CN102552928A - Double-stage targeted drug delivery system curing brain tumor and preparation method of same - Google Patents
Double-stage targeted drug delivery system curing brain tumor and preparation method of same Download PDFInfo
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- CN102552928A CN102552928A CN201010594309XA CN201010594309A CN102552928A CN 102552928 A CN102552928 A CN 102552928A CN 201010594309X A CN201010594309X A CN 201010594309XA CN 201010594309 A CN201010594309 A CN 201010594309A CN 102552928 A CN102552928 A CN 102552928A
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Abstract
The invention belongs to the technical field of biological medicines, and relates to a targeted drug delivery system curing brain tumor, in particular to a polymer nanoparticle used for brain tumor drug delivery and modified by a carrier system-Angiopep-2, and to a preparation method of the polymer nanoparticle. The system takes protein receptors relevant to low density lipoprotein receptors which are highly expressed on BBB and brain glioma cells as target points, and builds a double-stage targeted nano drug delivery system through LRP receptor-mediated BBB targeting and glioma targeting. The targeted drug delivery system can improve the solubility of PTX and prolongs the in vivo circulation time; moreover, PTX has a sustained release effect when being released from a carrier; carrier material is nontoxic and biodegradable, and has good biocompatibility; and the system can effectively solve the problem of blood-brain-tumor barrier during the chemotherapy of brain glioma, and can be applied to the treatment of the brain glioma.
Description
Technical field
The invention belongs to the biological medicine technology field; Relate to targeting drug delivery system; Be specifically related to a kind of twin-stage targeting drug delivery system that is used to treat brain tumor, pass polymer nanoparticle that the carrier system of medicine-Angiopep-2 modifies and preparation method thereof in particular for brain tumor.
Background technology
Cerebral glioma is one of clinical most common tumor, and research shows that it is to come from neuroectodermal tumor, accounts for 40%～50% of intracranial tumor sickness rate.Because glioma is pernicious infiltrative growth, and is grown in the brain important structure more, like Basal ganglia, the Rolandic fissure district, thalamus, positions such as brain stem, not only operation is difficult to cut entirely, and postoperative is prone to recurrence.World Health Organization's data show, glioblastoma are the 2nd causes of death of tumor patient below 34 years old, are 35～54 years old patient's the 3rd causes of death.In the whole world, annual malignant glioma unfeelingly seizes 18～600,000 young and middle-aged people's valuable life.
Astrocytoma (Astrocytoma) is modal glioma, accounts for 70 ~ 80% of glioma, and it is interior Anywhere to be grown in brain or spinal cord.Research shows that greatly how long adult's astrocytoma at brain, and child's astrocytoma is then often long at XIAONAO and brain stem.With regard to the grade of malignancy of tumor; Astrocytoma can be divided into following level Four: the first order-Mao shape astrocytoma (Pilocytic Astrocytoma); The second level-astrocytoma (Astrocytoma) belongs to low malignant tumor, the third level-poorly differentiated astrocytoma (Anaplastic Astrocytoma; AA), the fourth stage-pleiomorphism spongioblastoma (Glioblastoma Multiform; GBM) belong to malignant tumor
[1]
The growth characteristic of glioma is infiltrative growth, does not have obvious boundary with normal cerebral tissue, and majority is not limited to a cerebral lobe; Outside cerebral tissue, be finger-like and deeply destroy cerebral tissue, optimum partially person's poor growth, the course of disease is longer; From symptom to consultation time occurring average 2 years, pernicious person's tumor bulk-growth is fast, and the course of disease is short; Majority is within 3 months when occurring symptom to prescription on individual diagnosis, and 70-80% is many within half a year.
Operative treatment is based on the growth characteristic of glioma; Can not excise fully in theory; Be grown in then not performing the operation that the tumor of significant points such as brain stem has, reduce tumor cell quantity, alleviate lotus tumor symptom, temporarily reduce intracranial pressure, accomplish four diagnosis and treatment purposes such as pathologic diagnosis of tumor so the therapeutic purposes of operation can only be confined to reduce gross tumor volume.Yet operation but can activate the oncocyte that is in hibernation and gets into the propagation phase rapidly, causes postoperative malignancy of tumor degree upgrading in a short time and recurs.
No matter the auxiliary radiotherapy of postoperative is still put into practice from theory; All be proved the malignant glioma poor effect; Because only when radiological dose reaches 73～80Gy; Could form effectively glioma cell and kill and wound, and the tolerant dosage of normal cerebral tissue only has 60Gy, in fact this dosage be the radiotherapy to fragile cerebral tissue itself.
Chemotherapy is a vital ring in the many treatment links after the glioma excision clinically, and its success or failure are great to patient's quality of life and influence prognosis.Malignant glioma extensively soaks into growth and oncocyte existing away from primary tumor, has determined its postoperative should carry out system's chemotherapy, is difficult to all to reach especially that chemotherapy is present important clinically auxiliary treatment means under the situation of curative effect in operation and radiotherapy.The chemotherapy of this cancer at present makes much progress; But postoperative is auxiliary be through the distinct issues of Venous system chemotherapy; Because cerebral blood flow is 1/5 of a systemic blood flow, has the special construction of brain blood brain barrier (BBB) simultaneously, the chemical therapeutic effect of cerebral glioma is very undesirable.Therefore, the targeted therapy method of research cerebral glioma seems particularly important.
Microvascular endothelial gap in the normal structure is fine and close, structural integrity; Macromole and lipid granule are difficult for seeing through blood vessel wall; And solid tumor is organized that medium vessels is abundant, blood vessel wall gap broad, poor structural integrity, and the lymphatic return disappearance causes macromole class material and lipid granule to have selectivity high-permeability and anelasticity; This phenomenon is known as the high-permeability and the retention effect of solid tumor tissue, is called for short EPR effect (enhanced permeability and retention effect).The pathologic structure characteristics of solid tumor tissue make the macromole anticarcinogen have passive targeting property or characteristic optionally to solid tumor, and more distribution is arranged in tumor tissues after the whole body administration, are called the passive targeting of solid tumor again; And the micromolecule anticarcinogen can freely pass through the blood vessel wall of normal structure and tumor tissues; Consistent in normal structure with drug distribution in the tumor tissues; Be to cause one of anticarcinogenic effect poor selectivity, major reason that toxic and side effects is stronger, do not possess the passive target effect.Based on the oncotherapy strategy of the EPR characteristic of solid tumor tissue, can increase the targeting property of cancer therapy drug to a great extent, improve drug effect, alleviate toxic and side effects.
Nanoparticle is one type of more drug carrier system of passing that is used for the brain tumor treatment of research at present, the main because surface of nanoparticle easily functionalization (like the PEGization extension body internal recycle time), make the nanoparticle entering tumor locus that carries chemotherapeutics and strengthen chemotherapy effect by the EPR effect of tumor.But the passive target effect of depending merely on this tumor is difficult to the chemotherapy effect of breakthrough raising cerebral glioma, mainly is because 1) the inner cell of cerebral glioma is fine and close, special physiological environment such as interstice's pressure is big, anoxia have hindered chemotherapeutics entering; 2) cerebral glioma is infiltrative growth, and not obvious, the abundant BBB of tumor EPR effect that soaks into normal cerebral tissue has hindered the infiltration of chemotherapeutics.In the clinical treatment, be badly in need of solving the simultaneous blood-brain of cerebral glioma chemotherapy-tumor barrier (Blood Brain Tumor Barrier, problem BBTB) at present.
Discover that Angiopep-2 is that a kind of molecular weight is the micromolecule polypeptide of 2.4K, it can be with the transhipment through the receptor-mediated remarkable increase BBB of LRP, and its brain penetrating power is 10 times of Transfferin.Angiopep-2 and paclitaxel (PTX) conjugate ANG1005 gets into anti-cerebral glioma in the U.S. first phase clinical research.But this conjugate is poorly water-soluble still, and is the same with Taxol during application, need be by polyoxyethylene castor oil and ethanol as solubilizing agent, so still there are shortcomings such as sensitization, toxicity and body-internal-circulation time is short.
Summary of the invention
The objective of the invention is to overcome the defective and the deficiency of prior art; A kind of targeting drug delivery system of treating brain tumor is provided, is specifically related to a kind of new brain tumor that is used for and passs the polymer nanoparticle that the carrier system of medicine-Angiopep-2 modifies and (be called for short: ANG-NP-PTX) and preparation method thereof.
The targeting drug delivery system of treatment brain tumor of the present invention; LDH receptor related protein (LRP) receptor with equal high expressed on blood brain barrier (BBB) and the brain glioblastoma cell is an action target spot, makes up through receptor-mediated BBB targeting of LRP and glioma targeting twin-stage targeted nano delivery system.
Particularly, the invention provides the PEG-PCL-PTX nanoparticle (abbreviation: ANG-NP-PTX) that Angiopep-2 modifies.
Targeting drug delivery system of the present invention prepares through following method:
At first, a certain amount of MePEG-PCL, Male-PEG-PCL and PTX be dissolved in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion; Secondly, be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min; Once more, 40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, and abandoning supernatant promptly gets NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm; Then, the NP-PTX for preparing is disperseed with a certain amount of HEPES liquid (pH 7.0) piping and druming, add the Angiopep-2 of amount of calculation, inflated with nitrogen, lucifuge are hatched under the room temperature; At last, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm makes ANG-NP-PTX.
Among the present invention, PTX and MePEG-PCL:Male-PEG-PCL=4～20 : 1,
Among the present invention, Male and Angiopep-2 mol ratio are 1～10 : 1
The targeted system that the present invention is used to treat brain tumor has carried out Evaluation in Vivo and in Vitro:
Angiopep-2 modification PEG-PCL paclitaxel targeted nano granule to the present invention's preparation has carried out vitro drug release test, cytotoxicity test, cellular uptake test, inside and outside twin-stage targeting Journal of Sex Research; The result shows; After Angiopep-2 modifies, significantly do not change the behavior of PTX release in vitro, but significantly increase the cytotoxicity of U87 MG cell; Increase the picked-up ability of U87 MG cell, and result proof in inside and outside demonstrates twin-stage targeting property.
1. extracorporeal releasing test
ANG-NP-PTX of the present invention system extracorporeal releasing test is adopted following dialysis:
Getting 1 ml nanoparticle solution, to join molecular cut off be in 1.4 ten thousand the preparatory swelling bag filter, to tighten bag mouth, drops into 37 ℃ of constant temperature, in 100 rpm, the 39 mL1M sodium salicylate solution; Regularly from release medium, take out 0.2 mL, add 37 ℃ of blank release medium of equivalent simultaneously; Sample P TX concentration HPLC method is measured, and calculating cumulative discharges percentage rate F (t) (%); The result sees Fig. 1.
As shown in Figure 1; NP-PTX and ANG-NP-PTX are respectively 49.2 % and 50.1 % (P>0.05) in the cumulative release amount of 12 h; 96 h cumulative release amounts are respectively 77.2 % and 79.1 % (P>0.05), and visible two kinds of nanoparticles all present biphase release behavior, preceding 12 rapid release; The back is zero level basically and discharges, and drug release behavior is similar.
The result shows that for ANG-NP-PTX, the finishing of Angiopep-2 does not influence the release behavior of PTX.
2. cell toxicity test
Adopt mtt assay to measure ANG-NP-PTX to U87 MG cells in vitro inhibition test, the result is as shown in Figure 2.
As shown in Figure 2, Taxol, three kinds of preparations of NP-PTX and ANG-NP-PTX are to the IC of U87 MG
50Value is 225 ng/mL, 248 ng/mL and 66 ng/mL, and the result shows that the nanoparticle that Angiopep-2 modifies enlarges markedly the cytotoxicity (p of PTX to U87 MG<0.01), to go into born of the same parents' intake relevant with the receptor-mediated increase of the LRP PTX of ANG-NPPTX through U87 MG surface expression for it.
3. cellular uptake
Through carrier material being carried out be used for investigating after rhodamine isothiocyanate (RBITC) fluorescent labeling the qualitative picked-up situation of cell of carrier system,
The result shows (like Fig. 4 and shown in Figure 5); In 30-120 min; ANG-NP group fluorescence intensity (B among Fig. 4, D, F) obviously is better than NP group (A among Fig. 4, C, E), and this kind picked-up situation is suppressed (as shown in Figure 5) by free Angiopep-2 of 200 μ g/ml and Aprotinin.The result also shows, also can suppress the picked-up of ANG-NP under 4 ℃ of conditions, and explaining that Angiopep-2 modifies increases the picked-up of U87 MG cell to nanoparticle, and this kind picked-up is receptor-mediated through LRP, has energy dependence.
4. the evaluation of external twin-stage targeting property
Employing BCEC-U87 MG co-culture model comes the physiologic barrier of cerebral glioma in the analogue body, estimates the twin-stage targeting of ANG-NP/PTX, and the result sees.
The result shows (as shown in Figure 5); ANG-NP/PTX to U87 MG cell inhibitory rate significantly greater than Taxol and NP/PTX (p < 0.01); This is because BCEC and U87 MG express the LRP receptor; ANG-NP/>PTX increases the dual-target result of BBB transhipment and U87 MG picked-up through LRP mediation, and this twin-stage targeting effect can be competed inhibition (p < 0.01) by the part of free Angiopep-2 of 200 μ g/>mL and Aprotinin LRP receptor.
5. twin-stage targeting property evaluation in the body
Shown in the A among Fig. 6, the nanoparticle of DIR labelling is behind the tail intravenously administrable, and the ANG-NP group obviously is better than the NP group in the brain fluorescence intensity, shows through having increased the brain tropism of nanoparticle after the ANG modification.Isolated organ fluorescence distribution behind 24 h shows (seeing the B among Fig. 6), and the fluorescence intensity of ANG-NP at the brain tumor position obviously is better than the NP group, and presents full brain distribution.
The result shows 1) nanoparticle modified of ANG increases the permeability of blood brain barrier; 2) nanoparticle of ANG modification is increased in accumulating of glioma position and is detained; Therefore, ANG-NP demonstrates certain twin-stage targeting property in vivo.
Experimental result shows that targeted system of the present invention can increase the dissolubility of PTX, the extension body internal recycle time; Has twin-stage target function through receptor-mediated BBB of seeing through of LRP and glioma cell; And; PTX discharges from carrier and has slow releasing function, and nontoxic, the biodegradable and good biocompatibility of carrier material effectively solves the simultaneous blood-brain of cerebral glioma chemotherapy-tumor barrier (Blood Brain Tumor Barrier; BBTB) problem can be applicable to treat cerebral glioma.
The advantage of targeting drug delivery system of the present invention has:
1. through adopting nanotechnology, improve the dissolubility of PTX greatly, avoid the use of the safety issue that organic solvent causes in the process.
2. through being prepared into nanoparticle, utilize the nanometer size effect of nanoparticle and the EPR effect of glioma, help PTX in the tumor locus enrichment.
Prolong PTX circulation time in vivo 3.PEG change long-circulating nanoparticles, increase PTX, reduce reticuloendothelial system phagocytic, reduce PTX system toxic and side effects to the accumulative chance of tumor locus.
4.Angiopep-2 after modifying,, strengthen PTX to the apoptotic effect of tumor cell and the capture ability of G2/M through LRP mediation the having increased picked-up of tumor cell to nanosystems.
5.Angiopep-2 after modifying, increase the ability that nanosystems penetrates blood brain barrier through the LRP mediation.
6.Angiopep-2 after modifying, realize a targeting head base, " two targets " function of BBB and tumor twin-stage targeting through the LRP mediation.
For the ease of understanding, below will describe in detail microemulsion drug-supplying system of the present invention through concrete accompanying drawing and embodiment.What need particularly point out is; Instantiation and accompanying drawing only are in order to explain; Obviously those of ordinary skill in the art can explain according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in the scope of the present invention.
Description of drawings
Fig. 1 is a PTX release in vitro curve chart (n=3) among the present invention.
Fig. 2 is U87 MG cell survival curve figure (n=3) among the present invention.
Fig. 3 is the picked-up fluorogram of U87 MG cell among the present invention; Wherein, Figure A is 30 min with figure B action time, and figure C is 60 min with figure D action time, and figure E is 120 min with figure F action time; Figure A, figure C, figure E are the picked-up situation of NP, and figure B, figure D, figure F are the picked-up situation to ANG-NP.
Fig. 4 is the picked-up fluorogram of U87 MG cell under different rejection conditions among the present invention.
Fig. 5 is the cell viability figure (n=3) of U87 MG in co-culture model among the present invention.
Fig. 6 is the comparison diagram of the twin-stage targeting of ANG-NP/PTX among the present invention, wherein, figure A be lotus U87 MG glioma mice through the time fluorescence distribution figure, figure B is a mice isolated organ fluorogram after 24 hours.
The specific embodiment
Will
PTX and MePEG-PCL:Male-PEG-PCL=20 : 1 join in the dichloromethane and dissolve , adding 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 3 : 1), inflated with nitrogen, lucifuge are hatched 8 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 62.4 ± 4.5, and the ANG number is 7 ± 3 on each nanoparticle.
Will
PTX and MePEG-PCL:Male-PEG-PCL=15 : 1 join in the dichloromethane and dissolve , adding 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 3 : 1), inflated with nitrogen, lucifuge are hatched 8 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 84.5 ± 6.8, and the ANG number is 25 ± 5 on each nanoparticle.
Embodiment 3
Will
PTX and MePEG-PCL:Male-PEG-PCL=9 : 1 join in the dichloromethane and dissolve , adding 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 3 : 1), inflated with nitrogen, lucifuge are hatched 8 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 124.2 ± 8.2, and the ANG number is 54 ± 9 on each nanoparticle.
Embodiment 4
With PTX with
MePEG-PCL:Male-PEG-PCL=4 : 1 join in the dichloromethane and dissolve , adding 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 3 : 1), inflated with nitrogen, lucifuge are hatched 8 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 614.7 ± 102.8, and the ANG number is 85 ± 23 on each nanoparticle.
Embodiment 5
Will
PTX and MePEG-PCL:Male-PEG-PCL=9 : 1Add
Go in dichloromethane to dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 10 : 1), inflated with nitrogen, lucifuge are hatched 8 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 73.4 ± 3.1, and the ANG number is 14 ± 4 on each nanoparticle.
Embodiment 6
PTX and MePEG-PCL:Male-PEG-PCL=9 : 1 are joined in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 5 : 1), inflated with nitrogen, lucifuge are hatched 8 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 93.5 ± 4.8, and the ANG number is 34 ± 6 on each nanoparticle.
Embodiment 7
PTX and MePEG-PCL:Male-PEG-PCL=9 : 1 are joined in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 3 : 1), inflated with nitrogen, lucifuge are hatched 8 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 113.5 ± 7.2, and the ANG number is 62 ± 8 on each nanoparticle.
Embodiment 8
PTX and MePEG-PCL:Male-PEG-PCL=9 : 1 are joined in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 1 : 1), inflated with nitrogen, lucifuge are hatched 8 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 713.5 ± 172.4, and the ANG number is 79 ± 24 on each nanoparticle.
Embodiment 9
PTX and MePEG-PCL:Male-PEG-PCL=9 : 1 are joined in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 1 : 1), inflated with nitrogen, lucifuge are hatched 4 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 74.3 ± 1.1, and the ANG number is 8 ± 2 on each nanoparticle.
PTX and MePEG-PCL:Male-PEG-PCL=9 : 1 are joined in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 1 : 1), inflated with nitrogen, lucifuge are hatched 6 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 91.5 ± 4.7, and the ANG number is 26 ± 7 on each nanoparticle.
Embodiment 11
PTX and MePEG-PCL:Male-PEG-PCL=9 : 1 are joined in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 1 : 1), inflated with nitrogen, lucifuge are hatched 8 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 103.2 ± 7.2, and the ANG number is 52 ± 6 on each nanoparticle.
Embodiment 12
PTX and MePEG-PCL:Male-PEG-PCL=9 : 1 are joined in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 1 : 1), inflated with nitrogen, lucifuge are hatched 12 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 184.3 ± 10.2, and the ANG number is 67 ± 6 on each nanoparticle.
Embodiment 13
PTX and MePEG-PCL:Male-PEG-PCL=9 : 1 are joined in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water (O/W) Emulsion.Be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min.40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm.Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 1 : 1), inflated with nitrogen, lucifuge are hatched 16 h under the room temperature.Abandoning supernatant promptly gets ANG-NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm.Recording particle diameter is 694.3 ± 102.1, and the ANG number is 88 ± 32 on each nanoparticle.
The result of above embodiment shows, the targeted system that the present invention is used to treat brain tumor increases the dissolubility of PTX, extension body internal recycle time; Has twin-stage target function through receptor-mediated BBB of seeing through of LRP and glioma cell; And; PTX discharges from carrier and has slow releasing function, and nontoxic, the biodegradable and good biocompatibility of carrier material effectively solves the simultaneous blood-brain of cerebral glioma chemotherapy-tumor barrier (Blood Brain Tumor Barrier; BBTB) problem can be applicable to treat cerebral glioma.
Claims (4)
1. a targeting drug delivery system of treating brain tumor is characterized in that, described targeting drug delivery system is the PEG-PCL-PTX nanoparticle that Angiopep-2 modifies, and it prepares through following method:
At first, a certain amount of MePEG-PCL, Male-PEG-PCL and PTX be dissolved in the dichloromethane dissolve, add 1% sodium cholate aqueous solution, 200 W under the ice bath, 60 s are interrupted ultrasonic formation oil-in-water O/W Emulsion;
Secondly, be distributed to 0.5% sodium cholate aqueous solution and magnetic agitation 5 min; Once more, 40 ℃ of rotary evaporations are not removed dichloromethane to there being bubble, and abandoning supernatant gets NP-PTX behind 4 ℃ of centrifugal 60 min of 12000 rpm;
Then, the NP-PTX for preparing is disperseed with a certain amount of HEPES liquid pH 7.0 piping and druming, add the Angiopep-2 of amount of calculation, inflated with nitrogen, lucifuge are hatched under the room temperature;
Disperse with a certain amount of HEPES liquid (pH 7.0) piping and druming, add Angiopep-2 (Male and Angiopep-2 mol ratio are 1 : 1),
At last, abandoning supernatant behind 4 ℃ of centrifugal 60 min of 12000 rpm makes the ANG-NP-PTX nanoparticle.
2. by the targeting drug delivery system of the described treatment brain tumor of claim 1, it is characterized in that, in the described method for preparing, PTX and MePEG-PCL:Male-PEG-PCL=4～20 : 1.
3. by the targeting drug delivery system of the described treatment brain tumor of claim 1, it is characterized in that in the described method for preparing, Male among the described Angiopep-2 and Angiopep-2 mol ratio are 1～10 : 1.
4. the purposes of the targeting drug delivery system of the treatment brain tumor of claim 1 in treatment cerebral glioma medicine.
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