CN111494318A - Tumor targeting and reduction sensitive composite micelle and preparation method and application thereof - Google Patents
Tumor targeting and reduction sensitive composite micelle and preparation method and application thereof Download PDFInfo
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- CN111494318A CN111494318A CN202010347892.8A CN202010347892A CN111494318A CN 111494318 A CN111494318 A CN 111494318A CN 202010347892 A CN202010347892 A CN 202010347892A CN 111494318 A CN111494318 A CN 111494318A
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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)、Polyethylene glycol-stearylamine (cRGD-PEG-C) containing tumor targeting peptide18) The composite micelle provided by the invention can improve the water solubility of CE L, quickly release CE L after entering tumor cells in a targeted manner, improve the killing power of the tumor cells and reduce the toxic and side effects of the CE L.
Description
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 (Astrologer, CE L) is a pentacyclic triterpenoid monomer compound extracted from root bark of celastrus and celastrus plants of Celastraceae, and has wide pharmacological activity, and can play roles of anti-inflammation, antioxidation, antirheumatic and antitumor by regulating various molecular targets such as INF-a, NF-kappa B, COX-2, VEGF, Akt and the like.
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, a tumor necrosis factor receptor-associated factor, belongs to the tumor necrosis factor-associated factor family.TRAF 6, which is unique in signal transduction and can not only transduce signals associated with the TNF superfamily, but also act as an important adaptor protein of the Toll-like receptor (T L R)/interleukin 1 receptor I L-1R (TIR) superfamily. many studies have now found that there is abnormal expression and activation of TRAF6 protein in a variety of tumor tissues such as lung cancer, multiple myeloma, ovarian cancer, colon cancer, glioma, and breast cancer. in ovarian cancer, TRAF6 is expressed in significantly higher amounts than in paracancerous normal tissues TRAF6 expression is an important indicator of patients with ovarian cancer, and compared to TRAF 6-negative expressing patients, the five-year survival rate of positively expressing patients is significantly reduced.
The polymer containing hydrophilic groups and hydrophobic groups can form micelles with a core-shell structure in an aqueous solution, insoluble drugs can be wrapped by hydrophobic cores, enzyme damage is avoided, and the bioavailability of the drugs is improved, hydrophilic shells can be prevented from being phagocytized by mononuclear phagocytes, and the in-vivo circulation time of the drugs is prolonged.
The invention discloses a silk fibroin nanoparticle, which comprises silk fibroin and an active drug, wherein the silk fibroin loads the active drug, and the active drug is selected from triptolide or tripterine.the silk fibroin nanoparticle improves the water solubility of triptolide and tripterine and can effectively resist the proliferation of cancer cells.
The linear or cyclic polypeptide containing RGD sequence is recognition site of several kinds of integrins, and can be bonded on the surface of micelle, so that it can obviously raise the uptake of ovarian cancer cell to target micelle, if the stimuli-sensitive site is introduced into amphiphilic copolymer, said micelle system can quickly respond and release medicine in the tumor cell, and the microenvironment in the tumor cell has obvious difference, in particular its pH value and reduced Glutathione (GSH) concentration, and the pH value of microenvironment around the tumor is changed in gradient, and the pH value of peripheral normal tissue is 7.4, the local extracellular pH value of tumor is 6.8, and the intracellular inclusion pH value is 5.0-6.5, and the researchers can develop several pH-responsive micelles by introducing condensed aldehyde, orthoester and hydrazine bond into the polymer, and the intracellular GSH concentration (0.5-10 mmol/L) is several hundred times of extracellular disulfide bond (2-352-L), and can be stably existed in the condition of human body under the condition of high concentration (gSH-3520) of normal disulfide bond.
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 glycol stearamide (TIP-PEG-C) coupled to TIP peptide18) 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 drug18The drug-loaded micelle can effectively identify and enter ovarian cancer cells through the active targeting effect mediated by cRGD polypeptide, 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:
Preferably, the step 1 specifically comprises:
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 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 tripterine (CE L),adding methanol for dissolving, spin-drying, and adding distilled water for hydration to obtain the drug-loaded micelle with tumor targeting and reduction sensitivity.
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 entrap tripterine CE L serving as a water-insoluble antitumor drug, and the targeting peptide cRGD on the surface of the micelle has α over-expressed with the surface of ovarian cancer cellsvβ5The 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-C18Break and quick release of the entrapped drug CE L-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) CE L-Supported micelle mPEG-C18/CEL(D)、mPEG-SS-C18/CEL(E)、 cRGD/TIP/mPEG-SS-C18TEM image of/CE L (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 CE L and three drug-loaded micelles (B) on ovarian cancer cells a 2780.
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 sensitivity18The drug-loaded micelle can effectively identify and enter ovarian cancer cells through the active targeting effect mediated by cRGD polypeptide, and is caused by mPEG-SS-C under the intracellular reductive condition18Break and quick release of the entrapped drug CE L-PEG-C18In a single molecular state, the polypeptide binds to TRAF6 protein in cell fluid and inhibits the expression of downstream protein, thereby promoting apoptosis and inhibitingCell proliferation is controlled, and the curative effect of resisting ovarian cancer is synergistically improved.
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 CH2Cl2Mixing the two solutions, stirring and reacting for 1-48 h, dropping the reaction solution into a precipitator after the reaction is finished to obtain white precipitate, and repeatedly re-precipitating and purifying the precipitate by the 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) to obtain a solution with a solution concentration of 0.001-100 mmol/L, stirring and reacting for 1-48 h, filtering to remove solids in the reaction solution after the reaction is finished, and reserving the filtrate18-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 CH2Cl2Dropwise adding the solution into the filtrate in the previous step, stirring and reacting for 1-48 h, concentrating the reaction solution after the reaction is finished, and dropwise adding a precipitator to obtain a white precipitate, repeatedly re-precipitating and purifying the precipitate by using the 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 of0.001-100 mmol/L solution, maleic imide polyethylene glycol succinimidyl ester (Mal-PEG-NHS) dissolved in CHCl2Mixing the two solutions, adding a small amount of Triethylamine (TEA), stirring and reacting for 1-48 h under the protection of nitrogen, concentrating the reaction solution after the reaction is finished, and dripping a precipitator into the reaction solution to obtain white precipitates, repeatedly re-precipitating and purifying the precipitates by using the 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 2m L distilled water, reacting for 1-48 h at room temperature under the protection of nitrogen after mixing, transferring the solution into a dialysis bag with the molecular weight cutoff of 2000 after the reaction is finished, putting the dialysis bag into the distilled water for dialysis for 1-48 h, filtering, and freeze-drying to obtain the 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 2m L distilled water, stirring in dark place, and freeze-drying to obtain the blank micelle cRGD/TIP/mPEG-SS-C with tumor targeting 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 in total dissolved in 2m L anhydrous methanol, of which 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 percent18Weighing CE L to be dissolved in absolute methanol to prepare a solution with the concentration of 0.1-100 mg/L, mixing a methanol solution of 300 mu L CE L with the polymer methanol solution, spin-drying, adding 2m L distilled water, after hydration and shaking, centrifuging at 4000rpm for 15min, taking the 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 method18/CE L, and monofunctional drug-loaded micelles mPEG-SS-C with only reducing sensitivity18/CEL。
5. Determining the morphology, the particle size and the distribution of the drug-loaded micelle:
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.
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 CE L structure are easy to generate Michael addition reaction with sulfhydryl group on DTT, and Dithiothreitol (DTT) is contained in drug release medium simulating intracellular environment, in the in vitro drug release link, drug Doxorubicin (DOX) which does not react with DTT is selected) As a model drug, two DOX-loaded micelles are respectively prepared, namely, the non-functional 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 low in the considered concentration range, and the three blank micelles are proved to have good biological safety as drug carriers.
3. Anti-tumor activity study of free drug and three drug-loaded micelles by using MMT method to detect free CE L and mPEG-C18/CEL、mPEG-SS-C18/CEL、cRGD/TIP/mPEG-SS-C18The results show that the free CE L and the three drug-loaded micelles have growth inhibition effect on the A2780 cells, and compared with the free CE L, the three drug-loaded micelles show stronger cytotoxicity18The cell survival rate of the/CE L group was lower than that of mPEG-C18group/CE L, cRGD/TIP/mPEG-SS-C18Cell survival ratio mPEG-SS-C of/CE L group18the/CE L 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 2m L anhydrous dichloromethane (CH)2Cl2) In the solution, 100mg of mPEG-NHS is dissolved in 1m L 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-C183.62ppm (a, -CH)2-CH2-in mPEG units) is mPEG backbone-CH2CH2Oscillation peak of H in O-3.42 ppm (d, CH)3O-in mPEG units) is a characteristic peak of terminal methoxyl of mPEG; 1.26ppm (b, -CH)2-in C18units) and 0.87ppm (c, -CH)3in C18units) are each C18Characteristic peaks for methylene and terminal methyl groups in the segment. Signals from both PEG and C18 components were observed on nuclear magnetic spectra (FIGS. 1-2) demonstrating the formation of block copolymers.
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 35m of L 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 35m L CH2Cl2Dropwise adding the solution into the above filtrate, stirring for reaction for 24 hr to obtain reaction solution 1, weighing 528mg DCC and 29mg DMAP, dissolving in reaction solution 1, stirring for reaction for 3 hr, filtering, collecting filtrate, weighing 1.17g methoxypolyethylene glycol (mPEG-OH) and dissolving in 5m L 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-C183.62ppm (a, -CH)2-CH2-in mPEG units) and 3.42ppm (d, CH)3O-in mPEG units) are respectively mPEG skeleton-CH2CH2Characteristic peaks for O-and terminal methoxy; 2.88ppm (f, -CH)2CH2SSCH2CH2-)，2.58ppm(g， -CH2CH2SSCH2CH2-) is a characteristic peak of methylene at two sides of a disulfide bond; 2.98(e, -NHCH)2-in C18units),＝1.26ppm(b，-(CH2)16-CH3in C18units),＝0.87ppm(c，-(CH2)16-CH3in 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 2m L CH2Cl2In 100mg of maleimide polyethylene glycol succinimidyl ester (Mal-PEG-NHS) dissolved in 2m L 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 of cRGD, dissolving in 2m L distilled water, 46mg of Mal-PEG-C18Dissolving in 2m L distilled water, mixing, reacting at room temperature for 24h under nitrogen protection, transferring the solution into a dialysis bag with cut-off molecular weight of 2000 after the reaction is finished, putting the dialysis bag into distilled water for dialysis for 24h, 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), 6.61, 6.94ppm (j, k, -Ar-H-in cRGD) is characteristic peak of H on the phenyl ring of tyrosine in cRGD. In fig. 1-5(B), the signal for cRGD (═ 6.61, 6.94ppm), PEG (═ 3.62ppm), and C were observed181.26, 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)
The preparation method is the same as above, and the polymer TIP-PEG-C can be obtained by replacing cRGD with TIP18。
FIGS. 4(A) and (B) are TIP and TIP-PEG-C, respectively18Nuclear magnetic spectrum of (a). In fig. 4(a), 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), signals for TIP (═ 6.59, 6.95ppm), PEG (═ 3.62ppm), and C were observed181.26, 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-C18Weighing three polymers at a ratio of 70% to 12% to 18%, dissolving 10mg in 2m L distilled water, stirring in dark place, and freeze drying to obtain the bifunctional blank micelle cRGD/TIP/mPEG-SS-C with tumor targeting and reduction sensitivity18。
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:
dropping a drop of blank micelle aqueous solution on a copper net coated with a carbon film, dyeing by 0.2% (W/V) phosphotungstic acid, drying at room temperature, observing the appearance under a Transmission Electron Microscope (TEM), placing 1m L micelle aqueous solution in a sample cell, and measuring the particle size and the distribution of the micelle in the aqueous solution by using 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-C18Weighing three polymers according to the proportion of 70 percent to 12 percent to 18 percent, dissolving 10mg of the three polymers in 2m L absolute methanol, dissolving 2mg of CE L in 2m L absolute methanol to prepare a solution with the concentration of 1 mg/L, mixing a 300 mu L CE L methanol solution with the polymer methanol solution, spinning, adding 2m L distilled water, after hydration and 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。
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 method18/CE L, and monofunctional drug-loaded micelles mPEG-SS-C with only reducing sensitivity18/CEL。
5. Determining the morphology, the particle size and the distribution of the drug-loaded micelle:
dropping a drop of the drug-loaded micelle aqueous solution on a copper net coated with a carbon film, dyeing by 0.2% (W/V) phosphotungstic acid, drying at room temperature, observing the appearance under a Transmission Electron Microscope (TEM), placing 1m L micelle aqueous solution in a sample cell, and measuring the particle size and the distribution of the micelle in the aqueous solution by using 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-C18CE L-loaded micelle mPEG-C18/CEL、mPEG-SS-C18/CEL、cRGD/TIP/mPEG-SS-C18Average particle size of/CE L and distribution thereof
Example 4
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:
respectively weighing two DOX-loaded micelles, namely cRGD/TIP/mPEG-SS-C18(DOX) and mPEG-C18Adding 2m L distilled water to prepare a solution, transferring the solution into a dialysis bag, fastening two ends of the dialysis bag, placing the dialysis bag into a dissolution tube, adding 5m L drug release medium into each dissolution tube, placing the dissolution tube into a water bath at 37 ℃ for constant-temperature stirring, taking out the drug release medium in the tube according to preset time, supplementing the equivalent drug release medium, wherein the sampling time points are 0, 1, 2, 4, 6, 8, 10, 12, 24, 48 and 60h respectively, measuring the absorbance A of the taken sample by an ultraviolet spectrophotometer, calculating the drug cumulative release rate, and drawing an in-vitro drug release curve.
The results are shown in FIG. 6 in the DTT-containingIn a drug release medium, mPEG-C is added when the drug is released for 1h18(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 A2780 cells were treated with 1 × 104The density of each cell was inoculated into a 96-well plate, 1m L DMEM containing 10% FBS was added and incubated for 24 hours, then a series of blank micelle solutions of different concentrations (concentration of 31.25, 62.5, 125, 250, 500, 1000 μ g/m L, respectively) were added, and the incubation was continued in an incubator for 48 hours, after which 20 μ L MTT solution was added and the incubation was continued for 4 hours, after the MTT solution was discarded, the DMSO solution was added to dissolve MTT-formazan crystals, and the absorbance value at 570nm was measured, as shown in fig. 7A, the cell survival rate was above 90% when the three blank micelles were at a concentration of 31.25 to 125 μ g/m L, the cell survival rate was still above 82% when the blank was 1000 μ g/m L, and the micelle blank was less cytotoxic in the range examined (31.25 to 1000 μ g/m L), indicating that the three micelles had good biosafety as drug carrier materials.
3. Anti-tumor activity study of free drug and three drug-loaded micelles by using MMT method to detect free CE L and mPEG-C18/CEL、mPEG-SS-C18/CEL、cRGD/TIP/mPEG-SS-C18Growth inhibition of ovarian cancer cells A2780 by three drug-loaded micelles of/CE L A2780 cells are treated with 1 × 104The cells were seeded at a density per well in 96-well plates and incubated in 1m L DMEM containing 10% FBSAfter 24 hours, a series of different concentrations of free CE L and three micellar solutions (CE L concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2. mu.g/m L, respectively) were added, and the mixture was incubated in an incubator for 48 hours, followed by addition of 20. mu. L MTT solution and further incubation for 4 hours, after discarding the MTT solution, MTT-formazan crystals were solubilized by addition of DMSO solution, and the absorbance at 570nm was measured.
The results are shown in FIG. 7B, where both free CE L and three drug-loaded micelles had growth inhibition on A2780 cells, and the inhibition was dose-dependent, three drug-loaded micelles showed greater cytotoxicity than free CE L, and four formulations, free CE L, mPEG-C18/CEL、 mPEG-SS-C18/CEL、cRGD/TIP/mPEG-SS-C18IC of/CE L502.35. mu.g/m L, 2.10. mu.g/m L, 1.32. mu.g/m L and 0.50. mu.g/m L, respectively.
When the CE L concentration is the same, mPEG-SS-C18The cell survival rate of the/CE L group was lower than that of mPEG-C18the/CE L group, because the intracellular Glutathione (GSH) concentration is much higher than the extracellular concentration when mPEG-SS-C18After CE L enters cells, under the action of GSH-SS-is broken, the micelle structure is destroyed, CE L is promoted to be rapidly released from the interior of the micelle, an effective dose is reached in a short time, and the toxicity to tumor cells is enhanced18Cell survival ratio mPEG-SS-C of/CE L group18the/CE L group was smaller and had the highest cytotoxicity, since the cRGD peptide could specifically bind α on the surface of tumor cellsνβ5Integrins, 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, resulting in disintegration of micelle structure and rapid release of CE L encapsulated therein18Dispersed 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 (8)
1. A tumor targeting and reduction sensitive composite micelle, which is characterized by comprising the following components:
1) polymer mPEG-SS-C18；
2) Polymer cRGD-PEG-C18；
3) Polymer TIP-PEG-C18。
2. 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-C18And tripterine (CE L) to prepare the drug-loaded micelle with tumor targeting and reduction sensitivity.
3. The method for preparing the tumor targeting and reduction sensitive composite micelle according to claim 2, wherein the step 1 specifically comprises the following steps:
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.
4. The method for preparing the tumor targeting and reduction sensitive composite micelle according to claim 2, wherein the step 2 specifically comprises the following steps:
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, adding a proper amount of distilled water, transferring into a dialysis bag with the molecular weight cutoff of 2000, putting the dialysis bag into the distilled water for dialysis, and freeze-drying the solution to obtain Mal-PEG-C18A polymer;
step 22, weighing cRGD polypeptide and Mal-PEG-C18Dissolving polymer in distilled water, stirring for reaction, and transferring the reaction solution after reactionTransferring into dialysis bag with cut-off molecular weight of 2000, dialyzing 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.
5. The method for preparing the tumor targeting and reduction sensitive composite micelle according to claim 2, wherein the step 3 specifically comprises the following steps: preparing medicine-carrying micelle by thin film-hydration method, and mixing polymer mPEG-SS-C18、cRGD-PEG-C18、TIP-PEG-C18And mixing with tripterine (CE L), dissolving with methanol, spin drying, and adding distilled water for hydration to obtain the drug-loaded micelle with tumor targeting and reduction sensitivity.
6. The method for preparing tumor targeting and reduction sensitive composite micelle according to claim 3, wherein the organic solvent added in step 1 is Tetrahydrofuran (THF) or CH2Cl2。
7. The method for preparing tumor-targeting and reduction-sensitive composite micelle according to claim 3, wherein the precipitating agent added in step 1 is cold diethyl ether or n-hexane.
8. The application of the tumor targeting and reduction sensitive composite micelle is characterized in that: can be used for treating or preventing ovarian cancer, lung cancer, multiple myeloma, ovarian cancer, colon cancer, glioma, and breast cancer.
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