CN103623430A - 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 title 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 good biocompatibility, nontoxic, good encystation and the performance of film forming, be widely used in pharmacy, medical engineering material and modernization industry field.In the U.S., PLGA authenticates by FDA, formally as pharmaceutic adjuvant, is included into American Pharmacopeia.Become one of carrier material enjoying in recent years favor, be widely used in pharmaceutical carrier and medical bio engineering material field.Finally be degraded in vivo CO
2and H
2o.PLGA can effectively carry the bioactive substances such as protein, antibody, polypeptide, medicine and nucleic acid by certain technology and enter cell, thereby becomes the carrier that people pay close attention to.
Angiopep-2 is one of the most emerging novel targeted functional group, and it is the small peptide that 19 aminoacid of threonine-phenylalanine-phenylalanine-tyrosine-Gly-Gly-Vitro By Serine/arginine-glycine-lysine-arginine-agedoite-agedoite-phenylalanine-lysine-TE-Glu-Tyr form.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, can effectively 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 first phase clinical research that the conjugate GRN1005 Zheng U.S. of a molecule Angiopep-2 and three molecule paclitaxels enters anti-cerebral glioma.But this conjugate is poorly water-soluble still, the same with Texol during application, need to be by polyoxyethylene castor oil and ethanol as solubilizing agent, so still there is the shortcomings such as large and body-internal-circulation time of toxic and side effects is short.
The micromolecule such as doxorubicin hydrochloride, docetaxel antitumor drug is the substrate of brain capillary endothelial cell p-glycoprotein, thereby cannot see through blood brain barrier, enters brain essence and then the treatment cerebral tumor.Therefore by certain targeting group and nano-carrier, can overcome blood brain barrier delivering drugs 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 larger, is a dazzling nova in the disease treatments such as tumor.The performance of genomic medicine curative effect need to can be transported into cell by specific support.
In tumour medicine research, utilize fluorescent probe labeled drug, can be directly live body level observe medicine to tumor whether targeting, best targeting time and medicine in the accumulation of other organ-tissues of animal, be conducive to research worker in the best time, carry out fabric analysis.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.The fluorescent probe that is usually used in living imaging, great majority are traditional organic dye molecule, for example indocyanine green, phthalocyanine, methylene blue, rhodamine, Fluorescein isothiocyanate (FITC), and these organic dye molecules have some shortcomings of itself, as poor in light stability, the defect such as the low and targeting of sensitivity is poor, thereby limited it and further applied.By targeted nano granule carrier, can overcome above-mentioned defect, the diagnosis of tumor and targeted therapy are had to clear superiority.
For example, in order to realize the treatment of tumor, just independent chemotherapeutics often needs very heavy dose of antitumor curative effect that has, yet also to have brought certain toxicity, the cardiac toxicity of amycin simultaneously.Therefore chemotherapeutics becomes an important technical of oncotherapy with the use in conjunction of other treatment technology.The targeting of so how realizing 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 present invention's object is just to provide preparation method and the application that a kind of targeting based on Poly(D,L-lactide-co-glycolide carries drug delivery system nanoparticle altogether, can effectively solve the preparation difficult problem that the targeting based on Poly(D,L-lactide-co-glycolide carries drug delivery system nanoparticle altogether, and realize the application problem in cerebral tumor target therapeutic agent.
The technical scheme that the present invention solves is, utilize the active amino on chitosan to connect Angiopep-2, first chitosan reacts with 3-maleimide propanoic acid N-hydroxy-succinamide, make the amino terminal of chitosan there is maleimide, react with the Angiopep-2 of end sulfhydrylation again, obtain chitosan-angiopep-2 polymer, the preparation that the targeting based on PLGA carries drug delivery system altogether in preparation by this polymer applications.The scheme of Targeted PLGA transmission system nanoparticle that chemotherapeutics and genomic medicine are carried in preparation is altogether, PLGA, chemotherapeutics and adjuvant are dissolved in organic solvent, is prepared into oil phase; By surfactant and chitosan-angiopep-2 polymer dissolution in without RNA(ribonucleic acid, as follows) in the water of enzyme, be prepared into water, oil phase adds in water, and magnetic agitation or revolve to steam and remove organic solvent is removed free drug through centrifugal or dialysis, make PLGA drug-carrying nanometer particle, genomic medicine is added in nanoparticle solution, and the targeting obtaining based on PLGA copolymer carries drug delivery system nanoparticle altogether, and this nanoparticle can be effective in the medicine of preparation treatment cerebral tumor targeting.The scheme of Targeted PLGA transmission system nanoparticle that chemotherapeutics and fluorescent probe are carried in preparation is altogether, PLGA, chemotherapeutics, fluorescent probe and adjuvant are dissolved in organic solvent, is prepared into oil phase; By surfactant and chitosan-angiopep-2 polymer dissolution in the water without RNA enzyme, oil phase adds in water, magnetic agitation or revolve to steam and remove organic solvent, removes free drug finally by centrifugal or dialysis, obtains carrying altogether the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe.
The inventive method is reliable and stable, product result of use is good, can be used as medicine carrier system altogether, can transmit micromolecule chemicals and genomic medicine simultaneously, or transmit chemotherapeutics and fluorescent probe enters brain by blood brain barrier simultaneously, performance is worked in coordination with the targeted therapy cerebral tumor or the targeted therapy cerebral tumor and is diagnosed the effect in one, is the innovation on treatment of brain tumor medicine, has huge economic and social benefit.
The specific embodiment
Below in conjunction with concrete condition and embodiment, the specific embodiment of the present invention is elaborated.
By the technical scheme that provides of 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 to volumetric concentration is in 20% acetic acid solution 8mL, at power, is 300-500W ultrasonic dissolution 30min, with 5M(, is 5mol/L) NaOH solution adjust pH to 6.0, obtain chitosan solution;
B, in chitosan solution, add 3-maleimide propanoic acid-N-hydroxy succinic acid imines ester 5mg, under nitrogen protection, 30 ℃, 100r/min magnetic agitation reaction 48h, after having reacted, it is that the bag filter of 8KD-14KD is at the PBS 60h that dialyses that reactant liquor is placed in molecular cut off, remove unreacted 3-maleimide propanoic acid-N-hydroxy succinic acid imines ester, obtain dialysis solution;
C, then in dialysis solution, add the Angiopep-2 2mg of sulfhydrylation, under nitrogen protection, 35 ℃, 100-200r/min stirring reaction 24h, obtain the chitosan that Angiopep-2 modifies, the bag filter that is placed in again molecular cut off and the is 8KD-14KD 48h that dialyses, remove free Angiopep-2, at-80 ℃ of lyophilisation 72h, obtain chitosan-angiopep-2 polymer, standby in-20 ℃ of storages;
The Angiopep-2 of described sulfhydrylation is that carboxyl terminal is that the angiopep-2 of cysteine or angiopep-2 react a kind of in the sulfhydrylation angiopep-2 obtaining with Traut ' s.The angiopep-2 that described carboxyl terminal is connected with cysteine is that, when synthetic 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 obtains after reacting with Traut ' s is, Angiopep-2 5mg is dissolved in 20mL ultra-pure water, add 10mg Traut's reagent, 50 ℃ of ultrasonic 1h, make the end of Angiopep-2 have sulfydryl, the bag filter that is then placed in molecular cut off and is 8KD-14kD, at the PBS 48h that dialyses, is removed free angiopep-2 and Traut ' s, dialysis solution, at-80 ℃ of lyophilisation 72h, obtains the Angiopep-2 of sulfhydrylation;
(2), preparation carries the Targeted PLGA nanoparticle of chemotherapeutics and genomic medicine altogether, method is:
A, PLGA copolymer, chemotherapeutics and adjuvant are dissolved in to (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 a kind of in 25 ︰ 75,50 ︰ 50,75 ︰ 25, and the molecular weight of PLGA is 17kD, 50KD, a kind of in 100kD.Described chemotherapeutics is a kind of 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 polymer dissolution, in pH value, be 6 without in RNA enzyme water, be prepared into water (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 without RNA enzyme water, be, first will in water, add the pyrocarbonic acid diethyl ester of 1 ‰ volumes, 25 ℃, 500r/min magnetic agitation 12h, sterilizing 30min under 121 ℃, 102.9kPa condition, is 6 with acetic acid adjust pH, to 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 water and oil phase is 4-10) in water, obtains load positive charge PLGA copolymer drug-carrying nanometer particle, through stirring or revolving to steam, remove organic solvent, must remove the PLGA copolymer drug-carrying nanometer particle solution after organic solvent;
D, will remove PLGA copolymer drug-carrying nanometer particle solution after organic solvent through the centrifugal free drug of removing, or free drug is removed in dialysis, again genomic medicine is added in nanoparticle solution, obtain the targeted drug transmission system nanoparticle that carries altogether chemotherapeutics and genomic medicine;
(3) carry altogether the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe, method is:
A, PLGA copolymer, chemotherapeutics and adjuvant are dissolved in to (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 a kind of in 25 ︰ 75,50 ︰ 50,75 ︰ 25, and the molecular weight of PLGA is 17kD, 50KD, a kind of in 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 a kind of in indocyanine green, phthalocyanine, Coumarin-6, Fluorescein isothiocyanate (FITC), methylene blue, rhodamine.
B, by surfactant and chitosan-angiopep-2 polymer dissolution, 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 water;
Described pH value is that 6 the enzyme water without RNA is, first will in water, add the pyrocarbonic acid diethyl ester of 1 ‰ volumes, and 25 ℃, 500r/min magnetic agitation 12h are sterilizing 30min under 6,121 ℃, 102.9kPa condition with acetic acid adjust pH, and to 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 water and oil phase is 4-10) in water, through stirring or revolving to steam, remove organic solvent, must remove the Targeted PLGA copolymer drug-carrying nanometer particle solution that carries altogether chemotherapeutics and fluorescent probe of organic solvent, through the centrifugal free drug of removing, or dialyse and remove free drug, obtain the targeted drug transmission system nanoparticle that carries altogether chemotherapeutics and fluorescent probe finally;
The Targeted PLGA copolymer nano particle that carries altogether chemotherapeutics and gene, as medicine, is effective to treatment of brain tumor, realizes the application of Targeted PLGA copolymer nano particle in treatment cerebral tumor medicine carry altogether chemotherapeutics and gene;
Carry altogether the Targeted PLGA copolymer nano particle of chemotherapeutics and fluorescent probe, 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 carry altogether chemotherapeutics and fluorescent probe;
The described cerebral tumor is U87, U251 glioma cell, hair shape astrocytoma, astrocytoma, poorly differentiated astrocytoma, a kind of 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 to volumetric concentration is in 20% acetic acid solution 8mL, at power, is 500W ultrasonic dissolution 30min, with 5M(, is 5mol/L) NaOH solution adjust pH to 6.0, obtain chitosan solution;
B, in chitosan solution, add 3-maleimide propanoic acid-N-hydroxy succinic acid imines ester 5mg, under nitrogen protection, 30 ℃, 100r/min magnetic agitation reaction 48h, after having reacted, the bag filter that reactant liquor is placed in molecular cut off 8kD-14kD is at the 1000mL PBS 60h that dialyses, remove unreacted 3-maleimide propanoic acid-N-hydroxy succinic acid imines 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, the bag filter that is placed in molecular cut off and is 8kD-14kD, at the 1000mLPBS 48h that dialyses, is removed free angiopep-2 and Traut ' s, dialysis solution, at-80 ℃ of lyophilisation 72h, obtains the angiopep-2 of sulfhydrylation;
D, in step b, in dialysis solution, add the Angiopep-2 2mg of the sulfhydrylation that step c prepares; under nitrogen protection, 35 ℃, 100-200r/min stirring reaction 24h; obtain the chitosan that Angiopep-2 modifies; the bag filter that is placed in molecular cut off again and is 8kD-14kD is at the 1000mLPBS 48h that dialyses; remove free Angiopep-2; at-80 ℃ of lyophilisation 72h, obtain chitosan-angiopep-2 polymer, in-20 ℃ of storages.
Embodiment 2
Synthesizing of chitosan-angiopep-2 polymer
1) take chitosan 20mg, add 8mL20% acetic acid solution, 500W ultrasonic dissolution, adjusts pH to 6.0 with 5M NaOH;
2) in above-mentioned chitosan solution, add 3-maleimide propanoic acid N-hydroxy succinic acid imines ester 5mg, under nitrogen protection, heat 30 ° of C, 100r/min magnetic agitation reaction 48h, after having reacted, reactant liquor is placed in the bag filter of molecular cut off 8kD-14kD and removes unreacted 3-maleimide propanoic acid N-hydroxy succinic acid imines ester in 1000mLPBS dialysis, dialysis 60h;
3) take out the solution in bag filter; adding end is that Angiopep-2(carboxyl terminal in synthetic Angiopep-2 of cysteine adds a cysteine); under nitrogen protection, heated and stirred is fully reacted it; it is that the bag filter of 8kD-14kD is at the 1000mLPBS 48h that dialyses that the chitosan that Angiopep-2 modifies is placed in molecular cut off; to remove free Angiopep-2; after finally-80 ° of C lyophilization 72h, be stored in-20 ℃ of refrigerators.
Example 3
Carry altogether the preparation of the Targeted PLGA nanoparticle of doxorubicin hydrochloride and EGFR siRNA
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, after dissolve complete, add chitosan-Angiopep-2 62.5mg, ultrasonic dissolution is complete, as water;
3) get 5mL doxorubicin hydrochloride stock solution, add in 10mLPLGA storing solution (10mg/mL), ultrasonic 1min, as oil phase, oil phase with the speed of 1mL/min dropwise under stirring condition (100rpm) add in 60mL water, Probe Ultrasonic Searching (power 200W, working time 3s then, 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 is 6 without the aqueous dispersion of RNA enzyme, and-80 ℃ of lyophilization 72h obtain the nanoparticle of lyophilizing.During use, use without RNA enzyme and disperse.Use Nano-ZS90 type laser particle size analyzer to measure, refractive index is set to 1.590, absorptance is set to 0.010, temperature setting is set to 25 ℃, measurement pattern is set to automatically, using Z average statistics value as measurement result, each horizontal nanoparticle solution is all prepared 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 setting is set to 25 ℃, and measurement pattern is set to automatically, acquired results is that particle diameter is 100～200nm, and current potential is+38mV～+ 18mv;
5) nanoparticle and the EGFR siRNA mixture of preparation different quality ratio, under room temperature, hatch 20min, by the band brightness of agarose gel electrophoresis swimming lane and complete both ratios of load siRNA of location positioning nanoparticle, nanoparticle and siRNA mass ratio are 30～90 o'clock, and nanoparticle is 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 altogether the preparation of the Targeted PLGA nanoparticle of docetaxel and EGFR siRNA
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, docetaxel concentration is 3mg/mL;
2) take 1g bovine serum albumin, add 100mL pH value be 6 without in RNA enzyme water, after dissolve complete, with second acid for adjusting pH to 6.0, add the about 62.5mg of chitosan-angiopep-2, ultrasonic dissolution is complete, as water;
3) get 15mL oil phase with the speed of 1mL/min dropwise under stirring condition (100rpm) add in 60mL water, Probe Ultrasonic Searching (power 200W, working time 3s, intermittent time 6s, work times 20 times) then.At room temperature magnetic agitation 6h removes organic solvent-acetone and methanol.With the centrifugal 60min of 12000rpm, abandoning supernatant obtains nanoparticle;
4) nanoparticle is 6 with pH value without the aqueous dispersion of RNA enzyme, and-80 ℃ of lyophilization 72h obtain the nanoparticle of lyophilizing.During use, use without the aqueous dispersion of RNA enzyme.Use Nano-ZS90 type laser particle size analyzer to measure, refractive index is set to 1.590, absorptance is set to 0.010, temperature setting is set to 25 ℃, measurement pattern is set to automatically, using Z average statistics value as measurement result, each horizontal nanoparticle solution is all prepared 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 setting is set to 25 ℃, and measurement pattern is set to automatically, acquired results is that particle diameter is 100～200nm, and current potential is+38mV～+ 18mv;
5) nanoparticle and the siRNA mixture of preparation different quality ratio, under room temperature, hatch 20min, by the band brightness of agarose gel electrophoresis swimming lane and complete both ratios of load siRNA of location positioning nanoparticle, nanoparticle and siRNA mass ratio are 30～90, and nanoparticle is 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 altogether the preparation of the Targeted PLGA nanoparticle of docetaxel and Coumarin-6
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, 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, after dissolve complete, with second acid for adjusting pH to 6.0, add the about 62.5mg of chitosan-angiopep-2, ultrasonic dissolution is complete, as water;
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 stirring condition (100rpm) add in 60mL water, Probe Ultrasonic Searching (power 200W, working time 3s, intermittent time 6s, work times 20 times) then.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 disperses with ultra-pure water, and-80 ℃ of lyophilization 72h obtain the nanoparticle of lyophilizing.During use, with ultra-pure water, disperse.Use Nano-ZS90 type laser particle size analyzer to measure, refractive index is set to 1.590, absorptance is set to 0.010, temperature setting is set to 25 ℃, measurement pattern is set to automatically, using Z average statistics value as measurement result, each horizontal nanoparticle solution is all prepared 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 setting is set to 25 ℃, and measurement pattern is set to automatically, acquired results is that particle diameter is 100～200nm, and current potential is+38mV～+ 18mv.
Example 6
Carry altogether the preparation of the Targeted PLGA nanoparticle of docetaxel and indocyanine green
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, 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, after dissolve complete, with second acid for adjusting pH to 6.0, add the about 62.5mg of chitosan-angiopep-2, ultrasonic dissolution is complete, as water;
4) get indocyanine green stock solution (0.1mg/mL) 5mL, 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 stirring condition (100rpm) add in 60mL water, 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 disperses with ultra-pure water, and-80 ℃ of lyophilization 72h obtain the nanoparticle of lyophilizing.During use, with ultra-pure water, disperse.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 setting is set to 25 ℃, and measurement pattern is set to automatically, usings Z average statistics value as measurement result.Each horizontal nanoparticle solution is all prepared 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 setting is set to 25 ℃, and measurement pattern is set to automatically, and acquired results is that particle diameter is 100～200nm, and current potential is+38mV～+ 18mv.
Indocyanine green is strong especially near infrared extinction ability, and the light for 780nm, has bibliographical information, and the light absorbing ability of indocyanine green of equal in quality is more than 7 times of SWCN, is the more than 8500 times of nanometer gold bar.The PLGA targeted nano granule that carries altogether docetaxel and indocyanine green in the present embodiment can effectively carry indocyanine green and docetaxel to enter brain, by outside 808nm laser performance indocyanine green thermotherapy effect, to obtaining the effect of thermotherapy and chemotherapy combined treatment tumor.
The Targeted PLGA nanoparticle picked-up of carrying altogether chemotherapeutics and fluorescent probe enters brain tumor cell U87MG or U251MG and sees through blood brain barrier, realizes the treatment of the cerebral tumor;
The Targeted PLGA nanoparticle that carries altogether chemotherapeutics and fluorescent probe suppresses tumor cell in vitro U87MG or U251MG, realizes the treatment of the cerebral tumor;
The Targeted PLGA nanoparticle that carries altogether chemotherapeutics and genomic medicine or carry altogether chemotherapeutics and fluorescent probe suppresses in-vivo tumour A, realizes the treatment of the cerebral tumor; Described tumor A is that the described cerebral tumor is U87, U251 glioma cell, hair shape astrocytoma, astrocytoma, poorly differentiated astrocytoma, a kind of in pleiomorphism neuroblastoma.
The evaluation that above-mentioned Targeted PLGA nanoparticle picked-up of carrying altogether chemotherapeutics and fluorescent probe enters brain tumor cell U87MG or U251 adopts laser confocal microscope, observe brain tumor cell U87MG or U251MG through carrying altogether the Targeted PLGA nanoparticle effect 1h of chemotherapeutics and fluorescent probe, 2h, intracellular fluorescence distribution situation after 3h.
The evaluation that the above-mentioned Targeted PLGA nanoparticle that carries altogether chemotherapeutics and fluorescent probe sees through blood brain barrier ability adopts the micro-and living imaging technology of laser co-focusing.Mouse tail vein injection carries the Targeted PLGA nanoparticle 1h of chemotherapeutics and fluorescent probe altogether, and 2h, takes out cerebral tissue after 3h, prepare frozen section, directly in living imaging instrument, observes medicine in the distribution situation of brain.Or with the 4' of 1 μ g/mL, 6-diamidino-2-phenylindone (4', 6-diamidino-2-phenylindole, DAPI) dyeing 15min, with the PBS of pH7.4, rinse twice, naturally dry, under laser confocal microscope, observe cerebral tissue Chinese medicine distribution situation.
The evaluation that the above-mentioned Targeted PLGA nanoparticle that carries altogether chemotherapeutics and fluorescent probe sees through blood brain barrier ability can adopt living imaging technology.Mouse tail vein injection carries the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe altogether, injection 1h, and 2h, after 3h, by mouse anesthesia, is placed in living imaging instrument and observes medicine in the distribution situation of brain.Brain fluorescence is more much brighter, and the ability that medicine sees through blood brain barrier is stronger.
The inventive method is reliable and stable, products obtained therefrom quality is good, have and can transmit micromolecule chemicals and genomic medicine simultaneously, or transmit chemotherapeutics and fluorescent probe enters brain by blood brain barrier simultaneously, performance is worked in coordination with the targeted therapy cerebral tumor or the targeted therapy cerebral tumor and is diagnosed the effect in one, the Targeted PLGA nanoparticle that carries altogether the Targeted PLGA nanoparticle of chemotherapeutics and genomic medicine or carry altogether chemotherapeutics and fluorescent probe can enter brain by more drug delivery, make the medicine that can not see through blood brain barrier can see through blood brain barrier, reduce medicine in the distribution of periphery, improve brain drug level, increase the safety of medicinal application, and obtained useful technique effect through test, related tests data is as follows:
1, the investigation that drug-loading system transhipment enters U87 MG cell
Because requiring medicine, cells were tested by flow cytometry siRNA transfection efficiency there is fluorescence, therefore in implementing the process of 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 to hyclone (FBS) 10%, and in the MEM culture medium of mycillin mixed liquor 1%, condition of culture is 37 ℃, 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 ℃, at the bottom of being paved with hole to cell monolayer (6 hole flat underside).Discard culture medium, by depletion of blood nonreactive culture medium, clean cell hole 2 times, add the Targeted PLGA nanoparticle that carries altogether doxorubicin hydrochloride and EGFR siRNA, wherein the concentration of doxorubicin hydrochloride is 3.6 μ g/mL, and siRNA concentration is 50nmol/L.Cultivate after 5h, each hole cleans twice with PBS, with not containing the trypsin digestion cell 2min of EDTA, adds the culture medium that contains serum to stop digestion, collecting cell, and 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 the transfection efficiency to doxorubicin hydrochloride and siRNA at FL2 and FL1 passage difference analysis of cells.
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 sees through the situation test that blood brain barrier enters brain
Get normal BALB/c-nu mice, in tail vein injection example 5, carry altogether the Targeted PLGA nanoparticle (Coumarin-6 dosage 0.16mg/kg) of docetaxel and Coumarin-6.At predetermined point of time (1h after injection, 2h and 3h) cut off thoracic cavity after adopting chloral hydrate anesthesia mice, it is fast that action is wanted, the 20mL syringe connecting through left ventricle insert head sword-shaped needle, scalp acupuncture blunt, from with 45 ℃ of direction inserting needles of long axis of body, needle point inserts in ascending aorta, can see, it is soft that action is wanted, cut off the right heart simultaneously, push after 20mL normal saline has pushed away and change rapidly 4 ℃ of paraformaldehyde 20mL.Cut off scalp, take out brain, with the embedding of OCT glue, be placed in-20 ℃ of pre-coolings, fixedly cerebral tissue, cuts into slices with freezing microtome at-20 ℃, and slice thickness 5 μ m, are placed on microscope slide.DAPI lucifuge dyeing 15min with 1 μ g/mL, cleans three times to remove unnecessary DAPI with the PBS of pH7.4.At laser co-focusing fiber Microscopic observation nanoparticle, enter the situation of brain 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 the amount that nanoparticle sees through blood brain barrier is more.Result shows, at 1h, and 2h, 3h, drug-loading system all has distribution at brain, and 2h and 3h nanoparticle are 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
U87MG brain glioblastoma cell (being provided by Shanghai cell bank) is used as to cancerous cell to be investigated.U87MG cell culture is being contained to hyclone (FBS) 10%, and in the MEM culture medium of mycillin mixed liquor 1%, condition of culture is 37 ℃, 5% CO
2, within every 2～3 days, go down to posterity once.Collect logarithmic (log) phase cell, adjust 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, (edge hole is filled with aseptic PBS).Be placed in 5% CO
2hatch 24 h for 37 ℃, at the bottom of being paved with hole to cell monolayer (96 hole flat underside), add the Targeted PLGA nanoparticle that carries altogether doxorubicin hydrochloride and EGFR siRNA in example 3, wherein doxorubicin hydrochloride concentration 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, take and does not add drug delivery system of the present invention as matched group, and it is 4～6 that multiple hole is set.Cell plates are placed in 5%CO
2in incubator, hatch 48 h, stop cultivating, sucking-off pastille culture medium, every hole is washed 2 times with 150 μ l PBS, adds 10% TCA 200 μ l of pre-cooling, places 1 h for 4 ℃.Outwell fixative, every hole is washed 5 times with deionized water, dries air drying.Every hole adds the SRB solution of 100 μ l, and standing placement 10 min do not wash 5 times air drying with 1% acetic acid with protein bound SRB.In conjunction with the non-buffering of 150 μ l 10 mmol/L Tris alkali dissolution for SRB.At 565 nm places, measure the OD value in every hole.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 deduction blank group.
Result shows, after drug effect 48h, it is 0.18 μ g/mL that drug-loading system is respectively 43%(doxorubicin hydrochloride concentration to the cell inhibitory rate of U87 cell, EGFR siRNA concentration is 50nmol/L), 65%(doxorubicin hydrochloride concentration is 0.36 μ g/mL, EGFR siRNA concentration is 50nmol/L), 76%(doxorubicin hydrochloride concentration 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 approval.
1) in super-clean bench, with pentobarbital sodium (40mg/kg), anaesthetize BALB/c-nu nude mice.
2) first with iodophor disinfection, treat field of operation, with operating scissors, at front cranium place, cut off scalp, 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.At IVC Animal House, raise after 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, the Targeted PLGA nanoparticle group of carrying altogether doxorubicin hydrochloride and EGFR siRNA, wherein doxorubicin hydrochloride dosage is 3mg/kg, the dosage of EGFR siRNA is 1.2mg/kg.Every one day, be administered once, administration 5 times, records the survival period of 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 altogether the Targeted PLGA nanoparticle treated animal of doxorubicin hydrochloride and EGFR siRNA is respectively 18 ± 0.8 days, 2.1 ± 2.0 days, 19 ± 3.0 days, 28 ± 2.7 days.Experimental result shows, the Targeted PLGA nanoparticle group of carrying altogether doxorubicin hydrochloride and EGFR siRNA can significantly improve the median survival interval (comparing P<0.05 with normal saline) of animal, and the Targeted PLGA nanoparticle of year doxorubicin hydrochloride, the animal median survival interval of carrying the Targeted PLGA nanoparticle group of EGFR siRNA is compared and all be there is no significant difference with normal saline.
Above-mentioned showing, the invention provides a kind of targeted drug based on PLGA and carry altogether transmission system, preparation method and the application in treatment of brain tumor.Targeted drug based on PLGA of the present invention carries transmission system altogether, very low to the toxicity of organism, and physics and chemical stability are good, and quality is good, and the condition of preparation easily meets, and raw material sources are abundant, and cost is low.Targeted drug transmission system based on PLGA, Angiopep-2 is as target head, this nanoparticle drug-loading system can see through blood brain barrier, test result shows no matter be external or body in well generation and the development of inhibition tumor cell and tumor tissues, there is the effect of the fine anti-cerebral tumor.Expection can be loaded chemotherapeutics and genomic medicine or combine and load chemotherapeutics and fluorescent probe for being combined, and is that one on treatment of brain tumor medicine innovated greatly.Compared with prior art there is following outstanding useful technique effect:
1) chitosan that Angiopep-2 of the present invention modifies is prepared the PLGA nanoparticle drug-loading system of lotus positive electricity jointly as a kind of novel carrier material and PLGA, can load various chemotherapeutics and genomic medicine simultaneously, has 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 see through blood brain barrier, and inside and outside all has the effect of anti-cerebral glioma.
4) the Targeted PLGA nanoparticle that carries altogether chemotherapeutics and fluorescent probe provided by the invention not only can be realized the targeted therapy of the cerebral tumor, and can realize vivo tumor target tracing, through clinical 116 patient with brain tumors application, except 1 people is without positive effect, all the other have all obtained useful curative effect in various degree, and effective percentage reaches more than 99%, and getting well of its effect do not expected, have actual clinical meaning, economic and social benefit is huge.
Claims (8)
1. the preparation method that the targeting based on Poly(D,L-lactide-co-glycolide carries drug delivery system nanoparticle altogether, it is characterized in that, utilize the active amino on chitosan to connect Angiopep-2, first chitosan reacts with 3-maleimide propanoic acid N-hydroxy-succinamide, make the amino terminal of chitosan there is maleimide, react with the sulfydryl of the Angiopep-2 of end sulfhydrylation again, obtain chitosan-angiopep-2 polymer, the preparation that this polymer is carried to drug delivery system altogether for the targeting based on PLGA, 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, surfactant and chitosan-angiopep-2 polymer dissolution are had or not in RNA enzyme water in adding, become water, oil phase adds in water, magnetic agitation or revolve to steam and remove organic solvent, through centrifugal or dialysis, remove free drug, make PLGA drug-carrying nanometer particle, genomic medicine is added in nanoparticle solution, the targeting obtaining based on PLGA copolymer carries drug delivery system nanoparticle altogether, the method of Targeted PLGA nanoparticle that chemotherapeutics and fluorescent probe are carried in preparation is altogether, PLGA, chemotherapeutics, fluorescent probe and adjuvant are dissolved in organic solvent, is prepared into oil phase, by surfactant and chitosan-angiopep-2 polymer dissolution in the water without RNA enzyme, oil phase adds in water, magnetic agitation or revolve to steam and remove organic solvent, removes free drug through centrifugal or dialysis, obtains carrying altogether the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe.
2. the preparation method that the targeting based on Poly(D,L-lactide-co-glycolide according to claim 1 carries drug delivery system nanoparticle altogether, is characterized in that, by following steps, is realized:
(1), the preparation of chitosan-angiopep-2 polymer, method is:
A, chitosan 20mg is added to mass concentration is in 20% acetic acid solution 8mL, at power, is 300-500W ultrasonic dissolution 30min, with 5M NaOH solution adjust pH to 6.0, obtains chitosan solution;
B, in chitosan solution, add 3-maleimide propanoic acid-N-hydroxy succinic acid imines ester 5mg, under nitrogen protection, 30 ℃, 100r/min magnetic agitation reaction 48h, after having reacted, it is that the bag filter of 8KD-14KD is at the PBS 60h that dialyses that reactant liquor is placed in molecular cut off, remove unreacted 3-maleimide propanoic acid-N-hydroxy succinic acid imines ester, obtain dialysis solution;
C, then in dialysis solution, add the Angiopep-2 2mg of sulfhydrylation, under nitrogen protection, 35 ℃, 100-200r/min stirring reaction 24h, obtain the chitosan that Angiopep-2 modifies, the bag filter that is placed in again molecular cut off and the is 8KD-14KD 48h that dialyses, remove free Angiopep-2, at-80 ℃ of lyophilisation 72h, obtain chitosan-angiopep-2 polymer, standby in-20 ℃ of storages;
The Angiopep-2 of described sulfhydrylation is that carboxyl terminal is a kind of in the sulfhydrylation angiopep-2 obtaining after the angiopep-2 of cysteine or angiopep-2 react with Traut ' s; The angiopep-2 that described carboxyl terminal is connected with cysteine is that a molecule angiopep-2 carboxyl terminal connects the cysteine of a molecule by chemical reaction when synthetic angiopep-2; The angiopep-2 of the sulfhydrylation that described angiopep-2 obtains after reacting with Traut ' s is, Angiopep-2 5mg is dissolved in 20mL ultra-pure water, add 10mg Traut's reagent, 50 ℃ of ultrasonic 1h, make the end of Angiopep-2 have sulfydryl, the bag filter that is then placed in molecular cut off and is 8KD-14kD, at the PBS 48h that dialyses, is removed free angiopep-2 and Traut ' s, dialysis solution, at-80 ℃ of lyophilisation 72h, obtains the Angiopep-2 of sulfhydrylation;
(2), preparation carries the Targeted PLGA nanoparticle of chemotherapeutics and genomic medicine altogether, method is:
A, PLGA copolymer, chemotherapeutics and adjuvant are dissolved in organic solvent, 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 a kind of in 25 ︰ 75,50 ︰ 50,75 ︰ 25, and the molecular weight of PLGA is 17kD, 50KD, a kind of in 100kD; Described chemotherapeutics is a kind of 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 polymer dissolution, in pH value, be 6 without in RNA enzyme water, be prepared into water, surfactant concentration is 5-50mg/ml, and the concentration of chitosan-angiopep-2 is 0.02-2mg/ml;
Described pH value be 6 without RNA enzyme water, be, first will in water, add the pyrocarbonic acid diethyl ester of 1 ‰ volumes, 25 ℃, 500r/min magnetic agitation 12h, be sterilizing 30min under 6,121 ℃, 102.9kPa condition with second acid for adjusting pH; 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 water, the volume ratio of water and oil phase is 4-10, obtains load positive charge PLGA copolymer drug-carrying nanometer particle, through stirring or revolving to steam, removes organic solvent, must remove the PLGA copolymer drug-carrying nanometer particle solution after organic solvent;
D, will remove organic solvent after PLGA copolymer drug-carrying nanometer particle solution through the centrifugal free drug of removing, or free drug is removed in dialysis, again genomic medicine is added in nanoparticle solution, obtain the targeted drug transmission system nanoparticle that carries altogether chemotherapeutics and genomic medicine;
(3) carry altogether the Targeted PLGA nanoparticle of chemotherapeutics and fluorescent probe, method is:
A, PLGA copolymer, chemotherapeutics and adjuvant are dissolved in organic solvent, 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 a kind of in 25 ︰ 75,50 ︰ 50,75 ︰ 25, and the molecular weight of PLGA is 17kD, 50KD, a kind of in 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 a kind of in indocyanine green, phthalocyanine, Coumarin-6, Fluorescein isothiocyanate (FITC), methylene blue, rhodamine;
B, by surfactant and chitosan-angiopep-2 polymer dissolution, 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 water;
Described pH value is that 6 the enzyme water without RNA is, first will in water, add the pyrocarbonic acid diethyl ester of 1 ‰ volumes, and 25 ℃, 500r/min magnetic agitation 12h are sterilizing 30min under 6,121 ℃, 102.9kPa condition with acetic acid adjust pH, and to 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 water, the volume ratio of water and oil phase is 4-10, through stirring or revolving to steam, remove organic solvent, must remove the Targeted PLGA copolymer drug-carrying nanometer particle solution that carries altogether chemotherapeutics and fluorescent probe of organic solvent, through the centrifugal free drug of removing, or dialyse and remove free drug, obtain the targeted drug transmission system nanoparticle that carries altogether chemotherapeutics and fluorescent probe.
3. the preparation method that the targeting based on Poly(D,L-lactide-co-glycolide according to claim 1 carries drug delivery system nanoparticle altogether, is characterized in that, the preparation of described chitosan-angiopep-2 polymer, and method is:
A, chitosan 20mg is added to mass concentration is in 20% acetic acid solution 8mL, at power, is 500W ultrasonic dissolution 30min, with 5M NaOH solution adjust pH to 6.0, obtains chitosan solution;
B, in chitosan solution, add 3-maleimide propanoic acid-N-hydroxy succinic acid imines ester 5mg, under nitrogen protection, 30 ℃, 100r/min magnetic agitation reaction 48h, after having reacted, the bag filter that reactant liquor is placed in molecular cut off 8kD-14kD is at the 1000mL PBS 60h that dialyses, remove unreacted 3-maleimide propanoic acid-N-hydroxy succinic acid imines 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 ℃, make the end of Angiopep-2 have sulfydryl, the bag filter that is placed in molecular cut off and is 8kD-14kD, at the 1000mLPBS 48h that dialyses, is removed free angiopep-2 and Traut ' s, dialysis solution, at-80 ℃ of lyophilisation 72h, obtains the angiopep-2 of sulfhydrylation;
D, in step b, in dialysis solution, add the Angiopep-2 2mg of the sulfhydrylation that step c prepares; under nitrogen protection, 35 ℃, 100-200r/min stirring reaction 24h; obtain the chitosan that Angiopep-2 modifies; the bag filter that is placed in molecular cut off again and is 8kD-14kD is at the 1000mLPBS 48h that dialyses; remove free Angiopep-2; at-80 ℃ of lyophilisation 72h, obtain chitosan-angiopep-2 polymer, in-20 ℃ of storages.
4. the preparation method that the targeting based on Poly(D,L-lactide-co-glycolide according to claim 1 carries drug delivery system nanoparticle altogether, 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, adjusts pH to 6.0 with 5M NaOH;
2) in above-mentioned chitosan solution, add 3-maleimide propanoic acid N-hydroxy succinic acid imines ester 5mg, under nitrogen protection, heat 30 ° of C, 100r/min magnetic agitation reaction 48h, after having reacted, reactant liquor is placed in the bag filter of molecular cut off 8kD-14kD and removes unreacted 3-maleimide propanoic acid N-hydroxy succinic acid imines ester in 1000mLPBS dialysis, dialysis 60h;
3) take out the solution in bag filter; adding end is the Angiopep-2 of cysteine; under nitrogen protection, heated and stirred is fully reacted it; it is that the bag filter of 8kD-14kD is at the 1000mLPBS 48h that dialyses 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 ℃ of refrigerators.
5. the preparation method that the targeting based on Poly(D,L-lactide-co-glycolide according to claim 1 carries drug delivery system nanoparticle altogether, 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 in the aqueous solution that 100mL contains volume 1 ‰ DEPC, after dissolve complete, with second acid for adjusting pH to 6.0, add chitosan-Angiopep-2 62.5mg, ultrasonic dissolution is complete, as water;
3) get 5mL doxorubicin hydrochloride stock solution, add in 10mLPLGA storing solution ultrasonic 1min, as oil phase, speed oil phase with 1mL/min dropwise adds in 60mL water under 100rpm stirring condition, Probe Ultrasonic Searching then, 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 preparation method that the targeting based on Poly(D,L-lactide-co-glycolide according to claim 1 carries drug delivery system nanoparticle altogether, 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, after dissolve complete, with second acid for adjusting pH to 6.0, add the about 62.5mg of chitosan-angiopep-2, ultrasonic dissolution is complete, as water;
3) get 15mL oil phase dropwise adds in 60mL water 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.
7. the targeting of the Poly(D,L-lactide-co-glycolide that described in claim 1 prepared by method carries drug delivery system nanoparticle altogether, the application in treatment cerebral tumor medicine.
8. the targeting of Poly(D,L-lactide-co-glycolide according to claim 6 carries the application of drug delivery system nanoparticle in treatment cerebral tumor medicine altogether, it is characterized in that, the application of the Targeted PLGA copolymer nano particle that carries altogether chemotherapeutics and gene in treatment cerebral tumor medicine, or the application of the Targeted PLGA copolymer nano particle that carries altogether chemotherapeutics and fluorescent probe in treatment of brain tumor, diagnostic medicine.
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| CN111956627B (en) * | 2020-09-25 | 2022-02-18 | 郑州大学 | Preparation method and application of drug compound with nano targeting effect for improving II type diabetes pancreatic microenvironment |
| CN112472819A (en) * | 2020-11-30 | 2021-03-12 | 西安交通大学 | Polysaccharide-based nanoparticle carrying adriamycin and indocyanine green together, and preparation method and application thereof |
| CN112472819B (en) * | 2020-11-30 | 2022-04-22 | 西安交通大学 | Polysaccharide-based nanoparticle carrying adriamycin and indocyanine green together, and preparation method and application thereof |
| CN115825442A (en) * | 2021-11-23 | 2023-03-21 | 中国人民解放军总医院第一医学中心 | Application of perovskite nanocrystalline in preparation of probe for tumor diagnosis or treatment |
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