CN103263672B - Preparation method and application of nanometer particles of taxane drugs - Google Patents
Preparation method and application of nanometer particles of taxane drugs Download PDFInfo
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Abstract
The invention discloses a preparation method of a nanometer preparation of taxane drugs. The nanometer particles prepared by utilizing an emulsion diffusion method comprise the taxane drugs, copolymer carrier materials and emulgators. The preparation method comprises the steps of: dissolving the taxane drugs and the copolymer carrier materials into an organic phase mutually soluble with water, then dropwise adding the solution into a mixed solution of water in which the emulgators are dissolved and ethanol, stirring and mixing to form emulsion, and forming nanometer particles after removing organic solvents and solidifying. According to the nanometer particles prepared by utilizing the preparation method, the particle sizes are very uniform, the average particle size is 80-120 nm, the encapsulation efficiency is high, the stability is good, the preparation process is simple and controllable, and the scaled-up production is liable. The nanometer particles prepared by utilizing the preparation method can be applied to the treatment of malignant tumors and have obvious tumor inhibition effects.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, relate to the polymer nanoparticle that bag carries insoluble anti-tumor medicament, be specifically related to a kind of preparation method and application of the nanoparticle containing taxanes insoluble anti-tumor medicament.
Background technology
Taxone mainly comprises paclitaxel and Docetaxel, is current one of most effective antitumour medicine clinically.Paclitaxel (paclitaxel) is separated a kind of tetracyclic diterpene compound obtained from the bark of Ramulus et folium taxi cuspidatae of the Pacific Ocean, and molecular formula is C
47h
51o
14n, molecular weight is 853.9, water insoluble.U.S. FDA approval listing in 1992, its mechanism of action is combined with cellular tubulin, promotes tubulin polymerization, to resist depolymerization, to block mitosis, thus Tumor suppression growth, inducing apoptosis of tumour cell.Clinical research has confirmed that paclitaxel is at the multiple entity tumor for the treatment of, comprises breast carcinoma, advanced ovarian cancer, pulmonary carcinoma, brain and the aspect such as tumor colli and acute leukemia, has significant effect.The non-cell toxicity precursor compound 10-deacylated tRNA base Tetraol II that Docetaxel (docetaxel) is extracted by Taxus baccata obtains through semi-synthetic, within 1998, obtains U.S. FDA approval listing.The mechanism of action of Docetaxel is similar to paclitaxel, but anti-tumor activity is the doubly a lot of of paclitaxel, has obvious therapeutic effect to the cancer such as breast carcinoma, nonsmall-cell lung cancer.
Its water solublity of taxone lower (about 1 ~ 10 μ g/mL), therefore used clinically paclitaxel injection and Docetaxol injection all need to use surfactant polyoxyethylene Oleum Ricini (Cremophor EL) and polysorbate 80 (Tween 80) and cosolvent ethanol to reach the object of dissolved substance respectively.Existing listing dosage form is paclitaxel injection (commodity are called " taxol "), with poly-hydroxyethyl Oleum Ricini (Cremophor EL): ethanol=1: 1 for solvent, concentration is 6mg/mL, normal saline or dextran dilution 5 ~ 20 times during clinical practice, once quiet note 3h, dosage is 135mg/m
2.Polyoxyethylene castor oil has been proved to be and can have caused serious anaphylaxis, neurotoxicity, nephrotoxicity and hypotension etc.In addition, polyoxyethylene castor oil also can form molecule parcel taxane molecule in blood, affects drug molecule and spreads between tissue, reduce antitumous effect.The Docetaxel injection (commodity are called " taxotere ") of current listing, the double solvent of Tween 80 and dehydrated alcohol is adopted to be prepared from, the use of Tween 80 easily causes the anaphylaxis of serious patient, before chemotherapy, patient often needs to take or inject Claritin (corticoid and bisfentidine) in advance, in infusion process, also need to connect microfilter and enter patient blood blood circulation with the crystal fine grain preventing medicine from producing in dilution, these all bring great risk and inconvenience to clinical practice.
For eliminating the untoward reaction of existing taxone dosage form, increase dissolubility, the stability of taxoid drug, reduce toxicity, domestic and international researcher expands the research of a large amount of taxone novel form, comprises cyclodextrin clathrate, micelle, liposome, albumin etc.Although these novel forms improve the deficiency of existing preparation to a certain extent, all there is respective defect, as: there is high nephrotoxicity in cyclodextrin itself; Liposomal formulation envelop rate is low, unstable, easy to be revealed; Albumin paclitaxel complicated process of preparation etc.These are difficult to industrialized development and the reagent clinical practice of the above-mentioned new formulation of the drawbacks limit overcome.
Use polymeric material to prepare nanoparticle to be in recent years subject to people as anti-cancer medicament carrier and to pay close attention to widely.Polymer support has slow releasing medicine, reduces drug toxicity, promotes that medicine is in effects such as tumor locus absorptions.Adjuvant for the preparation of nano particle preparations mostly is high molecular degradable polymer, polyester is one of most widely used Biodegradable polymer material, and conventional has polylactic acid (PLA), polyglycolic acid (PGA), PLGA (PLGA) and poly-caprolactone (PCL) etc.Wherein nontoxic, the nonirritant of PLGA, has good biocompatibility, biodegradable and absorb, and its catabolite can participate in body metabolism, and therefore PLGA is widely used in pharmacy, pharmaceutical engineering material by U.S. FDA approval.By regulating lactic acid and the ratio of glycolic and the molecular weight of PLGA in PLGA, the polymer with different degradation rate can be obtained, thus meet the demand with the nano-particle of different release in vitro character.
Long circulating has very important significance for long-acting the working of nanoparticle.Be easy to by macrophage identification due to PLGA nanoparticle and engulf, circulation time is shorter in vivo, often can not play enough drug effects, use Polyethylene Glycol (polyethylene glycol, PEG) modify and make nanoparticle escape opsonic action in body to increase the circulation time in nanometer plastochondria, become the focus preparing long-circulating nanoparticles in recent years.The long circulating of surface nature to nanoparticle of nanoparticle plays very important effect, and surface will have enough hydrophilic that the opsonification in human body just can better be avoided to be removed from blood by nanoparticle.The finishing of long-circulating nanoparticles is generally obtained by physical absorption or some hydrophilic high molecular materials of chemical bonding on the surface of common nanoparticle, common hydrophilic high molecular material has polyoxyethylene (PEO), polyvidone (PVP), methoxy poly (ethylene glycol) (mPEG), Polyethylene Glycol (PEG), three pear esters 80, poloxamer, water solublity tocopherol (TPGS) etc., and these materials all can reach and improve macrocyclic object.Compared with common nanoparticle, the half-life of long-circulating nanoparticles in blood circulation significantly improves, and is therefore also called " stealth " nanoparticle.
Polyethylene glycol 1000 vitamin E succinic acid ester (TPGS) is the soluble derivative of vitamin E, formed by the carboxyl of vitamin e succinate (VES) and Polyethylene Glycol (PEG) 1000 esterification, relative molecular mass is about 1513, loaded " American Pharmacopeia ".TPGS is produced by Eastman company of the U.S. and is gone on the market the earliest, now be widely used in abroad in preparation research, as the carrier of solubilizing agent, absorption enhancer, emulsifying agent, plasticizer and slightly water-soluble and fat-soluble medicine transmission system, as the carrier of solid dispersion, dosing eyes, the carrier etc. of intranasal administration.TPGS is due to the hydrophilic interaction of PEG chain segment in addition, is also often used as the face finish material of " stealth " nanoparticle.
The conventional method preparing nanoparticle has emulsion process and nanoprecipitation method.The emulsion process of a lot of improvement of development in recent years and nanoprecipitation method, preparation technology has very large improvement, as we disclose a kind of reverse dropping aqueous phase to prepare long-circulating paclitaxel or docetaxel nano-particle preparation method (CN 102772368 and CN 102772369) to the mode of oil phase.Generally speaking, the technology just preparing long circulating taxane nanoparticle this respect is at present also immature, does not still have the preparation technology of a set of maturation to prepare desirable long-circulating nanoparticles, and finally realizes scale expanding production.
Summary of the invention
For above shortcomings in prior art, technical problem to be solved by this invention is the preparation method providing a kind of taxone nano particle preparations, the taxanes nanometer formulation prepared by this method can long circulating, toxic and side effects is low, antitumor curative effect is high, can intravenous injection.
The invention discloses a kind of nanoparticle of taxone.The nanoparticle adopting emulsion diffusion method to prepare comprises taxone, copolymerization carrier material and emulsifying agent.Taxone and copolymerization carrier material are dissolved in the organic facies of dissolving each other with water, are then added drop-wise to and are dissolved with in the water of emulsifying agent and the mixed solution of ethanol, be uniformly mixed formation emulsion, after removing organic solvent, solidification, form nanoparticle.Long circulating taxone nanoparticle prepared by the present invention can be applicable to the treatment of malignant tumor, has obvious tumor killing effect.
Technical problem to be solved by this invention is to provide a kind of brand-new polymer nanoparticle preparation method, because the existence of organic solvent two in emulsion diffusion method improves the diffusion rate of organic solvent at aqueous phase, and method is easy, the nanoparticle particle size distribution prepared is very even.
The present invention seeks to be achieved through the following technical solutions:
A kind of taxone nano particle preparations, comprises taxone, copolymer and emulsifying agent in preparation.
Wherein, described taxone is paclitaxel or Docetaxel; Described copolymer is polylactic acid (PLA), Poly(D,L-lactide-co-glycolide (PLGA) or PLGA-PEG-PLGA (PEG-PLA), PEG-PLGA copolymer (PEG-PLGA), or both mixture arbitrarily, described copolymer and the part by weight of paclitaxel are 5: 1 to 20: 1, are particularly preferably 10: 1.
Described emulsifying agent is watermiscible vitamin E (TPGS) or Solutol HS15 (HS15) or both mixture, is preferably TPGS.
The present invention utilizes TPGS or HS15 to form the Shell Materials of one deck protection nanoparticle as emulsifying agent; and we are on technique Shang Lingpixin road; the emulsion diffusion method containing two organic solvent is adopted to prepare uniform particle sizes, envelop rate is high, drug loading is high novel paclitaxel or docetaxel nano-particle; size controlling is in 80 ~ 120 nanometers, and the entrapment efficiency of nanoparticle can reach 80% to 95%.
Present invention also offers the preparation method of above-mentioned taxanes nano particle preparations, comprise the steps:
Taxone and copolymer are dissolved in as oil phase in organic solvent by i, as aqueous phase in the cosolvent of the water-soluble and ethanol of newborn solvent;
Oil phase instills in the cosolvent of water and ethanol by ii under shear action, obtains the emulsion of blue-opalescent;
Iii removes organic solvent.
Wherein, the described organic solvent one that to be organic solvent be in acetone, oxolane or mixture, be preferably acetone, the volume ratio of oil phase and aqueous phase is 1: 1 to 1: 3.
In traditional emulsion-solvent evaporation method, the volatilization of the formation of emulsion droplets and solvent needs greatly by the effect of external force, as ultrasonic, homogenize and the effect of negative pressure, the inhomogeneities of external force condition can cause the uneven of particle size distribution to a great extent.Have employed emulsion diffusion method in the present invention, is the improvement to conventional emulsion-solvent evaporation method, employs the solvent dissolved each other with water, as acetone etc.First by taxone and the water-soluble solvent dissolved each other of copolymer during preparation, then join and be dissolved in the mixed solution containing ethanol and water (ratio 1: 1) containing emulsifying agent as TPGS, form emulsion.Emulsifying agent is adsorbed on emulsion droplet surface, can prevent intergranular gathering from occurring.Water intersolubility solvent is as affine in acetone in copolymer material; and ethanol is affine in emulsifying agent; due to the dispersal behavior that acetone solvent is spontaneous; in preparation process; the ethanol first outside aqueous phase diffusion from emulsion droplets of parent's emulsifying agent; inner emulsion surface tension is reduced; particle diameter reduces rapidly; the acetone solvent of close copolymer outwards aqueous phase diffusion further subsequently; because acetone is not strong to emulsifying agent affinity; the increase of its concentration makes emulsifying agent flocculate on emulsion droplets surface, thus is adsorbed on nanoparticle surface formation protective layer.The coupling of two kinds of different affinity solvents uses the dispersion effect that improve efficiently emulsion, make emulsion droplets can spontaneous formation reducing, only need to stir stably to obtain the polymer drug-carried nanoparticle that particle diameter is less, be evenly distributed, also improve parcel efficiency, so emulsion-diffusion method has the prospect of industrial application.
Effect of nano-paclitaxel disclosed by the invention, because its size controlling is at 80-120nm, can be used for intravenous injection, surfactant for decorated nanometer grain is TPGS or HS15, wherein TPGS is the soluble derivative of vitamin E, be widely used in food and medicine industry, high to the safety of human body.The PEG chain segment of TPGS or HS15 is that the long circulating of nanoparticle provides condition, the sterically hindered of nanoparticle surface can be increased, nanoparticle contacts each other and tails off, and also just avoids the agglomeration between nanoparticle, adds the stability of nanoparticle in cyclic process.TPGS has certain effect to the drug resistance of inhibition tumor cell also tool according to another report, and what the molecular structure of its uniqueness can suppress medicine pumps effect, and TPGS has inhibitory action to P-glycoprotein.HS15 is a kind of novel high-performance, extremely hypotoxic solubilizing agent or emulsifying agent, and its advantageous exists: 1) high solubility emulsifiability ability; 2) low histamine release effect; 3) low haemolysis; In addition HS15 also has higher physiological tolerance, lower viscosity, and can adopt steam sterilization, and without the need to the aseptic manufacturing process of costliness, current HS15 takes in Deutscher Arzneibucs, and at America & Canada, oneself passes through the authorization for human injection.
The nanometer formulation of taxoid drug of the present invention, taxanes pharmaceutical pack is wrapped in the copolymer, solve well existing taxone because dissolubility is low can only with an organic solvent, the problem of the dangerous material such as polyoxyethylene castor oil, Tween 80.In addition, taxone nano-particle composition of the present invention can keep good stability in production, storage and transport and administration process, and produces drug effect entering this nanoparticle after in body and can discharging medicine rapidly.Nano-particle composition cost of the present invention is low, and stability is high, and safety is good, meets the requirement of clinical application, meets the requirement of large-scale production, possesses good market prospect.
The present invention also investigates the antitumor performance of nanoparticle, with mice notch graft tumor for model, compare the difference of the tumor killing effect between effect of nano-paclitaxel under same dose prepared by this experiment and market sale anti-tumor agent taxol (Taxol).After effect of nano-paclitaxel administration, the paclitaxel of Nano grade and contact tumor tissue also permeate to its depths, specific killing is carried out to tumor tissues, because the nanoparticulate materials of EPR effect particle diameter between 50-200nm is easily assembled at tumor locus, play the effect of drug accumulation, better can kill and wound tumor, the existence due to shells such as TPGS makes nanoparticle can discharge medicine slowly, plays good slow release effect.
Accompanying drawing explanation
Fig. 1 is the grain size distribution that bag carries the nanoparticle of paclitaxel
Fig. 2 is the scanning electron microscope (SEM) photograph that bag carries the nanoparticle of paclitaxel: (A) low power enlarged drawing; (B) high magnification map
Fig. 3 is the In-vitro release curves that bag carries the nanoparticle of paclitaxel
Fig. 4 is the impact that bag carries the suppression ratio of the cell in vitro strain of the nanoparticle of paclitaxel
Fig. 5 is the pharmacokinetic profile that bag carries the nanoparticle of paclitaxel
Fig. 6 is the impact that bag carries nanoparticle internal organs of the body distribution in A549 Model in Nude Mice of fluorescent probe: (A) mouse systemic figure; (B) main organs scattergram; (C) nanoparticle is in the fluorescence cartogram of main organs;
Fig. 7 is that the nanoparticle of bag year paclitaxel affects people A549 Nude Mice gross tumor volume
Fig. 8 wraps the nanoparticle carrying paclitaxel transplants A549 tumor nude mouse body weight variable effect to people
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention through a step, but not limit the present invention in any form.
The preparation of embodiment 1:TPGS emulsifying effect of nano-paclitaxel
80mg PLGA (50: 50, molecular weight 45000) and 8mg paclitaxel are dissolved in as oil phase in 20mL acetone solvent, and 240mgTPGS is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; Oil phase is contained the mixed solution of TPGS with the instillation of the speed of 1mL/min, under 300r/min stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, nano-emulsion being transferred to rotavapor under vacuum and rotate removing organic solvent, obtain nanoparticle.The mean diameter that dynamic Laser scatterometer records is 105.1 ± 2.3nm, and as shown in Figure 1, particle size distribution FACTOR P DI is 0.047 ± 0.025 to particle size distribution, and the envelop rate of nanoparticle is 92.5 ± 2.2%.Carry out morphology analysis with the nanoparticle of transmission electron microscope to preparation, Fig. 2 illustrates that taxol nanoparticle particle diameter prepared by the present embodiment is at about 100nm, and distributes very evenly.
The preparation of embodiment 2:TPGS emulsifying effect of nano-paclitaxel
80mg PLGA (50: 50, molecular weight 15000) and 8mg paclitaxel are dissolved in as oil phase in 20mL acetone solvent, and 240mgTPGS is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; Oil phase is contained the mixed solution of TPGS with the instillation of the speed of 1mL/min, under 300r/min stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, nano-emulsion being transferred to rotavapor under vacuum and rotate removing organic solvent, obtain nanoparticle.The mean diameter of dynamic Laser scatterometer test is 102.3 ± 2.4nm, and particle size distribution FACTOR P DI is 0.073 ± 0.012, and the envelop rate of nanoparticle is 85.2 ± 2.4%.
The preparation of embodiment 3:TPGS emulsifying effect of nano-paclitaxel
80mg PLGA (50: 50, molecular weight 100000) and 8mg paclitaxel are dissolved in as oil phase in 20mL acetone solvent, and 240mgTPGS is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; Oil phase is contained the mixed solution of TPGS with the instillation of the speed of 1mL/min, under 300r/min stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, nano-emulsion being transferred to rotavapor under vacuum and rotate removing organic solvent, obtain nanoparticle.The mean diameter of dynamic Laser scatterometer test is 109.1 ± 2.2nm, and particle size distribution FACTOR P DI is 0.062 ± 0.018, and the envelop rate of nanoparticle is 86.4 ± 2.1%.
The preparation of embodiment 4:TPGS emulsifying effect of nano-paclitaxel
100mg PEG5000-PLGA (50: 50, molecular weight 28000) and 10mg paclitaxel are dissolved in as oil phase in 20mL acetone solvent, and 120mgTPGS is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; By oil phase with the speed of 1.5mL/min instillation mixed solution, under stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, nano-emulsion being transferred to rotavapor under vacuum and rotate removing organic solvent, obtain nanoparticle.The mean diameter of dynamic Laser scatterometer test is 112.1 ± 2.5nm, and particle size distribution FACTOR P DI is 0.092 ± 0.032, and the envelop rate of nanoparticle is 81.5 ± 4.2%.
The preparation of embodiment 5:TPGS emulsifying effect of nano-paclitaxel
50mg PEG5000-PLGA (50: 50, molecular weight 15000), 50mg PLGA (50: 50, molecular weight 45000) and 12mg paclitaxel be dissolved in as oil phase in 20mL acetone solvent, 240mgTPGS is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; By oil phase with the speed of 1mL/min instillation mixed solution, under 300r/min stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, Emulsion being transferred to rotavapor under vacuum and rotate removing organic solvent, obtain nanoparticle.The mean diameter of dynamic Laser scatterometer test is 106.1 ± 4.2nm, and particle size distribution FACTOR P DI is 0.092 ± 0.045, and the envelop rate of nanoparticle is 84.2 ± 3.8%.
The preparation of embodiment 6:TPGS emulsifying effect of nano-paclitaxel
100mg PLGA (50: 50, molecular weight 45000) and 10mg paclitaxel are dissolved in as oil phase in 20mL tetrahydrofuran solvent, and 240mgTPGS is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; By oil phase with the speed of 1mL/min instillation mixed solution, under 300r/min stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, nano-emulsion being transferred to rotavapor under vacuum and rotate removing ethanol and oxolane, obtain nanoparticle.The mean diameter of dynamic Laser scatterometer test is 96.1 ± 8.9nm, and particle size distribution FACTOR P DI is 0.105 ± 0.037, and the envelop rate of nanoparticle is 87.1 ± 4.5%.
The preparation of embodiment 7:HS15 emulsifying effect of nano-paclitaxel
80mg PLGA (50: 50, molecular weight 45000) and 8mg paclitaxel are dissolved in as oil phase in 20mL acetone solvent, and 240mgHS15 is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; By oil phase with the speed of 1mL/min instillation mixed solution, under 300r/min stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, Emulsion being transferred to rotavapor under vacuum and rotate or through solution recycle system removing organic solvent, just obtain nanoparticle.The mean diameter of dynamic Laser scatterometer test is 104.5 ± 2.6nm, and particle size distribution FACTOR P DI is 0.083 ± 0.029, and the envelop rate of nanoparticle is 96.5 ± 2.1%.
The preparation of embodiment 8:TPGS and HS15 emulsifying effect of nano-paclitaxel
160mg PLGA (50: 50, molecular weight 45000) and 15mg paclitaxel are dissolved in as oil phase in 20mL acetone solvent, 120mgTPGS and 120mg HS15 is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; By oil phase with the speed of 1mL/min instillation mixed solution, under 300r/min stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, Emulsion being transferred to rotavapor under vacuum and rotate, obtain nanoparticle.The mean diameter of dynamic Laser scatterometer test is 121.7 ± 4.3nm, and particle size distribution FACTOR P DI is 0.073 ± 0.032, and the envelop rate of nanoparticle is 81.1 ± 5.2%.
The preparation of embodiment 9:TPGS emulsifying docetaxel nano-particle
40mg PLGA (50: 50, molecular weight 45000) and 4mg Docetaxel are dissolved in as oil phase in 20mL acetone solvent, and 240mgTPGS is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; By oil phase with the speed of 1mL/min instillation mixed solution, under 300r/min stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, Emulsion being transferred to rotavapor under vacuum and rotate removing organic solvent, just obtain nanoparticle.The mean diameter of dynamic Laser scatterometer test is 104.4 ± 2.1nm, and particle size distribution FACTOR P DI is 0.098 ± 0.026, and the envelop rate of nanoparticle is 94.5 ± 2.2%.
The preparation of embodiment 10:TPGS emulsifying docetaxel nano-particle
80mg PEG5000-PLA (50: 50, molecular weight 45000) and 8mg paclitaxel are dissolved in as oil phase in 20mL acetone solvent, and 120mgTPGS is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; By oil phase to be injected into fast in mixed solution with the speed of 10mL/min with syringe, under 500r/min stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, Emulsion being transferred to rotavapor under vacuum and rotate removing organic solvent, just obtain nanoparticle.The mean diameter of dynamic Laser scatterometer test is 110.5 ± 4.5nm, and particle size distribution FACTOR P DI is 0.12 ± 0.031, and the envelop rate of nanoparticle is 85.3 ± 3.2%.The preparation of embodiment 11:HS15 emulsifying docetaxel nano-particle
80mg PLGA and 8mg paclitaxel are dissolved in as oil phase in 20m acetone solvent, and 180mgHS15 is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; By oil phase with the speed of 1mL/min instillation mixed solution, under 300r/min stirring at low speed, form nattier blue nano-emulsion, after being added dropwise to complete, Emulsion being transferred to rotavapor under vacuum and rotate removing organic solvent, obtain nanoparticle.The mean diameter of dynamic Laser scatterometer test is 145.1 ± 3.2nm, and particle size distribution FACTOR P DI is 0.082 ± 0.041, and the envelop rate of nanoparticle is 87.5 ± 2.8%.
The preparation of embodiment 12:TPGS emulsifying docetaxel nano-particle
80mg PLGA and 8mg paclitaxel are dissolved in as oil phase in 20mL tetrahydrofuran solvent, and 80mgTPGS is dissolved in 40mL water and ethanol contend ratio is form mixed solution in the cosolvent of 1: 1; Oil phase syringe is injected mixed solution fast with the speed of 10mL/min, under 500r/min stirring at low speed, forms nattier blue nano-emulsion, after being added dropwise to complete, Emulsion being transferred to rotavapor under vacuum and rotate removing organic solvent, just obtain long-circulating nanoparticles.The mean diameter of dynamic Laser scatterometer test is 156.2 ± 3.8nm, and particle size distribution FACTOR P DI is 0.075 ± 0.024, and the envelop rate of nanoparticle is 82.5 ± 4.2%.
Embodiment 13: the mensuration of nanoparticle envelop rate
Liquid phase high efficiency chromatography is adopted to survey the content of paclitaxel: chromatographic column: Agilent C18 post; Mobile phase: acetonitrile: water (50: 50 (v: v)); Determined wavelength 227nm; Flow velocity 1.0mL/min; Sample size 20 μ L.Get paclitaxel or Docetaxel standard solution that concentration is 0.05 ~ 50ug/mL respectively, test according to chromatographic condition, with peak area, paclitaxel or Docetaxel concentration are carried out curve fitting, set up regression equation.
The nanoparticle suspension obtained is crossed the film of 0.22um, the suspension after mistake film is added the acetonitrile breakdown of emulsion of equal volume, the solution obtained by breakdown of emulsion measures the content of paclitaxel or Docetaxel according to chromatographic condition.The nanoparticle suspension simultaneously got without any process adds the acetonitrile breakdown of emulsion of same volume, surveys the content of paclitaxel or Docetaxel according to HPLC condition:
Amount × 100% that the amount/input medicine of the medicine of envelop rate (%)=nanoparticle encapsulating is total
The average envelop rate of gained nanoparticle is 80 ~ 95%.
Embodiment 14: the vitro drug release containing taxol drug nanoparticle is tested
The drug-carrying nanometer particle prepared (embodiment 1, embodiment 2 and embodiment 3) is dissolved in appropriate PBS buffer (pH7.4, containing 0.5% tween 80), is diluted to paclitaxel 1mg/mL, mixing.Get 5mL and be placed in bag filter, tighten bag filter.Bag filter is put into 50mL PBS buffer (pH7.4, containing tween 80 0.5%), 37 DEG C, 100r/min, get the outer PBS liquid 0.5mL of bag filter in different time points.Measure content of taxol (chromatographic column: ODS (Lichrospher-C18,250 × 4.6mm, 5 μm) respectively; Mobile phase: methanol-acetonitrile-water (30: 40: 32); Flow velocity: 1.0mL/min, determined wavelength: 226nm; Column temperature: 30 DEG C), the results are shown in Figure 3.
Embodiment 15: the vitro cytotoxicity experiment of nanoparticle
Human lung cancer cell A549 is cultivated in advance before toxicity test, at exponential phase cells, cell is inoculated in 96 orifice plates with the quantity of every hole 6000 cell, to the nanoparticle prepared by embodiment 1 and embodiment 1 not drug-carrying nanometer particle and market sale anti-tumor agent taxol carry out external inhibiting tumor assay, ensure that the concentration of medicine between drug-carrying nanometer particle and taxol is consistent.Cell continues cultivation 48 hours, using cell survival rate as evaluation index, compares the anti-tumor activity between them, the results are shown in Figure 4.
Embodiment 16: bag carries the interior medicine dynamics experiment of effect of nano-paclitaxel
The PLGA nanoparticle that the bag obtained to embodiment 1 carries paclitaxel carries out pharmacokinetic studies in body, take taxol as positive control.Get healthy female sd inbred rats (200 ± 10) grams totally 8, be divided into 2 groups at random, often organize 4, fasting one night before administration.Respectively with the dosage of 10mg/kg to often organizing rat tail vein administration, after 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1,2,4,8,12,24 hour, get blood 200 μ L respectively, 5000rpm centrifugalize.The content of taxol in blood plasma is detected by high performance liquid chromatography after acetonitrile treatment.Fig. 5 is the Drug-time curve obtained, and compared with taxol, effect of nano-paclitaxel prepared by embodiment 1, has long circulating action.
Embodiment 17: bag carries nanoparticle internal organs of the body distribution experiments in A549 Model in Nude Mice of fluorescent probe
80mg PLGA (50: 50, molecular weight 45000) and 0.8mg fluorescent probe DiR (1, the two octadecyl-3 of 1-, 3,3,3 tetramethyl iodate) be dissolved in as oil phase in 20mL acetone solvent, it is form cosolvent phase in the cosolvent of 1: 1 that 240mgTPGS is dissolved in 40mL water with ethanol contend ratio; By oil phase with the speed of 1mL/min instillation cosolvent phase, under 300r/min stirring at low speed, form nano-emulsion, after being added dropwise to complete, Emulsion being transferred to rotavapor under vacuum and rotate removing organic solvent, obtain nanoparticle.
A549 cell inoculation nude mouse, when gross tumor volume grows to about 500mm
3be divided into 3 groups at random, often organize 3 mouse, after tail vein injection, respectively at 2 hours, 8 hours, 24 hours points, living imaging is carried out to mouse systemic, sees Fig. 6 A.Then after being put to death by nude mice, take out main internal organs (heart, liver, spleen, lung, kidney) and tumor, by the distribution situation of nanoparticle in organizing like this of mice living imaging systems axiol-ogy parcel DiR fluorescence, see Fig. 6 B.Nanometer has obvious gathering at tumor locus, has passive target function.According to the fluorescence intensity signals that living imaging instrument records, carry out statistical analysis, obtain quantitative fluorescence data, see Fig. 6 C.
Embodiment 18: the antitumor zoopery of effect of nano-paclitaxel
In order to better evaluate the antitumor performance of our nanoparticle, below use effect of nano-paclitaxel (embodiment 1 and embodiment 7) with existing clinical paclitaxel injection (taxol), on murine lung cancer cell A549 cell line subcutaneous tumors model, observation and comparison has been carried out, shown in 7 to tumor killing effect.
Experimental result show: effect of nano-paclitaxel dosage form (dose of paclitaxel is 10mg/kg) compared with paclitaxel injection dosage form (content of taxol is 10mg/kg), the effect that effect of nano-paclitaxel has good Tumor suppression to grow to A549 cell line subcutaneous tumors model.In addition, paclitaxel nano dosage form does not significantly affect (Fig. 8) to Mouse Weight change, illustrates that new paclitaxel nano dosage form toxicity is very low.
Claims (7)
1. prepare a method for taxone nanometer formulation, it is characterized in that the method comprises the following steps:
Taxone and copolymer are dissolved in as oil phase in organic solvent by i, as aqueous phase in the water-soluble cosolvent formed with ethanol of emulsifying agent;
Oil phase is added dropwise to aqueous phase by ii under stirring at low speed, obtains the emulsion of blue-opalescent;
Iii uses Rotary Evaporators to remove organic solvent,
Wherein, the volume ratio of described oil phase and described aqueous phase is 1: 1 to 1: 3, and described oil phase and water are total to molten.
2. preparation method according to claim 1, wherein said organic solvent is a kind of in acetone, oxolane or both mixture.
3. preparation method according to claim 2, wherein said organic solvent is acetone.
4. preparation method according to claim 1, wherein said taxone is paclitaxel or Docetaxel.
5. preparation method according to claim 1, wherein said copolymerization carrier material is polylactic acid, Poly(D,L-lactide-co-glycolide or PLGA-PEG-PLGA, PEG-PLGA copolymer, or both mixture arbitrarily; Described copolymer and the part by weight of taxone are 5: 1 to 20: 1.
6. preparation method according to claim 1, wherein said emulsifying agent is watermiscible vitamin E (TPGS), Solutol HS15 (HS15) or both mixture.
7. preparation method according to claim 1, the ratio of wherein said water and described ethanol is 1: 1.
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