CN103006539A - Polymeric micelle medicine composition and preparation method thereof - Google Patents
Polymeric micelle medicine composition and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a polymeric micelle medicine composition encapsulating hydrophobic anti-tumour drug and multidrug resistance reversal agent pluronic at the same time and a preparation method thereof. The polymeric nano micelle composition contains polyethylene glycol-polyester segmented copolymer (or derivative thereof), tumour multidrug resistance reversal agent pluronic and anti-tumour drug. The functional anti-tumour drug nano micelle provided by the invention can effectively reverse multidrug resistance phenomenon of tumour, achieves the aim of targeted high-efficiency tumour treatment and has greater advantage compared with a common anti-tumour drug polymeric micelle preparation.
Description
Technical field
The invention belongs to medical technical field, relate to a kind of polymer micelle composition that wraps carrying anti-tumor medicine and multidrug resistance reversing agent and preparation method thereof, this polymer micelle composition has the characteristics of reverse multiple drug resistance of tumor.
Background technology
Malignant tumor is commonly encountered diseases and the frequently-occurring disease of serious threat human life health, has become to cause human dead primary disease in the world wide.At present, the clinical treatment of malignant tumor is still take chemotherapy as main, yet the water solublity extreme difference of most of antitumor drug has brought great inconvenience for exploitation and the clinical use of its preparation.For example, all added the dissolubility that a large amount of surfactants increases medicine in the preparation of the taxane anti-tumor medicament of listing.Paclitaxel injection taxol (Taxol) is that medicine is dissolved in the mixed solvent of polyoxyethylene castor oil and dehydrated alcohol (1:1).Docetaxel injection taxotere (Taxotere) is with medicine dissolution (40 g/L) in the surfactant tween 80, and then packing is diluted with the dilution of 13% ethanol water and 5% glucose solution before the clinical use again.
Discharge histamine during surfactant polyoxyethylene Oleum Ricini degradation in vivo, can cause the side effect such as severe allergic reaction and bone marrow depression, even threat to life.For preventing anaphylaxis, use clinically the front necessary injection cortex steroid hormone of paclitaxel injection and or antihistaminic class medicine.Nonetheless, still there is anaphylaxis in various degree in a large amount of patients.In addition, the plasticizer divinyl hexyl phthalate in the polyoxyethylene castor oil solubilized transfusion device causes toxic reaction, and can cause preparation with diluent (such as normal saline) when mixing paclitaxel separate out precipitation.The surfactant tween 80 has hemolytic and stickiness is large, also can produce obvious anaphylaxis after the injection.For these reasons, the clinical practice of antitumor drug paclitaxel and Docetaxel preparation is extremely restricted.
In addition, most antitumor drug are performed poor to polytype recurrent tumor treatment, present obvious multidrug resistance phenomenon.The generation of tumor multi-medicine drug-resistant mainly is because the overexpression (especially P-glycoprotein) of tumor cell abc transport protein family, so that antitumor drug is effluxed by the drug-resistant tumor cell, reduce the drug level in the tumor cell, caused clinical therapeutic efficacy not good.The multidrug resistance phenomenon that produces in the chemotherapy of tumors process has become the main cause of chemotherapy of tumors failure.Therefore, how to develop the hydrophobicity that can overcome antitumor drug and the preparation of multi-drug resistance of the tumor and become clinically urgent need to solve the problem.
Polymer micelle is the drug-loading system for insoluble drug that developed recently gets up, and it has nuclear-shell-like structure, and its center is hydrophobic parts, can be used as the storage storehouse of insoluble drug, and shell is hydrophilic parts, can make the micellar structure stable existence.In addition, the hydrophilic outer shell of polymer micelle can make its removing of avoiding in vivo reticuloendothelial system (RES) and mononuclear phagocyte system (MPS), gives the long cycle performance of polymer micelle.The micelle of low particle diameter (being generally less than 100nm) can be easier of the tumor locus enrichment by means of the blood vessel infiltration and the retention effect (EPR effect) that strengthen, and tumor is had targeting.Polymer micelle shows obvious advantage as the delivery vehicles of insoluble drug in stability and targeting.Compare with surfactant (polyoxyethylene castor oil and tween 80) commonly used, the carrier that the compatibility of polymer micelle applying biological and biodegradable are good, safety is higher.
Chinese patent CN 1214818C discloses a kind of amphipathic ethylene glycol-polyester block copolymer micelle, wherein, the hydrophobicity polyester block is selected from polylactic acid, polyglycolic acid, poly-(lactic acid-ethanol), poly-(6-caprolactone) and their mixture, the hydrophilic block is Polyethylene Glycol and derivant thereof, and the mass ratio of hydrophilic block and hydrophobic block is greater than 1:1.02.This type of polymer micelle can be used as the carrier of paclitaxel, effectively increase the dissolubility of paclitaxel in aqueous solution, the intrafascicular content of paclitaxel polymer latex is lower than 30%, and the copolymer micelle aqueous stability of bag year paclitaxel is relatively poor, and the leakage of 72 hours medicines can reach 30%.
Chinese patent CN 1296097C discloses a kind of amphipathic ethylene glycol of similar-polyester block copolymer micelle, wherein, the hydrophobicity polyester block is selected from polylactic acid, polyglycolic acid, poly-(lactic acid-ethanol), the hydrophilic block is Polyethylene Glycol, is that with the disclosed polyethylene glycol-ester block copolymer of CN 1214818C patent difference the mass ratio of hydrophilic block and hydrophobic block in the copolymer is less than 1:1.Patent attempt to obtain metastable micelle medicine carrying system, but its stability is still bad by increasing the molecular weight of hydrophobic polyester block in the polyethylene glycol-ester block copolymer.Chinese patent CN 100589843C discloses a kind of mixed polymer micelle administration system, and this system increases the stability of polymer micelle by adding phospholipid by forming mixed micelle with the polyethylene glycol-ester block copolymer.
At present, researcheres adopt various polymer micelle carriers to improve the dissolubility of hydrophobic anticancer drug in aqueous medium, and increase the stability of micelle administration system.But the polyethylene glycol-esters polymer micelle administration system that possesses the function of reverse multiple drug resistance of tumor there is not yet report.
Summary of the invention
The unexpected discovery of this patent mixed polyethylene glycol-lipin polymer (or derivatives thereof) with multidrug resistance reversing agent pluronic polymer and antitumor drug, can be prepared into the micelle administration system of parcel multidrug resistance reversing agent pluronic and antitumor drug, have the significantly function of reverse multiple drug resistance of tumor.The above-mentioned polyethylene glycol-esters micelle administration system that possesses the reverse multiple drug resistance of tumor function there is not yet any report.
The purpose of this invention is to provide polymer micelle composition and the preparation method of a series antineoplastic medicament, said composition has the function of obvious reverse multiple drug resistance of tumor, and good stability.
Polymer micelle composition of the present invention is by two or more polyethylene glycol-ester block copolymer (or derivatives thereof), pluronic polymer and antitumor drug form, has nuclear-shell-like structure, its center is hydrophobic parts, can be used as the storage storehouse of insoluble drug, shell is hydrophilic parts, can make the micellar structure stable existence, consist of the micelle hydrophilic area and include but not limited to Polyethylene Glycol (PEG), mono methoxy polyethylene glycol (mPEG) and derivant thereof consist of the micelle hydrophobic region and include but not limited to polyoxypropylene, polylactic acid, polyglycolic acid, poly (lactic acid-glycolic acid), poly-(e-caprolactone) and derivant thereof.Wherein, the polyethylene glycol-ester block copolymer belongs to prior art, including, but not limited to diblock copolymer and triblock copolymer.Preferred amphipathic ethylene glycol-polyester copolymer is mono methoxy polyethylene glycol-copolymer of poly lactic acid (mPEG-PDLLA) and PLA-PEG-PLA copolymer (PLLLA-PEG-PDLLA).
Block copolymer structure is as follows:
Two block types:
R wherein
1C
1-20Alkyl is preferably C
1-5Alkyl;
R
2Hydrogen or methyl;
M is the integer greater than 2, preferred 10-3000;
N, i and j are the integers greater than 2, preferred 2-2000;
Three block types:
Wherein R is hydrogen or methyl;
R
3And R
4Separately independently hydrogen or methyl;
M, n and j are as mentioned above;
X and y are the integers greater than 2.
The multidrug resistance reversing agent pluronic is including, but not limited to pluronic L61(Ploronic L61), pluronic P85 (Ploronic P85), pluronic P105(Ploronic P105), Pluronic F68 (Ploronic F68) and pluronic F127(Ploronic F127) preferred pluronic polymer is pluronic P85, pluronic P105, pluronic F127 and pluronic L61.
The polymer micelle composition of bag carrying anti-tumor medicine of the present invention and multidrug resistance reversing agent, the mass ratio of amphipathic ethylene glycol-polyester block copolymer wherein or its mixture and pluronic polymer or its mixture is 100/0.01-1/100, is preferably 100/0.1-100/30.
The polymer micelle composition of bag carrying anti-tumor medicine of the present invention and multidrug resistance reversing agent, wherein hydrophilic area polyethyleneglycol derivative and polyoxyethylene deriv refer to adopt some organ the hydrophilic block of polymer is terminal, tissue, cell, organelle has the molecular chemistry of pathoklisis to modify the derivant that obtains, comprise protein or polypeptide with tissue or cell-specific, antibody, somatomedin, vitamin and analog thereof are (such as folic acid, biotin etc.), polysaccharide, glycopeptide or glycoprotein, hormone, cofactor and other have some molecule of special affinity, preferred antibody to tumor cell, somatomedin and vitamin and analog thereof.
The polymer micelle composition of bag carrying anti-tumor medicine of the present invention and multidrug resistance reversing agent, wherein hydrophobic region biodegradable polyesters derivant refers to the terminal chemical substance that adopts some to have reactivity of the hydrophobic block of mono methoxy polyethylene glycol-polyester copolymer is modified the derivant that obtains, comprise cholesterol, deoxycholic acid, short chain saturated alkane etc., preferred cholesterol and deoxycholic acid.
The polymer micelle composition of bag carrying anti-tumor medicine of the present invention and multidrug resistance reversing agent, antitumor drug comprises the anticarcinogens such as taxanes, amycin, epirubicin, daunorubicin, ammonia first dish purine, teniposide, camptothecin, is preferably the cancer therapy drugs such as taxanes, amycin class, etoposide class.
The polymer micelle composition of bag carrying anti-tumor medicine of the present invention and multidrug resistance reversing agent, can prepare by the following method: solvent evaporation method, lyophilization, film forming redissolution dispersion method, dialysis and dissolution method, preferred solvent volatility process, lyophilization and film forming redissolution dispersion method.
Solvent evaporation method may further comprise the steps:
(1) polymeric blends in medicine and the claim 1 is dissolved in the organic solvent;
(2) water droplet is added in the organic solvent, or organic solvent is dropped in the water;
(3) fling to organic solvent, get the evenly micelle of clarification.
Lyophilization may further comprise the steps
(1) polymeric blends in medicine and the claim 1 is dissolved in the solvent;
(2) add an amount of freeze drying protectant, remove moisture through lyophilization, get the polymer micelle that dry bag carries hydrophobic antitumor drug;
(3) the lyophilizing micelle that makes is measured on demand added water, after the hydration vibration, be recovered to micellar solution.
Film forming redissolution dispersion method may further comprise the steps
(1) polymeric blends in medicine and the claim 1 is dissolved in the organic solvent;
(2) above mixture is rotated in a vacuum volatilize, get the mixture film of polymer and medicine;
(3) add appropriate amount of deionized water, redissolution namely gets the evenly micellar solution of clarification.
Polymer micelle composition by said method preparation can be even by the high pressure breast, high speed homogenization or the technique such as ultrasonic reduce micelle particle diameter and particle size distribution.This polymer micelle composition also can further be prepared into suitable formulations such as lyophilization, spray drying, reduction vaporization etc. through certain preparation process, such as injection, oral formulations etc.
Polymer micelle composition of the present invention compared with prior art, innovative point is: micelle composition can effectively overcome multidrug resistance phenomenon and the good stability of tumor.The function of micelle composition reverse multiple drug resistance of tumor can be proven by inhibitory action and the interior drug resistance breast cancer transplantable tumor experimental result of animal body to breast carcinoma mdr cell (MCF-7/Adr).The stability of micelle composition can be proven by the measurement result of micelle particle diameter and medicament contg.
Computing formula:
Description of drawings
Fig. 1 is mono methoxy polyethylene glycol-polylactic-acid block copolymer
1H-NMR collection of illustrative plates (solvent: CDCL
3, interior mark: TMS) (embodiment 1).
Fig. 2 is the gpc chromatogram of mono methoxy polyethylene glycol-polylactic-acid block copolymer.(post: Styragel HR(Waters, U.S.A)), flow velocity: 1ml/min, eluant: oxolane).Mw=4482, Mn=4085, polydispersity 1.097(embodiment 1).
Fig. 3 is parcel different proportion pluronic paclitaxel micelle particle size distribution figure (embodiment 6).
Fig. 4 wraps up different model pluronic paclitaxel micelle particle size distribution figure (embodiment 6).
Fig. 5 is for wrapping respectively the cumulative release curve (embodiment 8) of the micellar preparation that is loaded with paclitaxel and teniposide.
Fig. 6 is the cumulative release curve (embodiment 9) of parcel different proportion and model pluronic formulation for paclitaxel.
Fig. 7 is content and the micelle change of size (embodiment 10) that room temperature was placed after paclitaxel micelle (mPEG2000-PDLLA1750/P85 10:3) redissolved.
Fig. 8 is that the different pluronic ratios of parcel of film forming redissolution dispersion method preparation and the paclitaxel micelle room temperature of model are placed change of size (embodiment 11).
Fig. 9 micellar solution, dried frozen aquatic products and the rear product appearance (embodiment 11) of redissolving.
Figure 10 A is that different formulation for paclitaxel are to cell toxicant and the MCF-7 drug resistance of MCF-7 sensitive cells; B is that different formulation for paclitaxel are to the cell toxicant of MCF-7 mdr cell.
Figure 11 A is to the therapeutic effect of transplanted tumor model behind the different paclitaxel micelle administrations; B is the body weight change (embodiment 13) of nude mice during the different paclitaxel micelle administrations.
The specific embodiment
The below is the embodiment of this patent, but following embodiment does not limit the interest field of this patent.
Synthesizing of embodiment 1 mono methoxy polyethylene glycol-polylactic acid section copolymer (mPEG2000-PDLLA2482)
Get mono methoxy polyethylene glycol (Mw 2000) 4g, place 120 ℃ of lower vacuum dryings 3 hours, and added 6g lactide monomer and 5 μ l stannous iso caprylates (be equivalent to monomer total amount 0.063%) in polymerization pipe, evacuation 30 minutes, tube sealing was in 140 ℃ of lower polymerizations 6 hours.The product that polymerization is complete is dissolved in the dichloromethane of 20ml, splashes in the excessive ether of pre-cooling, makes the product precipitation under-20 ℃.With sedimentation and filtration, and wash with ether.Then repetitive operation 3 times with filtering residue reduced pressure at room temperature 48 hours, obtains the higher block copolymer mPEG-PDLLA(productive rate of purity and is about 78%).Nuclear magnetic resonance result is seen Fig. 1, and the gel permeation chromatography of polymer the results are shown in Figure 2.
Synthesizing of embodiment 2 mono methoxy polyethylene glycols-polylactic acid section copolymer (mPEG5000-PDLLA3034)
Get mono methoxy polyethylene glycol (Mw 5000) 3g, place 120 ℃ of lower vacuum dryings 3 hours, and added 7g lactide monomer and 5 μ l stannous iso caprylates (be equivalent to monomer total amount 0.063%) in polymerization pipe, evacuation 30 minutes, tube sealing was in 140 ℃ of lower polymerizations 6 hours.The product that polymerization is complete is dissolved in the dichloromethane of 20ml, splashes in the excessive ether of pre-cooling, makes the product precipitation under-20 ℃.With sedimentation and filtration, and wash with ether.Then repetitive operation 3 times with filtering residue reduced pressure at room temperature 48 hours, obtains the higher block copolymer mPEG-PDLLA(productive rate of purity and is about 88%).
The mPEG2000/5000-PDLLA block copolymer structure is as follows:
Synthesizing of embodiment 3 PLA-PEG-PLA block copolymers (PDLLA550-PEG2000-PDLLA550)
Taking polyethylene glycol (Mw 2000) 6g placed 120 ℃ of lower vacuum dryings 3 hours, added the 4g lactide monomer, 5 μ l stannous iso caprylates (be equivalent to monomer total amount 0.063%) in polymerization pipe, evacuation 30 minutes, tube sealing was in 140 ℃ of lower polymerizations 6 hours.The product that polymerization is complete is dissolved in the dichloromethane of 20ml, splashes in the excessive ether of pre-cooling, makes the product precipitation under-20 ℃.With sedimentation and filtration, and wash with ether.Then repetitive operation 3 times with filtering residue reduced pressure at room temperature 48 hours, obtains block copolymer PDLLA-PEG-PDLLA(productive rate and is about 85%).
Synthesizing of embodiment 4 PLA-PEG-PLA block copolymers (PDLLA750-PEG5000-PDLLA750)
Taking polyethylene glycol (Mw 5000) 5g placed 120 ℃ of lower vacuum dryings 3 hours, added the 5g lactide monomer, 5 μ l stannous iso caprylates (be equivalent to monomer total amount 0.063%) in polymerization pipe, evacuation 30 minutes, tube sealing was in 140 ℃ of lower polymerizations 6 hours.The product that polymerization is complete is dissolved in the dichloromethane of 20ml, splashes in the excessive ether of pre-cooling, makes the product precipitation under-20 ℃.With sedimentation and filtration, and wash with ether.Then repetitive operation 3 times with filtering residue reduced pressure at room temperature 48 hours, obtains block copolymer PDLLA-PEG-PDLLA(productive rate and is about 93%).
The PDLLA-PEG2000/5000-PDLLA block copolymer structure is as follows:
Embodiment 5: mono methoxy polyethylene glycol-PLGA block copolymer (mPEG2000-PLGA3200) synthetic
Get mono methoxy polyethylene glycol (Mw 2000) 4g, place 120 ℃ of lower vacuum dryings 3 hours, and added 4.8g lactide and 1.2g glycolide monomer and 5 μ l stannous iso caprylates (be equivalent to monomer total amount 0.063%) in polymerization pipe, evacuation 30 minutes, tube sealing was in 140 ℃ of lower polymerizations 6 hours.The product that polymerization is complete is dissolved in the dichloromethane of 20ml, splashes in the excessive ether of pre-cooling, makes the product precipitation under-20 ℃.With sedimentation and filtration, and wash with ether.Then repetitive operation 3 times with filtering residue reduced pressure at room temperature 48 hours, obtains block copolymer mPEG-PLGA(productive rate and is about 70%).
The structure of mPEG-PLGA block copolymer is as follows:
Embodiment 6: bag carries the preparation of the polymer micelle carrier of paclitaxel
(1) dissolution method
1. get polymer (mPEG2000-PDLLA2482) 10mg synthetic among the embodiment 1, pluronic polymer (Pluronic P85) 3mg, paclitaxel 5mg, be dissolved in 0.9% sodium chloride injection (3ml), ultrasonic Treatment 20 minutes, restir 4 hours by 0.22 μ m filter membrane, namely gets and clarifies the slightly mixed polymer micellar solution of opalescence.The content of paclitaxel detects (chromatographic column: Diamonsil C18(diamond) 5 μ, 200 * 4.6mm mobile phase: acetonitrile/water=55:45 (v/v)) with high performance liquid chromatography.The micelle carrier by formula calculates envelop rate and the drug loading of paclitaxel, the results are shown in Table 1.
2. step is with 1. identical, and different is to get polymer (mPEG5000-PDLLA3034) 10mg synthetic among the embodiment 2, pluronic polymer (Pluronic P105) 3mg, paclitaxel 5mg.
3. step is with 1. identical, and different is to get polymer (PDLLA550-PEG2000-PDLLA550) 10mg synthetic among the embodiment 3, pluronic polymer (Pluronic L61) 3mg, paclitaxel 5mg.
4. step is with 1. identical, and different is to get polymer (mPEG2000-PLGA3200) 10mg synthetic among the embodiment 5, pluronic polymer (Pluronic F127) 3mg, paclitaxel 5mg.
(2) solvent evaporation method
1. get polymer (mPEG2000-PDLLA1750) 10mg synthetic among the embodiment 1, pluronic polymer (Pluronic P85) 3mg, paclitaxel 5mg, be dissolved in the acetonitrile (1ml), slowly be added dropwise in 0.9% normal saline, stir, until acetonitrile volatilizees fully, by 0.22 μ m filter membrane, namely get clarification and with the mixed polymer micellar solution of light blue opalescence.The micelle carrier the results are shown in Table 1 to envelop rate and the drug loading of paclitaxel.
2. step is with 1. identical, and different is to get polymer (mPEG5000-PDLLA3034) 10mg synthetic among the embodiment 2, pluronic polymer (Pluronic P105) 3mg, paclitaxel 5mg.
3. step is with 1. identical, and different is to get polymer (PDLLA550-PEG2000-PDLLA550) 10mg synthetic among the embodiment 3, pluronic polymer (Pluronic L61) 3mg, paclitaxel 5mg.
4. step is with 1. identical, and different is to get polymer (mPEG2000-PLGA3200) 10mg synthetic among the embodiment 5, pluronic polymer (Pluronic F127) 2mg, paclitaxel 5mg.
(3) film forming redissolution dispersion method
1. get polymer (mPEG2000-PDLLA2482) 10mg synthetic among the embodiment 1, pluronic polymer (Pluronic P85) 3mg, paclitaxel 5mg, be dissolved in the acetonitrile (3ml), in 50 ℃ of lower rotary evaporation in vacuo, form the pharmaceutical polymer substrate of homogeneous transparent, add again 0.9% sodium chloride 3ml, vortex mixed by 0.22 μ m filter membrane, namely gets clarification and with the mixed polymer micellar solution of light blue opalescence.The particle size determination result of gained micellar preparation such as Fig. 3, Fig. 4.The micelle carrier the results are shown in Table 1 to envelop rate and the drug loading of paclitaxel.
2. step is with 1. identical, and different is to get polymer (mPEG2000-PDLLA2482) 10mg synthetic among the embodiment 1, pluronic polymer (Pluronic P85) 2mg, paclitaxel 5mg.
3. step is with 1. identical, and different is to get polymer (mPEG2000-PDLLA2482) 10mg synthetic among the embodiment 1, pluronic polymer (Pluronic P85) 1mg, paclitaxel 5mg.
4. step is with 1. identical, and different is to get polymer (mPEG2000-PDLLA1750) 10mg synthetic among the embodiment 1, pluronic polymer (Pluronic L61) 3mg, paclitaxel 5mg.
5. step is with 1. identical, and different is to get polymer (mPEG2000-PDLLA1750) 10mg synthetic among the embodiment 1, pluronic polymer (Pluronic F127) 3mg, paclitaxel 5mg.
6. step is with 1. identical, and different is to get polymer (mPEG5000-PDLLA3034) 10mg synthetic among the embodiment 1, pluronic polymer (Pluronic L61) 3mg, paclitaxel 5mg.
7. step is with 1. identical, and different is to get polymer (mPEG5000-PDLLA3034) 10mg synthetic among the embodiment 2, pluronic polymer (Pluronic P105) 3mg, paclitaxel 5mg.
8. step is with 1. identical, and different is to get polymer (PDLLA550-PEG2000-PDLLA550) 10mg synthetic among the embodiment 3, pluronic polymer (Pluronic L61) 3mg, paclitaxel 5mg.
9. step is with 1. identical, and different is to get polymer (mPEG2000-PLGA3200) 10mg synthetic among the embodiment 5, pluronic polymer (Pluronic F127) 3mg, paclitaxel 5mg.
(4) lyophilization
1. get polymer (mPEG2000-PDLLA1750) 10mg synthetic among the embodiment 1; each 2mg of pluronic polymer (Pluronic P85); paclitaxel 5mg; be dissolved in the ethanol (2ml), add an amount of freeze drying protectant, remove solvent through lyophilization; get the polymer micelle that dry bag carries hydrophobic antitumor drug; use the front lyophilizing micelle that makes is measured on demand to add water, after the hydration vibration, be recovered to micelle.The micelle carrier the results are shown in Table 1 to envelop rate and the drug loading of paclitaxel.
2. step is with 1. identical, and different is to get polymer (mPEG5000-PDLLA3034) 10mg synthetic among the embodiment 2, pluronic polymer (Pluronic P85) 3mg, paclitaxel 5mg.
3. step is with 1. identical, and different is to get polymer (PDLLA550-PEG2000-PDLLA550) 10mg synthetic among the embodiment 3, pluronic polymer (Pluronic P85) 3mg, paclitaxel 5mg.
4. step is with 1. identical, and different is to get polymer (mPEG2000-PLGA3200) 10mg synthetic among the embodiment 5, pluronic polymer (Pluronic P85) 3mg, paclitaxel 5mg.
The different preparation method micelle of table 1. carrier is to envelop rate and the drug loading of paclitaxel
Embodiment 7: bag carries the preparation of the polymer micelle carrier of teniposide
(1) dialysis
Get each 10mg of polymer (mPEG2000-PDLLA1750 and mPEG5000-PLGA3200), each 2mg of pluronic polymer (Pluronic P85), teniposide 5mg, pack in the bag filter of molecular cut off 3500, place the aqueous solution that contains other adding ingredient, dialysis 72h by 0.22 μ m filter membrane, namely gets and includes the slightly micelle of pale blue opalescence of clear.The micelle carrier the results are shown in Table 2 to envelop rate and the drug loading of teniposide.
(2) solvent evaporation method
Get each 10mg of polymer (mPEG2000-PDLLA1750 and mPEG5000-PLGA3200), each 2mg of pluronic polymer (Pluronic L61), teniposide 5mg, be dissolved in the dichloromethane, slowly be added dropwise in 0.9% normal saline that is added with lactose and mannitol, stir, until dichloromethane volatilizees fully, by 0.22 μ m filter membrane, namely get clarification and with the mixed polymer micellar solution of light blue opalescence.The micelle carrier the results are shown in Table 2 to envelop rate and the drug loading of teniposide.
The different preparation method micelle of table 2. carrier is to envelop rate and the drug loading of teniposide
Embodiment 8: drug release determination
Respectively preparation bag among embodiment 6 and the embodiment 7 is loaded with micelle (the mPEG2000-PDLLA2482/Pluronic P85 of paclitaxel and teniposide, 10/3, w/w) preparation 2ml, the molecular cut off of packing into is in 3500 the bag filter, place the dialysis medium, stirred 48 hours, different time points is got the burst size that its medium is measured different pharmaceutical.The result as shown in Figure 5, by release profiles as can be known, the block copolymer micelle that wraps up simultaneously Pluronic P85 and paclitaxel has certain slow releasing function, and in 4h, prominent releasing is lower than 4%.
Embodiment 9: the paclitaxel micelle drug release determination of parcel different proportion and model pluronic
Get respectively the parcel different proportion of film forming redissolution dispersion method preparation among the embodiment 6 and the formulation for paclitaxel 2ml of model pluronic, step is with embodiment 8, and the result as shown in Figure 6.
Embodiment 10: Stability Determination
To be loaded with by the bag of lyophilization preparation among the embodiment 6 micelle (the mPEG2000-PDLLA1750/ Pluronic P85 of paclitaxel, 10/3, w/w) after lyophilized powder redissolves, under room temperature, leave standstill, get a certain amount of preparation respectively at different time points and measure the content of taxol that wraps up in its particle diameter and the micelle, the result shows that this micellar preparation is stable in 48h as shown in Figure 7.
Embodiment 11: the stability of the paclitaxel micelle of parcel different proportion and model pluronic
Will be by the parcel different proportion of film forming redissolution dispersion method preparation among the embodiment 6 and the paclitaxel micellar preparation dried frozen aquatic products of model pluronic, with under room temperature, leaving standstill after the water for injection redissolution, get a certain amount of preparation respectively at different time points and measure its change of size, the result as shown in Figure 8.The micellar solution outward appearance as shown in Figure 9 after micellar solution, dried frozen aquatic products and the redissolution.
Embodiment 12: drug-carrying polymer micelle compositions cellulotoxic experiment
MCF-7 sensitivity and the mdr cell of trophophase of taking the logarithm respectively is inoculated in 96 well culture plates, every hole inoculation 115 * 10
4The cell suspension 200 μ L of μ g/mL.Experiment is established 11 groups altogether, every group of 6 holes: matched group is that RPMI RPMI-1640, positive group are paclitaxel mixed micelle solution (the mPEG2000-PDLLA2482/Pluronic L61 10/0.5 of preparation among the embodiment 4 for paclitaxel/polyoxyethylene castor oil injection, experimental group, w/w) or not the paclitaxel micelle (mPEG2000-PDLLA2482) that contains pluronic, positive group and experimental group are established respectively 100,50,20,10 and 5 μ g/mL concentration.Behind the cell attachment 24h, the above-mentioned culture fluid of respectively organizing is covered on the cell, add 5 mg/mL MTT, 20 μ L in the every hole of 24,48 and 72 h and cultivate 4h, exhaust culture fluid, every hole adds dimethyl sulfoxine (DMSO) solution 150 μ L again, shake 15min after placing 15～20 min, measure the trap (OD) at 490 nm places with microplate reader, and calculate IC
50Value.Result such as Figure 10, wherein the mixed micelle group is for the IC of mdr cell and sensitive cells
50Value raises minimum, illustrates in contrast to the paclitaxel solution group and do not contain pluronic micelle group, has more superior reversal of multidrug resistance of tumor cells effect.
Anti-drug resistance breast cancer transplantable tumor experiment in the embodiment 13 drug-carrying polymer micelle compositions
Female BALB/c nude mice (body weight 16-18g) is available from Shenyang Pharmaceutical University zoopery center, and animal is raised under aseptic condition, operates under the aseptic condition.With 1.0 * 10
7Individual MCF-7/Adr cell is suspended in the 200 μ l serum-free mediums, is subcutaneously injected into the nude mice oxter.Observe the oxter tumor growing state of nude mice every day, and the volume of record tumor.
Behind the inoculated tumour cell the 19th day, gross tumor volume reaches 150～180mm
3, can drug treatment.Postvaccinal nude mice is divided into 5 groups at random, 6 every group.After giving nude inoculation MCF-7/Adr cell the 20th, 22,24,26,28 and 30 day, give respectively normal saline, tail vein injection and do not contain pluronic paclitaxel micelle (mPEG2000-PDLLA1750), oral pluronic paclitaxel micelle, tail vein injection or oral paclitaxel micelle (the mPEG2000-PLA1750/Pluronic P85 that comprises pluronic (Pluronic P85) of not containing, 10/0.5, w/w).The dosage of paclitaxel is 10mg/kg.Measure size and the nude mice body weight of tumor every day.
The tumor bearing nude mice of each processed group is execution in the 35th day behind tumor inoculation, and the length of measurement tumor and wide adopts the tumor control rate after formula calculates administration.The toxicity of each administration group is estimated in the variation of nude mice body weight.Result such as Figure 11.
Claims (12)
1. polymer micelle composition that wraps simultaneously carrying anti-tumor medicine and multidrug-resistance reversal agent pluronic, it is characterized in that: compositions is comprised of two or more amphipathic ethylene glycol-polyester block copolymer (or derivatives thereof) micelle carriers, multidrug resistance reversing agent pluronic and antitumor drug.
2. polymer micelle carrier according to claim 1, it is characterized in that: wherein one or more amphipathic nature polyalcohols are two blocks or three block polyethylene glycol-ester copolymer (or derivatives thereof) or its mixture, it is comprised of hydrophilic block and hydrophobic block, and hydrophilic block can be selected from the Polyethylene Glycol or derivatives thereof; Hydrophobic block can be selected from Biodegradable polyester: polylactic acid, polyglycolic acid, poly-(lactic acid-ethanol), poly-(e-caprolactone), poly-(lactic acid-e-caprolactone), poly-(glycolic-e-caprolactone) or derivatives thereof.
3. amphipathic nature polyalcohol according to claim 2, it is characterized in that: the molecular weight of hydrophilic block Polyethylene Glycol or derivatives thereof is preferably 1000-5000 between 500-20000; The mass ratio of hydrophilic block and hydrophobic block polyester is preferably 3/7-7/3 between 1/9-9/1.
4. multidrug resistance reversing agent pluronic according to claim 1 is characterized in that: be comprised of one or more pluronic or derivatives thereofs with reverse multiple drug resistance of tumor function.
5. multidrug resistance reversing agent pluronic according to claim 1, it is characterized in that: it is comprised of hydrophilic block and hydrophobic block, and hydrophilic block can be selected from the polyoxyethylene or derivatives thereof; Hydrophobic block is selected from polyoxypropylene.
6. polymer micelle composition according to claim 1, it is characterized in that: the quality percentage composition of one or more multidrug resistance reversing agent pluronic or derivatives thereofs is 0.01%-50% in the compositions, is preferably 0.1%-30%.
7. polymer micelle composition according to claim 1, it is characterized in that: the quality percentage composition of antitumor drug is 0.01%-50% in the compositions, is preferably 1%-30%.
8. polymer micelle composition according to claim 1 is characterized in that: by the following method preparation, solvent evaporation method, lyophilization, film forming redissolution dispersion method, dialysis and dissolution method, preferred solvent volatility process, lyophilization and film forming redissolution dispersion method.
9. the polymer micelle composition preparation method of bag carrying anti-tumor medicine according to claim 8 is characterized in that: prepare by following steps:
(1) polymeric blends in medicine and the claim 1 is dissolved in the organic solvent;
(2) aqueous solution is dropped in the organic solvent, or organic solvent is dropped in the aqueous solution;
(3) fling to organic solvent, get the evenly micelle of clarification.
10. the polymer micelle composition preparation method of bag carrying anti-tumor medicine according to claim 8 is characterized in that: prepare by following steps:
(1) polymeric blends in medicine and the claim 1 is dissolved in the solvent;
(2) add an amount of freeze drying protectant, remove moisture through lyophilization, get the polymer micelle that dry bag carries hydrophobic antitumor drug;
(3) the lyophilizing micelle that makes is measured on demand added aqueous solution, be recovered to micellar solution.
11. the polymer micelle composition preparation method of bag carrying anti-tumor medicine according to claim 10; it is characterized in that: freeze drying protectant comprises: glucose, lactose, galactose, fructose, dextran, sucrose, mannitol, sorbitol, xylose, xylitol, maltose, trehalose, polyvinylpyrrolidone, hydroxypropyl beta cyclodextrin etc.; adding proportion 0.001-70%, preferred 0.1-40%.
12. the polymer micelle composition preparation method of bag carrying anti-tumor medicine according to claim 8 is characterized in that: prepare by following steps:
(1) polymeric blends in medicine and the claim 1 is dissolved in the organic solvent;
(2) above mixture rotation is volatilized solvent, get polymer and medicament mixed thin film;
(3) add an amount of aqueous solution, redissolution namely gets the micellar solution of clarification.
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