CN104096237A - Pluronics-PTX (Paclitaxel) amphiphilic macromolecular prodrug and micelle preparation thereof - Google Patents
Pluronics-PTX (Paclitaxel) amphiphilic macromolecular prodrug and micelle preparation thereof Download PDFInfo
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Abstract
The invention discloses a Pluronics-PTX (Paclitaxel) amphiphilic macromolecular prodrug which is prepared by bonding Pluronics and PTX through an ester bond. The prodrug can be self-assembled in water to form micelle which takes PTX as a hydrophobic core and takes Pluronics as a hydrophilic shell, the content of a PTX drug in a preparation is increased, the problem of low solubility of the PTX drugs in water is solved, and further, the final preparation is free of a solubilizer and an organic solvent, so that the safety of drug use is improved. An active targeting factor can be connected to a Pluronics-terminated hydroxyl to provide the drug with an active targeting function. Further, the defects of poor water solubility, serious allergic reaction and the like of the conventional PTX injection are overcome.
Description
Technical field
The present invention relates to a kind of Pluronics-paclitaxel amphiphilic macromolecular prodrug and micellar preparation thereof, and their preparation method, pharmaceutical chemistry technical field belonged to.
Background technology
Paclitaxel (Paclitaxel, PTX) is a kind of tetracyclic diterpene compound extracting from the bark of Pacific Ocean Ramulus et folium taxi cuspidatae the earliest, is one of anticancer choice drug, and chemical constitution is shown in Fig. 1.Paclitaxel antineoplastic mechanism is: in the cell mitogen phase, increase the dimeric quantity of microtubule and polymerization speed, promote the polymerization of microtubule, suppress the depolymerization of kytoplasm microtubule simultaneously, thereby inhibition cell mitogen, make cancerous cell stop at G2 phase and M phase, reach the object of inhibition tumor cell growth.At present, paclitaxel is the choice drug for the treatment of clinically ovarian cancer and breast carcinoma, and therapeutic index is high.In addition, it also has good curative effect to pulmonary carcinoma, the brain cancer, nonsmall-cell lung cancer, rectal cancer etc.
Paclitaxel anticancer therapeutic is good, but some untoward reaction have limited its clinical practice, comprise poorly water-soluble, the half-life is short and body in non-specific distribution etc.The formulation for paclitaxel of listing has two kinds at present: paclitaxel injection
with paclitaxel albumin nano granular
injection.
to using polyoxyethylene castor oil and dehydrated alcohol (volume ratio is 1:1) as solvent, dissolve paclitaxel preparation and injection, for intravenous drip administration.But this carrier system can cause a series of untoward reaction, as anaphylaxis, neutrophilic granulocytopenia, bone marrow depression etc., the processing etc. that needs before administration to desensitize, process is loaded down with trivial details.Paclitaxel albumin nano granular injection goes on the market, although this administration nano-drug administration system has numerous advantages, yet
expensive, increased patient's financial burden.Therefore, develop the trend that new low toxicity drug delivery sys tems becomes research.At present researcher to paclitaxel carrier systematic research mainly based on following two aspects: paclitaxel drug administration system carrier is selected and initiatively targeting research, to increase the active targeting of medicine, making medicine reach maximum at tumor locus accumulates, the effect of killing and wounding that increases tumor reduces the toxicity of killing and wounding to surrounding normal cell simultaneously.
In recent years, the research and development of polymer-bound medicine attract wide attention.So-called " polymer-bound medicine " also referred to as polymeric prodrugs, refers to drug molecule covalently boundly to high molecular polymer, and it is in vivo under physiological condition, and drug molecule dissociates out from high molecular polymer, performance curative effect.Insoluble drug as paclitaxel and docetaxel and amphipathy macromolecule polymer-bound after, still keep amphipathic, can self assembly in aqueous medium be nano-micelle, medicine is protected to micelle kernel.Because medicine is covalently bonded on polymer, avoided because of the unstable burst drug release causing in kinetics, thereby improved the bioavailability of medicine, reduce the toxic and side effects of medicine simultaneously.
Summary of the invention
For above-mentioned prior art, for overcoming the deficiencies in the prior art, the invention provides the covalently bound amphiphilic macromolecular prodrug being prepared from of a kind of Pluronics and paclitaxel, this prodrug is by polyoxyethylene-poly-oxypropylene polyoxyethylene (PEO-b-PPO-b-PEO, commodity are called Pluronics, purchased from BASF AG) triblock copolymer and paclitaxel form by ester bond bonding, and it also can connect initiatively targeting factor on Pluronics terminal hydroxy group makes it initiatively target function of tool.This prodrug can self assembly formation be take paclitaxel as hydrophobic inner core in water, Pluronics is the micelle of hydrophilic outer shell, not only improved the content of taxol drug in preparation, solved the low problem of the dissolubility of taxone in water, and final preparation is not containing solubilizing agent and organic solvent, increased the safety of medication.
The present invention is achieved by the following technical solutions:
A Pluronics-paclitaxel amphiphilic macromolecular prodrug is to be formed by ester bond bonding by polyoxyethylene-poly-oxypropylene polyoxyethylene (PEO-b-PPO-b-PEO, commodity are called Pluronics, purchased from BASF AG) triblock copolymer and paclitaxel.It also can connect initiatively targeting factor on Pluronics terminal hydroxy group makes it initiatively target function of tool.
The chemical constitution of described polyoxyethylene-poly-oxypropylene polyoxyethylene (Pluronics) is as follows:
Wherein, the integer that n is 1～100, the integer that m is 15～100.
Described active targeting factor; refer to can with human body in specific normal or pathological tissues, organ, cell or cell in specific part produce the material that specific binding, the aminoacid of take are ultimate unit, include but not limited to the antibody protein class materials such as the protein-based and VEGFR2 of polypeptide class, the folic acid etc. such as NGR, RGD.
The preparation method of described Pluronics-paclitaxel amphiphilic macromolecular prodrug, comprises the following steps:
(1) terminal hydroxy group of Pluronics is converted to end carboxyl or 2 ' or 7 hydroxyl of paclitaxel is converted to carboxyl:
Under catalyst exists, in organic solvent, Pluronics or paclitaxel are at room temperature carried out to esterification with anhydride, acquisition contains holds the Pluronics of carboxyl or the paclitaxel of carboxyl modified.
Described catalyst, includes but not limited to pyridine, triethylamine, DMAP, N-hydroxy thiosuccinimide (NHS) etc., is used alone or as a mixture, and its consumption is 0.5～2 times of hydroxyl molal quantity of Pluronics or paclitaxel.
Described organic solvent, includes but not limited to Isosorbide-5-Nitrae-dioxane, dichloromethane, acetone, oxolane, dimethyl sulfoxide or dimethyl formamide equal solvent, is used alone or as a mixture.
Described anhydride, includes but not limited to succinic anhydride, maleic anhydride or dihydroxy acetic acid acid anhydride, and its consumption is 0.5～1.5 times of Pluronics, is 1～2 times of paclitaxel molal quantity.
(2) 2 ' or 7 hydroxyl generation esterification of the end carboxyl of Pluronics and paclitaxel, or the carboxyl generation esterification of the terminal hydroxy group of Pluronics and paclitaxel, forms macromolecule prodrugs of paclitaxel:
In organic solvent, under condensing agent and catalyst exist, 2 ' or 7 hydroxyl of the end carboxyl of Pluronics and paclitaxel carried out to esterification, or the terminal hydroxy group of Pluronics and the carboxyl of paclitaxel carry out esterification, acquisition macromolecule prodrugs of paclitaxel.
Described organic solvent, includes but not limited to Isosorbide-5-Nitrae-dioxane, dichloromethane, acetone, oxolane, dimethyl sulfoxide or dimethyl formamide equal solvent, is used alone or as a mixture.
Described condensing agent, include but not limited to dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylamino-propyl) phosphinylidyne diimine or N, N '-DIC etc., are used alone or as a mixture, and its consumption is 1～2 times of carboxyl molal quantity of Pluronics or paclitaxel.
Described catalyst, includes but not limited to pyridine, triethylamine or DMAP etc., is used alone or as a mixture, and its consumption is 0.5～2 times of carboxyl molal quantity of Pluronics or paclitaxel.
The consumption of described paclitaxel is 0.5～1.5 times of Pluronics polymer molal quantity.
Described carboxylated paclitaxel consumption is 0.5～1.5 times of Pluronics polymer molal quantity.
Further, in described step (1), after having reacted, first use saturated NaHCO
3wash away unreacted anhydride, the hydrochloric acid of 0.1M washes away catalyst; Then add anhydrous magnesium sulfate drying to spend the night (8～12h), dried product carries out separation and purification through silica gel column chromatography technology, obtains the taxol drug that white solid crystal material is carboxyl modified.
A kind of Pluronics-paclitaxel amphiphilic macromolecular prodrug micellar preparation, by following methods, prepared: adopt physical method to process Pluronics-paclitaxel amphiphilic macromolecular prodrug, i.e. self assembly forms Pluronics-paclitaxel amphiphilic macromolecular prodrug micellar preparation.
Described physical method includes but not limited to the methods such as dialysis, film dispersion method, emulsified solvent-volatility process or the nanometer sedimentation method.
Further, and to adding 0.5～20% in the micellar preparation making (unit: freeze drying protectant mg/mL), filtration sterilization, moisture is removed in lyophilization, obtains Pluronics-paclitaxel macromolecular prodrug lyophilizing micellar preparation.
Described freeze drying protectant includes but not limited to lactose, mannitol, sucrose, trehalose, glucose, maltose, glucosan, albumin, sorbitol, fructose, dextran, aminoacid, amino acid salts, phosphate or Polyethylene Glycol etc., is used alone or as a mixture.
Vascular system and basement membrane are the primary barriers that medicine transports in vivo.Microvascular endothelial gap densification in normal structure, structural integrity, macromole and lipid granule are difficult for seeing through blood vessel wall.The blood vessel of tumor tissues is different from normal structure, showing as blood vessel increases rapidly, adventitial cell lacks, basement membrane distortion, lymph pipeline return-flow system is damaged, a large amount of vascular permeability regulators generate, cause tumor vessel to increase the permeability of macromolecular substances and macromolecular substances is trapped in the increase of accumulating of tumor tissues, the selectivity that this tumor tissues has macromolecular substances, high-permeability and anelasticity are called EPR effect (enhanced permeability and retention effect).Pluronics-paclitaxel micelle particle diameter prepared by the present invention is 80～120nm, and it has hydrophilic shell, can prolong drug at the circulation time of blood circulation, therefore can be by EPR effect passive target in tumor tissues.In addition, the terminal hydroxy group of Pluronics is being converted to end carboxyl, or during the hydroxyl generation esterification of the end carboxyl of Pluronics and paclitaxel, the consumption of controlling anhydride or paclitaxel is 0.5～1.5 times of Pluronics polymer molal quantity, make Pluronics reserve part terminal hydroxy group, be used for connecting initiatively targeting factor, be expected to reach the initiatively object of targeted delivery of drugs.
Pluronics-paclitaxel amphiphilic macromolecular prodrug of the present invention and micellar preparation thereof, compare with existing taxol drug preparation, and advantage is as follows:
(1) final products are containing organic solvent and solubilizing agent, and by the Pluronics of good biocompatibility, as pharmaceutical carrier, safety is good.
(2) chemical connection method carrying medicament, drug loading is high.Medicine is connected by covalent bond with carrier, and the release of medicine must have been avoided, because of the unstable burst drug release phenomenon causing in kinetics, having improved the bioavailability of medicine through the effect of relevant enzyme in body, has reduced the toxic and side effects of medicine.
(3) the micellar preparation particle diameter of preparing is even, and size, at 80～120nm, can optionally concentrate in tumor tissues by EPR effect, thereby can increase the therapeutic index of paclitaxel.By its surface being carried out to the modification of targeting factor, be expected to obtain the initiatively micellar preparation of targeting of tumor tissues.
Accompanying drawing explanation
Fig. 1: the chemical structural formula of paclitaxel, in figure, " 2 ' ", " 7 " represent position that can esterified hydroxy groups.
Fig. 2: the synthetic route schematic diagram of Pluronics-paclitaxel amphiphilic macromolecular prodrug (take embodiment 1 as example).
Fig. 3: the H-NMR spectrogram contrast of Pluronics-paclitaxel conjugates, paclitaxel and Pluronics polymer, wherein, (a) paclitaxel; (b) Pluronics polymer; (c) Pluronics-paclitaxel.
Fig. 4: the outside drawing of Pluronics-paclitaxel micelle, wherein, (a) paclitaxel api; (b) Pluronics-paclitaxel micelle; (c) the blank micelle of Pluronics.
Fig. 5: the transmission electron microscope photo of the micelle that Pluronics-paclitaxel forms by self assembly in aqueous medium.
Fig. 6: the particle size distribution figure of Pluronics-paclitaxel amphiphilic macromolecular prodrug micelle.
Fig. 7: the cytotoxicity figure of Pluronics-paclitaxel micelle (A549 and B16 cell).
The cellular uptake figure of the fluorescently-labeled Pluronics-paclitaxel of Fig. 8: FITC and FA-Pluronics-paclitaxel micelle (A549 and B16 cell), wherein, (a) B16 cell; (b) A549 cell.
Fig. 9: mice relative body weight temporal evolution curve chart.
The specific embodiment
Embodiment elaborates to the present invention below: the present embodiment is implemented take technical solution of the present invention under prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1 preparation Pluronics-paclitaxel amphiphilic macromolecular prodrug and micellar preparation (synthetic route as shown in Figure 2) thereof
(1) in the eggplant-shape bottle of 50mL, take 300mg paclitaxel and be dissolved in 10mL anhydrous methylene chloride, add successively succinic anhydride (48mg), DMAP (DMAP, 30mg), under nitrogen protection, stirring at room 24h.Dichloromethane evaporate to dryness is removed, added 10mL ethyl acetate, fully dissolve, then add 10mL dilute hydrochloric acid (mass concentration 10.5%) to carry out pickling, stir 10min, organic facies is collected in extraction, and pickling is 2 times repeatedly.The ethyl acetate layer that contains product spends the night (12h) with anhydrous magnesium sulfate drying, and rotary evaporation is concentrated, the method using a dry method on a sample, and silicagel column separation and purification product, obtains the paclitaxel of carboxyl modified.
(2) in the eggplant-shape bottle of 50mL, the paclitaxel of carboxyl modified (100mg) is dissolved in to 10mL anhydrous methylene chloride, under condition of ice bath, add successively 1-ethyl-3-(3-dimethylamino-propyl) phosphinylidyne diimine (EDC) (34mg), DMAP (10.9mg) and Pluronic P123 (500mg), room temperature reaction 48h.Dialysis 48h, removes dichloromethane, and lyophilization obtains P123-PTX amphiphilic macromolecular prodrug solid.
(3) folic acid (0.03g) is dissolved in to 10mL anhydrous dimethyl sulphoxide (DMSO), add successively N-hydroxy thiosuccinimide (NHS) (0.32g), 1-ethyl-3-(3-dimethylamino-propyl) phosphinylidyne diimine (EDC) (0.53g) and triethylamine (0.2mL), stir 4h.Take P123-PTX (0.4g), join in flask, room temperature reaction 48h.Reactant liquor is transferred in the bag filter of MWCO=3500Da, the 48h that dialyses in distilled water, after DMSO eliminates, lyophilization obtains FA-P123-PTX product solid.
(4) respectively 100mg P123-PTX and FA-P123-PTX amphiphilic macromolecular prodrug lyophilized solid are dissolved in 2mL oxolane (THF), dropwise splash in the distilled water of vigorous stirring, gained mixed solution is packed in processed bag filter (molecular cut off is 3500Da), be placed in 500mL distilled water, dialysis 48h, every 4h changes water one time, removes THF, prepares P123-PTX and FA-P123-PTX micellar solution.
In order to verify formation, targeting, cytotoxicity, the preparation toxicity of the correct synthetic and micellar preparation of Pluronics-paclitaxel amphiphilic macromolecular prodrug.Take embodiment 1 as example, and Fig. 2 is the synthetic route schematic diagram of Pluronics-paclitaxel amphiphilic macromolecular prodrug; Fig. 3 is the H of Pluronics-paclitaxel amphiphilic macromolecular prodrug (P123-PTX arriving that step (3) is prepared)
1nMR structure verification figure (compares P123-PTX conjugate with Pluronic P123 raw material (seeing Fig. 3 b)
1in H-NMR spectrum (seeing Fig. 3 c), there are a series of new absworption peaks, wherein comparatively feature be the peak feature comparatively of multiplet (δ 7.0-8.0) on paclitaxel phenyl ring and δ 8.1 (d, 2H), just in time with paclitaxel
1h-NMR composes (see that Fig. 3 is a) corresponding, known paclitaxel is successfully connected on Pluronic P123).
Fig. 4 is the outside drawing of Pluronics-paclitaxel micelle (the P123-PTX micellar solution arriving that step (4) is prepared), and in figure, a is paclitaxel api, is white suspension; B is the P123 micellar solution of not medicine carrying, and because particle diameter only has 20～30nm, state is clear; C is Pluronics-paclitaxel solution, presents clear state, manifests blue-opalescent.
Fig. 5 is the electron microscopic morphology figure of Pluronics-paclitaxel micelle (the P123-PTX micellar solution arriving that step (4) is prepared), in figure, shows, Pluronics-paclitaxel micelle is spherical shape or near-spherical, and size is more even, good dispersion.
Fig. 6 is the particle size distribution figure of Pluronics-paclitaxel micelle (the P123-PTX micellar solution arriving that step (4) is prepared), in figure, shows, particle diameter is (102.63 ± 7.36) nm.
Fig. 7 be Pluronics-paclitaxel micelle (step (4) prepared to P123-PTX micellar solution) cytotoxicity figure (A549 and B16 cell are subject cell), in figure, show, along with the Enrichment of paclitaxel, the cell inhibitory rate of A549 and B16 cell all increases.Experimental technique: the vitro cytotoxicity that adopts mtt assay research paclitaxel micelle.The A549 of exponential phase and B16 cell line are inoculated into respectively in 96 well culture plates to density about 4 * 10
3individual/hole, cultivates after 24h the P123-PTX micellar solution of variable concentrations.Continue to cultivate after 72h, every hole adds after MTT solution, adopts microplate reader to measure the trap value (A) in every hole.Calculate inhibitory rate of cell growth and half suppression ratio (IC50).
Fig. 8 be the fluorescently-labeled non-targeted Pluronics-paclitaxel micelle of FITC (step (4) prepared to P123-PTX micellar solution) and the picked-up of folate-targeted Pluronics-paclitaxel micelle (the FA-P123-PTX micellar solution arriving that step (4) is prepared) in A549 and B16 cell, in figure, show, in 0 to 4 hour, along with incubation time extends, cell increases the picked-up of micelle, and positive rate increases.In the B16 of folacin receptor positive cell, modified with folic acid can obviously promote the endocytosis of cell to micelle.At 3 time points, with non-targeted-FITC labelling micellar phase ratio, in its cell of targeting-FITC labelling micelle, fluorescence positive rate all has difference (P ﹤ 0.05).In the A549 of folacin receptor feminine gender cell, because the folacin receptor of cell surface expression is less, therefore cell does not rely on folacin receptor mediated endocytic pathway to the picked-up of two kinds of micelles, two kinds of micelles uptake ratio there was no significant difference (P>0.05) in this cell.Illustrate that non-targeted-FITC labelling micelle has better cell-targeting efficiency to folacin receptor positive cell than targeting-FITC labelling micelle in identical incubation time.
Fig. 9 be tail vein injection Pluronics-paclitaxel micelle (the P123-PTX micellar solution arriving that step (4) is prepared) (normal saline and
in contrast), mice relative body weight temporal evolution curve, shows in figure, and the demonstration of mice relative body weight change curve, compares with normal saline administration group, and the Mouse Weight of Pluronics-paclitaxel micelle group decreases, but
group Mouse Weight reduces than Pluronics-paclitaxel micelle group obvious (P<0.01).Because body weight change can be used for judging the toxic and side effects of preparation, so the toxic action of Pluronics-paclitaxel micelle is much smaller than commercially available injection
there is the value for clinical administration.Experimental technique: adopt the female Kunming kind white mice of 18-22g, be divided at random 6 groups, 8 every group, be respectively normal saline group (Normal saline, N.S.), paclitaxel injection
group (
20mg PTX/kg) and P123-PTX group (P123-PTX micelles, 20mg PTX/kg) adopt tail vein injection administration, be administered once weekly, successive administration 3 weeks.After administration, every other day record Mouse Weight.
Embodiment 2
(1) in the eggplant-shape bottle of 50mL, 2g Pluronic P123 and 34mg succinic anhydride are dissolved in 15mL anhydrous dimethyl formamide, then add successively 0.072mL triethylamine (TEA) and 0.08g DMAP, room temperature reaction 24h.Reactant liquor is packed in processed bag filter (molecular cut off is 3500Da), be placed in 500mL distilled water, dialysis 48h, every 4h changes water one time, removes dimethyl formamide, and lyophilization obtains the Pluronic P123 solid product of band edge carboxyl.
(2) in the eggplant-shape bottle of 50mL, the Pluronic P123 of 0.5g band edge carboxyl is dissolved in to anhydrous dimethyl sulphoxide, after polymer dissolution, under condition of ice bath, add 0.036g dicyclohexylcarbodiimide (DCC), 0.023g DMAP and 0.071g paclitaxel, room temperature reaction 48h, filter the precipitation generating in course of reaction, filtrate packs in processed bag filter (molecular cut off is 3500Da), be placed in 500mL distilled water, dialysis 72h, every 4h changes water one time, remove dimethyl sulfoxide, lyophilization obtains P123-PTX amphiphilic macromolecular prodrug solid.
(3) take 150mg P123-PTX amphiphilic macromolecular prodrug, add 2mL acetonitrile, ultrasonic, fully dissolve, 37 ℃ of water-baths, decompression rotary evaporation, eliminates acetonitrile, forms one deck homogeneous film, and room temperature vacuum drying spends the night, and removes residual organic solvent.37 ℃ of heating in water bath 10min, add the synthermal normal saline of 5mL, and stirring at room aquation 2h crosses 0.22 μ m microporous filter membrane, obtains the P123-PTX amphiphilic macromolecular prodrug micellar solution of clarification.Add 0.2g mannose as freeze drying protectant, be sub-packed in aseptic cillin bottle every 2mL; lyophilization, obtains P123-PTX amphiphilic macromolecular prodrug micelle freeze-drying preparation, and this lyophilized formulations adds 2mL normal saline through jolting; can in 1min, redissolve completely, gained micellar solution clear.
Embodiment 3
(1) in the eggplant-shape bottle of 50mL, take 100mg paclitaxel and be dissolved in the anhydrous THF of 5mL, add successively maleic anhydride (17mg), DMAP (10mg), under nitrogen protection, stirring at room 36h.THF evaporate to dryness is removed, added 15mL ethyl acetate, fully dissolve, then add 20mL dilute hydrochloric acid solution to carry out pickling, stir 30min, organic facies is collected in extraction, and pickling is 2 times repeatedly.The ethyl acetate layer that contains product spends the night with anhydrous magnesium sulfate drying, and rotary evaporation is concentrated, the method using a dry method on a sample, and silicagel column separation and purification product, obtains the paclitaxel of carboxyl modified.
(2) in the eggplant-shape bottle of 50mL, the paclitaxel of carboxyl modified (0.2g) is dissolved in to 10mL anhydrous 1,4-dioxane, under condition of ice bath, add successively N, N '-DIC (DIC) (0.056g), DMAP (0.031g) and Pluronic F68 (2.065g), room temperature reaction 48h.Dialysis 72h, removes Isosorbide-5-Nitrae-dioxane, and lyophilization obtains F68-PTX amphiphilic macromolecular prodrug solid.
(3) 200mg F68-PTX amphiphilic macromolecular prodrug lyophilized solid is dissolved in 3mL THF, dropwise splash in the distilled water of vigorous stirring, gained mixed solution is packed in processed bag filter (molecular cut off is 3500Da), be placed in 500mL distilled water, dialysis 48h, every 4h changes water one time, removes THF, prepares F68-PTX amphiphilic macromolecular prodrug micellar solution.
Embodiment 4
(1) in the eggplant-shape bottle of 50mL, 1g Pluronic F127 and 10mg succinic anhydride are dissolved in the anhydrous Isosorbide-5-Nitrae-dioxane of 15mL, then add successively 0.033mL TEA and 0.029g DMAP, room temperature reaction 12h.Reactant liquor is packed in processed bag filter (molecular cut off is 3500Da), be placed in 500mL distilled water, dialysis 48h, every 4h changes water one time, removes Isosorbide-5-Nitrae-dioxane, and lyophilization obtains the Pluronic F127 solid product of carboxyl modified.
(2) in the eggplant-shape bottle of 50mL, the Pluronic F127 of 0.5g carboxyl modified is dissolved in to 10mL anhydrous dimethyl formamide, after polymer dissolution, is cooled to 0 ℃, then add 0.023g EDC, 0.014g DMAP and 0.035g paclitaxel, room temperature reaction 36h.Reactant liquor packs in processed bag filter (molecular cut off is 3500Da), is placed in 500mL distilled water, dialysis 72h, and every 4h changes water one time, removes dimethyl formamide, and lyophilization obtains Pluronic F127 solid.
(3) take 300mg F127-PTX conjugate and be dissolved in 3mL acetone, dropwise splash in 10mL distilled water, stirring at room, until remove acetone completely, crosses 0.22 μ m microporous filter membrane, obtains the 127-PTX amphiphilic macromolecular prodrug conjugate micellar solution of clarification.
Embodiment 5
(1) in the eggplant-shape bottle of 50mL, take 200mg paclitaxel and be dissolved in 8mL anhydrous methylene chloride, add successively succinic anhydride (37mg), DMAP (DMAP, 45mg), under nitrogen protection, stirring at room 36h.Dichloromethane evaporate to dryness is removed, added 20mL ethyl acetate, fully dissolve, then add 10mL dilute hydrochloric acid solution to carry out pickling, stir 30min, organic facies is collected in extraction, and pickling is 2 times repeatedly.The ethyl acetate layer that contains product spends the night with anhydrous magnesium sulfate drying, and rotary evaporation is concentrated, the method using a dry method on a sample, and silicagel column separation and purification product, obtains the paclitaxel of carboxyl modified.
(2) in the eggplant-shape bottle of 50mL, the paclitaxel of carboxyl modified (0.2g) is dissolved in to 10mL anhydrous methylene chloride, under condition of ice bath, add successively EDC (0.084g), DMAP (0.054g) and Pluronic P105 (1.193g), room temperature reaction 48h.Dialysis 96h, removes dichloromethane, and lyophilization obtains P105-PTX amphiphilic macromolecular prodrug solid.
(3) in the eggplant-shape bottle of 50mL, 0.5g P105-PTX amphiphilic macromolecular prodrug and 0.036g folic acid are dissolved in to 5mL anhydrous dimethyl sulphoxide altogether, under condition of ice bath, add add successively N-hydroxy thiosuccinimide (NHS) (0.32g), 1-ethyl-3-(3-dimethylamino-propyl) phosphinylidyne diimine (EDC) (0.53g) and triethylamine (0.2mL), lucifuge, room temperature reaction 48h.Lucifuge dialysis 72h, removes dimethyl sulfoxide, and lyophilization obtains folic acid-P105-PTX solid.
(4) 0.5g folic acid-P105-PTX conjugate lyophilized solid is dissolved in 5mL dimethyl sulfoxide, dropwise splash in the distilled water of vigorous stirring, gained mixed solution is packed in processed bag filter (molecular cut off is 3500Da), be placed in 500mL distilled water, dialysis 96h, every 4h changes water one time, removes dimethyl sulfoxide, prepare folic acid-P105-PTX conjugate micellar solution, preparation process is wanted lucifuge.
Claims (10)
1. a Pluronics-paclitaxel amphiphilic macromolecular prodrug, is characterized in that: be to be formed by ester bond bonding by polyoxyethylene-poly-oxypropylene polyoxyethylene triblock copolymer and paclitaxel.
2. the preparation method of Pluronics-paclitaxel amphiphilic macromolecular prodrug claimed in claim 1, is characterized in that: comprise the following steps:
(1) terminal hydroxy group of Pluronics is converted to end carboxyl or 2 ' or 7 hydroxyl of paclitaxel is converted to carboxyl:
Under catalyst exists, in organic solvent, Pluronics or paclitaxel are at room temperature carried out to esterification with anhydride, acquisition contains holds the Pluronics of carboxyl or the paclitaxel of carboxyl modified;
(2) 2 ' or 7 hydroxyl generation esterification of the end carboxyl of Pluronics and paclitaxel, or the carboxyl generation esterification of the terminal hydroxy group of Pluronics and paclitaxel, forms macromolecule prodrugs of paclitaxel:
In organic solvent, under condensing agent and catalyst exist, 2 ' or 7 hydroxyl of the end carboxyl of Pluronics and paclitaxel carried out to esterification, or the terminal hydroxy group of Pluronics and the carboxyl of paclitaxel carry out esterification, acquisition macromolecule prodrugs of paclitaxel.
3. the preparation method of Pluronics-paclitaxel amphiphilic macromolecular prodrug claimed in claim 2, it is characterized in that: in described step (1), (2), catalyst is selected from any or the arbitrarily several combination in pyridine, triethylamine, DMAP, N-hydroxy thiosuccinimide, and its consumption is 0.5～2 times of hydroxyl/carboxyl molal quantity of Pluronics or paclitaxel.
4. the preparation method of Pluronics-paclitaxel amphiphilic macromolecular prodrug claimed in claim 2, it is characterized in that: in described step (1), (2), organic solvent is selected from any or the arbitrarily several combination in Isosorbide-5-Nitrae-dioxane, dichloromethane, acetone, oxolane, dimethyl sulfoxide or dimethyl formamide.
5. the preparation method of Pluronics-paclitaxel amphiphilic macromolecular prodrug claimed in claim 2, it is characterized in that: in described step (1), anhydride is selected from any or the arbitrarily several combination in succinic anhydride, maleic anhydride or dihydroxy acetic acid acid anhydride, and its consumption is 1～2 times of hydroxyl molal quantity of Pluronics or paclitaxel.
6. the preparation method of Pluronics-paclitaxel amphiphilic macromolecular prodrug claimed in claim 2, it is characterized in that: in described step (2), condensing agent is selected from dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylamino-propyl) phosphinylidyne diimine or N, any in N '-DIC or arbitrarily several combinations, its consumption is 1～2 times of carboxyl molal quantity of Pluronics or paclitaxel.
7. a Pluronics-paclitaxel amphiphilic macromolecular prodrug micellar preparation, it is characterized in that: by following methods, prepared: employing physical method is processed the Pluronics-paclitaxel amphiphilic macromolecular prodrug described in claim 1, i.e. self assembly forms Pluronics-paclitaxel amphiphilic macromolecular prodrug micellar preparation.
8. Pluronics-paclitaxel amphiphilic macromolecular prodrug micellar preparation according to claim 7, is characterized in that: described physical method is dialysis, film dispersion method, emulsified solvent-volatility process or the nanometer sedimentation method.
9. according to the Pluronics-paclitaxel amphiphilic macromolecular prodrug micellar preparation described in claim 7 or 8; it is characterized in that: in the micellar preparation making, add 0.5～20% (unit: freeze drying protectant mg/mL); filtration sterilization; moisture is removed in lyophilization, obtains Pluronics-paclitaxel macromolecular prodrug lyophilizing micellar preparation.
10. Pluronics-paclitaxel amphiphilic macromolecular prodrug micellar preparation according to claim 9, is characterized in that: described freeze drying protectant is selected from any or several combination arbitrarily in lactose, mannitol, sucrose, trehalose, glucose, maltose, glucosan, albumin, sorbitol, fructose, dextran, aminoacid, amino acid salts, phosphate or Polyethylene Glycol.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104288778A (en) * | 2014-10-22 | 2015-01-21 | 河北工业大学 | Method for perfecting hydrophilia of docetaxel |
| CN111317715A (en) * | 2018-12-13 | 2020-06-23 | 四川大学 | Golgi-targeted mucopolysaccharide nano-micelle and preparation method and application thereof |
| CN111615405A (en) * | 2017-11-17 | 2020-09-01 | 国家科学研究中心 | Polymeric prodrugs and subcutaneous and/or intramuscular administration thereof |
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2014
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Non-Patent Citations (1)
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| 李敏: ""紫杉醇-Pluronic P123复合型胶束给药系统的研究"", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104288778A (en) * | 2014-10-22 | 2015-01-21 | 河北工业大学 | Method for perfecting hydrophilia of docetaxel |
| CN104288778B (en) * | 2014-10-22 | 2016-12-07 | 河北工业大学 | One improves the hydrophilic method of Docetaxel |
| CN111615405A (en) * | 2017-11-17 | 2020-09-01 | 国家科学研究中心 | Polymeric prodrugs and subcutaneous and/or intramuscular administration thereof |
| CN111615405B (en) * | 2017-11-17 | 2023-11-28 | 国家科学研究中心 | Polymeric prodrugs and subcutaneous and/or intramuscular administration thereof |
| CN111317715A (en) * | 2018-12-13 | 2020-06-23 | 四川大学 | Golgi-targeted mucopolysaccharide nano-micelle and preparation method and application thereof |
| CN111317715B (en) * | 2018-12-13 | 2021-09-21 | 四川大学 | Golgi-targeted mucopolysaccharide nano-micelle and preparation method and application thereof |
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