CN112439076A - Preparation of reduction responsive nano micelle medicine - Google Patents
Preparation of reduction responsive nano micelle medicine Download PDFInfo
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- CN112439076A CN112439076A CN201910800315.7A CN201910800315A CN112439076A CN 112439076 A CN112439076 A CN 112439076A CN 201910800315 A CN201910800315 A CN 201910800315A CN 112439076 A CN112439076 A CN 112439076A
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- A—HUMAN NECESSITIES
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
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- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
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Abstract
The invention mainly relates to the field of drug carriers for cancer treatment, in particular to a method for preparing a reduction-responsive nano micelle drug (TPGS) with long circulation in vivo, small toxic and side effects and high chemotherapy efficiency3350‑SS‑DTX&TPGS3350-FOL). Docetaxel (DTX) is hydrophobic and unstable in the aqueous phase. TPGS3350Is a derivative of vitamin E, which contains PEG 3350. DTX and TPGS Using disulfide bond3350Chaining to form TPGS3350-SS-DTX copolymer, followed by TPGS with targeting function3350And mixing FOL, and preparing the reduction-responsive nano micelle medicine by a thin film hydration method. The nanometer micelle has high stability, and can promote long circulation in vivo. Also has multiple functions of active targeting function, rapid release of intracellular drugs and the like, thereby realizing effective treatment on tumors and lower toxic and side effects.
Description
Technical Field
The invention belongs to the field of anti-cancer medicaments, and relates to preparation of reduction responsive TPGS3350-SS-DTX&TPGS3350FOL anticancer nano-micelle medicine.
Background
In recent years, malignant tumors are highly developed, and the treatment of the malignant tumors becomes a hot problem for research. The traditional chemotherapeutic drugs have poor water solubility, large toxic and side effects and poor selectivity, so that the development of a new dosage form of the chemotherapeutic drugs is urgently needed. Docetaxel (DTX) is hydrophobic and unstable in the aqueous phase. TPGS3350Is a kind of vitaminA derivative of element E comprising PEG 3350. DTX and TPGS Using disulfide bond3350Chaining to form TPGS3350-SS-DTX copolymer, followed by TPGS with targeting function3350And mixing FOL, and preparing the reduction-responsive nano micelle medicine by a thin film hydration method. The nano micelle has high stability in the water phase, wherein TPGS3350Can prolong the in vivo circulation time of the nano micelle drug, the folic acid molecules can actively target tumor cells so as to improve the drug delivery efficiency, and the disulfide bonds in the micelle can be cut by high-concentration glutathione in the tumor cells so as to realize the rapid release of the drug in cells. Therefore, the nano micelle drug has multiple functions of active targeting, reduction responsiveness, rapid intracellular drug release and the like, and realizes high transfer efficiency of the drug, thereby improving the treatment effect of chemotherapy and reducing the toxic and side effects of the drug.
Docetaxel is a commonly used antitumor drug in clinic, but has poor water solubility and poor in-vivo stability. At present, no TPGS exists3350Formation of TPGS with disulfide bond, docetaxel3350-SS-DTX compound is then conjugated with folic acid to form a novel compound TPGS3350-SS-DTX&TPGS3350-report of FOL.
The reductive responsive anticancer nano micelle has the functions of active targeting, in-vivo long circulation and rapid release of intracellular drugs, and provides a new dosage form selection for the treatment of cancers.
Disclosure of Invention
The invention adopts TPGS with good biocompatibility3350A carrier material linked to Docetaxel (DTX) by a disulfide bond to synthesize reduction-responsive TPGS3350-SS-DTX followed by targeting of TPGS3350FOL mixing, and preparing the reduction responsive nano micelle drug with multiple functions of active targeting, in-vivo long circulation, rapid intracellular drug release and the like by a thin film hydration method. The nano micelle improves the water solubility and selectivity of docetaxel, enhances the chemotherapy effect of docetaxel and reduces the toxic and side effects of docetaxel.
1. Has active targeting, in vivo long circulation and quick release of intracellular medicineThe preparation of the reduction responsive anticancer nano micelle medicine with multiple functions comprises the following contents: reduction responsive copolymer TPGS3350-SS-DTX preparation, and TPGS3350-SS-DTX&TPGS3350Preparing FOL nano micelle medicine.
2. In the invention, the surface morphology of the micelle is represented by a Transmission Electron Microscope (TEM), and the micelle is synthesized and is a nano particle similar to a sphere. In the present invention, the Critical Micelle Concentration (CMC) of the micelle preparation was determined by fluorescence spectroscopy using DPH as a hydrophobic fluorescent probe, and these data obtained indicate TPGS3350-SS-DTX&TPGS3350FOL formed homogeneous micelles with a core-shell structure, with a measured CMC of 0.128mg/ml, and the low CMC of the micelle formulation ensured the stability of these core-shell micelle structures both in vitro and in vivo. In the present invention, in vitro cytotoxicity experiments were performed with TPGS at different time periods3350-SS-DTX&TPGS3350FOL micelles showed significant cytotoxicity differences against murine melanoma cells B16. In the invention, the mice are injected with micelle medicine in tail vein after the murine melanoma B16 cells are injected subcutaneously, the weight and tumor volume changes of the mice are measured every time within 20 days from the beginning of treatment to the end of treatment, the weight and volume changes of the mice in each group are compared, and TPGS is finished on the 20 th day3350-SS-DTX&TPGS3350The Folate micellar group can significantly inhibit the growth of melanoma, and tumors grow more slowly.
Drawings
FIG. 1.TPGS3350-SS-DTX&TPGS3350TEM image of FoL nanomicelle drug transmission electron microscope
FIG. 2.TPGS3350-SS-DTX&TPGS3350In vitro cytotoxicity of FoL nanomicelle drugs on melanoma B16 cells within 24 hours
Detailed Description
The invention is further illustrated by the following examples:
example 1
TPGS3350-NH2The preparation of (1):
1) weighing alpha-TOS, diamino polyethylene glycol, N-hydroxysuccinimide and N, N' -dicyclohexylcarbodiimide (the molar ratio is 1: 1.2: 2) to be dissolved in dichloromethane, adding triethylamine, and placing in a three-neck flask;
2) introducing nitrogen into the solution obtained in the step 1), and reacting for two days at room temperature;
2) filtering the solution reacted in the step 2), removing reaction byproducts, and adding the solution into anhydrous ether to obtain white precipitate;
3) centrifuging the product obtained in the step 3), pouring out liquid, and collecting white precipitate;
4) transferring the white precipitate obtained in the step 4) into a dialysis bag, and dialyzing to remove unreacted substances;
5) freeze-drying the sample treated in the step 5) to obtain TPGS3350-NH2A white powder;
example 2
TPGS3350-COOH preparation:
1) TPGS 3350-NH2(1mmol), DCC (1.2mmol), NHS (1.2mmol) and glutaric acid (1.2mmol) were weighed out and dissolved in 10mL DMSO
2) Introducing nitrogen into the solution obtained in the step 1) at room temperature for reaction for 24 hours;
3) filtering the solution reacted in the step 2) by using a 0.45-micrometer membrane to remove reaction byproducts to obtain filtrate;
4) transferring the filtrate obtained in the step 3) into a dialysis bag (MWCO 3350), dialyzing for 24 hours by using dimethyl sulfoxide as a dialysate, transferring into a large beaker using distilled water as a dialysate after dialysis is finished, dialyzing for 48 hours, and changing water every 4 hours to dialyze out an organic reagent;
5) transferring the material in the dialysis bag in the step 4) to a culture dish, and freeze-drying to obtain pure white solid TPGS3350-COOH；
Example 3
TPGS3350-preparation of Fol:
1) weighing folic acid (Folate), N-hydroxysuccinimide (NHS) and DCC, mixing and dissolving in 30ml DMSO at a molar ratio of 1: 1.2, reacting in dark for 6 hours, and filtering with 0.22 μm membrane to remove byproduct DCU;
2) adding 1.2 times of molar ratio of ethylenediamine and DMAP into the solution obtained in the step 1), reacting, precipitating, centrifuging, washing, and vacuum-drying to remove the organic solvent.
3) Mixing the precipitate obtained in the step 2) with TPGS3350-COOH solution, deionized water and 1.2 times molar amount of EDC and carboxyl groups of NHS activated TPGS 3350, reacting under alkaline conditions and filtering;
4) dialyzing the filtrate obtained in the step 3) with water to remove unreacted aminated folic acid and TPGS3350-COOH, freeze-drying to give TPGS3350-FOL；
Example 4
TPGS3350Preparation of SS-DTX:
1) mixing 2g of DTDP and 7mL of acetyl chloride, heating in a water bath, carrying out rotary evaporation, adding 6 times of cold ether, stirring, centrifuging, precipitating and drying to obtain solid DTDPA;
2) DTDPA and TPGS obtained in the step 1)3350-NH2NHS and DCC are dissolved in anhydrous DMF according to the molar ratio of 1: 2, 2 times of triethylamine is taken and dripped into the reaction, the mixture is stirred in a water bath, filtered by a filter membrane, washed and centrifuged;
3) transferring the product obtained in the step 2) into a dialysis bag (MWCO 3350), using DMF as a dialysate, and after 24 hours of dialysis, replacing the dialysate with an aqueous solution for dialysis for 48 hours and replacing water every 4 hours. Freeze drying to obtain solid TPGS3350-SS-COOH；
4) Reacting the solid obtained in the step 3), Docetaxel (DTX) and DMAP according to the molar ratio of 1: 2, adding 2 times of triethylamine, then adding 5ml of anhydrous DMF, stirring the reaction in a water bath at 45 ℃ for 36 hours, and filtering after the reaction is finished;
5) adding excessive cold diethyl ether into the filtrate obtained in the step 4) for precipitation, then carrying out low-temperature centrifugation, washing the precipitate, and carrying out vacuum drying to obtain a white product TPGS3350-SS-DTX；
Example 5
TPGS3350-SS-DTX&TPGS3350-FOL micelle preparation:
1) by making thinMembrane hydration method, weighing TPGS3350-SS-DTX and TPGS3350Folate weight ratio 5: 1 in a 25mL round-bottom flask, 2-3mL of dichloromethane were added,
2) ultrasonically dissolving the substance obtained in the step 1), and magnetically stirring to obtain a solution;
3) transferring the solution obtained in the step 2) into a rotary evaporator, removing the organic solvent by rotary evaporation until a layer of film appears in the round-bottom flask, adding secondary deionized water, performing ultrasonic treatment for 5min, and then magnetically stirring at 400rpm for 30min to obtain the polymer micelle.
4) Transferring the polymer micelle obtained in the step 3) to a syringe, filtering the polymer micelle into a vial by using a 0.45 mu m filter, and filtering the polymer micelle to remove residual substances which are not formed to obtain TPGS3350-SS-DTX&TPGS3350-FOL micelles.
Claims (4)
1. The preparation of reducing responsive anticancer nano micelle medicine with multiple functions of active targeting, in vivo long circulation and rapid release of intracellular medicine includes the following contents: reduction responsive copolymer TPGS3350-SS-DTX preparation, and TPGS3350-SS-DTX&TPGS3350Preparing FOL nano micelle medicine.
2. The preparation of nanomicelle pharmaceuticals according to claim 1, resulting in nanomicelle characterized by: the nano micelle obtained by the film hydration method has uniform grain diameter and good dispersibility.
3. The preparation of nanomicelle pharmaceuticals according to claim 1, wherein the nanomicelle is obtained by: the surfactant and the free drug are washed with secondary distilled water three times, each time at 10000 r/min, and centrifuged for 15 min.
4. The preparation of nanomicelle pharmaceuticals according to claim 1, wherein the nanomicelle is obtained by: the final nano particles are obtained by freeze drying water, the re-solubility is good, and ethanol or Tween 80 is not needed for solubilization.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106176602A (en) * | 2016-07-21 | 2016-12-07 | 中国科学院海洋研究所 | A kind of targeting is in the Docetaxel chitosan nano-micelle of stomach organization and preparation method and application |
| CN107349188A (en) * | 2017-07-06 | 2017-11-17 | 天津工业大学 | The preparation method of the sensitive intelligent nano-medicament carriers of pH |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106176602A (en) * | 2016-07-21 | 2016-12-07 | 中国科学院海洋研究所 | A kind of targeting is in the Docetaxel chitosan nano-micelle of stomach organization and preparation method and application |
| CN107349188A (en) * | 2017-07-06 | 2017-11-17 | 天津工业大学 | The preparation method of the sensitive intelligent nano-medicament carriers of pH |
Non-Patent Citations (1)
| Title |
|---|
| 雷帅权: ""对肿瘤微环境响应的抗癌纳米药物的研究"", 《中国优秀博硕士学位论文全文数据库(硕士) 工程科技Ⅰ辑》 * |
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