentration was increased to 133. Mu.g/mL, the cell viability was still as high as 100%, indicating that LMWH itself did not affect cell activity. The results in FIG. 7B show that the cell viability of the LMWH-COF and LMWH-GS5-COF nanocarriers was about 100% without significant cytotoxicity.

A549 cytotoxicity evaluation of vector:a549 cells were cultured in RPMI 1640 medium (containing 10% fetal bovine serum) at 37 ℃,5% CO ₂ Culturing in a constant temperature and humidity incubator. Selecting cells in logarithmic phase for experiment, the cells in logarithmic phase were grown at 2×10 ⁴ The density of each/well was inoculated in a 96-well plate and cultured for 24 hours. After removing the upper layer of culture solution, 200. Mu.L of LMWH-COF and LMWH-GS5-COF samples of different concentrations were added, respectively, while a blank group (containing only the culture solution) and a control group (containing cells and the culture solution) were set. The experiment was set up at 8 different concentrations of 0.0067, 0.033, 0.067, 0.33, 0.67, 1.33, 13.3 and 133. Mu.g.mL, respectively ^-1 After a further incubation period of 24 hours in the incubator, 15. Mu.L of CCK-8 solution was added to each well and incubation was continued for 1.5 hours at 37 ℃. And (3) stopping culturing, detecting an absorbance value at 450nm by adopting an enzyme-labeled instrument, and calculating the cell survival rate according to a formula (1).

FIG. 8 is an A549 cytotoxicity evaluation of LMWH-GS5-COF and LMWH-COF in example 2, showing that the concentrations of the LMWH-COF and LMWH-GS5-COF nanocarriers were 0.0067-13.3. Mu.g.multidot.mL ^-1 The cell activity is basically close to 100% in the range, and no obvious cytotoxicity exists.

Example 3

(1) Loaded doxorubicin hydrochloride

Preparation of doxorubicin hydrochloride-loaded double modified vector composition: 12mg of doxorubicin hydrochloride (DOX) was weighed and dissolved in 2mL of pure water, sonicated for 10min to dissolve, then 30mg of the LMWH-GS5-COF nanoparticle prepared in example 2 was added, the molar ratio of the drug to the LMWH-GS5-COF nanoparticle was 1:1, stirred at 25℃at 300rpm in the absence of light for 24 hours, and drug-loaded was incubated. After completion of drug loading, centrifugation was carried out at 4000rpm for 5min, and washing was carried out with pure water to remove free DOX, to obtain a lower layer doxorubicin hydrochloride-loaded double modified vector composition (RCLD).

The drug loading rate of the medicine is measured by a fluorescence spectrophotometry, a proper amount of RCLD is weighed and dispersed in pure water, 1M sodium hydroxide solution is added for dissolution, 1M hydrochloric acid with the same volume is added for neutralization, the fluorescence intensity of DOX is measured at Ex=470 nm and Em=598 nm of the solution, and the drug loading rate is measured according to a formula (2). The results indicated that the RCLD drug loading was 15%.

Preparation of doxorubicin hydrochloride-loaded low molecular weight heparin-grafted carrier composition without GS 5: 12mg of DOX is weighed and dissolved in 2mL of pure water, ultrasonic treatment is carried out for 10min to dissolve the DOX, 30mg of LMWH-COF nano particles prepared in the example 2 are respectively added, the molar ratio of the drug to the LMWH-COF is 1:1, the stirring is carried out for 24h at 25 ℃ in the dark at 300rpm, and the drug carrying is incubated. After completion of drug loading, centrifugation at 4000rpm for 5min, washing with pure water to remove free DOX, a low molecular heparin-grafted carrier Composition (CLD) without RGD was obtained with doxorubicin hydrochloride as the lower layer.

The drug-loading rate of the CLD is measured by a fluorescence spectrophotometry, a proper amount of the CLD is weighed and dispersed in pure water, 1M sodium hydroxide solution is added for dissolution, 1M hydrochloric acid with the same volume is added for neutralization, the fluorescence intensity of DOX is measured at Ex=470 nm and Em=598 nm of the solution, and the drug-loading rate is measured according to a formula (2). The results indicated a CLD drug loading of 15%.

(2) In vitro release investigation of doxorubicin-loaded vectors

And (3) examining the drug release behavior of the RCLD nanoparticles under the conditions of different concentrations of reduced Glutathione (GSH) by adopting a dynamic membrane dialysis bag method. 5mg of a 5mL dispersion of RCLD (DOX dose of 750. Mu.g) was filled into dialysis bags (cut-off molecular weight of 14 kDa). Each dialysis bag was immersed in PBS (ph=7.4, 100 mL) with different GSH concentrations (0, 1, 10 mM) and incubated in a shaker at 75rpm and 37 ℃, 5mL of release medium was collected at predetermined time points (0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 72 h) and supplemented with an equal volume of medium. The amount of DOX released was determined for the samples using fluorescence spectrophotometry at ex=470 nm and em=598 nm.

FIG. 9 is an in vitro release evaluation of RCLD in example 3, showing that compared to RCLD release of 50% in PBS release medium without GSH for 72h, RCLD nanoparticles can rapidly release DOX in 10mM GSH release medium, 50% drug in the first 12h, and 70% drug in 72h, due to high concentration GSH cleaving the connection between heparin and COF, accelerating DOX release. In addition, in a release medium containing 1mM GSH and not containing GSH, the release behaviors are similar, which shows that RCLD nanoparticles have good stability in vivo circulation, and can avoid the premature release of the drug before reaching the tumor site.

(3) Cytotoxicity evaluation of two doxorubicin-loaded carrier compositions

Cytotoxicity of the above two doxorubicin-loaded vector composition nanoparticles (RCLD and CLD) on B16F10 tumor cells was examined using the CCK-8 method. B16F10 cells were cultured in DMEM medium (containing 10% fetal bovine serum) at 37 ℃ with 5% CO ₂ Culturing in a constant temperature and humidity incubator. Selecting cells in logarithmic phase for experiment, the cells in logarithmic phase were grown at 5×10 ³ The density of each well was inoculated in a 96-well plate with a final volume of 200. Mu.L per well and cultured for 12 hours. Removing culture solution, adding DOX and above two carrier compositions RCLD or CLD loaded with doxorubicin respectively, setting total concentration to 8 different concentrations, and setting final concentration of DOX to 0.1, 0.2, 0.5, 1, 2, 5, 10 and 20μg.mL according to DOX drug loading conversion ^-1 . A blank (containing only culture) and a control (cells and culture) were set at the same time. After a further incubation period of 24 hours in the incubator, 15. Mu.L of CCK-8 solution was added to each well and incubation was continued for 2 hours at 37 ℃. The culture was terminated, absorbance (a) at 450nm was measured using a microplate reader, and cell viability (n=6) was calculated according to the formula of example 2.

FIG. 10 shows cytotoxicity evaluation of CLD and RCLD in example 3. The results in fig. 10 show that: RCLD has strong cytotoxicity, can obviously inhibit the growth of tumor cells in vitro, and has weaker cytotoxicity compared with free DOX.

(4) Evaluation of migration and invasion inhibition of tumor cells by two doxorubicin-loaded carrier compositions

Wound healing experiments with two doxorubicin hydrochloride-loaded carrier compositions: B16F10 cells were seeded into 6-well plates and when the cells grew to fill 90% of the bottom area of the wells, a wound was drawn with 200 μl of sterile pipette tips and rinsed once with PBS to form a scratch free of cells. Subsequently, the cells were incubated with PBS, free DOX, free LMWH, COF prepared in example 1, RCLD prepared in example 3, or CLD at 37 ℃ for 24h. Photographs were taken at 0h and 24h observation using an inverted light microscope, respectively, and wound healing rates were calculated.

Fig. 11A and 11C are representative pictures of the wound healing experiments and the wound healing rate analysis (n=3) of CLD and RCLD in example 3. * P <0.001.

The results in fig. 11A and 11C show that B16F10 cells of the PBS control group were almost full of scratches after 24h incubation, with a wound healing rate of 82.3%. The wound healing rate of LMWH group was significantly reduced compared to PBS group, indicating that LMWH can reduce the migration capacity of B16F10 cells. The wound healing rate of the DOX group was also reduced due to the cytocidal effect of DOX. In addition, the combined action of LMWH and DOX shows the strongest inhibition of the migration of tumor cells by RCLD or CLD nanoparticles.

Transwell cell invasion experiments of two doxorubicin-loaded vector compositions: B16F10 cells in exponential growth phase were digested, the cell suspension was centrifuged at 1000rpm for 3min, the supernatant was discarded, and the lower cells were dispersed in medium containing 0.5% fbs. Coating matrigel on upper layer of transwell chamber, adding 1×10 ⁵ For B16F10 cells, 100. Mu.L of samples were added, DOX, LMWH-COF prepared in example 2 and LMWH-GS5-COF, respectively, and RCLD or CLD prepared in example 3 (to give a final DOX concentration of 0.5. Mu.g/mL). 600. Mu.L of medium containing 20% FBS was added as a chemokine to the lower layer of the chamber. After 48h incubation at 37 ℃, the culture solution in the upper chamber was aspirated, matrigel and cells in the upper chamber were rubbed off with a cotton swab, and washed 2 times with PBS. The cell membranes were fixed with 4% paraformaldehyde for 10min at room temperature and washed 2 times with pbs. After the film was air-dried, it was stained with 0.1% crystal violet at room temperature for 30min, and the cells that invaded the lower layer were stained with crystal violet. After the cell membranes were air dried, the cells under the membrane were photographed under an inverted microscope at 5-fold magnification and qualitatively observed for cells that had invaded the lower layer of the membrane. Then, crystal violet on the lower membrane was dissolved with 33% acetic acid, OD was measured at 570nm, cells invading the lower membrane were quantitatively studied, and the effect of two doxorubicin-loaded carrier compositions on the ability of cells to invade was evaluated.

Fig. 11B and 11D are representative pictures and quantitative relative invasiveness rates (n=3) of CLD and RCLD cell invasion experiments. * P <0.001.

The results in fig. 11B and 11D show that RCLD or CLD can significantly inhibit invasion of B16F10 tumor cells, and has better inhibitory effect than the same dose of DOX.

(5) Pharmacodynamics study of two carrier compositions loaded with doxorubicin hydrochloride

Lung cancer targeting and biodistribution studies of two doxorubicin hydrochloride-loaded carrier compositions: CLD and RCLD nanoparticles were fluorescently labeled using Cy5 probes. And then constructing a metastatic melanin lung cancer model to research lung cancer targeting and biological distribution of the above two carrier compositions loaded with doxorubicin in vivo. Specifically, 2×10 ⁵ The individual B16F10 cells were dispersed in 100. Mu.L of PBS and tail vein injected into C57BL/6 mice to construct a melanoma lung cancer metastasis model. On day 15 after injection of B16F10 cells, mice were injected i.v. with PBS, cy 5-labeled RCLD or CLD nanoparticles (n=3), respectively. After 2h of injection of RCLD or CLD nanoparticles, mice were anesthetized with isoflurane and targeted to lung tumors with IVIS in vivo. Mice were then sacrificed, heart, liver, spleen, lung and kidney organs were dissected out and fluorescence imaging was performed ex vivo to study the in vivo biodistribution of the two doxorubicin-loaded carrier compositions above nanoparticles.

FIG. 12 shows lung cancer targeting and biodistribution of the CLD and the RCLD of example 3, wherein FIG. 12A shows the in vivo distribution of the RCLD and the CLD in B16F10 lung cancer metastasis, FIG. 12B shows fluorescence intensity analysis of lung tissue, C shows ex vivo imaging of mouse organs, and FIG. 12D shows quantitative fluorescence intensity analysis of the RCLD and the CLD in heart, liver, spleen, lung and kidney of mice.

The results of fig. 12A-D demonstrate that CLD achieves high targeting of lung cancer without GS5 surface functionalization, because COF vectors have high lung targeting functions themselves. After GS5 modification, the fluorescence intensity of RCLD group in lung is 1.9 times that of CLD group, and the RCLD group shows more efficient lung cancer targeting ability due to double targeting effect of COF carrier and GS 5. Furthermore, RCLD nanoparticles are distributed mainly in the lungs after intravenous injection, with fluorescence intensity in lung tissue 5.8 times that of the liver.

Evaluation of lung cancer treatment effect of two doxorubicin hydrochloride-loaded carrier compositions:

two doxorubicin hydrochloride-loaded carrier compositions treat a549 metastatic lung cancer: will be 1X 10 ⁶ The individual A549 cells were dispersed in 200. Mu.L PBS, and the tail vein was injected into BALB/c mice to construct a human lung cancer metastasis model. On day 7 after injection of a549 cells, mice were randomized into 6 groups, given PBS, free DOX 2.5 (dose 2.5 mg/kg), free LMWH, CLD (dose 1mg/kg of DOX), RCLD (dose 1mg/kg and 0.5mg/kg of DOX, respectively), once every 3 days, for 5 consecutive administrations. Mice were monitored daily for body weight changes throughout the course of the experiment. On day 32, mice were sacrificed, lung tissue was dissected out, the number of lung tumor metastasis nodules was counted, and a photograph was taken. Simultaneous H of lung tissue and other major organs &E staining analysis.

FIG. 13 shows the antitumor effects of CLD and RCLD in the A549 lung cancer model in example 3. (a) lung pictures collected from an a549 lung cancer mouse model; (B) a treatment regimen for RCLD; (C) Quantitative analysis of lung a549 metastatic nodule number (n=5); (D) H & E staining analysis of lung tissue. * p <0.05, < p <0.001, ns means no significant difference between the two groups.

The results in FIGS. 13A-D show that DOX-loaded CLD and RCLD groups exhibit a strong anti-tumor effect, not only because LMWH is capable of inhibiting migration and invasion of cancer cells, but DOX is also capable of directly killing tumor cells. It is worth mentioning that the RCLD group (DOX dose of 1 mg/kg) has the best therapeutic effect on lung cancer, and almost no metastatic nodules of the lung can be seen in the RCLD group (DOX dose of 1 mg/kg), and the targeting distribution and residence time of RCLD at tumor sites can be increased due to RGD target modification. Even though the number of pulmonary metastasis nodules in the DOX 2.5 group (DOX dose of 2.5 mg/kg) was similar to that in the RCLD 0.5 group (DOX dose of 0.5 mg/kg) (Panel C), the H & E staining results showed that the size of the pulmonary metastasis nodules in the RCLD 0.5 group was significantly smaller than in the DOX group (Panel D).

FIG. 14 shows the change in body weight of A549-loaded BALB/c mice in example 3. Wherein, the body weight of each group of mice except DOX 2.5 dose group is not obviously different. The mice in the DOX 2.5 dose group had decreased body weight, probably due to DOX toxicity.

Two doxorubicin hydrochloride-loaded carrier compositions treat pulmonary metastatic lung cancer of B16F10 melanoma: digesting B16F10 cells in exponential growth phase, centrifuging the cell suspension at 1000rpm for 3min, removing supernatant, adding sterile PBS into the lower layer cell to give a cell concentration of 2×10 ⁵ And each mL. 100 mu L of the cell suspension is sucked by an insulin syringe, and the tail vein is injected into a C57BL/6 mouse body to construct a mouse melanoma lung metastasis model. On day 2 of tumor cell inoculation, mice were randomly divided into 6 groups: PBS group, LMWH group, 2.5mg/kg DOX group, CLD group (DOX dose is 1 mg/kg), RCLD group (DOX dose is 1mg/kg and 0.5mg/kg respectively), and 200. Mu.L of the drug was administered by tail vein injection once every three days. Mice were kept normally after dosing, mice were weighed daily, animals were sacrificed five times after dosing, lung tissue was dissected out, photographed and lung transfer area was calculated using Image-Pro-Plus software.

FIG. 15 shows the therapeutic effect of RCLD and CLD in the B16F10 metastatic lung cancer model in example 3. (A) Lung pictures collected from B16F10 metastatic lung cancer mouse models; (B) a treatment regimen for RCLD; (C) area of B16F10 metastatic lung cancer (n=5). * P <0.01, p <0.001, ns represents no significant difference between the two groups.

The results in fig. 15 show that: the RCLD group (DOX dose of 1 mg/kg) can significantly inhibit lung metastasis of melanoma, and almost no lung metastasis nodule can be seen in the administration group, and the lung metastasis area is reduced by 70%, which results from the synergistic antitumor effect of LMWH and DOX.

The results in fig. 16 show that: free DOX showed significant cardiotoxicity, while major organs of the RCLD group did not undergo structural changes, indicating that multiple treatments given to RCLD did not cause acute toxicity or tissue necrosis of the organs, as RCLD targeted drug delivery platforms reduced dose and cardiotoxicity of DOX.

(6) Evaluation of in vivo safety of two doxorubicin-loaded carrier compositions: respectively to healthy male C57BL/6 mouse tailsPBS, free DOX (dose of 2.5 mg/kg), free LMWH, CLD (dose of DOX of 1 mg/kg), RCLD (dose of DOX of 1mg/kg and 0.5mg/kg, respectively) were administered intravenously, once every 3 days, and 5 times in succession. 24h after the last administration with EDTAK ₂ Whole blood was collected for anticoagulation, blood routine examination was performed, and serum was collected for biochemical analysis.

Fig. 17 shows the blood cell analysis and serum biochemical analysis (n=4) of the mice after five intravenous injection of RCLD and CLD in example 3. * p <0.05, < p <0.01, < p <0.001.

The results in fig. 17 show that there was no significant change in the white blood cell, red blood cell and platelet counts of the mice, indicating that the delivery system of the two doxorubicin-loaded carrier compositions has good biosafety.

Example 4

LMWH-GS5-COF carries different antitumor drugs: respectively weighing a proper amount of antitumor drugs such as paclitaxel, capecitabine, gemcitabine, carboplatin, oxaliplatin, gefitinib, topotecan and the like, adding a proper amount of solvent, carrying out ultrasonic treatment for 10min to dissolve the antitumor drugs, then respectively adding the LMWH-GS5-COF prepared in the example 2, wherein the molar ratio of the drugs to the LMWH-GS5-COF is 2:1, stirring at 400rpm for 24h at 37 ℃, and incubating for drug loading. After drug loading is completed, centrifuging at 4000rpm for 5min, washing off free drugs on the surface by using an incubation solvent, drying to obtain lower-layer drug loading LMWH-GS5-COF nano-particles, and measuring the drug loading by using a high performance liquid phase method, wherein the drug loading is shown in Table 1.

TABLE 1 drug loading rate of LMWH-GS5-COF loaded antitumor drugs

The carrier composition loaded with the anti-tumor drug in the embodiment has higher drug loading rate, stronger cytotoxicity in vitro and good anti-lung cancer efficacy in vivo.

Examples 5 to 18

Preparation of tumor targeting polypeptide grafted COF carrier: the COF and the functional polypeptide (RGD peptide, NGR peptide) in the equimolar ratio in example 1 were weighed at a molar ratio of 1:1, placed in a round bottom flask, added into a certain volume of first solvent (DMF, ACN), stirred uniformly, then added with a certain amount of activator a (DMAP, EDC, TEA), placed on a magnetic stirrer at 37 ℃ and stirred at 400rpm for 24 hours, so that the COF and the functional polypeptide were fully coupled. After the reaction, centrifuging at 4000rpm for 5min, washing with the same volume of the first solvent and pure water for 2 times respectively, and freeze-drying at-50 ℃ for 24h to obtain the functional polypeptide modified COF, wherein the specific preparation scheme is shown in Table 2.

Table 2 preparation of tumor targeting polypeptide grafted COF vectors

The functional polypeptide composition in the above examples is cubic and has a particle size of about 100-500nm. The mass percentage of the crosslinked cyclodextrin organic framework COF to the functional polypeptide measured by an HPLC method is 1:0.001-0.1.

Examples 19 to 49

Activation of COF and preparation of LMWH-COF were performed as shown in Table 3.

Activation of GS 5-COF: an amount of activated linker arm (N, N '-disuccinimide carbonate (DSC), N' -Carbonyldiimidazole (CDI), succinyl chloride) was weighed, sonicated in a third solvent (acetonitrile (ACN), formamide, dimethylformamide (DMF)), and added with GS5-COF powder in a molar ratio to an activation catalyst (triethylamine (TEA), pyridine, N-hydroxysuccinimide (NHS)), and reacted at a first temperature (25 ℃,40 ℃, 60 ℃) for a first time (6 h, 12h, 48 h).

Preparation of LMWH-COF: 300mg of LMWH-CYS is weighed, and a proper amount of fourth solvent (acetonitrile (ACN), dimethylformamide (DMF) and formamide) is added for dissolution, and the solution is dissolved under magnetic stirring to obtain an LMWH-CYS solution. The activated GS5-COF or COF solution was added to the LMWH-CYS solution and reacted at a second temperature (25 ℃,40 ℃, 60 ℃) for a second time (12 h, 24h, 48 h) to give a crude LMWH-COF product. In order to remove unreacted micromolecules and byproducts thereof, the crude product is dialyzed for three days by acetonitrile/water mixed solvent (4:1, v:v), and then is dialyzed for two days by pure water continuously, and is centrifuged and freeze-dried at-50 ℃ to obtain the LMWH-COF.

TABLE 3 activation of COF and preparation of LMWH-COF

The final product LMWH-COF in the above examples has a percentage of low molecular heparin LMWH of 0.1-1% as measured by toluidine blue method, and the LMWH-COF composition is in cubic form and has a particle size of about 100-500nm.

Example 50

(1) Topotecan hydrochloride loading

Preparation of topotecan hydrochloride-loaded cyclodextrin organic framework composition: 100mg of topotecan hydrochloride (TPT) is weighed and dissolved in 2.5mL of pure water, the solution is made by ultrasonic treatment for 10min, 34mg of cyclodextrin organic framework COF nano particles prepared in the example 1 are added, the molar ratio of the drug to the LMWH-GS5-COF nano particles is 2:1, the stirring is carried out for 12h at 37 ℃ in the dark at 300rpm, and the drug is incubated. After completion of drug loading, centrifugation at 4000rpm for 5min was carried out, and washing with pure water was carried out to remove free TPT, to obtain a lower layer topotecan hydrochloride-loaded double modified carrier composition (TPT@COF).

The drug loading of the drug is determined by ultraviolet spectrophotometry. An appropriate amount of TPT@COF was weighed and dissolved in 0.1M sodium hydroxide solution, the liquid was filtered through a 0.22 μm filter membrane and the absorbance was measured at 422nm, and the amount of TPT loaded was determined by the formula (2) of example 3. The results showed that the drug loading of TPT@COF was 12%.

Preparation of topotecan hydrochloride-loaded double modified vector composition: 20mg of TPT is weighed and dissolved in 2mL of pure water, ultrasonic treatment is carried out for 10min to dissolve the TPT, 34mg of LMWH-GS5-COF nanoparticle prepared in example 2 is added, the molar ratio of the drug to the LMWH-GS5-COF nanoparticle is 2:1, the drug is stirred for 12h at 25 ℃ in the dark at 300rpm, and the drug is incubated. After completion of drug loading, centrifugation at 4000rpm for 5min, washing with pure water to remove free TPT, a double modified carrier composition (RCLT) of the underlying topotecan hydrochloride was obtained.

The drug loading of the drug is determined by ultraviolet spectrophotometry. An appropriate amount of RCLT was weighed and dissolved in a 0.1M sodium hydroxide solution, the liquid was filtered through a 0.22 μm filter membrane, and the absorbance was measured at 422nm, and the TPT drug loading was measured by the formula (2) of example 3. The results indicated that the RCLT drug loading was 18%.

Preparation of a low molecular weight heparin-grafted carrier composition with topotecan hydrochloride free of GS 5: 20mg of TPT is weighed and dissolved in 2mL of pure water, ultrasonic treatment is carried out for 10min to dissolve the TPT, 34mg of LMWH-COF nano particles prepared in the example 2 are respectively added, the molar ratio of the drug to the LMWH-COF is 1:2, the mixture is stirred for 14h at 25 ℃ in the dark at 300rpm, and the drug is incubated. After completion of drug loading, centrifugation at 4000rpm for 5min, washing with pure water to remove free TPT, a low molecular heparin-grafted carrier Composition (CLT) without RGD was obtained with the underlying topotecan hydrochloride.

The drug loading of the drug is determined by ultraviolet spectrophotometry. An appropriate amount of CLT was weighed and dissolved in 0.1M sodium hydroxide solution, and the liquid was filtered through a 0.22 μm filter membrane, and the absorbance was measured at 422nm, whereby the TPT drug loading was measured by the formula (2) of example 3. The results indicated that the CLT drug loading was 17%.

(2) In vitro release investigation of topotecan-loaded vectors

In vitro release of topotecan hydrochloride-loaded cyclodextrin organic framework composition: a quantity of tpt@cof nanoparticles was dispersed in 40mL of phosphate buffer (PBS, ph=7.4) and incubated at 50rpm and 37 ℃ for different times in a dark environment, 1mL of supernatant was collected at predetermined time points (0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 h) and an equivalent volume of release medium was replenished. The drug content released by the nanoparticles was assessed by using the high performance liquid phase method (n=3).

Drug release behavior of topotecan hydrochloride-loaded double-modified carrier composition under conditions of different concentrations of reduced Glutathione (GSH): a quantity of RCLT nanoparticles was dispersed in 40mL of PBS (ph=7.4) at different GSH concentrations (0, 1, 10 mM) and incubated at 50rpm and 37 ℃ for different times in a dark environment, 1mL of supernatant was collected at predetermined time points (0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 h) and supplemented with an equivalent volume of release medium. The drug content released by the nanoparticles was assessed by using the high performance liquid phase method (n=3).

FIG. 18 is an in vitro release evaluation of TPT@COF and RCLT in example 50, showing that the early release of TPT@COF is faster and about 50% of the drug is released within 12 hours, which is probably caused by burst release of TPT adsorbed on the outer surface of the COF; the later period has better slow release effect, and is favorable for maintaining the drug concentration in the treatment window. Compared with 48h release of RCLT in PBS release medium without GSH, RCLT nanoparticles release 50% of drug in 12h faster in 1mM GSH release medium, and 62% of drug in 48 h; TPT can be rapidly released in 10mM GSH release medium, 60% of the drug is released in the first 1h, and 80% of the drug is released in 12h, which is attributed to the fact that high concentration GSH cuts off the connection between heparin and COF, so that TPT release is accelerated, and RCLT has obvious redox responsive release characteristics.

(3) Cytotoxicity evaluation of topotecan-loaded carrier compositions

Cytotoxicity of the above topotecan-loaded carrier composition nanoparticles (TPT@COF, RCLT and CLT) on B16F10 tumor cells was examined by using the CCK-8 method. B16F10 cells were cultured in DMEM medium (containing 10% fetal bovine serum) at 37 ℃ with 5% CO ₂ Culturing in a constant temperature and humidity incubator. Selecting cells in logarithmic phase for experiment, the cells in logarithmic phase were grown at 2×10 ⁴ The density of each well was inoculated in 96-well plates, and the final volume of each well was 200. Mu.L, and cultured for 24 hours. Removing culture solution, adding TPT, TPT@COF, RCLT or CLT respectively, setting total concentration to 8 different concentrations, and setting final concentration of TPT to 0.01, 0.1, 1, 2, 5, 10, 20 and 50μg.mL according to the drug loading conversion of TPT ^-1 . A blank (containing only culture) and a control (cells and culture) were set at the same time. After a further incubation period of 24 hours in the incubator, 15. Mu.L of CCK-8 solution was added to each well and incubation was continued for 1.5 hours at 37 ℃. The culture was terminated, absorbance (a) at 450nm was measured using a microplate reader, and cell viability (n=6) was calculated according to formula (1) of example 2.

Fig. 19 is an evaluation of B16F10 cytotoxicity of topotecan loaded carrier composition of example 50, showing that: TPT@COF, RCLT and CLT have stronger cytotoxicity, and can obviously inhibit the growth of tumor cells in vitro.

(4) Topotecan hydrochloride-loaded carrier composition for treating lung metastatic lung cancer of B16F10 melanoma

Tumor inhibition evaluation of topotecan hydrochloride-loaded cyclodextrin organic framework composition: digesting B16F10 cells in exponential growth phase, centrifuging the cell suspension at 1000rpm for 3min, removing supernatant, adding sterile PBS into the lower layer cell to give a cell concentration of 2×10 ⁵ And each mL. 100 mu L of the cell suspension is sucked by an insulin syringe, and the tail vein is injected into a C57BL/6 mouse body to construct a mouse melanoma lung metastasis model. On day 3 of tumor cell inoculation, mice were randomly divided into 6 groups: physiological saline group (Normal saline), 5mg/kg TPT group, TPT@COF group (TPT dose is 2.5mg/kg and 1mg/kg respectively), and 200. Mu.L of the drug was administered by tail vein injection once every three days for 5 times. Mice were kept normally after dosing, mice were weighed daily, animals were sacrificed after 20 th seed tumor, lung tissue was dissected out, photographed and lung transfer area was calculated using Image-Pro-Plus software.

Tumor inhibition evaluation of topotecan hcl-loaded double modified vector composition: digesting B16F10 cells in exponential growth phase, centrifuging the cell suspension at 1000rpm for 3min, removing supernatant, adding sterile PBS into the lower layer cell to give a cell concentration of 2×10 ⁵ And each mL. 100 mu L of the cell suspension is sucked by an insulin syringe, and the tail vein is injected into a C57BL/6 mouse body to construct a mouse melanoma lung metastasis model. On day 3 of tumor cell inoculation, mice were randomly divided into 6 groups: physiological saline group (Normal saline), LMWH group, 5mg/kg TPT group, RLCT group (TPT dose is 2.5mg/kg and 1mg/kg respectively), and 200. Mu.L of the drug was administered by tail vein injection once every three days for 5 times. Mice were kept normally after dosing, mice were weighed daily, animals were sacrificed after 20 th seed tumor, lung tissue was dissected out, photographed and lung transfer area was calculated using Image-Pro-Plus software.

FIG. 20 shows the therapeutic effects of TPT@COF and RCLT in B16F10 metastatic lung cancer model in example 50. (A) Lung pictures collected in RCLT treated B16F10 metastatic lung cancer mouse model; (B) treatment regimen of tpt@cof and RCLT; (C) area of RCLT treated B16F10 metastatic lung cancer; (D) Lung pictures collected in a tpt@cof treated B16F10 metastatic lung cancer mouse model; (E) Area of B16F10 metastatic lung cancer treated with tpt@cof (n=5). * P <0.01, p <0.001, ns represents no significant difference between the two groups.

The results of fig. 20A and 20C show that: RCLT 2.5 group (TPT dose of 2.5 mg/kg) can significantly inhibit lung metastasis of melanoma, and almost no lung metastasis nodule can be seen in administration group, and lung metastasis area of control group is reduced by 67%, resulting from synergistic antitumor effect of LMWH and TPT. The treatment effect of the RCLT 1 group (TPT dose of 1 mg/kg) is equivalent to that of the free drug TPT group.

The results of fig. 20D and 20E show that: the treatment effect of the TPT@COF1 group (the TPT dosage is 1 mg/kg) is equivalent to that of the free medicine TPT 5mg/kg group. Whereas the tpt@cof2.5 group (TPT dose of 2.5 mg/kg) significantly inhibited lung metastasis of melanoma, the lung metastasis area of the control group was reduced by 52%, mainly due to lung targeting of COF.

FIG. 21 shows the body weight change of C57BL/6 mice treated with TPT@COF or RCLT in B16F 10. (A) Weight change in RCLT-treated C57BL/6 mice bearing B16F 10; (B) Weight change in the C57BL/6 mice treated with TPT@COF in B16F 10.

Figure 21 shows that the mice body weight changes were not evident during tpt@cof or RCLT treatment, indicating that the treatment regimen was better safe.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. An LMWH-polypeptide-COF composition, comprising the following components:

1) Crosslinking cyclodextrin organic frameworks COFs;

3) A low molecular heparin LMWH covalently linked to the cross-linked cyclodextrin organic backbone COF;

the polypeptide is selected from the group consisting of: integrin binding peptide (RGD), CD13 metallopeptidase binding peptide (NGR), or a combination thereof;

The low molecular heparin LMWH and the crosslinked cyclodextrin organic framework COF are connected as follows:

a2 Covalent connection is carried out between the amino group at one end of the cystamine modified low molecular heparin LMWH obtained in the step a 1) and the hydroxyl group on the surface of the crosslinked cyclodextrin organic framework COF;

the mass ratio of the crosslinked cyclodextrin organic framework COF to the polypeptide is 1:0.001-0.1;

the mass ratio of the cross-linked cyclodextrin organic framework COF to the low molecular heparin LMWH is 1:0.001-0.05.

2. The composition of claim 1, wherein the polypeptide is an integrin binding peptide (RGD).

3. The composition of claim 1, wherein the low molecular heparin LMWH is selected from the group consisting of: low molecular heparin, low molecular heparin sodium, low molecular heparin calcium, or a combination thereof.

4. The composition of claim 1, wherein the mass ratio of the cross-linked cyclodextrin organic backbone COF to the polypeptide is 1:0.01-0.1; and/or

The mass ratio of the cross-linked cyclodextrin organic framework COF to the low molecular heparin LMWH is 1:0.005-0.02.

5. A method of preparing the composition of claim 1, comprising the steps of:

4) Preparing an LMWH-polypeptide-COF composition comprising the steps of: reacting the cystamine modified low molecular heparin LMWH obtained in step 2) and the activated polypeptide modified cross-linked cyclodextrin organic backbone COF obtained in step 3) in a fourth solvent at a second temperature for a second time to obtain the composition of claim 1.

6. The method of claim 5, wherein the method has one or more features selected from the group consisting of:

5) In the cystamine modified low-molecular heparin LMWH, the feeding mass ratio of cystamine to the low-molecular heparin LMWH is 1:10-20;

9) The molar ratio of the polypeptide modified crosslinked cyclodextrin organic framework COF, the activated connecting arm and the activated catalyst is 1:1-10:1-7;

10 The first temperature is 10-100 ℃;

11 The first time is 3-50h;

13 The second temperature is 10-100 ℃;

14 The second time is 8-60h.

7. A pharmaceutically active ingredient, wherein said pharmaceutically active ingredient comprises:

(1) The LMWH-polypeptide-COF composition of claim 1 as a pharmaceutical carrier; and

8. A pharmaceutical composition comprising:

(a) A pharmaceutically active ingredient according to claim 7; and

(b) A pharmaceutically acceptable carrier.

9. A process for the preparation of a pharmaceutically active ingredient as claimed in claim 7, comprising the steps of: (i) The LMWH-polypeptide-COF composition of claim 1 and an antitumor drug are mixed as a drug carrier, and the antitumor drug is carried on the drug carrier, thereby obtaining the pharmaceutically active ingredient of claim 7.

10. Use of an LMWH-polypeptide-COF composition according to claim 1 and/or a pharmaceutically active ingredient according to claim 7 for the preparation of a medicament for the prevention and/or treatment of tumors or a medicament for targeting the lung.