CN111494318B

CN111494318B - Tumor targeting and reduction sensitive composite micelle and preparation method and application thereof

Info

Publication number: CN111494318B
Application number: CN202010347892.8A
Authority: CN
Inventors: 华小黎; 于丽秀; 杨玉; 廖婧; 黄璞; 陈东生
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2020-04-28
Filing date: 2020-04-28
Publication date: 2022-04-12
Anticipated expiration: 2040-04-28
Also published as: CN111494318A

Abstract

The invention discloses a tumor targeting and reduction sensitive composite micelle, a preparation method and an application thereof, wherein the composite micelle is composed of three block polymers according to a certain proportion, and comprises polyethylene glycol-stearylamine (mPEG-SS-C) containing disulfide bonds₁₈) Polyethylene glycol-stearylamine (TIP-PEG-C) containing cell-penetrating peptide₁₈) And polyethylene glycol-stearylamine (cRGD-PEG-C) containing tumor targeting peptide₁₈). The micelle inner core can entrap hydrophobic anti-tumor drug tripterine (CEL), targets ovarian cancer cells through the cRGD peptide on the micelle surface, is efficiently taken up by the ovarian cancer cells under the mediation effect of the TIP peptide, and is broken in a reduction environment in cells to quickly release drugs. The composite micelle provided by the invention can improve the water solubility of CEL, quickly release CEL after target entering tumor cells, improve the killing power of the tumor cells and reduce the toxic and side effects of CEL.

Description

Tumor targeting and reduction sensitive composite micelle and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a tumor targeting and reduction sensitive composite micelle and a preparation method and application thereof.

Background

Ovarian cancer is the most common malignancy in women over the age of 40, except for breast cancer. In recent years, the incidence of ovarian cancer has increased year by year. Mortality rates continue to rise as most patients are diagnosed at a middle or late stage. At present, the main clinical treatment method of ovarian cancer is surgery and chemotherapy, and the combined use of platinum and paclitaxel is the standard chemotherapy scheme of ovarian cancer, so that although the survival time of a patient can be prolonged, the recurrence rate is high, the drug resistance problem is serious, and the 5-year survival rate is about 30%. Therefore, it is urgent to find a more effective treatment method for reversing the drug resistance of ovarian cancer.

Tripterine (CEL) is a pentacyclic triterpene monomer compound extracted from root bark of plant of Celastraceae, Tripterygium and Celastrus. Tripterine has wide pharmacological activity, and can exert antiinflammatory, antioxidant, antirheumatic and antitumor effects by regulating multiple molecular targets such as INF-a, NF-kappa B, COX-2, VEGF, Akt, etc. The erygiene can be used as a natural antitumor drug to inhibit the growth of various tumor cells such as ovarian cancer, prostatic cancer, lung cancer, liver cancer, leukemia, glioma and the like. However, tripterine has poor water solubility and low stability, and has various toxic and side effects, such as cardiotoxicity, hepatotoxicity, neurotoxicity, hematological toxicity and the like, which seriously affect the clinical application and curative effect.

For example, patent application publication No. CN110623963A discloses a pharmaceutical composition for treating ovarian cancer and its application. The pharmaceutical composition contains kaempferol and tripterine as active ingredients. In the invention, by combining kaempferol and tripterine, kaempferol can remarkably improve the anti-tumor curative effect of tripterine by blocking cell cycle, inducing apoptosis, inhibiting cell proliferation and migration and the like, and shows remarkable synergistic effect in inhibiting the growth of ovarian cancer cells. However, the problems of poor water solubility, low stability and various toxic and side effects of tripterine are not considered and solved.

TRAF6 belongs to the family of TRAFs. TRAF6 is unique in signal transduction, not only can transduce signals associated with the TNF superfamily, but also can act as an important adaptor protein for the Toll-like receptor (TLR)/interleukin 1 receptor IL-1R (TIR) superfamily. Many studies now find that the abnormal expression and activation of TRAF6 protein are present in various tumor tissues such as lung cancer, multiple myeloma, ovarian cancer, colon cancer, glioma and breast cancer. In ovarian cancer, the expression level of TRAF6 was significantly higher than in paracancerous normal tissues. TRAF6 expression is an important indicator of ovarian cancer patient prognosis, and the five-year survival rate of positively expressed patients is significantly reduced compared to TRAF 6-negatively expressed patients. If the expression level of TRAF6 in cancer cells could be reduced, it would play a key role in tumor suppression. Research shows that the core motif (RKIPTEDEY, TIP peptide) of the TRAF6 decoy peptide can be specifically targeted and combined with TRAF6, and inhibit the NF-kB signal pathway at the downstream of the TRAF6, thereby promoting tumor cell apoptosis, inhibiting cell proliferation and migration and the like. However, polypeptides often have the disadvantages of immunogenicity, short half-life and the like in vivo, and the PEGylation of polypeptides can effectively increase the half-life and reduce the immunogenicity due to the surface shielding effect. In addition, the polypeptide is introduced into the polymer and forms a nano micelle system, which is also beneficial to the enrichment of the polypeptide at the tumor site.

The polymer containing hydrophilic groups and hydrophobic groups can form micelle with a core-shell structure in an aqueous solution, and the hydrophobic core can wrap insoluble drugs, so that the damage of enzyme is avoided, and the bioavailability of the drugs is improved; the hydrophilic shell can avoid phagocytosis by mononuclear phagocyte, and prolong the in vivo circulation time of the medicine. At present, some micelles are developed to improve the water solubility of tripterine, and Li and the like uses polyethylene glycol-b-polycaprolactone copolymer (PEG-b-PC) to load tripterine (NP), SO that the water solubility of free drugs can be improved, and the growth of human retinoblastoma cells SO-Rb50 can be inhibited and the apoptosis of the human retinoblastoma cells is induced. In the prior art, PEG-PCL is used as a carrier to prepare a drug-loaded micelle nano-tripterine with a spherical structure, and the nano-micelle can not only effectively increase the water solubility of the tripterine, but also effectively reduce the body fat and the weight of a diet-induced obese mouse. After 21 days of equivalent dose treatment, the free drug caused anal inflammation in animals, while the nanomicelle treated group did not show any sign of inflammation.

Patent publication No. CN107041875B discloses a silk fibroin nanoparticle, which comprises silk fibroin and an active drug, wherein the silk fibroin is loaded with the active drug, and the active drug is selected from triptolide or erygium wilfordii. The silk protein nanoparticles improve the water solubility of triptolide and tripterine, and can effectively resist the proliferation of cancer cells. The silk protein nanoparticles carrying Triptolide (TPL) or tripterine (CL) improve the water solubility of triptolide and tripterine, and promote the TPL and CL to be passively accumulated in cancer tissues. However, the invention promotes the triptolide and the tripterine to be passively accumulated in the cancer tissue by improving the water solubility of the cancer treatment drugs, but does not realize the active targeting accumulation in the cancer tissue, and cannot well inhibit the growth of the cancer cells. Secondly, the silk fibroin of the silk fibroin nanoparticle is loaded with active drugs, but does not contain drug ingredients which can promote the release of the active drugs in ovarian cancer cells and inhibit the expression of TRAF 6.

The integrin family plays an important role in the signaling, proliferation, apoptosis, invasion, metastasis and vascular growth of tumor cells, and is highly expressed on various malignant tumor cells including ovarian cancer. The linear or annular polypeptide containing the RGD sequence is a recognition site of various integrins, and is bonded to the surface of a micelle, so that the uptake of targeted micelles by ovarian cancer cells can be obviously improved. If a stimulation sensitive site is introduced into the amphiphilic copolymer, the micelle system can rapidly respond and release the drug in tumor cells. The microenvironment in tumor cells is significantly different from that of normal cells, especially the pH value and the concentration of reduced Glutathione (GSH). The pH of the microenvironment around the tumor is changed in a gradient manner, the pH of the surrounding normal tissues is 7.4, the extracellular pH of local tumor cells is 6.8, the pH of intracellular inclusion bodies is 5.0-6.5, and researchers develop various pH responsive micelles by introducing acetal, orthoester, hydrazine bonds and the like into the polymer. In addition, the concentration of GSH in tumor cells (0.5-10mmol/L) is more than several hundred times of that in cells outside the cells (2-20. mu. mol/L). Disulfide bonds are very stable in the normal physiological environment of the human body, but can be cleaved in the presence of high intracellular concentrations of gSH.

The invention aims to prepare three polymers, namely methoxy polyethylene glycol-SS-stearamide (mPEG-SS-C) with reduction sensitivity₁₈) Polyethylene glycol stearamide (cRGD-PEG-C) coupled with tumor targeting group cRGD peptide₁₈) Polyethylene bis to which TIP peptide is coupledAlcohol stearamide (TIP-PEG-C)₁₈) The drug-loaded micelle cRGD/TIP/mPEG-SS-C with dual functions of tumor targeting property and reduction sensitivity is prepared by mixing the three polymers in a certain proportion by taking erygigo erythrogenin as an anti-tumor drug₁₈and/CEL. Through the active targeting effect mediated by cRGD polypeptide, the drug-loaded micelle can effectively identify and enter ovarian cancer cells, and is caused by mPEG-SS-C under the intracellular reductive condition₁₈The entrapped medicine tripterine, TIP-PEG-C is quickly released by the broken string₁₈In a single molecular state, the polypeptide is combined with TRAF6 protein in cell sap and inhibits the expression of downstream protein, thereby promoting cell apoptosis, inhibiting cell proliferation and synergistically improving the anti-tumor curative effect. So far, no reports about the preparation of tripterine-loaded composite micelles by using three polymers as carrier materials and the application of the tripterine-loaded composite micelles in resisting ovarian cancer are found.

Disclosure of Invention

Technical problem to be solved

The invention aims to provide a tumor targeting and reduction sensitive composite micelle and a preparation method and application thereof, and aims to solve the problems that the cancer treatment medicament in the prior art cannot realize active targeting accumulation in cancer tissues, cannot well inhibit the growth of cancer cells, and does not contain a medicinal component capable of promoting the release of an active medicament in the cancer cells and inhibiting the expression of TRAF 6.

(II) technical scheme

In order to solve the problems that the cancer treatment medicine in the prior art can not realize active targeting accumulation in cancer tissues, can not well inhibit the growth of cancer cells and does not contain a medicine component capable of promoting the release of an active medicine in the cancer cells and inhibiting the expression of TRAF6, the invention provides the following technical scheme:

a tumor targeting and reduction sensitive composite micelle, which comprises the following components:

1) polymer mPEG-SS-C₁₈；

2) Polymer cRGD-PEG-C₁₈；

3) Polymer TIP-PEG-C₁₈。

A preparation method of tumor targeting and reduction sensitive composite micelle comprises the following steps:

step 1, synthesizing a polymer mPEG-SS-C₁₈；

Step 2, synthesizing polymer cRGD-PEG-C₁₈、TIP-PEG-C₁₈；

Step 3, mixing the polymer mPEG-SS-C₁₈、cRGD-PEG-C₁₈、TIP-PEG-C₁₈And the drug-loaded micelle is combined with eryciceline (CEL) to prepare the drug-loaded micelle with tumor targeting and reduction sensitivity.

Preferably, the step 1 specifically comprises:

step 11, weighing 3,3 '-dithiodipropionic acid, N, N' -Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP), dissolving in an organic solvent, stirring for reaction, filtering, and keeping a filtrate;

step 12, weighing stearylamine (ODA), dissolving in an organic solvent, dripping the filtrate obtained in the step 11, stirring for reaction, and purifying to obtain an intermediate product 1;

step 13, weighing the intermediate product 1, dissolving the intermediate product 1 and N, N' -Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP) in an organic solvent, stirring for reaction, filtering, and keeping a filtrate;

step 14, weighing polyethylene glycol (mPEG), dissolving in an organic solvent, dripping the filtrate obtained in the step 13 into the organic solvent, stirring for reaction, filtering after the reaction is finished, concentrating the filtrate, dripping the filtrate into a precipitator to obtain a white solid, collecting the precipitate, drying, adding water to dissolve the white solid, transferring the white solid into a dialysis bag with the molecular weight cutoff of 2000, putting the dialysis bag into distilled water for dialysis, and freeze-drying the solution to obtain mPEG-SS-C₁₈A polymer.

Preferably, the step 2 specifically comprises:

step 21, weighing stearylamine (ODA) and maleimide-polyethylene glycol-succinimide succinimidyl ester (Mal-PEG-NHS) to prepare two solutions respectively, dropwise adding one solution into the other solution, adding Triethylamine (TEA), stirring for reaction, spinning off the reaction solution after the reaction is finished, and addingTransferring into dialysis bag with cut-off molecular weight of 2000, dialyzing in distilled water, and lyophilizing to obtain Mal-PEG-C₁₈A polymer;

step 22, weighing cRGD polypeptide and Mal-PEG-C₁₈Dissolving polymer in distilled water, stirring for reaction, transferring reaction liquid into dialysis bag with cut-off molecular weight of 2000, dialyzing the dialysis bag in distilled water, and lyophilizing to obtain cRGD-PEG-C₁₈A polymer;

step 23, weighing TIP peptide and Mal-PEG-C₁₈Dissolving polymer in distilled water, stirring for reaction, transferring reaction solution into dialysis bag with cut-off molecular weight of 2000, dialyzing in distilled water, and lyophilizing to obtain TIP-PEG-C₁₈A polymer.

Preferably, the step 3 specifically comprises: preparing medicine-carrying micelle by thin film-hydration method, and mixing polymer mPEG-SS-C₁₈、cRGD-PEG-C₁₈、TIP-PEG-C₁₈And combining with tripterine (CEL), dissolving with methanol, spin-drying, and hydrating with distilled water to obtain the tumor-targeting and reduction-sensitive drug-loaded micelle.

Preferably, the organic solvent added in step 1 is Tetrahydrofuran (THF) or CH₂Cl₂。

Preferably, the precipitating agent added in the step 1 is cold diethyl ether or n-hexane.

An application of tumor-targeting and reduction-sensitive composite micelle in treating or preventing ovarian cancer, lung cancer, multiple myeloma, ovarian cancer, colon cancer, glioma and breast cancer is disclosed.

(III) advantageous effects

Compared with the prior art, the invention provides a tumor targeting and reduction sensitive composite micelle, a preparation method and an application thereof, and the invention has the following beneficial effects:

1. the composite micelle can encapsulate tripterine CEL serving as a water-insoluble anti-tumor drug, and the targeting peptide cRGD on the surface of the micelle has alpha overexpressed with the surface of ovarian cancer cells_vβ₅The ability of integrins to bind specifically. By regulating cRGD-PEG-C₁₈The composite micelle with the optimal targeting performance can be obtained.

2. The composite micelle contains a polymer mPEG-SS-C₁₈、TIP-PEG-C₁₈The composite micelle enters ovarian cancer cells and is subjected to intracellular reductive condition due to mPEG-SS-C₁₈To rapidly release the encapsulated drug CEL, TIP-PEG-C₁₈In the cell sap in a monomolecular state, the TIP peptide can be combined with TRAF6 protein and inhibit the expression of downstream protein, thereby promoting apoptosis and inhibiting cell proliferation and synergistically improving the anti-ovarian cancer curative effect.

Drawings

FIG. 1 is polymer mPEG-C₁₈Nuclear magnetic spectrum of (a);

FIG. 2 is polymer mPEG-SS-C₁₈Nuclear magnetic spectrum of (a);

FIG. 3 shows the polypeptide cRGD and the polymer cRGD-PEG-C₁₈Nuclear magnetic spectrum of (a);

FIG. 4 is a polypeptide TIP and a polymer TIP-PEG-C₁₈Is/are as follows¹An HnmR map;

FIG. 5 is blank micelle mPEG-C₁₈(A)、mPEG-SS-C₁₈(B)、cRGD/TIP/mPEG-SS-C₁₈(C) CEL micelle loaded mPEG-C₁₈/CEL(D)、mPEG-SS-C₁₈/CEL(E)、 cRGD/TIP/mPEG-SS-C₁₈Transmission electron micrographs of/CEL (F);

FIG. 6 shows that DOX micelle loaded cRGD/TIP/mPEG-SS-C₁₈(DOX) and mPEG-C₁₈The drug release profile of DOX in simulated intracellular environment;

FIG. 7 is a graph of cytotoxicity of three blank micelles (A), free CEL and three drug-loaded micelles (B) on ovarian cancer cells A2780.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A tumor targeting and reduction sensitive composite micelle is prepared by mixing tripterine as antitumor drug with three polymers at a certain ratio to obtain reduction sensitive methoxypolyethylene glycol-SS-stearamide (mPEG-SS-C)₁₈) Polyethylene glycol stearamide (cRGD-PEG-C) coupled with tumor targeting group cRGD peptide₁₈) Polyethylene glycol stearamide (TIP-PEG-C) coupled with TIP peptide₁₈) Can prepare the difunctional drug-loaded micelle cRGD/TIP/mPEG-SS-C with tumor targeting and reduction sensitivity₁₈and/CEL. Through the active targeting effect mediated by cRGD polypeptide, the drug-loaded micelle can effectively identify and enter ovarian cancer cells, and is caused by mPEG-SS-C under the intracellular reductive condition₁₈To rapidly release the encapsulated drug CEL, TIP-PEG-C₁₈The single molecule is combined with TRAF6 protein in the cell sap and inhibits the expression of downstream protein, thereby promoting the apoptosis and inhibiting the cell proliferation, and synergistically improving the anti-ovarian cancer curative effect.

The preparation method of the composite micelle with tumor targeting and reduction sensitivity comprises the following steps:

1. preparation of amphiphilic Polymer:

1.1 mPEG-C₁₈synthesis of (2)

Weighing stearyl amine (C)₁₈-NH₂) Dissolving in anhydrous dichloromethane (CH)₂Cl₂) In the solution with the concentration of 0.01-100 mmol/L, mPEG-NHS is further weighed and dissolved in anhydrous CH₂Cl₂And mixing the two solutions with the solution concentration of 0.01-100 mmol/L, and stirring for reacting for 1-48 h. After the reaction is finished, the reaction liquid is dripped into a precipitator to obtain white precipitate. Repeatedly re-precipitating and purifying the precipitate by using a precipitator to obtain mPEG-C₁₈。

1.2 mPEG-SS-C₁₈Synthesis of (2)

Weighing 3,3 '-dithiodipropionic acid (COOH-SS-COOH), N, N' -Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP), dissolving in Tetrahydrofuran (THF),the solution with the concentration of 0.001-100 mmol/L is stirred and reacted for 1-48 h. After the reaction, the solid in the reaction solution was removed by filtration, and the filtrate was reserved for further use. Weighing C₁₈-NH₂Dissolved in CH₂Cl₂And dropwise adding the solution into the filtrate, and stirring for reaction for 1-48 hours to obtain a reaction solution 1. Weighing DCC and DMAP, dissolving the DCC and DMAP in the reaction solution 1, stirring for reaction for 1-48 h, filtering, and keeping the filtrate. Dissolving methoxypolyethylene glycol (mPEG-OH) in CH₂Cl₂And (3) dropwise adding the solution into the filtrate obtained in the previous step, and stirring for reacting for 1-48 h, wherein the solution concentration is 0.001-100 mmol/L. After the reaction is finished, the reaction solution is concentrated and then is dripped into a precipitator to obtain white precipitate. Repeatedly re-precipitating and purifying the precipitate by using a precipitator to obtain mPEG-SS-C₁₈。

1.3 cRGD-PEG-C₁₈、TIP-PEG-C₁₈Synthesis of (2)

1.3.1 Mal-PEG-C₁₈Synthesis of (2)

Weighing C₁₈-NH₂Dissolved in CH₂Cl₂In the solution with the concentration of 0.001-100 mmol/L, maleimide polyethylene glycol succinimide ester (Mal-PEG-NHS) is dissolved in CHCl₂And mixing the two solutions, adding a small amount of Triethylamine (TEA) into the mixture after the two solutions are mixed, and stirring and reacting for 1-48 hours under the protection of nitrogen. After the reaction is finished, the reaction solution is concentrated and then is dripped into a precipitator to obtain white precipitate. Repeatedly re-precipitating and purifying the precipitate by using a precipitator to obtain Mal-PEG-C₁₈。

1.3.2 cRGD-PEG-C₁₈Synthesis of (2)

Weighing cRGD, dissolving in distilled water, and adding Mal-PEG-C₁₈Dissolving the mixture in 2mL of distilled water, mixing, and reacting at room temperature for 1-48 h under the protection of nitrogen. And transferring the solution into a dialysis bag with the molecular weight cutoff of 2000 after the reaction is finished, and then putting the dialysis bag into distilled water for dialysis for 1-48 h. Filtering, and freeze-drying to obtain cRGD-PEG-C₁₈。

1.3.3 TIP-PEG-C₁₈Synthesis of (2)

The preparation method is the same as above, and the polymer TIP-PEG-C can be obtained by replacing cRGD with TIP₁₈。

2. Preparation of blank micelle:

weighing mPEG-SS-C according to proportion₁₈、cRGD-PEG-C₁₈、TIP-PEG-C₁₈Dissolving the three polymers in 2mL of distilled water, stirring in the dark, and freeze-drying to obtain the bifunctional blank micelle cRGD/TIP/mPEG-SS-C with tumor targeting property and reduction sensitivity₁₈. Wherein mPEG-SS-C₁₈The proportion of the cRGD-PEG-C is 50 percent to 100 percent₁₈The proportion of the TIP-PEG-C is 1 to 25 percent₁₈The proportion of the active ingredients is 1 to 25 percent.

Respectively using 10mg of mPEG-C₁₈Or 10mg of mPEG-SS-C₁₈The mixture of the three polymers is replaced, and the nonfunctional blank micelle mPEG-C without tumor targeting and reduction sensitivity is prepared by the same method₁₈And monofunctional blank micelle mPEG-SS-C with only reduction sensitivity₁₈。

3. And (3) determining the morphology, the particle size and the distribution of blank micelles:

the morphology of the drug-loaded micelle was observed with a Transmission Electron Microscope (TEM), and the particle size and distribution thereof were measured with a light scattering particle size analyzer.

4. Preparing a drug-loaded micelle:

weighing mPEG-SS-C according to proportion₁₈、cRGD-PEG-C₁₈、TIP-PEG-C₁₈Three polymers, 10mg total dissolved in 2mL of anhydrous methanol, where mPEG-SS-C₁₈The proportion of the cRGD-PEG-C is 50 percent to 100 percent₁₈The proportion of the TIP-PEG-C is 1 to 25 percent₁₈The proportion of the active ingredients is 1 to 25 percent. Weighing CEL, dissolving in absolute methanol, and preparing into a solution with the concentration of 0.1-100 mg/L. Mixing 300 mu LCEL methanol solution with the polymer methanol solution, spin-drying, adding 2mL of distilled water, hydrating, shaking, centrifuging at 4000rpm for 15min, collecting supernatant, and freeze-drying to obtain the targeted and reduction-sensitive bifunctional drug-loaded micelle cRGD/TIP/mPEG-SS-C₁₈/CEL。

Respectively using 10mg of mPEG-C₁₈Or 10mg of mPEG-SS-C₁₈The mixture of the three polymers is replaced, and the nonfunctional drug-loaded micelle mPEG-C without tumor targeting and reduction sensitivity is prepared by the same method₁₈CEL, and monofunctional drug-loaded micelle mPEG-SS-C with only reduction sensitivity₁₈/CEL。

5. Determining the morphology, the particle size and the distribution of the drug-loaded micelle:

Example 2

The application of the tumor-targeting and reduction-sensitive composite micelle is specifically used for treating or preventing ovarian cancer, lung cancer, multiple myeloma, ovarian cancer, colon cancer, glioma and breast cancer, and the experimental results are as follows:

1. in vitro drug release studies of drug loaded micelles:

because unsaturated double bonds on A ring and B ring in the CEL structure are easy to generate Michael addition reaction with sulfydryl on DTT, and Dithiothreitol (DTT) is contained in a drug release medium simulating intracellular environment, in an in-vitro drug release link, adriamycin (DOX) which does not react with DTT is selected as a model drug to prepare two kinds of micelles loading DOX, namely nonfunctional micelle mPEG-C₁₈/DOX, dual-functional micelle cRGD/TIP/mPEG-SS-C₁₈DOX, and the drug loading was determined. The PBS solution containing 10mM DTT simulates the intracellular environment, and the controlled release performance of the drugs of the two drug-loaded micelles in the simulated intracellular environment is examined.

The results show that in DTT-containing drug delivery media, mPEG-C is reacted with₁₈cRGD/TIP/mPEG-SS-C compared with DOX₁₈The cumulative amount of drug released was higher for/DOX. This is because DTT breaks-SS-in the reduction-sensitive polymer, resulting in disintegration of the micelle structure and promotion of drug release. The result proves that the bifunctional composite micelle can realize the destruction of the micelle structure and quickly release the medicine under the stimulation of the reduction environment in the cell.

2. Cytotoxicity of blank micelles: detection of mPEG-C by MMT method₁₈、mPEG-SS-C₁₈、 cRGD/TIP/mPEG-SS-C₁₈Cytotoxicity of three blank micelles on ovarian cancer cells a 2780. The results show that the toxicity of the three blank micelles to A2780 cells is very high in the range of the investigated concentrationLow, indicating that the three blank micelles have good biological safety as drug carriers.

3. The antitumor activity of the free drug and three drug-loaded micelles is studied: detection of free CEL and mPEG-C by MMT₁₈/CEL、mPEG-SS-C₁₈/CEL、cRGD/TIP/mPEG-SS-C₁₈The three drug-loaded micelles have the growth inhibition effect on ovarian cancer cells A2780. The results show that both free CEL and the three drug-loaded micelles have growth inhibitory effects on a2780 cells, and the three drug-loaded micelles exhibit greater cytotoxicity than free CEL. When the CEL concentration is the same, mPEG-SS-C₁₈The cell survival rate of the/CEL group is lower than that of mPEG-C₁₈cRGD/TIP/mPEG-SS-C of/CEL group₁₈Cell survival ratio mPEG-SS-C of/CEL group₁₈the/CEL group was smaller and had the highest cytotoxicity.

Example 3

A preparation method of a composite micelle with tumor targeting and reduction sensitivity comprises the following steps:

1. preparation of amphiphilic Polymer:

1.1 mPEG-C₁₈synthesis of (2)

80mg of stearyl amine (C) are weighed out₁₈-NH₂) Dissolved in 2mL of anhydrous dichloromethane (CH)₂Cl₂) In addition, 100mg mPEG-NHS was dissolved in 1mL anhydrous CH₂Cl₂And mixing the two solutions, and stirring for reaction for 24 hours. After the reaction, the reaction solution was dropped into cold ether to obtain a white precipitate. Repeatedly re-precipitating and purifying the precipitate by using a precipitator to obtain mPEG-C₁₈。

FIG. 1 is mPEG-C₁₈δ is 3.62ppm (a, -CH)₂-CH₂-in mPEG units) is mPEG backbone-CH₂CH₂H peak in O-with δ being 3.42ppm (d, CH)₃O-in mPEG units) is a characteristic peak of terminal methoxyl of mPEG; δ 1.26ppm (b, -CH)₂-in C₁₈units) and δ 0.87ppm (c, -CH)₃ in C₁₈units) are each C₁₈Characteristic peaks for methylene and terminal methyl groups in the segment. Signals of two components, PEG and C18, were observed on NMR spectra (FIGS. 1-2), demonstrating block copolymerizationAnd (4) forming the substance.

1.2 mPEG-SS-C₁₈Synthesis of (2)

1.05g of 3,3 '-dithiodipropionic acid (COOH-SS-COOH), 0.45g of 0.45g N, N' -Dicyclohexylcarbodiimide (DCC), 0.02g of 4-Dimethylaminopyridine (DMAP) were weighed out and dissolved in 35mL of Tetrahydrofuran (THF) or CH₂Cl₂The reaction was stirred for 24 h. After the reaction is finished, filtering to remove the solid in the reaction solution, and reserving the filtrate for standby. Weighing 1.35g C₁₈-NH₂Dissolved in 35mL CH₂Cl₂This solution was added dropwise to the above filtrate, and the reaction was stirred for 24 hours to obtain a reaction solution 1. 528mg of DCC and 29mg of DMAP were weighed out and dissolved in the reaction solution 1, and after stirring and reacting for 3 hours, the filtrate was filtered and retained. 1.17g of methoxypolyethylene glycol (mPEG-OH) was dissolved in 5mL of CH₂Cl₂The solution was added dropwise to the filtrate of the previous step, and the reaction was stirred for 24 hours. After the reaction is finished, the reaction liquid is concentrated and then is dripped into cold ether or n-hexane to obtain white precipitate. Repeatedly re-precipitating and purifying the precipitate by using a precipitator to obtain mPEG-SS-C₁₈。

FIG. 2 is mPEG-SS-C₁₈δ is 3.62ppm (a, -CH)₂-CH₂-in mPEG units) and δ 3.42ppm (d, CH)₃O-in mPEG units) are respectively mPEG skeleton-CH₂CH₂Characteristic peaks for O-and terminal methoxy; delta 2.88ppm (f, -CH)₂CH₂SSCH₂CH₂-)，2.58ppm(g， -CH₂CH₂SSCH₂CH₂-) is a characteristic peak of methylene at two sides of a disulfide bond; delta 2.98(e, -NHCH)_2-in C₁₈ units),δ＝1.26ppm(b，-(CH₂₎₁₆-CH₃ in C₁₈ units),δ＝0.87ppm(c，-(CH₂₎₁₆-CH₃ in C₁₈units) are each C₁₈Methylene and terminal methyl characteristic peaks of the segment. NMR spectra (FIGS. 1-3) showed not only PEG and C₁₈The signals of the two components, and also of the disulfide-bond para-methylene group, demonstrate the structure of the-SS-containing block copolymer. In addition, in the condensation reaction experiment, white by-products, namely DCU is generated in the clear reaction solution, and the phenomenon also indicates that DCC condensation reaction occurs.

1.3 cRGD-PEG-C₁₈、TIP-PEG-C₁₈Synthesis of (2)

1.3.1 Mal-PEG-C₁₈Synthesis of (2)

Weighing 13.5mg of C₁₈-NH₂Dissolved in 2mL CH₂Cl₂In the solution, 100mg of maleimide polyethylene glycol succinimidyl ester (Mal-PEG-NHS) was dissolved in 2mL of CHCl₂After mixing the two solutions, a small amount of Triethylamine (TEA) is added, and the mixture is stirred and reacted for 24 hours under the protection of nitrogen. After the reaction is finished, the reaction solution is concentrated and then is dripped into cold ether to obtain white precipitate. Repeatedly re-precipitating and purifying the precipitate by using a precipitator to obtain Mal-PEG-C₁₈。

1.3.2 cRGD-PEG-C₁₈Synthesis of (2)

Weighing 12mg cRGD, dissolving in 2mL distilled water, 46mg Mal-PEG-C₁₈Dissolved in 2mL of distilled water, mixed and reacted at room temperature for 24h under nitrogen protection. After the reaction is finished, transferring the solution into a dialysis bag with the molecular weight cutoff of 2000, and then putting the dialysis bag into distilled water for dialysis for 24 hours. Filtering, and freeze-drying to obtain cRGD-PEG-C₁₈。

FIGS. 3(A) and (B) are cRGD and cRGD-PEG-C, respectively₁₈Nuclear magnetic spectrum of (a). In fig. 1-5(a), δ ═ 6.61, 6.94ppm (j, k, -Ar-H-in cRGD) are characteristic peaks of H on the phenyl ring of tyrosine in cRGD. In fig. 1-5(B), the signal of cRGD (δ ═ 6.61, 6.94ppm), the signal of PEG (δ ═ 3.62ppm), and C were observed₁₈Is equal to 1.26, 0.87 ppm. cRGD, PEG and C₁₈The signal of (a) is simultaneously present, indicating that the cRGD-PEG-C₁₈The preparation was successful. According to the peak area ratio of j to a of 1:240, cRGD-PEG-C can be calculated₁₈The grafting rate of the medium cRGD is 37.5%.

1.3.3 TIP-PEG-C₁₈Synthesis of (2)

FIGS. 4(A) and (B) are TIP and TIP-PEG-C, respectively₁₈Nuclear magnetic spectrum of (a). In fig. 4(a), δ 6.59, 6.95ppm (m, n, -Ar-H-in TIP) are characteristic peaks of H on the phenyl ring of tyrosine in TIP. In FIG. 4(B), it can be observed thatSignal for TIP (δ 6.59, 6.95ppm), signal for PEG (δ 3.62ppm), and C₁₈Is equal to 1.26, 0.87 ppm. TIP, PEG and C₁₈The simultaneous presence of the signal(s) indicates that TIP-PEG-C is present₁₈The preparation was successful. According to the peak area ratio of m to a of 1:445, TIP-PEG-C can be calculated₁₈The graft ratio of medium TIP was 20.2%.

2. Preparation of blank micelle:

according to mPEG-SS-C₁₈：cRGD-PEG-C₁₈：TIP-PEG-C₁₈70%: 12%: weighing three polymers according to the proportion of 18 percent, dissolving 10mg of the three polymers in 2mL of distilled water, stirring the three polymers in the dark, and then freezing and drying the mixture to obtain the bifunctional blank micelle cRGD/TIP/mPEG-SS-C with tumor targeting and reduction sensitivity₁₈。

a drop of blank micelle aqueous solution is dropped on a copper net coated with a carbon film, dyed by 0.2 percent (W/V) phosphotungstic acid and dried at room temperature, and the appearance of the blank micelle aqueous solution is observed under a Transmission Electron Microscope (TEM). 1mL of micelle aqueous solution is placed in a sample cell, and the particle size and the distribution of the micelle in the aqueous solution are measured by a light scattering particle size analyzer.

As shown in Table 1 and FIGS. 5A-C, the three blank micelles have regular spherical shape, round appearance and uniform dispersion, and the mean particle diameters of the non-functional, monofunctional and bifunctional blank micelles are 290.6 + -9.50 nm, 0.259 + -0.013 nm and 269.4 + -10.75 nm respectively, and the particle diameter distributions are 0.202 + -0.052, 224.3 + -8.90 and 0.248 + -0.034 respectively.

4. Preparing a drug-loaded micelle:

according to mPEG-SS-C₁₈：cRGD-PEG-C₁₈：TIP-PEG-C₁₈70%: 12%: three polymers were weighed out at a ratio of 18%, and 10mg in total was dissolved in 2mL of anhydrous methanol. 2mg of CEL was weighed out and dissolved in 2mL of anhydrous methanol to prepare a solution having a concentration of 1 mg/L. Mixing 300 mu LCEL methanol solution with the polymer methanol solution, spin-drying, adding 2mL of distilled water, hydrating, shaking, centrifuging at 4000rpm for 15min, taking supernatant, and freeze-drying to obtain the targeted and reduction-sensitive bifunctional drug-loaded micelle cRGD/TIP/mPEG-SS-C₁₈/CEL。

a drop of drug-loaded micelle aqueous solution is dropped on a copper net coated with a carbon film, dyed by 0.2 percent (W/V) phosphotungstic acid and dried at room temperature, and the appearance of the solution is observed under a Transmission Electron Microscope (TEM). 1mL of micelle aqueous solution is placed in a sample cell, and the particle size and the distribution of the micelle in the aqueous solution are measured by a light scattering particle size analyzer.

As shown in table 1 and fig. 5D-F, the three drug-loaded micelles are regular spheres, round in appearance, and uniform in dispersion, and the mean particle sizes of the non-functional, monofunctional, and bifunctional drug-loaded micelles are 282.0 ± 1.70nm, 304.1 ± 5.70nm, and 215.2 ± 1.10nm, respectively, and the particle size distributions are 0.39 ± 0.023, 0.38 ± 0.006, and 0.28 ± 0.016, respectively.

TABLE 1 blank micelle mPEG-C₁₈、mPEG-SS-C₁₈、cRGD/TIP/mPEG-SS-C₁₈And supported CEL micelle mPEG-C₁₈/CEL、mPEG-SS-C₁₈/CEL、cRGD/TIP/mPEG-SS-C₁₈Average particle size of/CEL and distribution thereof

Example 4

1. in vitro drug release studies of drug loaded micelles:

respectively weighing two DOX-loaded micelles, namely cRGD/TIP/mPEG-SS-C₁₈(DOX) and mPEG-C₁₈and/DOX (the content of each group of DOX is consistent), adding 2mL of distilled water to prepare a solution, transferring the solution into a dialysis bag, fastening two ends of the dialysis bag, and placing the dialysis bag into a dissolution tube. 5mL of drug release medium was added to each dissolution tube, and the dissolution tubes were placed in a water bath at 37 ℃ and stirred at constant temperature. Taking out the drug release medium in the tube according to the preset time, and supplementing the same amount of the drug release medium, wherein the sampling time points are respectively 0, 1, 2, 4, 6, 8, 10, 12, 24, 48 and 60 hours. And (3) measuring the absorbance A of the taken sample by an ultraviolet spectrophotometer, calculating the cumulative release rate of the drug, and drawing an in vitro drug release curve.

The results are shown in FIG. 6, where mPEG-C is released 1h in the DTT-containing drug release medium₁₈(DOX) and cRDG/TIP/mPEG-SS-C₁₈The cumulative release of the drug by/DOX is 18% and 29% respectively; at 12h, the release amounts of the two drug-loaded micelles reach 49% and 76% respectively; when the drug release time is 60h, mPEG-C₁₈The cumulative release amount of/DOX is 52%, cRGD/TIP/mPEG-SS-C₁₈The cumulative release amount of DOX in DOX reaches 81 percent, which is 1.62 times of the release amount of the non-functional micelle. This is because DTT breaks-SS-in the reduction-sensitive polymer, resulting in disintegration of the micelle structure and promotion of drug release. These results demonstrate that the bifunctional complex micelle can realize the destruction of micelle structure and release drug rapidly under the stimulation of intracellular reducing environment.

2. Cytotoxicity study of blank micelles: detection of three blank micelles mPEG-C by MMT method₁₈、mPEG-SS-C₁₈、cRGD/TIP/mPEG-SS-C₁₈Cytotoxicity to ovarian cancer cells a 2780. A2780 cells at 1X 10⁴Density of cells/wellTo a 96-well plate, 1mL of DMEM containing 10% FBS was added and incubated for 24 hours, and then a series of blank micellar solutions (at concentrations of 31.25, 62.5, 125, 250, 500, 1000. mu.g/mL) at different concentrations were added and incubated in an incubator for 48 hours, after which 20. mu.L of MTT solution was added and incubation was continued for 4 hours. After discarding the MTT solution, MTT-formazan crystals were dissolved by adding DMSO solution, and the absorbance at 570nm was measured. As shown in FIG. 7A, the cell viability was 90% or more when the concentration of the three blank micelles was 31.25 to 125. mu.g/mL, and was still 82% or more when the concentration of the blank micelles was 1000. mu.g/mL. The cytotoxicity of the blank micelle in the investigated range (31.25-1000 mug/mL) is small, which indicates that the three micelles have good biological safety as the drug carrier material.

3. The antitumor activity of the free drug and three drug-loaded micelles is studied: detection of free CEL and mPEG-C by MMT₁₈/CEL、mPEG-SS-C₁₈/CEL、cRGD/TIP/mPEG-SS-C₁₈The three drug-loaded micelles have the growth inhibition effect on ovarian cancer cells A2780. A2780 cells at 1X 10⁴The cells were plated at a density of one well in 96-well plates, incubated for 24 hours in 1mL DMEM containing 10% FBS, followed by a series of free CEL and three micellar solutions at different concentrations (CEL concentrations 0.1, 0.2, 0.4, 0.8, 1.6, 3.2. mu.g/mL), incubated for 48 hours in an incubator, and then 20. mu. LMTT solution was added and incubation continued for 4 hours. After discarding the MTT solution, MTT-formazan crystals were dissolved by adding DMSO solution, and the absorbance at 570nm was measured.

The results are shown in fig. 7B, where both free CEL and the three drug-loaded micelles had growth inhibition on a2780 cells, and the inhibition was dose-dependent. The three drug loaded micelles showed stronger cytotoxicity compared to free CEL. Four preparations, namely free CEL, mPEG-C₁₈/CEL、 mPEG-SS-C₁₈/CEL、cRGD/TIP/mPEG-SS-C₁₈IC of/CEL₅₀2.35. mu.g/mL, 2.10. mu.g/mL, 1.32. mu.g/mL, 0.50. mu.g/mL, respectively.

When the CEL concentration is the same, mPEG-SS-C₁₈The cell survival rate of the/CEL group is lower than that of mPEG-C₁₈CEL group, due to high intracellular Glutathione (GSH) concentrationThe degree is much higher than that of extracellular mPEG-SS-C₁₈After CEL enters cells, under the action of GSH, SS-is broken, the micelle structure is destroyed, CEL is promoted to be rapidly released from the interior of the micelle, effective dose is reached in a short time, and toxicity to tumor cells is enhanced. cRGD/TIP/mPEG-SS-C₁₈Cell survival ratio mPEG-SS-C of/CEL group₁₈the/CEL group was smaller and had the highest cytotoxicity, since cRGD peptide could specifically bind to alpha on the surface of tumor cells_νβ₅Integrins, increase the uptake of micelles by tumor cells. mPEG-SS-C after micelles have entered tumor cells₁₈The part is broken under the action of GSH, so that the micelle structure is disintegrated, and CEL encapsulated in the micelle structure is rapidly released. TIP-PEG-C₁₈Dispersed in the cell sap in a monomolecular state, and after being specifically combined with TRAF6 protein in cells, downstream signal channels such as NF-kB and the like are inhibited, and synergistic anti-tumor effects such as cell apoptosis induction, cell proliferation inhibition and the like are generated.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A preparation method of tumor targeting and reduction sensitive composite micelle is characterized by comprising the following steps:

step 1, synthesizing a polymer mPEG-SS-C₁₈；

Step 2, synthesizing polymer cRGD-PEG-C₁₈、TIP-PEG-C₁₈；

Step 3, mixing the polymer mPEG-SS-C₁₈、cRGD-PEG-C₁₈、TIP-PEG-C₁₈Combining with tripterine to prepare the drug-loaded micelle with tumor targeting and reduction sensitivity;

wherein the polymer mPEG-SS-C₁₈、cRGD-PEG-C₁₈、TIP-PEG-C₁₈The proportion of the three components satisfies: mPEG-SS-C₁₈：cRGD-PEG-C₁₈：TIP-PEG-C₁₈=70%：12%：18%。

2. The method for preparing the tumor targeting and reduction sensitive composite micelle according to claim 1, wherein the step 1 specifically comprises the following steps:

step 1-1, weighing 3,3 '-dithiodipropionic acid, N, N' -dicyclohexylcarbodiimide and 4-dimethylaminopyridine, dissolving in an organic solvent, stirring for reaction, filtering, and keeping a filtrate;

step 1-2, weighing stearylamine, dissolving the stearylamine in an organic solvent, dripping the filtrate obtained in the step 11 into the organic solvent, stirring the mixture for reaction, and purifying the reaction product to obtain an intermediate product 1;

step 1-3, weighing the intermediate product 1, dissolving the intermediate product 1 and N, N' -dicyclohexylcarbodiimide, 4-dimethylaminopyridine in an organic solvent, stirring for reaction, filtering, and keeping a filtrate;

step 1-4, weighing polyethylene glycol, dissolving in an organic solvent, dripping the filtrate obtained in step 13 into the organic solvent, stirring for reaction, filtering after the reaction is finished, concentrating the filtrate, dripping the filtrate into a precipitator to obtain a white solid, collecting the precipitate, drying, adding water to dissolve the white solid, transferring the white solid into a dialysis bag with the molecular weight cutoff of 2000, placing the dialysis bag into distilled water for dialysis, and freeze-drying the solution to obtain the polyethylene glycol freeze-dried solutionmPEG-SS-C₁₈A polymer.

3. The method for preparing the tumor targeting and reduction sensitive composite micelle according to claim 1, wherein the step 2 specifically comprises the following steps:

step 2-1, weighing stearylamine and maleimide-polyethylene glycol-succinimide ester, preparing two solutions respectively, adding one solution into the other solution dropwise, adding triethylamine, stirring for reaction, drying reaction liquid after the reaction is finished, adding a proper amount of distilled water, transferring the reaction liquid into a dialysis bag with cut-off molecular weight of 2000, placing the dialysis bag into distilled water for dialysis, and freeze-drying the solution to obtain Mal-PEG-C₁₈A polymer;

step 2-2, weighing cRGD polypeptide and Mal-PEG-C₁₈Dissolving polymer in distilled water, stirring for reaction, transferring reaction solution into dialysis bag with cut-off molecular weight of 2000, dialyzing the dialysis bag in distilled water, and lyophilizing to obtain cRGD-PEG-C₁₈A polymer;

step 2-3, weighing TIP peptide and Mal-PEG-C₁₈Dissolving polymer in distilled water, stirring for reaction, transferring reaction solution into dialysis bag with cut-off molecular weight of 2000, dialyzing in distilled water, and lyophilizing to obtain TIP-PEG-C₁₈A polymer.

4. The method for preparing tumor targeting and reduction sensitive composite micelle according to claim 1, wherein the step 3 specifically comprises: preparing medicine-carrying micelle by thin film-hydration method, and mixing polymer mPEG-SS-C₁₈、cRGD-PEG-C₁₈、TIP-PEG-C₁₈And combining with tripterine, dissolving with methanol, spin-drying, and hydrating with distilled water to obtain the drug-loaded micelle with tumor targeting and reduction sensitivity.

5. The method for preparing tumor targeting and reduction sensitive composite micelle according to claim 2, wherein the organic solvent added in step 1Is tetrahydrofuran or CH₂Cl₂。

6. The method for preparing tumor-targeting and reduction-sensitive composite micelle according to claim 2, wherein the precipitating agent added in step 1 is cold diethyl ether or n-hexane.