CN103623430B - Preparation method and application of targeted co-supported drug delivery system nano-particles based on polylactic-co-glycolic acid - Google Patents
Preparation method and application of targeted co-supported drug delivery system nano-particles based on polylactic-co-glycolic acid Download PDFInfo
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Abstract
The invention relates to a preparation method and application of targeted co-supported drug delivery system nano-particles based on polylactic-co-glycolic acid (PLGA), which can effectively solve the problem of preparing the targeted co-supported drug delivery system nano-particles based on polylactic-co-glycolic acid, and realizes the application in cerebral tumor targeted treatment medicines. The method comprises the following steps: connecting Angiopep-2 by utilizing the reactive amino group on chitosan: reacting chitosan with 3-maleimidopropionic acid N-hydroxysuccinimide to enable the amino terminal of chitosan to have maleimide; reacting with terminal-sulfhydrylated Angiopepe-2 to obtain a chitosan-angiopep-2 polymer. The polymer can be applied to preparation of a PLGA-based targeted co-supported drug delivery system. The method is stable and reliable, the product has a good using effect, small-molecular chemical drugs and genetic drugs can be delivered at the same time, or a fluorescent probe can pass through a blood brain barrier and enters the brain while chemotherapeutic drugs are delivered, and targeted cerebral tumor treatment can be synergized or targeted cerebral tumor treatment and diagnosis can be integrated into a whole.
Description
Technical field
The present invention relates to medicine, particularly a kind of targeting based on Poly(D,L-lactide-co-glycolide carries preparation method and the application of drug delivery system nanoparticle altogether.
Background technology
Poly(D,L-lactide-co-glycolide (poly lactic-co-glycolic acid, PLGA, or claim PLGA), by two kinds of monomers---lactic acid and hydroxyacetic acid are polymerized, it is a kind of degradable functional polymer organic compound, there is the performance of good biocompatibility, nontoxic, good encystation and film forming, be widely used in pharmacy, medical engineering material and modernization industrial circle.At U.S. PLGA by FDA certification, formally included into American Pharmacopeia as pharmaceutic adjuvant.Become one of carrier material enjoying favor in recent years, be widely used in pharmaceutical carrier and medical bio engineering material field.Finally be degraded to CO in vivo
2and H
2o.PLGA effectively can carry the bioactive substances such as protein, antibody, polypeptide, medicine and nucleic acid by certain technology and enter cell, thus becomes the carrier of people's concern.
Angiopep-2 is one of the most emerging novel targeted functional group, and it is the small peptide of threonine-phenylalanine-phenylalanine-tyrosine-Gly-Gly-Vitro By Serine/arginine-glycine-lysine-arginine-Asn-Asn-Phe-Lys-TE-Glu-Tyr 19 aminoacid composition.Research shows, the LDH receptor related protein of Angiopep-2 and brain capillary endothelial cell and the equal high expressed of glioma cell has affinity, effectively can improve it across blood brain barrier and cerebral glioma transport efficacy.Research report, Angiopep-2 is the several times of transferrins in the distribution volume of brain parenchymal cell.The conjugate GRN1005 of a molecule Angiopep-2 and three molecule paclitaxel is just entering the first phase clinical research of anti-cerebral glioma in the U.S..But this conjugate still poorly water-soluble, the same with Texol during application, need by polyoxyethylene castor oil and ethanol as solubilizing agent, so still there is the shortcomings such as the large and circulation time in vivo of toxic and side effects is short.
The small molecule, anti-tumor drug such as doxorubicin hydrochloride, docetaxel is the substrate of brain capillary endothelial cell p-glycoprotein, cannot through blood brain barrier thus enter brain essence so that treatment the cerebral tumor.Therefore can overcome blood brain barrier delivering drugs by certain targeting group and nano-carrier and enter brain.
RNA perturbation technique is applied for many years in gene therapy, siRNA, shRNA, the medicines such as antisense oligonucleotide have the feature of high specific and low toxic and side effects, and small quantities of nucleic acids fragment gets final product effective reticent genes of interest, suppression ratio is comparatively large, is a dazzling nova in the disease treatments such as tumor.The performance of genomic medicine curative effect needs can be transported into cell by specific support.
In tumour medicine research, utilize fluorescent probe labeled drug, targeting, best targeting time and medicine, in the accumulation of other organ-tissues of animal, are conducive to the time of research worker in the best, carry out fabric analysis directly can to observe drug on tumor in live body level.The fluorescent probe such as indocyanine green, phthalocyanine not only can be used in living imaging but also can absorb near infrared light, and the electronics in molecule is from ground state transition to excite state, and the molecule of excited state discharges heat subsequently, has thermotherapy effect.Be usually used in the fluorescent probe of living imaging, great majority are traditional organic dye molecule, such as indocyanine green, phthalocyanine, methylene blue, rhodamine, Fluorescein isothiocyanate (FITC), and these organic dye molecules have some shortcomings of itself, as light stability is poor, the defects such as sensitivity low and targeting difference, thus limit it and further apply.Above-mentioned defect can be overcome by targeted nano granule carrier, to the diagnosis of tumor and targeted therapy, there is clear superiority.
In order to realize the treatment of tumor, just independent chemotherapeutics often needs very heavy dose ofly have antitumor curative effect, but also bring certain toxicity, the cardiac toxicity of such as amycin simultaneously.Therefore chemotherapeutics becomes an important technical of oncotherapy with the use in conjunction of other treatment technology.The targeting so how realized based on PLGA copolymer carries drug delivery system altogether, and realizes the application in cerebral tumor target therapeutic agent, and so far there are no is publicly reported.
Summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, the object of the present invention is just to provide preparation method and application that a kind of targeting based on Poly(D,L-lactide-co-glycolide carries drug delivery system nanoparticle altogether, the targeting that can effectively solve based on Poly(D,L-lactide-co-glycolide carries a preparation difficult problem for drug delivery system nanoparticle altogether, and realizes the application problem in cerebral tumor target therapeutic agent.
The technical scheme that the present invention solves is, the active amino on chitosan is utilized to connect Angiopep-2, first chitosan and 3-maleimidopropionic acid N-hydroxy-succinamide ester react, the amino terminal of chitosan is made to have maleimide, react with the Angiopep-2 of terminal sulfhydryl group again, obtain chitosan-Angiopep-2 polymer, by this polymer applications in preparing the preparation of carrying drug delivery system based on the targeting of PLGA altogether.The scheme that the Targeted PLGA transmission system nanoparticle of chemotherapeutics and genomic medicine is carried in preparation is altogether dissolved in organic solvent by PLGA, chemotherapeutics and adjuvant, is prepared into oil phase; By surfactant and chitosan-Angiopep-2 dissolution of polymer in without RNA(ribonucleic acid, as follows) in the water of enzyme, be prepared into aqueous phase, oil phase adds in aqueous phase, magnetic agitation or revolve steam removing organic solvent, through centrifugal or dialysis removing free drug, make PLGA drug-carrying nanometer particle, added by genomic medicine in nanoparticle solution, the targeting obtained based on PLGA copolymer carries drug delivery system nanoparticle altogether, and this nanoparticle can be effective to prepare in the medicine for the treatment of cerebral tumor targeting.The scheme that the Targeted PLGA transmission system nanoparticle of chemotherapeutics and fluorescent probe is carried in preparation is altogether dissolved in organic solvent by PLGA, chemotherapeutics, fluorescent probe and adjuvant, is prepared into oil phase; By surfactant and chitosan-Angiopep-2 dissolution of polymer in the water without RNA enzyme, oil phase adds in aqueous phase, magnetic agitation or revolve steam removing organic solvent, finally by centrifugal or dialysis removing free drug, carried the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe altogether.
The inventive method is reliable and stable, product result of use is good, can as medicine carrier system altogether, small-molecule chemical medicine and genomic medicine can be transmitted simultaneously, or transmission chemotherapeutics and fluorescent probe enter brain by blood brain barrier simultaneously, play the collaborative targeted therapy cerebral tumor or the targeted therapy cerebral tumor and the effect diagnosed in one, be the innovation on treatment of brain tumor medicine, have huge economic and social benefit.
Detailed description of the invention
Below in conjunction with concrete condition and embodiment, the specific embodiment of the present invention is elaborated.
Provide technical scheme by foregoing invention content part, the present invention in the specific implementation, is realized by following steps:
(1), the preparation of chitosan-Angiopep-2 polymer, method is:
A, chitosan 20mg is added volumetric concentration is in the acetic acid solution 8mL of 20%, is 300-500W ultrasonic dissolution 30min, with 5M(and 5mol/L at power) NaOH solution adjust pH to 6.0, obtain chitosan solution;
B, in chitosan solution, add 3-maleimidopropionic acid N-hydroxy-succinamide ester 5mg, under nitrogen protection, 30 DEG C, 100r/min magnetic agitation reaction 48h, after having reacted, it is that the bag filter of 8KD-14KD to be dialysed 60h at PBS that reactant liquor is placed in molecular cut off, remove unreacted 3-maleimidopropionic acid N-hydroxy-succinamide ester, obtain dialysis solution;
C, in dialysis solution, then add the Angiopep-2 2mg of sulfhydrylation, under nitrogen protection, 35 DEG C, 100-200r/min stirring reaction 24h, obtain the chitosan that Angiopep-2 modifies, being placed in molecular cut off is again that the bag filter of 8KD-14KD is dialysed 48h, the Angiopep-2 that removing is free, at-80 DEG C of lyophilisation 72h, obtain chitosan-Angiopep-2 polymer, for subsequent use in-20 DEG C of storages;
One in the sulfhydrylation Angiopep-2 that the Angiopep-2 of the Angiopep-2 of described sulfhydrylation to be carboxyl terminal be cysteine or Angiopep-2 and Traut ' s is obtained by reacting.The Angiopep-2 that described carboxyl terminal is connected with cysteine is, when synthesizing Angiopep-2, a molecule Angiopep-2 carboxyl terminal connects the cysteine of a molecule by chemical reaction; The Angiopep-2 of the sulfhydrylation that described Angiopep-2 and Traut ' s obtains after reacting is, Angiopep-2 5mg is dissolved in 20mL ultra-pure water, add 10mg Traut's reagent, 50 DEG C of ultrasonic 1h, make the end of Angiopep-2 have sulfydryl, being then placed in molecular cut off is that the bag filter of 8KD-14kD to be dialysed 48h at PBS, Angiopep-2 and the Traut ' s that removing is free, dialysis solution, at-80 DEG C of lyophilisation 72h, obtains the Angiopep-2 of sulfhydrylation;
(2), prepare the Targeted PLGA nanoparticle carrying chemotherapeutics and genomic medicine altogether, method is:
A, PLGA copolymer, chemotherapeutics and adjuvant are dissolved in (concentration of PLGA copolymer is 5mg-50mg/ml, and the concentration of chemotherapeutics is 1mg/ml-10mg/ml, and the volumetric concentration of adjuvant is 0.5%-1%) in organic solvent, are prepared into oil phase;
Described organic solvent is one or more the compositions in acetone, ethyl acetate, dichloromethane, acetonitrile, methanol; Described PLGA copolymer is the copolymer of polylactide, Acetic acid, hydroxy-, bimol. cyclic ester, and the weight ratio of Ju Bing Jiao Zhi ︰ Acetic acid, hydroxy-, bimol. cyclic ester is be one in 25 ︰ 75,50 ︰ 50,75 ︰ 25, and the molecular weight of PLGA is the one in 17kD, 50KD, 100kD.Described chemotherapeutics is the one in doxorubicin hydrochloride, paclitaxel, docetaxel, cisplatin, carboplatin, daunorubicin, 5-fluorouracil; Described adjuvant is one or more the compositions in sorbester p17, polysorbas20, Tween 80;
B, by surfactant and chitosan-Angiopep-2 dissolution of polymer in pH value be 6 without in RNA enzyme water, be prepared into aqueous phase (surfactant concentration is 5mg/ml-50mg/ml, and the concentration of chitosan-Angiopep-2 is 0.02mg/ml-2mg/ml);
Described pH value be 6 be first will add the pyrocarbonic acid diethyl ester of 1 ‰ volumes in water without RNA enzyme water, 25 DEG C, 500r/min magnetic agitation 12h, 121 DEG C, sterilizing 30min under 102.9kPa condition are 6 with acetic acid adjust pH, become pH value be 6 without RNA enzyme water; Described surfactant is one or more compositions of PLURONICS F87, poloxamer188, bovine serum albumin, polyvinyl alcohol, sodium cholate, lecithin;
C, oil phase is added dropwise to (volume ratio of aqueous phase and oil phase is 4-10) in aqueous phase, obtains load positive charge PLGA copolymer drug-carrying nanometer particle, steam removing organic solvent through stirring or revolving, the PLGA copolymer drug-carrying nanometer particle solution after organic solvent must be removed;
D, by removing organic solvent after PLGA copolymer drug-carrying nanometer particle solution through centrifugal removing free drug, or dialysis removing free drug, again genomic medicine is added in nanoparticle solution, obtain the targeted drug delivery system nanoparticle carrying chemotherapeutics and genomic medicine altogether;
(3) carry the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe altogether, method is:
A, PLGA copolymer, chemotherapeutics and adjuvant are dissolved in (concentration of PLGA copolymer is 5mg-50mg/ml, and the concentration of chemotherapeutics is 1mg/ml-10mg/ml, and the volumetric concentration of adjuvant is 0.5%-1%) in organic solvent, are prepared into oil phase;
Described organic solvent is one or more the compositions in acetone, ethyl acetate, dichloromethane, acetonitrile, methanol; Described PLGA copolymer is the copolymer of polylactide, Acetic acid, hydroxy-, bimol. cyclic ester, and the weight ratio of Ju Bing Jiao Zhi ︰ Acetic acid, hydroxy-, bimol. cyclic ester is be one in 25 ︰ 75,50 ︰ 50,75 ︰ 25, and the molecular weight of PLGA is the one in 17kD, 50KD, 100kD.Described chemotherapeutics is one or more compositions of doxorubicin hydrochloride, paclitaxel, docetaxel, cisplatin, carboplatin, daunorubicin, 5-fluorouracil; Described adjuvant is one or more the compositions in sorbester p17, polysorbas20, Tween 80; Described fluorescent probe is the one in indocyanine green, phthalocyanine, Coumarin-6, Fluorescein isothiocyanate (FITC), methylene blue, rhodamine.
B, by surfactant and chitosan-Angiopep-2 dissolution of polymer in pH value be 6 without (surfactant concentration is 5mg/ml-50mg/ml, and the concentration of chitosan-Angiopep-2 is 0.02mg/ml-2mg/ml) in RNA enzyme water, be prepared into aqueous phase;
Described pH value be 6 the enzyme water without RNA be first will add the pyrocarbonic acid diethyl ester of 1 ‰ volumes in water, 25 DEG C, 500r/min magnetic agitation 12h, with acetic acid adjust pH be 6,121 DEG C, sterilizing 30min under 102.9kPa condition, become pH value be 6 without RNA enzyme water; Described surfactant is one or more compositions of PLURONICS F87, poloxamer188, bovine serum albumin, polyvinyl alcohol, sodium cholate, lecithin;
C, oil phase is added dropwise to (volume ratio of aqueous phase and oil phase is 4-10) in aqueous phase, removing organic solvent is steamed through stirring or revolving, the Targeted PLGA copolymer drug-carrying nanometer particle solution carrying chemotherapeutics and fluorescent probe altogether of organic solvent must be removed, through centrifugal removing free drug, or dialysis removing free drug, obtain the targeted drug delivery system nanoparticle carrying chemotherapeutics and fluorescent probe altogether finally;
Carry the Targeted PLGA copolymer nano particle of chemotherapeutics and gene altogether, as medicine, be effective to treatment of brain tumor, realize the application of Targeted PLGA copolymer nano particle in treatment cerebral tumor medicine of carrying chemotherapeutics and gene altogether;
Carry the Targeted PLGA copolymer nano particle of chemotherapeutics and fluorescent probe altogether, as medicine, be effective to chemotherapy and fluorescent probe in treatment of brain tumor, diagnosis, realize the application of Targeted PLGA copolymer nano particle in treatment of brain tumor, diagnostic medicine of carrying chemotherapeutics and fluorescent probe altogether;
The described cerebral tumor is U87, U251 glioma cell, hair shape astrocytoma, astrocytoma, poorly differentiated astrocytoma, the one in pleiomorphism neuroblastoma.
In order to further describe specific embodiment of the invention scheme, spy provides following examples:
Embodiment 1
(1), the preparation of chitosan-Angiopep-2 polymer, method is:
A, chitosan 20mg is added volumetric concentration is in the acetic acid solution 8mL of 20%, is 500W ultrasonic dissolution 30min, with 5M(and 5mol/L at power) NaOH solution adjust pH to 6.0, obtain chitosan solution;
B, in chitosan solution, add 3-maleimidopropionic acid N-hydroxy-succinamide ester 5mg, under nitrogen protection, 30 DEG C, 100r/min magnetic agitation reaction 48h, after having reacted, the bag filter that reactant liquor is placed in molecular cut off 8kD-14kD to be dialysed 60h at 1000mL PBS, remove unreacted 3-maleimidopropionic acid N-hydroxy-succinamide ester, obtain dialysis solution;
C, take Angiopep-2 5mg and be dissolved in 20mL ultra-pure water, add 10mg Traut's reagent, ultrasonic 1h, temperature is 50 ° of C, make the end of Angiopep-2 have sulfydryl, being placed in molecular cut off is that the bag filter of 8kD-14kD to be dialysed 48h at 1000mLPBS, Angiopep-2 and the Traut ' s that removing is free, dialysis solution, at-80 DEG C of lyophilisation 72h, obtains the Angiopep-2 of sulfhydrylation;
The Angiopep-2 2mg of the sulfhydrylation that step c prepares is added in d, in stepb dialysis solution; under nitrogen protection, 35 DEG C, 100-200r/min stirring reaction 24h; obtain the chitosan that Angiopep-2 modifies; being placed in molecular cut off is again that the bag filter of 8kD-14kD to be dialysed 48h at 1000mLPBS; the Angiopep-2 that removing is free; at-80 DEG C of lyophilisation 72h, obtain chitosan-Angiopep-2 polymer, in-20 DEG C of storages.
Embodiment 2
The synthesis of chitosan-Angiopep-2 polymer
1) take chitosan 20mg, add 8mL20% acetic acid solution, 500W ultrasonic dissolution, adjust pH to 6.0 with 5M NaOH;
2) in above-mentioned chitosan solution, add 3-maleimidopropionic acid N-hydroxy-succinamide ester 5mg, heat 30 ° of C under nitrogen protection, 100r/min magnetic agitation reaction 48h, after having reacted, reactant liquor is placed in the bag filter of molecular cut off 8kD-14kD at the unreacted 3-maleimidopropionic acid N-hydroxy-succinamide ester of 1000mLPBS dialysis removing, dialysis 60h;
3) solution in bag filter is taken out; add end be cysteine Angiopep-2(synthesis Angiopep-2 in carboxyl terminal add a cysteine); heated and stirred makes it fully react under nitrogen protection; it is that the bag filter of 8kD-14kD to be dialysed 48h at 1000mLPBS that the chitosan that Angiopep-2 modifies is placed in molecular cut off; to remove free Angiopep-2; finally after-80 ° of C lyophilization 72h, be stored in-20 DEG C of refrigerators.
Example 3
Carry the preparation of the Targeted PLGA nanoparticle of doxorubicin hydrochloride and EGFR siRNA altogether
1) take 100mgPLGA(Ju Bing Jiao Zhi ︰ Acetic acid, hydroxy-, bimol. cyclic ester=50 ︰ 50, molecular weight is 17kD), be dissolved in 10mL acetone, prepare PLGA stock solution (10mg/mL), take 15mg doxorubicin hydrochloride, be dissolved in 5mL methanol, obtain doxorubicin hydrochloride stock solution (3mg/mL);
2) take 1g bovine serum albumin, add 100mLpH value be 6 without in RNA enzyme aqueous solution, dissolve completely, add chitosan-Angiopep-2 62.5mg, ultrasonic dissolution is complete, as aqueous phase;
3) 5mL doxorubicin hydrochloride stock solution is got, add in 10mLPLGA storing solution (10mg/mL), ultrasonic 1min, as oil phase, oil phase with the speed of 1mL/min dropwise under agitation (100rpm) add in 60mL aqueous phase, then Probe Ultrasonic Searching (power 200W, working time 3s, intermittent time 6s, work times 20 times).At room temperature magnetic agitation 6h removes organic solvent-acetone and methanol.With the centrifugal 60min of 12000rpm, abandoning supernatant obtains nanoparticle;
4) nanoparticle pH value be 6 without the aqueous dispersion of RNA enzyme ,-80 DEG C of lyophilization 72h obtain the nanoparticle of lyophilizing.With disperseing without RNA enzyme during use.Nano-ZS90 type laser particle size analyzer is used to measure, refractive index is set to 1.590, absorptance is set to 0.010, temperature is set to 25 DEG C, measurement pattern is set to automatically, using Z average statistical value as measurement result, each horizontal nano grain solution all prepares 3 parts, measures three times, gets the meansigma methods of three measured values as measurement result for every part, dielectric constant is set to 79, coefficient of viscosity is set to 0.8872, and temperature is set to 25 DEG C, and measurement pattern is set to automatically, acquired results is particle diameter is 100 ~ 200nm, and current potential is+38mV ~+18mv;
5) nanoparticle and the EGFR siRNA mixture of different quality ratio is prepared, incubated at room temperature 20min, the ratio of complete both the load siRNA of nanoparticle is determined by the band brightness of agarose gel electrophoresis swimming lane and position, when nanoparticle and siRNA mass ratio are 30 ~ 90, nanoparticle can load siRNA completely;
SiRNA sequence in above-mentioned steps 5 is:
Sense5′-3′ GUG UGU AAC GGA AUA GGU ATT
Ansense5′-3′ UAC CUA UUC CGU UAC ACA CTT。
Example 4
Carry the preparation of the Targeted PLGA nanoparticle of docetaxel and EGFR siRNA altogether
1) take 150mgPLGA(polylactide: Acetic acid, hydroxy-, bimol. cyclic ester=50:50, molecular weight is 17kD) and 45mg docetaxel, be dissolved in 15mL acetone, as oil phase, PLGA concentration is 10mg/mL, and docetaxel concentration is 3mg/mL;
2) take 1g bovine serum albumin, add 100mL pH value be 6 without in RNA enzyme water, dissolve completely, with second acid for adjusting pH to 6.0, add chitosan-Angiopep-2 and be about 62.5mg, ultrasonic dissolution is complete, as aqueous phase;
3) get 15mL oil phase with the speed of 1mL/min dropwise under agitation (100rpm) add in 60mL aqueous phase, then Probe Ultrasonic Searching (power 200W, working time 3s, intermittent time 6s, work times 20 times).At room temperature magnetic agitation 6h removes organic solvent-acetone and methanol.With the centrifugal 60min of 12000rpm, abandoning supernatant obtains nanoparticle;
4) nanoparticle pH value be 6 without the aqueous dispersion of RNA enzyme ,-80 DEG C of lyophilization 72h obtain the nanoparticle of lyophilizing.Use without the aqueous dispersion of RNA enzyme during use.Nano-ZS90 type laser particle size analyzer is used to measure, refractive index is set to 1.590, absorptance is set to 0.010, temperature is set to 25 DEG C, measurement pattern is set to automatically, using Z average statistical value as measurement result, each horizontal nano grain solution all prepares 3 parts, measures three times, gets the meansigma methods of three measured values as measurement result for every part, dielectric constant is set to 79, coefficient of viscosity is set to 0.8872, and temperature is set to 25 DEG C, and measurement pattern is set to automatically, acquired results is particle diameter is 100 ~ 200nm, and current potential is+38mV ~+18mv;
5) nanoparticle and the siRNA mixture of different quality ratio is prepared, incubated at room temperature 20min, the ratio of complete both the load siRNA of nanoparticle is determined by the band brightness of agarose gel electrophoresis swimming lane and position, nanoparticle and siRNA mass ratio are 30 ~ 90, and nanoparticle can load siRNA completely;
SiRNA sequence in above-mentioned steps 5 is:
Sense5′-3′ GUG UGU AAC GGA AUA GGU ATT
Ansense5′-3′ UAC CUA UUC CGU UAC ACA CTT。
Example 5
Carry the preparation of the Targeted PLGA nanoparticle of docetaxel and Coumarin-6 altogether
1) take 100mgPLGA(polylactide: Acetic acid, hydroxy-, bimol. cyclic ester=50:50, molecular weight is 17kD) and 30mg docetaxel, be dissolved in acetone, prepare the stock solution of PLGA and docetaxel, PLGA concentration is 10mg/mL, and docetaxel concentration is 3mg/mL;
2) take 1mg Coumarin-6, be dissolved in 10mL acetone, obtain Coumarin-6 stock solution (0.1mg/mL);
3) take 1g bovine serum albumin, add in 100mL ultra-pure water, dissolve completely, with second acid for adjusting pH to 6.0, add chitosan-Angiopep-2 and be about 62.5mg, ultrasonic dissolution is complete, as aqueous phase;
4) get Coumarin-6 stock solution (0.1mg/mL) 5mL, add in (1) in made PLGA and docetaxel stock solution 1mL, the ultrasonic 1min of 500W, as oil phase.Oil phase with the speed of 1mL/min dropwise under agitation (100rpm) add in 60mL aqueous phase, then Probe Ultrasonic Searching (power 200W, working time 3s, intermittent time 6s, work times 20 times).At room temperature magnetic agitation 6h removes organic solvent-acetone and methanol, and with the centrifugal 60min of 12000rpm, abandoning supernatant obtains nanoparticle;
5) nanoparticle ultra-pure water disperses, and-80 DEG C of lyophilization 72h obtain the nanoparticle of lyophilizing.Disperse with ultra-pure water during use.Nano-ZS90 type laser particle size analyzer is used to measure, refractive index is set to 1.590, absorptance is set to 0.010, temperature is set to 25 DEG C, measurement pattern is set to automatically, using Z average statistical value as measurement result, each horizontal nano grain solution all prepares 3 parts, measures three times, gets the meansigma methods of three measured values as measurement result for every part, dielectric constant is set to 79, coefficient of viscosity is set to 0.8872, and temperature is set to 25 DEG C, and measurement pattern is set to automatically, acquired results is particle diameter is 100 ~ 200nm, and current potential is+38mV ~+18mv.
Example 6
Carry the preparation of the Targeted PLGA nanoparticle of docetaxel and indocyanine green altogether
1) take 100mgPLGA(polylactide: Acetic acid, hydroxy-, bimol. cyclic ester=50:50, molecular weight is 17kD) and 30mg docetaxel, be dissolved in acetone, prepare the stock solution of PLGA and docetaxel, PLGA concentration is 10mg/mL, and docetaxel concentration is 3mg/mL;
2) take 1mg indocyanine green, be dissolved in 10mL methanol, obtain indocyanine green stock solution (0.1mg/mL);
3) take 1g bovine serum albumin, add in 100mL ultra-pure water, dissolve completely, with second acid for adjusting pH to 6.0, add chitosan-Angiopep-2 and be about 62.5mg, ultrasonic dissolution is complete, as aqueous phase;
4) indocyanine green stock solution (0.1mg/mL) 5mL is got, add in PLGA stock solution made in (1) and docetaxel stock solution 10mL, the ultrasonic 1min of 500W, as oil phase, oil phase with the speed of 1mL/min dropwise under agitation (100rpm) add in 60mL aqueous phase, then Probe Ultrasonic Searching (power 200W, working time 3s, intermittent time 6s, work times 20 times), at room temperature magnetic agitation 6h removes organic solvent-acetone and methanol, and with the centrifugal 60min of 12000rpm, abandoning supernatant obtains nanoparticle;
5) nanoparticle ultra-pure water disperses, and-80 DEG C of lyophilization 72h obtain the nanoparticle of lyophilizing.Disperse with ultra-pure water during use.Use Nano-ZS90 type laser particle size analyzer to measure, refractive index is set to 1.590, and absorptance is set to 0.010, and temperature is set to 25 DEG C, and measurement pattern is set to automatically, using Z average statistical value as measurement result.Each horizontal nano grain solution all prepares 3 parts, measures three times, gets the meansigma methods of three measured values as measurement result for every part.Dielectric constant is set to 79, and coefficient of viscosity is set to 0.8872, and temperature is set to 25 DEG C, and measurement pattern is set to automatically, and acquired results is particle diameter is 100 ~ 200nm, and current potential is+38mV ~+18mv.
Indocyanine green is strong especially near infrared absorbing ability, and for the light of 780nm, have bibliographical information, the light absorbing ability of indocyanine green of equal in quality is more than 7 times of SWCN, is more than 8500 times of nanometer gold bar.The PLGA targeted nano granule carrying docetaxel and indocyanine green in the present embodiment altogether can effectively carry indocyanine green and docetaxel to enter brain, indocyanine green thermotherapy effect is played, to the effect of thermotherapy and chemotherapy combined treatment tumor can be obtained by outside 808nm laser.
The Targeted PLGA nanoparticle picked-up of carrying chemotherapeutics and fluorescent probe altogether enters brain tumor cell U87MG or U251MG and through blood brain barrier, realizes the treatment of the cerebral tumor;
The Targeted PLGA nanoparticle carrying chemotherapeutics and fluorescent probe altogether suppresses tumor cell in vitro U87MG or U251MG, realizes the treatment of the cerebral tumor;
The Targeted PLGA nanoparticle carrying chemotherapeutics and genomic medicine altogether or carry chemotherapeutics and fluorescent probe altogether suppresses in-vivo tumour A, realizes the treatment of the cerebral tumor; Described tumor A is the described cerebral tumor is U87, U251 glioma cell, hair shape astrocytoma, astrocytoma, poorly differentiated astrocytoma, the one in pleiomorphism neuroblastoma.
The evaluation that above-mentioned Targeted PLGA nanoparticle picked-up of carrying chemotherapeutics and fluorescent probe altogether enters brain tumor cell U87MG or U251 adopts laser confocal microscope, observe brain tumor cell U87MG or U251MG through carrying the Targeted PLGA nanoparticle effect 1h of chemotherapeutics and fluorescent probe altogether, intracellular fluorescence distribution situation after 2h, 3h.
The above-mentioned Targeted PLGA nanoparticle carrying chemotherapeutics and fluorescent probe altogether adopts the micro-and bioluminescence imaging technology of laser co-focusing through the evaluation of blood brain barrier ability.Mouse tail vein injection carries the Targeted PLGA nanoparticle 1h of chemotherapeutics and fluorescent probe altogether, takes out cerebral tissue, prepare frozen section after 2h, 3h, directly in living imaging instrument, observes the distribution situation of medicine at brain.Or with the 4' of 1 μ g/mL, 6-diamidino-2-phenylindone (4', 6-diamidino-2-phenylindole, DAPI) dye 15min, rinse twice with the PBS of pH7.4, naturally dry, under laser confocal microscope, observe cerebral tissue Chinese medicine distribution situation.
The above-mentioned Targeted PLGA nanoparticle carrying chemotherapeutics and fluorescent probe altogether can adopt bioluminescence imaging technology through the evaluation of blood brain barrier ability.Mouse tail vein injection carries the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe altogether, after injection 1h, 2h, 3h, by mouse anesthesia, is placed in living imaging instrument and observes the distribution situation of medicine at brain.Brain fluorescence is more much brighter, and the ability of agent permeates therethrough blood brain barrier is stronger.
The inventive method is reliable and stable, products obtained therefrom quality is good, have can transmit small-molecule chemical medicine and genomic medicine simultaneously, or transmission chemotherapeutics and fluorescent probe enter brain by blood brain barrier simultaneously, play the collaborative targeted therapy cerebral tumor or the targeted therapy cerebral tumor and the effect diagnosed in one, more drug delivery can be entered brain by the Targeted PLGA nanoparticle carrying chemotherapeutics and genomic medicine altogether or the Targeted PLGA nanoparticle carrying chemotherapeutics and fluorescent probe altogether, making can not can through blood brain barrier through the medicine of blood brain barrier, reduce the distribution of medicine in periphery, improve brain drug level, increase the safety of medicinal application, and achieve useful technique effect through test, related tests data is as follows:
1, drug-loading system transhipment enters the investigation of U87 MG cell
Because cells were tested by flow cytometry siRNA transfection efficiency requires that medicine has fluorescence, therefore in the process implementing this example, siRNA in example 3 in drug-loading system changes the siRNA of CF 5(6)-Carboxyfluorescein (FAM) labelling into, and base composition is identical with the EGFR siRNA in example 3.
U87 MG cell culture is being contained hyclone (FBS) 10%, and in the MEM culture medium of mycillin mixed liquor 1%, condition of culture is 37 DEG C, 5% CO
2, within every 2 ~ 3 days, go down to posterity once.Collect logarithmic (log) phase cell, with there being blood to adjust concentration of cell suspension without antibody, the 6 every holes of orifice plate add 2 mL, and bed board makes cell to be measured adjust density to 2 × 10
5individual/hole.Be placed in 5% CO
2, hatch 24 h for 37 DEG C, be paved with (6 hole flat underside) at the bottom of hole to cell monolayer.Discard culture medium, clean cell hole 2 times by depletion of blood nonreactive culture medium, add the Targeted PLGA nanoparticle carrying doxorubicin hydrochloride and EGFR siRNA altogether, wherein the concentration of doxorubicin hydrochloride is 3.6 μ g/mL, and siRNA concentration is 50nmol/L.After cultivating 5h, each hole PBS cleans twice, and with the trypsin digestion cell 2min not containing EDTA, the culture medium added containing serum stops digestion, and collecting cell, the centrifugal 5min of 1000rpm, supernatant discarded, overhangs cell with PBS, uses BD Accuri
tMc6 flow cytometer detects.First data analysis application Flow Jo7.6.1 software carry out fluorescence compensation, then FL2 and FL1 passage respectively analysis of cells to the transfection efficiency of doxorubicin hydrochloride and siRNA.
Experimental result shows, in drug-loading system, the transfection efficiency of siRNA is 79%, and the transfection efficiency of doxorubicin hydrochloride is 85%.
2, nanoparticle system enters the situation test of brain through blood brain barrier
Get normal BALB/c-nu mice, in tail vein injection example 5, carry the Targeted PLGA nanoparticle (Coumarin-6 dosage 0.16mg/kg) of docetaxel and Coumarin-6 altogether.At predetermined point of time (the rear 1h of injection, 2h and 3h) adopt chloral hydrate anesthesia mice after cut off thoracic cavity, action wants fast, through the 20mL syringe that left ventricle insert head sword-shaped needle connects, and scalp acupuncture blunt, from with long axis of body 45 DEG C of direction inserting needles, needle point inserts in ascending aorta, can see, action wants soft, cut off the right heart simultaneously, push after 20mL normal saline has pushed away and change 4 DEG C of paraformaldehyde 20mL rapidly.Cut off scalp, take out brain, with the embedding of OCT glue, be placed in-20 DEG C of pre-coolings, fixing cerebral tissue, cut into slices with freezing microtome at-20 DEG C, slice thickness 5 μm, is placed on microscope slide.With the DAPI lucifuge dyeing 15min of 1 μ g/mL, clean three times to remove unnecessary DAPI with the PBS of pH7.4.Enter the situation of brain at laser co-focusing fiber Microscopic observation nanoparticle and take pictures.
Green fluorescence bright spot in brain tissue slice is more, and color is darker, and perinuclear bright spot is more much darker, and nanoparticle is more through the amount of blood brain barrier.Result shows, at 1h, 2h, 3h, drug-loading system all has distribution at brain, and 2h and 3h nanoparticle is in the distribution showed increased of brain.In 2h and 3h brain, the distribution of nanoparticle does not have notable difference.
3, nanoparticle anticancer experiment in vitro
By U87MG brain glioblastoma cell (being provided by Shanghai cell bank) as cancerous cell to be investigated.U87MG cell culture is being contained hyclone (FBS) 10%, and in the MEM culture medium of mycillin mixed liquor 1%, condition of culture is 37 DEG C, 5% CO
2, within every 2 ~ 3 days, go down to posterity once.Collect logarithmic (log) phase cell, adjustment concentration of cell suspension, the 96 every holes of orifice plate add 200 μ l, and bed board makes cell to be measured adjust density to 6 × 10
3individual/hole, (the aseptic PBS of edge hole fills).Be placed in 5% CO
2hatch 24 h for 37 DEG C, (96 hole flat underside) at the bottom of hole is paved with to cell monolayer, add the Targeted PLGA nanoparticle carrying doxorubicin hydrochloride and EGFR siRNA altogether in example 3, wherein doxorubicin hydrochloride strengths concentration is 0.18 μ g/mL, 0.36 μ g/mL, 0.78 μ g/mL, the concentration of EGFR siRNA is 50nmol/L, and not add drug delivery system of the present invention for matched group, arranging multiple hole is 4 ~ 6.Cell plates are placed in 5%CO
2hatch 48 h in incubator, stop cultivating, sucking-off pastille culture medium, every hole 150 μ l PBS wash 2 times, add 10% TCA 200 μ l of pre-cooling, place 1 h for 4 DEG C.Outwell fixative, every hole deionized water washes 5 times, dries, air drying.Every hole adds the SRB solution of 100 μ l, leaves standstill placement 10 min, does not wash 5 times, air drying with protein bound SRB 1% acetic acid.In conjunction with SRB 150 μ l 10 mmol/L non-buffered Tris alkali dissolutions.The OD value in every hole is measured at 565 nm places.The computing formula of suppression ratio: suppression ratio=1-experimental group OD value/matched group OD value, wherein experimental group and matched group are the value after deducting blank group.
Result shows, after drug effect 48h, it is 0.18 μ g/mL that the cell inhibitory rate of drug-loading system to U87 cell is respectively 43%(doxorubicin hydrochloride strengths, EGFR siRNA concentration is 50nmol/L), 65%(doxorubicin hydrochloride strengths is 0.36 μ g/mL, EGFR siRNA concentration is 50nmol/L), 76%(doxorubicin hydrochloride strengths is 0.78 μ g/mL, EGFR siRNA concentration is 50nmol/L).
4, the application of drug-loading system in treatment of brain tumor
All zooperies are all carried out according to the guide of the assessment of Ethics Committee of Zhengzhou University and accreditation.
1) in super-clean bench, BALB/c-nu nude mice is anaesthetized with pentobarbital sodium (40mg/kg).
2) first treat field of operation with iodophor disinfection, cut off scalp with operating scissors at front cranium place, at skull precontract 3mm, right about 2mm inserting needle 5mm, extracts 1mm, with speed injection U87MG tumor cell suspension 10 μ l(1 × 10 of 1 μ l/min
7).After injection, stop 10min, extract pin.
3) suture operation otch, uses iodophor disinfection field of operation.After IVC Animal House raises 7 days, be divided into four groups, group is respectively normal saline group, carry the Targeted PLGA nanoparticle group of doxorubicin hydrochloride, carry the Targeted PLGA nanoparticle group of EGFR siRNA, carry the Targeted PLGA nanoparticle group of doxorubicin hydrochloride and EGFR siRNA altogether, wherein doxorubicin hydrochloride dosage is the dosage of 3mg/kg, EGFR siRNA is 1.2mg/kg.Be administered once every one day, administration 5 times, the survival period of record animal, utilizes GraphPad Prism 5.0 to calculate the median survival interval of each treated animal.
Experimental result, normal saline group, carry the Targeted PLGA nanoparticle group of doxorubicin hydrochloride, carry the Targeted PLGA nanoparticle group of EGFR siRNA, the median survival interval of carrying the Targeted PLGA nanoparticle treated animal of doxorubicin hydrochloride and EGFR siRNA is altogether respectively 18 ± 0.8 days, 2.1 ± 2.0 days, 19 ± 3.0 days, 28 ± 2.7 days.Experimental result shows, carry the median survival interval (compared with normal saline P<0.05) that doxorubicin hydrochloride and the Targeted PLGA nanoparticle group of EGFR siRNA can significantly improve animal altogether, and carry the Targeted PLGA nanoparticle of doxorubicin hydrochloride, the animal median survival interval of carrying the Targeted PLGA nanoparticle group of EGFR siRNA does not all have significant difference compared with normal saline.
Above-mentionedly to show, the invention provides a kind of targeted drug based on PLGA and carry transmission system, preparation method and the application in treatment of brain tumor altogether.Targeted drug based on PLGA of the present invention carries transmission system altogether, very low to the toxicity of organism, and well, quality is good, and the condition of preparation easily meets, abundant raw material source, and cost is low for physics and chemical stability.Based on the targeted drug delivery system of PLGA, Angiopep-2 is as target head, this nanoparticle drug-loading system can through blood brain barrier, no matter test result is external or can the well generation of inhibition tumor cell and tumor tissues and development in body if showing, have the effect of the fine anti-cerebral tumor.Expection may be used for combining loading chemotherapeutics and genomic medicine or combining loads chemotherapeutics and fluorescent probe, is that on treatment of brain tumor medicine innovates greatly.Compared with prior art there is following outstanding Advantageous Effects:
1) the PLGA nanoparticle drug-loading system of lotus positive electricity prepared jointly by the chitosan that Angiopep-2 of the present invention modifies as a kind of novel carrier material and PLGA, can load various chemotherapeutics and genomic medicine simultaneously, have broad spectrum activity.
2) the equal biodegradable of targeted nano granule drug-loading system material therefor based on PLGA provided by the invention, has no side effect, good biocompatibility, there is slow release and targeting, cheap and easy to get, good product quality, use safety, cost is low, is only 1/3 of similar drugs cost.
3) the targeted nano granule drug-loading system based on PLGA provided by the invention can through blood brain barrier, and inside and outside all has the effect of anti-cerebral glioma.
4) the Targeted PLGA nanoparticle carrying chemotherapeutics and fluorescent probe altogether provided by the invention not only can realize the targeted therapy of the cerebral tumor, and vivo tumor target tracing can be realized, through clinical 116 patient with brain tumors application, except 1 people is without except positive effect, all the other all achieve beneficial therapeutic effect in various degree, and effective percentage reaches more than 99%, and getting well of its effect was not expected, have actual clinical meaning, economic and social benefit is huge.
Claims (8)
1. the targeting based on Poly(D,L-lactide-co-glycolide carries the preparation method of drug delivery system nanoparticle altogether, it is characterized in that, the active amino on chitosan is utilized to connect Angiopep-2, first chitosan and 3-maleimidopropionic acid N-hydroxy-succinamide ester react, the amino terminal of chitosan is made to have maleimide, react with the sulfydryl of the Angiopep-2 of terminal sulfhydryl group again, obtain chitosan-Angiopep-2 polymer, this polymer is used for the preparation of carrying drug delivery system based on the targeting of PLGA altogether, the method that the Targeted PLGA nanoparticle of chemotherapeutics and genomic medicine is carried in preparation is altogether, PLGA, chemotherapeutics and adjuvant are dissolved in organic solvent, be prepared into oil phase, by surfactant and chitosan-Angiopep-2 dissolution of polymer in adding with or without in RNA enzyme water, become aqueous phase, oil phase adds in aqueous phase, magnetic agitation or revolve steam removing organic solvent, through centrifugal or dialysis removing free drug, make PLGA drug-carrying nanometer particle, added by genomic medicine in nanoparticle solution, the targeting obtained based on PLGA copolymer carries drug delivery system nanoparticle altogether, the method that the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe is carried in preparation is altogether dissolved in organic solvent by PLGA, chemotherapeutics, fluorescent probe and adjuvant, is prepared into oil phase, by surfactant and chitosan-Angiopep-2 dissolution of polymer in the water without RNA enzyme, oil phase adds in aqueous phase, magnetic agitation or revolve steam removing organic solvent, through centrifugal or dialysis removing free drug, carried the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe altogether.
2. the targeting based on Poly(D,L-lactide-co-glycolide according to claim 1 carries the preparation method of drug delivery system nanoparticle altogether, it is characterized in that, is realized by following steps:
(1), the preparation of chitosan-Angiopep-2 polymer, method is:
A, chitosan 20mg is added mass concentration is in the acetic acid solution 8mL of 20%, is 300-500W ultrasonic dissolution 30min, with 5M NaOH solution adjust pH to 6.0, obtains chitosan solution at power;
B, in chitosan solution, add 3-maleimidopropionic acid N-hydroxy-succinamide ester 5mg, under nitrogen protection, 30 DEG C, 100r/min magnetic agitation reaction 48h, after having reacted, it is that the bag filter of 8KD-14KD to be dialysed 60h at PBS that reactant liquor is placed in molecular cut off, remove unreacted 3-maleimidopropionic acid N-hydroxy-succinamide ester, obtain dialysis solution;
C, in dialysis solution, then add the Angiopep-2 2mg of sulfhydrylation, under nitrogen protection, 35 DEG C, 100-200r/min stirring reaction 24h, obtain the chitosan that Angiopep-2 modifies, being placed in molecular cut off is again that the bag filter of 8KD-14KD is dialysed 48h, the Angiopep-2 that removing is free, at-80 DEG C of lyophilisation 72h, obtain chitosan-Angiopep-2 polymer, for subsequent use in-20 DEG C of storages;
One in the sulfhydrylation Angiopep-2 obtained after the Angiopep-2 of the Angiopep-2 of described sulfhydrylation to be carboxyl terminal be cysteine or Angiopep-2 and Traut ' s reacts; The Angiopep-2 that described carboxyl terminal is connected with cysteine is, when synthesizing Angiopep-2, molecule Angiopep-2 carboxyl terminal connects the cysteine of a molecule by chemical reaction; The Angiopep-2 of the sulfhydrylation that described Angiopep-2 and Traut ' s obtains after reacting is, Angiopep-2 5mg is dissolved in 20mL ultra-pure water, add 10mg Traut's reagent, 50 DEG C of ultrasonic 1h, make the end of Angiopep-2 have sulfydryl, being then placed in molecular cut off is that the bag filter of 8KD-14kD to be dialysed 48h at PBS, Angiopep-2 and the Traut ' s that removing is free, dialysis solution, at-80 DEG C of lyophilisation 72h, obtains the Angiopep-2 of sulfhydrylation;
(2), prepare the Targeted PLGA nanoparticle carrying chemotherapeutics and genomic medicine altogether, method is:
A, be dissolved in organic solvent by PLGA copolymer, chemotherapeutics and adjuvant, the concentration of PLGA copolymer is 5mg-50mg/ml, and the concentration of chemotherapeutics is 1mg/ml-10mg/ml, and the volumetric concentration of adjuvant is 0.5%-1%, is prepared into oil phase;
Described organic solvent is one or more the compositions in acetone, ethyl acetate, dichloromethane, acetonitrile, methanol; Described PLGA copolymer is the copolymer of polylactide, Acetic acid, hydroxy-, bimol. cyclic ester, and the weight ratio of Ju Bing Jiao Zhi ︰ Acetic acid, hydroxy-, bimol. cyclic ester is the one in 25 ︰ 75,50 ︰ 50,75 ︰ 25, and the molecular weight of PLGA is the one in 17kD, 50KD, 100kD; Described chemotherapeutics is the one in doxorubicin hydrochloride, paclitaxel, docetaxel, cisplatin, carboplatin, daunorubicin, 5-fluorouracil; Described adjuvant is one or more the compositions in sorbester p17, polysorbas20, Tween 80;
B, by surfactant and chitosan-Angiopep-2 dissolution of polymer in pH value be 6 without in RNA enzyme water, be prepared into aqueous phase, surfactant concentration is 5-50mg/ml, and the concentration of chitosan-Angiopep-2 is 0.02-2mg/ml;
Described pH value be 6 be first will add the pyrocarbonic acid diethyl ester of 1 ‰ volumes in water without RNA enzyme water, 25 DEG C, 500r/min magnetic agitation 12h, with second acid for adjusting pH be 6,121 DEG C, sterilizing 30min under 102.9kPa condition; Described surfactant is one or more compositions of PLURONICS F87, poloxamer188, bovine serum albumin, polyvinyl alcohol, sodium cholate, lecithin;
C, be added dropwise in aqueous phase by oil phase, the volume ratio of aqueous phase and oil phase is that (4-10) ︰ 1, obtains load positive charge PLGA copolymer drug-carrying nanometer particle, steams removing organic solvent, must remove the PLGA copolymer drug-carrying nanometer particle solution after organic solvent through stirring or revolving;
D, organic solvent will be removed after PLGA copolymer drug-carrying nanometer particle solution through centrifugal removing free drug, or dialysis removing free drug, again genomic medicine is added in nanoparticle solution, obtain the targeted drug delivery system nanoparticle carrying chemotherapeutics and genomic medicine altogether;
(3) carry the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe altogether, method is:
A, be dissolved in organic solvent by PLGA copolymer, chemotherapeutics, fluorescent probe and adjuvant, the concentration of PLGA copolymer is 5-50mg/ml, and the concentration of chemotherapeutics is 1-10mg/ml, and the volumetric concentration of adjuvant is 0.5-1%, is prepared into oil phase;
Described organic solvent is one or more the compositions in acetone, ethyl acetate, dichloromethane, acetonitrile, methanol; Described PLGA copolymer is the copolymer of polylactide, Acetic acid, hydroxy-, bimol. cyclic ester, and the weight ratio of Ju Bing Jiao Zhi ︰ Acetic acid, hydroxy-, bimol. cyclic ester is be one in 25 ︰ 75,50 ︰ 50,75 ︰ 25, and the molecular weight of PLGA is the one in 17kD, 50KD, 100kD; Described chemotherapeutics is one or more compositions of doxorubicin hydrochloride, paclitaxel, docetaxel, cisplatin, carboplatin, daunorubicin, 5-fluorouracil; Described adjuvant is one or more the compositions in sorbester p17, polysorbas20, Tween 80; Described fluorescent probe is the one in indocyanine green, phthalocyanine, Coumarin-6, Fluorescein isothiocyanate, methylene blue, rhodamine;
B, by surfactant and chitosan-Angiopep-2 dissolution of polymer in pH value be 6 without in RNA enzyme water, surfactant concentration is 5-50mg/ml, and the concentration of chitosan-Angiopep-2 is 0.02-2mg/ml, is prepared into aqueous phase;
Described pH value be 6 the enzyme water without RNA be first will add the pyrocarbonic acid diethyl ester of 1 ‰ volumes in water, 25 DEG C, 500r/min magnetic agitation 12h, with acetic acid adjust pH be 6,121 DEG C, sterilizing 30min under 102.9kPa condition, become pH value be 6 without RNA enzyme water; Described surfactant is one or more compositions of PLURONICS F87, poloxamer188, bovine serum albumin, polyvinyl alcohol, sodium cholate, lecithin;
C, oil phase is added dropwise in aqueous phase, the volume ratio of aqueous phase and oil phase is (4-10) ︰ 1, removing organic solvent is steamed through stirring or revolving, the Targeted PLGA copolymer drug-carrying nanometer particle solution carrying chemotherapeutics and fluorescent probe altogether of organic solvent must be removed, through centrifugal removing free drug, or dialysis removing free drug, obtain the targeted drug delivery system nanoparticle carrying chemotherapeutics and fluorescent probe altogether.
3. the targeting based on Poly(D,L-lactide-co-glycolide according to claim 1 carries the preparation method of drug delivery system nanoparticle altogether, it is characterized in that, the preparation of described chitosan-Angiopep-2 polymer, and method is:
A, chitosan 20mg is added mass concentration is in the acetic acid solution 8mL of 20%, is 500W ultrasonic dissolution 30min, with 5M NaOH solution adjust pH to 6.0, obtains chitosan solution at power;
B, in chitosan solution, add 3-maleimidopropionic acid N-hydroxy-succinamide ester 5mg, under nitrogen protection, 30 DEG C, 100r/min magnetic agitation reaction 48h, after having reacted, the bag filter that reactant liquor is placed in molecular cut off 8kD-14kD to be dialysed 60h at 1000mL PBS, remove unreacted 3-maleimidopropionic acid N-hydroxy-succinamide ester, obtain dialysis solution;
C, take Angiopep-2 5mg and be dissolved in 20mL ultra-pure water, add 10mg Traut's reagent, ultrasonic 1h, temperature is 50 DEG C, make the end of Angiopep-2 have sulfydryl, being placed in molecular cut off is that the bag filter of 8kD-14kD to be dialysed 48h at 1000mLPBS, Angiopep-2 and the Traut ' s that removing is free, dialysis solution, at-80 DEG C of lyophilisation 72h, obtains the Angiopep-2 of sulfhydrylation;
The Angiopep-2 2mg of the sulfhydrylation that step c prepares is added in d, in stepb dialysis solution; under nitrogen protection, 35 DEG C, 100-200r/min stirring reaction 24h; obtain the chitosan that Angiopep-2 modifies; being placed in molecular cut off is again that the bag filter of 8kD-14kD to be dialysed 48h at 1000mLPBS; the Angiopep-2 that removing is free; at-80 DEG C of lyophilisation 72h, obtain chitosan-Angiopep-2 polymer, in-20 DEG C of storages.
4. the targeting based on Poly(D,L-lactide-co-glycolide according to claim 1 carries the preparation method of drug delivery system nanoparticle altogether, it is characterized in that, the preparation of described chitosan-Angiopep-2 polymer, and method is:
1) take chitosan 20mg, add 8mL20% acetic acid solution, 500W ultrasonic dissolution, adjust pH to 6.0 with 5M NaOH;
2) in above-mentioned chitosan solution, add 3-maleimidopropionic acid N-hydroxy-succinamide ester 5mg, heat 30 ° of C under nitrogen protection, 100r/min magnetic agitation reaction 48h, after having reacted, reactant liquor is placed in the bag filter of molecular cut off 8kD-14kD at the unreacted 3-maleimidopropionic acid N-hydroxy-succinamide ester of 1000mLPBS dialysis removing, dialysis 60h;
3) solution in bag filter is taken out; add the Angiopep-2 that end is cysteine; heated and stirred makes it fully react under nitrogen protection; it is that the bag filter of 8kD-14kD to be dialysed 48h at 1000mLPBS that the chitosan that Angiopep-2 modifies is placed in molecular cut off; to remove free Angiopep-2;-80 ° of C lyophilization 72h, are stored in-20 DEG C of refrigerators.
5. the targeting based on Poly(D,L-lactide-co-glycolide according to claim 1 carries the preparation method of drug delivery system nanoparticle altogether, it is characterized in that:
1) take 100mgPLGA, Ju Bing Jiao Zhi ︰ Acetic acid, hydroxy-, bimol. cyclic ester=50 ︰ 50, molecular weight is 17kD, be dissolved in 10mL acetone, prepare PLGA stock solution 10mg/mL, take 15mg doxorubicin hydrochloride, be dissolved in 5mL methanol, obtain doxorubicin hydrochloride stock solution 3mg/mL;
2) take 1g bovine serum albumin, add 100mL and contain in the aqueous solution of volume 1 ‰ DEPC, dissolve completely, with second acid for adjusting pH to 6.0, add chitosan-Angiopep-2 62.5mg, ultrasonic dissolution is complete, as aqueous phase;
3) get 5mL doxorubicin hydrochloride stock solution, add in 10mLPLGA storing solution, ultrasonic 1min, as oil phase, oil phase is dropwise added in 60mL aqueous phase with the speed of 1mL/min under 100rpm stirring condition, then Probe Ultrasonic Searching, power 200W, working time 3s, intermittent time 6s, work times 20 times, at room temperature magnetic agitation 6h removes organic solvent-acetone and methanol, with the centrifugal 60min of 12000rpm, abandoning supernatant obtains nanoparticle.
6. the targeting based on Poly(D,L-lactide-co-glycolide according to claim 1 carries the preparation method of drug delivery system nanoparticle altogether, it is characterized in that:
1) take 150mgPLGA, polylactide: Acetic acid, hydroxy-, bimol. cyclic ester=50:50, molecular weight is 17kD, and 45mg docetaxel, is dissolved in 15mL acetone, and as oil phase, PLGA concentration is 10mg/mL, and docetaxel concentration is 3mg/mL;
2) take 1g bovine serum albumin, add in 100mL DEPC water, dissolve completely, with second acid for adjusting pH to 6.0, add chitosan-Angiopep-2 62.5mg, ultrasonic dissolution is complete, as aqueous phase;
3) get 15mL oil phase dropwise to add under 100rpm stirring condition in 60mL aqueous phase with the speed of 1mL/min, then Probe Ultrasonic Searching, power 200W, working time 3s, intermittent time 6s, work times 20 times, at room temperature magnetic agitation 6h removes organic solvent-acetone, with the centrifugal 60min of 12000rpm, abandoning supernatant obtains nanoparticle.
7. the targeting of Poly(D,L-lactide-co-glycolide that prepared by method described in claim 1 carries the application of drug delivery system nanoparticle in preparation treatment cerebral tumor medicine altogether.
8. the targeting of Poly(D,L-lactide-co-glycolide according to claim 7 carries the application of drug delivery system nanoparticle in preparation treatment cerebral tumor medicine altogether, it is characterized in that, described medicine is the Targeted PLGA copolymer nano particle carrying chemotherapeutics and genomic medicine altogether, or carries the Targeted PLGA copolymer nano particle of chemotherapeutics and fluorescent probe altogether.
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