Be used for improving nanoparticle of rapamycin bioavailability and preparation method thereofTechnical field
The present invention relates to biomaterial and field of nanometer technology, be specifically related to a kind of have suitable envelop rate and drug loading, high biological safety, and can slowly discharge nanoscopic drug carriers of drug rapamycin and preparation method thereof.
Background technology
Rapamycin (Rapamycin) is the strong immunosuppressive drug with low nephrotoxicity, is widely used in transplant operation.Simultaneously it can effectively suppress again propagation and the transfer ability of vascular smooth muscle cell after blood vessel injury, thereby reduce the generation of vascular restenosis, and it can also promote myocardium damaged place cardiomyocyte proliferation, thereby very large application prospect is arranged aspect treating cardiovascular disease.But, due to the low-solubility of himself and the administering mode of present stage, greatly limited performance and the range of application of its drug effect.
Nano medicament carrying system has become the study hotspot in pharmaceutics and modern biomedical field as a kind of effective means of optimizing drug effect.Study more nano medicament carrying system and comprise nanometer liposome, nanosphere, nanocapsule, solid lipid nanoparticle and polymer capsule etc.At present, the study hotspot of nano medicament carrying system mainly concentrates on the exploitation for the diagnosis of cancer and targeted therapy preparation, and the part preparation has been arranged, and the stage enters clinical trial or comes into the market by experiment.cardiovascular disease is the same with cancer, human health in serious threat, but because part disease pathomechanism is explained imperfection, lack the reasons such as efficient targeting effect group, drug-loading system research for the cardiovascular disease diagnosis medicine lags far behind cancer, and many medicines are because poorly water-soluble, absorbance is low, bioavailability is low, the factors such as oral administration difficulty are not yet well applied, at present, the research of rapamycin nano-carrier still is in the starting stage both at home and abroad, domestic do not have a listing of effective rapamycin sustained-release oral preparation, in view of the good therapeutic effect of this medicine in many diseases field, accord with the demands of the market and clinical needs for a kind of suitable nano-carrier of this medicine exploitation.
This patent uses emulsion-solvent evaporation method to prepare the PEG-PLGA Nano microsphere and carries rapamycin, and similarly research has no report.PLGA and PEG are the high molecular polymer that possesses biological safety of FDA approval, and both synthetic block polymer vivo degradation product has no side effect to body.nanoparticle with the emulsion-solvent evaporation method preparation, internal layer is the core that is formed by physical action by hydrophobicity PLGA block and medicine, skin is hydrophilic PEG hydrated sheath, this hydrated sheath not only can solve the water solublity problem of insoluble drug rapamycin, can also help nano-particle to hide engulfing of reticuloendothelial system, reduce the adhesive attraction of serum albumin, thereby the time of staying of prolong drug in blood circulation, be conducive to the exploitation of oral slow-releasing preparation, avoided the high request of intravenous drug preparation Surfactant and dissolvent residual, potential biological safety risk and the inconvenience on administering mode.Because the PEG end can be active group, can further modify aglucon or other targeting group at nano grain surface by simple chemical reaction, reach the purpose of targeting pathological tissues, organ, thereby improve the drug level of lesions position, reduce the toxic and side effects of medicine to other histoorgan.In addition, PLGA is as a kind of biodegradable polymer, can slow degradation be the small-molecule substance of inanimate object toxicity in cyclic process, discharge simultaneously the medicine of parcel in degradation process, realize the slow release of medicine, can also control the rate of release of medicine by the ratio of lactic acid and hydroxyacetic acid in change PLGA, in the hope of meeting the administration requirements of different pharmaceutical, improve absorbance and the bioavailability of medicine, reach the maximization of drug effect.
Summary of the invention
Technical problem: the objective of the invention is by the nanometer synthetic technology, build the nano medicament carrying system for drug rapamycin, optimize existing drug administration mode, solve the problem of rapamycin water solublity, absorbability, targeting, bioavailability existence.The advantages such as another object of the present invention is to provide a kind of method for preparing above-mentioned rapamycin nano medicament carrying system, makes it have good reproducibility, and is easy to operate, simple, with low cost.
Technical scheme: in order to realize the foregoing invention purpose, the nanoparticle be used to improving the rapamycin bioavailability of the present invention is:
Use polyethylene glycol-polylactic acid hydroxyl ethanol acid copolymer PEG-PLGA as carrier, wherein the PEG end is methoxyl group, amino or carboxyl active group, form the internal package rapamycin with nucleocapsid structure by emulsion-solvent evaporation method, outside hydrophilic particle diameter is the nanoparticle of 100～120nm.
The preparation method of the nanoparticle be used to improving the rapamycin bioavailability of the present invention comprises the following steps:
1) take described copolymer p EG-PLGA and rapamycin, fully be dissolved in acetone respectively;
2) magnetic agitation with the acetone soln of described copolymer p EG-PLGA with after the acetone soln of rapamycin is mixed homogeneously, obtain organic liquid mixture, this organic liquid mixture is splashed in water under magnetic agitation, keep continuing to stir 30～60min, obtain the mixing material of organic facies and water;
3) with step 2) gained mixing material ultrasonic emulsification instrument supersound process 1～2min;
4) vacuum drying 3～6h removes acetone;
5) with step 4) remove acetone after remaining liq be transferred to the centrifugal free drug of removing in centrifuge tube;
6) shift supernatant to ultrafiltration and concentration nano-particle in super filter tube, residual acetone is removed in the ultra-pure water washing;
7) the gained concentrated solution uses the freezer dryer lyophilizing.
Wherein:
Step 1) in, copolymer p EG-PLGA and the rapamycin concentration ratio that feeds intake is 9-11: 1, and use acetone as solvent, the volume ratio of dissolving PEG-PLGA and rapamycin acetone used is 2: 1.
Step 2) in, the volume of water is 2: 1 with the ratio of organic liquid mixture volume, and the speed that organic facies splashes into water is 0.5mL/min.
Beneficial effect: the invention has the advantages that: adopt simple emulsion-solvent evaporation method to prepare nanoparticle, the method is with low cost, do not need super clean experimental situation, favorable repeatability, and can prepare in enormous quantities, have the advantages such as easy row convenient and simple for operation, with low cost and favorable repeatability, these advantages can be widely used in the nanoparticle preparation of various hydrophobic medicine.The nanoparticle good water solubility of preparation, have good biocompatibility and biological safety, and can be active group effectively prolong drug release time, and PEG end, is conducive to realize future target administration and prepares diagnosis and treatment one body preparation.
The specific embodiment
The present invention is described further below in conjunction with specific embodiment.
The preparation of rapamycin medicament-carried nano granule (RAPA-NPs): precision takes 100mg PEG-PLGA and 5mg rapamycin, fully is dissolved in respectively in 4mL and 2mL acetone.Magnetic agitation with the organic facies mix homogeneously after, with the speed of 0.5mL/min, the organic facies mixed liquor is splashed into 12mL aqueous phase under magnetic agitation, keep middling speed to continue to stir 30min.Gained liquid is removed organic facies with the dry 3h of the ultrasonic 1min of ultrasonic emulsification instrument (3s opens for instrument parameters: Φ 3 gear levers, 20%, 20 ℃ of power, and 1s closes) final vacuum.Liquid rotating is moved to centrifugal free drug (parameter: 3000rpm, 25 ℃, the 15min of removing in centrifuge tube, * 2), shift supernatant to ultrafiltration and concentration nano-particle (parameter: 4000rpm, 35 ℃ in super filter tube, 15min, * 2), residual organic solvent is removed in the ultra-pure water washing.Concentrated solution remain in 4 ℃ standby, get part and add 5% (w/v) sucrose as the freeze drying protectant mix homogeneously, lyophilizing in freezer dryer, weighing quality.
Nanoparticle size characterizes: use Particle Size Analyzer and dynamic light scattering to measure particle size and dispersibility, acquired results coincide, and the mean diameter that records is 112.25 ± 5.56nm, and the coefficient of dispersion is 0.248.
Vitro drug release: configure certain density RAPA-NPs solution 2mL, solvent is PBS: ethanol=9: 1, and put into 37 ℃ of calorstats and hatch, respectively at 4h, 7h, 20h, 30h, 42h, 52h, 70h takes out centrifugal (10000rpm, 25 ℃, 30min), draw supernatant 1.5mL and be stored in 4 ℃ of concentration to be measured, add the 1.5mL solvent and continue to hatch in nanoparticle precipitate.Use ultraviolet spectrophotometer to measure rapamycin in the supernatant of hatching the different time acquisition and, at the light absorption value at 277nm place, by the linear fit Equation for Calculating, go out concentration, draw the drug release curve, and carry out match, determine drug release model and equation thereof.Experimental result shows that within 20h, release amount of medicine arrives 40%, and slow release duration＞90h, show and can play good medicament slow release effect afterwards, and pharmaceutical release time-concentration relationship meets Higuchi and discharge model, meets the experiment expected results.
The cytotoxicity test: use CCK-8 test kit and schwann cell to carry out the cytotoxicity test, administration concentration is respectively 0.002,0.02,0.2,2,20 μ g/mL, and the continuous culture observation of cell is active.Test result shows, the rapamycin nanoparticle can suppress cell-proliferation activity to a certain extent, and inhibition strengthens with concentration in certain concentration range, but do not observe the reduction of cell quantity, show that this nano-particle does not have cell killing toxicity, has good biological safety.